In JoVE (1)

Other Publications (122)

Articles by Roberto Kolter in JoVE

Other articles by Roberto Kolter on PubMed

Root Colonization by Pseudomonas Putida: Love at First Sight

Microbiology (Reading, England). Feb, 2002  |  Pubmed ID: 11832496

Alcohol-induced Delay of Viability Loss in Stationary-phase Cultures of Escherichia Coli

Journal of Bacteriology. Jun, 2002  |  Pubmed ID: 12003929

During prolonged incubation in stationary phase Escherichia coli undergoes starvation-induced differentiation, resulting in highly resistant cells. In rich medium with high amino acid content further incubation of cultures at high cell density leads to the generation of a population of cells no longer able to form colonies. The viability loss is due to some component of spent medium, active at high pH and high cell density, and can be prevented either by keeping the pH close to neutrality, by washing off the nonsalt components of the medium, or by keeping the saturating cell density low. Exposure to short-chain n-alcohols within a specific time window in stationary phase also prevents viability loss, in an rpoS-dependent fashion. The development of stress resistance, a hallmark of stationary-phase cells, is affected following alcohol treatment, as is the response to extracellular factors in spent medium. Alcohols seem to block cells in an early phase of starvation-induced differentiation, most likely by interfering with processes important for regulation of sigma(s) such as cell density signals and sensing the nutrient content of the medium.

Pseudomonas-Candida Interactions: an Ecological Role for Virulence Factors

Science (New York, N.Y.). Jun, 2002  |  Pubmed ID: 12077418

Bacterial-fungal interactions have great environmental, medical, and economic importance, yet few have been well characterized at the molecular level. Here, we describe a pathogenic interaction between Pseudomonas aeruginosa and Candida albicans, two opportunistic pathogens. P. aeruginosa forms a dense biofilm on C. albicans filaments and kills the fungus. In contrast, P. aeruginosa neither binds to nor kills yeast-form C. albicans. Several P. aeruginosa virulence factors that are important in disease are involved in the killing of C. albicans filaments. We propose that many virulence factors studied in the context of human infection may also have a role in bacterial-fungal interactions.

Why Are Bacteria Refractory to Antimicrobials?

Current Opinion in Microbiology. Oct, 2002  |  Pubmed ID: 12354553

The incidence of antibiotic resistance in pathogenic bacteria is rising. Antibiotic resistance can be achieved via three distinct routes: inactivation of the drug, modification of the target of action, and reduction in the concentration of drug that reaches the target. It has long been recognized that specific antibiotic resistance mechanisms can be acquired through mutation of the bacterial genome or by gaining additional genes through horizontal gene transfer. Recent attention has also brought to light the importance of different physiological states for the survival of bacteria in the presence of antibiotics. It is now apparent that bacteria have complex, intrinsic resistance mechanisms that are often not detected in the standard antibiotic sensitivity tests performed in clinical laboratories. The development of resistance in bacteria found in surface-associated aggregates or biofilms, owing to these intrinsic mechanisms, is paramount.

Identification and Characterization of a Vibrio Cholerae Gene, MbaA, Involved in Maintenance of Biofilm Architecture

Journal of Bacteriology. Feb, 2003  |  Pubmed ID: 12562809

The formation of biofilms is thought to play a key role in the environmental survival of the marine bacterium Vibrio cholerae. Although the factors involved in V. cholerae attachment to abiotic surfaces have been extensively studied, relatively little is known about the mechanisms involved in the subsequent maturation of the biofilms. Here we report the identification of a novel gene, which we have named mbaA (for maintenance of biofilm architecture), that plays a role in the formation and maintenance of the highly organized three-dimensional architecture of V. cholerae El Tor biofilms. We demonstrate that although the absence of mbaA does not significantly affect the initial attachment of cells onto the surface, it leads to the formation of biofilms that lack the typical structure, including the pillars of cells separated by fluid-filled channels that are evident in mature wild-type biofilms. Microscopic analysis indicates that the absence of mbaA leads to an increase in the amount of extracellular matrix material in the biofilms. The predicted mbaA product is a member of a family of regulatory proteins, containing GGDEF and EAL domains, suggesting that MbaA regulates the synthesis of some component of the biofilm matrix.

Bacterial Evolution Through the Selective Loss of Beneficial Genes. Trade-offs in Expression Involving Two Loci

Genetics. Aug, 2003  |  Pubmed ID: 12930738

The loss of preexisting genes or gene activities during evolution is a major mechanism of ecological specialization. Evolutionary processes that can account for gene loss or inactivation have so far been restricted to one of two mechanisms: direct selection for the loss of gene activities that are disadvantageous under the conditions of selection (i.e., antagonistic pleiotropy) and selection-independent genetic drift of neutral (or nearly neutral) mutations (i.e., mutation accumulation). In this study we demonstrate with an evolved strain of Escherichia coli that a third, distinct mechanism exists by which gene activities can be lost. This selection-dependent mechanism involves the expropriation of one gene's upstream regulatory element by a second gene via a homologous recombination event. Resulting from this genetic exchange is the activation of the second gene and a concomitant inactivation of the first gene. This gene-for-gene expression tradeoff provides a net fitness gain, even if the forfeited activity of the first gene can play a positive role in fitness under the conditions of selection.

Genes Involved in Matrix Formation in Pseudomonas Aeruginosa PA14 Biofilms

Molecular Microbiology. Feb, 2004  |  Pubmed ID: 14731271

Pseudomonas aeruginosa forms diverse matrix-enclosed surface-associated multicellular assemblages (biofilms) that aid in its survival in a variety of environments. One such biofilm is the pellicle that forms at the air-liquid interface in standing cultures. We screened for transposon insertion mutants of P. aeruginosa PA14 that were unable to form pellicles. Analysis of these mutants led to the identification of seven adjacent genes, named pel genes, the products of which appear to be involved in the formation of the pellicle's extracellular matrix. In addition to being required for pellicle formation, the pel genes are also required for the formation of solid surface-associated biofilms. Sequence analyses predicted that three pel genes encode transmembrane proteins and that five pel genes have functional homologues involved in carbohydrate processing. Microscopic and macroscopic observations revealed that wild-type P. aeruginosa PA14 produces a cellulase-sensitive extracellular matrix able to bind Congo red; no extracellular matrix was produced by the pel mutants. A comparison of the carbohydrates produced by the wild-type strain and pel mutants suggested that glucose was a principal component of the matrix material. Together, these results suggest that the pel genes are responsible for the production of a glucose-rich matrix material required for the formation of biofilms by P. aeruginosa PA14.

Burkholderia Spp. Alter Pseudomonas Aeruginosa Physiology Through Iron Sequestration

Journal of Bacteriology. Apr, 2004  |  Pubmed ID: 15060040

Pseudomonas aeruginosa and members of the Burkholderia cepacia complex often coexist in both the soil and the lungs of cystic fibrosis patients. To gain an understanding of how these different species affect each other's physiology when coexisting, we performed a screen to identify P. aeruginosa genes that are induced in the presence of Burkholderia: A random gene fusion library was constructed in P. aeruginosa PA14 by using a transposon containing a promoterless lacZ gene. Fusion strains were screened for their ability to be induced in the presence of Burkholderia strains in a cross-streak assay. Three fusion strains were induced specifically by Burkholderia species; all three had transposon insertions in genes known to be iron regulated. One of these fusion strains, containing a transposon insertion in gene PA4467, was used to characterize the inducing activity from Burkholderia: Biochemical and genetic evidence demonstrate that ornibactin, a siderophore produced by nearly all B. cepacia strains, can induce P. aeruginosa PA4467. Significantly, PA4467 is induced early in coculture with an ornibactin-producing but not an ornibactin-deficient B. cepacia strain, indicating that ornibactin can be produced by B. cepacia and detected by P. aeruginosa when the two species coexist.

Genes Involved in Formation of Structured Multicellular Communities by Bacillus Subtilis

Journal of Bacteriology. Jun, 2004  |  Pubmed ID: 15175311

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.

Escherichia Coli Evolution During Stationary Phase

Research in Microbiology. Jun, 2004  |  Pubmed ID: 15207864

The process of evolution by natural selection has been known for a century and a half, yet the mechanics of selection are still poorly understood. In most cases where natural selection has been studied, the genetic and physiological bases of fitness variation that result in population changes were not identified, leaving only a partial understanding of selection. Starved cultures of the bacterium Escherichia coli present a model system with which to address the genetic and physiological bases of natural selection. This is a model system that also reflects the prevalent state of bacteria in the natural world; due to intense competition for nutrients, microorganisms spend the majority of their lives under starvation conditions. Genetic analyses of a single survivor of starvation identified four adaptive mutations(1). Investigation of these mutations has revealed insights into the molecular and physiological bases of evolution during prolonged starvation stress.

Two Genetic Loci Produce Distinct Carbohydrate-rich Structural Components of the Pseudomonas Aeruginosa Biofilm Matrix

Journal of Bacteriology. Jul, 2004  |  Pubmed ID: 15231777

Pseudomonas aeruginosa forms biofilms, which are cellular aggregates encased in an extracellular matrix. Molecular genetics studies of three common autoaggregative phenotypes, namely wrinkled colonies, pellicles, and solid-surface-associated biofilms, led to the identification of two loci, pel and psl, that are involved in the production of carbohydrate-rich components of the biofilm matrix. The pel gene cluster is involved in the production of a glucose-rich matrix material in P. aeruginosa strain PA14 (L. Friedman and R. Kolter, Mol. Microbiol. 51:675-690, 2004). Here we investigate the role of the pel gene cluster in P. aeruginosa strain ZK2870 and identify a second genetic locus, termed psl, involved in the production of a mannose-rich matrix material. The 11 predicted protein products of the psl genes are homologous to proteins involved in carbohydrate processing. P. aeruginosa is thus able to produce two distinct carbohydrate-rich matrix materials. Either carbohydrate-rich matrix component appears to be sufficient for mature biofilm formation, and at least one of them is required for mature biofilm formation in P. aeruginosa strains PA14 and ZK2870.

The SAT1 Flipper, an Optimized Tool for Gene Disruption in Candida Albicans

Gene. Oct, 2004  |  Pubmed ID: 15474295

The construction of Candida albicans mutants by targeted gene disruption usually depends on the use of nutritional markers for the selection of prototrophic transformants from auxotrophic host strains, but it is becoming increasingly evident that this strategy may cause difficulties in the interpretation of mutant phenotypes. Here, we describe a new method for inactivating both alleles of a target gene in C. albicans wild-type strains to obtain homozygous null mutants. The SAT1 flipping method relies on the use of a cassette that contains a dominant nourseothricin resistance marker (caSAT1) for the selection of integrative transformants and a C. albicans-adapted FLP gene that allows the subsequent excision of the cassette, which is flanked by FLP target sequences, from the genome. Two rounds of integration/excision generate homozygous mutants that differ from the wild-type parent strain only by the absence of the target gene, and reintegration of an intact gene copy for complementation of mutant phenotypes is performed in the same way. Transformants are obtained after only 1 day of growth on a selective medium, and integration into the target locus occurs with high specificity after adding homologous flanking sequences on both sides of the cassette. FLP-mediated excision of the SAT1 flipper cassette is achieved by simply growing the transformants for a few hours in medium without selective pressure, and nourseothricin-sensitive (NouS) derivatives can easily be identified by their slower growth on indicator plates containing a low concentration of nourseothricin. We demonstrate the use of the system by deleting the OPT1 gene, which encodes an oligopeptide transporter, in the C. albicans model strain SC5314. The null mutants became resistant to the toxic peptide KLLEth, and reintroduction of an intact OPT1 copy restored susceptibility. The SAT1 flipping method provides a highly efficient method for gene disruption in C. albicans wild-type strains, which eliminates currently encountered problems in the genetic analysis of this important human fungal pathogen.

A Pseudomonas Aeruginosa Quorum-sensing Molecule Influences Candida Albicans Morphology

Molecular Microbiology. Dec, 2004  |  Pubmed ID: 15554963

Candida albicans is an opportunistic pathogen that is commonly found as a member of the human microflora. The ability of C. albicans to alter its cellular morphology has been associated with its virulence; yeast cells are more prevalent in commensal interactions whereas filamentous cells appear important in opportunistic infections. C. albicans encounters a multitude of other microbial species in the host environment and it is likely that they impact the C. albicans transition between virulent and non-virulent states. Here, we report that C. albicans morphology is significantly affected by the presence of Pseudomonas aeruginosa, another opportunistic pathogen. In a screen using a C. albicans HWP1-lacZ strain to indicate regions of filamentous growth, we identified P. aeruginosa mutants incapable of inhibiting C. albicans filamentation. Through these studies, we found that 3-oxo-C12 homoserine lactone, a cell-cell signalling molecule produced by P. aeruginosa, was sufficient to inhibit C. albicans filamentation without affecting fungal growth rates. Both microscopic analysis and real-time reverse transcription polymerase chain reaction analysis of morphology-specific markers confirmed that filamentation was suppressed by 200 microM 3-oxo-C12 homoserine lactone. Structurally related compounds with a 12-carbon chain length, e.g. C12-acyl homoserine lactone and dodecanol also affected C. albicans filamentation at similar concentrations. In contrast, other acylated homoserine lactones of different chain lengths did not affect fungal morphology. The activity of 3OC12HSL is compared with that of farnesol, a C. albicans-produced molecule also with a C12-backbone. The effects that bacteria have on the morphology of C. albicans represents one of the ways by which bacteria can influence the behaviour of fungal cells.

Surfacing Views of Biofilm Biology

Trends in Microbiology. Jan, 2005  |  Pubmed ID: 15639623

Biofilms: the Matrix Revisited

Trends in Microbiology. Jan, 2005  |  Pubmed ID: 15639628

Microbes often construct and live within surface-associated multicellular communities known as biofilms. The precise structure, chemistry and physiology of the biofilm all vary with the nature of its resident microbes and local environment. However, an important commonality among biofilms is that their structural integrity critically depends upon an extracellular matrix produced by their constituent cells. Extracellular matrices might be as diverse as biofilms, and they contribute significantly to the organization of the community. This review discusses recent advances in our understanding of the extracellular matrix and its role in biofilm biology.

Pseudomonas-Saccharomyces Interactions: Influence of Fungal Metabolism on Bacterial Physiology and Survival

Journal of Bacteriology. Feb, 2005  |  Pubmed ID: 15659672

Fungal-bacterial interactions are ubiquitous, yet their molecular basis is only poorly understood. In this study, a novel beneficial interaction between a strain of Pseudomonas putida and the fungus Saccharomyces cerevisiae was identified. When the bacteria were incubated alone in grape juice or in synthetic medium containing various concentrations of glucose, they lost viability rapidly during stationary phase. However, when the bacteria were incubated in these media in the presence of the fungus, their stationary phase survival improved dramatically. On agar plates containing glucose, the beneficial effects of the fungus were manifested in robust bacterial growth and exopolysaccharide production that led to visible mucoidy. In contrast, bacteria grew poorly and were nonmucoid in such media in the absence of the fungus. By using the available S. cerevisiae deletion library, yeast mutants that were unable to mediate this beneficial interaction were identified. These mutants revealed that the beneficial effect on bacterial physiology and survival was mediated by the ability of the fungus to metabolize the available glucose and consequent effects on the medium's pH. In natural environments where the concentration of glucose is high, it is likely that the presence of fungi has had profound beneficial effects on the physiology and survival of certain P. putida strains throughout their natural history.

A Master Regulator for Biofilm Formation by Bacillus Subtilis

Molecular Microbiology. Feb, 2005  |  Pubmed ID: 15661000

Wild strains of Bacillus subtilis are capable of forming architecturally complex communities of cells known as biofilms. Critical to biofilm formation is the eps operon, which is believed to be responsible for the biosynthesis of an exopolysaccharide that binds chains of cells together in bundles. We report that transcription of eps is under the negative regulation of SinR, a repressor that was found to bind to multiple sites in the regulatory region of the operon. Mutations in sinR bypassed the requirement in biofilm formation of two genes of unknown function, ylbF and ymcA, and sinI, which is known to encode an antagonist of SinR. We propose that these genes are members of a pathway that is responsible for counteracting SinR-mediated repression. We further propose that SinR is a master regulator that governs the transition between a planktonic state in which the bacteria swim as single cells in liquid or swarm in small groups over surfaces, and a sessile state in which the bacteria adhere to each other to form bundled chains and assemble into multicellular communities.

Virulence and the Environment: a Novel Role for Vibrio Cholerae Toxin-coregulated Pili in Biofilm Formation on Chitin

Journal of Bacteriology. May, 2005  |  Pubmed ID: 15866944

The toxin-coregulated pilus (TCP) of Vibrio cholerae is required for intestinal colonization and cholera toxin acquisition. Here we report that TCP mediates bacterial interactions required for biofilm differentiation on chitinaceous surfaces. We also show that undifferentiated TCP- biofilms have reduced ecological fitness and, thus, that chitin colonization may represent an ecological setting outside the host in which selection for a host colonization factor may take place.

Thieves, Assassins and Spies of the Microbial World

Nature Cell Biology. Oct, 2005  |  Pubmed ID: 16195747

Genetically Encoded Short Peptide Tag for Versatile Protein Labeling by Sfp Phosphopantetheinyl Transferase

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2005  |  Pubmed ID: 16236721

An 11-residue peptide with the sequence DSLEFIASKLA was identified from a genomic library of Bacillus subtilis by phage display as an efficient substrate for Sfp phosphopantetheinyl transferase-catalyzed protein labeling by small molecule-CoA conjugates. We name this peptide the "ybbR tag," because part of its sequence is derived from the ybbR ORF in the B. subtilis genome. The site of Sfp-catalyzed ybbR tag labeling was mapped to the underlined Ser residue, and the ybbR tag was found to have a strong tendency for adopting an alpha-helical conformation in solution. Here we demonstrate that the ybbR tag can be fused to the N or C termini of target proteins or inserted in a flexible loop in the middle of a target protein for site-specific protein labeling by Sfp. The short size of the ybbR tag and its compatibility with various target proteins, the broad substrate specificity of Sfp for labeling the ybbR tag with small-molecule probes of diverse structures, and the high specificity and efficiency of the labeling reaction make Sfp-catalyzed ybbR tag labeling an attractive tool for expanding protein structural and functional diversities by posttranslational modification.

Mutations That Activate the Silent Bgl Operon of Escherichia Coli Confer a Growth Advantage in Stationary Phase

Journal of Bacteriology. Dec, 2005  |  Pubmed ID: 16291664

Wild-type strains of Escherichia coli are unable to utilize aromatic beta-glucosides such as arbutin and salicin because the major genetic system that encodes the functions for their catabolism, the bgl operon, is silent and uninducible. We show that strains that carry an activated bgl operon exhibit a growth advantage over the wild type in stationary phase in the presence of the rpoS819 allele that causes attenuated rpoS regulon expression. Our results indicate a possible evolutionary advantage in retaining the silent bgl operon by wild-type bacteria.

Mycobacterial Biofilms: a Greasy Way to Hold It Together

Cell. Dec, 2005  |  Pubmed ID: 16325571

Microorganisms growing on surfaces can form biofilms under certain conditions. In this issue of Cell, Ojha et al. (2005) investigate biofilm formation in mycobacteria. They identify new cell-wall components that are required for the formation of architecturally complex mature biofilms in these bacteria and the surprising involvement of a chaperone protein in this process.

Targets of the Master Regulator of Biofilm Formation in Bacillus Subtilis

Molecular Microbiology. Feb, 2006  |  Pubmed ID: 16430695

Wild strains of the spore-forming bacterium Bacillus subtilis are capable of forming architecturally complex communities of cells. The formation of these biofilms is mediated in part by the 15-gene exopolysaccharide operon, epsA-O, which is under the direct negative control of the SinR repressor. We report the identification of an additional operon, yqxM-sipW-tasA, that is required for biofilm formation and is under the direct negative control of SinR. We now show that all three members of the operon are required for the formation of robust biofilms and that SinR is a potent repressor of the operon that acts by binding to multiple sites in the promoter region. Genome-wide analysis of SinR-controlled transcription indicates that the epsA-O and yqxM-sipW-tasA operons constitute many of the most strongly controlled genes in the SinR regulon. These findings reinforce the view that SinR is a master regulator for biofilm formation and further suggest that a principal biological function of SinR is to govern the assembly of complex multicellular communities.

A Major Protein Component of the Bacillus Subtilis Biofilm Matrix

Molecular Microbiology. Feb, 2006  |  Pubmed ID: 16430696

Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

Activity Screening of Carrier Domains Within Nonribosomal Peptide Synthetases Using Complex Substrate Mixtures and Large Molecule Mass Spectrometry

Biochemistry. Feb, 2006  |  Pubmed ID: 16460000

For screening a pool of potential substrates that load carrier domains found in nonribosomal peptide synthetases, large molecule mass spectrometry is shown to be a new, unbiased assay. Combining the high resolving power of Fourier transform mass spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin, and enterobactin biosynthetic pathways as proof of principle, preferred substrates are readily identified from substrate pools. Furthermore, this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the pksN and pksJ genes that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate.

Microbial Sciences: the Superficial Life of Microbes

Nature. May, 2006  |  Pubmed ID: 16710410

Spontaneous Reversion of Mycobacterium Abscessus from a Smooth to a Rough Morphotype is Associated with Reduced Expression of Glycopeptidolipid and Reacquisition of an Invasive Phenotype

Microbiology (Reading, England). Jun, 2006  |  Pubmed ID: 16735722

Mycobacterium abscessus is an increasingly important cause of human disease; however, virulence determinants are largely uncharacterized. Previously, it was demonstrated that a rough, wild-type human clinical isolate (390R) causes persistent, invasive infection, while a smooth isogenic mutant (390S) has lost this capability. During serial passage of 390S, a spontaneous rough revertant was obtained, which was named 390V. This revertant regained the ability to cause persistent, invasive infection in human monocytes and the lungs of mice. Glycopeptidolipid (GPL), which plays a role in environmental colonization, was present in abundance in the cell wall of 390S, and was associated with sliding motility and biofilm formation. In contrast, a marked reduction in the amount of GPL in the cell wall of 390R and 390V was correlated with cord formation, a property associated with mycobacterial virulence. These results indicate that the ability to switch between smooth and rough morphologies may allow M. abscessus to transition between a colonizing phenotype and a more virulent, invasive form.

Interactions Between Streptomyces Coelicolor and Bacillus Subtilis: Role of Surfactants in Raising Aerial Structures

Journal of Bacteriology. Jul, 2006  |  Pubmed ID: 16788200

Using mixed-species cultures, we have undertaken a study of interactions between two common spore-forming soil bacteria, Bacillus subtilis and Streptomyces coelicolor. Our experiments demonstrate that the development of aerial hyphae and spores by S. coelicolor is inhibited by surfactin, a lipopeptide surfactant produced by B. subtilis. Current models of aerial development by sporulating bacteria and fungi postulate a role for surfactants in reducing surface tension at air-liquid interfaces, thereby removing the major barrier to aerial growth. S. coelicolor produces SapB, an amphipathic peptide that is surface active and required for aerial growth on certain media. Loss of aerial hyphae in developmental mutants can be rescued by addition of purified SapB. While a surfactant from a fungus can substitute for SapB in a mutant that lacks aerial hyphae, not all surfactants have this effect. We show that surfactin is required for formation of aerial structures on the surface of B. subtilis colonies. However, in contrast to this positive role, our experiments reveal that surfactin acts antagonistically by arresting S. coelicolor aerial development and causing altered expression of developmental genes. Our observations support the idea that surfactants function specifically for a given organism regardless of their shared ability to reduce surface tension. Production of surfactants with antagonistic activity could provide a powerful competitive advantage during surface colonization and in competition for resources.

Biofilm Formation by a Bacillus Subtilis Strain That Produces Gamma-polyglutamate

Microbiology (Reading, England). Sep, 2006  |  Pubmed ID: 16946274

The extracellular matrix produced by Bacillus subtilis B-1, an environmental strain that forms robust floating biofilms, was purified, and determined to be composed predominantly of gamma-polyglutamate (gamma-PGA), with a molecular mass of over 1,000 kDa. Both biofilm formation and gamma-PGA production by B. subtilis B-1 increased with increasing Mn(2+) or glycerol concentration. gamma-PGA was produced in a growth-associated manner in standing culture, and floating biofilms were formed. However, gamma-PGA was produced in a non-growth-associated manner in shaking culture conditions. When B. subtilis B-1 was grown in a microaerated culture system, floating biofilm formation and gamma-PGA production were significantly retarded, suggesting that oxygen depletion is involved in the initial steps of floating biofilm formation in standing culture. Proteomic analysis of membrane proteins demonstrated that flagellin, oligopeptide permease and Vpr protease precursor were the major proteins produced by cells in a floating biofilm and a colony.

Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains Via Phosphopantetheinyl Elimination Reactions During Tandem Mass Spectrometry

Biochemistry. Oct, 2006  |  Pubmed ID: 17042494

With the emergence of drug resistance and the genomic revolution, there has been a renewed interest in the genes that are responsible for the generation of bioactive natural products. Secondary metabolites of one major class are biosynthesized at one or more sites by ultralarge enzymes that carry covalent intermediates on phosphopantetheine arms. Because such intermediates are difficult to characterize in vitro, we have developed a new approach for streamlined detection of substrates, intermediates, and products attached to a phosphopantetheinyl arm of the carrier site. During vibrational activation of gas-phase carrier domains, facile elimination occurs in benchtop and Fourier-transform mass spectrometers alike. Phosphopantetheinyl ejections quickly reduce >100 kDa megaenzymes to <1000 Da ions for structural assignment of intermediates at <0.007 Da mass accuracy without proteolytic digestion. This "top down" approach quickly illuminated diverse acyl intermediates on the carrier domains of the nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs) found in the biosynthetic pathways of prodigiosin, pyoluteorin, mycosubtilin, nikkomycin, enterobactin, gramicidin, and several proteins from the orphan pksX gene cluster from Bacillus subtilis. By focusing on just those regions undergoing covalent chemistry, the method delivered clean proof for the reversible dehydration of hydroxymethylglutaryl-S-PksL via incorporation of 2H or 18O from the buffer. The facile nature of this revised assay will allow diverse laboratories to spearhead their NRPS-PKS projects with benchtop mass spectrometers.

Bacillus Subtilis Genome Diversity

Journal of Bacteriology. Feb, 2007  |  Pubmed ID: 17114265

Microarray-based comparative genomic hybridization (M-CGH) is a powerful method for rapidly identifying regions of genome diversity among closely related organisms. We used M-CGH to examine the genome diversity of 17 strains belonging to the nonpathogenic species Bacillus subtilis. Our M-CGH results indicate that there is considerable genetic heterogeneity among members of this species; nearly one-third of Bsu168-specific genes exhibited variability, as measured by the microarray hybridization intensities. The variable loci include those encoding proteins involved in antibiotic production, cell wall synthesis, sporulation, and germination. The diversity in these genes may reflect this organism's ability to survive in diverse natural settings.

A Singular Enzymatic Megacomplex from Bacillus Subtilis

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2007  |  Pubmed ID: 17190806

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

The Identification of Bacillaene, the Product of the PksX Megacomplex in Bacillus Subtilis

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2007  |  Pubmed ID: 17234808

The approximately 80-kb pksX gene cluster in Bacillus subtilis encodes an unusual hybrid polyketide/nonribosomal peptide synthase that has been linked to the production of the uncharacterized antibiotic bacillaene. Multiple copies of this synthase, each similar in size to the ribosome, assemble into a single organelle-like complex with a mass of tens to hundreds of megadaltons. The resource requirements of the assembled megacomplex suggest that bacillaene has an important biological role. By coupling a differential NMR spectroscopic technique with genetically manipulated strains of B. subtilis, we were able to characterize the structure of this unusual secondary metabolite, which could not be predicted by using bioinformatic analysis. We report that bacillaene is a linear molecule with two amide bonds: the first links an alpha-hydroxy carboxylic acid to a omega-amino carboxylic acid containing a conjugated hexaene, and the second links the hexaene-containing carboxylic acid to an (omega-1) amino carboxylic acid containing a conjugated triene. Knowledge of bacillaene's structure has enabled us to annotate the pksX gene cluster and should facilitate the study of bacillaene's biosynthesis as well as its biological role in B. subtilis.

Genome-wide High-throughput Mining of Natural-product Biosynthetic Gene Clusters by Phage Display

Chemistry & Biology. Mar, 2007  |  Pubmed ID: 17379145

We have developed a phage-display method for high-throughput mining of bacterial gene clusters encoding the natural-product biosynthetic enzymes, polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). This method uses the phosphopantetheinyl transferase activity of Sfp to specifically biotinylate NRPS and PKS carrier-protein domains expressed from a library of random genome fragments fused to a gene encoding a phage coat protein. Subsequently, the biotinylated phages are enriched through selection on streptavidin-coated plates. Using this method, we isolated phage clones from the multiple NRPS and PKS gene clusters encoded in the genomes of Bacillus subtilis and Myxococcus xanthus. Due to the rapid and unambiguous identification of carrier domains, this method will provide an efficient tool for high-throughput cloning of NRPS and PKS gene clusters from many individual bacterial genomes and multigenome environmental DNA.

Flagellar Motility is Critical for Listeria Monocytogenes Biofilm Formation

Journal of Bacteriology. Jun, 2007  |  Pubmed ID: 17416647

The food-borne pathogen Listeria monocytogenes attaches to environmental surfaces and forms biofilms that can be a source of food contamination, yet little is known about the molecular mechanisms of its biofilm development. We observed that nonmotile mutants were defective in biofilm formation. To investigate how flagella might function during biofilm formation, we compared the wild type with flagellum-minus and paralyzed-flagellum mutants. Both nonmotile mutants were defective in biofilm development, presumably at an early stage, as they were also defective in attachment to glass during the first few hours of surface exposure. This attachment defect could be significantly overcome by providing exogenous movement toward the surface via centrifugation. However, this centrifugation did not restore mature biofilm formation. Our results indicate that it is flagellum-mediated motility that is critical for both initial surface attachment and subsequent biofilm formation. Also, any role for L. monocytogenes flagella as adhesins on abiotic surfaces appears to be either minimal or motility dependent under the conditions we examined.

Quorum-sensing Regulation of the Biofilm Matrix Genes (pel) of Pseudomonas Aeruginosa

Journal of Bacteriology. Jul, 2007  |  Pubmed ID: 17496081

Quorum sensing (QS) has been previously shown to play an important role in the development of Pseudomonas aeruginosa biofilms (D. G. Davies et al., Science 280:295-298, 1998). Although QS regulation of swarming and DNA release has been shown to play important roles in biofilm development, regulation of genes directly involved in biosynthesis of biofilm matrix has not been described. Here, transcription of the pel operon, essential for the production of a glucose-rich matrix exopolysaccharide, is shown to be greatly reduced in lasI and rhlI mutants. Chemical complementation of the lasI mutant with 3-oxo-dodecanoyl homoserine lactone restores pel transcription to the wild-type level and biofilm formation ability. These findings thus connect QS signaling and transcription of genes responsible for biofilm matrix biosynthesis.

Microbiology. Deadly Priming

Science (New York, N.Y.). Oct, 2007  |  Pubmed ID: 17962544

Thinking About Bacillus Subtilis As a Multicellular Organism

Current Opinion in Microbiology. Dec, 2007  |  Pubmed ID: 17977783

Initial attempts to use colony morphogenesis as a tool to investigate bacterial multicellularity were limited by the fact that laboratory strains often have lost many of their developmental properties. Recent advances in elucidating the molecular mechanisms underlying colony morphogenesis have been made possible through the use of undomesticated strains. In particular, Bacillus subtilis has proven to be a remarkable model system to study colony morphogenesis because of its well-characterized developmental features. Genetic screens that analyze mutants defective in colony morphology have led to the discovery of an intricate regulatory network that controls the production of an extracellular matrix. This matrix is essential for the development of complex colony architecture characterized by aerial projections that serve as preferential sites for sporulation. While much progress has been made, the challenge for future studies will be to determine the underlying mechanisms that regulate development such that differentiation occurs in a spatially and temporally organized manner.

Bistability and Biofilm Formation in Bacillus Subtilis

Molecular Microbiology. Jan, 2008  |  Pubmed ID: 18047568

Biofilms of Bacillus subtilis consist of long chains of cells that are held together in bundles by an extracellular matrix of exopolysaccharide and the protein TasA. The exopolysaccharide is produced by enzymes encoded by the epsA-O operon and the gene encoding TasA is located in the yqxM-sipW-tasA operon. Both operons are under the control of the repressor SinR. Derepression is mediated by the antirepressor SinI, which binds to SinR with a 1:1 stoichiometry. Paradoxically, in medium promoting derepression of the matrix operons, the overall concentration of SinR in the culture greatly exceeded that of SinI. We show that under biofilm-promoting conditions sinI, which is under the control of the response regulator Spo0A, was expressed only in a small subpopulation of cells, whereas sinR was expressed in almost all cells. Activation of Spo0A is known to be subject to a bistable switch, and we infer that SinI reaches levels sufficient to trigger matrix production only in the subpopulation of cells in which Spo0A is active. Additionally, evidence suggests that sinI is expressed at intermediate, but not low or high, levels of Spo0A activity, which may explain why certain nutritional conditions are more effective in promoting biofilm formation than others.

Control of Cell Fate by the Formation of an Architecturally Complex Bacterial Community

Genes & Development. Apr, 2008  |  Pubmed ID: 18381896

Bacteria form architecturally complex communities known as biofilms in which cells are held together by an extracellular matrix. Biofilms harbor multiple cell types, and it has been proposed that within biofilms individual cells follow different developmental pathways, resulting in heterogeneous populations. Here we demonstrate cellular differentiation within biofilms of the spore-forming bacterium Bacillus subtilis, and present evidence that formation of the biofilm governs differentiation. We show that motile, matrix-producing, and sporulating cells localize to distinct regions within the biofilm, and that the localization and percentage of each cell type is dynamic throughout development of the community. Importantly, mutants that do not produce extracellular matrix form unstructured biofilms that are deficient in sporulation. We propose that sporulation is a culminating feature of biofilm formation, and that spore formation is coupled to the formation of an architecturally complex community of cells.

A Novel Regulatory Protein Governing Biofilm Formation in Bacillus Subtilis

Molecular Microbiology. Jun, 2008  |  Pubmed ID: 18430133

Production of an extracellular matrix is a hallmark of biofilm formation. In the spore-forming bacterium Bacillus subtilis, the matrix consists of an exopolysaccharide, which is specified by the epsA-O operon, and a secreted protein TasA, which is encoded by the yqxM-sipW-tasA operon. Past and present evidence establish that the epsA-O and yqxM-sipW-tasA operons are controlled by the repressor proteins SinR and AbrB. Here, we report the identification of a novel regulatory protein Slr that promotes transcription of the yqxM-sipW-tasA operon but is not needed for expression of the epsA-O operon. We further show that the gene for Slr is itself under the negative control of SinR and AbrB. These findings reveal that matrix production is governed by an intricate network involving the interplay of negatively and positively acting regulatory proteins.

Ecology and Genomics of Bacillus Subtilis

Trends in Microbiology. Jun, 2008  |  Pubmed ID: 18467096

Bacillus subtilis is a remarkably diverse bacterial species that is capable of growth within many environments. Recent microarray-based comparative genomic analyses have revealed that members of this species also exhibit considerable genomic diversity. The identification of strain-specific genes might explain how B. subtilis has become so broadly adapted. The goal of identifying ecologically adaptive genes could soon be realized with the imminent release of several new B. subtilis genome sequences. As we embark upon this exciting new era of B. subtilis comparative genomics we review what is currently known about the ecology and evolution of this species.

A Keystone Predator Controls Bacterial Diversity in the Pitcher-plant (Sarracenia Purpurea) Microecosystem

Environmental Microbiology. Sep, 2008  |  Pubmed ID: 18479443

The community of organisms inhabiting the water-filled leaves of the carnivorous pitcher-plant Sarracenia purpurea includes arthropods, protozoa and bacteria, and serves as a model system for studies of food web dynamics. Despite the wealth of data collected by ecologists and zoologists on this food web, very little is known about the bacterial assemblage in this microecosystem. We used terminal restriction fragment length polymorphism (T-RFLP) analysis to quantify bacterial diversity within the pitchers as a function of pitcher size, pH of the pitcher fluid and the presence of the keystone predator in this food web, larvae of the pitcher-plant mosquito Wyeomyia smithii. Results were analysed at two spatial scales: within a single bog and across three isolated bogs. Pitchers were sterile before they opened and composition of the bacterial assemblage was more variable between different bogs than within bogs. Measures of bacterial richness and diversity were greater in the presence of W. smithii and increased with increasing pitcher size. Our results suggest that fundamental ecological concepts derived from macroscopic food webs can also be used to predict the bacterial assemblages in pitcher plants.

Generation of Multiple Cell Types in Bacillus Subtilis

FEMS Microbiology Reviews. Jan, 2009  |  Pubmed ID: 19054118

Bacillus subtilis is a Gram-positive bacterium that is well known for its ability to differentiate into metabolically inactive spores that are highly resistant to environmental stresses. In fact, populations of genetically identical B. subtilis comprise numerous distinct cell types. In addition to spores, cells can become genetically competent, motile, produce extracellular matrix or degradative enzymes, or secrete toxins that allow them to cannibalize their neighbors. Many of the cell fates listed above appear to be mutually exclusive. In this review, we discuss how individual cells within a population control their gene expression to ensure that proper regulation of differentiation occurs. These different cell fates are regulated by an intricate network that relies primarily on the activity of three major transcriptional regulators: Spo0A, DegU, and ComK. While individual cells must choose distinct cell fates, the population as a whole exhibits a spectrum of phenotypes whose diversity may increase fitness.

Structurally Diverse Natural Products That Cause Potassium Leakage Trigger Multicellularity in Bacillus Subtilis

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2009  |  Pubmed ID: 19114652

We report a previously undescribed quorum-sensing mechanism for triggering multicellularity in Bacillus subtilis. B. subtilis forms communities of cells known as biofilms in response to an unknown signal. We discovered that biofilm formation is stimulated by a variety of small molecules produced by bacteria--including the B. subtilis nonribosomal peptide surfactin--that share the ability to induce potassium leakage. Natural products that do not cause potassium leakage failed to induce multicellularity. Small-molecule-induced multicellularity was prevented by the addition of potassium, but not sodium or lithium. Evidence is presented that potassium leakage stimulates the activity of a membrane protein kinase, KinC, which governs the expression of genes involved in biofilm formation. We propose that KinC responds to lowered intracellular potassium concentration and that this is a quorum-sensing mechanism that enables B. subtilis to respond to related and unrelated bacteria.

A Widely Conserved Gene Cluster Required for Lactate Utilization in Bacillus Subtilis and Its Involvement in Biofilm Formation

Journal of Bacteriology. Apr, 2009  |  Pubmed ID: 19201793

We report that catabolism of l-lactate in Bacillus subtilis depends on the previously uncharacterized yvfV-yvfW-yvbY (herein renamed lutABC) operon, which is inferred to encode three iron-sulfur-containing proteins. The operon is under the dual control of a GntR-type repressor (LutR, formerly YvfI) and the master regulator for biofilm formation SinR and is induced during growth in response to l-lactate. Operons with high similarity to lutABC are present in the genomes of a variety of gram-positive and gram-negative bacteria, raising the possibility that LutABC is a widely conserved and previously unrecognized pathway for the utilization of l-lactate or related metabolites.

New Developments in Microbial Interspecies Signaling

Current Opinion in Microbiology. Apr, 2009  |  Pubmed ID: 19251475

There is a growing appreciation that in addition to well-documented intraspecies quorum sensing systems, small molecules act as signals between microbes of different species. This review will focus on how bacterial small molecules modulate these interspecies interactions. We will particularly emphasize complex relationships such as those between microbes and insects, interactions resulting in non-antagonistic outcomes (i.e. developmental and morphological processes), how co-culture can lead to the discovery of new small molecules, and the use of known compounds to evoke unexpected responses and mediate crosstalk between microbes.

Interspecies Chemical Communication in Bacterial Development

Annual Review of Microbiology. 2009  |  Pubmed ID: 19566421

Our view of bacteria, from the earliest observations through the heyday of antibiotic discovery, has shifted dramatically. We recognize communities of bacteria as integral and functionally important components of diverse habitats, ranging from soil collectives to the human microbiome. To function as productive communities, bacteria coordinate metabolic functions, often requiring shifts in growth and development. The hallmark of cellular development, which we characterize as physiological change in response to environmental stimuli, is a defining feature of many bacterial interspecies interactions. Bacterial communities rely on chemical exchanges to provide the cues for developmental change. Traditional methods in microbiology focus on isolation and characterization of bacteria in monoculture, separating the organisms from the surroundings in which interspecies chemical communication has relevance. Developing multispecies experimental systems that incorporate knowledge of bacterial physiology and metabolism with insights from biodiversity and metagenomics shows great promise for understanding interspecies chemical communication in the microbial world.

Paracrine Signaling in a Bacterium

Genes & Development. Jul, 2009  |  Pubmed ID: 19605685

Cellular differentiation is triggered by extracellular signals that cause target cells to adopt a particular fate. Differentiation in bacteria typically involves autocrine signaling in which all cells in the population produce and respond to the same signal. Here we present evidence for paracrine signaling in bacterial populations-some cells produce a signal to which only certain target cells respond. Biofilm formation in Bacillus involves two centrally important signaling molecules, ComX and surfactin. ComX triggers the production of surfactin. In turn, surfactin causes a subpopulation of cells to produce an extracellular matrix. Cells that produced surfactin were themselves unable to respond to it. Likewise, once surfactin-responsive cells commenced matrix production, they no longer responded to ComX and could not become surfactin producers. Insensitivity to ComX was the consequence of the extracellular matrix as mutant cells unable to make matrix responded to both ComX and surfactin. Our results demonstrate that extracellular signaling was unidirectional, with one subpopulation producing a signal and a different subpopulation responding to it. Paracrine signaling in a bacterial population ensures the maintenance, over generations, of particular cell types even in the presence of molecules that would otherwise cause those cells to differentiate into other cell types.

Cannibalism Enhances Biofilm Development in Bacillus Subtilis

Molecular Microbiology. Nov, 2009  |  Pubmed ID: 19775247

Cannibalism is a mechanism to delay sporulation in Bacillus subtilis. Cannibal cells express the skf and sdp toxin systems to lyse a fraction of their sensitive siblings. The lysed cells release nutrients that serve to feed the community, effectively delaying spore formation. Here we provide evidence that the subpopulation of cells that differentiates into cannibals is the same subpopulation that produces the extracellular matrix that holds cells together in biofilms. Cannibalism and matrix formation are both triggered in response to the signalling molecule surfactin. Nutrients released by the cannibalized cells are preferentially used by matrix-producing cells, as they are the only cells expressing resistance to the Skf and Sdp toxins. As a result this subpopulation increases in number and matrix production is enhanced when cannibalism toxins are produced. The cannibal/matrix-producing subpopulation is also generated in response to antimicrobials produced by other microorganisms and may thus constitute a defense mechanism to protect B. subtilis from the action of antibiotics in natural settings.

Paralogous Antirepressors Acting on the Master Regulator for Biofilm Formation in Bacillus Subtilis

Molecular Microbiology. Nov, 2009  |  Pubmed ID: 19788541

Matrix production during biofilm formation by Bacillus subtilis is governed by a gene control circuit at the heart of which are three dedicated regulatory proteins, the antirepressor SinI, the repressor SinR and the downstream regulator SlrR. Matrix production is triggered by the synthesis of SinI, which binds to and inactivates SinR, thereby derepressing genes for matrix production as well as the gene for SlrR. Recently, two additional regulators of matrix genes were identified: SlrA, which was reported to be an activator of SlrR, and YwcC, a repressor of SlrA synthesis (Kobayashi, 2008). We present evidence indicating that SlrA, which is a paralogue of SinI, is like SinI, an antirepressor that binds to, and inactivates, SinR. We also show that SlrA does not activate SlrR for expression of matrix genes. Instead, SlrR binds to, and inhibits the activity of, SlrA. Thus, the YwcC-SlrA-SinR-SlrR pathway is a negative feedback loop in which SlrA indirectly stimulates the synthesis of SlrR, and SlrR, in turn, inhibits the activity of SlrA. Finally, we report that under standard laboratory conditions SlrA makes only a small contribution to the expression of genes for matrix production. We propose that in response to an unknown signal recognized by the YwcC repressor, SlrA transiently boosts matrix production.

Bacillus Subtilis Spreads by Surfing on Waves of Surfactant

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2009  |  Pubmed ID: 19826092

The bacterium Bacillus subtilis produces the molecule surfactin, which is known to enhance the spreading of multicellular colonies on nutrient substrates by lowering the surface tension of the surrounding fluid, and to aid in the formation of aerial structures. Here we present experiments and a mathematical model that demonstrate how the differential accumulation rates induced by the geometry of the bacterial film give rise to surfactant waves. The spreading flux increases with increasing biofilm viscosity. Community associations are known to protect bacterial populations from environmental challenges such as predation, heat, or chemical stresses, and enable digestion of a broader range of nutritive sources. This study provides evidence of enhanced dispersal through cooperative motility, and points to nonintuitive methods for controlling the spread of biofilms.

Extracellular Signals That Define Distinct and Coexisting Cell Fates in Bacillus Subtilis

FEMS Microbiology Reviews. Mar, 2010  |  Pubmed ID: 20030732

The soil-dwelling bacterium Bacillus subtilis differentiates into distinct subpopulations of specialized cells that coexist within highly structured communities. The coordination and interplay between these cell types requires extensive extracellular communication driven mostly by sensing self-generated secreted signals. These extracellular signals activate a set of sensor kinases, which respond by phosphorylating three major regulatory proteins, Spo0A, DegU and ComA. Each phosphorylated regulator triggers a specific differentiation program while at the same time repressing other differentiation programs. This allows a cell to differentiate in response to a specific cue, even in the presence of other, possibly conflicting, signals. The sensor kinases involved respond to an eclectic group of extracellular signals, such as quorum-sensing molecules, natural products, temperature, pH or scarcity of nutrients. This article reviews the cascades of cell differentiation pathways that are triggered by sensing extracellular signals. We also present a tentative developmental model in which the diverse cell types sequentially differentiate to achieve the proper development of the bacterial community.

Amyloid Fibers Provide Structural Integrity to Bacillus Subtilis Biofilms

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2010  |  Pubmed ID: 20080671

Bacillus subtilis forms biofilms whose constituent cells are held together by an extracellular matrix. Previous studies have shown that the protein TasA and an exopolysaccharide are the main components of the matrix. Given the importance of TasA in biofilm formation, we characterized the physicochemical properties of this protein. We report that purified TasA forms fibers of variable length and 10-15 nm in width. Biochemical analyses, in combination with the use of specific dyes and microscopic analyses, indicate that TasA forms amyloid fibers. Consistent with this hypothesis, TasA fibers required harsh treatments (e.g., formic acid) to be depolymerized. When added to a culture of a tasA mutant, purified TasA restored wild-type biofilm morphology, indicating that the purified protein retained biological activity. We propose that TasA forms amyloid fibers that bind cells together in the biofilm.

Bacterial Community Succession and Chemical Profiles of Subtidal Biofilms in Relation to Larval Settlement of the Polychaete Hydroides Elegans

The ISME Journal. Jun, 2010  |  Pubmed ID: 20090788

Earlier studies have shown that biofilms can mediate the larval settlement of the polychaete Hydroides elegans and that changes in the bacterial community structure and density of biofilms often alter the larval settlement response. However, the chemical cues that mediate this response remain unknown. In this study, both successional changes in the bacterial community structure and the chemical profiles of subtidal biofilms are described and related to the larval settlement response. Multispecies biofilms were developed on polystyrene Petri dishes and granite rock in the subtidal zone over a period of 20 days. The effects of the substratum and age on the bacterial community structure and chemical profiles of the biofilms were evaluated with two molecular methods (microarray (PhyloChip) and denaturing gradient gel electrophoresis) and with gas chromatography-mass spectrometry, respectively. Both age and substratum altered the bacterial community structures and chemical profiles of the biofilms. Age had a greater effect in shaping the bacterial community structure than did the substratum. In contrast, the type of substratum more strongly affected the chemical profile. Extracts of biofilms of different ages, which developed on different substrata, were tested for the settlement of H. elegans larvae. The extracts induced larval settlement in a biofilm-age-dependent manner, and extracts originating from different substrata of the same age showed no differences in larval settlement. Our results suggest that the larval settlement response cannot be predicted by the overall chemical composition of the biofilm alone.

Relationship Between Cystic Fibrosis Respiratory Tract Bacterial Communities and Age, Genotype, Antibiotics and Pseudomonas Aeruginosa

Environmental Microbiology. May, 2010  |  Pubmed ID: 20192960

Polymicrobial bronchopulmonary infections in cystic fibrosis (CF) cause progressive lung damage and death. Although the arrival of Pseudomonas aeruginosa often heralds a more rapid rate of pulmonary decline, there is significant inter-individual variation in the rate of decline, the causes of which remain poorly understood. By coupling culture-independent methods with ecological analyses, we discovered correlations between bacterial community profiles and clinical disease markers in respiratory tracts of 45 children with CF. Bacterial community complexity was inversely correlated with patient age, presence of P. aeruginosa and antibiotic exposure, and was related to CF genotype. Strikingly, bacterial communities lacking P. aeruginosa were much more similar to each other than were those containing P. aeruginosa, regardless of antibiotic exposure. This suggests that community composition might be a better predictor of disease progression than the presence of P. aeruginosa alone and deserves further study.

An Epigenetic Switch Governing Daughter Cell Separation in Bacillus Subtilis

Genes & Development. Apr, 2010  |  Pubmed ID: 20351052

Growing cells of Bacillus subtilis are a bistable mixture of individual motile cells in which genes for daughter cell separation and motility are ON, and chains of sessile cells in which these genes are OFF. How this ON/OFF switch is controlled has been mysterious. Here we report that a complex of the SinR and SlrR proteins binds to and represses genes involved in cell separation and motility. We also report that SinR and SlrR constitute a double-negative feedback loop in which SinR represses the gene for SlrR (slrR), and, by binding to (titrating) SinR, SlrR prevents SinR from repressing slrR. Thus, SlrR indirectly derepresses its own gene, creating a self-reinforcing loop. Finally, we show that, once activated, the loop remains locked in a high SlrR state in which cell separation and motility genes are OFF for extended periods of time. SinR and SlrR constitute an epigenetic switch for controlling genes involved in cell separation and motility.

D-amino Acids Trigger Biofilm Disassembly

Science (New York, N.Y.). Apr, 2010  |  Pubmed ID: 20431016

Bacteria form communities known as biofilms, which disassemble over time. In our studies outlined here, we found that, before biofilm disassembly, Bacillus subtilis produced a factor that prevented biofilm formation and could break down existing biofilms. The factor was shown to be a mixture of D-leucine, D-methionine, D-tyrosine, and D-tryptophan that could act at nanomolar concentrations. D-amino acid treatment caused the release of amyloid fibers that linked cells in the biofilm together. Mutants able to form biofilms in the presence of D-amino acids contained alterations in a protein (YqxM) required for the formation and anchoring of the fibers to the cell. D-amino acids also prevented biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa. D-amino acids are produced by many bacteria and, thus, may be a widespread signal for biofilm disassembly.

The Virulence Regulator PrfA Promotes Biofilm Formation by Listeria Monocytogenes

Journal of Bacteriology. Aug, 2010  |  Pubmed ID: 20511507

Listeria monocytogenes is a food-borne facultative intracellular pathogen. It is widespread in the environment and has several distinct life-styles. The key transcriptional activator PrfA positively regulates L. monocytogenes virulence genes to mediate the transition from extracellular, flagellum-propelled cell to intracellular pathogen. Here we report the first evidence that PrfA also has a significant positive impact on extracellular biofilm formation. Mutants lacking prfA were defective in surface-adhered biofilm formation. The DeltaprfA mutant exhibited wild-type flagellar motility, and its biofilm defect occurred after initial surface adhesion. We also observed that mutations that led to the constitutive expression of PrfA-dependent virulence genes had a minimal impact on biofilm formation. Furthermore, biofilm development was enhanced in a mutant encoding a PrfA protein variant unable to fully transition from the extracellular form to the virulent, intracellular activity conformation. These results indicate that PrfA positively regulates biofilm formation and suggest that PrfA has a global role in modulating the life-style of L. monocytogenes. The requirement of PrfA for optimal biofilm formation may provide selective pressure to maintain this critical virulence regulator when L. monocytogenes is outside host cells in the environment.


Cold Spring Harbor Perspectives in Biology. Jul, 2010  |  Pubmed ID: 20519345

The ability to form biofilms is a universal attribute of bacteria. Biofilms are multicellular communities held together by a self-produced extracellular matrix. The mechanisms that different bacteria employ to form biofilms vary, frequently depending on environmental conditions and specific strain attributes. In this review, we emphasize four well-studied model systems to give an overview of how several organisms form biofilms: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Using these bacteria as examples, we discuss the key features of biofilms as well as mechanisms by which extracellular signals trigger biofilm formation.

Common Biosynthetic Origins for Polycyclic Tetramate Macrolactams from Phylogenetically Diverse Bacteria

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2010  |  Pubmed ID: 20547882

A combination of small molecule chemistry, biosynthetic analysis, and genome mining has revealed the unexpected conservation of polycyclic tetramate macrolactam biosynthetic loci in diverse bacteria. Initially our chemical analysis of a Streptomyces strain associated with the southern pine beetle led to the discovery of frontalamides A and B, two previously undescribed members of this antibiotic family. Genome analyses and genetic manipulation of the producing organism led to the identification of the frontalamide biosynthetic gene cluster and several biosynthetic intermediates. The biosynthetic locus for the frontalamides' mixed polyketide/amino acid structure encodes a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), which resembles iterative enzymes known in fungi. No such mixed iterative PKS-NRPS enzymes have been characterized in bacteria. Genome-mining efforts revealed strikingly conserved frontalamide-like biosynthetic clusters in the genomes of phylogenetically diverse bacteria ranging from proteobacteria to actinomycetes. Screens for environmental actinomycete isolates carrying frontalamide-like biosynthetic loci led to the isolation of a number of positive strains, the majority of which produced candidate frontalamide-like compounds under suitable growth conditions. These results establish the prevalence of frontalamide-like gene clusters in diverse bacterial types, with medicinally important Streptomyces species being particularly enriched.

KinD is a Checkpoint Protein Linking Spore Formation to Extracellular-matrix Production in Bacillus Subtilis Biofilms

MBio. Apr, 2010  |  Pubmed ID: 20689749

Bacillus subtilis cells form multicellular biofilm communities in which spatiotemporal regulation of gene expression occurs, leading to differentiation of multiple coexisting cell types. These cell types include matrix-producing and sporulating cells. Extracellular matrix production and sporulation are linked in that a mutant unable to produce matrix is delayed for sporulation. Here, we show that the delay in sporulation is not due to a growth advantage of the matrix-deficient mutant under these conditions. Instead, we show that the link between matrix production and sporulation is through the Spo0A signaling pathway. Both processes are regulated by the phosphorylated form of the master transcriptional regulator Spo0A. When cells have low levels of phosphorylated Spo0A (Spo0A~P), matrix genes are expressed; however, at higher levels of Spo0A~P, sporulation commences. We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes. This is due to the activity of one of the components of the Spo0A phosphotransfer network, KinD. A deletion of kinD suppresses the sporulation defect of matrix mutants, while its overproduction delays sporulation. Our data indicate that KinD displays a dual role as a phosphatase or a kinase and that its activity is linked to the presence of extracellular matrix in the biofilms. We propose a novel role for KinD in biofilms as a checkpoint protein that regulates the onset of sporulation by inhibiting the activity of Spo0A until matrix, or a component therein, is sensed.

Functional Microdomains in Bacterial Membranes

Genes & Development. Sep, 2010  |  Pubmed ID: 20713508

The membranes of eukaryotic cells harbor microdomains known as lipid rafts that contain a variety of signaling and transport proteins. Here we show that bacterial membranes contain microdomains functionally similar to those of eukaryotic cells. These membrane microdomains from diverse bacteria harbor homologs of Flotillin-1, a eukaryotic protein found exclusively in lipid rafts, along with proteins involved in signaling and transport. Inhibition of lipid raft formation through the action of zaragozic acid--a known inhibitor of squalene synthases--impaired biofilm formation and protein secretion but not cell viability. The orchestration of physiological processes in microdomains may be a more widespread feature of membranes than previously appreciated.

Comparative Analyses of the Bacterial Microbiota of the Human Nostril and Oropharynx

MBio. 2010  |  Pubmed ID: 20802827

The nose and throat are important sites of pathogen colonization, yet the microbiota of both is relatively unexplored by culture-independent approaches. We examined the bacterial microbiota of the nostril and posterior wall of the oropharynx from seven healthy adults using two culture-independent methods, a 16S rRNA gene microarray (PhyloChip) and 16S rRNA gene clone libraries. While the bacterial microbiota of the oropharynx was richer than that of the nostril, the oropharyngeal microbiota varied less among participants than did nostril microbiota. A few phyla accounted for the majority of the bacteria detected at each site: Firmicutes and Actinobacteria in the nostril and Firmicutes, Proteobacteria, and Bacteroidetes in the oropharynx. Compared to culture-independent surveys of microbiota from other body sites, the microbiota of the nostril and oropharynx show distinct phylum-level distribution patterns, supporting niche-specific colonization at discrete anatomical sites. In the nostril, the distribution of Actinobacteria and Firmicutes was reminiscent of that of skin, though Proteobacteria were much less prevalent. The distribution of Firmicutes, Proteobacteria, and Bacteroidetes in the oropharynx was most similar to that in saliva, with more Proteobacteria than in the distal esophagus or mouth. While Firmicutes were prevalent at both sites, distinct families within this phylum dominated numerically in each. At both sites there was an inverse correlation between the prevalences of Firmicutes and another phylum: in the oropharynx, Firmicutes and Proteobacteria, and in the nostril, Firmicutes and Actinobacteria. In the nostril, this inverse correlation existed between the Firmicutes family Staphylococcaceae and Actinobacteria families, suggesting potential antagonism between these groups.

Biofilms in Lab and Nature: a Molecular Geneticist's Voyage to Microbial Ecology

International Microbiology : the Official Journal of the Spanish Society for Microbiology. Mar, 2010  |  Pubmed ID: 20890834

This article reviews the latest findings on how extracellular signaling controls cell fate determination during the process of biofilm formation by Bacillus subtilis in the artificial setting of the laboratory. To complement molecular genetic approaches, surface-associated communities in settings as diverse as the pitcher plant Sarracenia purpurea and the human lung were investigated. The study of the pitcher plant revealed that the presence or absence of a mosquito larva in the pitcher plant controlled bacterial diversity in the ecosystem inside the pitcher plant. Through the analysis of the respiratory tract microbiota of humans suffering from cystic fibrosis (CF) a correlation between lung function and bacterial community diversity was found. Those that had lungs in good condition had also more diverse communities, whereas patients harboring Pseudomonas aeruginosa-the predominant CF pathogen-in their lungs had less diverse communities. Further studies focused on interspecies and intraspecies relationships at the molecular level in search for signaling molecules that would promote biofilm formation. Two molecules were found that induced biofilm formation in B. subtilis: nystatin-released by other species-and surfactin-released by B. subtilis itself. This is a role not previously known for two molecules that were known for other activities-nystatin as an antifungal and surfactin as a surfactant. In addition, surfactin was found to also trigger cannibalism under starvation. This could be a strategy to maintain the population because the cells destroyed serve as nutrients for the rest. The path that led the author to the study of microbial biofilms is also described.

Reversal of an Epigenetic Switch Governing Cell Chaining in Bacillus Subtilis by Protein Instability

Molecular Microbiology. Oct, 2010  |  Pubmed ID: 20923420

Bacillus subtilis forms long chains of cells during growth and biofilm formation. Cell separation is mediated by autolysins, whose genes are under the negative control of a heteromeric complex composed of the proteins SinR and SlrR. Formation of the SinR-SlrR complex is governed by a self-reinforcing, double-negative feedback loop in which SinR represses the gene for SlrR and SlrR, by forming the SinR-SlrR complex, titrates SinR and prevents it from repressing slrR. The loop is a bistable switch and exists in a SlrR(LOW) state in which autolysin genes are on, and a SlrR(HIGH) state in which autolysin genes are repressed by SinR-SlrR. Cells in the SlrR(LOW) state are driven into the SlrR(HIGH) state by SinI, an antirepressor that binds to and inhibits SinR. However, the mechanism by which cells in the SlrR(HIGH) state revert back to the SlrR(LOW) state is unknown. We report that SlrR is proteolytically unstable and present evidence that self-cleavage via a LexA-like autopeptidase and ClpC contribute to its degradation. Cells producing a self-cleavage-resistant mutant of SlrR exhibited more persistent chaining during growth and yielded biofilms with enhanced structural complexity. We propose that degradation of SlrR allows cells to switch from the SlrR(HIGH) to the SlrR(LOW) state.

Potassium Sensing Histidine Kinase in Bacillus Subtilis

Methods in Enzymology. 2010  |  Pubmed ID: 20946851

The soil-dwelling organism Bacillus subtilis is able to form multicellular aggregates known as biofilms. It was recently reported that the process of biofilm formation is activated in response to the presence of various, structurally diverse small-molecule natural products. All of these small-molecule natural products made pores in the membrane of the bacterium, causing the leakage of potassium cations from the cytoplasm of the cell. The potassium cation leakage was sensed by the membrane histidine kinase KinC, triggering the genetic pathway to the production of the extracellular matrix that holds cells within the biofilm. This chapter presents the methodology used to characterize the leakage of cytoplasmic potassium as the signal that induces biofilm formation in B. subtilis via activation of KinC. Development of novel techniques to monitor activation of gene expression in microbial populations led us to discover the differentiation of a subpopulation of cells specialized to produce the matrix that holds all cells together within the biofilm. This phenomenon of cell differentiation was previously missed by conventional techniques used to monitor transcriptional gene expression.

The Biocide Chlorine Dioxide Stimulates Biofilm Formation in Bacillus Subtilis by Activation of the Histidine Kinase KinC

Journal of Bacteriology. Dec, 2010  |  Pubmed ID: 20971918

Bacillus subtilis forms biofilms in response to signals that remain poorly defined. We report that biofilm formation is stimulated by sublethal doses of chlorine dioxide (ClO(2)), an extremely effective and fast-acting biocide. ClO(2) accelerated biofilm formation in B. subtilis as well as in other bacteria, suggesting that biofilm formation is a widely conserved response to sublethal doses of the agent. Biofilm formation depends on the synthesis of an extracellular matrix that holds the constituent cells together. We show that the transcription of the major operons responsible for the matrix production in B. subtilis, epsA-epsO and yqxM-sipW-tasA, was enhanced by ClO(2), in a manner that depended on the membrane-bound kinase KinC. Activation of KinC appeared to be due to the ability of ClO(2) to collapse the membrane potential. Importantly, strains unable to make a matrix were hypersensitive to ClO(2), indicating that biofilm formation is a defensive response that helps protect cells from the toxic effects of the biocide.

Letter to the Editor

Journal of Microbiology & Biology Education. 2010  |  Pubmed ID: 23653692

Spatial Regulation of Histidine Kinases Governing Biofilm Formation in Bacillus Subtilis

Journal of Bacteriology. Feb, 2011  |  Pubmed ID: 21097618

Bacillus subtilis is able to form architecturally complex biofilms on solid medium due to the production of an extracellular matrix. A master regulator that controls the expression of the genes involved in matrix synthesis is Spo0A, which is activated by phosphorylation via a phosphorelay involving multiple histidine kinases. Here we report that four kinases, KinA, KinB, KinC, and KinD, help govern biofilm formation but that their contributions are partially masked by redundancy. We show that the kinases fall into two categories and that the members of each pair (one pair comprising KinA and KinB and the other comprising KinC and KinD) are partially redundant with each other. We also show that the kinases are spatially regulated: KinA and KinB are active principally in the older, inner regions of the colony, and KinC and KinD function chiefly in the younger, outer regions. These conclusions are based on the morphology of kinase mutants, real-time measurements of gene expression using luciferase as a reporter, and confocal microscopy using a fluorescent protein as a reporter. Our findings suggest that multiple signals from the older and younger regions of the colony are integrated by the kinases to determine the overall architecture of the biofilm community.

Shear Stress Increases the Residence Time of Adhesion of Pseudomonas Aeruginosa

Biophysical Journal. Jan, 2011  |  Pubmed ID: 21244830

Although ubiquitous, the processes by which bacteria colonize surfaces remain poorly understood. Here we report results for the influence of the wall shear stress on the early-stage adhesion of Pseudomonas aeruginosa PA14 on glass and polydimethylsiloxane surfaces. We use image analysis to measure the residence time of each adhering bacterium under flow. Our main finding is that, on either surface, the characteristic residence time of bacteria increases approximately linearly as the shear stress increases (∼0-3.5 Pa). To investigate this phenomenon, we used mutant strains defective in surface organelles (type I pili, type IV pili, or the flagellum) or extracellular matrix production. Our results show that, although these bacterial surface features influence the frequency of adhesion events and the early-stage detachment probability, none of them is responsible for the trend in the shear-enhanced adhesion time. These observations bring what we believe are new insights into the mechanism of bacterial attachment in shear flows, and suggest a role for other intrinsic features of the cell surface, or a dynamic cell response to shear stress.

Tracing the Domestication of a Biofilm-forming Bacterium

Journal of Bacteriology. Apr, 2011  |  Pubmed ID: 21278284

Over the course of more than a century of laboratory experimentation, Bacillus subtilis has become "domesticated," losing its ability to carry out many behaviors characteristic of its wild ancestors. One such characteristic is the ability to form architecturally complex communities, referred to as biofilms. Previous work has shown that the laboratory strain 168 forms markedly attenuated biofilms compared with the wild strain NCIB3610 (3610), even after repair of a mutation in sfp (a gene involved in surfactin production) previously known to impair biofilm formation. Here, we show that in addition to the sfp mutation, mutations in epsC, swrA, and degQ are necessary and sufficient to explain the inability of the laboratory strain to produce robust biofilms. Finally, we show that the architecture of the biofilm is markedly influenced by a large plasmid present in 3610 but not 168 and that the effect of the plasmid can be attributed to a gene we designate rapP. When rapP is introduced into 168 together with wild-type alleles of sfp, epsC, swrA, and degQ, the resulting repaired laboratory strain forms biofilms that are as robust as and essentially indistinguishable in architecture from those of the wild strain, 3610. Thus, domestication of B. subtilis involved the accumulation of four mutations and the loss of a plasmid-borne gene.

Evidence That Metabolism and Chromosome Copy Number Control Mutually Exclusive Cell Fates in Bacillus Subtilis

The EMBO Journal. Apr, 2011  |  Pubmed ID: 21326214

Bacillus subtilis chooses between matrix production and spore formation, which are both controlled by the regulator Spo0A~P. We report that metabolism and chromosome copy number dictate which fate is adopted. Conditions that favour low Spo0A~P levels promote matrix production, whereas conditions favouring high levels trigger sporulation. Spo0A~P directs the synthesis of SinI, an antirepressor for the SinR repressor of matrix genes. The regulatory region of sinI contains an activator site that Spo0A~P binds strongly and operators that bind Spo0A~P weakly. Evidence shows that low Spo0A~P levels turn sinI ON and high levels turn sinI OFF and instead switch sporulation ON. Cells in which sinI and sinR were transplanted from their normal position near the chromosome replication terminus to positions near the origin and cells that harboured an extra copy of the genes were blocked in matrix production. Thus, matrix gene expression is sensitive to the number of copies of sinI and sinR. Because cells at the start of sporulation have two chromosomes and matrix-producing cells one, chromosome copy number could contribute to cell-fate determination.

The Jekyll-and-Hyde Chemistry of Phaeobacter Gallaeciensis

Nature Chemistry. Apr, 2011  |  Pubmed ID: 21430694

Emiliania huxleyi, an environmentally important marine microalga, has a bloom-and-bust lifestyle in which massive algal blooms appear and fade. Phaeobacter gallaeciensis belongs to the roseobacter clade of α-Proteobacteria, the populations of which wax and wane with that of E. huxleyi. Roseobacter are thought to promote algal growth by biosynthesizing and secreting antibiotics and growth stimulants (auxins). Here we show that P. gallaeciensis switches its secreted small molecule metabolism to the production of potent and selective algaecides, the roseobacticides, in response to p-coumaric acid, an algal lignin breakdown product that is symptomatic of aging algae. This switch converts P. gallaeciensis into an opportunistic pathogen of its algal host.

Metal-enhanced Fluorescence to Quantify Bacterial Adhesion

Advanced Materials (Deerfield Beach, Fla.). Mar, 2011  |  Pubmed ID: 21433096

Will Biofilm Disassembly Agents Make It to Market?

Trends in Microbiology. Jul, 2011  |  Pubmed ID: 21458996

Nearly 12 years after promising results suggested that antibiofilm agents might be developed into novel therapeutics, there are no such products on the market. In our opinion, the reasons for this have been predominantly economic. Recent developments, however, suggest that there could still be emerging opportunities for the developments of such products.

An Accessory Protein Required for Anchoring and Assembly of Amyloid Fibres in B. Subtilis Biofilms

Molecular Microbiology. Jun, 2011  |  Pubmed ID: 21477127

Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibres, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibres. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibres to the cell wall and to assemble TasA into fibres. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibres can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibres. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibres that are not anchored to the cell.

Structure and Biosynthesis of Amychelin, an Unusual Mixed-ligand Siderophore from Amycolatopsis Sp. AA4

Journal of the American Chemical Society. Aug, 2011  |  Pubmed ID: 21699219

Actinobacteria generate a large number of structurally diverse small molecules with potential therapeutic value. Genomic analyses of this productive group of bacteria show that their genetic potential to manufacture small molecules exceeds their observed ability by roughly an order of magnitude, and this revelation has prompted a number of studies to identify members of the unknown majority. As a potential window into this cryptic secondary metabolome, pairwise assays for developmental interactions within a set of 20 sequenced actinomycetes were carried out. These assays revealed that Amycolatopsis sp. AA4, a so-called "rare" actinomycete, produces a novel siderophore, amychelin, which alters the developmental processes of several neighboring streptomycetes. Using this phenotype as an assay, we isolated amychelin and solved its structure by NMR and MS methods coupled with an X-ray crystallographic analysis of its Fe-complex. The iron binding affinity of amychelin was determined using EDTA competition assays, and a biosynthetic cluster was identified and annotated to provide a tentative biosynthetic scheme for amychelin.

Characterization of Planctomyces Limnophilus and Development of Genetic Tools for Its Manipulation Establish It As a Model Species for the Phylum Planctomycetes

Applied and Environmental Microbiology. Aug, 2011  |  Pubmed ID: 21724885

Planctomycetes represent a remarkable clade in the domain Bacteria because they play crucial roles in global carbon and nitrogen cycles and display cellular structures that closely parallel those of eukaryotic cells. Studies on Planctomycetes have been hampered by the lack of genetic tools, which we developed for Planctomyces limnophilus.

Antibiotics As Signal Molecules

Chemical Reviews. Sep, 2011  |  Pubmed ID: 21786783

Inhibitory Effects of D-amino Acids on Staphylococcus Aureus Biofilm Development

Journal of Bacteriology. Oct, 2011  |  Pubmed ID: 21856845

Biofilms are communities of cells held together by a self-produced extracellular matrix typically consisting of protein, exopolysaccharide, and often DNA. A natural signal for biofilm disassembly in Bacillus subtilis is certain D-amino acids, which are incorporated into the peptidoglycan and trigger the release of the protein component of the matrix. D-amino acids also prevent biofilm formation by the related Gram-positive bacterium Staphylococcus aureus. Here we employed fluorescence microscopy and confocal laser scanning microscopy to investigate how D-amino acids prevent biofilm formation by S. aureus. We report that biofilm formation takes place in two stages, initial attachment to surfaces, resulting in small foci, and the subsequent growth of the foci into large aggregates. D-amino acids did not prevent the initial surface attachment of cells but blocked the subsequent growth of the foci into larger assemblies of cells. Using protein- and polysaccharide-specific stains, we have shown that D-amino acids inhibited the accumulation of the protein component of the matrix but had little effect on exopolysaccharide production and localization within the biofilm. We conclude that D-amino acids act in an analogous manner to prevent biofilm development in B. subtilis and S. aureus. Finally, to investigate the potential utility of D-amino acids in preventing device-related infections, we have shown that surfaces impregnated with D-amino acids were effective in preventing biofilm growth.

Roseobacticides: Small Molecule Modulators of an Algal-bacterial Symbiosis

Journal of the American Chemical Society. Nov, 2011  |  Pubmed ID: 21928816

Marine bacteria and microalgae engage in dynamic symbioses mediated by small molecules. A recent study of Phaeobacter gallaeciensis, a member of the large roseobacter clade of α-proteobacteria, and Emiliania huxleyi, a prominent member of the microphytoplankton found in large algal blooms, revealed that an algal senescence signal produced by E. huxleyi elicits the production of novel algaecides, the roseobacticides, from the bacterial symbiont. In this report, the generality of these findings are examined by expanding the number of potential elicitors. This expansion led to the identification of nine new members of the roseobacticide family, rare bacterial troponoids, which provide insights into both their biological roles and their biosynthesis. The qualitative and quantitative changes in the levels of roseobacticides induced by the additional elicitors and the elicitors' varied efficiencies support the concept of host-targeted roseobacticide production. Structures of the new family members arise from variable substituents at the C3 and C7 positions of the roseobacticide core as the diversifying elements and suggest that the roseobacticides result from modifications and combinations of aromatic amino acids. Together these studies support a model in which algal senescence converts a mutualistic bacterial symbiont into an opportunistic parasite of its hosts.

Extracellular Signaling and Multicellularity in Bacillus Subtilis

Current Opinion in Microbiology. Dec, 2011  |  Pubmed ID: 22024380

Bacillus subtilis regulates its ability to differentiate into distinct, co-existing cell types in response to extracellular signaling molecules produced either by itself, or present in its environment. The production of molecules by B. subtilis cells, as well as their response to these signals, is not uniform across the population. There is specificity and heterogeneity both within genetically identical populations as well as at the strain-level and species-level. This review will discuss how extracellular signaling compounds influence B. subtilis multicellularity with regard to matrix-producing cannibal differentiation, germination, and swarming behavior, as well as the specificity of the quorum-sensing peptides ComX and CSF. It will also highlight how imaging mass spectrometry can aid in identifying signaling compounds and contribute to our understanding of the functional relationship between such compounds and multicellular behavior.

High-throughput Assay for Bacterial Adhesion on Acellular Dermal Matrices and Synthetic Surgical Materials

Plastic and Reconstructive Surgery. Nov, 2011  |  Pubmed ID: 22030489

There has been increasing use of synthetic and acellular dermal matrix materials in surgery, ranging from breast reconstruction to hernia repairs. There is a paucity of data on how acellular dermal matrix compares with other surgical materials as a substrate for bacterial adhesion, the first step in formation biofilm, which occurs in prosthetic wound infections. The authors have designed a high-throughput assay to evaluate Staphylococcus aureus adherence on various synthetic and biologically derived materials.

Interspecies Interactions That Result in Bacillus Subtilis Forming Biofilms Are Mediated Mainly by Members of Its Own Genus

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2011  |  Pubmed ID: 22074846

Many different systems of bacterial interactions have been described. However, relatively few studies have explored how interactions between different microorganisms might influence bacterial development. To explore such interspecies interactions, we focused on Bacillus subtilis, which characteristically develops into matrix-producing cannibals before entering sporulation. We investigated whether organisms from the natural environment of B. subtilis--the soil--were able to alter the development of B. subtilis. To test this possibility, we developed a coculture microcolony screen in which we used fluorescent reporters to identify soil bacteria able to induce matrix production in B. subtilis. Most of the bacteria that influence matrix production in B. subtilis are members of the genus Bacillus, suggesting that such interactions may be predominantly with close relatives. The interactions we observed were mediated via two different mechanisms. One resulted in increased expression of matrix genes via the activation of a sensor histidine kinase, KinD. The second was kinase independent and conceivably functions by altering the relative subpopulations of B. subtilis cell types by preferentially killing noncannibals. These two mechanisms were grouped according to the inducing strain's relatedness to B. subtilis. Our results suggest that bacteria preferentially alter their development in response to secreted molecules from closely related bacteria and do so using mechanisms that depend on the phylogenetic relatedness of the interacting bacteria.

In-depth Characterization Via Complementing Culture-independent Approaches of the Microbial Community in an Acidic Hot Spring of the Colombian Andes

Microbial Ecology. Jan, 2012  |  Pubmed ID: 21947461

The microbial community of a Colombian high mountain hot spring, El Coquito, was analyzed using three different culture-independent assessments of 16S ribosomal RNA genes: clone libraries, pyrosequencing of the V5-V6 hypervariable region, and microarray. This acidic spring had a diverse community composed mainly of Bacteria that shared characteristics with those from other hot springs and extreme acidic environments. The microbial community was dominated by Proteobacteria, Firmicutes, and Planctomycetes and contained chemotrophic bacteria potentially involved in cycling of ferrous and sulfur-containing minerals and phototrophic organisms, most of which were eukaryotic micro-algae. Despite the presence of a large proportion of novel, unclassified sequences, the taxonomic profiles obtained with each strategy showed similarities at higher taxonomic levels. However, some groups, such as Spirochaetes and Aquificae, were identified using only one methodology, and more taxa were detected with the gene array, which also shared more groups with the pyrosequencing data. Overall, the combined use of different approaches provided a broader view of the microbial community in this acidic hot spring.

Osmotic Spreading of Bacillus Subtilis Biofilms Driven by an Extracellular Matrix

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2012  |  Pubmed ID: 22232655

Bacterial biofilms are organized communities of cells living in association with surfaces. The hallmark of biofilm formation is the secretion of a polymeric matrix rich in sugars and proteins in the extracellular space. In Bacillus subtilis, secretion of the exopolysaccharide (EPS) component of the extracellular matrix is genetically coupled to the inhibition of flagella-mediated motility. The onset of this switch results in slow expansion of the biofilm on a substrate. Different strains have radically different capabilities in surface colonization: Flagella-null strains spread at the same rate as wild type, while both are dramatically faster than EPS mutants. Multiple functions have been attributed to the EPS, but none of these provides a physical mechanism for generating spreading. We propose that the secretion of EPS drives surface motility by generating osmotic pressure gradients in the extracellular space. A simple mathematical model based on the physics of polymer solutions shows quantitative agreement with experimental measurements of biofilm growth, thickening, and spreading. We discuss the implications of this osmotically driven type of surface motility for nutrient uptake that may elucidate the reduced fitness of the matrix-deficient mutant strains.

Whole-genome Sequences of Bacillus Subtilis and Close Relatives

Journal of Bacteriology. May, 2012  |  Pubmed ID: 22493193

We sequenced four strains of Bacillus subtilis and the type strains for two closely related species, Bacillus vallismortis and Bacillus mojavensis. We report the high-quality Sanger genome sequences of B. subtilis subspecies subtilis RO-NN-1 and AUSI98, B. subtilis subspecies spizizenii TU-B-10(T) and DV1-B-1, Bacillus mojavensis RO-H-1(T), and Bacillus vallismortis DV1-F-3(T).

Biosynthesis of Piperazic Acid Via N5-hydroxy-ornithine in Kutzneria Spp. 744

Chembiochem : a European Journal of Chemical Biology. May, 2012  |  Pubmed ID: 22522643

Which came first? We have investigated the biosynthesis of the piperazic acid (Piz) building blocks in the kutzneride family of metabolites. The flavin-dependent oxygenase KtzI was shown to convert ornithine to N(5)-OH-Orn. LC-MS/MS showed (13)C(5)-labeled versions of these two amino acids to be direct precursors of piperazic acid in vivo.

A Self-produced Trigger for Biofilm Disassembly That Targets Exopolysaccharide

Cell. Apr, 2012  |  Pubmed ID: 22541437

Biofilms are structured communities of bacteria that are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often have a limited lifespan, disassembling as nutrients become exhausted and waste products accumulate. D-amino acids were previously identified as a self-produced factor that mediates biofilm disassembly by causing the release of the protein component of the matrix in Bacillus subtilis. Here we report that B. subtilis produces an additional biofilm-disassembly factor, norspermidine. Dynamic light scattering and scanning electron microscopy experiments indicated that norspermidine interacts directly and specifically with exopolysaccharide. D-amino acids and norspermidine acted together to break down existing biofilms and mutants blocked in the production of both factors formed long-lived biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B. subtilis, Escherichia coli, and Staphylococcus aureus.

A Massively Spectacular View of the Chemical Lives of Microbes

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2012  |  Pubmed ID: 22711837

A Bacillus Subtilis Sensor Kinase Involved in Triggering Biofilm Formation on the Roots of Tomato Plants

Molecular Microbiology. Aug, 2012  |  Pubmed ID: 22716461

The soil bacterium Bacillus subtilis is widely used in agriculture as a biocontrol agent able to protect plants from a variety of pathogens. Protection is thought to involve the formation of bacterial communities - biofilms - on the roots of the plants. Here we used confocal microscopy to visualize biofilms on the surface of the roots of tomato seedlings and demonstrated that biofilm formation requires genes governing the production of the extracellular matrix that holds cells together. We further show that biofilm formation was dependent on the sensor histidine kinase KinD and in particular on an extracellular CACHE domain implicated in small molecule sensing. Finally, we report that exudates of tomato roots strongly stimulated biofilm formation ex planta and that an abundant small molecule in the exudates, (L) -malic acid, was able to stimulate biofilm formation at high concentrations in a manner that depended on the KinD CACHE domain. We propose that small signalling molecules released by the roots of tomato plants are directly or indirectly recognized by KinD, triggering biofilm formation.

Mixing and Matching Siderophore Clusters: Structure and Biosynthesis of Serratiochelins from Serratia Sp. V4

Journal of the American Chemical Society. Aug, 2012  |  Pubmed ID: 22830960

Interrogation of the evolutionary history underlying the remarkable structures and biological activities of natural products has been complicated by not knowing the functions they have evolved to fulfill. Siderophores-soluble, low molecular weight compounds-have an easily understood and measured function: acquiring iron from the environment. Bacteria engage in a fierce competition to acquire iron, which rewards the production of siderophores that bind iron tightly and cannot be used or pirated by competitors. The structures and biosyntheses of "odd" siderophores can reveal the evolutionary strategy that led to their creation. We report a new Serratia strain that produces serratiochelin and an analog of serratiochelin. A genetic approach located the serratiochelin gene cluster, and targeted mutations in several genes implicated in serratiochelin biosynthesis were generated. Bioinformatic analyses and mutagenesis results demonstrate that genes from two well-known siderophore clusters, the Escherichia coli enterobactin cluster and the Vibrio cholera vibriobactin cluster, were shuffled to produce a new siderophore biosynthetic pathway. These results highlight how modular siderophore gene clusters can be mixed and matched during evolution to generate structural diversity in siderophores.

Osmotic Pressure Can Regulate Matrix Gene Expression in Bacillus Subtilis

Molecular Microbiology. Oct, 2012  |  Pubmed ID: 22882172

Many bacteria organize themselves into structurally complex communities known as biofilms in which the cells are held together by an extracellular matrix. In general, the amount of extracellular matrix is related to the robustness of the biofilm. Yet, the specific signals that regulate the synthesis of matrix remain poorly understood. Here we show that the matrix itself can be a cue that regulates the expression of the genes involved in matrix synthesis in Bacillus subtilis. The presence of the exopolysaccharide component of the matrix causes an increase in osmotic pressure that leads to an inhibition of matrix gene expression. We further show that non-specific changes in osmotic pressure also inhibit matrix gene expression and do so by activating the histidine kinase KinD. KinD, in turn, directs the phosphorylation of the master regulatory protein Spo0A, which at high levels represses matrix gene expression. Sensing a physical cue such as osmotic pressure, in addition to chemical cues, could be a strategy to non-specifically co-ordinate the behaviour of cells in communities composed of many different species.

Galactose Metabolism Plays a Crucial Role in Biofilm Formation by Bacillus Subtilis

MBio. 2012  |  Pubmed ID: 22893383

Galactose is a common monosaccharide that can be utilized by all living organisms via the activities of three main enzymes that make up the Leloir pathway: GalK, GalT, and GalE. In Bacillus subtilis, the absence of GalE causes sensitivity to exogenous galactose, leading to rapid cell lysis. This effect can be attributed to the accumulation of toxic galactose metabolites, since the galE mutant is blocked in the final step of galactose catabolism. In a screen for suppressor mutants restoring viability to a galE null mutant in the presence of galactose, we identified mutations in sinR, which is the major biofilm repressor gene. These mutations caused an increase in the production of the exopolysaccharide (EPS) component of the biofilm matrix. We propose that UDP-galactose is the toxic galactose metabolite and that it is used in the synthesis of EPS. Thus, EPS production can function as a shunt mechanism for this toxic molecule. Additionally, we demonstrated that galactose metabolism genes play an essential role in B. subtilis biofilm formation and that the expressions of both the gal and eps genes are interrelated. Finally, we propose that B. subtilis and other members of the Bacillus genus may have evolved to utilize naturally occurring polymers of galactose, such as galactan, as carbon sources.

Interspecies Modulation of Bacterial Development Through Iron Competition and Siderophore Piracy

Molecular Microbiology. Nov, 2012  |  Pubmed ID: 22931126

While soil-dwelling actinomycetes are renowned for secreting natural products, little is known about the roles of these molecules in mediating actinomycete interactions. In a previous co-culture screen, we found that one actinomycete, Amycolatopsis sp. AA4, inhibited aerial hyphae formation in adjacent colonies of Streptomyces coelicolor. A siderophore, amychelin, mediated this developmental arrest. Here we present genetic evidence that confirms the role of the amc locus in the production of amychelin and in the inhibition of S. coelicolor development. We further characterize the Amycolatopsis sp. AA4 - S. coelicolor interaction by examining expression of developmental and iron acquisition genes over time in co-culture. Manipulation of iron availability and/or growth near Amycolatopsis sp. AA4 led to alterations in expression of the critical developmental gene bldN, and other key downstream genes in the S. coelicolor transcriptional cascade. In Amycolatopsis sp. AA4, siderophore genes were downregulated when grown near S. coelicolor, leading us to find that deferrioxamine E, produced by S. coelicolor, could be readily utilized by Amycolatopsis sp. AA4. Collectively these results suggest that competition for iron via siderophore piracy and species-specific siderophores can alter patterns of gene expression and morphological differentiation during actinomycete interactions.

Identification of Proteins Likely to Be Involved in Morphogenesis, Cell Division, and Signal Transduction in Planctomycetes by Comparative Genomics

Journal of Bacteriology. Dec, 2012  |  Pubmed ID: 23002222

Members of the Planctomycetes clade share many unusual features for bacteria. Their cytoplasm contains membrane-bound compartments, they lack peptidoglycan and FtsZ, they divide by polar budding, and they are capable of endocytosis. Planctomycete genomes have remained enigmatic, generally being quite large (up to 9 Mb), and on average, 55% of their predicted proteins are of unknown function. Importantly, proteins related to the unusual traits of Planctomycetes remain largely unknown. Thus, we embarked on bioinformatic analyses of these genomes in an effort to predict proteins that are likely to be involved in compartmentalization, cell division, and signal transduction. We used three complementary strategies. First, we defined the Planctomycetes core genome and subtracted genes of well-studied model organisms. Second, we analyzed the gene content and synteny of morphogenesis and cell division genes and combined both methods using a "guilt-by-association" approach. Third, we identified signal transduction systems as well as sigma factors. These analyses provide a manageable list of candidate genes for future genetic studies and provide evidence for complex signaling in the Planctomycetes akin to that observed for bacteria with complex life-styles, such as Myxococcus xanthus.

Calcium Causes Multimerization of the Large Adhesin LapF and Modulates Biofilm Formation by Pseudomonas Putida

Journal of Bacteriology. Dec, 2012  |  Pubmed ID: 23042991

LapF is a large secreted protein involved in microcolony formation and biofilm maturation in Pseudomonas putida. Its C-terminal domain shows the characteristics of proteins secreted through a type I secretion system and includes a predicted calcium binding motif. We provide experimental evidence of specific binding of Ca(2+) to the purified C-terminal domain of LapF (CLapF). Calcium promotes the formation of large aggregates, which disappear in the presence of the calcium chelator EGTA. Immunolocalization of LapF also shows the tendency of this protein to accumulate in vivo in certain extracellular regions. These findings, along with results showing that calcium influences biofilm formation, lead us to propose a model in which P. putida cells interact with each other via LapF in a calcium-dependent manner during the development of biofilms.

Catecholate Siderophores Protect Bacteria from Pyochelin Toxicity

PloS One. 2012  |  Pubmed ID: 23071628

Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.

Old Meets New: Using Interspecies Interactions to Detect Secondary Metabolite Production in Actinomycetes

Methods in Enzymology. 2012  |  Pubmed ID: 23084935

Actinomycetes, a group of filamentous, Gram-positive bacteria, have long been a remarkable source of useful therapeutics. Recent genome sequencing and transcriptomic studies have shown that these bacteria, responsible for half of the clinically used antibiotics, also harbor a large reservoir of gene clusters, which have the potential to produce novel secreted small molecules. Yet, many of these clusters are not expressed under common culture conditions. One reason why these clusters have not been linked to a secreted small molecule lies in the way that actinomycetes have typically been studied: as pure cultures in nutrient-rich media that do not mimic the complex environments in which these bacteria evolved. New methods based on multispecies culture conditions provide an alternative approach to investigating the products of these gene clusters. We have recently implemented binary interspecies interaction assays to mine for new secondary metabolites and to study the underlying biology of interactinomycete interactions. Here, we describe the detailed biological and chemical methods comprising these studies.

Histones Join the Fight Against Bacteria Inside Cells

ELife. 2012  |  Pubmed ID: 23150800

Experiments on Drosophila have shown that the histones that are normally bound to lipid droplets inside cells can be released to provide protection against infection.

Biocontrol of Tomato Wilt Disease by Bacillus Subtilis Isolates from Natural Environments Depends on Conserved Genes Mediating Biofilm Formation

Environmental Microbiology. Mar, 2013  |  Pubmed ID: 22934631

Bacillus subtilis and other Bacilli have long been used as biological control agents against plant bacterial diseases but the mechanisms by which the bacteria confer protection are not well understood. Our goal in this study was to isolate strains of B. subtilis that exhibit high levels of biocontrol efficacy from natural environments and to investigate the mechanisms by which these strains confer plant protection. We screened a total of 60 isolates collected from various locations across China and obtained six strains that exhibited above 50% biocontrol efficacy on tomato plants against the plant pathogen Ralstonia solanacearum under greenhouse conditions. These wild strains were able to form robust biofilms both in defined medium and on tomato plant roots and exhibited strong antagonistic activities against various plant pathogens in plate assays. We show that plant protection by those strains depended on widely conserved genes required for biofilm formation, including regulatory genes and genes for matrix production. We provide evidence suggesting that matrix production is critical for bacterial colonization on plant root surfaces. Finally, we have established a model system for studies of B. subtilis-tomato plant interactions in protection against a plant pathogen.

Biofilm Inhibitors That Target Amyloid Proteins

Chemistry & Biology. Jan, 2013  |  Pubmed ID: 23352144

Bacteria establish stable communities, known as biofilms, that are resistant to antimicrobials. Biofilm robustness is due to the presence of an extracellular matrix, which for several species-among them Bacillus subtilis-includes amyloid-like protein fibers. In this work, we show that B. subtilis biofilms can be a simple and reliable tool for screening of molecules with antiamyloid activity. We identified two molecules, AA-861 and parthenolide, which efficiently inhibited biofilms by preventing the formation of amyloid-like fibers. Parthenolide also disrupted pre-established biofilms. These molecules also impeded the formation of biofilms of other bacterial species that secrete amyloid proteins, such as Bacillus cereus and Escherichia coli. Furthermore, the identified molecules decreased the conversion of the yeast protein New1 to the prion state in a heterologous host, indicating the broad range of activity of the molecules.

Sticking Together: Building a Biofilm the Bacillus Subtilis Way

Nature Reviews. Microbiology. Mar, 2013  |  Pubmed ID: 23353768

Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long served as a robust model organism to examine the molecular mechanisms of biofilm formation, and a number of studies have revealed that this process is regulated by several integrated pathways. In this Review, we focus on the molecular mechanisms that control B. subtilis biofilm assembly, and then briefly summarize the current state of knowledge regarding biofilm disassembly. We also discuss recent progress that has expanded our understanding of B. subtilis biofilm formation on plant roots, which are a natural habitat for this soil bacterium.

Synthesis and Activity of Biomimetic Biofilm Disruptors

Journal of the American Chemical Society. Feb, 2013  |  Pubmed ID: 23406351

Biofilms are often associated with human bacterial infections, and the natural tolerance of biofilms to antibiotics challenges treatment. Compounds with antibiofilm activity could become useful adjuncts to antibiotic therapy. We used norspermidine, a natural trigger for biofilm disassembly in the developmental cycle of Bacillus subtilis , to develop guanidine and biguanide compounds with up to 20-fold increased potency in preventing biofilm formation and breaking down existing biofilms. These compounds also were active against pathogenic Staphylococcus aureus . An integrated approach involving structure-activity relationships, protonation constants, and crystal structure data on a focused synthetic library revealed that precise spacing of positively charged groups and the total charge at physiological pH distinguish potent biofilm inhibitors.

Bacterial Flagella Explore Microscale Hummocks and Hollows to Increase Adhesion

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2013  |  Pubmed ID: 23509269

Biofilms, surface-bound communities of microbes, are economically and medically important due to their pathogenic and obstructive properties. Among the numerous strategies to prevent bacterial adhesion and subsequent biofilm formation, surface topography was recently proposed as a highly nonspecific method that does not rely on small-molecule antibacterial compounds, which promote resistance. Here, we provide a detailed investigation of how the introduction of submicrometer crevices to a surface affects attachment of Escherichia coli. These crevices reduce substrate surface area available to the cell body but increase overall surface area. We have found that, during the first 2 h, adhesion to topographic surfaces is significantly reduced compared with flat controls, but this behavior abruptly reverses to significantly increased adhesion at longer exposures. We show that this reversal coincides with bacterially induced wetting transitions and that flagellar filaments aid in adhesion to these wetted topographic surfaces. We demonstrate that flagella are able to reach into crevices, access additional surface area, and produce a dense, fibrous network. Mutants lacking flagella show comparatively reduced adhesion. By varying substrate crevice sizes, we determine the conditions under which having flagella is most advantageous for adhesion. These findings strongly indicate that, in addition to their role in swimming motility, flagella are involved in attachment and can furthermore act as structural elements, enabling bacteria to overcome unfavorable surface topographies. This work contributes insights for the future design of antifouling surfaces and for improved understanding of bacterial behavior in native, structured environments.

Bacillus Subtilis Biofilm Induction by Plant Polysaccharides

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2013  |  Pubmed ID: 23569226

Bacillus subtilis is a plant-beneficial Gram-positive bacterium widely used as a biofertilizer. However, relatively little is known regarding the molecular processes underlying this bacterium's ability to colonize roots. In contrast, much is known about how this bacterium forms matrix-enclosed multicellular communities (biofilms) in vitro. Here, we show that, when B. subtilis colonizes Arabidopsis thaliana roots it forms biofilms that depend on the same matrix genes required in vitro. B. subtilis biofilm formation was triggered by certain plant polysaccharides. These polysaccharides served as a signal for biofilm formation transduced via the kinases controlling the phosphorylation state of the master regulator Spo0A. In addition, plant polysaccharides are used as a source of sugars for the synthesis of the matrix exopolysaccharide. The bacterium's response to plant polysaccharides was observed across several different strains of the species, some of which are known to have beneficial effects on plants. These observations provide evidence that biofilm genes are crucial for Arabidopsis root colonization by B. subtilis and provide insights into how matrix synthesis may be triggered by this plant.

Respiration Control of Multicellularity in Bacillus Subtilis by a Complex of the Cytochrome Chain with a Membrane-embedded Histidine Kinase

Genes & Development. Apr, 2013  |  Pubmed ID: 23599347

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.

Isolation, Characterization, and Aggregation of a Structured Bacterial Matrix Precursor

The Journal of Biological Chemistry. Jun, 2013  |  Pubmed ID: 23632024

Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some β-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.

Interspecies Interactions Stimulate Diversification of the Streptomyces Coelicolor Secreted Metabolome

MBio. 2013  |  Pubmed ID: 23963177

Soils host diverse microbial communities that include filamentous actinobacteria (actinomycetes). These bacteria have been a rich source of useful metabolites, including antimicrobials, antifungals, anticancer agents, siderophores, and immunosuppressants. While humans have long exploited these compounds for therapeutic purposes, the role these natural products may play in mediating interactions between actinomycetes has been difficult to ascertain. As an initial step toward understanding these chemical interactions at a systems level, we employed the emerging techniques of nanospray desorption electrospray ionization (NanoDESI) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry to gain a global chemical view of the model bacterium Streptomyces coelicolor interacting with five other actinomycetes. In each interaction, the majority of secreted compounds associated with S. coelicolor colonies were unique, suggesting an idiosyncratic response from S. coelicolor. Spectral networking revealed a family of unknown compounds produced by S. coelicolor during several interactions. These compounds constitute an extended suite of at least 12 different desferrioxamines with acyl side chains of various lengths; their production was triggered by siderophores made by neighboring strains. Taken together, these results illustrate that chemical interactions between actinomycete bacteria exhibit high complexity and specificity and can drive differential secondary metabolite production.

Whole-Genome Sequences of 94 Environmental Isolates of Bacillus Cereus Sensu Lato

Genome Announcements. 2013  |  Pubmed ID: 24092776

Bacillus cereus sensu lato is a species complex that includes the anthrax pathogen Bacillus anthracis and other bacterial species of medical, industrial, and ecological importance. Their phenotypes of interest are typically linked to large plasmids that are closely related to the anthrax plasmids pXO1 and pXO2. Here, we present the draft genome sequences of 94 isolates of B. cereus sensu lato, which were chosen for their plasmid content and environmental origins.

D-amino Acids Indirectly Inhibit Biofilm Formation in Bacillus Subtilis by Interfering with Protein Synthesis

Journal of Bacteriology. Dec, 2013  |  Pubmed ID: 24097941

The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of D-leucine, D-methionine, D-tryptophan, and D-tyrosine and was reported to inhibit biofilm formation via the incorporation of these D-amino acids into the cell wall. Here, we show that L-amino acids were able to specifically reverse the inhibitory effects of their cognate D-amino acids. We also show that D-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding D-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of D-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of D-amino acids without losing the ability to incorporate at least one noncanonical D-amino acid, D-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of D-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.

Eco-evolutionary Feedbacks Drive Species Interactions

The ISME Journal. May, 2014  |  Pubmed ID: 24304674

In the biosphere, many species live in close proximity and can thus interact in many different ways. Such interactions are dynamic and fall along a continuum between antagonism and cooperation. Because interspecies interactions are the key to understanding biological communities, it is important to know how species interactions arise and evolve. Here, we show that the feedback between ecological and evolutionary processes has a fundamental role in the emergence and dynamics of species interaction. Using a two-species artificial community, we demonstrate that ecological processes and rapid evolution interact to influence the dynamics of the symbiosis between a eukaryote (Saccharomyces cerevisiae) and a bacterium (Rhizobium etli). The simplicity of our experimental design enables an explicit statement of causality. The niche-constructing activities of the fungus were the key ecological process: it allowed the establishment of a commensal relationship that switched to ammensalism and provided the selective conditions necessary for the adaptive evolution of the bacteria. In this latter state, the bacterial population radiates into more than five genotypes that vary with respect to nutrient transport, metabolic strategies and global regulation. Evolutionary diversification of the bacterial populations has strong effects on the community; the nature of interaction subsequently switches from ammensalism to antagonism where bacteria promote yeast extinction. Our results demonstrate the importance of the evolution-to-ecology pathway in the persistence of interactions and the stability of communities. Thus, eco-evolutionary dynamics have the potential to transform the structure and functioning of ecosystems. Our results suggest that these dynamics should be considered to improve our understanding of beneficial and detrimental host-microbe interactions.

Functional Analysis of the Accessory Protein TapA in Bacillus Subtilis Amyloid Fiber Assembly

Journal of Bacteriology. Apr, 2014  |  Pubmed ID: 24488317

Bacillus subtilis biofilm formation relies on the assembly of a fibrous scaffold formed by the protein TasA. TasA polymerizes into highly stable fibers with biochemical and morphological features of functional amyloids. Previously, we showed that assembly of TasA fibers requires the auxiliary protein TapA. In this study, we investigated the roles of TapA sequences from the C-terminal and N-terminal ends and TapA cysteine residues in its ability to promote the assembly of TasA amyloid-like fibers. We found that the cysteine residues are not essential for the formation of TasA fibers, as their replacement by alanine residues resulted in only minor defects in biofilm formation. Mutating sequences in the C-terminal half had no effect on biofilm formation. However, we identified a sequence of 8 amino acids in the N terminus that is key for TasA fiber formation. Strains expressing TapA lacking these 8 residues were completely defective in biofilm formation. In addition, this TapA mutant protein exhibited a dominant negative effect on TasA fiber formation. Even in the presence of wild-type TapA, the mutant protein inhibited fiber assembly in vitro and delayed biofilm formation in vivo. We propose that this 8-residue sequence is crucial for the formation of amyloid-like fibers on the cell surface, perhaps by mediating the interaction between TapA or TapA and TasA molecules.

Altered Desferrioxamine-mediated Iron Utilization is a Common Trait of Bald Mutants of Streptomyces Coelicolor

Metallomics : Integrated Biometal Science. Aug, 2014  |  Pubmed ID: 24788337

Streptomyces coelicolor is an important model organism for developmental studies of filamentous GC-rich actinobacteria. The genetic characterization of mutants of S. coelicolor blocked at the vegetative mycelium stage, the so-called bald (bld) mutants that are unable to erect spore-forming aerial hyphae, has opened the way to discovering the molecular basis of development in actinomycetes. Desferrioxamine (DFO) production and import of ferrioxamines (FO; iron-complexed DFO) are key to triggering morphogenesis of S. coelicolor and we show here that growth of S. coelicolor on the reference medium for Streptomyces developmental studies is fully dependent on DFO biosynthesis. UPLC-ESI-MS analysis revealed that all bld mutants tested are affected in DFO biosynthesis, with bldA, bldJ, and ptsH mutants severely impaired in DFO production, while bldF, bldK, crr and ptsI mutants overproduce DFO. Morphogenesis of bldK and bldJ mutants was recovered by supplying exogenous iron. Transcript analysis showed that the bldJ mutant is impaired in expression of genes involved in the uptake of FO, whereas transcription of genes involved in both DFO biosynthesis and FO uptake is increased in bldK mutants. Our study allows proposing altered DFO production and/or FO uptake as a novel phenotypic marker of many S. coelicolor bld mutants, and strengthens the role of siderophores and iron acquisition in morphological development of actinomycetes.

Sporulation During Growth in a Gut Isolate of Bacillus Subtilis

Journal of Bacteriology. Dec, 2014  |  Pubmed ID: 25225273

Sporulation by Bacillus subtilis is a cell density-dependent response to nutrient deprivation. Central to the decision of entering sporulation is a phosphorelay, through which sensor kinases promote phosphorylation of Spo0A. The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. subtilis sporulates with high efficiency during growth, bypassing the cell density, nutritional, and other signals that normally make sporulation a post-exponential-phase response. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain. This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK, coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B. subtilis isolates, presumably as a form of survival and/or propagation in the gut environment.

Hybrid Biosynthesis of Roseobacticides from Algal and Bacterial Precursor Molecules

Journal of the American Chemical Society. Oct, 2014  |  Pubmed ID: 25295497

Roseobacticides regulate the symbiotic relationship between a marine bacterium (Phaeobacter inhibens) and a marine microalga (Emiliania huxleyi). This relationship can be mutualistic, when the algal host provides food for the bacteria and the bacteria produce growth hormones and antibiotics for the algae, or parasitic, when the algae senesce and release p-coumaric acid. The released p-coumaric acid causes the bacteria to synthesize roseobacticides, which are nM-μM toxins for the algae. We examined the biosynthesis of roseobacticides and report that all roseobacticide precursors play critical roles during the mutualist phase of the symbiosis. Roseobacticides are biosynthesized from the algal growth promoter, the major food molecule provided by the algal cells, and the algal senescence signal that initiates the mutualist-to-parasite switch. Thus, molecules that are beneficial during mutualism are diverted to the synthesis of toxins during parasitism. A plausible mechanism for assembling roseobacticides from these molecules is proposed.

Structural and Functional Analysis of Bacillus Subtilis YisP Reveals a Role of Its Product in Biofilm Production

Chemistry & Biology. Nov, 2014  |  Pubmed ID: 25308276

YisP is involved in biofilm formation in Bacillus subtilis and has been predicted to produce C30 isoprenoids. We determined the structure of YisP and observed that it adopts the same fold as squalene and dehydrosqualene synthases. However, the first aspartate-rich motif found in essentially all isoprenoid synthases is aspartate poor in YisP and cannot catalyze head-to-head condensation reactions. We find that YisP acts as a phosphatase, catalyzing formation of farnesol from farnesyl diphosphate, and that it is the first phosphatase to adopt the fold seen in the head-to-head prenyl synthases. Farnesol restores biofilm formation in a Δyisp mutant and modifies lipid membrane structure similarly to the virulence factor staphyloxanthin. This work clarifies the role of YisP in biofilm formation and suggests an intriguing possibility that many of the YisP-like homologs found in other bacteria may also have interesting products and functions.

Mutualistic Interaction Between Salmonella Enterica and Aspergillus Niger and Its Effects on Zea Mays Colonization

Microbial Biotechnology. Nov, 2014  |  Pubmed ID: 25351041

Salmonella Typhimurium inhabits a variety of environments and is able to infect a broad range of hosts. Throughout its life cycle, some hosts can act as intermediates in the path to the infection of others. Aspergillus niger is a ubiquitous fungus that can often be found in soil or associated to plants and microbial consortia. Recently, S. Typhimurium was shown to establish biofilms on the hyphae of A. niger. In this work, we have found that this interaction is stable for weeks without a noticeable negative effect on either organism. Indeed, bacterial growth is promoted upon the establishment of the interaction. Moreover, bacterial biofilms protect the fungus from external insults such as the effects of the anti-fungal agent cycloheximide. Thus, the Salmonella-Aspergillus interaction can be defined as mutualistic. A tripartite gnotobiotic system involving the bacterium, the fungus and a plant revealed that co-colonization has a greater negative effect on plant growth than colonization by either organism in dividually. Strikingly, co-colonization also causes a reduction in plant invasion by S. Typhimurium. This work demonstrates that S. Typhimurium and A. niger establish a mutualistic interaction that alters bacterial colonization of plants and affects plant physiology.

New Tools for Comparing Microscopy Images: Quantitative Analysis of Cell Types in Bacillus Subtilis

Journal of Bacteriology. Feb, 2015  |  Pubmed ID: 25448819

Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed.

On the Evolution of Bacterial Multicellularity

Current Opinion in Microbiology. Apr, 2015  |  Pubmed ID: 25597443

Multicellularity is one of the most prevalent evolutionary innovations and nowhere is this more apparent than in the bacterial world, which contains many examples of multicellular organisms in a surprising array of forms. Due to their experimental accessibility and the large and diverse genomic data available, bacteria enable us to probe fundamental aspects of the origins of multicellularity. Here we discuss examples of multicellular behaviors in bacteria, the selective pressures that may have led to their evolution, possible origins and intermediate stages, and whether the ubiquity of apparently convergent multicellular forms argues for its inevitability.

Directed Natural Product Biosynthesis Gene Cluster Capture and Expression in the Model Bacterium Bacillus Subtilis

Scientific Reports. 2015  |  Pubmed ID: 25807046

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning "plug-and-play" approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

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