Bacteria develop a broad range of phage resistance mechanisms, such as prevention of phage adsorption and CRISPR/Cas system, to survive phage predation. In this study, Pseudomonas aeruginosa PA1 strain was infected with lytic phage PaP1, and phage-resistant mutants were selected. A high percentage (~30%) of these mutants displayed red pigmentation phenotype (Red mutant). Through comparative genomic analysis, one Red mutant PA1r was found to have a 219.6?kb genomic fragment deletion, which contains two key genes hmgA and galU related to the observed phenotypes. Deletion of hmgA resulted in the accumulation of a red compound homogentisic acid; while A galU mutant is devoid of O-antigen, which is required for phage adsorption. Intriguingly, while the loss of galU conferred phage resistance, it significantly attenuated PA1r in a mouse infection experiment. Our study revealed a novel phage resistance mechanism via chromosomal DNA deletion in P. aeruginosa.
The oral opportunistic pathogen Fusobacterium nucleatum is known to interact with a large number of different bacterial species residing in the oral cavity. It adheres to a variety of Gram-positive bacteria, including oral streptococci via the arginine-inhibitable adhesin RadD. In this study, we describe a novel protein encoded by the predicted open reading frame FN1253 that appears to play a role in interspecies interactions of F. nucleatum, particularly with oral streptococci and related Gram-positive species. We designated FN1253 as aid1 (Adherence Inducing Determinant 1). Expression analyses demonstrated that this gene was induced in F. nucleatum single species biofilms, while the presence of representative members of the oral microbiota known to adhere to F. nucleatum triggered its suppression. Inactivation as well as overexpression of aid1 affected the ability of F. nucleatum to coaggregate with oral streptococci and the closely related Enterococcus faecalis, but not other Gram-positive oral species tested. Furthermore, overexpression of aid1 led to a drastic change in the structure of dual species biofilms of F. nucleatum with oral streptococci. Aid1 function was abolished in the presence of arginine and found to be dependent on RadD. Interestingly, differential expression of aid1 did not affect messenger RNA and protein levels of RadD. These findings indicate that RadD-mediated adhesion to oral streptococci involves more complex cellular processes than the simple interaction of adhesins on the surface of partner strains. Aid1 could potentially play an important role in facilitating RadD-mediated interaction with oral streptococci by increasing binding specificity of F. nucleatum to other microbial species.
The classic organization by Socransky and coworkers categorized the oral bacteria of the subgingival plaque into different complexes. Treponema denticola, Porphyromonas gingivalis and Tannerella forsythia are grouped into the red complex that is highly correlated with periodontal disease. Socransky's work closely associates red with orange complex species such as Fusobacterium nucleatum and Prevotella intermedia but not with members of the other complexes. While the relationship between species contained by these complexes is in part supported by their ability to physically attach to each other, the physiological consequences of these interactions and associations are less clear. In this study, we employed T. denticola as a model organism to analyze contact-dependent responses to interactions with species belonging to the same complex (P. gingivalis and T. forsythia), the closely associated orange complex (using F. nucleatum and P. intermedia as representatives) and the unconnected yellow complex (using Streptococcus sanguinis and S. gordonii as representatives). RNA was extracted from T. denticola alone as well as after pairwise co-incubation for 5 hrs with representatives of the different complexes, and the respective gene expression profiles were determined using microarrays. Numerous genes related to motility, metabolism, transport, outer membrane and hypothetical proteins were differentially regulated in T. denticola in the presence of the tested partner species. Further analysis revealed a significant overlap in the affected genes and we identified a general response to the presence of other species, those specific to two of the three complexes as well as individual complexes. Most interestingly, many predicted major antigens (e.g. flagella, Msp, CTLP) were suppressed in responses that included red complex species indicating that the presence of the most closely associated species induces immune-evasive strategies. In summary, the data presented here provide an in-depth understanding of the transcriptional responses triggered by contact-dependent interactions between microorganisms inhabiting the periodontal pocket.
Dentures are often colonized with a variety of microorganisms, including Candida albicans, that contribute to denture stomatitis. Several in vitro models have been previously established to study denture-related microbial colonization and evaluate treatment efficacy of denture cleansers; however, those models typically fail to appreciate the complex topology and heterogeneity of denture surfaces and lack effective ways to accurately measure microbial colonization. The purpose of this study was to study microbial colonization with a new model system based on real dentures, to more realistically mimic in vivo conditions.
It is well established that host-associated microbial communities can interfere with the colonization and establishment of microbes of foreign origins, a phenomenon often referred to as bacterial interference or colonization resistance. However, due to the complexity of the indigenous microbiota, it has been extremely difficult to elucidate the community colonization resistance mechanisms and identify the bacterial species involved. In a recent study, we have established an in vitro mice oral microbial community (O-mix) and demonstrated its colonization resistance against an Escherichia coli strain of mice gut origin. In this study, we further analyzed the community structure of the O-mix by using a dilution/regrowth approach and identified the bacterial species involved in colonization resistance against E. coli. Our results revealed that, within the O-mix there were three different types of bacterial species forming unique social structure. They act as Sensor, Mediator and Killer, respectively, and have coordinated roles in initiating the antagonistic action and preventing the integration of E. coli. The functional role of each identified bacterial species was further confirmed by E. coli-specific responsiveness of the synthetic communities composed of different combination of the identified players. The study reveals for the first time the sophisticated structural and functional organization of a colonization resistance pathway within a microbial community. Furthermore, our results emphasize the importance of Facilitation or positive interactions in the development of community-level functions, such as colonization resistance.The ISME Journal advance online publication, 3 October 2013; doi:10.1038/ismej.2013.172.
Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.
Nutrient or niche-based competition among bacteria is a widespread phenomenon in the natural environment. Such interspecies interactions are often mediated by secreted soluble factors and/or direct cell-cell contact. As ubiquitous soil bacteria, Myxococcus species are able to produce a variety of bioactive secondary metabolites to inhibit the growth of other competing bacterial species. Meanwhile, Myxococcus spp. also exhibit sophisticated predatory behavior, an extreme form of competition that is often stimulated by close contact with prey cells and largely depends on the availability of solid surfaces. Myxococcus spp. can also be isolated from aquatic environments. However, studies focusing on the interaction between Myxococcus and other bacteria in such environments are still limited. In this study, using the well-studied Myxococcus xanthus DK1622 and Escherichia coli as model interspecies interaction pair, we demonstrated that in an aqueous environment, M. xanthus was able to kill E. coli in a cell contact-dependent manner and that the observed contact-dependent killing required the formation of co-aggregates between M. xanthus and E. coli cells. Further analysis revealed that exopolysaccharide (EPS), type IV pilus, and lipopolysaccharide mutants of M. xanthus displayed various degrees of attenuation in E. coli killing, and it correlated well with the mutants reduction in EPS production. In addition, M. xanthus showed differential binding ability to different bacteria, and bacterial strains unable to co-aggregate with M. xanthus can escape the killing, suggesting the specific nature of co-aggregation and the targeted killing of interacting bacteria. In conclusion, our results demonstrated EPS-mediated, contact-dependent killing of E. coli by M. xanthus, a strategy that might facilitate the survival of this ubiquitous bacterium in aquatic environments.
Confocal laser scanning microscopy (CLSM) is a useful research tool to explore the exopolysaccharides (EPS) in bacterial biofilms. Here, we describe the analysis of different biofilms of Myxococcus xanthusformed in a modified chamber slide system with CLSM. In conjunction with several specific fluorescent probes, the EPS within M. xanthusbiofilms can be visualized and analyzed in situ.
The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.
The development of multispecies oral microbial communities involves complex intra- and interspecies interactions at various levels. The ability to adhere to the resident bacteria or the biofilm matrix and overcome community resistance are among the key factors that determine whether a bacterium can integrate into a community. Fusobacterium nucleatum is a prevalent Gram-negative oral bacterial species that is able to adhere to a variety of oral microbes and has been implicated in playing an important role in the establishment of multispecies oral microbial community. However, the majority of experiments thus far has focused on the physical adherence between two species as measured by in vitro co-aggregation assays, while the community-based effects on the integration of F. nucleatum into multispecies microbial community remains to be investigated. In this study, we focus on community integration of F. nucleatum. We demonstrated using an established in vitro mice oral microbiota (O-mix) that the viability of F. nucleatum was significantly reduced upon addition to the O-mix due to cell contact-dependent induction of hydrogen peroxide (H(2)O(2)) production by oral community. Interestingly, this inhibitory effect was significantly alleviated when F. nucleatum was allowed to adhere to its known interacting partner species (such as Streptococcus sanguinis) prior to addition. Furthermore, this aggregate formation-dependent protection was absent in the F. nucleatum mutant strain ?Fn1526 that is unable to bind to a number of Gram-positive species. More importantly, this protective effect was also observed during integration of F. nucleatum into a human salivary microbial community (S-mix). These results suggest that by adhering to other oral microbes, F. nucleatum is able to mask the surface components that are recognized by H(2)O(2) producing oral community members. This evasion strategy prevents detection by antagonistic oral bacteria and allows integration into the developing oral microbial community.
Type IV pili (TFP) and exopolysaccharides (EPS) are important components for social behaviors in Myxococcus xanthus, including gliding motility and fruiting body formation. Although specific interactions between TFP and EPS have been proposed, there have as yet been no direct observations of these interactions under native conditions. In this study, we found that a truncated PilA protein (PilACt) containing only the C-terminal domain (amino acids 32-208) is sufficient for EPS binding in vitro. Furthermore, an enhanced green fluorescent protein (eGFP) and PilACt fusion protein were constructed and used to label the native EPS in M. xanthus. Under confocal laser scanning microscope, the eGFP-PilACt-bound fruiting bodies, trail structures and biofilms exhibited similar patterns as the wheat germ agglutinin lectin-labeled EPS structures. This study showed that eGFP-PilACt fusion protein was able efficiently to label the EPS of M. xanthus, providing evidence for the first time of the direct interaction between the PilA protein and EPS under native conditions.
This study investigated the bacterial communities residing in the apical portion of human teeth with apical periodontitis in primary and secondary infections by using a culture-independent molecular biology approach.
Type IV pili (TFP) are membrane-anchored filaments with a number of important biological functions. In the model organism Myxococcus xanthus, TFP act as molecular engines that power social (S) motility through cycles of extension and retraction. TFP filaments consist of several thousand copies of a protein called PilA or pilin. PilA contains an N-terminal ?-helix essential for TFP assembly and a C-terminal globular domain important for its activity. The role of the PilA sequence and its structure-function relationship in TFP-dependent S motility remain active areas of research. In this study, we identified an M. xanthus PilA mutant carrying an alanine to valine substitution at position 32 in the ?-helix, which produced structurally intact but retraction-defective TFP. Characterization of this mutant and additional single-residue variants at this position in PilA demonstrated the critical role of alanine 32 in PilA stability, TFP assembly and retraction.
Identifying essential factors in cellular interactions and organized movement of cells is important in predicting behavioral phenotypes exhibited by many bacterial cells. We chose to study Myxococcus xanthus, a soil bacterium whose individual cell behavior changes while in groups, leading to spontaneous formation of aggregation center during the early stage of fruiting body development. In this paper, we develop a cell-based computational model that solely relies on experimentally determined parameters to investigate minimal elements required to produce the observed social behaviors in M. xanthus. The model verifies previously known essential parameters and identifies one novel parameter, the active turning, which we define as the ability and tendency of a cell to turn to a certain angle without the presence of any obvious external factors. The simulation is able to produce both gliding pattern and spontaneous aggregation center formation as observed in experiments. The model is tested against several known M. xanthus mutants and our modification of parameter values relevant for the individual mutants produces good phenotypic agreements. This outcome indicates the strong predictive potential of our model for the social behaviors of uncharacterized mutants and their expected phenotypes during development.
Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as "bait": Fusobacterium nucleatum (F. nucleatum) whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans (S. mutans) whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the "bait" oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The "prey" bacterial mixtures (including known species or natural saliva samples) were added, unbound cells were washed off after the incubation period and the remaining cells were eluted using 0.2 mol x L(-1) glycine. Genomic DNA was extracted, subjected to 16S rRNA PCR amplification and separation of the resulting PCR products by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F. nucleatum adhered to a variety of bacterial species including uncultivable and uncharacterized ones. This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.
Dental caries (tooth decay) is caused by a specific group of cariogenic bacteria, like Streptococcus mutans, which convert dietary sugars into acids that dissolve the mineral in tooth structure. Killing cariogenic bacteria is an effective way to control or prevent tooth decay. In a previous study, we discovered a novel compound (Glycyrrhizol A), from the extraction of licorice roots, with strong antimicrobial activity against cariogenic bacteria. In the current study, we developed a method to produce these specific herbal extracts in large quantities, and then used these extracts to develop a sugar-free lollipop that effectively kills cariogenic bacteria like Streptococcus mutans. Further studies showed that these sugar-free lollipops are safe and their antimicrobial activity is stable. Two pilot human studies indicate that a brief application of these lollipops (twice a day for ten days) led to a marked reduction of cariogenic bacteria in oral cavity among most human subjects tested. This herbal lollipop could be a novel tool to promote oral health through functional foods.
Finding unique peptides to target specific biological surfaces is crucial to basic research and technology development, though methods based on biological arrays or large libraries limit the speed and ease with which these necessary compounds can be found. We reasoned that because biological surfaces, such as cell surfaces, mineralized tissues, and various extracellular matrices have unique molecular compositions, they present unique physicochemical signatures to the surrounding medium which could be probed by peptides with appropriately corresponding physicochemical properties. To test this hypothesis, a naïve pilot library of 36 peptides, varying in their hydrophobicity and charge, was arranged in a two-dimensional matrix and screened against various biological surfaces. While the number of peptides in the matrix library was very small, we obtained "hits" against all biological surfaces probed. Sequence refinement of the "hits" led to peptides with markedly higher specificity and binding activity against screened biological surfaces. Genetic studies revealed that peptide binding to bacteria was mediated, at least in some cases, by specific cell-surface molecules, while examination of human tooth sections showed that this method can be used to derive peptides with highly specific binding to human tissue.
Myxococcus xanthus belongs to the delta class of the proteobacteria and is notable for its complex life-style with social behaviors and relatively large genome. Although previous observations have suggested the existence of horizontal gene transfer in M. xanthus, its ability to take up exogenous DNA via natural transformation has not been experimentally demonstrated. In this study, we achieved natural transformation in M. xanthus using the autonomously replicating myxobacterial plasmid pZJY41 as donor DNA. M. xanthus exopolysaccharide (EPS) was shown to be an extracellular barrier for transformation. Cells deficient in EPS production, e.g., mutant strains carrying ?difA or ?epsA, became naturally transformable. Among the inner barriers to transformation were restriction-modification systems in M. xanthus, which could be partially overcome by methylating DNA in vitro using cell extracts of M. xanthus prior to transformation. In addition, the incubation time of DNA with cells and the presence of divalent magnesium ion affected transformation frequency of M. xanthus. Furthermore, we also observed a potential involvement of the type IV pilus system in the DNA uptake machinery of M. xanthus. The natural transformation was totally eliminated in the ?pilQ/epsA and ?tgl/epsA mutants, and null mutation of pilB or pilC in an ?epsA background diminished the transformation rate. Our study, to the best of our knowledge, provides the first example of a naturally transformable species among deltaproteobacteria.
Social motility (S motility), the coordinated movement of large cell groups on agar surfaces, of Myxococcus xanthus requires type IV pili (TFP) and exopolysaccharides (EPS). Previous models proposed that this behavior, which only occurred within cell groups, requires cycles of TFP extension and retraction triggered by the close interaction of TFP with EPS. However, the curious observation that M. xanthus can perform TFP-dependent motility at a single-cell level when placed onto polystyrene surfaces in a highly viscous medium containing 1% methylcellulose indicated that "S motility" is not limited to group movements. In an apparent further challenge of the previous findings for S motility, mutants defective in EPS production were found to perform TFP-dependent motility on polystyrene surface in methylcellulose-containing medium. By exploring the interactions between pilin and surface materials, we found that the binding of TFP onto polystyrene surfaces eliminated the requirement for EPS in EPS(-) cells and thus enabled TFP-dependent motility on a single cell level. However, the presence of a general anchoring surface in a viscous environment could not substitute for the role of cell surface EPS in group movement. Furthermore, EPS was found to serve as a self-produced anchoring substrate that can be shed onto surfaces to enable cells to conduct TFP-dependent motility regardless of surface properties. These results suggested that in certain environments, such as in methylcellulose solution, the cells could bypass the need for EPS to anchor their TPF and conduct single-cell S motility to promote exploratory movement of colonies over new specific surfaces.
Bacterially induced cell death in human lymphocytes is an important virulence factor for pathogenic bacteria. Previously discovered mechanisms of bacterially induced cell death are predominantly based on the transfer of bacterial proteins to the target host cell, such as the toxins secreted through type I, II, and VI secretion systems or effector proteins injected through type III, IV, and Vb secretion systems. Here, we report a mechanism employed by the Gram-negative oral pathogen Fusobacterium nucleatum for cell death induction of human lymphocytes via two outer membrane proteins (OMPs), Fap2 and RadD, which share regions homologous to autotransporter secretion systems (type Va secretion systems). Genetic and physiological studies established that inactivation of the two OMPs led to significantly reduced ability to trigger cell death in Jurkat cells, while the corresponding double mutant was almost completely attenuated. Additional biochemical and molecular analyses demonstrated that cell-free F. nucleatum membranes are sufficient to induce cell death in Jurkat cells, suggesting that no active process or effector protein transfer was necessary to induce eukaryotic cell death.
Dental biofilms are complex communities composed largely of harmless bacteria. Certain pathogenic species including Streptococcus mutans (S. mutans) can become predominant when host factors such as dietary sucrose intake imbalance the biofilm ecology. Current approaches to control S. mutans infection are not pathogen-specific and eliminate the entire oral community along with any protective benefits provided. Here, we tested the hypothesis that removal of S. mutans from the oral community through targeted antimicrobial therapy achieves protection against subsequent S. mutans colonization.
The objective of this study was to compare the antimicrobial activity of commercially available antiseptic mouthrinses against saliva-derived plaque biofilms in static and flow-through biofilm systems in vitro.
Myxococcus xanthus is a Gram-negative bacterium capable of complex developmental processes involving vegetative swarming and fruiting body formation. Social (S-) gliding motility, one of the two motility systems used by M. xanthus, requires at least two cell surface structures: type IV pili (TFP) and extracellular polysaccharides (EPS). Extended TFP that are composed of thousands of copies of PilA retract upon binding to EPS and thereby pull the cell forward. TFP also act as external sensor to regulate EPS production. In this study, we generated a random PilA mutant library and identified one derivative, SW1066, which completely failed to undergo developmental processes. Detailed characterization revealed that SW1066 produced very little EPS but wild-type amounts of PilA. These mutated PilA subunits, however, are unable to assemble into functional TFP despite their ability to localize to the membrane. By preventing the mutated PilA of SW1066 to translocate from the cytoplasm to the membrane, fruiting body formation and EPS production were restored to the levels observed in mutant strains lacking PilA. This apparent connection between PilA membrane accumulation and reduction in surface EPS implies that specific cellular PilA localization are required to maintain the EPS level necessary to sustain normal S-motility in M. xanthus.
The periodontal pathogen T. denticola resides in a stressful environment rife with challenges, the human oral cavity. Knowledge of the stress response capabilities of this invasive spirochete is currently very limited. Whole genome expression profiles in response to different suspected stresses including heat shock, osmotic downshift, oxygen and blood exposure were examined. Most of the genes predicted to encode conserved heat shock proteins (HSPs) were found to be induced under heat and oxygen stress. Several of these HSPs also seem to be important for survival in hypotonic solutions and blood. In addition to HSPs, differential regulation of many genes encoding metabolic proteins, hypothetical proteins, transcriptional regulators and transporters was observed in patterns that could betoken functional associations. In summary, stress responses in T. denticola exhibit many similarities to the corresponding stress responses in other organisms but also employ unique components including the induction of hypothetical proteins.
Within the same human gastrointestinal tract, substantial differences in the bacterial species that inhabit oral cavity and intestinal tract have been noted. Previous research primarily attributed the differences to the influences of host environments and nutritional availabilities ("host habitat" effect). Our recent study indicated that, other than the host habitat effect, an existing microbial community could impose a selective pressure on incoming foreign bacterial species independent of host-mediated selection ("community selection" effect). In this study, we employed in vitro microbial floras representing microorganisms that inhabit the oral cavities and intestinal tract of mice in combination with Escherichia coli as a model intestinal bacterium and demonstrated that E. coli displays a striking community preference. It thrived when introduced into the intestinal microbial community and survived poorly in the microbial flora of foreign origin (oral community). A more detailed examination of this phenomenon showed that the oral community produced oxygen-free radicals in the presence of wild-type E. coli while mutants deficient in lipopolysaccharides (LPS) did not trigger significant production of these cell-damaging agents. Furthermore, mutants of E. coli defective in the oxidative stress response experienced a more drastic reduction in viability when cocultivated with the oral flora, while the exogenous addition of the antioxidant vitamin C was able to rescue it. We concluded that the oral-derived microbial community senses the E. coli LPS and kills the bacterium with oxygen-free radicals. This study reveals a new mechanism of community invasion resistance employed by established microflora to defend their domains.
The gastrointestinal (GI) tract is home to trillions of microbes. Within the same GI tract, substantial differences in the bacterial species that inhabit the oral cavity and intestinal tract have been noted. While the influence of host environments and nutritional availability in shaping different microbial communities is widely accepted, we hypothesize that the existing microbial flora also plays a role in selecting the bacterial species that are being integrated into the community. In this study, we used cultivable microbial communities isolated from different parts of the GI tract of mice (oral cavity and intestines) as a model system to examine this hypothesis. Microbes from these two areas were harvested and cultured using the same nutritional conditions, which led to two distinct microbial communities, each with about 20 different species as revealed by PCR-based denaturing gradient gel electrophoresis analysis. In vitro community competition assays showed that the two microbial floras exhibited antagonistic interactions toward each other. More interestingly, all the original isolates tested and their closely related species displayed striking community preferences: They persisted when introduced into the bacterial community of the same origin, while their viable count declined more than three orders of magnitude after 4 days of coincubation with the microbial flora of foreign origin. These results suggest that an existing microbial community might impose a selective pressure on incoming foreign bacterial species independent of host selection. The observed inter-flora interactions could contribute to the protective effect of established microbial communities against the integration of foreign bacteria to maintain the stability of the existing communities.
Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals, creation of an acidic environment favors growth of acid-enduring and acid-generating species, which causes further reduction in the plaque pH. In this study, we developed a prototype antimicrobial peptide capable of achieving high activity exclusively at low environmental pH to target bacterial species like Streptococcus mutans that produce acid and thrive under the low pH conditions detrimental for tooth integrity. The features of clavanin A, a naturally occurring peptide rich in histidine and phenylalanine residues with pH-dependent antimicrobial activity, served as a design basis for these prototype acid-activated peptides (AAPs). Employing the major cariogenic species S. mutans as a model system, the two AAPs characterized in this study exhibited a striking pH-dependent antimicrobial activity, which correlated well with the calculated charge distribution. This type of peptide represents a potential new way to combat dental caries.
A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central bridging organisms in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine-inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine-binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum. Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD) demonstrated significantly decreased co-aggregation with representatives of the gram-positive early oral colonizers. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation.
Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.
One intriguing discovery in modern microbiology is the extensive presence of extracellular DNA (eDNA) within biofilms of various bacterial species. Although several biological functions have been suggested for eDNA, including involvement in biofilm formation, the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood. In the biofilms formed by Myxococcus xanthus, a Gram-negative soil bacterium with complex morphogenesis and social behaviors, DNA was found within both extracted and native extracellular matrices (ECM). Further examination revealed that these eDNA molecules formed well organized structures that were similar in appearance to the organization of exopolysaccharides (EPS) in ECM. Biochemical and image analyses confirmed that eDNA bound to and colocalized with EPS within the ECM of starvation biofilms and fruiting bodies. In addition, ECM containing eDNA exhibited greater physical strength and biological stress resistance compared to DNase I treated ECM. Taken together, these findings demonstrate that DNA interacts with EPS and strengthens biofilm structures in M. xanthus.
The Clp/HSP100 family of molecular chaperones is ubiquitous in both prokaryotes and eukaryotes. These proteins play important roles in refolding, disaggregating and degrading proteins damaged by stress. As a subclass of the Clp/HSP100 family, ClpB has been shown to be involved in various stress responses as well as other functions in bacteria. In the present study, we investigated the role of a predicted ClpB-encoding gene, MXAN5092, in the stress response during vegetative growth and development of Myxococcus xanthus. Transcriptional analysis confirmed induction of this clpB homologue under different stress conditions, and further phenotypic analysis revealed that an in-frame deletion mutant of MXAN5092 was more sensitive to various stress treatments than the wild-type strain during vegetative growth. Moreover, the absence of the MXAN5092 gene resulted in decreased heat tolerance of myxospores, indicating the involvement of this clpB homologue in the stress response during the development of myxospores. The M. xanthus recombinant ClpB (MXAN5092) protein also showed a general chaperone activity in vitro. Overall, our genetic and phenotypic analysis of the predicted ATP-dependent chaperone protein ClpB (MXAN5092) demonstrated that it functions as a chaperone protein and plays an important role in cellular stress tolerance during both vegetative growth and development of M. xanthus.
The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 µm) with a pronounced peak at 1.5 µm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phylotypes which cannot be characterized in vitro.
Exopolysaccharide (EPS) of Myxococcus xanthus is a well-regulated cell surface component. In addition to its known functions for social motility and fruiting body formation on solid surfaces, EPS has also been proposed to play a role in multi-cellular clumping in liquid medium, though this phenomenon has not been well studied. In this report, we confirmed that M. xanthus clumps formed in liquid were correlated with EPS levels and demonstrated that the EPS encased cell clumps exhibited biofilm-like structures. The clumps protected the cells at physiologically relevant EPS concentrations, while cells lacking EPS exhibited significant reduction in long-term viability and resistance to stressful conditions. However, excess EPS production was counterproductive to vegetative growth and viable cell recovery declined in extended late stationary phase as cells became trapped in the matrix of clumps. Therefore, optimal EPS production by M. xanthus is important for normal physiological functions in liquid.
Many human microbial infectious diseases including dental caries are polymicrobial in nature. How these complex multi-species communities evolve from a healthy to a diseased state is not well understood. Although many health- or disease-associated oral bacteria have been characterized in vitro, their physiology within the complex oral microbiome is difficult to determine with current approaches. In addition, about half of these species remain uncultivated to date with little known besides their 16S rRNA sequence. Lacking culture-based physiological analyses, the functional roles of uncultivated species will remain enigmatic despite their apparent disease correlation. To start addressing these knowledge gaps, we applied a combination of Magnetic Resonance Spectroscopy (MRS) with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities from healthy children for in vitro temporal monitoring of metabolites and identification of metabolically active and inactive bacterial species.
It is a well-recognized fact that the composition of human salivary microbial community is greatly affected by its nutritional environment. However, most studies are currently focused on major carbon or nitrogen sources with limited attention to trace elements like essential mineral ions. In this study, we examined the effect of iron availability on the bacterial profiles of an in vitro human salivary microbial community as iron is an essential trace element for the survival and proliferation of virtually all microorganisms. Analysis via a combination of PCR with denaturing gradient gel electrophoresis demonstrated a drastic change in species composition of an in vitro human salivary microbiota when iron was scavenged from the culture medium by addition of the iron chelator 2,2-bipyridyl. This shift in community profile was prevented by the presence of excessive ferrous iron (Fe(2+)). Most interestingly, under iron deficiency, the in vitro grown salivary microbial community became dominated by several hemolytic bacterial species, including Streptococcus spp., Gemella spp., and Granulicatella spp. all of which have been implicated in infective endocarditis. These data provide evidence that iron availability can modulate host-associated oral microbial communities, resulting in a microbiota with potential clinical impact.
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