Selective digestive decontamination (SDD) is an infection prevention measure for critically ill patients in intensive care units (ICUs) that aims to eradicate opportunistic pathogens from the oropharynx and intestines, while sparing the anaerobic flora, by the application of non-absorbable antibiotics. Selection for antibiotic-resistant bacteria is still a major concern for SDD. We therefore studied the impact of SDD on the reservoir of antibiotic resistance genes (i.e. the resistome) by culture-independent approaches.
A previous study of prokaryotic genomes identified large reservoirs of putative mobile promoters (PMPs), that is, homologous promoter sequences associated with nonhomologous coding sequences. Here we extend this data set to identify the full complement of mobile promoters in sequenced prokaryotic genomes. The expanded search identifies nearly 40,000 PMP sequences, 90% of which occur in noncoding regions of the genome. To gain further insight from this data set, we develop a birth-death-diversification model for mobile genetic elements subject to sequence diversification; applying the model to PMPs we are able to quantify the relative importance of duplication, loss, horizontal gene transfer (HGT), and diversification to the maintenance of the PMP reservoir. The model predicts low rates of HGT relative to the duplication and loss of PMP copies, rapid dynamics of PMP families, and a pool of PMPs that exist as a single copy in a genome at any given time, despite their mobility. We report evidence of these "singletons" at high frequencies in prokaryotic genomes. We also demonstrate that including selection, either for or against PMPs, was not necessary to describe the observed data.
The use of in silico simulations as a basis for designing artificial biological systems (and experiments to characterize them) is one of the tangible differences between Synthetic Biology and "classical" Genetic Engineering. To this end, synthetic biologists have adopted approaches originating from the traditionally non-biological fields of Nonlinear Dynamics and Systems & Control Theory. However, due to the complex molecular interactions affecting the emergent properties of biological systems, mechanistic descriptions of even the simplest genetic circuits (transcriptional feedback oscillators, bi-stable switches) produced by these methods tend to be either oversimplified, or numerically intractable. More comprehensive and realistic models can be approximated by constructing "toy" genetic circuits that provide the experimenter with some degree of control over the transcriptional dynamics, and allow for experimental set-ups that generate reliable data reflecting the intracellular biochemical state in real time. To this end, we designed two genetic circuits (basic and tunable) capable of exhibiting synchronized oscillatory green fluorescent protein (GFP) expression in small populations of Escherichia coli cells. The functionality of the basic circuit was verified microscopically. High-level visualizations of computational simulations were analyzed to determine whether the reliability and utility of a synchronized transcriptional oscillator could be enhanced by the introduction of chemically inducible repressors.
Quantifying patterns of adaptive divergence between taxa is a major goal in the comparative and evolutionary study of prokaryote genomes. When applied appropriately, the McDonald-Kreitman (MK) test is a powerful test of selection based on the relative frequency of non-synonymous and synonymous substitutions between species compared to non-synonymous and synonymous polymorphisms within species. The webserver ODoSE (Ortholog Direction of Selection Engine) allows the calculation of a novel extension of the MK test, the Direction of Selection (DoS) statistic, as well as the calculation of a weighted-average Neutrality Index (NI) statistic for the entire core genome, allowing for systematic analysis of the evolutionary forces shaping core genome divergence in prokaryotes. ODoSE is hosted in a Galaxy environment, which makes it easy to use and amenable to customization and is freely available at www.odose.nl.
Horizontal gene transfer constitutes a powerful and innovative force in evolution, but often little is known about the actual origins of transferred genes. Sequence alignments are generally of limited use in tracking the original donor, since still only a small fraction of the total genetic diversity is thought to be uncovered. Alternatively, approaches based on similarities in the genome specific relative oligonucleotide frequencies do not require alignments. Even though the exact origins of horizontally transferred genes may still not be established using these compositional analyses, it does suggest that compositionally very similar regions are likely to have had a common origin. These analyses have shown that up to a third of large acquired gene clusters that reside in the same genome are compositionally very similar, indicative of a shared origin. This brings us closer to uncovering the original donors of horizontally transferred genes, and could help in elucidating possible regulatory interactions between previously unlinked sequences.
Microbial genomes do not merely evolve through the slow accumulation of mutations, but also, and often more dramatically, by taking up new DNA in a process called horizontal gene transfer. These innovation leaps in the acquisition of new traits can take place via the introgression of single genes, but also through the acquisition of large gene clusters, which are termed Genomic Islands. Since only a small proportion of all the DNA diversity has been sequenced, it can be hard to find the appropriate donors for acquired genes via sequence alignments from databases. In contrast, relative oligonucleotide frequencies represent a remarkably stable genomic signature in prokaryotes, which facilitates compositional comparisons as an alignment-free alternative for phylogenetic relatedness. In this project, we test whether Genomic Islands identified in individual bacterial genomes have a similar genomic signature, in terms of relative dinucleotide frequencies, and can therefore be expected to originate from a common donor species.
Chthoniobacter flavus Ellin428 is the first isolate from the class Spartobacteria of the bacterial phylum Verrucomicrobia. C. flavus Ellin428 can metabolize many of the saccharide components of plant biomass but is incapable of growth on amino acids or organic acids other than pyruvate.
"Pedosphaera parvula" Ellin514 is an aerobically grown verrucomicrobial isolate from pasture soil. It is one of the few cultured representatives of subdivision 3 of the phylum Verrucomicrobia. Members of this group are widespread in terrestrial environments.
Bacteria of the deeply branching phylum Verrucomicrobia are rarely cultured yet commonly detected in metagenomic libraries from aquatic, terrestrial, and intestinal environments. We have sequenced the genome of Opitutus terrae PB90-1, a fermentative anaerobe within this phylum, isolated from rice paddy soil and capable of propionate production from plant-derived polysaccharides.
Victivallis vadensis ATCC BAA-548 represents the first cultured representative from the novel phylum Lentisphaerae, a deep-branching bacterial lineage. Few cultured bacteria from this phylum are known, and V. vadensis therefore represents an important organism for evolutionary studies. V. vadensis is a strictly anaerobic sugar-fermenting isolate from the human gastrointestinal tract.
Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on D-sorbitol, small amounts of D-maltose or D-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by D-maltose or D-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on D-maltose and ?-xylosidase D on D-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra D-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of D-xylose or D-maltose. Furthermore, D-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers D-maltose and D-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by D-maltose or D-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for D-xylose induction, D-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation.
The human gastrointestinal tract contains a complex community of microbes, fulfilling important health-promoting functions. However, this vast complexity of species hampers the assignment of responsible organisms to these functions. Recently, Akkermansia muciniphila, a new species from the deeply branched phylum Verrucomicrobia, was isolated from the human intestinal tract based on its capacity to efficiently use mucus as a carbon and nitrogen source. This anaerobic resident is associated with the protective mucus lining of the intestines.
Mucins are a family of heavily glycosylated proteins that are the major organic components of the mucus layer, the protective layer covering the epithelial cells in many human and animal organs, including the entire gastro-intestinal tract. Microbes that can associate with mucins benefit from this interaction since they can get available nutrients, experience physico-chemical protection and adhere, resulting in increased residence time. Mucin-degrading microorganisms, which often are found in consortia, have not been extensively characterized as mucins are high molecular weight glycoproteins that are hard to study because of their size, complexity and heterogeneity. The purpose of this review is to discuss how advances in mucus and mucin research, and insight in the microbial ecology promoted our understanding of mucin degradation. Recent insight is presented in mucin structure and organization, the microorganisms known to use mucin as growth substrate, with a specific attention on Akkermansia muciniphila, and the molecular basis of microbial mucin degradation owing to availability of genome sequences.
Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.
The importance of Dehalobacter species in bioremediation as dedicated degraders of chlorinated organics has been well recognized. However, still little is known about Dehalobacters full genomic repertoires, including the genes involved in dehalogenation. Here we report the first insights into the genome sequence of Dehalobacter sp. E1 that grows in strict co-culture with Sedimentibacter sp. B4. Based on the co-culture metagenome and the genome of strain B4 (4.2?Mbp) we estimate the genome sequence of strain E1 to be 2.6?Mbp. Ten putative reductive dehalogenase homologue (Rdh)-encoding gene clusters were identified. One cluster has a putative tetrachloroethene Rdh-encoding gene cluster, similar to the pceABCT operon previously identified in Dehalobacter restrictus. Metagenome analysis indicated that the inability of strain E1 to synthesize cobalamin, an essential cofactor of reductive dehalogenases, is complemented by Sedimentibacter. The metagenomic exploration described here maps the extensive dechlorinating potential of Dehalobacter, and paves way for elucidation of the interactions with its co-cultured Sedimentibacter.
Anecdotal evidence shows promoters being reused separate from their downstream gene, thus providing a mechanism for the efficient and rapid rewiring of a genes transcriptional regulation. We have identified over 4000 groups of highly similar promoters using a conservative sequence similarity search in all fully sequenced prokaryotic genomes. About 6% of those groups are shared between bacteria from different taxonomic depth, including different genera, families, orders, classes and even phyla. Database searches against known mobile elements and RNA motifs have indicated that regulatory motifs such as riboswitches could be moved around on putative mobile promoters.
Synthetic biology is an emerging field that combines molecular biology with engineering principles, which requires abstraction levels applied to a modular biological componentry. The Registry of Standard Biological Parts harbours such a repository of standardized parts, and thereby facilitates the combination of complex molecular modules to novel genetic circuits and devices. However, since finding the best parts for a pre-determined genetic design can be time consuming, we devised the Constructor, a web tool that recommends the smallest number of cloning steps for pre-designed circuits, and implements user-defined quality checks.We present the Constructor ( http://www.systemsbiology.nl/the_constructor) as a constructive web tool that simplifies the in silico assembly of pre-designed gene circuitries from standard parts, reducing both planning and subsequent cloning time.
Transcriptional activation or rewiring of silent genes is an important, yet poorly understood, phenomenon in prokaryotic genomes. Anecdotal evidence coming from experimental evolution studies in bacterial systems has shown the promptness of adaptation upon appropriate selective pressure. In many cases, a partial or complete promoter is mobilized to silent genes from elsewhere in the genome. We term hereafter such recruited regulatory sequences as Putative Mobile Promoters (PMPs) and we hypothesize they have a large impact on rapid adaptation of novel or cryptic functions. Querying all publicly available prokaryotic genomes (1362) uncovered >4000 families of highly conserved PMPs (50 to 100 long with ?80% nt identity) in 1043 genomes from 424 different genera. The genomes with the largest number of PMP families are Anabaena variabilis (28 families), Geobacter uraniireducens (27 families) and Cyanothece PCC7424 (25 families). Family size varied from 2 to 93 homologous promoters (in Desulfurivibrio alkaliphilus). Some PMPs are present in particular species, but some are conserved across distant genera. The identified PMPs represent a conservative dataset of very recent or conserved events of mobilization of non-coding DNA and thus they constitute evidence of an extensive reservoir of recyclable regulatory sequences for rapid transcriptional rewiring.
At the junction between systems and synthetic biology, genome streamlining provides a solid foundation both for increased understanding of cellular circuitry, and for the tailoring of microbial chassis towards innovative biotechnological applications. Iterative genomic deletions (targeted and random) helps to generate simplified, stabilized and predictable genomes, whereas multiplexing genome engineering reveals a broad functional genetic diversity. The decrease in oligo and gene synthesis costs promises effective combinatorial tools for the generation of chassis based on streamlined and tractable genomes. Here we review recent progresses in streamlining genomes through recombineering techniques aiming to generate insights into cellular mechanisms and responses towards the design and assembly of streamlined genome chassis together with new cellular modules in diverse biotechnological applications.
Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms.
CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference.
Cumulative site-directed mutagenesis is of limited suitability for the global analysis of the gene functions in the microbes cellular network. In order to simplify and stabilize the genome of the soil bacterium Pseudomonas putida, we developed a recyclable three-step excision method based on the combination of customized mini-transposons and the FLP-FRT site-specific recombination system. To demonstrate the powerful potential of these tools, we first established insertion mutant libraries that allow users to study gene functions with respect either to phenotypic characteristics (single insertions) or to their involvement in predicted networks (double insertions). Based on these libraries, we generated as a proof-of-principle, single-deletion mutants lacking ~4.1% of the genome (~3.7% of the gene repertoire). A cyclical application of the method generated four double-deletion mutants of which a maximum of ~7.4% of the chromosome (~6.9% of the gene count) was excised. This procedure demonstrates a new strategy for rapid genome streamlining and gain of new insights into the molecular interactions and regulations.
We sought to assess whether the concept of relative entropy (information capacity), could aid our understanding of the process of horizontal gene transfer in microbes. We analyzed the differences in information capacity between prokaryotic chromosomes, genomic islands (GI), phages, and plasmids. Relative entropy was estimated using the Kullback-Leibler measure.
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