Regulation of horizontal gene transfer by intercellular peptide signaling in Bacillus subtilis

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006."February 2006."Includes bibliographical references.Horizontal gene transfer plays an important role in bacterial evolution. Although acquisition of foreign DNA can be beneficial to cells, it can also be detrimental. Therefore, cells that possess mechanisms to regulate horizontal gene transfer likely have a competitive advantage. Similarly, several mobile genetic elements possess mechanisms that regulate transfer. This regulation maintains stability and promotes dissemination of the element, thereby ensuring its survival. Elucidating the mechanisms that regulate transfer should provide insights into conditions that favor horizontal gene transfer and bacterial evolution. This thesis describes the regulation of two means of horizontal gene transfer in the gram-positive bacterium Bacillus subtilis. Under certain conditions, B. subtilis cells undergo differentiation into competent cells capable of acquiring foreign DNA from the environment. A variety of factors regulate competence development. Initiation of genetic competence is controlled by a transcription factor, ComA, that also activates expression of genes that encode degradative enzymes, antibiotics, and secreted products important for biofilm formation.(cont.) Three signaling peptides were known to stimulate the activity of ComA. I have characterized a fourth signaling peptide that stimulates the activity of ConmA and have shown that intercellular peptide signaling modulates the timing and levels of ComA-dependent gene expression in response to different cellular cues. B. subtilis cells also contain a mobile genetic element known as ICEBsl (integrative and conjugative element B. subtilis #1). ICEBsl is normally integrated in the chromosome of the host cell. Under certain conditions, ICEBsl excises from the chromosome and transfers through a self-encoded conjugative apparatus to recipient cells. Both the global DNA damage response and intercellular peptide signaling regulate excision and transfer of ICEBsl. The global DNA damage response stimulates ICEBsl excision and transfer and likely provides a mechanism for the ICEBsl element to escape a distressed host cell for a more suitable host. Intercellular peptide signaling limits excision and transfer of ICEBsl to conditions when successful dissemination to cells lacking ICEBsl is most likely to occur. The ICEBsl-encoded proteins that regulate excision and transfer in response to intercellular peptide signaling and the global DNA damage response are encoded by other mobile genetic elements, indicating that this may be a conserved mechanism regulating their dissemination.by Jennifer M. Auchtung.Ph.D

Cultivation of stable, reproducible microbial communities from different fecal donors using minibioreactor arrays (MBRAs)

Background: Continuous-flow culture models are one tool for studying complex interactions between members of human fecal microbiotas because they allow studies to be completed during an extended period of time under conditions where pH, nutrient availability, and washout of waste products and dead cells can be controlled. Because many of the existing well-validated continuous-flow models are large and complex, we were interested in developing a simpler continuous-flow system that would allow microbial community dynamics to be examined in higher throughput while still maintaining complex microbial communities. To this end, we developed minibioreactor arrays (MBRAs), small volume bioreactors (15 ml) that allow simultaneous cultivation of up to 48 microbial communities in a single anaerobic chamber. Results: We used MBRA to characterize the microbial community dynamics of replicate reactors inoculated from three different human fecal donors and reactors seeded with feces pooled from these three donors. We found that MBRA could be used to efficiently cultivate complex microbial communities that were a subset of the initial fecal inoculum (15–25 % of fecal OTUs initially observed). After an initial acclimation period of approximately 1 week, communities in each reactor stabilized and exhibited day-to-day variation similar to that observed in stable mouse fecal communities. Replicate reactors were predominately populated by shared core microbial communities; variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units (OTUs). Consistent with differences between fecal donors, MBRA communities present in reactors seeded with different fecal samples had distinct composition and structure. Conclusions: From these analyses, we conclude that MBRAs can be used to cultivate communities that recapitulate key features of human fecal communities and are a useful tool to facilitate higher-throughput studies of the dynamics of these communities

Modulation of the ComA-Dependent Quorum Response in \u3ci\u3eBacillus subtilis\u3c/i\u3e by Multiple Rap Proteins and Phr Peptides

In Bacillus subtilis, extracellular peptide signaling regulates several biological processes. Secreted Phr signaling peptides are imported into the cell and act intracellularly to antagonize the activity of regulators known as Rap proteins. B. subtilis encodes several Rap proteins and Phr peptides, and the processes regulated by many of these Rap proteins and Phr peptides are unknown. We used DNA microarrays to characterize the roles that several rap-phr signaling modules play in regulating gene expression. We found that rapK-phrK regulates the expression of a number of genes activated by the response regulator ComA. ComA activates expression of genes involved in competence development and the production of several secreted products. Two Phr peptides, PhrC and PhrF, were previously known to stimulate the activity of ComA. We assayed the roles that PhrC, PhrF, and PhrK play in regulating gene expression and found that these three peptides stimulate ComA-dependent gene expression to different levels and are all required for full expression of genes activated by ComA. The involvement of multiple Rap proteins and Phr peptides allows multiple physiological cues to be integrated into a regulatory network that modulates the timing and magnitude of the ComA response

Prebiotics enhance persistence of fermented-food associated bacteria in \u3ci\u3ein vitro\u3c/i\u3e cultivated fecal microbial communities

It is well established that the gastrointestinal (GI) microbiota plays a major role in human health. Dietary interventions, and consumption of fermented foods that contain live microbes, in particular, are among the approaches being investigated to modulate the GI microbiota and improve health. However, the persistence of fermented food-associated bacteria (FAB) within the GI tract is typically limited by host factors that limit colonization and competition with autochthonous microbes. In this research, we examined if the addition of prebiotics, dietary substrates that are selectively metabolized by microbes to improve health, would enhance the persistence of FAB. We evaluated the persistence of bacteria from three live microbe-containing fermented foods— kefir, sausage, and sauerkraut—in fecal microbial communities from four healthy adults. Fecal communities were propagated in vitro and were inoculated with fermented food-associated microbes from kefir, sausage, or sauerkraut at ~107 CFU/mL. Communities were diluted 1:100 every 24 h into fresh gut simulation medium to simulate microbial community turnover in the GI tract. We measured the persistence of Lactobacillaceae from fermented foods by quantitative PCR (qPCR) and the persistence of other FAB through 16S rRNA gene sequencing. FAB were unable to persist in vitro, reaching undetectable levels within 96 h. Addition of prebiotics, including xylooligosaccharides and a mixture of fructooligosaccharides and galactooligosaccharides enhanced the persistence of some species of FAB, but the level of persistence varied by fecal donor, fermented food, and prebiotic tested. Addition of prebiotics also increased the relative abundance of Bifidobacterium species, which most likely originated from the fecal microbiota. Collectively, our results support previous in vivo studies demonstrating the transient nature of FAB in the GI tract and indicate that consumption of prebiotics may enhance their persistence

Investigating Colonization of the Healthy Adult Gastrointestinal Tract by Fungi

A wide diversity of fungi have been detected in the human gastrointestinal (GI) tract with the potential to provide or influence important functions. However, many of the fungi most commonly detected in stool samples are also present in food or the oral cavity. Therefore, to recognize which gut fungi are likely to have a sustained influence on human health, there is a need to separate transient members of the GI tract from true colonizers. To identify colonizing fungi, the eukaryotic rRNA operon’s second internal transcribed spacer (ITS2) was sequenced from the stool, saliva, and food of healthy adults following consumption of different controlled diets. Unlike most bacterial 16S rRNA genes, the only fungal ITS2 operational taxonomic units (OTUs) detected in stool DNA across multiple diets were also present in saliva and/or food. Additional analyses, including culture-based approaches and sequencing of the 18S rRNA gene, ITS2 cDNA, and DNA extracted using alternative methods, failed to detect additional fungi. Two abundant fungi, Saccharomyces cerevisiae and Candida albicans, were examined further in healthy volunteers. Saccharomyces became undetectable in stool when a S. cerevisiae-free diet was consumed, and the levels of C. albicans in stool were dramatically reduced by more frequent cleaning of teeth. Extremely low fungal abundance, the inability of fungi to grow under conditions mimicking the distal gut, and evidence from analysis of other public datasets further support the hypothesis that fungi do not routinely colonize the GI tracts of healthy adults

Humanized microbiota mice as a model of recurrent \u3ci\u3eClostridium difficile\u3c/i\u3e disease

Background: Clostridium difficile disease is the leading antibiotic-associated cause of diarrhea and nosocomial acquired infection in the western world. The per annum burden in the USA alone amounts to 250,000 cases with 14,000 ascribed deaths and medical costs in excess of a billion dollars. Novel models for the study of C. difficile infection are therefore pertinent. Results: Germ free C57BL/6 mice gavaged with a healthy human fecal microbiota maintained a stable “humanized” microbiota over multiple generations when housed under specific pathogen-free (SPF) conditions. As with mice containing a conventional microbiota, treatment with a five-antibiotic cocktail followed by a single dose of clindamycin renders the animals susceptible to C. difficile infection (CDI). Interestingly, after recovery from the initial CDI infection, a single intraperitoneal injection of clindamycin is sufficient to induce CDI relapse. Relapse of CDI can be induced up to 35 days postinfection after recovery from the initial infection, and multiple episodes of relapse can be induced. Conclusions: This model enables the study of recurrent C. difficile disease in a host containing a human-derived microbiota. Probiotic treatments using human-derived microbes, either prophylactic or curative, can be tested within the model. The identification and testing of human-derived microbial communities within a humanized microbiota mouse model may enable a higher rate of successful transfer of bacteria-based treatments from the lab to human patients due to the microbes involved initiating from, and being adapted to, the human GI tract

A high-throughput LC-MS/MS method for the measurement of the bile acid/salt content in microbiome-derived sample sets

Due to the physicochemical properties of bile acids/salts (i.e., hydrophobic and ionizable), the application of reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods are ideally suited for the measurement of these compounds in a host of microbiologically-relevant matrices. Here, we provide a detailed bioanalytical protocol that contains several modifications of a method previously described by Wegner et al. [1]. Briefly, this modified method exhibits the following advantages for the measurement of cholic acid (CA), taurocholic acid (TCA), and deoxycholic acid (DCA) in microbiome-relevant sample matrices: i) fecal sample processing has been streamlined by the elimination of lyophilization and manual homogenization steps; ii) the Sciex 6500 QTRAP hybrid triple-quadrupole/linear ion trap mass spectrometer has sufficient sensitivity to perform the measurement of bile acids/salts in negative ion mode – ammonium adducts of bile acids/salts are not required for detection; and, iii) assay throughput has been boosted by more than 5-fold by shortening the chromatographic duty cycle of a single sample injection from 45 min to 8.4 min. Recently, the method was used to perform 508 sequential injections (72 calibration standards, 52 blank-internal standard sample, and 368 MiniBioReactor Array (MBRA)-derived samples) from four separate batches over a 4-day time period

Divergence in Gene Regulation Contributes to Sympatric Speciation of \u3ci\u3eShewanella baltica\u3c/i\u3e Strains

Niche partitioning and sequence evolution drive genomic and phenotypic divergence, which ultimately leads to bacterial diversification. This study investigated the genomic composition of two Shewanella baltica clades previously identified through multilocus sequencing typing and recovered from the redox transition zone in the central Baltic Sea. Comparative genomic analysis revealed significantly higher interclade than intraclade genomic dissimilarity and that a subset of genes present in clade A were associated with potential adaptation to respiration of sulfur compounds present in the redox transition zone. The transcriptomic divergence between two representative strains of clades A and D, OS185 and OS195, was also characterized and revealed marked regulatory differences. We found that both the transcriptional divergence of shared genes and expression of strain-specific genes led to differences in regulatory patterns between strains that correlate with environmental redox niches. For instance, under anoxic conditions of respiratory nitrate ammonification, OS185—the strain isolated from a nitrate-rich environment—upregulated nearly twice the number of shared genes upregulated by OS195—the strain isolated from an H2S-containing anoxic environment. Conversely, OS195 showed stronger induction of strain-specific genes, especially those associated with sulfur compound respiration, under thiosulfate-reducing conditions. A positive association between the level of transcriptional divergence and the level of sequence divergence for shared genes was also noted. Our results provide further support for the hypothesis that genomic changes impacting transcriptional regulation play an important role in the diversification of ecologically distinct populations

Efficient Gene Transfer in Bacterial Cell Chains

Horizontal gene transfer contributes to evolution and the acquisition of new traits. In bacteria, horizontal gene transfer is often mediated by conjugative genetic elements that transfer directly from cell to cell. Integrative and conjugative elements (ICEs; also known as conjugative transposons) are mobile genetic elements that reside within a host genome but can excise to form a circle and transfer by conjugation to recipient cells. ICEs contribute to the spread of genes involved in pathogenesis, symbiosis, metabolism, and antibiotic resistance. Despite its importance, little is known about the mechanisms of conjugation in Gram-positive bacteria or how quickly or frequently transconjugants become donors. We visualized the transfer of the integrative and conjugative element ICEBs1 from a Bacillus subtilis donor to recipient cells in real time using fluorescence microscopy. We found that transfer of DNA from a donor to a recipient appeared to occur at a cell pole or along the lateral cell surface of either cell. Most importantly, we found that when acquired by 1 cell in a chain, ICEBs1 spread rapidly from cell to cell within the chain by additional sequential conjugation events. This intrachain conjugation is inherently more efficient than conjugation that is due to chance encounters between individual cells. Many bacterial species, including pathogenic, commensal, symbiotic, and nitrogen-fixing organisms, harbor ICEs and grow in chains, often as parts of microbial communities. It is likely that efficient intrachain spreading is a general feature of conjugative DNA transfer and serves to amplify the number of cells that acquire conjugative mobile genetic elements

Effects of tobacco smoke and electronic cigarette vapor exposure on the oral and gut microbiota in humans: a pilot study

Background: The use of electronic cigarettes (ECs) has increased drastically over the past five years, primarily as an alternative to smoking tobacco cigarettes. However, the adverse effects of acute and long-term use of ECs on the microbiota have not been explored. In this pilot study, we sought to determine if ECs or tobacco smoking are associated with differences in the oral and gut microbiota, in comparison to non-smoking controls. Methods: We examined a human cohort consisting of 30 individuals: 10 EC users, 10 tobacco smokers, and 10 controls. We collected cross-sectional fecal, buccal swabs, and saliva samples from each participant. All samples underwent V4 16S rRNA gene sequencing. Results: Tobacco smokers had significantly different bacterial profiles in all sample types when compared to controls, and in feces and buccal swabs when compared to EC users. The most significant associations were found in the gut, with a higher relative abundance of Prevotella (P = 0.006) and lowered Bacteroides (P = 0.036) in tobacco smokers. The Shannon diversity was also significantly reduced (P = 0.009) in fecal samples collected from tobacco smokers compared to controls. No significant difference was found in the alpha diversity, beta-diversity or taxonomic relative abundances between EC users and controls. Discussion: The current pilot data demonstrate that tobacco smoking is associated with signicant differences in the oral and gut microbiome in humans. However, validation in larger cohorts and greater understanding of the short and long-term impact of EC use on microbiota composition and function is warranted