Jennifer M. Auchtung

Towards Environmental Systems Biology of Shewanella

Bacteria of the genus Shewanella are known for their versatile electron-accepting capacities, which allow them to couple the decomposition of organic matter to the reduction of the various terminal electron acceptors that they encounter in their stratified environments. Owing to their diverse metabolic capabilities, shewanellae are important for carbon cycling and have considerable potential for the remediation of contaminated environments and use in microbial fuel cells. Systems-level analysis of the model species Shewanella oneidensis MR-1 and other members of this genus has provided new insights into the signal-transduction proteins, regulators, and metabolic and respiratory subsystems that govern the remarkable versatility of the shewanellae.

Comparative systems biology across an evolutionary gradient within the Shewanella genus

To what extent genotypic differences translate to phenotypic variation remains a poorly understood issue of paramount importance for several cornerstone concepts of microbiology including the species definition. Here, we take advantage of the completed genomic sequences, expressed proteomic profiles, and physiological studies of 10 closely related Shewanella strains and species to provide quantitative insights into this issue. Our analyses revealed that, despite extensive horizontal gene transfer within these genomes, the genotypic and phenotypic similarities among the organisms were generally predictable from their evolutionary relatedness. The power of the predictions depended on the degree of ecological specialization of the organisms evaluated. Using the gradient of evolutionary relatedness formed by these genomes, we were able to partly isolate the effect of ecology from that of evolutionary divergence and to rank the different cellular functions in terms of their rates of evolution. Our ranking also revealed that whole-cell protein expression differences among these organisms, when the organisms were grown under identical conditions, were relatively larger than differences at the genome level, suggesting that similarity in gene regulation and expression should constitute another important parameter for (new) species description. Collectively, our results provide important new information toward beginning a systems-level understanding of bacterial species and genera.

Identification and characterization of int (integrase), xis (excisionase) and chromosomal attachment sites of the integrative and conjugative element ICEBs1 of Bacillus subtilis

ICEBs1 is an integrative and conjugative element (conjugative transposon) integrated into trnS‐leu2 in Bacillus subtilis. In response to DNA damage or high concentrations of potential mating partners, ICEBs1 can excise and transfer to various recipients, including other species. We found that excision of ICEBs1 occurs by site‐specific recombination within 60 bp direct repeats that mark the junctions between ICEBs1 and chromosomal DNA. Excision required two ICEBs1 genes, int (integrase, ydcL), predicted to encode a tyrosine recombinase similar to that of phage lambda, and xis (excisionase, sacV). Ectopic expression of xis was sufficient to induce excision of ICEBs1, indicating that regulation of xis transcription by DNA damage and peptide signalling normally controls excision. Int, but not Xis, was needed for site‐specific integration. We found that in the absence of the primary bacterial attachment site (attB) in trnS‐leu2, ICEBs1 integrated in secondary attachment sites that are similar to a 17 bp sequence in attB. In the absence of int, ICEBs1 could recombine into the chromosome by RecA‐dependent homologous recombination, provided ICEBs1 contained a region of sequence identity to a chromosomal locus.

Unprecedented levels of horizontal gene transfer among spatially co-occurring Shewanella bacteria from the Baltic Sea.

High-throughput sequencing studies during the last decade have uncovered that bacterial genomes are very diverse and dynamic, resulting primarily from the frequent and promiscuous horizontal gene exchange that characterizes the bacterial domain of life. However, a robust understanding of the rates of genetic exchange for most bacterial species under natural conditions and the influence of the ecological settings on the rates remain elusive, severely limiting our view of the microbial world. Here, we analyzed the complete genomic sequences and expressed transcriptomes of several Shewanella baltica isolates recovered from different depths in the Baltic Sea and found that isolates from more similar depths had exchanged a larger fraction of their core and auxiliary genome, up to 20% of the total, compared with isolates from more different depths. The exchanged genes seem to be ecologically important and contribute to the successful adaptation of the isolates to the unique physicochemical conditions of the depth. Importantly, the latter genes were exchanged in very recent past, presumably as an effect of isolates seasonal migration across the water column, and reflected sexual speciation within the same depth. Therefore, our findings reveal that genetic exchange in response to environmental settings may be surprisingly rapid, which has important broader impacts for understanding bacterial speciation and evolution and for modeling bacterial responses to human-induced environmental impacts.

Epidemic Clostridium difficile Strains Demonstrate Increased Competitive Fitness Compared to Nonepidemic Isolates

ABSTRACT Clostridium difficile infection is the most common cause of severe cases of antibiotic-associated diarrhea (AAD) and is a significant health burden. Recent increases in the rate of C. difficile infection have paralleled the emergence of a specific phylogenetic clade of C. difficile strains (ribotype 027; North American pulsed-field electrophoresis 1 [NAP1]; restriction endonuclease analysis [REA] group BI). Initial reports indicated that ribotype 027 strains were associated with increased morbidity and mortality and might be hypervirulent. Although subsequent work has raised some doubt as to whether ribotype 027 strains are hypervirulent, the strains are considered epidemic isolates that have caused severe outbreaks across the globe. We hypothesized that one factor that could lead to the increased prevalence of ribotype 027 strains would be if these strains had increased competitive fitness compared to strains of other ribotypes. We developed a moderate-throughput in vitro model of C. difficile infection and used it to test competition between four ribotype 027 clinical isolates and clinical isolates of four other ribotypes (001, 002, 014, and 053). We found that ribotype 027 strains outcompeted the strains of other ribotypes. A similar competitive advantage was observed when two ribotype pairs were competed in a mouse model of C. difficile infection. Based upon these results, we conclude that one possible mechanism through which ribotype 027 strains have caused outbreaks worldwide is their increased ability to compete in the presence of a complex microbiota.

Dietary trehalose enhances virulence of epidemic Clostridium difficile

Clostridium difficile disease has recently increased to become a dominant nosocomial pathogen in North America and Europe, although little is known about what has driven this emergence. Here we show that two epidemic ribotypes (RT027 and RT078) have acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose. RT027 strains contain a single point mutation in the trehalose repressor that increases the sensitivity of this ribotype to trehalose by more than 500-fold. Furthermore, dietary trehalose increases the virulence of a RT027 strain in a mouse model of infection. RT078 strains acquired a cluster of four genes involved in trehalose metabolism, including a PTS permease that is both necessary and sufficient for growth on low concentrations of trehalose. We propose that the implementation of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to hypervirulence.

Biology of ICEBs1, an integrative and conjugative element in Bacillus subtilis

Horizontal gene transfer plays a profound role in bacterial evolution by propelling the rapid transfer of genes and gene cassettes. Integrative and conjugative elements (ICEs) are one important mechanism driving horizontal gene transfer. ICEs, also known as conjugative transposons, reside on the host chromosome but can excise to form a conjugative DNA circle that is capable of transfer to other cells. Analysis of the large number of completed bacterial genome sequences has revealed many previously unrecognized ICEs, including ICEBs1, found in the Gram-positive model bacterium Bacillus subtilis. The discovery of ICEBs1 in an organism with such an impressive array of molecular tools for genetics and molecular biology was fortuitous. Significant insights into ICE biology have resulted since its discovery <15years ago. In this review, we describe aspects of ICEBs1 biology, such as excision, conjugative transfer, and reintegration, likely to be conserved across many ICEs. We will also highlight some of the more unexpected aspects of ICEBs1 biology, such as its ability to undergo plasmid-like replication after excision and its ability to mobilize plasmids lacking dedicated mobilization functions. A molecular understanding of ICEBs1 has led to additional insights into signals and mechanisms that promote horizontal gene transfer and shape bacterial evolution.

Genome Sequencing of Five Shewanella baltica Strains Recovered from the Oxic-Anoxic Interface of the Baltic Sea

Here we describe five Shewanella baltica genomes recovered from the same sample, as well as 12 years apart from the same sampling station. These genomes expand the collection of previously sequenced S. baltica strains and represent a valuable resource for assessing the role of environmental settings on genome adaptation.

Shewanella oneidensis MR-1 Chemotaxis in a Diffusion Gradient Chamber

To obtain a systems-level understanding of Shewanella biology and ecology, the influence of electron acceptor availability on Shewanellas growth, metabolism, and transport needs to be elucidated. The diffusion gradient chamber (DGC) is an experimental tool developed to study population-level microbial growth and motility in response to concentration gradients. In this paper, the response of populations of Shewanella oneidensis MR-1 cells to an applied single gradient of the electron acceptor fumarate and applied opposing gradients of fumarate and nitrate, also an electron acceptor, were studied in the DGC. Mathematical models capable of predicting cellular growth and chemotaxis under the influence of gradients were used to analyze the results. Examining wild-type cells grown in a single gradient of fumarate, we found that MR-1 cells formed a chemotactic band that migrated up the electron acceptor gradient essentially as predicted by the model. The predicted velocity of the chemotactic cell band advancing toward the chemoattractant source (0.139 cm/h, R(2) = 0.996) closely matched that measured in the DGC (0.134 cm/h, R(2) = 0.997). Investigating the impact of opposing gradients of nitrate and fumarate on the chemotactic behaviors of S. oneidensis MR-1 fumarate reductase and nitrate reductase mutants, we found that the DGC was able to separate these mutants based upon their abilities to use the available electron acceptors in accordance with model predictions. Differences in the ability of Shewanella species to respond to and use available electron acceptors is thought to play an important role in their ecology. Therefore, these results validate the use of the DGC system to measure and simulate Shewanella chemotaxis in response to electron acceptor gradients and establish it as a research tool to help elucidate Shewanellas role in environmental processes.

Next-generation probiotics targeting Clostridium difficile through precursor-directed antimicrobial biosynthesis

ABSTRACT Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. Clostridium difficile infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified Lactobacillus reuteri to be a promising candidate for adjunct therapy. Human-derived L. reuteri bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the pocR gene locus were potent reuterin producers, with L. reuteri 17938 inhibiting C. difficile growth at a level on par with the level of growth inhibition by vancomycin. Targeted pocR mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas treatment with either glycerol or L. reuteri alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with L. reuteri elicited changes in the composition and function of the human microbial community that preferentially targets C. difficile outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of C. difficile, and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials.