High-throughput sequencing reveals suppressors of Vibrio cholerae rpoE mutations: one fewer porin is enough

Analyses of suppressor mutations have been extremely valuable in understanding gene function. However, techniques for mapping suppressor mutations are not available for most bacterial species. Here, we used high-throughput sequencing technology to identify spontaneously arising suppressor mutations that enabled disruption of rpoE (which encodes σE) in Vibrio cholerae, the agent of cholera. The alternative sigma factor σE, which is activated by envelope stress, promotes expression of factors that help preserve and/or restore cell envelope integrity. In Escherichia coli, rpoE is an essential gene that can only be disrupted in the presence of additional suppressor mutations. Among a panel of independent V. cholerae rpoE mutants, more than 75% contain suppressor mutations that reduce production of OmpU, V. cholerae’s principal outer membrane porin. OmpU appears to be a key determinant of V. cholerae’s requirement for and production of σE. Such dependence upon a single factor contrasts markedly with regulation of σE in E. coli, in which numerous factors contribute to its activation and none is dominant. We also identified a suppressor mutation that differs from all previously described suppressors in that it elevates, rather than reduces, σE’s activity. Finally, analyses of a panel of rpoE mutants shed light on the mechanisms by which suppressor mutations may arise in V. cholerae

A Multiorgan Trafficking Circuit Provides Purifying Selection of Listeria monocytogenes Virulence Genes.

Listeria monocytogenes can cause a life-threatening illness when the foodborne pathogen spreads beyond the intestinal tract to distant organs. Many aspects of the intestinal phase of L. monocytogenes pathogenesis remain unknown. Here, we present a foodborne infection model using C57BL/6 mice that have been pretreated with streptomycin. In this model, as few as 100 L. monocytogenes CFU were required to cause self-limiting enterocolitis, and systemic dissemination followed previously reported routes. Using this model, we report that listeriolysin O (LLO) and actin assembly-inducing protein (ActA), two critical virulence determinants, were necessary for intestinal pathology and systemic spread but were dispensable for intestinal growth. Sequence tag-based analysis of microbial populations (STAMP) was used to investigate the within-host population dynamics of wild-type and LLO-deficient strains. The wild-type bacterial population experienced severe bottlenecks over the course of infection, and by 5 days, the intestinal population was highly enriched for bacteria originating from the gallbladder. In contrast, LLO-deficient strains did not efficiently disseminate and gain access to the gallbladder, and the intestinal population remained diverse. These findings suggest that systemic spread and establishment of a bacterial reservoir in the gallbladder imparts an intraspecies advantage in intestinal occupancy. Since intestinal L. monocytogenes is ultimately released into the environment, within-host population bottlenecks may provide purifying selection of virulence genes.IMPORTANCE Listeria monocytogenes maintains capabilities for free-living growth in the environment and for intracellular replication in a wide range of hosts, including livestock and humans. Here, we characterized an enterocolitis model of foodborne L. monocytogenes infection. This work highlights a multiorgan trafficking circuit and reveals a fitness advantage for bacteria that successfully complete this cycle. Because virulence factors play critical roles in systemic dissemination and multiple bottlenecks occur as the bacterial population colonizes different tissue sites, this multiorgan trafficking circuit likely provides purifying selection of virulence genes. This study also serves as a foundation for future work using the L. monocytogenes-induced enterocolitis model to investigate the biology of L. monocytogenes in the intestinal environment

Identification of 17 Pseudomonas aeruginosa sRNAs and prediction of sRNA-encoding genes in 10 diverse pathogens using the bioinformatic tool sRNAPredict2

sRNAs are small, non-coding RNA species that control numerous cellular processes. Although it iswidely accepted that sRNAs are encoded by most if not all bacteria, genome-wide annotations for sRNA-encoding genes have been conducted in only a few of the nearly 300 bacterial species sequenced to date. To facilitate the efficient annotation of bacterial genomes for sRNA-encoding genes, we developed a program, sRNAPredict2, that identifies putative sRNAs by searching for co-localization of genetic features commonly associated with sRNA-encoding genes. Using sRNAPredict2, we conducted genome-wide annotations for putative sRNA-encoding genes in the intergenic regions of 11 diverse pathogens. In total, 2759 previously unannotated candidate sRNA loci were predicted. There was considerable range in the number of sRNAs predicted in the different pathogens analyzed, raising the possibility that there are species-specific differences in the reliance on sRNA-mediated regulation. Of 34 previously unannotated sRNAs predicted in the opportunistic pathogen Pseudomonas aeruginosa, 31 were experimentally tested and 17 were found to encode sRNA transcripts. Our findings suggest that numerous genes have been missed in the current annotations of bacterial genomes and that, by using improved bioinformatic approaches and tools, much remains to be discovered in ‘intergenic’ sequences

Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition

The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein interactions in live bacteria. As proof of concept and to develop a new tool for studies of bacterial chemotaxis, fragments of Renilla luciferase (Rluc) were fused to the chemotaxis-associated response regulator CheY3 and its phosphatase CheZ in the enteric pathogen Vibrio cholerae. Luciferase activity was dependent on the presence of both CheY3 and CheZ fusion proteins, demonstrating the specificity of the assay. Furthermore, enzyme activity was markedly reduced in V. cholerae chemotaxis mutants, suggesting that this approach can measure defects in chemotactic signaling. However, attempts to measure changes in dynamic CheY3-CheZ interactions in response to various chemoeffectors were undermined by nonspecific inhibition of the full-length luciferase. These observations reveal an unexpected limitation of split Rluc complementation that may have implications for existing data and highlight the need for great caution when evaluating small molecule effects on dynamic protein-protein interactions using the split luciferase technology

Mobile Antibiotic Resistance Encoding Elements Promote Their Own Diversity

Integrating conjugative elements (ICEs) are a class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome. The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties. These elements contribute to the spread of antibiotic resistance genes in several gram-negative bacteria including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found evidence that the genomes of these elements have been shaped by inter–ICE recombination. We developed a high throughput semi-quantitative method to explore the genetic determinants involved in hybrid ICE formation. Recombinant ICE formation proved to be relatively frequent, and to depend on host (recA) and ICE (s065 and s066) loci, which can independently and potentially cooperatively mediate hybrid ICE formation. s065 and s066, which are found in all SXT/R391 ICEs, are orthologues of the bacteriophage λ Red recombination genes bet and exo, and the s065/s066 recombination system is the first Red-like recombination pathway to be described in a conjugative element. Neither ICE excision nor conjugative transfer proved to be essential for generation of hybrid ICEs. Instead conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics. Thus, ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes

PprA Contributes to Deinococcus radiodurans Resistance to Nalidixic Acid, Genome Maintenance after DNA Damage and Interacts with Deinococcal Topoisomerases

PprA is known to contribute to Deinococcus radiodurans' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA's role(s) in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans's capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB) as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal), an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA) in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB

Endopeptidase-Mediated Beta Lactam Tolerance

In many bacteria, inhibition of cell wall synthesis leads to cell death and lysis. The pathways and enzymes that mediate cell lysis after exposure to cell wall-acting antibiotics (e.g. beta lactams) are incompletely understood, but the activities of enzymes that degrade the cell wall (‘autolysins’) are thought to be critical. Here, we report that Vibrio cholerae, the cholera pathogen, is tolerant to antibiotics targeting cell wall synthesis. In response to a wide variety of cell wall- acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical. Genetic analyses revealed that paradoxically, V. cholerae survival via sphere formation required the activity of D,D endopeptidases, enzymes that cleave the cell wall. Other autolysins proved dispensable for this process. Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate - sphere formation vs. lysis – after treatment with antibiotics that target cell wall synthesis

Comparative RNA-Seq based dissection of the regulatory networks and environmental stimuli underlying Vibrio parahaemolyticus gene expression during infection

Vibrio parahaemolyticus is the leading worldwide cause of seafood-associated gastroenteritis, yet little is known regarding its intraintestinal gene expression or physiology. To date, in vivo analyses have focused on identification and characterization of virulence factors—e.g. a crucial Type III secretion system (T3SS2)—rather than genome-wide analyses of in vivo biology. Here, we used RNA-Seq to profile V. parahaemolyticus gene expression in infected infant rabbits, which mimic human infection. Comparative transcriptomic analysis of V. parahaemolyticus isolated from rabbit intestines and from several laboratory conditions enabled identification of mRNAs and sRNAs induced during infection and of regulatory factors that likely control them. More than 12% of annotated V. parahaemolyticus genes are differentially expressed in the intestine, including the genes of T3SS2, which are likely induced by bile-mediated activation of the transcription factor VtrB. Our analyses also suggest that V. parahaemolyticus has access to glucose or other preferred carbon sources in vivo, but that iron is inconsistently available. The V. parahaemolyticus transcriptional response to in vivo growth is far more widespread than and largely distinct from that of V. cholerae, likely due to the distinct ways in which these diarrheal pathogens interact with and modulate the environment in the small intestine