Mark J. Mandel

A single regulatory gene is sufficient to alter bacterial host range.

Microbial symbioses are essential for the normal development and growth of animals. Often, symbionts must be acquired from the environment during each generation, and identification of the relevant symbiotic partner against a myriad of unwanted relationships is a formidable task. Although examples of this specificity are well-documented, the genetic mechanisms governing it are poorly characterized. Here we show that the two-component sensor kinase RscS is necessary and sufficient for conferring efficient colonization of Euprymna scolopes squid by bioluminescent Vibrio fischeri from the North Pacific Ocean. In the squid symbiont V. fischeri ES114, RscS controls light-organ colonization by inducing the Syp exopolysaccharide, a mediator of biofilm formation during initial infection. A genome-level comparison revealed that rscS, although present in squid symbionts, is absent from the fish symbiont V. fischeri MJ11. We found that heterologous expression of RscS in strain MJ11 conferred the ability to colonize E. scolopes in a manner comparable to that of natural squid isolates. Furthermore, phylogenetic analyses support an important role for rscS in the evolution of the squid symbiosis. Our results demonstrate that a regulatory gene can alter the host range of animal-associated bacteria. We show that, by encoding a regulator and not an effector that interacts directly with the host, a single gene can contribute to the evolution of host specificity by switching ‘on’ pre-existing capabilities for interaction with animal tissue.

Transcriptional patterns in both host and bacterium underlie a daily rhythm of anatomical and metabolic change in a beneficial symbiosis.

Mechanisms for controlling symbiont populations are critical for maintaining the associations that exist between a host and its microbial partners. We describe here the transcriptional, metabolic, and ultrastructural characteristics of a diel rhythm that occurs in the symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri. The rhythm is driven by the host’s expulsion from its light-emitting organ of most of the symbiont population each day at dawn. The transcriptomes of both the host epithelium that supports the symbionts and the symbiont population itself were characterized and compared at four times over this daily cycle. The greatest fluctuation in gene expression of both partners occurred as the day began. Most notable was an up-regulation in the host of >50 cytoskeleton-related genes just before dawn and their subsequent down-regulation within 6 h. Examination of the epithelium by TEM revealed a corresponding restructuring, characterized by effacement and blebbing of its apical surface. After the dawn expulsion, the epithelium reestablished its polarity, and the residual symbionts began growing, repopulating the light organ. Analysis of the symbiont transcriptome suggested that the bacteria respond to the effacement by up-regulating genes associated with anaerobic respiration of glycerol; supporting this finding, lipid analysis of the symbionts’ membranes indicated a direct incorporation of host-derived fatty acids. After 12 h, the metabolic signature of the symbiont population shifted to one characteristic of chitin fermentation, which continued until the following dawn. Thus, the persistent maintenance of the squid–vibrio symbiosis is tied to a dynamic diel rhythm that involves both partners.

Escherichia coli starvation diets: essential nutrients weigh in distinctly.

Bacterial growth is often limited by availability of nutrients. Soil, water, and even host environments such as macrophages can be nutrient poor, lacking essential building blocks for growth, including carbon, nitrogen, and phosphorus. The ratios of these elements differ according to the local

Starvation for Different Nutrients in Escherichia coli Results in Differential Modulation of RpoS Levels and Stability

Levels of RpoS increase upon glucose starvation in Escherichia coli, which leads to the transcription of genes whose products combat a variety of stresses. RpoS stability is a key level of control in this process, as SprE (RssB)-mediated degradation is inhibited under glucose starvation. Starvation for ammonia or phosphate also results in increased stress resistance and induction of RpoS-dependent genes. However, we demonstrate that RpoS levels following ammonia starvation are only slightly increased compared to growing cells and are 10-fold below the levels observed under glucose or phosphate limitation. This difference is largely due to regulated proteolysis of RpoS, as its stability in ammonia-starved cells is intermediate between that in logarithmic-phase cells and glucose-starved cells. Use of an rpoS construct that is devoid of the genes native transcriptional and translational control regions reveals that stability differences are sufficient to explain the different levels of RpoS observed in logarithmic phase, ammonia starvation, and glucose starvation. Under phosphate starvation, however, rpoS translation is increased. The cellular response to nutrient limitation is much more complex than previously appreciated, as there is not simply one response that is activated by starvation for any essential nutrient. Our data support the hypothesis that SprE activity is the key level at which ammonia and glucose starvation signals are transmitted to RpoS, and they suggest that carbon source and/or energy limitation are necessary for full inactivation of the SprE pathway.

Genome-Wide Identification of Acinetobacter baumannii Genes Necessary for Persistence in the Lung

ABSTRACT Acinetobacter baumannii is a Gram-negative bacterium that causes diseases such as pneumonia, bacteremia, and soft tissue infections in hospitalized patients. Relatively little is known about how A. baumannii causes these infections. Thus, we used insertion sequencing (INSeq), a combination of transposon mutagenesis and massively parallel next-generation sequencing, to identify novel virulence factors of A. baumannii. To this end, we generated a random transposon mutant library containing 150,000 unique insertions in A. baumannii strain ATCC 17978. The INSeq analysis identified 453 genes required for growth in rich medium. The library was then used in a murine pneumonia model, and the relative levels of abundance of mutants before and after selection in the mouse were compared. When genes required for growth in rich medium were removed from the analysis, 157 genes were identified as necessary for persistence in the mouse lung. Several of these encode known virulence factors of A. baumannii, such as OmpA and ZnuB, which validated our approach. A large number of the genes identified were predicted to be involved in amino acid and nucleotide metabolism and transport. Other genes were predicted to encode an integration host factor, a transmembrane lipoprotein, and proteins involved in stress response and efflux pumps. Very few genes, when disrupted, resulted in an increase in A. baumannii numbers during host infection. The INSeq approach identified a number of novel virulence determinants of A. baumannii, which are candidate targets for therapeutic interventions. IMPORTANCE A. baumannii has emerged as a frequent cause of serious infections in hospitals and community settings. Due to increasing antibiotic resistance, alternative approaches, such as antivirulence strategies, are desperately needed to fight A. baumannii infections. Thorough knowledge of A. baumannii pathogenicity is essential for such approaches but is currently lacking. With the increasingly widespread use of massively parallel sequencing, a class of techniques known as transposon insertion sequencing has been developed to perform comprehensive virulence screens of bacterial genomes in vivo. We have applied one of these approaches (INSeq) to uncover novel virulence factors in A. baumannii. We identified several such factors, including those predicted to encode amino acid and nucleotide metabolism proteins, an integration host factor protein, stress response factors, and efflux pumps. These results greatly expand the number of A. baumannii virulence factors and uncover potential targets for antivirulence treatments. A. baumannii has emerged as a frequent cause of serious infections in hospitals and community settings. Due to increasing antibiotic resistance, alternative approaches, such as antivirulence strategies, are desperately needed to fight A. baumannii infections. Thorough knowledge of A. baumannii pathogenicity is essential for such approaches but is currently lacking. With the increasingly widespread use of massively parallel sequencing, a class of techniques known as transposon insertion sequencing has been developed to perform comprehensive virulence screens of bacterial genomes in vivo. We have applied one of these approaches (INSeq) to uncover novel virulence factors in A. baumannii. We identified several such factors, including those predicted to encode amino acid and nucleotide metabolism proteins, an integration host factor protein, stress response factors, and efflux pumps. These results greatly expand the number of A. baumannii virulence factors and uncover potential targets for antivirulence treatments.

AinS Quorum Sensing Regulates the Vibrio fischeri Acetate Switch

The marine bacterium Vibrio fischeri uses two acyl-homoserine lactone (acyl-HSL) quorum-sensing systems. The earlier signal, octanoyl-HSL, produced by AinS, is required for normal colonization of the squid Euprymna scolopes and, in culture, is necessary for a normal growth yield. In examining the latter requirement, we found that during growth in a glycerol/tryptone-based medium, wild-type V. fischeri cells initially excrete acetate but, in a metabolic shift termed the acetate switch, they subsequently utilize the acetate, removing it from the medium. In contrast, an ainS mutant strain grown in this medium does not remove the excreted acetate, which accumulates to lethal levels. The acetate switch is characterized by the induction of acs, the gene encoding acetyl coenzyme A (acetyl-CoA) synthetase, leading to uptake of the excreted acetate. Wild-type cells induce an acs transcriptional reporter 25-fold, coincident with the disappearance of the extracellular acetate; in contrast, the ainS mutant did not display significant induction of the acs reporter. Supplementation of the medium of an ainS mutant with octanoyl-HSL restored normal levels of acs induction and acetate uptake. Additional mutant analyses indicated that acs regulation was accomplished through the regulator LitR but was independent of the LuxIR quorum-signaling pathway. Importantly, the acs mutant of V. fischeri has a competitive defect when colonizing the squid, indicating the importance of proper control of acetate metabolism in the light of organ symbiosis. This is the first report of quorum-sensing control of the acetate switch, and it indicates a metabolic connection between acetate utilization and cell density.

Squid-Derived Chitin Oligosaccharides Are a Chemotactic Signal during Colonization by Vibrio fischeri

ABSTRACT Chitin, a polymer of N-acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the family Vibrionaceae (“vibrios”), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacterium Vibrio fischeri is the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes. The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbionts nutritional requirements. V. fischeri cells are harvested from seawater during each host generation, and V. fischeri is the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria. V. fischeri uses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone.

Comparative genomics-based investigation of resequencing targets in Vibrio fischeri: Focus on point miscalls and artefactual expansions

BackgroundSequence closure often represents the end-point of a genome project, without a system in place for subsequent improvement and refinement. Building on the genome project of Vibrio fischeri ES114, we used a comparative approach to identify and investigate genes that had a high likelihood of sequence error.ResultsComparison of the V. fischeri ES114 genome with that of conspecific strain MJ11 identified 82 target loci in ES114 as containing likely errors, and thus of high-priority for resequencing. Analysis of the targets identified 75 loci in which an error had occurred, resulting in the correction of 10,457 base pairs to generate the new ES114 genomic sequence. A majority of the inaccurate loci involved frameshift errors, correction of which fused adjacent ORFs. Although insertions/deletions are thought to be rare in microbial genome assemblies, fourteen of the loci contained extraneous sequence of over 300 bp, likely due to imperfect contig ends that were misassembled in tandem rather than as overlapping segments. Additionally we updated the entire genome annotation with 113 new features including previously uncalled protein-coding genes, regulatory RNA genes and operon leader peptides, and we analyzed the transcriptional apparatus encoded by ES114.ConclusionWe demonstrate that errors in microbial genome sequences, thought to largely be confined to point mutations, may also consist of other prevalent large-scale rearrangements such as insertions. Ongoing genome quality control and annotation programs are necessary to accompany technological advancements in data generation. These updates further advance V. fischeri as an important model for understanding intercellular communication and colonization of animal tissue.

Crl Facilitates RNA Polymerase Holoenzyme Formation

The Escherichia coli Crl protein has been described as a transcriptional coactivator for the stationary-phase sigma factor sigma(S). In a transcription system with highly purified components, we demonstrate that Crl affects transcription not only by the Esigma(S) RNA polymerase holoenzyme but also by Esigma(70) and Esigma(32). Crl increased transcription dramatically but only when the sigma concentration was low and when Crl was added to sigma prior to assembly with the core enzyme. Our results suggest that Crl facilitates holoenzyme formation, the first positive regulator identified with this mechanism of action.

Global discovery of colonization determinants in the squid symbiont Vibrio fischeri

Significance Animals form associations with bacteria that play important roles in host development and fitness. The mechanisms by which animals horizontally acquire their bacterial partners from the environment are poorly understood. To address this question, we take advantage of a natural symbiosis between the luminous Gram-negative bacterium Vibrio fischeri and its squid host, Euprymna scolopes. We applied the insertion sequencing global approach and identified 380 colonization determinants in V. fischeri. Characterization of the factors revealed novel biofilm regulation and beneficial colonization factors at the cell envelope. To our knowledge, our study is the first global functional analysis in V. fischeri and expands opportunities for systems biology approaches at the host microbe interface in a valuable reductionist model of microbiota colonization. Animal epithelial tissue becomes reproducibly colonized by specific environmental bacteria. The bacteria (microbiota) perform critical functions for the host’s tissue development, immune system development, and nutrition; yet the processes by which bacterial diversity in the environment is selected to assemble the correct communities in the host are unclear. To understand the molecular determinants of microbiota selection, we examined colonization of a simplified model in which the light organ of Euprymna scolopes squid is colonized exclusively by Vibrio fischeri bacteria. We applied high-throughput insertion sequencing to identify which bacterial genes are required during host colonization. A library of over 41,000 unique transposon insertions was analyzed before and after colonization of 1,500 squid hatchlings. Mutants that were reproducibly depleted following squid colonization represented 380 genes, including 37 that encode known colonization factors. Validation of select mutants in defined competitions against the wild-type strain identified nine mutants that exhibited a reproducible colonization defect. Some of the colonization factors identified included genes predicted to influence copper regulation and secretion. Other mutants exhibited defects in biofilm development, which is required for aggregation in host mucus and initiation of colonization. Biofilm formation in culture and in vivo was abolished in a strain lacking the cytoplasmic chaperone DnaJ, suggesting an important role for protein quality control during the elaboration of bacterial biofilm in the context of an intact host immune system. Overall these data suggest that cellular stress responses and biofilm regulation are critical processes underlying the reproducible colonization of animal hosts by specific microbial symbionts.