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A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae.

ABSTRACT: Defined mutant libraries allow for efficient genome-scale screening and provide a convenient collection of mutations in almost any nonessential gene of interest. Here, we present a near-saturating transposon insertion library in Vibrio cholerae strain C6706, a clinical isolate belonging to the O1 El Tor biotype responsible for the current cholera pandemic. Automated sequencing analysis of 23,312 mutants allowed us to build a 3,156-member subset library containing a representative insertion in every disrupted ORF. Because uncharacterized mutations that affect motility have shown utility in attenuating V. cholerae live vaccines, we used this genome-wide subset library to define all genes required for motility and to further assess the accuracy and purity of the library. In this screen, we identified the hypothetical gene VC2208 (flgT) as essential for motility. Flagellated cells were very rare in a flgT mutant, and transcriptional analysis showed it was specifically stalled at the class III/IV assembly checkpoint of the V. cholerae flagellar regulatory system. Because FlgT is predicted to have structural homology to TolB, a protein involved in determining outer membrane architecture, and the sheath of the V. cholerae flagellum appears to be derived from the cell's outer membrane, FlgT may play a direct role in flagellar sheath formation.

SUBMITTER: Cameron DE

PROVIDER: S-EPMC2438431 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae.

Cameron D Ewen DE Urbach Jonathan M JM Mekalanos John J JJ

Proceedings of the National Academy of Sciences of the United States of America 20080623 25

Defined mutant libraries allow for efficient genome-scale screening and provide a convenient collection of mutations in almost any nonessential gene of interest. Here, we present a near-saturating transposon insertion library in Vibrio cholerae strain C6706, a clinical isolate belonging to the O1 El Tor biotype responsible for the current cholera pandemic. Automated sequencing analysis of 23,312 mutants allowed us to build a 3,156-member subset library containing a representative insertion in ev ...[more]

PMID: 18574146

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Project description:Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a Vibrio fischeri mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in alr, glr (murI), glmS, several heme biosynthesis genes, and ftsA, as well as a mutant disrupted 14 bp upstream of ftsQ Mutants with insertions in ftsA or upstream of ftsQ were recovered by addition of Mg2+ to LBS, but their cell morphology and motility were affected. The ftsA mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in glmS, alr, or glr was recovered with N-acetylglucosamine (NAG), d-alanine, or d-glutamate, respectively. We hypothesized that NAG, d-alanine, or d-glutamate might be available to V. fischeri in the Euprymna scolopes light organ; however, none of these mutants colonized the host effectively. In contrast, hemA and hemL mutants, which are auxotrophic for δ-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide Bradyrhizobium symbionts with ALA, but they contrast with virulence phenotypes of hemA mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment.IMPORTANCE By supplementing a rich yeast-based medium, we were able to recover V. fischeri mutants with insertions in conditionally essential genes, and further characterization of these mutants provided new insights into this bacterium's symbiotic environment. Most notably, we show evidence that the squid host can provide V. fischeri with enough ALA to support its growth in the light organ, paralleling the finding that legumes provide Bradyrhizobium ALA in symbiotic nodules. Taken together, our results show how a simple method of augmenting already rich media can expand the reach and utility of defined mutant libraries.

Project description:Cholera disease is caused by Vibrio cholerae infecting the lining of the small intestine and results in severe diarrhea. V. cholerae's swimming motility is known to play a crucial role in pathogenicity and may aid the bacteria in crossing the intestinal mucus barrier to reach sites of infection, but the exact mechanisms are unknown. The cell can be either pushed or pulled by its single polar flagellum, but there is no consensus on the resulting repertoire of motility behaviors. We use high-throughput three-dimensional (3D) bacterial tracking to observe V. cholerae swimming in buffer, in viscous solutions of the synthetic polymer PVP, and in mucin solutions that may mimic the host environment. We perform a statistical characterization of its motility behavior on the basis of large 3D trajectory data sets. We find that V. cholerae performs asymmetric run-reverse-flick motility, consisting of a sequence of a forward run, reversal, and a shorter backward run, followed by a turn by approximately 90°, called a flick, preceding the next forward run. Unlike many run-reverse-flick swimmers, V. cholerae's backward runs are much shorter than its forward runs, resulting in an increased effective diffusivity. We also find that the swimming speed is not constant but subject to frequent decreases. The turning frequency in mucin matches that observed in buffer. Run-reverse-flick motility and speed fluctuations are present in all environments studied, suggesting that these behaviors also occur in natural aquatic habitats as well as the host environment. IMPORTANCE Cholera disease produces vomiting and severe diarrhea and causes approximately 100,000 deaths per year worldwide. The disease is caused by the bacterium Vibrio cholerae colonizing the lining of the small intestine. V. cholerae's ability to swim is known to increase its infectivity, but the underlying mechanisms are not known. One possibility is that swimming aids in crossing the protective mucus barrier that covers the lining of the small intestine. Our work characterizing how V. cholerae swims in environments that mimic properties of the host environment may advance the understanding of how motility contributes to infection.

Project description:Motility is required for many bacterial pathogens to reach and colonize target sites. Vibrio cholerae traverses a thick mucus barrier coating the small intestine to reach the underlying epithelium. We screened a transposon library in motility medium containing mucin to identify factors that influence mucus transit. Lesions in structural genes of the type VI secretion system (T6SS) were among those recovered. Two-dimensional (2D) and 3D single-cell tracking was used to compare the motility behaviors of wild-type cells and a mutant that collectively lacked three essential T6SS structural genes (T6SS-). In the absence of mucin, wild-type and T6SS- cells exhibited similar speeds and run-reverse-flick (RRF) swimming patterns, in which forward-moving cells briefly backtrack before stochastically reorienting (flicking) in a new direction upon resuming forward movement. We show that mucin induced T6SS expression and activity in wild-type bacteria but significantly decreased their swimming speed and flicking, yielding curvilinear or near-surface circular traces for many cells. Conversely, mucin slowed T6SS- cells to a lesser extent, and many continued to flick and produce RRF-like traces. ΔcheY3 cells, which exclusively swim in the forward direction and thus cannot flick, also produced curvilinear traces with or without mucin present and, on occasion, near-surface circular traces in the presence of mucin. The dependence of flicking on swimming speed suggested that mucin-induced T6SS activity further decreased V. cholerae motility and thereby reduced flicking probability during reverse-to-forward transitions. We propose that this encourages cells to continue on their current trajectory rather than reorienting, which may benefit those tracking toward the epithelial surface.IMPORTANCEV. cholerae deploys an arsenal of virulence factors as it attempts to traverse a protective mucus layer and reach the epithelial surface of the distal small intestine. The T6SS used to cull bacterial competition during infection is induced by mucus. We show that this activity may serve an additional purpose by further decreasing motility in the presence of mucin, thereby reducing the probability of speed-dependent, near-perpendicular directional changes. We posit that this encourages cells to maintain course rather than change direction, which may aid those attempting to reach and colonize the epithelial surface.

Project description:UnlabelledThe entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions.ImportanceThe relationship between Photorhabdus and entomopathogenic nematode Heterorhabditis represents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding of Photorhabdus as both an insect pathogen and a nematode symbiont.

Dataset Information

A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae.

Publications

A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae.

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