Gene Fitness Landscapes of Vibrio cholerae at Important Stages of Its Life Cycle

Kamp, Heather D.; Patimalla-Dipali, Bharathi; Lazinski, David W.; Wallace-Gadsden, Faith; Camilli, Andrew

doi:10.1371/journal.ppat.1003800

Cited by 212 publications

(183 citation statements)

References 44 publications

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“…Deletion of these transporters was found to affect V. cholerae growth under zinc-deficient conditions, including in in vivo adult and infant mouse models, particularly in the presence of the gut microbiota. A recent transposon sequencing screening study indicates that ZnuABC contributes to V. cholerae colonization in the infant rabbit model (25), supporting the notion that zinc transporters are important for the V. cholerae-host interaction. A few bacterial species have been reported to have more than one zinc uptake system (2).…”

Section: Resultsmentioning

confidence: 79%

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Dual Zinc Transporter Systems in Vibrio cholerae Promote Competitive Advantages over Gut Microbiome

et al. 2015

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Zinc is an essential trace metal required for numerous cellular processes in all forms of life. In order to maintain zinc homeostasis, bacteria have developed several transport systems to regulate its uptake. In this study, we investigated zinc transport systems in the enteric pathogen Vibrio cholerae, the causative agent of cholera. Bioinformatic analysis predicts that two gene clusters, VC2081 to VC2083 (annotated as zinc utilization genes znuABC) and VC2551 to VC2555 (annotated as zinc-regulated genes zrgABCDE), are regulated by the putative zinc uptake regulator Zur. Using promoter reporter and biochemical assays, we confirmed that Zur represses znuABC and zrgABCDE promoters in a Zn 2؉ -dependent manner. Under Zn 2؉ -limiting conditions, we found that mutations in either the znuABC or zrgABCDE gene cluster affect bacterial growth, with znuABC mutants displaying a more severe growth defect, suggesting that both ZnuABC and ZrgABCDE are involved in Zn 2؉ uptake and that ZnuABC plays the predominant role. Furthermore, we reveal that ZnuABC and ZrgABCDE are important for V. cholerae colonization in both infant and adult mouse models, particularly in the presence of other intestinal microbiota. Collectively, our studies indicate that these two zinc transporter systems play vital roles in maintaining zinc homeostasis during V. cholerae growth and pathogenesis. Metal ions are required for many crucial biological processes and are necessary for the survival of living organisms, including bacteria (1). For example, zinc is an essential cofactor for enzymatic reactions, DNA synthesis, and gene expression (2). One study has shown that over 3% of Escherichia coli proteins contain zinc (3). Bacteria have therefore evolved sophisticated systems to control their intracellular zinc concentrations in response to zinc fluctuations in the environment. One system utilized by nearly all bacteria is ZnuABC, a high-affinity zinc uptake system belonging to the ATP binding cassette (ABC) transporter family (4). Three proteins constitute this system: ZnuA, a periplasmic Zn 2ϩ binding protein that captures and delivers zinc to ZnuB, which serves as an inner membrane channel, and ZnuC, an ATPase that provides the energy needed for zinc transport (4). On the other hand, zinc levels in bacteria need to be tightly regulated, as excess zinc has deleterious effects on cells, such as prevention of Mn 2ϩ intracellular accumulation (5) and inhibition of enzymes (6). Zinc transport genes are generally controlled by Zur, a member of the Fur protein family of metal-dependent transcriptional regulators (7). Under zinc-replete conditions, Zur binds free Zn 2ϩ . The Zur-Zn complex then binds to the promoter of znuABC, thus blocking the binding of RNA polymerase (8). Under zinc-deficient conditions, the zinc binding sites of Zur are unoccupied, leading to the destabilization of Zur and the inability to bind and repress znuABC transcription, thus allowing zinc acquisition. In some bacteria, in addition to repressing Zn 2ϩ uptake transporters, Zur can a...

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Section: Resultsmentioning

confidence: 79%

“…V. cholerae El Tor C6706 (25) was used as the wild-type strain in this study. In-frame deletions were constructed by cloning the regions flanking the target genes into suicide vector pWM91 containing a sacB counterselectable marker (26).…”

Section: Methodsmentioning

confidence: 99%

Dual Zinc Transporter Systems in Vibrio cholerae Promote Competitive Advantages over Gut Microbiome

et al. 2015

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“…Heme is a potential iron source in the intestine and was reported to be present within the cecal fluid of the rabbit intestine (64,65); however, to our knowledge, this has not been assessed in infant mice. Disruption of the three heme receptors in V. cholerae, which prevented heme transport, did not result in a loss of fitness during in vivo competition with the wild type (24).…”

Section: Discussionmentioning

confidence: 93%

Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae

et al. 2016

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Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, thrives in both marine environments and the human host. To do so, it must encode the tools necessary to acquire essential nutrients, including iron, under these vastly different conditions. A number of V. cholerae iron acquisition systems have been identified; however, the precise role of each system is not fully understood. To test the roles of individual systems, we generated a series of mutants in which only one of the four systems that support iron acquisition on unsupplemented LB agar, Feo, Fbp, Vct, and Vib, remains functional. Analysis of these mutants under different growth conditions showed that these systems are not redundant. The strain carrying only the ferrous iron transporter Feo grew well at acidic, but not alkaline, pH, whereas the ferric iron transporter Fbp promoted better growth at alkaline than at acidic pH. A strain defective in all four systems (null mutant) had a severe growth defect under aerobic conditions but accumulated iron and grew as well as the wild type in the absence of oxygen, suggesting the presence of an additional, unidentified iron transporter in V. cholerae. In support of this, the null mutant was only moderately attenuated in an infant mouse model of infection. While the null mutant used heme as an iron source in vitro, we demonstrate that heme is not available to V. cholerae in the infant mouse intestine. The Gram-negative bacterium Vibrio cholerae has an absolute requirement for iron (1). Despite the prevalence of iron within its environmental niches and human host, most of this iron is not readily available. Iron occurs naturally as oxidized (ferric) or reduced (ferrous) iron (2). Ferric iron(III), found in oxygenated environments at neutral to alkaline pH, has very low water solubility and forms large insoluble complexes. Studies of ocean water have found iron to be the limiting nutrient for microbial growth (3). Soluble, ferrous iron(II) is more prevalent under the anoxic conditions that V. cholerae may encounter while colonizing the human host. However, access to iron in the host is limited due to competition with other microbes on host surfaces, as well as sequestration by high-affinity iron-binding host proteins such as transferrin and lactoferrin (4-7). Despite the challenges of obtaining iron in these various environments, V. cholerae is proficient in growth in both marine and host environments.The V. cholerae genome encodes multiple iron acquisition systems, which each transport a specific form of iron and may thus optimize iron acquisition in the various niches that V. cholerae inhabits (8-12). These include genes for the synthesis (vib) and utilization (viu) of vibriobactin, a siderophore that is secreted into the environment, where it binds ferric iron with extremely high affinity (9,(13)(14)(15). Ferrivibriobactin is bound by the outer membrane receptor ViuA (16,17) and is transported across the outer membrane in a process that requires either of two energy transduction systems, To...

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“…2), during growth in the host intestine, NlpD's role cannot be filled by EnvC. The increased importance of NlpD for in vivo growth is corroborated by two recent transposon insertion sequencing studies that revealed that nlpD mutants (but not envC mutants) have reduced fitness in the infant rabbit model of cholera (25,37).…”

Section: Bacth Analysis Suggests That Multiple Interactions Occur Amomentioning

confidence: 96%

Cell Separation in Vibrio cholerae Is Mediated by a Single Amidase Whose Action Is Modulated by Two Nonredundant Activators

et al. 2014

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Synthesis and hydrolysis of septal peptidoglycan (PG) are critical processes at the conclusion of cell division that enable separation of daughter cells. Cleavage of septal PG is mediated by PG amidases, hydrolytic enzymes that release peptide side chains from the glycan strand. Most gammaproteobacteria, including Escherichia coli, encode several functionally redundant periplasmic amidases. However, members of the Vibrio genus, including the enteric pathogen Vibrio cholerae, encode only a single PG amidase, AmiB. Here, we show that V. cholerae AmiB is crucial for cell division and growth. Genetic and biochemical analyses indicated that AmiB is regulated by two activators, EnvC and NlpD, at least one of which is required for AmiB's localization to the cell division site. Localization of the activators (and thus of AmiB) is dependent upon the cell division protein FtsN. These factors mediate septal PG cleavage in E. coli as well; however, their precise roles vary between the two organisms in a number of ways. Notably, even though V. cholerae EnvC and NlpD appear to be functionally redundant under most growth conditions tested, NlpD is specifically required for intestinal colonization in the infant mouse model of cholera and for V. cholerae resistance against bile salts, perhaps due to environmental regulation of AmiB or its activators. Collectively, our findings reveal that although the cellular components that enable cleavage of septal PG appear to be generally conserved between E. coli and V. cholerae, they can be combined into diverse functional regulatory networks.

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Gene Fitness Landscapes of Vibrio cholerae at Important Stages of Its Life Cycle

Cited by 212 publications

References 44 publications

Dual Zinc Transporter Systems in Vibrio cholerae Promote Competitive Advantages over Gut Microbiome

Dual Zinc Transporter Systems in Vibrio cholerae Promote Competitive Advantages over Gut Microbiome

Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae

Cell Separation in Vibrio cholerae Is Mediated by a Single Amidase Whose Action Is Modulated by Two Nonredundant Activators

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