Impact of Na+-Translocating NADH:Quinone Oxidoreductase on Iron Uptake and nqrM Expression in Vibrio cholerae

ABSTRACT The Na+ ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is a membrane-bound respiratory enzyme which harbors flavins and Fe-S clusters as redox centers. The NQR is the main producer of the sodium motive force (SMF) and drives energy-dissipating processes such as flagellar rotation, substrate uptake, ATP synthesis, and cation-proton antiport. The NQR requires for its maturation, in addition to the six structural genes nqrABCDEF, a flavin attachment gene, apbE, and the nqrM gene, presumably encoding a Fe delivery protein. We here describe growth studies and quantitative real-time PCR for the V. cholerae O395N1 wild-type (wt) strain and its mutant Δnqr and ΔubiC strains, impaired in respiration. In a comparative proteome analysis, FeoB, the membrane subunit of the uptake system for Fe2+ (Feo), was increased in V. cholerae Δnqr. In this study, the upregulation was confirmed on the mRNA level and resulted in improved growth rates of V. cholerae Δnqr with Fe2+ as an iron source. We studied the expression of feoB on other respiratory enzyme deletion mutants such as the ΔubiC mutant to determine whether iron transport is specific to the absence of NQR resulting from impaired respiration. We show that the nqr operon comprises, in addition to the structural nqrABCDEF genes, the downstream apbE and nqrM genes on the same operon and demonstrate induction of the nqr operon by iron in V. cholerae wt. In contrast, expression of the nqrM gene in V. cholerae Δnqr is repressed by iron. The lack of functional NQR has a strong impact on iron homeostasis in V. cholerae and demonstrates that central respiratory metabolism is interwoven with iron uptake and regulation. IMPORTANCE Investigating strategies of iron acquisition, storage, and delivery in Vibrio cholerae is a prerequisite to understand how this pathogen thrives in hostile, iron-limited environments such as the human host. In addition to highlighting the maturation of the respiratory complex NQR, this study points out the influence of NQR on iron metabolism, thereby making it a potential drug target for antibiotics.

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Complex Iron Uptake by the Putrebactin-Mediated and Feo Systems in Shewanella oneidensis

Applied and Environmental Microbiology ◽

10.1128/aem.01752-18 ◽

2018 ◽

Vol 84 (20) ◽

Cited By ~ 3

Author(s):

Lulu Liu ◽

Shisheng Li ◽

Sijing Wang ◽

Ziyang Dong ◽

Haichun Gao

Keyword(s):

Iron Uptake ◽

Iron Homeostasis ◽

Shewanella Oneidensis ◽

Special Role ◽

Uptake System ◽

Biological Processes ◽

Content Type ◽

Iron Uptake System ◽

Reducing Bacteria ◽

Metal Reducing Bacteria

ABSTRACT Shewanella oneidensis is an extensively studied bacterium capable of respiring minerals, including a variety of iron ores, as terminal electron acceptors (EAs). Although iron plays an essential and special role in iron respiration of S. oneidensis, little has been done to date to investigate the characteristics of iron transport in this bacterium. In this study, we found that all proteins encoded by the pub-putA-putB cluster for putrebactin (S. oneidensis native siderophore) synthesis (PubABC), recognition-transport of Fe3+-putrebactin across the outer membrane (PutA), and reduction of ferric putrebactin (PutB) are essential to putrebactin-mediated iron uptake. Although homologs of PutA are many, none can function as its replacement, but some are able to work with other bacterial siderophores. We then showed that Fe2+-specific Feo is the other primary iron uptake system, based on the synthetical lethal phenotype resulting from the loss of both iron uptake routes. The role of the Feo system in iron uptake appears to be more critical, as growth is significantly impaired by the absence of the system but not of putrebactin. Furthermore, we demonstrate that hydroxyl acids, especially α-types such as lactate, promote iron uptake in a Feo-dependent manner. Overall, our findings underscore the importance of the ferrous iron uptake system in metal-reducing bacteria, providing an insight into iron homeostasis by linking these two biological processes. IMPORTANCE S. oneidensis is among the first- and the best-studied metal-reducing bacteria, with great potential in bioremediation and biotechnology. However, many questions regarding mechanisms closely associated with those applications, such as iron homeostasis, including iron uptake, export, and regulation, remain to be addressed. Here we show that Feo is a primary player in iron uptake in addition to the siderophore-dependent route. The investigation also resolved a few puzzles regarding the unexpected phenotypes of the putA mutant and lactate-dependent iron uptake. By elucidating the physiological roles of these two important iron uptake systems, this work revealed the breadth of the impacts of iron uptake systems on the biological processes.

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Metabolic Reprogramming ofVibrio choleraeImpaired in Respiratory NADH Oxidation Is Accompanied by Increased Copper Sensitivity

Journal of Bacteriology ◽

10.1128/jb.00761-17 ◽

2018 ◽

Vol 200 (15) ◽

Cited By ~ 5

Author(s):

Charlotte Toulouse ◽

Kristina Metesch ◽

Jens Pfannstiel ◽

Julia Steuber

Keyword(s):

Vibrio Cholerae ◽

Pathogenic Bacteria ◽

Carbon Sources ◽

Copper Resistance ◽

Metabolic Reprogramming ◽

Deletion Strain ◽

Substrate Uptake ◽

Content Type ◽

Nadh:Quinone Oxidoreductase ◽

The Impact

ABSTRACTThe electrogenic, sodium ion-translocating NADH:quinone oxidoreductase (NQR) fromVibrio choleraeis frequent in pathogenic bacteria and a potential target for antibiotics. NQR couples the oxidation of NADH to the formation of a sodium motive force (SMF) and therefore drives important processes, such as flagellar rotation, substrate uptake, and energy-dissipating cation-proton antiport. We performed a quantitative proteome analysis ofV. choleraeO395N1 compared to its variant lacking the NQR using minimal medium with glucose as the carbon source. We found 84 proteins (regulation factor of ≥2) to be changed in abundance. The loss of NQR resulted in a decrease in the abundance of enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle and an increase in abundance of virulence factors AcfC and TcpA. Most unexpected, the copper resistance proteins CopA, CopG, and CueR were decreased in thenqrdeletion strain. As a consequence, the mutant exhibited diminished resistance to copper compared to the reference strain, as confirmed in growth studies using either glucose or mixed amino acids as carbon sources. We propose that the observed adaptations of thenqrdeletion strain represent a coordinated response which counteracts a drop in transmembrane voltage that challengesV. choleraein its different habitats.IMPORTANCEThe importance of the central metabolism for bacterial virulence has raised interest in studying catabolic enzymes not present in the host, such as NQR, as putative targets for antibiotics.Vibrio choleraelacking the NQR, which is studied here, is a model to estimate the impact of specific NQR inhibitors on the phenotype of a pathogen. Our comparative proteomic study provides a framework to evaluate the chances of success of compounds directed against NQR with respect to their bacteriostatic or bactericidal action.

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Investigating theCampylobacter jejuniTranscriptional Response to Host Intestinal Extracts Reveals the Involvement of a Widely Conserved Iron Uptake System

mBio ◽

10.1128/mbio.01347-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 4

Author(s):

Martha M. Liu ◽

Christine J. Boinett ◽

Anson C. K. Chan ◽

Julian Parkhill ◽

Michael E. P. Murphy ◽

...

Keyword(s):

Iron Uptake ◽

Bacterial Species ◽

Molecular Genetic ◽

Uptake System ◽

Transcriptional Responses ◽

Susceptible Animal ◽

Susceptible Host ◽

Altered Expression ◽

Content Type ◽

Iron Uptake System

ABSTRACTCampylobacter jejuniis a pathogenic bacterium that causes gastroenteritis in humans yet is a widespread commensal in wild and domestic animals, particularly poultry. Using RNA sequencing, we assessedC. jejunitranscriptional responses to medium supplemented with human fecal versus chicken cecal extracts and in extract-supplemented medium versus medium alone.C. jejuniexposed to extracts had altered expression of 40 genes related to iron uptake, metabolism, chemotaxis, energy production, and osmotic stress response. In human fecal versus chicken cecal extracts,C. jejunidisplayed higher expression of genes involved in respiration (fdhTU) and in known or putative iron uptake systems (cfbpA,ceuB,chuC, andCJJ81176_1649–1655[here designated1649–1655]). The1649–1655genes and downstream overlapping gene1656were investigated further. Uncharacterized homologues of this system were identified in 33 diverse bacterial species representing 6 different phyla, 21 of which are associated with human disease. The1649and1650(p19) genes encode an iron transporter and a periplasmic iron binding protein, respectively; however, the role of the downstream1651–1656genes was unknown. A Δ1651–1656deletion strain had an iron-sensitive phenotype, consistent with a previously characterized Δp19mutant, and showed reduced growth in acidic medium, increased sensitivity to streptomycin, and higher resistance to H2O2stress. In iron-restricted medium, the1651–1656andp19genes were required for optimal growth when using human fecal extracts as an iron source. Collectively, this implicates a function for the1649–1656gene cluster inC. jejuniiron scavenging and stress survival in the human intestinal environment.IMPORTANCEDirect comparative studies ofC. jejuniinfection of a zoonotic commensal host and a disease-susceptible host are crucial to understanding the causes of infection outcome in humans. These studies are hampered by the lack of a disease-susceptible animal model reliably displaying a similar pathology to human campylobacteriosis. In this work, we compared the phenotypic and transcriptional responses ofC. jejunito intestinal compositions of humans (disease-susceptible host) and chickens (zoonotic host) by using human fecal and chicken cecal extracts. The mammalian gut is a complex and dynamic system containing thousands of metabolites that contribute to host health and modulate pathogen activity. We identifiedC. jejunigenes more highly expressed during exposure to human fecal extracts in comparison to chicken cecal extracts and differentially expressed in extracts compared with medium alone, and targeted one specific iron uptake system for further molecular, genetic, and phenotypic study.

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Dopamine Is a Siderophore-Like Iron Chelator That PromotesSalmonella entericaSerovar Typhimurium Virulence in Mice

mBio ◽

10.1128/mbio.02624-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Stefanie Dichtl ◽

Egon Demetz ◽

David Haschka ◽

Piotr Tymoszuk ◽

Verena Petzer ◽

...

Keyword(s):

Bacterial Growth ◽

Iron Uptake ◽

Iron Homeostasis ◽

Iron Acquisition ◽

Lipocalin 2 ◽

Intracellular Iron ◽

Content Type ◽

Hepcidin Expression

ABSTRACTWe have recently shown that the catecholamine dopamine regulates cellular iron homeostasis in macrophages. As iron is an essential nutrient for microbes, and intracellular iron availability affects the growth of intracellular bacteria, we studied whether dopamine administration impacts the course ofSalmonellainfections. Dopamine was found to promote the growth ofSalmonellaboth in culture and within bone marrow-derived macrophages, which was dependent on increased bacterial iron acquisition. Dopamine administration to mice infected withSalmonella entericaserovar Typhimurium resulted in significantly increased bacterial burdens in liver and spleen, as well as reduced survival. The promotion of bacterial growth by dopamine was independent of the siderophore-binding host peptide lipocalin-2. Rather, dopamine enhancement of iron uptake requires both the histidine sensor kinase QseC and bacterial iron transporters, in particular SitABCD, and may also involve the increased expression of bacterial iron uptake genes. Deletion or pharmacological blockade of QseC reduced but did not abolish the growth-promoting effects of dopamine. Dopamine also modulated systemic iron homeostasis by increasing hepcidin expression and depleting macrophages of the iron exporter ferroportin, which enhanced intracellular bacterial growth.Salmonellalacking all central iron uptake pathways failed to benefit from dopamine treatment. These observations are potentially relevant to critically ill patients, in whom the pharmacological administration of catecholamines to improve circulatory performance may exacerbate the course of infection with siderophilic bacteria.IMPORTANCEHere we show that dopamine increases bacterial iron incorporation and promotesSalmonellaTyphimurium growth bothin vitroandin vivo. These observations suggest the potential hazards of pharmacological catecholamine administration in patients with bacterial sepsis but also suggest that the inhibition of bacterial iron acquisition might provide a useful approach to antimicrobial therapy.

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Iron Acquisition in Mycobacterium avium subsp. paratuberculosis

Journal of Bacteriology ◽

10.1128/jb.00922-15 ◽

2015 ◽

Vol 198 (5) ◽

pp. 857-866 ◽

Cited By ~ 10

Author(s):

Joyce Wang ◽

Jalal Moolji ◽

Alex Dufort ◽

Alfredo Staffa ◽

Pilar Domenech ◽

...

Keyword(s):

Mycobacterium Avium ◽

Iron Uptake ◽

Genomic Island ◽

Iron Acquisition ◽

Laboratory Data ◽

Uptake System ◽

Intracellular Iron ◽

Content Type ◽

Iron Uptake System ◽

Environmental Bacterium

ABSTRACTMycobacterium aviumsubsp.paratuberculosisis a host-adapted pathogen that evolved from the environmental bacteriumM. aviumsubsp.hominissuisthrough gene loss and gene acquisition. Growth ofM. aviumsubsp.paratuberculosisin the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system inM. aviumsubsp.paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed thatM. aviumsubsp.paratuberculosisis impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, inM. aviumsubsp.paratuberculosisgenes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on aM. aviumsubsp.paratuberculosis-specific genomic island, LSPP15. We obtained a transposon (Tn) mutant with a disruption in the LSPP15 geneMAP3776cfor targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775ctoMAP3772c[MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressingMAP3775-2cin wild-typeM. aviumsubsp.paratuberculosis. These data indicate that the horizontally acquired LSPP15 genes contribute to iron acquisition byM. aviumsubsp.paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen.IMPORTANCEMany microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception isMycobacterium aviumsubsp.paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely relatedM. aviumsubspecies, mycobactin production and iron uptake are different inM. aviumsubsp.paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSPP15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer inM. aviumsubsp.paratuberculosisevolution.

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Antibiotic Korormicin A Kills Bacteria by Producing Reactive Oxygen Species

Journal of Bacteriology ◽

10.1128/jb.00718-18 ◽

2019 ◽

Vol 201 (11) ◽

Cited By ~ 4

Author(s):

Adam Maynard ◽

Nicole L. Butler ◽

Takeshi Ito ◽

Adilson José da Silva ◽

Masatoshi Murai ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Pseudomonas Aeruginosa ◽

Vibrio Cholerae ◽

Pathogenic Bacteria ◽

Reactive Oxygen ◽

Direct Result ◽

Oxygen Species ◽

Gram Negative ◽

Content Type ◽

Nadh:Quinone Oxidoreductase

ABSTRACT Korormicin is an antibiotic produced by some pseudoalteromonads which selectively kills Gram-negative bacteria that express the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR.) We show that although korormicin is an inhibitor of Na+-NQR, the antibiotic action is not a direct result of inhibiting enzyme activity. Instead, perturbation of electron transfer inside the enzyme promotes a reaction between O2 and one or more redox cofactors in the enzyme (likely the flavin adenine dinucleotide [FAD] and 2Fe-2S center), leading to the production of reactive oxygen species (ROS). All Pseudoalteromonas contain the nqr operon in their genomes, including Pseudoalteromonas strain J010, which produces korormicin. We present activity data indicating that this strain expresses an active Na+-NQR and that this enzyme is not susceptible to korormicin inhibition. On the basis of our DNA sequence data, we show that the Na+-NQR of Pseudoalteromonas J010 carries an amino acid substitution (NqrB-G141A; Vibrio cholerae numbering) that in other Na+-NQRs confers resistance against korormicin. This is likely the reason that a functional Na+-NQR is able to exist in a bacterium that produces a compound that typically inhibits this enzyme and causes cell death. Korormicin is an effective antibiotic against such pathogens as Vibrio cholerae, Aliivibrio fischeri, and Pseudomonas aeruginosa but has no effect on Bacteroides fragilis and Bacteroides thetaiotaomicron, microorganisms that are important members of the human intestinal microflora. IMPORTANCE As multidrug antibiotic resistance in pathogenic bacteria continues to rise, there is a critical need for novel antimicrobial agents. An essential requirement for a useful antibiotic is that it selectively targets bacteria without significant effects on the eukaryotic hosts. Korormicin is an excellent candidate in this respect because it targets a unique respiratory enzyme found only in prokaryotes, the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR). Korormicin is synthesized by some species of the marine bacterium Pseudoalteromonas and is a potent and specific inhibitor of Na+-NQR, an enzyme that is essential for the survival and proliferation of many Gram-negative human pathogens, including Vibrio cholerae and Pseudomonas aeruginosa, among others. Here, we identified how korormicin selectively kills these bacteria. The binding of korormicin to Na+-NQR promotes the formation of reactive oxygen species generated by the reaction of the FAD and the 2Fe-2S center cofactors with O2.

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IurV, Encoded by ORF VCA0231, Is Involved in the Regulation of Iron Uptake Genes in Vibrio cholerae

Genes ◽

10.3390/genes11101184 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1184

Author(s):

Bernardo Sachman-Ruiz ◽

José Antonio Ibarra ◽

Paulina Estrada-de los Santos ◽

Alexia Torres Muñoz ◽

Begoña Giménez ◽

...

Keyword(s):

Vibrio Cholerae ◽

Iron Uptake ◽

Iron Homeostasis ◽

Iron Acquisition ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Reading Frame ◽

Upregulated Genes ◽

In Silico Analyses ◽

Genomic Neighborhood

The pathogen Vibrio cholerae has multiple iron acquisition systems which allow bacteria to exploit a variety of iron sources across the different environments on which it thrives. The expression of such iron uptake systems is highly regulated, mainly by the master iron homeostasis regulator Fur but also by other mechanisms. Recently, we documented that the expression of many of the iron-responsive genes is also modulated by riboflavin. Among them, the open reading frame VCA0231, repressed both by riboflavin and iron, encodes a putative transcriptional regulator of the AraC/XylS family. Nonetheless, the genes or functions affected by this factor are unknown. In the present study, a series of in silico analyses was performed in order to identify the putative functions associated with the product of VCA0231. The STRING database predicted many iron uptake genes as functional partners for the product of VCA0231. In addition, a genomic neighborhood analysis with the Enzyme Function Initiative tools detected many Pfam families involved in iron homeostasis genetically associated with VCA0231. Moreover, a phylogenetic tree showed that other AraC/XylS members known to regulate siderophore utilization in bacteria clustered together and the product of VCA0231 localized in this cluster. This suggested that the product of VCA0231, here named IurV, is involved in the regulation of iron uptake processes. RNAseq was performed to determine the transcriptional effects of a deletion in VCA0231. A total of 52 genes were overexpressed and 21 genes were downregulated in response to the iurV deletion. Among these, several iron uptake genes and other iron homeostasis-related genes were found. Six gene ontology (GO) functional terms were enriched in the upregulated genes, of which five were related to iron metabolism. The regulatory pattern observed in the transcriptomics of a subset of genes was independently confirmed by quantitative real time PCR analysis. The results indicate that IurV is a novel regulator of the AraC/XylS family involved in the repression of iron uptake genes. Whether this effect is direct or indirect remains to be determined.

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Central role of the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) in sodium bioenergetics of Vibrio cholerae

Biological Chemistry ◽

10.1515/hsz-2014-0204 ◽

2014 ◽

Vol 395 (12) ◽

pp. 1389-1399 ◽

Cited By ~ 18

Author(s):

Julia Steuber ◽

Petra Halang ◽

Thomas Vorburger ◽

Wojtek Steffen ◽

Georg Vohl ◽

...

Keyword(s):

Vibrio Cholerae ◽

Cytoplasmic Membrane ◽

Sea Water ◽

Three Dimensional ◽

Transport Processes ◽

Dimensional Structure ◽

Substrate Uptake ◽

Molecular Architecture ◽

Nadh:Quinone Oxidoreductase

Abstract Vibrio cholerae is a Gram-negative bacterium that lives in brackish or sea water environments. Strains of V. cholerae carrying the pathogenicity islands infect the human gut and cause the fatal disease cholera. Vibrio cholerae maintains a Na+ gradient at its cytoplasmic membrane that drives substrate uptake, motility, and efflux of antibiotics. Here, we summarize the major Na+-dependent transport processes and describe the central role of the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), a primary Na+ pump, in maintaining a Na+-motive force. The Na+-NQR is a membrane protein complex with a mass of about 220 kDa that couples the exergonic oxidation of NADH to the transport of Na+ across the cytoplasmic membrane. We describe the molecular architecture of this respiratory complex and summarize the findings how electron transport might be coupled to Na+-translocation. Moreover, recent advances in the determination of the three-dimensional structure of this complex are reported.

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The Vibrio cholerae Pst2 Phosphate Transport System Is Upregulated in Biofilms and Contributes to Biofilm-Induced Hyperinfectivity

Infection and Immunity ◽

10.1128/iai.06277-11 ◽

2012 ◽

Vol 80 (5) ◽

pp. 1794-1802 ◽

Cited By ~ 19

Author(s):

Benjamin Mudrak ◽

Rita Tamayo

Keyword(s):

Vibrio Cholerae ◽

Diarrheal Disease ◽

Phosphate Transport ◽

Uptake System ◽

Content Type ◽

Infant Mouse ◽

Membrane Complex ◽

Inner Membrane Complex ◽

Infectious Form

ABSTRACTVibrio choleraeis the causative agent of the deadly diarrheal disease cholera. As part of its life cycle,V. choleraepersists in marine environments, where it forms surface-attached communities commonly described as biofilms. Evidence indicates that these biofilms constitute the infectious form of the pathogen during outbreaks. Previous work has shown that biofilm-derivedV. choleraecells, even when fully dispersed from the biofilm matrix, are vastly more infectious than planktonic (free-living) cells. Here, we sought to identify factors that contribute to biofilm-induced hyperinfectivity inV. cholerae, and we present evidence for one aspect of the molecular basis of this phenotype. We identified proteins upregulated during growth in biofilms and determined their contributions to the hyperinfectivity phenotype. We found that PstS2, the periplasmic component of the Pst2 phosphate uptake system, was enriched in biofilms. Another gene in thepst2locus was transcriptionally upregulated in biofilms. Using the infant mouse model, we found that mutation of twopst2components resulted in impaired colonization. Importantly, deletion of the Pst2 inner membrane complex caused a greater colonization defect after growth in a biofilm compared to shaking culture. Based on these data, we propose thatV. choleraecells in biofilms upregulate the Pst2 system and therefore gain an advantage upon entry into the host. Further characterization of factors contributing to biofilm-induced hyperinfectivity inV. choleraewill improve our understanding of the transmission of the bacteria from natural aquatic habitats to the human host.

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