The Mycobacterium tuberculosis phosphate-specific transport (Pst) system controls gene expression in response to phosphate availability by inhibiting the activation of the SenX3-RegX3 two-component system under phosphate-rich conditions, but the mechanism of communication between these systems is unknown. In Escherichia coli, inhibition of the two-component system PhoR-PhoB under phosphate-rich conditions requires both the Pst system and PhoU, a putative adaptor protein. E. coli PhoU is also involved in the formation of persisters, a subpopulation of phenotypically antibiotic-tolerant bacteria. M. tuberculosis encodes two PhoU orthologs, PhoY1 and PhoY2. We generated phoY single- and double-deletion mutants and examined the expression of RegX3-regulated genes by quantitative reverse transcription-PCR (qRT-PCR). Gene expression was increased only in the ΔphoY1 ΔphoY2 double mutant and could be restored to the wild-type level by complementation with either phoY1 or phoY2 or by deletion of regX3. These data suggest that the PhoY proteins function redundantly to inhibit SenX3-RegX3 activation. We analyzed the frequencies of antibiotic-tolerant persister variants in the phoY mutants using several antibiotic combinations. Persister frequency was decreased at least 40-fold in the ΔphoY1 ΔphoY2 mutant compared to the frequency in the wild type, and this phenotype was RegX3 dependent. A ΔpstA1 mutant lacking a Pst system transmembrane component exhibited a similar RegX3-dependent decrease in persister frequency. In aerosol-infected mice, the ΔphoY1 ΔphoY2 and ΔpstA1 mutants were more susceptible to treatment with rifampin but not isoniazid. Our data demonstrate that disrupting phosphate sensing mediated by the PhoY proteins and the Pst system enhances the susceptibility of M. tuberculosis to antibiotics both in vitro and during infection.

cMycobacterium tuberculosis requires the phosphate-sensing signal transduction system Pst/SenX3-RegX3 to resist host immune responses. A ⌬pstA1 mutant lacking a Pst phosphate uptake system component is hypersensitive to diverse stress conditions in vitro and is attenuated in vivo due to constitutive expression of the phosphate starvation-responsive RegX3 regulon. Transcriptional profiling of the ⌬pstA1 mutant revealed aberrant expression of certain pe and ppe genes. PE and PPE proteins, defined by conserved N-terminal domains containing Pro-Glu (PE) or Pro-Pro-Glu (PPE) motifs, account for a substantial fraction of the M. tuberculosis genome coding capacity, but their functions are largely uncharacterized. Because some PE and PPE proteins localize to the cell wall, we hypothesized that overexpression of these proteins sensitizes M. tuberculosis to stress by altering cell wall integrity. To test this idea, we deleted pe and ppe genes that were overexpressed by ⌬pstA1 bacteria. Deletion of a single pe gene, pe19, suppressed hypersensitivity of the ⌬pstA1 mutant to both detergent and reactive oxygen species. Ethidium bromide uptake assays revealed increased envelope permeability of the ⌬pstA1 mutant that was dependent on PE19. The replication defect of the ⌬pstA1 mutant in NOS2 ؊/؊ mice was partially reversed by deletion of pe19, suggesting that increased membrane permeability due to PE19 overexpression sensitizes M. tuberculosis to host immunity. Our data indicate that PE19, which comprises only a 99-amino-acid PE domain, has a unique role in the permeability of the M. tuberculosis envelope that is regulated to resist stresses encountered in the host. O ver 15 years ago, the novel PE and PPE protein families were identified in the complete genome sequence of Mycobacterium tuberculosis; together, these proteins represent over 7% of the genome coding capacity (1). Despite the attention placed on these protein families, their functions remain largely uncharacterized. PE and PPE proteins are defined by conserved N-terminal domains of ϳ110 or ϳ180 amino acids that contain Pro-Glu (PE) or Pro-Pro-Glu (PPE) sequence motifs, respectively (1). Although PE and PPE proteins can be identified in the genomes of all sequenced members of the Mycobacterium genus, their expansion into large multiprotein families is restricted to the slow-growing pathogenic mycobacterial species, including M. tuberculosis, and associated with the expansion of the ESX type VII secretion systems (2). There is evidence that some PE and PPE proteins are exported to the bacterial cell surface or extracellular milieu in an ESX-dependent manner (3-6). ESX-dependent export requires specific sequences within the PE or PPE domain (7, 8), including a recently described YxxxD/E ESX secretion targeting motif located near the C terminus of the ϳ110-amino-acid PE domain (9).The 99 PE proteins and 69 PPE proteins encoded by the M. tuberculosis H37Rv genome can be further divided into subfamilies based on C-terminal sequence motifs (2). The PE_PGRS (polymorphic GC-...

Bacterial surface structures called pili have been studied extensively for their role as possible colonization factors. Most sequenced Vibrio genomes predict a variety of pili genes in these organisms, including several types of type IV pili. In particular, the mannose-sensitive hemagglutinin (MSHA) and the PilA pili, also known as the chitin-regulated pilus (ChiRP), are type IVa pili commonly found in Vibrio genomes and have been shown to play a role in the colonization of Vibrio species in the environment and/or host tissue. Here, we report sequence comparisons of two type IVa pilin subunit genes, mshA and pilA, and their corresponding amino acid sequences, for several strains from the three main human pathogenic Vibrio species, V. cholerae, V. parahaemolyticus, and V. vulnificus. We identified specific groupings of these two genes in V. cholerae, whereas V. parahaemolyticus and V. vulnificus strains had no apparent allelic clusters, and these genes were strikingly divergent. These results were compared with other genes from the MSHA and PilA operons as well as another Vibrio pili from the type IVb group, the toxin co-regulated pilus (TCP) from V. cholerae. Our data suggest that a selective pressure exists to cause these strains to vary their MSHA and PilA pilin subunits. Interestingly, V. cholerae strains possessing TCP have the same allele for both mshA and pilA. In contrast, V. cholerae isolates without TCP have polymorphisms in their mshA and pilA sequences similar to what was observed for both V. parahaemolyticus and V. vulnificus. This data suggests a possible linkage between host interactions and maintaining a highly conserved type IV pili sequence in V. cholerae. Although the mechanism underlying this intriguing diversity has yet to be elucidated, our analyses are an important first step towards gaining insights into the various aspects of Vibrio ecology.

The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na+-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na+-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na+-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na+ pump(s) can compensate for Na+ pumping activity of Na+-NQR. Overall, our study provides important insights into the contribution of Na+-NQR to V. cholerae physiology.

Vibrio parahaemolyticus can resist oyster depuration, suggesting that it possesses specific factors for persistence. We show that type I pili, type IV pili, and both flagellar systems contribute to V. parahaemolyticus persistence in Pacific oysters whereas type III secretion systems and phase variation do not.T he genus Vibrio consists of a group of bacteria that naturally inhabit aquatic environments worldwide. The human pathogen Vibrio parahaemolyticus is commonly found associated with shellfish, particularly oysters (1, 2). Depuration is a controlled process in which shellfish are placed into clean seawater to reduce the bacterial contaminants in their tissues. However, depuration is not very effective at reducing the numbers of V. parahaemolyticus cells in oysters (3), and little is known about the association of V. parahaemolyticus with oysters.Both type I and type IV pili are important for bacterial attachment to a variety of surfaces (4-6). V. parahaemolyticus encodes a homologue of type I pili that is most similar to the CsuA/B operon from Acinetobacter baumannii (7) and homologues of two wellstudied type IV pili from vibrios, the mannose-sensitive hemagglutinin (MSHA) and the chitin-regulated pilus (PilA) (http://img .jgi.doe.gov/) (8, 9). Vibrio vulnificus uses PilA for persistence in Crassostrea virginica (10, 11). Both type IV pili are involved in biofilm formation in V. parahaemolyticus (12). Flagella are often critical during early stages of bacterial colonization of a surface (13,14). V. parahaemolyticus possesses a single, polar flagellum used for movement in liquid (reviewed in reference 14) as well as peritrichous (lateral) flagella for surface movement (15-17 and reviewed in reference 18). Many bacterial pathogens use type III secretion systems (T3SSs) for survival in the host by injecting virulence factors directly into the host. V. parahaemolyticus encodes two T3SSs that exhibit different phenotypes during disease (19)(20)(21). V. parahaemolyticus also exhibits "phase variation" and can switch from an opaque (OP) to a translucent (TR) phenotype based on polysaccharide production (22). In this study, we examined the role of V. parahaemolyticus pili, flagella, phase variation, and T3SSs in persistence in the Pacific oyster, Crassostrea gigas.Oysters were collected from Oregon Oyster Farm (Newport, OR) and exposed to ϳ10 5 CFU/ml of V. parahaemolyticus for 16 to 18 h at room temperature (ϳ20°C) with a recirculating pump. Algae were added to the tank to facilitate uptake according to the manufacturer's recommendation (Phytoplex; Kent Marine). Depuration was conducted at 19 to 20°C for 48 to 72 h. At each time point, animals from each exposure tank were weighed, homogenized, and diluted with phosphate-buffered saline (PBS) for enumeration on tryptic soy agar (TSA) supplemented with 1.5% NaCl and either 100 g/ml streptomycin, 60 g/ml chloramphenicol, 10 g/ml phosphomycin, or 50 g/ml kanamycin, depending on the strain (see Table S1 in the supplemental material), to eliminate naturally occurring bac...

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To examine the possible physiological significance of Mrp, a multi-subunit cation/proton antiporter from Vibrio cholerae, a chromosomal deletion Δmrp of V. cholerae was constructed and characterized. The resulting mutant showed a consistent early growth defect in LB broth that became more evident at elevated pH of the growth medium and increasing Na+ or K+ loads. After 24 h incubation, these differences disappeared likely due to the concerted effort of other cation pumps in the mrp mutant. Phenotype MicroArray analyses revealed an unexpected systematic defect in nitrogen utilization in the Δmrp mutant that was complemented by using the mrpA'-F operon on an arabinose-inducible expression vector. Deletion of the mrp operon also led to hypermotility, observable on LB and M9 semi-solid agar. Surprisingly, Δmrp mutation resulted in wild-type biofilm formation in M9 despite a growth defect but the reverse was true in LB. Furthermore, the Δmrp strain exhibited higher susceptibility to amphiphilic anions. These pleiotropic phenotypes of the Δmrp mutant demonstrate how the chemiosmotic activity of Mrp contributes to the survival potential of V. cholerae despite the presence of an extended battery of cation/proton antiporters of varying ion selectivity and pH profile operating in the same membrane.

The human pathogen Vibrio parahaemolyticus is a leading cause of seafood-borne illness in the USA, and infections with V. parahaemolyticus typically result from eating raw or undercooked oysters. V. parahaemolyticus has been shown to be highly resistant to oyster depuration, suggesting that the bacterium possesses specific mechanisms or factors for colonizing oysters and persisting during depuration. In this study, we characterized eight different V. parahaemolyticus strains for differences in resistance to oyster depuration, biofilm formation, and motility. While each strain exhibited distinct phenotypes in the various assays, we determined that biofilm formation on abiotic surfaces, such as glass or plastic, does not directly correlate with bacterial retention in oysters during depuration. However, we did observe that the motility phenotype of a strain appeared to be a better indicator for persistence in the oyster. Further studies examining the molecular mechanisms underlying the observed colonization differences by these and other V. parahaemolyticus strains may provide beneficial insights into what critical factors are required for proficient colonization of the Pacific oyster.