A proteome reference map for Vibrio cholerae El Tor

A proteome reference map has been constructed for Vibrio cholerae El Tor, in the pI range of 4.0 to 7.0. The map is based on two-dimensional gels (2-D) and the identification, by peptide mass fingerprint, of proteins in 94 spots, corresponding to 80 abundant proteins. Two strains are compared, strain N16961 and a Latin American El Tor strain C3294. The consensus map contains 340 spots consistently seen with both strains grown in Luria-Bertani broth (LB) or minimal M9 medium. The results were obtained from nine gels run with 18 cm immobilized pH gradient strips and precast gels. The 2-D gels were anchored to real N16961 proteins identified by mass spectrometry. Various energy metabolism components and periplasmic ATP-binding cassette (ABC) transporter proteins were identified among the abundant proteins. Two isoforms of OmpU were found. Five operons are proposed and seven hypothetical proteins were experimentally confirmed. Comparisons are made with protein 2-D gels for a classical strain and to microarray analysis available for the N16961 El Tor strain. New results were obtained from the proteome analysis, indicating an abundance of periplasmic ABC transporter proteins not found in microarray studies.     

Steps in the development of a Vibrio cholerae El Tor biofilm

We report that, in a simple, static culture system, wild-type Vibrio cholerae El Tor forms a three-dimensional biofilm with characteristic water channels and pillars of bacteria. Furthermore, we have isolated and characterized transposon insertion mutants of V. cholerae that are defective in biofilm development. The transposons were localized to genes involved in (i) the biosynthesis and secretion of the mannose-sensitive haemagglutinin type IV pilus (MSHA); (ii) the synthesis of exopolysaccharide; and (iii) flagellar motility. The phenotypes of these three groups suggest that the type IV pilus and flagellum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotic surface, and exopolysaccharide is involved in the formation of three-dimensional biofilm architecture.     

El Tor霍乱弧菌双精氨酸转运系统功能单位的分析研究

本文旨在分析El Tor霍乱弧菌双精氨酸转运(Tat)系统的基因簇构成,对其功能进行分析,确定其最小功能单位.通过与大肠埃希菌Tat系统基因簇的基因同源性比较,推测霍乱国际测序标准株N16961的Tat系统基因簇构成;分别对霍乱弧菌Tat系统的基因簇进行单基因缺失、双基因缺失和全基因缺失,建立缺失株和回补株,并与野生株...     

Molecular cloning of the hemolysin determinant from Vibrio cholerae El Tor

Vibrio cholerae El Tor RV79 is phenotypically nonhemolytic; however, strongly hemolytic convertants are occasionally observed on blood agar plates. We have cloned DNA sequences corresponding to the hemolysin determinant from RV79 (Hly+) in the lambda L47.1 and pBR322 vectors. A 2.3-kilobase fragment of V. cholerae DNA was found to be necessary for hemolytic activity. This cloned DNA sequence was used as a probe in Southern blot hybridization analysis of chromosomal restriction digests of a variety of El Tor and classical biotype V. cholerae strains. In all cases, DNA fragments with the same electrophoretic mobilities hybridized to the Hly probe. The results presented demonstrate that the cloned hemolysin determinant is the hly locus. By using mutator vibriophage VcA-3 insertion to promote high-frequency transfer, the hly locus was mapped between arg and ilv on the V. cholerae RV79 chromosome.     

Amino acid metabolism and protein synthesis in streptomycin resistant Vibrio El Tor.

Metabolic activities in relation to protein synthesis and amino acid utilization are altered in Vibrio El Tor after development of resistance towards streptomycin. Efficiency of in vivo and in vitro protein synthesis is markedly reduced in streptomycin resistant Vibrio El Tor. The rate of incorporation of 14C-amino acids into protein, uptake of 14C-valine and oxidation of certain amino acids are also altered.     

A Role for the Mannose-Sensitive Hemagglutinin in Biofilm Formation by Vibrio cholerae El Tor

While much has been learned regarding the genetic basis of host-pathogen interactions, less is known about the molecular basis of a pathogen's survival in the environment. Biofilm formation on abiotic surfaces represents a survival strategy utilized by many microbes. Here it is shown that Vibrio cholerae El Tor does not use the virulence-associated toxin-coregulated pilus to form biofilms on borosilicate but rather uses the mannose-sensitive hemagglutinin (MSHA) pilus, which plays no role in pathogenicity. In contrast, attachment of V. cholerae to chitin is shown to be independent of the MSHA pilus, suggesting divergent pathways for biofilm formation on nutritive and nonnutritive abiotic surfaces.     

Polymyxin B Resistance in El Tor Vibrio cholerae Requires Lipid Acylation Catalyzed by MsbB

Antimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. In Vibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classical V. cholerae mutants resistant to polymyxin B and El Tor V. cholerae mutants sensitive to polymyxin B. Insertions in a gene annotated msbB (encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance of V. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated that msbB is required for resistance to all antimicrobial peptides tested. Mutation of msbB in a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B. msbB mutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of the msbB mutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase in V. cholerae.Pathogenic bacteria that colonize the digestive tract must overcome a variety of stresses imposed upon them by the host. Epithelial cells in the crypts of the intestinal lumen (Paneth cells and enterocytes) produce large amounts of antimicrobial peptides called defensins (16). Defensins, like most antimicrobial peptides, are thought to act by associating with the lipopolysaccharide (LPS) on the bacterial surface (through electrostatic interactions) and then permeabilizing the membranes, leading to cell death (37, 48). Gram-negative bacteria have developed a wide range of strategies to overcome the antimicrobial activity of these peptides, including production of proteases that degrade the peptides (41), production of secretory proteins that bind the peptides and prevent them from accessing their target (21), production of efflux systems that actively pump antimicrobial peptides back into the environment if they access the bacterial cytoplasm (36), and incorporation of positively charged groups into lipid A, which reduces the net anionic charge of the bacterial surface and decreases the affinity of the peptides for the membrane (10, 13, 14).LPS of Gram-negative bacteria is composed of three main parts: (i) the O-antigen polysaccharide (O-PS); (ii) the relatively conserved core polysaccharide (core-PS); and (iii) lipid A, the hydrophobic lipid component responsible for biological activities within the host (9, 25). The lipid A region of the LPS is anchored in the bacterial outer membrane, and the hydrophilic core-PS and O-PS project outward into the environment. LPS comprises 70% of the bacterial outer membrane and is the main surface-associated antigen recognized by the innate immune system. Toll-like receptors in the host recognize the lipid A portion of the LPS in association with MD2 and CD14 and stimulate inflammation to attract immune cells and clear bacterial infections (5, 27). The strong immune response to lipid A is the reason that LPS has historically been referred to as “endotoxin” (20). Some pathogens regulate the structure of their lipid A and its acylation patterns in order to adapt to the host environment, thereby contributing to greater fitness within the host (12, 31).Vibrio cholerae causes cholera, an epidemic diarrheal disease. Disease occurs when contaminated food or water is ingested, resulting in a voluminous secretory diarrhea that can lead to dehydration and death if left untreated. The V. cholerae species is not homogeneous, with distinctions made on the basis of serogroup, serotype, biotype, production of cholera toxin, and potential for epidemic spread. While more than 200 serogroups have been identified, only two of these, O1 and O139, are associated with epidemic cholera (33). V. cholerae O1 strains can be subdivided into two biotypes, classical and El Tor, which differ biochemically and clinically (3). The first six cholera pandemics were caused by the classical biotype, but the current (seventh) pandemic has been caused by the El Tor biotype (33). Classical strains typically cause a more severe disease, while El Tor strains cause less severe and sometimes even asymptomatic cases. However, El Tor strains appear to have increased fitness in the environment, which may be why they have largely replaced classical strains as the cause of disease in recent years (49).The subdivision into the classical or El Tor biotype is based on several laboratory tests (3). One of the commonly used tests is assessing sensitivity of the strain to the antimicrobial peptide polymyxin B. Classical strains are very sensitive to polymyxin B, while El Tor strains are relatively resistant. We hypothesize that differences in surface structures of the two biotypes are responsible for differential sensitivity. To test this and to determine the genetic basis of antimicrobial peptide resistance in V. cholerae, we carried out genetic screens to identify genes associated with resistance and sensitivity to polymyxin B in El Tor and classical V. cholerae, respectively. As a result of these screens, we chose to further characterize the role of msbB, a lipid IVA acyltransferase gene, with regard to antimicrobial peptide resistance and virulence in V. cholerae. We report that msbB contributes to resistance of El Tor strains to all antimicrobial peptides tested. Mutation of msbB in a classical strain led to significantly reduced innate resistance to several antimicrobial peptides, not including polymyxin B. While msbB mutants of both biotypes exhibit decreased colonization of infant mice, a more significant decrease was observed for the El Tor mutant. Mass spectrometry analysis confirmed that deletion of msbB from either biotype resulted in loss of an acyl chain, as expected. These results suggest that msbB from V. cholerae is required for wild-type antimicrobial peptide resistance and colonization. However, some biotype-specific phenotypes imply that the role of msbB may be different in each biotype.     

Comparison of chitin-induced natural transformation in pandemic Vibrio cholerae O1 El Tor strains

The human pathogen Vibrio cholerae serves as a model organism for many important processes ranging from pathogenesis to natural transformation, which has been extensively studied in this bacterium. Previous work has deciphered important regulatory circuits involved in natural competence induction as well as mechanistic details related to its DNA acquisition and uptake potential. However, since competence was first reported for V. cholerae in 2005, many researchers have struggled with reproducibility in certain strains. In this study, we therefore compare prominent seventh pandemic V. cholerae isolates, namely strains A1552, N16961, C6706, C6709, E7946, P27459, and the close relative MO10, for their natural transformability and decipher underlying defects that mask the high degree of competence conservation. Through a combination of experimental approaches and comparative genomics based on new whole-genome sequences and de novo assemblies, we identify several strain-specific defects, mostly in genes that encode key players in quorum sensing. Moreover, we provide evidence that most of these deficiencies might have recently occurred through laboratory domestication events or through the acquisition of mobile genetic elements. Lastly, we highlight that differing experimental approaches between research groups might explain more of the variations than strain-specific alterations.     

Genetic determination of the vibriocinogenicity trait in Vibrio cholerae of the El Tor biotype

In two different strains of cholera vibrios two recA-dependent plasmids, pVib I (1.9-2.2 Md) and pVib II (5.2-5.8 Md), have been detected. These plasmids determine the synthesis of vibriocin, coagulase and fibrinolysin, which has been established by the cotransformation of the DNA of plasmids pVib I and pBR322 and by the transfer mobilization with the use of plasmid RP4.     

Cellular localization and export of the soluble haemolysin of Vibrio cholerae El Tor

Summary The cellular location of the haemolysin of Vibrio cholerae El Tor strain 017 has been analyzed. This protein is found both in the periplasmic space and the extracellular medium in Vibrio cholerae. However, when the cloned gene, present on plasmid pPM431, is introduced into E. coli K-12 this protein remains localized predominantly in the periplasmic space with no activity detected in the extracellular medium. Mutants of E. coli K-12 (tolA and tolB) which leak periplasmic proteins mimic excretion and release the haemolysin into the growth medium. Secretion of haemolysin into the periplasm is independent of perA (envZ) and in fact, mutants in perA (envZ) harbouring pPM431 show hyperproduction of periplasmic haemolysin. These results in conjunction with those for other V. cholerae extracellular proteins suggest that although E. coli K-12 can secrete these proteins into the periplasm, it lacks a specific excretion mechanism, present in V. cholerae, for the release of soluble proteins into the growth medium.