Genetic analysis of the capsule polysaccharide (K antigen) and exopolysaccharide genes in pandemic Vibrio parahaemolyticus O3:K6
© Chen et al; licensee BioMed Central Ltd. 2010
Received: 28 May 2010
Accepted: 2 November 2010
Published: 2 November 2010
Pandemic Vibrio parahaemolyticus has undergone rapid changes in both K- and O-antigens, making detection of outbreaks more difficult. In order to understand these rapid changes, the genetic regions encoding these antigens must be examined. In Vibrio cholerae and Vibrio vulnificus, both O-antigen and capsular polysaccharides are encoded in a single region on the large chromosome; a similar arrangement in pandemic V. parahaemolyticus would help explain the rapid serotype changes. However, previous reports on "capsule" genes are controversial. Therefore, we set out to clarify and characterize these regions in pandemic V. parahaemolyticus O3:K6 by gene deletion using a chitin based transformation strategy.
We generated different deletion mutants of putative polysaccharide genes and examined the mutants by immuno-blots with O and K specific antisera. Our results showed that O- and K-antigen genes are separated in V. parahaemolyticus O3:K6; the region encoding both O-antigen and capsule biosynthesis in other vibrios, i.e. genes between gmhD and rjg, determines the K6-antigen but not the O3-antigen in V. parahaemolyticus. The previously identified "capsule genes" on the smaller chromosome were related to exopolysaccharide synthesis, not K-antigen.
Understanding of the genetic basis of O- and K-antigens is critical to understanding the rapid changes in these polysaccharides seen in pandemic V. parahaemolyticus. This report confirms the genetic location of K-antigen synthesis in V. parahaemolyticus O3:K6 allowing us to focus future studies of the evolution of serotypes to this region.
V. parahaemolyticus is a naturally occurring marine bacterium that has been recognized as an important food borne pathogen since a large outbreak occurred in Japan in 1950. Before 1996, no particular serotype of V. parahaemolyticus was associated with outbreaks. In that year, there was a major outbreak in Kolkata, India caused by strains with increased virulence and more than half of the patient isolates were serotype O3:K6 . These isolates quickly spread to other countries in Asia, followed by South America, Africa and the United States affecting tens of thousands people and resulting in the first known V. parahaemolyticus pandemic [2, 3]. Strains from early in the pandemic were all serotype O3:K6 [4, 5]. However, the pandemic strains have rapidly evolved to more than 20 serovariants including O3:K6, O4:K68, O1:K25, O1:KUT (K-untypable) and others . The pandemic isolates are closely related (clonal) as shown by pulse-field gel electrophoresis, ribotyping, and multilocus sequence typing (MLST). Therefore, new serotypes seem to have arisen from the original pandemic O3:K6 strain by changes occurring in both the K- (capsule) and the O-antigen. Understanding the mechanism underlying rapid serotype conversion may help us develop improved diagnostics for identifying isolates with pandemic potential.
Eleven O and 65 K serotypes are recognized in V. parahaemolyticus. The lipopolysaccharide (LPS) of most Gram-negative bacilli consists of lipid A, core polysaccharide and the highly variable O side chain (O-antigen). The capsular or K-antigen is composed of high molecular weight polysaccharide and forms a dense, high molecular weight coat outside of the bacterial cells. Encapsulated pathogens can become invasive and cause septicemia due to their increased resistance to phagocytosis and complement-mediated killing. K- and O- antigens are generally encoded in discreet loci; but, in limited studies in V. cholerae and V. vulnificus isolates, O-antigen and K-antigen have been shown to be co-located [6–8]. A third form of polysaccharide, the exopolysaccharide, is a loose slime outside the cell that forms an intercellular matrix in biofilms. In V. cholerae, this exopolysaccharide is expressed by cells that display a rugose (wrinkled) colony phenotype .
Genetic study of surface polysaccharides in V. parahaemolyticus is limited and controversial. Guvener et al. have proposed a locus on chromosome II, VPA1403-VPA1412, for capsular polysaccharide biosynthesis, but have not shown a correlation with the K-antigen . Comparison of restriction fragment polymorphisms of different serotypes led Okura et al to suggest a different locus, around VP214-VP237on chromosome I, for K-antigen genes and a region with homology to the LPS-core polysaccharide region for the O-antigen, but have not experimentally confirmed the function of these regions . To resolve this controversy, we have investigated these putative K-antigen genetic determinants in an epidemic O3:K6 isolate by construction of gene deletions.
Polysaccharide gene clusters in V. parahaemolyticus O3:K6
V. parahaemolyticu s strains used in this study
Deletion of VP0220 (wbfF) in region B
∆0220 plus complementation
∆0220 trans-complemented with VP0220
Deletion of VP0219-0237 in region B
Deletion of VP0215-0218 in region B
Deletion of VPA1403-1406 in region C
∆EPS plus complementation
∆EPS trans-complemented with VPA1403-1406
∆EPS plus empty vector
∆EPS with pBBR1-MCS2 empty vector
Deletion of wza, wzb and wzc (VPA1602-1604) genes in region D
Region B determines capsule (K-antigen)
K-antigen/Capsule genes of V. parahaemolyticu s O3:K6
putative regulator protein
capsule assembly protein
polysaccharide chain length determinant
dTDP-glucose 4,6 dehydratase
UDP-galactose phosphate transferase
similar to carbamoyl phosphate synthase
K-antigen processing genes
However, a K-antigen processing system similar to the O-antigen/capsule polysaccharide genes in V. cholerae O139 [13, 20, 21] is present in V. parahaemolyticus. VP0219-0221 are homologous to wbfE, wbfF and wzz genes in V. cholerae O139, sharing 49%, 69% and 54% amino acid identities. Therefore a similar capsule processing mechanism may exist for both taxa. We generated an in frame deletion of VP0220, the wbfF homolog. Mutant ∆0220 displayed an intermediate level of translucence. Immunoblots indicated that deletion of VP0220 did not affect O3 antigen synthesis (Figure 4). However, the midpoint of the K-antigen band shifted in this mutant, suggesting a role of VP0220 in the later stage of the K-antigen processing. Complementation of ∆0220 with over expressed wild type VP0220 gene restored mostly the pattern of the wild type K antigen (Figure 4). However, there was more reactive material away from the midpoint of the K-antigen band in the complemented mutant than the wild type (Figure 4), possibly due to the over expression of VP0220 or other reasons that remain unclear.
Other K-antigen region features
A complete set of genes of the rhamnose pathway rmlBADC are present in the K-antigen genes of V. parahaemolyticus. However, four open reading frames, VP0225-0228, are inserted between the rmlD and rmlC genes. Analysis of the GC percentage revealed that the average GC percentage in VP0225-0228 is lower than the rest of the genes in this operon (Figure 2). The unusual arrangement of the rhamnose gene order and the mosaic GC percentage pattern indicated that there was a recent recombination event in the K antigen genes.
Between gmhD and the K-antigen operon like genes, there are four genes (VP0215-0218) transcribed to the opposite direction (Figure 2). In frame deletion of these four genes led to the over expression of K-antigen polysaccharides (Figure 4), suggesting these genes may have a regulatory role in capsule expression.
Region C is the exopolysaccharide gene cluster (EPS)
The genetic region encoding the capsular polysaccharide, or K antigen in V. parahaemolyticus has been controversial, with two different investigators suggesting different loci [10, 11]. In our study, construction of gene deletions with confirmation of loss of binding K6-specific antiserum in immunoblots provided solid evidence that the region between genes gmhD and rjg (VP0215-0237) on chromosome I was the genetic determinant of the K6-antigen in the pandemic V. parahaemolyticus O3:K6 serotype. This antigen consists of high molecular weight polysaccharide that is located on the surface of the cell. Loss of this antigen resulted in a translucent colony morphology. These data are consistent with the K6 antigen being a typical vibrio capsular polysaccharide. Our study supports the location suggested by Okura et al as encoding the K-antigen . Although O-antigen genes have been identified in a conserved locus between genes gmhD and rjg in both V. cholerae and V. vulnificus, our study found that this locus in V. parahaemolyticus was not involved in O-antigen biosynthesis. We also showed that gene cluster referred to as "capsule" genes by Guvener et al (VPA1403-VPA1412) was not related to either K-antigen capsule polysaccharide or O-antigen but was instead related to exopolysaccharide production, which causes rugose phase variation. We suggest reserving the term "capsule" for K-antigen polysaccharides and referring to the rugose related polysaccharide exopolysaccharide.
Our understanding of the major surface polysaccharides in V. parahaemolyticus had been limited, in part, due to our limited ability to perform genetic manipulations in this species. Genetic manipulation of genes in V. parahaemolyticus was previously achieved by first cloning the DNA of interest into a suicide plasmid that cannot replicate in V. parahaemolyticus, propagating the plasmid in an E. coli host, then transferring the plasmid from E. coli to V. parahaemolyticus by conjugation, followed by counter selection against the E. coli host and screening for mutants of V. parahaemolyticus. The procedure is tedious and time consuming. There are few reports using electroporation in V. parahaemolyticus and no report of successful chemical transformation [24, 25]. We tested electroporation on V. parahaemolyticus and had limited success with plasmid DNA but no success with linear DNA (data not shown). Chemical transformation was also not successful. Therefore we sought alternative methods for targeted gene deletion in V. parahaemolyticus. Meibom et al. reported that V. cholerae became competent and took up foreign DNA when cultured with chitin . The chitin based transformation method was later successfully adapted for V. vulnificus. We modified the chitin based transformation technique and developed a rapid method to mutate genes in V. parahaemolyticus. On average, 150 mutants were obtained from each transformation. Since only one mutant is needed in most cases, this transformation efficiency will satisfy most deletion applications in V. parahaemolyticus.
Capsule biogenesis in E. coli is classified into 4 groups. Exportation of group 1 and 4 capsules rely on Wza proteins, while group 2 and 3 may rely on CPSM and CPST proteins . Previous research has shown that capsules in V. cholerae O31 and V. vulnificus have similarities to E. coli group 1- or group 4 capsules; with a wza gene inside the capsule gene cluster [6, 7, 19]. Genomic analysis also revealed that a wza gene was present in the putative capsule regions in the other published genomes of V. vulnificus and non-O1, non-O139 V. cholerae. In contrast, the wza gene was present in V. parahaemolyticus, but was not within the capsular polysaccharide region. Furthermore, mutagenesis of this gene showed it was not required for K antigen biosynthesis. Deletion of wbfF changed the pattern of the K-antigen seen on immunoblots, suggesting that this gene may play a role in assembly of the capsule. However, the processing of K-antigen by the wbfF gene and possibly the adjacent wzz gene, and the regulation role of the upstream genes will require further investigation.
In both V. cholerae and V. vulnificus the capsule and O-antigen genes lie in a region similar to the O-antigen region of enteric, such as E. coli, and that specific genes may be shared by both biosynthetic pathways [6, 7]. Pandemic V. parahaemolyticus has changed rapidly in both O and K types, leading to the hypothesis that the genetic determinants of O and K also share the same genetic locus thus allowing a single genetic event to alter the structure of both antigens. However, our finding is not consistent with this hypothesis. Our experiments clearly demonstrated that genes determining the K-antigen in pandemic V. parahaemolyticus were located in the region determining both surface polysaccharides in the other vibrios, but that the O-antigen genes are located elsewhere. From our data and Okura et al's observations on polysaccharide genes, we speculate that the region with homology to LPS core regions may be playing the role of O antigen. This speculation is consistent with the finding that the LPS in V. parahaemolyticus are rough type . Since the core genes are adjacent to the capsule genes, they could still be replaced in the same recombination event and give rise to both new O- and K-antigens. Analysis of putative O and K antigen genes in a different serotype O4:K68 revealed that these regions are distinct from those of O3:K6 serotype despite their highly similar genetic backbones  and suggested both the O and K regions were replaced during the serotype conversion (Chen et al: Comparative genomic analysis of Vibrio parahaemolyticus: serotype conversion and virulence, submitted).
Understanding of the genetic basis of O- and K-antigens is critical to understanding the rapid changes in these polysaccharides seen in pandemic V. parahaemolyticus. This is also important in understanding the virulence of V. parahaemolyticus as the O- and K-antigens represent major surface antigens responsible for protective immunity. In this study, we found the O and K genes were separated in V. parahaemolyticus but their locus maybe adjacent. This report also confirms the genetic location of K-antigen synthesis in V. parahaemolyticus O3:K6 allowing us to focus future studies of the evolution of serotypes to this region.
Bacterial strains and growth condition
At the time of this study, we didn't have access to the sequenced strain RIMD 2210633 and numerous studies showed that the pandemic strains of V. parahaemolyticus O3:K6 are highly clonal and homogenous in their genomes. In particular, the polysaccharide genes are almost identical in RIMD2210633 and two O3:K6 pandemic strains sequenced (Chen et al: Comparative genomic analysis of Vibrio parahaemolyticus: serotype conversion and virulence, submitted), and primers based on RIMD2210633 sequence successfully amplified target DNA from VP53, an O3:K6 pandemic strain isolated from a patient in Kolkata, India in 1996 , and subsequent sequence was confirmed to be identical to RIMD2210633. Thus all mutants were generated from V. parahaemolyticus VP53. Unless otherwise stated, bacteria were cultured in LB broth or LB agar at 37°C. Antibiotics were added in the following concentration when needed: chloramphenicol at 10 μg/ml, and Kanamycin at 50 μg/ml for Escherichia coli and 100 μg/ml for V. parahaemolyticus.
To induce rugose phenotype, a single colony was inoculated into 2 ml APW#3 broth , incubated at 37°C statically for 48 hours. Then 1 μl of culture was spotted on LB agar plate and incubated at 30°C for 48-72 hours. Pictures were taken when colony size reached about half centimeter.
Construction of Mutants
Genetic regions to be targeted and primer sequences were determined based on the annotation of V. parahaemolyticus genome RIMD2210633 (GenBank Accession BA000031 and BA000032). Several mutants, including a mutation deleting the entire K-antigen structural gene operon on chromosome I (VP0219-0237), several partial deletion mutations in the region on chromosome I (VP0215-0218 and VP0220 gene), and a deletion mutation of exopolysaccharide region in chromosome II (VPA1403-1406) as well as a deletion mutation in a separate region containing polysaccharide transport genes wza, wzb, and wzc were constructed (Table 1). Polymerase Chain Reaction (PCR) was performed using Taq DNA polymerase (Thermo Fisher, Waltham, MA). PCR products were purified on Qiagen PCR purification columns (Qiagen, Valencia, CA). Restriction enzymes were purchased from New England Biolabs (Ipswich, MA).
Primers used in this study
pKD3 forward 1
pKD3 reverse 1
pKD3 forward 2
pKD3 reverse 2
flanking sequence of VP0220 (wbfF) gene
flanking sequence of CPS region VP0219-0237
flanking sequence of VP0215-0218
flanking sequence of EPS genes VPA1403-1406
upstream forward $
flanking sequence of wza, wzb, wzc genes
Chitin based transformation
Transformations were performed using a modification of the chitin based method of Meibom , which has been successfully used in V. vulnificus. V. parahaemolyticus was cultured to an optical density of 0.4 - 0.6, pelleted and resuspended to an optical density of 0.2 in sterile seawater. Two ml of this culture were added to pieces of sterile crab shell in a culture plate and incubated at 30°C overnight. The next day, culture supernatant was discarded and 2 ml of fresh sea water, along with 2-4 μg of DNA that was prepared as described above, were added. The mixture was returned to 30°C and incubated overnight. Bacteria were then released from crab shells by vortexing and plated on chloramphenicol agar. Deletions in mutants were confirmed by PCR and sequencing.
Complementation of mutants
DNA fragments deleted in the ∆0220 or ∆EPS mutants were amplified from the wild type genomic DNA (primers listed in Table 3) and cloned into the BamHI and XmaI site of plasmid pBBR1-MCS2 . The resulting plasmids were propagated in E. coli S17pir and then mobilized d into V. parahaemolyticus mutants by conjugation as described previously. All constructs were confirmed by restriction enzyme digestion patterns and sequencing.
To prepare whole cell lysate for polysaccharide analysis, one ml of bacteria culture grown to an O.D. ~1.0 was pelleted, resuspended in 100 μl cell lysis solutions (200 μg/ml lysozyme, 50 μg/ml DNase, 100 μg/ml RNase A) per O.D. and incubated at 37°C for one hour. SDS was added to the solution to 0.5% final concentration and incubated for an additional 30 min. Pronase was then added to 100 μg/ml and samples were incubated at 37°C overnight. Samples were mixed with equal amount of sample buffer (Biorad), boiled for 10 min, separated in a 15% SDS polyacrylamide gel and then transferred to PVDF membranes (Bio-Rad, Hercules, CA). Cell fractions were prepared as described by Koga and Kawata . Briefly, bacteria were treated with lysis buffer (0.6 M sucrose, 100 μg/ml lysozyme, 2.5 mM EDTA and 50 mM Tris-HCl, pH 8.0) at 37°C for 20 min, and then centrifuged at 8000 g for 15 min. The supernatant represented the outer membrane fraction and the pellet represented the cytoplasmic fraction. Cell fraction samples were then treated with DNase and RNase followed by pronase. Aliquots equal to 1 × 108 cells were separated and blotted as described above. The membranes were blocked with 3% skim milk, and incubated with O3 or K6 specific typing sera (Denka Seiken, Japan), followed by binding with a secondary goat anti-rabbit antibody conjugated with alkaline phosphatase (Bio-Rad). Alkaline phosphatase activity was detected by GAR-AP detection kit (Bio-Rad).
Polysaccharides were stained by a combination of stains-all/silver-stain method adapted from . After electrophoresis, polyacrylamide gel was fixed following the fixative step as instructed by the silver stain plus kit (Biorad). The gel was then washed with water four times, 10 min each, to ensure the removal of SDS. The gel was stained for 2 hours with a solution containing 4 mg/ml stains-all (MP Biomedicals), 5% formamide, 25% isopropanol and 15 mM Tris-HCL, pH8.8. The gel was de-stained with water until background became clear (about 30 min). Silver stain was then performed following the staining and developing step as instructed by the silver stain plus kit.
Immuno-gold EM was performed in the Interdisciplinary Center for Biotechnology Research at the University of Florida. V. parahaemolyticus samples were treated by high-pressure freezing, followed by freeze-substitution, embedded in EPOXY resin and thin sectioned. Samples were then labeled with K6 antiserum, followed by gold-labeled secondary antibodies.
We thank G. Balakrish Nair and O. Colin Stine for their suggestions and supplying bacterial strains and Michael E. Kovach for providing plasmid pBBR1-MCS2. We also thank Paul Gulig for sharing his chitin based transformation protocol before publication and Lolia Fernandez for reading our manuscript.
- Fujino L, Okuno Y, Nakada D, Aoyama A, Fukai K, Mukai T, Uebo T: On the bacteriological examination of shirasu food poisoning. Med J Osaka Univ. 1953, 4: 299-304.Google Scholar
- Nair GB, Ramamurthy T, Bhattacharya SK, Dutta B, Takeda Y, Sack DA: Global dissemination of Vibrio parahaemolyticus serotype O3:K6 and its serovariants. Clin Microbiol Rev. 2007, 20 (1): 39-48. 10.1128/CMR.00025-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Nair GB, Hormazabal JC: The Vibrio parahaemolyticus pandemic. Rev Chilena Infectol. 2005, 22 (2): 125-130.PubMedGoogle Scholar
- Chowdhury NR, Chakraborty S, Ramamurthy T, Nishibuchi M, Yamasaki S, Takeda Y, Nair GB: Molecular evidence of clonal Vibrio parahaemolyticus pandemic strains. Emerg Infect Dis. 2000, 6 (6): 631-636. 10.3201/eid0606.000612.PubMed CentralView ArticlePubMedGoogle Scholar
- Chowdhury NR, Stine OC, Morris JG, Nair GB: Assessment of evolution of pandemic Vibrio parahaemolyticus by multilocus sequence typing. J Clin Microbiol. 2004, 42 (3): 1280-1282. 10.1128/JCM.42.3.1280-1282.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Nakhamchik A, Wilde C, Rowe-Magnus DA: Identification of a Wzy polymerase required for group IV capsular polysaccharide and lipopolysaccharide biosynthesis in Vibrio vulnificus. Infect Immun. 2007, 75 (12): 5550-5558. 10.1128/IAI.00932-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Y, Bystricky P, Adeyeye J, Panigrahi P, Ali A, Johnson JA, Bush CA, Morris JG, Stine OC: The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae NRT36S: genes are embedded in the LPS region. BMC Microbiol. 2007, 7: 20-10.1186/1471-2180-7-20.PubMed CentralView ArticlePubMedGoogle Scholar
- Comstock LE, Maneval D, Panigrahi P, Joseph A, Levine MM, Kaper JB, Morris JG, Johnson JA: The capsule and O antigen in Vibrio cholerae O139 Bengal are associated with a genetic region not present in Vibrio cholerae O1. Infect Immun. 1995, 63 (1): 317-323.PubMed CentralPubMedGoogle Scholar
- Yildiz FH, Schoolnik GK: Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. Proc Natl Acad Sci USA. 1999, 96 (7): 4028-4033. 10.1073/pnas.96.7.4028.PubMed CentralView ArticlePubMedGoogle Scholar
- Guvener ZT, McCarter LL: Multiple regulators control capsular polysaccharide production in Vibrio parahaemolyticus. J Bacteriol. 2003, 185 (18): 5431-5441. 10.1128/JB.185.18.5431-5441.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Okura M, Osawa R, Tokunaga A, Morita M, Arakawa E, Watanabe H: Genetic analyses of the putative O and K antigen gene clusters of pandemic Vibrio parahaemolyticus. Microbiol Immunol. 2008, 52 (5): 251-264. 10.1111/j.1348-0421.2008.00027.x.View ArticlePubMedGoogle Scholar
- Chen Y, Stine OC, Morris JG, Johnson JA: Genetic variation of capsule/LPS biogenesis in two serogroup O31 Vibrio cholerae isolates. FEMS Microbiol Lett. 2007, 273 (2): 133-139. 10.1111/j.1574-6968.2007.00808.x.View ArticlePubMedGoogle Scholar
- Comstock LE, Johnson JA, Michalski JM, Morris JG, Kaper JB: Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1. Mol Microbiol. 1996, 19 (4): 815-826. 10.1046/j.1365-2958.1996.407928.x.View ArticlePubMedGoogle Scholar
- Li M, Shimada T, Morris JG, Sulakvelidze A, Sozhamannan S: Evidence for the emergence of non-O1 and non-O139 Vibrio cholerae strains with pathogenic potential by exchange of O-antigen biosynthesis regions. Infect Immun. 2002, 70 (5): 2441-2453. 10.1128/IAI.70.5.2441-2453.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Manning PA, Heuzenroeder MW, Yeadon J, Leavesley DI, Reeves PR, Rowley D: Molecular cloning and expression in Escherichia coli K-12 of the O antigens of the Inaba and Ogawa serotypes of the Vibrio cholerae O1 lipopolysaccharides and their potential for vaccine development. Infect Immun. 1986, 53 (2): 272-277.PubMed CentralPubMedGoogle Scholar
- Yamasaki S, Shimizu T, Hoshino K, Ho ST, Shimada T, Nair GB, Takeda Y: The genes responsible for O-antigen synthesis of vibrio cholerae O139 are closely related to those of vibrio cholerae O22. Gene. 1999, 237 (2): 321-332. 10.1016/S0378-1119(99)00344-3.View ArticlePubMedGoogle Scholar
- Makino K, Oshima K, Kurokawa K, Yokoyama K, Uda T, Tagomori K, Iijima Y, Najima M, Nakano M, Yamashita A: Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae. Lancet. 2003, 361 (9359): 743-749. 10.1016/S0140-6736(03)12659-1.View ArticlePubMedGoogle Scholar
- Johnson JA, Panigrahi P, Morris JG: Non-O1 Vibrio cholerae NRT36S produces a polysaccharide capsule that determines colony morphology, serum resistance, and virulence in mice. Infect Immun. 1992, 60 (3): 864-869.PubMed CentralPubMedGoogle Scholar
- Wright AC, Powell JL, Kaper JB, Morris JG: Identification of a group 1-like capsular polysaccharide operon for Vibrio vulnificus. Infect Immun. 2001, 69 (11): 6893-6901. 10.1128/IAI.69.11.6893-6901.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Stroeher UH, Manning PA: Genetics of Vibrio cholerae O1 and O139 surface polysaccharides. 1999, Boca Raton, Fl.: CRC PressGoogle Scholar
- Stroeher UH, Parasivam G, Dredge BK, Manning PA: Novel Vibrio cholerae O139 genes involved in lipopolysaccharide biosynthesis. J Bacteriol. 1997, 179 (8): 2740-2747.PubMed CentralPubMedGoogle Scholar
- Ali A, Rashid MH, Karaolis DK: High-frequency rugose exopolysaccharide production by Vibrio cholerae. Appl Environ Microbiol. 2002, 68 (11): 5773-5778. 10.1128/AEM.68.11.5773-5778.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Xu M, Yamamoto K, Honda T, Ming X: Construction and characterization of an isogenic mutant of Vibrio parahaemolyticus having a deletion in the thermostable direct hemolysin-related hemolysin gene (trh). J Bacteriol. 1994, 176 (15): 4757-4760.PubMed CentralPubMedGoogle Scholar
- Wang H, Griffiths MW: Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation. Lett Appl Microbiol. 2009, 48 (3): 349-354. 10.1111/j.1472-765X.2008.02531.x.View ArticlePubMedGoogle Scholar
- Hamashima H, Iwasaki M, Arai T: A simple and rapid method for transformation of Vibrio species by electroporation. Methods Mol Biol. 1995, 47: 155-160.PubMedGoogle Scholar
- Meibom KL, Blokesch M, Dolganov NA, Wu CY, Schoolnik GK: Chitin induces natural competence in Vibrio cholerae. Science. 2005, 310 (5755): 1824-1827. 10.1126/science.1120096.View ArticlePubMedGoogle Scholar
- Gulig PA, Tucker MS, Thiaville PC, Joseph JL, Brown RN: USERTM friendly cloning coupled with chitin-based natural transformation enables rapid mutagenesis of Vibrio vulnificus. Appl Environ Microbiol. 2009, 75 (15): 4936-49. 10.1128/AEM.02564-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Whitfield C: Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu Rev Biochem. 2006, 75: 39-68. 10.1146/annurev.biochem.75.103004.142545.View ArticlePubMedGoogle Scholar
- Chun J, Grim CJ, Hasan NA, Lee JH, Choi SY, Haley BJ, Taviani E, Jeon YS, Kim DW, Lee JH: Comparative genomics reveals mechanism for short-term and long-term clonal transitions in pandemic Vibrio cholerae. Proc Natl Acad Sci USA. 2009, 106 (36): 15442-15447. 10.1073/pnas.0907787106.PubMed CentralView ArticlePubMedGoogle Scholar
- Iguchi T, Kondo S, Hisatsune K: Vibrio parahaemolyticus O serotypes from O1 to O13 all produce R-type lipopolysaccharide: SDS-PAGE and compositional sugar analysis. FEMS Microbiol Lett. 1995, 130 (2-3): 287-292. 10.1111/j.1574-6968.1995.tb07733.x.View ArticlePubMedGoogle Scholar
- Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000, 97 (12): 6640-6645. 10.1073/pnas.120163297.PubMed CentralView ArticlePubMedGoogle Scholar
- Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM: Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene. 1995, 166 (1): 175-176. 10.1016/0378-1119(95)00584-1.View ArticlePubMedGoogle Scholar
- Koga T, Kawata T: Isolation and characterization of the outer membrane from Vibrio parahaemolyticus. J Gen Microbiol. 1983, 129 (10): 3185-3196.PubMedGoogle Scholar
- Goldberg HA, Warner KJ: The staining of acidic proteins on polyacrylamide gels: enhanced sensitivity and stability of "Stains-all" staining in combination with silver nitrate. Anal Biochem. 1997, 251 (2): 227-233. 10.1006/abio.1997.2252.View ArticlePubMedGoogle Scholar
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