Suckling Mouse Intestine Research Articles

DegS regulates the aerobic metabolism of Vibrio cholerae via the ArcA-isocitrate dehydrogenase pathway for growth and intestinal colonization.

Aerobic respiration is the key driver of Vibrio cholerae proliferation and infection.Our previous transcriptome results suggested that degS knockout downregulates a few genes involved in NADH and ATP synthesis in the aerobic respiratory pathway. In this study, non-targeted metabolomics results showed that the differential metabolites affected by degS knockout were associated with aerobic respiration. Further results suggested that the key products of aerobic respiration, NADH and ATP, were reduced upon degS deletion and were not dependent on the classical σE pathway. The two-component system response factor aerobic respiration control A (ArcA) is involved in regulating NADH and ATP levels. qRT-PCR demonstrated that DegS negatively regulates the transcription of the arcA gene, which negatively regulates the expression of isocitrate dehydrogenase (ICDH), a key rate-limiting enzyme of the tricarboxylic acid cycle. NADH and ATP levels were partially restored with the knockout of the arcA gene in the ΔdegS strain, while levels were partially restored with overexpression of ICDH in the ΔdegS strain. In a growth experiment, compared to the ΔdegS strain, the growth rates of ΔdegSΔarcA and ΔdegS-overexpressed icdh strains (ΔdegS+icdh) were partially restored during the logarithmic growth period. Colonization of the intestines of suckling mice showed a significant reduction in the colonizing ability of the ΔdegS strain, similar colonizing ability of the ΔdegS::degS strain and the wild-type strain, and a partial recovery of the colonizing ability of the ΔdegS+icdh strain. Overall, these findings suggest that the DegS protease regulates the expression of ICDH through ArcA, thereby affecting the NADH and ATP levels of V. cholerae and its growth and intestinal colonization ability.

Mucosal and systemic neutralizing antibodies to norovirus induced in infant mice orally inoculated with recombinant rotaviruses

Rotaviruses (RVs) preferentially replicate in the small intestine and frequently cause severe diarrheal disease, and the following enteric infection generally induces variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid only-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens invivo. This is a newly available opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued replication-competent recombinant RRVs harboring bicistronic gene segment 7 that encodes the native RV nonstructuralprotein 3 (NSP3) protein and a human norovirus (HuNoV) VP1 protein or P domain from the predominant genotype GII.4. The rescued viruses expressed HuNoV VP1 or P protein in infected cells invitro and elicited systemic and local antibody responses to HuNoV and RRV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RRV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens.

In vivo fluid accumulation-inhibitory, anticolonization and anti-inflammatory and in vitro biofilm-inhibitory activities of methyl gallate isolated from Terminalia chebula against fluoroquinolones resistant Vibrio cholerae

Acute Vibrio cholerae infection triggers significant inflammatory response and immense fluid secretion in the intestine. In the present study, methyl gallate (MG) isolated from Terminalia chebula was evaluated to determine the in vivo fluid accumulation-inhibitory, anticolonization and anti-inflammatory and in vitro biofilm-inhibitory activities against multi-drug resistant (MDR) V. cholerae.Bacterial membrane-damaging and biofilm-inhibitory activities were determined by membrane perturbation and transmission electron microscopy (TEM); and microdilution assays, respectively. Fluid accumulation-inhibitory and anticolonization activities of MG (23.80–95.23 mg/kg body weight) were determined in 4–5 days old BALB/c mice with an incubation time of 18 h. The effect of MG (1, 50 and 500 mg/kg body weight) on intestinal inflammatory reaction induced by V. cholerae was studied by performing histology in Swiss albino mice.MIC and MBC of MG against the test strains were 32–64 and 64–256 μg/ml, respectively. MG showed the fluid accumulation-inhibitory activity with inhibition values of 42.86–89.08% at doses between 23.80 and 95.23 mg/kg body weight and significant anticolonization activity (p < 0.0001) against V. choleare in the suckling mouse intestine. MG (500 mg/kg body weight) significantly inhibited the inflammatory reactions induced by V. cholerae compared to the vehicle control. MG exhibited 70% minimum biofilm inhibition concentration of 64 μg/ml and bacterial membrane damaging activity at 1 × MBC. The results obtained in the present study suggest that MG has potential as an effective agent for the treatment of severe secretory and inflammatory diarrheal disease caused by MDR V. cholerae.

Flagellar motility, extracellular proteases and Vibrio cholerae detachment from abiotic and biotic surfaces

Vibrio cholerae of serogroups O1 and O139, the causative agent of Asiatic cholera, continues to be a major global health threat. This pathogen utilizes substratum-specific pili to attach to distinct surfaces in the aquatic environment and the human small intestine and detaches when conditions become unfavorable. Both attachment and detachment are critical to bacterial environmental survival, pathogenesis and disease transmission. However, the factors that promote detachment are less understood. In this study, we examine the role of flagellar motility and hemagglutinin/protease (HapA) in vibrio detachment from a non-degradable abiotic surface and from the suckling mouse intestine. Flagellar motility facilitated V. cholerae detachment from abiotic surfaces. HapA had no effect on the stability of biofilms formed on abiotic surfaces despite representing >50% of the proteolytic activity present in the extracellular matrix. We developed a balanced lethal plasmid system to increase the bacterial cyclic diguanylate (c-di-GMP) pool late in infection, a condition that represses motility and HapA expression. Increasing the c-di-GMP pool enhanced V. cholerae colonization of the suckling mouse intestine. The c-di-GMP effect was fully abolished in hapA isogenic mutants. These results suggest that motility facilitates detachment in a substratum-independent manner. Instead, HapA appears to function as a substratum-specific detachment factor.

An Outbreak of Diarrhea in Mandera, Kenya, Due to Escherichia coli Serogroup O-Nontypable Strain That Had a Coding Gene for Enteroaggregative E. coli Heat-Stable Enterotoxin 1.

In an outbreak of gastroenteritis in December 2009, in Mandera, Kenya, Escherichia coli O-nontypable (ONT) strain was isolated from stool specimens of patients (18/24, 75%). The E. coli ONT organisms could not be assigned to any of the recognized diarrheagenic groups of E. coli However, they possessed the enteroaggregative E. coli heat-stable enterotoxin-1 gene. The cell-free culture filtrates of the E. coli ONT strain isolated from the outbreak cases induced considerable amount of fluid accumulation in suckling mouse intestine, indicating production of an enterotoxic factor(s). These results identify E. coli that did not have any diarrheagenic characteristics except astA as the etiological agent of the diarrheal outbreak in Mandera. It is however considered necessary to characterize the fluid accumulation factor(s) to determine whether any novel toxins were responsible for the fluid accumulation. Moreover, it is important to study dissemination of strains producing the enterotoxic factor(s) to assess their public health significance distribution in the environment.

A D, D-carboxypeptidase is required for Vibrio cholerae halotolerance.

The biological roles of low molecular weight penicillin-binding proteins (LMW PBP) have been difficult to discern in Gram-negative organisms. In Escherichia coli, mutants lacking these proteins often have no phenotype, and cells lacking all seven LMW PBPs remain viable. In contrast, we report here that Vibrio cholerae lacking DacA-1, a PBP5 homologue, displays slow growth, aberrant morphology and altered peptidoglycan (PG) homeostasis in Luria-Bertani (LB) medium, as well as a profound plating defect. DacA-1 alone among V. cholerae's LMW PBPs is critical for bacterial growth; mutants lacking the related protein DacA-2 and/or homologues of PBP4 or PBP7 displayed normal growth and morphology. Remarkably, the growth and morphology of the dacA-1 mutant were unimpaired in LB media containing reduced concentrations of NaCl (100 mM or less), and also within suckling mice, a model host for the study of cholera pathogenesis. Peptidoglycan from the dacA-1 mutant contained elevated pentapeptide levels in standard and low salt media, and comparative analyses suggest that DacA-1 is V. cholerae's principal DD-carboxypeptidase. The basis for the dacA-1 mutant's halosensitivity is unknown; nonetheless, the mutant's survival in biochemically uncharacterized environments (such as the suckling mouse intestine) can be used as a reporter of low Na(+) content.

Characterization of an Enterovirus species E isolated from naturally infected bovine in China

Bovine enteroviruses, which belong to the Picornaviridae family, can cause clinical symptoms in cattle and are excreted in feces. In this study, a cytolytic virus was isolated from Madin-Darby bovine kidney (MDBK) cells from fecal samples of bovine with severe diarrhea and hemorrhagic intestinal mucosa that had been originally diagnosed with bovine viral diarrhea (BVD) by a bovine viral diarrhea virus Ag point-of-care test (IDEXX, American). Random priming PCR was used to amplify underlying viral sequences and identify the isolated virus. Phylogenetic analysis indicated that the isolated virus closely matches the EV-E2 species, which is different from other Chinese strains previously isolated. The newly identified virus was named HLJ-3531/2013. We infected the sulking mice with the isolated virus. Reverse-transcription PCR, hematoxylin and eosin (HE) staining, serum neutralization (SN) test, and virus isolation from various tissues revealed that HLJ-3531/2013 can infect the intestine, liver, and lung of suckling mice. The present work is the first to report the reproduction of clinical symptoms by an isolated virus in an experimental infection model of animals and lays a solid foundation for the development of the pathogenesis of bovine enteroviruses.

Role of Dual Flagella in the Pathogenesis of Vibrio parahaemolyticus

Vibrio parahaemolyticus possesses two flagella systems: polar and lateral flagella for swimming in liquid and swarming on solid surfaces or in viscous environments. To elucidate the pathogenic role of these dual flagella systems, we constructed single- and double-deletion mutants of the lafA and flhAB flagellum genes and investigated their biofilm formation, cell adhesion, and colonization of the small intestine of suckling mice. The double-mutant strain was more impaired in biofilm formation than either of the single-mutant strains. In addition, the lafA, flhAB, and double-mutant strains showed 40%, 45%, and 60%, respectively, lower adherence to HeLa cells than the wild-type strain. Moreover, the lafA, flhAB, and double-mutant strains exhibited 49%, 5.6 and 6.7 times, respectively, lower colonization in a competition assay than the wild-type strain. These findings indicated that polar flagella were more important than lateral flagella for the pathogenesis of V. parahaemolyticus.

The procollagen type III, alpha 1 (COL3A1) gene first intron expresses poly-A+RNA corresponding to multiple ESTs and putative miRNAs

The mouse COL3A1 first intron is 9684 bp. RNA's of approximately 1.6 and 3.0 kb were detected by Northern hybridization analysis of poly-A RNA from fetal mice and total RNA from suckling and adult mouse intestine using (32)P-labeled, anti-sense RNA synthesized from a mouse COL3A1 first intron, 5 prime region, 5.4 kb Xba I fragment (1655-7030 bp), recombinant plasmid (pPI5.4x). Expression of the 1.6 and 3.0 kb RNA's was significantly reduced in adult mouse intestine, indicating that these RNAs are developmentally regulated. "BLAST" analysis indicated that the mouse first intron 5 prime sequence has 94-100% identity to 13 mouse ESTs. These mouse first intron EST's lie within the 5.4 Xba I fragment of the mouse COL3A1 first intron. Two of the mouse first intron EST's have significant identity to known miRNA, mature sequences, mmu-miR-466f-3P, mmu-miR-1187, and mmu-miR-574-5P as well as others. Predicted targets for mmu-miR-466f-3P include COL1A1, COL19A1, COL11A2, COL4A1, and COL4A5 indicating that COL3A1 intronic miRNAs may regulate the expression of other collagen genes in development.

Attenuation of bacterial virulence by quorum sensing-regulated lysis

Genetically attenuated pathogenic bacteria have been extensively considered as vaccine candidates. However, insufficient attenuation has been a frequent limitation of this approach. Many pathogens use quorum sensing to escape host defense mechanism. Here, we hypothesized that quorum sensing can be manipulated to diminish pathogenesis. To test this hypothesis, we modified the quorum sensing circuitry of a live cholera vaccine strain to add a second layer of attenuation. Attenuation resulted from the expression of phage PhiX174 lysis gene E on a balanced lethal plasmid from the quorum sensing-regulated luxC promoter. For conditional expression of quorum sensing and positive selection in vivo, the host strain was deleted of its cqsA and thyA genes encoding cholera autoinducer 1 (CAI-1) synthase and thymidylate synthase, respectively. A recombinant cqsA gene expressed from the cholera toxin (CT) promoter and an active thyA gene was provided in trans. The resulting strain expressed CAI-1 in AKI cultures (CT permissive condition) but not in LB medium. Additionally, it expressed elevated biofilm in LB medium compared to AKI conditions where CAI-1 is synthesized to repress biofilm formation. Induction of lysis gene E by quorum sensing restricted growth to a lower cell density in AKI medium, the suckling mouse intestine or LB supplemented with exogenous CAI-1. Microscopic examination revealed the presence of Vibrio cholerae ghost cells at high cell density. Lysis was accompanied by the release of intracellular β-galactosidase to the culture medium. We conclude that it is possible to manipulate quorum sensing to attenuate a live vaccine vector and restrict its shedding to the environment and diminish its subsequent dissemination.

Ultrastructural changes in the small intestine of suckling mice, caused by Vibrio cholerae hemagglutinin/protease

Suckling mice aged 4-5 days were injected with Vibrio cholerae hemagglutinin/protease and ultrastructural changes in their small intestine were studied after 5 h. The preparation caused a statistically significant accumulation of fluid in the intestine, appearance of large gaps along cell-cell spaces in the villi and crypts, intense production and secretion of the mucus by goblet cells. The formation of interepithelial cavities was paralleled by vascular changes, supplemented by extravasal disorders caused by mast cell reaction. The role of enterochromaffin cells and lipofibroblasts, modulating the secretion in the intestine, is confirmed.

Global Gene Expression and Phenotypic Analysis of a Vibrio cholerae rpoH Deletion Mutant

Vibrio cholerae, the cause of cholera, can grow in a variety of environments outside of human hosts. During infection, this pathogen must adapt to significant environmental alterations, including the elevated temperature of the human gastrointestinal tract. Sigma(32), an alternative sigma factor encoded by rpoH, activates transcription of genes involved in the heat shock response in several bacterial species. Here, we assessed the role of sigma(32) in V. cholerae physiology. In aggregate, our findings suggest that sigma(32) promotes V. cholerae growth at temperatures ranging at least from 15 degrees C to 42 degrees C. Growth of the rpoH mutant was severely attenuated within the suckling mouse intestine, suggesting that sigma(32)-regulated genes are critical for V. cholerae adaptation to conditions within the gastrointestinal tract. We defined the V. cholerae RpoH regulon by comparing the whole-genome transcription profiles of the wild-type and rpoH mutant strains after a temperature up-shift. Most of the V. cholerae genes expressed in an RpoH-dependent manner after heat shock encode proteins that influence protein fate, such as proteases and chaperones, or are of unknown function. Bioinformatic analyses of the microarray data were used to define a putative sigma(32) consensus binding sequence and subsequently to identify genes that are likely to be directly regulated by RpoH in the whole V. cholerae genome.

Polyphosphate Stores Enhance the Ability of Vibrio cholerae To Overcome Environmental Stresses in a Low-Phosphate Environment

Vibrio cholerae, the causative agent of Asiatic cholera, has been reported to make large quantities of polyphosphate. Inorganic polyphosphate is a ubiquitous molecule with a variety of functions in prokaryotic and eukaryotic cells. We constructed a V. cholerae mutant with a deletion in the polyphosphate kinase (ppk) gene. The mutant was defective in polyphosphate biosynthesis. Deletion of ppk had no significant effect on production of cholera toxin, hemagglutinin/protease, motility, biofilm formation, and colonization of the suckling mouse intestine. The wild type and mutant had similar growth rates in rich and minimal medium and exhibited similar phosphate uptake and alkaline phosphatase induction. In contrast to ppk mutants from other gram-negative bacteria, the V. cholerae mutant survived prolonged starvation in LB medium and artificial seawater basal salts. The ppk mutant was significantly more sensitive to low pH, high salinity, and oxidative stress when it was cultured in low-phosphate minimal medium. The ppk mutant failed to induce catalase when it was downshifted to phosphorus-limiting conditions. Furthermore, the increased sensitivity of the ppk mutant to environmental stressors in phosphate-limited medium correlated with a diminished capacity to synthesize ATP from intracellular reservoirs. We concluded that polyphosphate protects V. cholerae from environmental stresses under phosphate limitation conditions. It has been proposed that toxigenic V. cholerae can survive in estuaries and brackish waters in which phosphorus and/or nitrogen can be a limiting nutrient. Thus, synthesis of large polyphosphate stores could enhance the ability of V. cholerae to survive in the aquatic environment.

Activation of the Vibrio cholerae SOS Response Is Not Required for Intestinal Cholera Toxin Production or Colonization

Cholera toxin, one of the main virulence factors of Vibrio cholerae, is encoded in the genome of CTXphi, a V. cholerae-specific lysogenic filamentous bacteriophage. Although the genes encoding cholera toxin, ctxAB, are known to have their own promoter, the toxin genes can also be transcribed from an upstream CTXphi promoter, PrstA. The V. cholerae SOS response to DNA damage induces the CTX prophage by stimulating gene expression initiating from PrstA. Here, we investigated whether ctxA mRNA levels increase along with the levels of the transcripts for the other CTXphi genes following stimulation of the V. cholerae SOS response. Treatment of V. cholerae with the SOS-inducing agent mitomycin C increased the level of ctxA mRNA approximately sevenfold, apparently by augmenting the activity of PrstA. However, using suckling mice as a model host, we found that intraintestinal ctxA transcription does not depend on PrstA. In fact, the suckling mouse intestine does not appear to be a potent inducer of the V. cholerae SOS response. Furthermore, alleviation of LexA-mediated repression of the V. cholerae SOS regulon was not required for V. cholerae growth in the suckling mouse intestine. Our observations suggest that pathogenicity of V. cholerae does not depend on its SOS response.

A Vibrio cholerae Relaxed ( relA ) Mutant Expresses Major Virulence Factors, Exhibits Biofilm Formation and Motility, and Colonizes the Suckling Mouse Intestine

We have constructed a relaxed mutant of El Tor biotype Vibrio cholerae strain C7258 by disruption of the RelA catalytic domain. The ability of the V. cholerae relaxed mutant to biosynthesize guanosine tetraphosphate and pentaphosphate was severely affected; the mutant showed a reduced growth rate in minimal medium that could be reversed by the addition of Casamino Acids, and it was thermosensitive. Contrary to published findings, the new relA mutant still produced significant cholera toxin and toxin-coregulated pilus. The V. cholerae relA mutant was motile, produced normal biofilms, and colonized the suckling mouse intestine. Our data suggest that levels of basal guanosine nucleotides pppGpp and ppGpp, rather than the availability of a stringent response, could influence expression of virulence factors, depending on strain and culture conditions. Production of hemagglutinin (HA)/protease, which requires HapR, RpoS, and the cyclic AMP receptor protein, was not strongly affected. Nevertheless, overexpression of RelA protein from an isopropyl-beta-d-thiogalactopyranoside-inducible promoter posttranscriptionally diminished production of HA/protease.

Characterization of the Small Untranslated RNA RyhB and Its Regulon inVibrio cholerae

Numerous small untranslated RNAs (sRNAs) have been identified in Escherichia coli in recent years, and their roles are gradually being defined. However, few of these sRNAs appear to be conserved in Vibrio cholerae, and both identification and characterization of sRNAs in V. cholerae remain at a preliminary stage. We have characterized one of the few sRNAs conserved between E. coli and V. cholerae: RyhB. Sequence conservation is limited to the central region of the gene, and RyhB in V. cholerae is significantly larger than in E. coli. As in E. coli, V. cholerae RyhB is regulated by the iron-dependent repressor Fur, and it interacts with the RNA-binding protein Hfq. The regulons controlled by RyhB in V. cholerae and E. coli appear to differ, although some overlap is evident. Analysis of gene expression in V. cholerae in the absence of RyhB suggests that the role of this sRNA is not limited to control of iron utilization. Quantitation of RyhB expression in the suckling mouse intestine suggests that iron availability is not limiting in this environment, and RyhB is not required for colonization of this mammalian host by V. cholerae.

Hfq is essential for Vibrio cholerae virulence and downregulates sigma expression.

Hfq is an RNA-binding protein that interacts with both small untranslated RNAs (sRNAs) and mRNAs to modulate gene expression post-transcriptionally. In Escherichia coli and Salmonella typhimurium, Hfq is required for efficient expression of the stationary phase sigma factor sigma(S), and consequently is critical for Salmonella virulence. We have found that Hfq is also essential for the virulence of Vibrio cholerae, as strains lacking hfq fail to colonize the suckling mouse intestine. Deletion of the V. cholerae hfq does not prevent production of sigma(S), nor does it prevent expression of TCP, V. cholerae's primary colonization factor. The expression and activity of the alternative sigma factor sigma(E) are dramatically increased in a V. cholerae hfq mutant. Comparison of the transcriptome of an hfq mutant with that of an rseA mutant, which also overexpresses sigma(E), revealed that sigma(E) controls approximately half the genes found to be upregulated in the hfq mutant. However, increased sigma(E) does not appear to account for this strain's reduced virulence. It is likely that sRNAs, in conjunction with Hfq, are critical regulators of V. cholerae pathogenicity.

Rotavirus-specific proteins are detected in murine macrophages in both intestinal and extraintestinal lymphoid tissues

Rotaviruses replicate in mature, villous epithelial cells of the mammalian small intestine. Although rotavirus has not been detected in plasma of infants with rotavirus-induced gastroenteritis, rotavirus particles and rotavirus genomic RNA have been detected in extraintestinal sites (e.g. cerebrospinal fluid). Using a murine rotavirus strain well adapted to growth in the small intestines of suckling mice, we found that macrophages (and to a lesser extent B cells) in gut-associated lymphoid tissue contained rotavirus-specific proteins, and that these antigen-containing cells travelled to sites distant to the intestine.

Disruption of the guanylyl cyclase-C gene leads to a paradoxical phenotype of viable but heat-stable enterotoxin-resistant mice.

Heat-stable enterotoxins (STa), which cause an acute secretory diarrhea, have been suggested to mediate their actions through the guanylyl cyclase-C (GC-C) receptor. The GC-C gene was disrupted by insertion of neo into exon 1 and subsequent homologous recombination. GC-C null mice contained no detectable GC-C protein. Intestine mucosal guanylyl cyclase activity was approximately 16-fold higher in wild-type mice than in the GC-C null mice, and STa-stimulable guanylyl cyclase activity was absent in the null animals. Thus, GC-C is the major cyclase activity present in the intestine, and also completely accounts for the STa-induced elevations of cGMP. Gavage with STa resulted in marked fluid accumulation within the intestine of wild-type and heterozygous suckling mice, but GC-C null animals were resistant. In addition, infection with enterotoxigenic bacteria that produce STa led to diarrhea and death in wild-type and heterozygous mice, while the null mice were protected. Cholera toxin, in contrast, continued to cause diarrhea in GC-C null mice, demonstrating that the cAMP signaling pathway remained intact. Markedly different diets (high carbohydrate, fat, or protein) or the inclusion of high salt (K+, Na+) in the drinking water or diet also did not severely affect the null animals. Given that GC-C is a major intestinal receptor in all mammals, the pressure to retain a functional GC-C in the face of diarrhea-inflicted mortality remains unexplained. Therefore, GC-C likely provides a protective effect against stressors not yet tested, possibly pathogens other than noninvasive enterotoxigenic bacteria.

A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139.

Vibrio cholerae O139 is the first non-O1 serogroup of V. cholerae to give rise to epidemic cholera. Apparently, this new serogroup arose from an El Tor O1 strain of V cholerae, but V. cholerae O139 is distinguishable from V. cholerae El Tor O1 by virtue of its novel antigenic structure and also its characteristic pattern of resistances to the antibiotics sulfamethoxazole, trimethoprim, streptomycin, and furazolidone. We found that the first three of these antibiotic resistances are carried on an approximately 62-kb self-transmissible, chromosomally integrating genetic element which we have termed the SXT element. This novel conjugative transposon-like element could be conjugally transferred from V. cholerae O139 to V cholerae O1 and Escherichia coli strains, where it integrated into the recipient chromosomes in a site-specific manner independent of recA. To study the potential virulence properties of the SXT element as well as to improve upon the live attenuated O139 vaccine strain Bengal-2, a large internal deletion in the SXT element was crossed on to the Bengal-2 chromosome. The resulting strain, Bengal-2.SXT(s), is sensitive to sulfamethoxazole and trimethoprim and colonizes the intestines of suckling mice as well as wild-type strains do, suggesting that the SXT element does not encode a colonization factor. Derivatives of Bengal-2.SXT(s) are predicted to be safe, antibiotic-sensitive, live attenuated vaccines for cholera due to the O139 serogroup.