Evidence suggests that a repertoire of Vibrio cholerae genes are differentially expressed in vivo, and regulation of virulence factors in vivo may follow a different pathway. Our work was aimed at characterization of in vivo-grown bacteria and identification of genes that are differentially expressed following infection by RNA arbitrarily primed (RAP)-PCR fingerprinting. The ligated rabbit ileal loop model was used. The motility of in vivo-grown bacteria increased by 350% over that of in vitro-grown bacteria. Also, the in vivo-grown cells were more resistant to killing by human serum. By using the RAP-PCR strategy, five differentially expressed transcripts were identified. Two in vitro-induced transcripts encoded polypeptides for the leucine tRNA synthatase and the 50S ribosomal protein, and the three in vivo-induced transcripts encoded the SucA and MurE proteins and a polypeptide of unknown function. MurE is a protein involved in the peptidoglycan biosynthetic pathway. The lytic profiles of in vivo-and in vitro-grown cells suspended in distilled water were compared; the former was found to be slightly less sensitive to lysis. Ultrathin sections of both cells observed under the transmission electron microscope revealed that in contrast to the usual wavy discontinuous membrane structure of the in vitro-grown cells, in vivo-grown cells had a more rigid, clearly visible double-layered structure. The V. cholerae murE gene was cloned and sequenced. The sequence contained an open reading frame of 1,488 nucleotides with its own ribosome-binding site. A plasmid containing the murE gene of V. cholerae was transformed into V. cholerae 569B, and a transformed strain, 569BME, containing the plasmid was obtained. Ultrathin sections of 569BME viewed under a transmission electron microscope revealed a slightly more rigid cell wall than that of wild-type 569B. When V. cholerae 569B and 569BME cells were injected separately into ligated rabbit ileal loops, the transformed cells had a preference for growth in the ileal loops versus laboratory conditions. Cholera is still a major public health problem in developing countries as well as in some developed countries. The causative organism, Vibrio cholerae, is a gram-negative curved rod, and the life-threatening watery diarrhea is largely due to the action of the secreted cholera toxin on the epithelium of the small intestine (8). Several lines of evidence suggest that a number of V. cholerae genes are differentially expressed in vivo following infection. The major virulence genes of V. cholerae O1 biotype Classical under the coordinate regulation of the transcriptional activator ToxR are maximally expressed in vitro. In contrast, in the intestinal lumen, conditions repress the expression of ToxR-controlled virulence factors (16,17). Thus, to induce the ToxR-controlled virulence genes in vivo, V. cholerae may recognize other unknown external signals in the host environment (20). V. cholerae cells are extremely sensitive to a wide variety of chemicals, particularly hydrophobic compoun...

The rate and extent of lysis of Vibrio cholerae cells under nongrowing conditions were dependent on the osmolarity of the growth medium. Gross alterations in cellular morphology were observed when V. chokrae cells were grown in media of high and low osmolarity. The rate of lysis of V. cholerae cells under nongrowing conditions increased after treatment with chloramphenicol. Chloramphenicol-treated V. cholerae 569B cells showed formation of sphaeroplast-like bodies in medium of high osmolarity, but not in low osmolarity. Changes in the osmolarity of the growth medium also regulated the expression of the outer membrane proteins. This regulation was abolished if V. cholerae cells were grown in Pi-depleted medium. Analysis of the lytic behavior and composition of outer membrane proteins of an osmotically fragile mutant strain revealed a similar dependence on the osmolarity of the growth medium. * Corresponding author. OmpC proteins (13, 36). The synthesis of the PhoE protein is derepressed upon phosphate starvation (25). It has also been suggested that the rigidity of the outer membranepeptidoglycan layer might also depend upon the specific major outer membrane protein present (13). It is in this context that the present report describes the changes in the lytic phenotype, cellular morphology, and composition of the outer membrane of a hypertoxinogenic strain (569B) of V. cholerae grown under various osmolarities and in phosphate-depleted (LP) growth medium. The lytic phenotype and cellular morphology were also affected drastically after treatment with chloramphenicol (CAM).

Controlled exposure of Leishmania dono ani promastigotes to hypotonic shock results in the formation of deflagellated unsealed ghosts of original polarity that largely retain the pellicular microtubular structure associated with plasma membrane of the parasite. Gentle shearing followed by suspension of the purified membrane in appropriate isotonic buffer containing Mg# + (4 mM) results in the formation of sealed everted vesicles. The presence of Mg# + (4 mM) appears to be essential for efficient sealing and also to prevent leakiness. ATP-dependent Ca# + accumulation can be demonstrated in these vesicles. K m values for Ca# + and ATP were 125 nM and 0n8 mM respectively. The accumulated Ca# + reaches a concentration of 1n1 mM. Ca# + uptake is completely inhibited by vanadate (40 µM) and several

Intracellular development of choleraphage Φ149 following infection of Vibrio cholerae and Vibrio eltor cells under different conditions was examined by thin-section electron microscopy. Degradation of the host DNA following infection and formation of mature phage particles inside the infected cells were demonstrated. The concatemeric DNA intermediate formed during intracellular replication of Φ149 DNA in permissive hosts was resolved. In confirmation of biochemical evidence, no concatemeric DNA intermediate was observed for infection in high-phosphate medium.

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