ABSTRACT
Aeromonas have been isolated from a wide variety of aquatic environments. However the number of Aeromonas in sea water is extremely small compared to that in fresh water. In in vitro culture, Aeromonas can grow in mediums containing NaCl at a concentration of 3.0%, this concentration corresponding to that of sea water. It is unclear why the number of Aeromonas is low in sea water. Exoproteins of bacteria are thought to be important for bacterial growth and survival in the environment. Previously, the present authors have shown that mediums containing 3.0% NaCl suppress production of two proteases, serine protease and metalloprotease. In this experiment, other exoproteins whose production is influenced by the amount of NaCl in the medium were analyzed. A protein whose production is repressed in medium containing 3.0% NaCl was found and purified. Biological assay of the purified protein showed that it degrades tributyrin and hydrolyzes para-nitrophenyl-fatty acylesters. These results show that the protein is a lipase. Subsequently, the nucleotide sequence of the gene encoding the lipase was determined and the amount of mRNA of the lipase gene in the cells measured. It was found that transcription of the gene is not inhibited by NaCl in the medium. This result indicates that the lipase might be synthesized, but the folding process to become an active structure does not progress smoothly in a medium containing 3.0% NaCl.
Subject(s)
Aeromonas/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Lipase/metabolism , Aeromonas/chemistry , Aeromonas/genetics , Aeromonas/isolation & purification , Amino Acid Sequence , Bacterial Proteins/genetics , Cloning, Molecular , Culture Media/metabolism , Gene Expression Regulation, Enzymologic , Lipase/chemistry , Lipase/genetics , Molecular Sequence Data , Protein Transport , Seawater/microbiology , Sequence Alignment , Sodium Chloride/metabolism , Substrate SpecificityABSTRACT
A non-invasive live transconjugant Shigella hybrid (LTSHΔstx) strain was constructed from a Shiga toxin gene deleted mutant of Shigella dysenteriae 1 by introducing a plasmid vector pPR1347 that carried a lipopolysaccharide biosynthesis gene (rfb and rfc) of Salmonella typhimurium. In guinea pigs, four successive oral administrations of LTSH Δstx showed complete protection against rectal challenge with wild type S. dysenteriae 1 strain. Exponential increase of the serum IgG and IgA titer against lipopolysaccharide of LTSH Δstx was observed during immunization, peaked on day 28 and remained at that level until day 35 after the initiation of the immunization. In intestinal lavage of the immunized animals, significant increase of IgA titer against lipopolysaccharide of LTSH Δstx was also observed. These data suggested that LTSH Δstx could be a useful candidate to induce protective immunity against S. dysenteriae 1 infection.
Subject(s)
Dysentery, Bacillary/immunology , Dysentery, Bacillary/prevention & control , Shigella dysenteriae/immunology , Animals , Antibodies, Bacterial/immunology , Disease Models, Animal , Dysentery, Bacillary/microbiology , Female , Guinea Pigs , Humans , Immunization , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Lipopolysaccharides/immunology , Male , Shigella dysenteriae/physiologyABSTRACT
A pair of primers targeting the hlyA gene for Vibrio cholerae which could distinguish the classical from El Tor biotypes was designed and combined with other specific primers for ompW, rfb complex, and virulence genes such as ctxA, toxR, and tcpI in a multiplex PCR (m-PCR) assay. This m-PCR correctly identified 39 V. cholerae from clinical, water and seafood samples. The efficiency of this multiplex PCR (m-PCR) was compared with conventional biochemical and serogrouping methods. One O139 and 25 O1 V. cholerae strains including 10 environmental strains harbored all virulence-associated genes except 1 clinical strain which only had toxR and hlyA genes. Thirteen environmental strains were classified as non-O1/non-O139 and had the toxR and hlyA genes only. The detection limit of m-PCR was 7 x 10(4) cfu/ml. The m-PCR test was reliable and rapid and reduced the identification time to 4 h.
Subject(s)
Bacterial Proteins/genetics , Cholera/virology , Hemolysin Proteins/genetics , Polymerase Chain Reaction/methods , Seafood/microbiology , Seawater/microbiology , Vibrio cholerae , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Typing Techniques/methods , DNA Primers , Hemolysin Proteins/chemistry , Humans , Malaysia , Molecular Sequence Data , Sensitivity and Specificity , Serotyping , Vibrio cholerae/classification , Vibrio cholerae/genetics , Vibrio cholerae/isolation & purification , Vibrio cholerae/pathogenicity , Virulence/geneticsABSTRACT
Vibrio cholerae O1 strains that are hybrids between the classical and El Tor biotypes were isolated during two consecutive years (2004-2005) from diarrheal patients in Mozambique. Similar variants isolated in Bangladesh and recently isolated El Tor strains were analyzed for genetic diversity. Pulsed-field gel electrophoresis (PFGE) analysis using the restriction enzyme NotI, resulted in 18-21 bands showed five closely related PFGE patterns that were distributed similarly in both years (2004-2005) among the 80 strains tested in Mozambique. Overall based on the PFGE patterns the hybrids indicated an El Tor lineage. The restriction patterns of whole-chromosomal DNA grouped the hybrid strains from Mozambique into a separate cluster from Bangladeshi clinical and environmental hybrid strains. A high molecular weight band of 398kb that contain rstR allele of the classical type was detected from all hybrid strains, which was absent in all conventional classical and El Tor strains. This band could be designated as a marker for the hybrid strains. This study suggests that hybrid strains from Mozambique are closely related to each other, different from Bangladeshi hybrid strains that are diverse in nature and all hybrid strains differed markedly from conventional classical and El Tor strains.
