Transcriptional analysis of the Aeromonas salmonicida S-layer protein gene vapA.

The vapA gene of Aeromonas salmonicida encodes the subunit of the surface protein array known as A-layer. Nucleotide sequence analysis of the 374 bp of DNA immediately upstream of vapA revealed two potential promoter sequences and other possible regulatory sequences. Sequencing and polymerase chain reaction analysis showed that the region was conserved in wild-type A. salmonicida. Primer extension and Northern (RNA) blot analysis showed that vapA transcription in A. salmonicida was directed predominantly by a distal promoter, P1, resulting in a 1.7-kb unit-length mRNA with an untranslated 181-nucleotide leader sequence which contained two predicted low-free-energy stem-loop structures. Northern analysis of cells grown at 15 degrees C showed that vapA transcript production peaked during the mid-log phase of growth (A600 = 0.25). At 15 degrees C, the half-life of the vapA mRNA was 22 min, while at 20 degrees C, the half-life was significantly shorter, 11 min. The amount of vapA transcript produced was reduced by growth in the presence of the DNA gyrase inhibitors nalidixic acid and novobiocin. Environmental factors such as growth temperature and atmospheric oxygen tension also affected the quantity of vapA mRNA. vapA transcript could not be detected in mutants which produced either low levels of full-length or truncated A protein or no detectable A protein.

Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida.

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.

Nucleotide sequences of the gnd genes from nine natural isolates of Escherichia coli: evidence of intragenic recombination as a contributing factor in the evolution of the polymorphic gnd locus.

Nine natural isolates of Escherichia coli were examined, and the sequence of the entire 1,404 bases of the gnd gene (6-phosphogluconate dehydrogenase, EC 1.1.1.44) was determined. These isolates, along with E. coli K-12, constitute 10 strains for analysis. (The sequence of the E. coli K-12 gnd gene is known.) A total of 184 sites were polymorphic, and up to 6% sequence divergence was observed between pairs of strains. The deduced amino acid sequences showed much more variation than had been shown by multilocus enzyme electrophoresis, and in addition the net charge calculated did not correlate strongly with electrophoretic mobility. A phylogenetic tree for the sequences that was based on maximum parsimony differed significantly from a tree for the same strains that was based on multilocus enzyme electrophoresis for 35 enzymes (R. K. Selander, D. A. Caugant, and T. S. Whittam, p. 1625-1648, in F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger, ed., Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 1987). These data, together with analysis of sequence variation between the strains, indicated that intragenic recombination and transfer of the whole of gnd have occurred in the evolution of these strains. There is evidence of one recombination event between E. coli and Salmonella typhimurium.

Cloning and expression of a Salmonella enteritidis fimbrin gene in Escherichia coli.

A gene bank of DNA from a human isolate of Salmonella enteritidis was constructed in the cosmid pHC79 in Escherichia coli HB101. Five clones containing 35- to 45-kilobase inserts of S. enteritidis DNA reacted in colony immunoblot assays with a polyclonal antiserum prepared against purified S. enteritidis fimbriae. Electron microscopy showed that none of the five fimbrin-producing clones produced fimbriae, yet radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis located the 14,400-molecular-weight S. enteritidis in the outer membrane fraction of three of the clones and in the periplasmic fraction of all five clones. By using an oligonucleotide probe homologous to the 5' region of the fimbrin structural gene, the fimbrin gene was located on a 5.3-kilobase HindIII fragment. In vitro transcription-translation analysis verified that this HindIII fragment subcloned into plasmid pTZ18R produced unprocessed S. enteritidis fimbrin of molecular weight 16,400. Dot blot hybridization against a selection of strains of the family Enterobacteriaceae indicated a limited distribution of the S. enteritidis fimbrin gene.

Purification and characterization of fimbriae from Salmonella enteritidis.

A human isolate of Salmonella enteritidis which displayed strong pellicle formation during static broth culture and mannose-sensitive hemagglutination produced fimbriae which were morphologically indistinguishable from type 1 fimbriae of members of the family Enterobacteriaceae. Fimbrin was purified to homogeneity, and the apparent molecular weight (Mr, 14,400) was markedly lower than that reported for the type 1 fimbrin of Salmonella typhimurium (Mr, 22,100). This fimbrin contained 40% hydrophobic amino acids and lacked cysteine. The sequence of the N-terminal 64 amino acids was determined, and sequence alignment revealed that although the 18 N-terminal residues of the S. enteritidis molecule shared considerable homology with Escherichia coli and S. typhimurium type 1 fimbrins, the S. enteritidis fimbrin lacked a 6- to 9-residue terminal sequence present in the other type 1 fimbrins and, after residue 18, shared little homology with the E. coli sequence. Antibodies raised to the purified S. enteritidis fimbrin bound to surface-exposed conformational epitopes on the native fimbriae and displayed pronounced serospecificity. These antibodies were used in the isolation of a nonfimbriated Tn10 insertion mutant which was unable to hemagglutinate.