Brian Noonan

Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori

Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the hosts life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organisms ability to cause different diseases or even be beneficial to the infected host, and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.

Molecular analysis of an A-protein secretion mutantof Aeromonas salmonicida reveals a surface layer-specific protein secretion pathway

The Aeromonas salmonicida Tn5 mutant, A449-TM1, is unable to secrete the surface layer protein (A-protein) through the outer membrane. Immunogold labeling of thin sections of A449-TM1, with polyclonal antisera against the A-protein, showed the accumulation of large quantities of A-protein in an enlarged periplasm. The majority of the labeled A-protein could be seen at the poles of the cells. The ability of A449-TM1 to secrete other extracellular proteins such as hemolysin and protease was not impaired by the Tn5 insertion, which indicates that the mutation in A449-TM1 interferes with a secretion pathway specifically for the translocation of the A-protein through the outer membrane. The mutant, A449-TM1, was shown to be avirulent for fish. A cosmid clone from a gene library of A449-TM1, which contains the Tn5 insertion from the chromosome, was used to identify a 1.4 kb SaII/ClaI fragment from immediately adjacent to the Tn5 insertion. This fragment was used to identify and clone a 4 kb HindIII fragment from a chromosomal DNA digest from the wild-type strain, A449. DNA sequence analysis of this clone identified an open reading frame (ORF) of 1656 bp. The deduced product of this ORF showed sequence similarity to a family of ATP-binding secretion proteins, but appeared to be phylogenetically distinct from these proteins, consistent with its participation in a secretory pathway specific for surface layer protein.

The leucine zipper of Aeromonas salmonicida AbcA is required for the transcriptional activation of the P2 promoter of the surface-layer structural gene, vapA, in Escherichia coli

The Aeromonas salmonicida AbcA protein is involved in the synthesis of the O‐polysaccharide side‐chains on the lipopolysaccharide and is also capable of enhancing the expression of the structural gene for the A‐layer, vapA, when cloned into Escherichia coli. The P2 promoter of the vapA gene of A. salmonicida was cloned into a promoter probe vector and expression in E. coli was monitored. The expression of P2::lacZ was shown to be increased when abcA was provided in trans. AbcA contains an N‐terminal ATP‐binding domain as well as a C‐terminal leucine zipper domain. Site‐directed mutagenesis has been used to show that the ATP‐binding domain is required for the synthesis of the O‐polysaccharide side‐chains, but not for the enhancement of vapA expression. Conversely, the leucine zipper is needed for the increase in vapA expression, but not for O‐polysaccharide side‐chain synthesis. This indicates that AbcA is a bifunctional protein that can influence the synthesis of the two principle antigenic components of the A. salmonicida cell surface.

The molecular biology of Aeromonas salmonicida

Abstract The fish pathogen, Aeromonas salmonicida, has been the focus of a number of molecular genetic studies designed to characterize the microorganism and its pathogenesis. The paracrystalline surface protein layer (A-layer) of A. salmonicida has been studied in considerable detail. The A-layer gene, vapA, has been cloned and sequenced and studies have been performed on its regulation. The secretion pathway specific for the A-layer subunits has also been partially characterized as has the general protein secretion pathway. Other genes involved in the biogenesis of the A. salmonicida surface include abcA, asoA and asoB. The abcA gene encodes a protein which is involved in lipopolysacharide O-chain synthesis and secretion and may have a role in the regulation of vapA gene expression. A. salmonicida also possesses plasmids of various sizes which exhibit a high degree of conservation and can encode antibiotic resistance elements. Insertion sequence elements have been identified in two strains of A. salmonicida and are capable of transposing within a cell to cause mutations that affect virulence. Molecular biology techniques have also been applied to the problem of detection of low levels of A. salmonicida in natural environments and carrier fish. The development of tools such as specific DNA probes and PCR primer pairs allows the detection of extremely low numbers of A. salmonicida even in the presence of high numbers of other bacteria. The development of vaccines against A. salmonicida has incorporated some new techniques such as the generation of specific mutations in the chromosome or the production of large quantities of particular proteins, such as the outer membrane porins, in expression systems. Another approach involves the use of an avirulent A. salmonicida strain as a shuttle system to express fragments of genes from viral pathogens with a view to providing protective immunity against multiple diseases with a single vaccine.

Molecular characterization of an Aeromonas salmonicida mutant with altered surface morphology and increased systemic virulence

The asoA gene of Aeromonas salmonicida is located approximately 7 kb downstream of the A‐layer structural gene, vapA. A 6 kb Bam HI fragment containing aso A was cloned and marker‐exchange mutagenesis using a kanamycin‐resistance cassette was performed to generate an aso A mutation in the low‐virulence strain A449L. When analysed by electron microscopy, the mutant A449L‐MB exhibited an altered surface morphology. Strands and blebs of membranous material were observed protruding from the disorganized cell surface. This material was shown to contain lipopolysaccharide and A‐layer subunit protein. The disorganization of the surface of A449L‐IV1B had no apparent effect on virulence when the bacteria were administered to rainbow trout (Oncorhynchus mykiss) by bath Immersion. However, when administered by intraperitoneal injection, the mutant A449L‐MB was found to exhibit significantly increased virulence. The predicted amino acid sequence of AsoA shows homology to a number of polytopic membrane proteins involved in translocation across the cytoplasmic membrane.

An Aeromonas salmonicida gene required for the establishment of infection in rainbow trout (Oncorhynchus mykiss).

The asoB gene of Aeromonas salmonicida is located approximately 9 kb downstream of the structural gene (vapA) for the surface layer (A-layer). The nucleotide sequence of asoB was determined and found to encode a putative polytopic cytoplasmic membrane protein which exhibited homology to a number of bacterial transport proteins. Allele exchange mutagenesis of asoB resulted in a mutant (A449-D) which was avirulent when administered by bath immersion. However, when administered by intraperitoneal injection, A449-D is as lethal as wild type. Characterization of the phenotype of A449-D showed that there were pleiotropic effects on VapA secretion, haemolysis and outer membrane protein composition. Mobilization of cloned asoB on a broad-host-range plasmid into A449-D resulted in the complementation of VapA translocation, haemolytic activity and virulence.

Erratum: Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori: Correction

This corrects the article DOI: 10.1038/16495

Localisation and Cloning of Genes Involved in the Export of the A-Protein of Aeromonas salmonicida

The paracrystalline surface protein array of Aeromonas salmonicida is composed of tetragonally arranged subunits of Mr = 50800 (Chu et al., 1991). The gene encoding the A-protein has recently been cloned (Belland and Trust, 1987) and sequenced (Chu et al., 1991), and studies on its 3 dimensional structure have been performed (Dooley et al., 1989). The A-layer is involved in the ability of A. salmonicida to produce disease in fish and it appears to protect the bacteria from the bacteriocidal effect of immune and non-immune serum (Ishiguro et al., 1981; Munn et al., 1982) as well as from phagocytic cells (Trust et al., 1983).