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BackgroundStrains of Helicobacter pylori carrying the virulence associated cag pathogenicity island (PAI) induce gastric epithelial synthesis of the chemokine interleukin-8 (IL-8), a neutrophil chemoattractant, and thereby a strong inflammatory response during chronic infection of the human gastric mucosa. Previous mutational analyses have shown that many genes in the cag PAI are needed to elicit IL-8 synthesis in gastric epithelial cells, and also that some genes are not involved.AimTo test the possibility that certain genes in the cag PAI also downregulate (modulate) the inflammatory response elicited by cag+ H pylori infection.MethodsCells of L5F11, a derivative of the Kato-3 gastric epithelial cell line that carries an engineered IL-8 promoter-luciferase reporter gene fusion, were cocultured with H pylori strain 26695 or with an isogenic mutant in which most of the cag PAI ORF 10 gene, an Agrobacterium virD4 homologue, was deleted. Luciferase activity was measured to assess IL-8 gene transcription and secreted IL-8 was measured by enzyme linked immunosorbent assay to assess synthesis and release of IL-8 protein from gastric epithelial cells.ResultsInactivation of ORF10 led to a 2.8-fold increase in IL-8 gene transcription and a 3.6-fold increase in IL-8 synthesis and secretion.ConclusionsThe results suggest that this VirD4 homologue participates in the control of inflammation that H pylori infection elicits by downregulating (modulating) the strong induction of IL-8 synthesis mediated by other cag encoded proteins.

We demonstrate here that the arbitrary primer polymerase-chain-reaction-based DNA fingerprinting method (also termed random amplified polymorphic DNA or RAPD) can be used to distinguish among strains of the avian pathogen Mycoplasma gallisepticum. Ten base oligonucleotide primers were used individually to prime DNA synthesis from genomic DNAs. Strain-specific arrays of DNA fragments were generated, which allowed us to identify and group isolates. Isolates of M. synoviae, M. gallinarumand M. inersyielded arrays of DNA fragments that differed markedly from those generated from the M. gallisepticumisolates using the same arbitrary primers. These results show that the RAPD fingerprinting method distinguishes genetically different strains of M. gallisepticumand indicates that it should be valuable for monitoring transmission of this pathogen.

We have developed a strategy for efficient sequence analysis of the genome of E.coli K-12 using insertions of a Tn5-derived mini-transposon into overlapping ordered λ phage clones to provide universal primer-binding sites, and PCR amplification of DNA segments adjacent to the insertions. Transposon-containing clones were selected by blue plaque formation on dnaBamber IacZamber E.coli strain. Insertion points every 0.5–1 kb were identified by ‘analytical PCR’ and segments between the transposon inserts and phage arms were amplified by ‘preparative PCR’ using one biotinylated and one non-biotinylated primer. Single strands of amplified DNA fragments were coupled to Streptoavidin-coated paramagnetic beads (Dynabeads M280) through their biotin tails, purified magnetically, and used as templates for fluorescence-based automatic nucleotide sequencing.