<i>Helicobacter pylori</i> with separate β‐ and β′‐subunits of RNA polymerase is viable and can colonize conventional mice

Raudonikiene, Aušra; Zakharova, Natalya; Su, Wan; Jeong, Jin Yong; Bryden, Louis; Hoffman, Paul S.; Berg, Douglas E.; Severinov, Konstantin

doi:10.1046/j.1365-2958.1999.01336.x

Cited by 18 publications

(24 citation statements)

References 22 publications

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“…We inserted a chloramphenicol acetyltransferase (cat) cassette lacking a transcriptional terminator (27,42) into the coding sequence of alpA. Polar effects on alpB expression were not expected, based on our own experience and that of other laboratories using the nonpolar cat cassette (27,42).…”

Section: Resultsmentioning

confidence: 99%

“…The alpA::cat strain (⌬AlpAB) was constructed by PCR amplifying DNA from strain 26695m, a motile variant of 26695, using primers omp20F and omp20R (see Table S1 in the supplemental material), cloning the alpA fragment into PstI and XbaI sites of pBluescript KS (yielding pBSalpA) and inserting a nonpolar (lacking transcriptional terminator) chloramphenicol acetyltransferase gene (27,42) into the native EcoRI site, located roughly in the middle (position 755 out of 1,548 nucleotides) of the alpA gene. H. pylori strains 26695m and SS1 were transformed with the plasmid containing the interrupted alpA gene using electroporation.…”

Section: Methodsmentioning

confidence: 99%

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Helicobacter pylori AlpA and AlpB Bind Host Laminin and Influence Gastric Inflammation in Gerbils

et al. 2011

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Helicobacter pylori persistently colonizes humans, causing gastritis, ulcers, and gastric cancer. Adherence to the gastric epithelium has been shown to enhance inflammation, yet only a few H. pylori adhesins have been paired with targets in host tissue. The alpAB locus has been reported to encode adhesins involved in adherence to human gastric tissue. We report that abrogation of H. pylori AlpA and AlpB reduces binding of H. pylori to laminin while expression of plasmid-borne alpA or alpB confers laminin-binding ability to Escherichia coli. An H. pylori strain lacking only AlpB is also deficient in laminin binding. Thus, we conclude that both AlpA and AlpB contribute to H. pylori laminin binding. Contrary to expectations, the H. pylori SS1 mutant deficient in AlpA and AlpB causes more severe inflammation than the isogenic wild-type strain in gerbils. Identification of laminin as the target of AlpA and AlpB will facilitate future investigations of host-pathogen interactions occurring during H. pylori infection.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Helicobacter pylori AlpA and AlpB Bind Host Laminin and Influence Gastric Inflammation in Gerbils

et al. 2011

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“…In addition, in vivo manipulation is unlikely to account for differences in colonization by the mutants. We and others have shown that some H. pylori mutants colonize with an efficiency equal to that of the wild type in spite of in vitro manipulations which were similar to the ones used in this study (6,7,31). In addition, we used pools of mutants and repeated the transformations at least twice to ensure that random genetic events in a single clone would not result in spurious loss of colonization potential.…”

Section: Discussionmentioning

confidence: 96%

In Vivo Complementation of ureB Restores the Ability of Helicobacter pylori To Colonize

et al. 2002

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The objective of this study was to determine (i) if complementation of ureB-negative Helicobacter pylori restores colonization and (ii) if urease is a useful reporter for promoter activity in vivo. Strains used were M6, M6⌬ureB, and 10 recombinant derivatives of M6 or M6⌬ureB in which urease expression was under the control of different H. pylori promoters. Mice were orally inoculated with either the wild type or one of the mutant strains, and colonization, in vivo urease activity, and extent of gastritis were determined. Of eight M6⌬ureB recombinants tested, four colonized mice. Of those, three had the highest in vitro urease activity of any of the recombinants, significantly different from that of the noncolonizing mutants. The fourth colonizing recombinant, with ureB under control of the cag-15 promoter, had in vitro urease activity which did not differ significantly from the noncolonizing strains. In vivo, urease activities of the four colonizing transformants and the wild-type control were indistinguishable. There were no differences in gastritis or epithelial lesions between mice infected with M6 and those infected with the transformants. These results demonstrate that recovery of urease activity can restore colonizing ability to urease-negative H. pylori. They also suggest that cag-15 is upregulated in vivo, as was previously suggested by demonstrating that it is upregulated upon contact with epithelial cells. Finally, our results suggest that total urease activity and colonization density do not contribute to gastritis due to H. pylori.

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“…It is important to note that the stem-loop structure that is just downstream of the open reading frame in many cat cassettes has been removed here. This cassette is considered nonpolar on distal gene expression, because its insertion between DNA segments encoding the ␤ and ␤Ј domains of the large ␤-␤Ј RNA polymerase subunit (normally fused in H. pylori) does not impair growth (44). An fdxA::aphA insertion allele that is probably polar on distal gene expression, because fdxA and aphA are in opposite orientations, was generated similarly by using the aphA cassette from the frxA::aphA allele.…”

Section: Methodsmentioning

confidence: 99%

The fdxA Ferredoxin Gene Can Down-Regulate frxA Nitroreductase Gene Expression and Is Essential in Many Strains of Helicobacter pylori

et al. 2003

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Very few examples of metabolic regulation are known in the gastric pathogen Helicobacter pylori. An unanticipated case was suggested, however, upon finding two types of metronidazole (Mtz)-susceptible strains: type I, in which frxA (which encodes a nitroreductase that contributes to Mtz susceptibility) is quiescent, and type II, in which frxA is well expressed. Here we report that inactivation of the fdxA ferredoxin gene (hp277) in type I strains resulted in high-level frxA expression (in effect, making them type II). However, fdxA null derivatives were obtained from only 6 of 32 type I strains tested that were readily transformed with an frxA::aphA marker. This suggested that fdxA is often essential. This essentiality was overcome in 4 of 20 strains by inactivating frxA, which suggested both that frxA overexpression is potentially deleterious and also that fdxA has additional, often vital roles. With type II strains, in contrast, fdxA null derivatives were obtained in 20 of 23 cases tested. Thus, fdxA is dispensable in most strains that normally exhibit (and tolerate) strong frxA expression. We propose that restraint of frxA expression helps maintain balanced metabolic networks in most type I strains, that other homeostatic mechanisms predominate in type II strains, and that these complex results constitute a phenotypic manifestation of H. pylori's great genetic diversity.

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Helicobacter pylori with separate β‐ and β′‐subunits of RNA polymerase is viable and can colonize conventional mice

Cited by 18 publications

References 22 publications

Helicobacter pylori AlpA and AlpB Bind Host Laminin and Influence Gastric Inflammation in Gerbils

Helicobacter pylori AlpA and AlpB Bind Host Laminin and Influence Gastric Inflammation in Gerbils

In Vivo Complementation of ureB Restores the Ability of Helicobacter pylori To Colonize

The fdxA Ferredoxin Gene Can Down-Regulate frxA Nitroreductase Gene Expression and Is Essential in Many Strains of Helicobacter pylori

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