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BACKGROUND - LuxS may function as a metabolic enzyme or as the synthase of a quorum sensing signalling molecule, auto-inducer-2 (AI-2); hence, the mechanism underlying phenotypic changes upon luxS inactivation is not always clear. In Helicobacter pylori, we have recently shown that, rather than functioning in recycling methionine as in most bacteria, LuxS (along with newly-characterised MccA and MccB), synthesises cysteine via reverse transsulphuration. In this study, we investigated whether and how LuxS controls motility of H. pylori, specifically if it has its effects via luxS-required cysteine metabolism or via AI-2 synthesis only.
RESULTS - We report that disruption of luxS renders H. pylori non-motile in soft agar and by microscopy, whereas disruption of mccAHp or mccBHp (other genes in the cysteine provision pathway) does not, implying that the lost phenotype is not due to disrupted cysteine provision. The motility defect of the DeltaluxSHp mutant was complemented genetically by luxSHp and also by addition of in vitro synthesised AI-2 or 4, 5-dihydroxy-2, 3-pentanedione (DPD, the precursor of AI-2). In contrast, exogenously added cysteine could not restore motility to the DeltaluxSHp mutant, confirming that AI-2 synthesis, but not the metabolic effect of LuxS was important. Microscopy showed reduced number and length of flagella in the DeltaluxSHp mutant. Immunoblotting identified decreased levels of FlaA and FlgE but not FlaB in the DeltaluxSHp mutant, and RT-PCR showed that the expression of flaA, flgE, motA, motB, flhA and fliI but not flaB was reduced. Addition of DPD but not cysteine to the DeltaluxSHp mutant restored flagellar gene transcription, and the number and length of flagella.
CONCLUSIONS - Our data show that as well as being a metabolic enzyme, H. pylori LuxS has an alternative role in regulation of motility by modulating flagellar transcripts and flagellar biosynthesis through production of the signalling molecule AI-2.
Furanone metabolites called AI-2 (autoinducer 2), used by some bacterial species for signaling and cell density-regulated changes in gene expression, are made while regenerating S-adenosyl methionine (SAM) after its use as a methyl donor. The luxS-encoded enzyme, in particular, participates in this activated methyl cycle by generating both a pentanedione, which is transformed chemically into these AI-2 compounds, and homocysteine, a precursor of methionine and SAM. Helicobacter pylori seems to contain the genes for this activated methyl cycle, including luxS, but not genes for AI-2 uptake and transcriptional regulation. Here we report that deletion of luxS in H. pylori reference strain SS1 diminished its competitive ability in mice and motility in soft agar, whereas no such effect was seen with an equivalent Delta luxS derivative of the unrelated strain X47. These different outcomes are consistent with H. pylori's considerable genetic diversity and are reminiscent of phenotypes seen after deletion of another nonessential metabolic gene, that encoding polyphosphate kinase 1. We suggest that synthesis of AI-2 by H. pylori may be an inadvertent consequence of metabolite flux in its activated methyl cycle and that impairment of this cycle and/or pathways affected by it, rather than loss of quorum sensing, is deleterious for some H. pylori strains. Also tenable is a model in which AI-2 affects other microbes in H. pylori's gastric ecosystem and thereby modulates the gastric environment in ways to which certain H. pylori strains are particularly sensitive.
LuxS plays a role in the synthesis of an extracellular signaling molecule, autoinducer 2 (AI-2). To analyze a possible role of AI-2 in regulating Helicobacter pylori gene expression, we constructed a panel of transcriptional reporter strains. We show that the expression of H. pylori flaA is growth phase dependent and that flaA transcription increases in association with increased culture density. Mutating the luxS gene eliminates growth-phase-dependent control of flaA, and this growth phase dependence is restored when the luxS mutant strain is complemented with the wild-type luxS gene.
Individual bacteria of numerous species can communicate and coordinate their actions via the production, release, and detection of extracellular signaling molecules. In this study, we used the Vibrio harveyi luminescence bioassay to determine whether Helicobacter pylori produces such a factor. Cell-free conditioned media from H. pylori strains 60190 and 26695 each induced >100-fold-greater luminescence in V. harveyi than did sterile culture medium. The H. pylori signaling molecule had a molecular mass of <10 kDa, and its activity was unaffected by heating to 80 degrees C for 5 min or protease treatment. The genome sequence of H. pylori 26695 does not contain any gene predicted to encode an acyl homoserine lactone synthase but does contain an orthologue of luxS, which is required for production of autoinducer-2 (AI-2) in V. harveyi. To evaluate the role of luxS in H. pylori, we constructed luxS null mutants derived from H. pylori 60190 and 26695. Conditioned media from the wild-type H. pylori strains induced >100-fold-greater luminescence in the V. harveyi bioassay than did conditioned medium from either mutant strain. Production of the signaling molecule was restored in an H. pylori luxS null mutant strain by complementation with a single intact copy of luxS placed in a heterologous site on the chromosome. In addition, Escherichia coli DH5alpha produced autoinducer activity following the introduction of an intact copy of luxS from H. pylori. Production of the signaling molecule by H. pylori was growth phase dependent, with maximal production occurring in the mid-exponential phase of growth. Transcription of H. pylori vacA also was growth phase dependent, but this phenomenon was not dependent on luxS activity. These data indicate that H. pylori produces an extracellular signaling molecule related to AI-2 from V. harveyi. We speculate that this signaling molecule may play a role in regulating H. pylori gene expression.