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Molecular characterization of flgM, a gene encoding a negative regulator of flagellin synthesis in Salmonella typhimurium.

ABSTRACT: The expression of flagellin in Salmonella typhimurium is coupled to the assembly of complete flagella. Mutations which disrupt this coupling define a gene, flgM, which represses the expression of the flagellin genes in strains with mutations in the basal body, switch, or hook flagellar gene (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:2301-2310, 1991). Complementation studies demonstrated that the flgM gene is contained within a 600-bp cloned DNA fragment. Sequence analysis revealed that this fragment carries a small open reading frame corresponding to a 97-amino-acid protein. The FlgM protein observed in a T7-mediated expression system showed an apparent molecular mass of 9.5 kDa, similar to the predicted size of 10.6 kDa. Upstream of the flgM coding region is a putative promoter sequence which shows strong homology to that thought to be recognized by the flagellin-specific sigma factor (FliA). Consistent with the use of this promoter in vivo, promoter mapping by primer extension demonstrated a transcriptional start site 11 bases downstream from the center of the putative -10 promoter element, which was dependent on functional FliA for full expression.

SUBMITTER: Gillen KL

PROVIDER: S-EPMC208980 | biostudies-other | 1991 Oct

REPOSITORIES: biostudies-other

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Molecular characterization of flgM, a gene encoding a negative regulator of flagellin synthesis in Salmonella typhimurium.

Gillen K L KL Hughes K T KT

Journal of bacteriology 19911001 20

The expression of flagellin in Salmonella typhimurium is coupled to the assembly of complete flagella. Mutations which disrupt this coupling define a gene, flgM, which represses the expression of the flagellin genes in strains with mutations in the basal body, switch, or hook flagellar gene (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:2301-2310, 1991). Complementation studies demonstrated that the flgM gene is contained within a 600-bp cloned DNA fragment. Sequence analysis revealed that th ...[more]

PMID: 1655712

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Project description:Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of KmNa (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.

Dataset Information

Molecular characterization of flgM, a gene encoding a negative regulator of flagellin synthesis in Salmonella typhimurium.

Publications

Molecular characterization of flgM, a gene encoding a negative regulator of flagellin synthesis in Salmonella typhimurium.

Similar Datasets

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