15661014

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3

In Listeria monocytogenes the promoter region of the svpA-srtB locus contains a well-conserved Fur box. We characterized the iron-regulation of this locus: real-time polymerase chain reaction analyses and anti-SvpA immunoblots showed that, in response to iron deprivation svpA transcription and SvpA production markedly increased (80-fold and 10-fold respectively), when initiated by either the addition of the iron chelator 2,2'-bipyridyl to BHI media, or by growth in iron-restricted minimal media. Green fluorescent protein (GFP) reporter constructs also showed increased activity of the svpA-srtB promoter in Escherichia coli (37-fold) and in L. monocytogenes (two- to threefold) when the bacteria were grown in iron-deficient conditions. A Deltafur mutant of L. monocytogenes constitutively synthesized SvpA, as well as GFP fused to the svpA-srtB promoter. Cellular fractionation data revealed that in iron-rich media wild-type SvpA was exclusively secreted to the culture supernatant. However, both the Deltafur derivative and wild-type L. monocytogenes grown in iron-deficient media anchored a fraction of the SvpA proteins (approximately 5%) to peptidoglycan, and produced a lower-molecular weight, wholly secreted form of SvpA. Together these data establish that iron availability controls transcription of the svpA-srtB locus (through Fur-mediated regulation), and attachment of SvpA to the cell wall (through SrtB-mediated covalent linkage). SvpA bears homology to IsdC, a haemin-binding protein of Staphylococcus aureus, and haemin bound to SvpA in solution. However, site-directed deletions of four structural genes and the promoter of the svpA-srtB locus did not impair haemin, haemoglobin or ferrichrome utilization in nutrition tests. We did not find strong evidence to support the notion that the svpA-srtB locus participates in haemin acquisition, as was reported for the homologous isd operon of S. aureus. Furthermore, the svpA-srtB mutant strains showed no significant attenuation of virulence in an intravenous mouse model system, but we found that the mutations reduced the persistence of L. monocytogenes in murine liver, spleen and intestines after oral administration.

eng

IM

MEDLINE

0950-382X

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NLM

927-40

pubmed-meshheading:15661014-Aminoacyltransferases, pubmed-meshheading:15661014-Animals, pubmed-meshheading:15661014-Bacterial Proteins, pubmed-meshheading:15661014-Base Sequence, pubmed-meshheading:15661014-Caco-2 Cells, pubmed-meshheading:15661014-Cell Line, pubmed-meshheading:15661014-Culture Media, pubmed-meshheading:15661014-Cysteine Endopeptidases, pubmed-meshheading:15661014-Disease Models, Animal, pubmed-meshheading:15661014-Female, pubmed-meshheading:15661014-Gene Deletion, pubmed-meshheading:15661014-Gene Expression Regulation, Bacterial, pubmed-meshheading:15661014-Humans, pubmed-meshheading:15661014-Iron, pubmed-meshheading:15661014-Listeria monocytogenes, pubmed-meshheading:15661014-Listeriosis, pubmed-meshheading:15661014-Membrane Proteins, pubmed-meshheading:15661014-Mice, pubmed-meshheading:15661014-Mice, Inbred BALB C, pubmed-meshheading:15661014-Molecular Sequence Data, pubmed-meshheading:15661014-Repressor Proteins, pubmed-meshheading:15661014-Virulence

The svpA-srtB locus of Listeria monocytogenes: fur-mediated iron regulation and effect on virulence.

Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural