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LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response
G V Plano1, S S Barve, S C Straley
1Department of Microbiology and Immunology, Chandler Medical Center, University of Kentucky, Lexington 40536.
Abstract:
Yersinia pestis, the etiologic agent of bubonic plague, contains a 75-kb virulence plasmid, called pCD1 in Y. pestis KIM. The low-Ca(2+)-response genes of Y. pestis regulate both bacterial growth and the expression of pCD1-encoded virulence determinants in response to temperature and the presence of Ca2+ or nucleotides. This study characterizes the nucleotide sequence and protein product of the lcrD locus. An lcrD mutant, in contrast to the parent Y. pestis, did not undergo growth restriction or induce strong expression of the V antigen when grown under conditions (37 degrees C, no Ca2+) expected to elicit maximal expression of pCD1 genes. DNA sequence analysis of the cloned lcrD locus showed a single open reading frame that could encode a protein with a molecular weight of 77,804 and a pI of 4.88. LcrD was identified as a 70-kDa inner membrane protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. LcrD membrane topology was investigated by using lcrD-phoA translational fusions generated with the transposon TnphoA. The alkaline phosphatase activities of the resultant hybrid proteins were consistent with a model predicting eight amino-terminal transmembrane segments that anchor a large cytoplasmic carboxyl-terminal domain to the inner membrane.
Insights
Yersinia pestis LcrD is an inner membrane protein crucial for regulating bacterial growth and virulence gene expression. Its absence prevents growth restriction and V antigen induction under specific conditions.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Pathogenesis
Background:
- Yersinia pestis causes bubonic plague and possesses a virulence plasmid (pCD1).
- Low-Ca(2+)-response genes in Y. pestis control bacterial growth and virulence factor expression based on environmental cues.
- The lcrD locus is involved in regulating these virulence factors.
Purpose of the Study:
- To characterize the nucleotide sequence and protein product of the lcrD locus in Yersinia pestis.
- To understand the role of LcrD in regulating bacterial growth and virulence gene expression.
Main Methods:
- DNA sequence analysis of the cloned lcrD locus.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis to identify LcrD protein.
- lcrD-phoA translational fusions with transposon TnphoA to determine membrane topology.
Main Results:
- A single open reading frame in the lcrD locus was identified, encoding a protein of 77,804 Da.
- LcrD was confirmed as a 70-kDa inner membrane protein.
- An lcrD mutant exhibited impaired growth restriction and reduced V antigen expression under conditions favoring virulence gene expression.
- Membrane topology studies suggested eight amino-terminal transmembrane segments anchoring a cytoplasmic carboxyl-terminal domain.
Conclusions:
- LcrD is a critical inner membrane protein regulating Yersinia pestis growth and virulence.
- The LcrD protein structure, with multiple transmembrane segments, is essential for its function in the bacterial cell envelope.
- Understanding LcrD function provides insights into Yersinia pestis pathogenesis and potential therapeutic targets.