Molecular cloning, sequencing, and expression of a chemoreceptor gene from Leptospirillum ferrooxidans.
We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both sigma 28-like and sigma 70-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.
Delgado
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M
Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
Toledo
H
H
Jerez
C A
CA
D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics*; D001426:Bacterial Proteins; D002628:Chemoreceptor Cells / Q000502:physiology*; D002633:Chemotaxis; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics*; D008565:Membrane Proteins / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins