MOLECULAR-CLONING, SEQUENCING, AND EXPRESSION OF A CHEMORECEPTOR GENEFROM LEPTOSPIRILLUM FERROOXIDANS

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferroox idans. This DNA contained an open reading frame for a 577-amino-acid p rotein 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, fe rrooxidans encoding a protein. The lcrI gene showed both sigma(28)-lik e and sigma(70)-like putative promoters. The LcrI deduced protein cont ained two hydrophobic regions most likely corresponding to the two tra nsmembrane regions present in all of the methyl-accepting chemotaxis p roteins (MCPs) which make them fold with both periplasmic and cytoplas mic domains. We have proposed a cytoplasmic domain for LcrI, which als o 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 sh owed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein tan MCP from Escherichia coli), co nfirming some degree of antigenic conservation. In addition, this 58-k Da protein was expressed in E, coli, being associated with its cytopla smic 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 al low the right conformation for the LcrI periplasmic domain.