FUNCTIONAL AND GENETIC-CHARACTERIZATION OF MCPC, WHICH ENCODES A 3RD METHYL-ACCEPTING CHEMOTAXIS PROTEIN IN BACILLUS-SUBTILIS

A 3135 bp DNA segment downstream of the spl gene on the Bacillus subti lis chromosome was cloned and its nucleotide sequence determined. An o pen reading frame capable of encoding a putative protein of 654 amino acids with a calculated molecular mass of 72.1 kDa was identified. The deduced amino acid sequence was similar to the McpA and McpB proteins of B. subtilis. McpA and McpB encode different methyl-accepting chemo taxis proteins (MCPs). A mutant strain containing an antibiotic resist ance DNA cassette inserted into the region containing the MCP-like rea ding frame suffered a complete loss of taxis to the amino acids cystei ne, proline, threonine, glycine, serine, lysine, valine and arginine. The open reading frame was designated mcpC. The wildtype and an mcpC m utant strain were analysed for their content of methylated proteins an d it was found that mcpC encodes a methylated membrane protein that ha s previously been designated H3. These results show that mcpC encodes a third MCP in B. subtilis. The transcription start site upstream of t he mcpC gene was determined by primer extension analysis and it was fo und to be preceded by a potential promoter sequence that is recognized by the sigma(D) form of RNA polymerase. The level of beta-galactosida se expressed from a transcriptional mcpC-lacZ fusion was increased thr eefold when cells entered the stationary phase. No beta-galactosidase could be detected in a sigD genetic background.