Identification of the cobalamin-dependent methionine synthase gene, metH, in Vibrio fischeri ATCC 7744 by sequencing using genomic DNA as a template.
To confirm the presence of cobalamin-dependent methionine synthase (CDMS) in luminous bacteria, which is a prerequisite for the substantiation of our proposals on the physiological function of the lux operon, we identified the CDMS gene (metH) in Vibrio fischeri ATCC 7744. Two partial metH sequences, one located near the 5'-terminus of the gene and the other near the 3'-terminus, were sequenced by a PCR based method. To design a new set of PCR primers located on the two flanking regions of the gene, the genomic DNA was sequenced by SUGDAT method (sequencing using genomic DNA as a template) upstream or downstream from the respective partial gene sequences. Subsequently a 4.2 kb DNA fragment containing the whole metH was amplified by PCR and sequenced. The number of amino acid residues comprising the protein (1226 amino acids) was comparable to those of known CDMSs. The deduced amino acid sequence showed 85, 74, 55, 31, 30, 52, or 52% identity with that of Vibrio cholerae, Escherichia coli, Deinococcus radiodurans, Synechocystis PCC6803, Mycobacterium tuberculosis, Caenorhabditis elegans or Homo sapiens, respectively. All the predicted amino acid residues for the binding of cobalamin and S-adenosylmethionine were conserved. In the regulatory region of the V. fischeri metH, the binding site of the met repressor, MetJ, was present, although the site is atypically not present in E. coli metH or Salmonella typhimurium metH. It was shown that nucleotide sequences, even long ones, can be determined without a cloning step, if only parts of the DNA fragment to be sequenced are amplified by PCR.
Kasai
S
S
Department of Bioapplied Chemistry, Faculty of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan. kasai@bioa.eng.osaka-cu.ac.jp
Yamazaki
T
T
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D004269:DNA, Bacterial; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase