The metC gene of Escherichia coli K-12 was cloned and the nucleotide sequence of the metC gene and its flanking regions was determined. The translation initiation codon was identified by sequencing the NH2-terminal part of 13-cystathionase, the MetC gene product. The meIC gene (1185 nucleotides) encodes a protein having 395 amino acid residues.The 5' noncoding region was found to contain a "Met box" homologous to sequences suggestive of operator structures upstream from other methionine genes that are controlled by the product of the pleiotropic regulatory metJ gene. The deduced amino acid sequence of (3-cystathionase showed extensive homology with that of the MetB protein (cystathionine y-synthase) that catalyzes the preceding step in methionine biosynthesis. The homology strongly suggests that the structural genes for the MetB and MetC proteins evolved from a common ancestral gene.

A total of 46 Borrelia burgdorferi sensu lato isolates that were isolated from patients with Lyme borreliosis and infected animals or were extracted from ticks of the genus lxodes were analyzed. Large restriction fragment patterns obtained after cleavage of genomic DNAs with Mlul were analyzed by pulsed-field gel electrophoresis (PFGE). To eliminate the contribution of plasmid DNA, only frmMents greater than 70 kb were used for the analysis. The results indicated that each of the 14 B. burgdorferi sensu stricto isolates were recognized by a band at 135 kbp, each of the 12 Borrelia garinii isolates by two bands (220 and 80 kbp), and each of the 20 Borrelia afzelii isolates by three bands (460, 320, and 90 kbp). Whereas differences in the PFGE patterns among B. burgdorferi sensu stricto isolates and B. garinii isolates were noted, B. afzelii isolates were all similar. Identification of isolates by PFGE correlates with their belonging to a given species within B.

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The Leptospira meyeri serovar semaranga metX gene was identified by complementation of an Escherichia coli metA mutant, i.e., devoid of homoserine O-succinyltransferase. However, the MetX protein exhibited a homoserine O-acetyltransferase activity in agreement with its similarity to homoserine O-acetyltransferases. Reverse transcription-PCR analysis demonstrated that metX is the second gene of an operon.The first step of the biosynthetic pathway leading to methionine is esterification of homoserine (6). However, the precursor differs according to the organism. In members of the family Enterobacteriaceae, O-succinyl-homoserine is formed by acylation of homoserine in the presence of O-succinyl-coenzyme A, catalyzed by homoserine O-succinyltransferase, the metA gene product (7,18). In gram-positive bacteria of the genus Bacillus and in fungi, esterification of homoserine occurs by an acetylation (3, 9, 13) catalyzed by a homoserine O-acetyltransferase.The genus Leptospira consists of two nomenspecies, Leptospira interrogans sensu lato (pathogenic) and Leptospira biflexa sensu lato (nonpathogenic), which belong to the order Spirochaetales (4). Previous studies indicated that Leptospira spp. synthesize most of their amino acids by the same biosynthetic pathways as those used by Escherichia coli (5).Our interest in methionine biosynthesis in Leptospira arose from the fact that radiotracer studies of biosynthetic pathways did not include any data for the methionine pathway (5). In this paper, we describe isolation and transcriptional organization of the metX gene, as well as the enzymatic characterization and regulation of its product, homoserine O-acetyltransferase.(This work represents a portion of a thesis submitted by P. Bourhy to the University of Paris VII, Paris, France, for the Ph.D.)Cloning of a DNA fragment from Leptospira meyeri which can complement an E. coli metA mutant. We had previously constructed a recombinant cosmid library and cloned the Leptospira metY gene, which complemented E. coli metB mutants (1). The size of the cloned DNA fragment (25 kbp) in the pb10 recombinant cosmid suggested that other methionine biosynthetic genes might be found on this fragment. In order to test this hypothesis, E. coli RC709 (metF63 pro-22; R. Clowes) and AB1932 (metA28 argH1 thi-1 lacY1 lacZ4 galK2 xyl-4 [or -5] tsx-6 F Ϫ ; E. A. Adelberg) were used for complementation experiments in supplemented M9 minimal medium (20) at 30°C. Both strains were electroporated with pb10. pb10 weakly complemented metA but not metF mutants. Further subcloning experiments allowed us to locate the Leptospira DNA able to complement E. coli metA and metB mutants more precisely within a 6.8-kbp fragment. Plasmid pb12 carrying a 6.8-kbp PstI fragment from pb10 in the pBR322 vector (22) still complemented E. coli metB and metA mutants (Fig.

A gene library of the Leptospira meyeri serovar semaranga strain Veldrat S.173 DNA has been constructed in a mobilizable cosmid with inserts of up to 40 kb. It was demonstrated that a Leptospira DNA fragment carrying metYcomplemented Escherichia coli strains carrying mutations inmetB. The latter gene encodes cystathionine γ-synthase, an enzyme which catalyzes the second step of the methionine biosynthetic pathway. The metY gene is 1,304 bp long and encodes a 443-amino-acid protein with a molecular mass of 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of theLeptospira metY product has a high degree of similarity to those of O-acetylhomoserine sulfhydrylases fromAspergillus nidulans and Saccharomyces cerevisiae. A lower degree of sequence similarity was also found with bacterial cystathionine γ-synthase. The L. meyeri metY gene was overexpressed under the control of the T7 promoter. MetY exhibits an O-acetylhomoserine sulfhydrylase activity. Genetic, enzymatic, and physiological studies reveal that the transsulfuration pathway via cystathionine does not exist in L. meyeri, in contrast to the situation found for fungi and some bacteria. Our results indicate, therefore, that the L. meyeri MetY enzyme is able to perform direct sulfhydrylation for methionine biosynthesis by using O-acetylhomoserine as a substrate.