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.
We present biochemical and genetic data to support the hypothesis that the Escherichia coli met repressor, MetJ, binds to synthetic and natural operator sequences in tandem arrays such that repression depends not only on the affinity of the DNA-protein interaction, but also on protein-protein contacts along the tandem array. This represents a novel form of regulatory switch. Furthermore, there seems to be homology between the organization of the met and trp operators.
Leptospira interrogans is a pathogenic bacterium with a low G+C content (34 to 39%). The restriction enzymes NotI, AscI, and SrfI cut the chromosome of L. interrogans serovar icterohaemorrhagiae into 13, 3, and 5 fragments separable by one- and two-dimensional pulsed-field gel electrophoresis (PFGE). The genome is composed of a circular 4.6-Mbp chromosome and a 0.35-Mbp extrachromosomal element. A physical map of the chromosome was constructed for NotI, AscI, and SrfI by using single and double digests, or partial NotI digests obtained at random or by cross-protection of NotI sites by FnuDII methylase, and linking clones. rRNA genes were found to be widely scattered on the chromosome.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.