Cloning and analysis of the leuB gene of Leptospira interrogans serovar pomona

Ding, Min; Yelton, David B.

doi:10.1099/00221287-139-5-1093

Cited by 15 publications

(7 citation statements)

References 19 publications

(23 reference statements)

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“…First, except for CimA, the enzymes of leucine biosynthesis and isoleucine biosynthesis via the pyruvate pathway are shared. Second, the enzymes of leucine biosynthesis in L. interrogans have the same function as their E. coli counterparts, as confirmed by previous work (12). We first confirmed that the cimA gene of L. interrogans was able to reverse the isoleucine requirement of an E. coli ilvA mutant, AB1255.…”

Section: Discussionsupporting

confidence: 75%

Isoleucine Biosynthesis in Leptospira interrogans Serotype lai Strain 56601 Proceeds via a Threonine-Independent Pathway

Zhang

Guo

et al. 2004

J Bacteriol

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Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded ␣-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower K m (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed ␤-methyl-D-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and ␤-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.

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Section: Discussionsupporting

confidence: 75%

Isoleucine Biosynthesis in Leptospira interrogans Serotype lai Strain 56601 Proceeds via a Threonine-Independent Pathway

Zhang

Guo

et al. 2004

J Bacteriol

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“…In fact, there is an inherent contradiction in this approach-genes are cloned from exotic organisms to discover new motifs in biology, and yet these genes are required to be expressed in Escherichia coli or another domesticated bacterium in order to be detected. The diversity of the organisms whose DNA has been successfully expressed in E. coli is surprising (16,33,45,57,(118)(119)(120), but heterologous expression remains a barrier to extracting the maximum information from functional metagenomics analyses.…”

Section: Functional Metagenomics Heterologous Expressionmentioning

confidence: 99%

Metagenomics: Application of Genomics to Uncultured Microorganisms

Handelsman

2004

Microbiol Mol Biol Rev

1,950

1,038

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Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na+(Li+)/H+ antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies

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“…Several amino acid biosynthetic genes have been cloned from L. interrogans (sensu stricto) (3,10,35). These genes were localized on the physical maps of both serovars.…”

Section: Resultsmentioning

confidence: 99%

Comparison of genetic maps for two Leptospira interrogans serovars provides evidence for two chromosomes and intraspecies heterogeneity

1993

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Genetic maps were constructed for Leptospira interrogans serovars icterohaemorrhagiae and pomona. Previously we independently constructed physical maps of the genomes for these two serovars. The genomes of both serovars consist of a large replicon (4.4 to 4.6 Mb) and a small replicon (350 kb). Genes were localized on the physical maps by using Southern blot analysis with specific probes. Among the probes used were genes encoding a variety of essential enzymes and genes usually found near bacterial chromosomal replication origins. Most of the essential genes are on the larger replicon of each serovar. However, the smaller replicons of both serovars contain the asd gene. The asd gene encodes aspartate 0-semialdehyde dehydrogenase, an enzyme essential in amino acid and cell wall biosyntheses. The finding that both L. interrogans replicons contain essential genes suggests that both replicons are chromosomes. Comparison of the genetic maps of the larger replicons of the two serovars showed evidence of large rearrangements. These data show that there is considerable intraspecies heterogeneity in L. interrogans.Leptospirosis is an anthropozoonose, a disease common among most animal species, including humans. Leptospirosis is caused by pathogenic members of the genus Leptospira. Until recently, all pathogenic leptospires were classified within the same species, Leptospira interrogans (sensu lato). Recent DNA homology studies have resulted in reclassification of L. interrogans with the formation of seven and L. weilii (34, 44). Because of extensive antigenic heterogeneity, each species is further divided according to antigenic composition into serogroups and serovars. Some serovars occur in more than one of the newly formed species (e.g., serovar hardjo is found in L. interrogans [sensu stricto] and L.

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Cloning and analysis of the leuB gene of Leptospira interrogans serovar pomona

Cited by 15 publications

References 19 publications

Isoleucine Biosynthesis in Leptospira interrogans Serotype lai Strain 56601 Proceeds via a Threonine-Independent Pathway

Isoleucine Biosynthesis in Leptospira interrogans Serotype lai Strain 56601 Proceeds via a Threonine-Independent Pathway

Metagenomics: Application of Genomics to Uncultured Microorganisms

Comparison of genetic maps for two Leptospira interrogans serovars provides evidence for two chromosomes and intraspecies heterogeneity

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