Cloning of dapD, aroD and asd of Leptospira interrogans serovar icterohaemorrhagiae, and nucleotide sequence of the asd gene

Baril, C; Richaud, Catherine; Fournié, Edith; Baranton, Guy; Saint, Girons Isabelle

doi:10.1099/00221287-138-1-47

Cited by 21 publications

(10 citation statements)

References 26 publications

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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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“…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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“…In the first genetic studies carried out in the 1990s, several Leptospira genes were isolated by the functional complementation of E. coli mutants. This method led to the identification of the L. biflexa recA gene 70 , the L. interrogans rfb genes 71 , and a number of amino acid biosynthesis genes, such as asd and trpE 72, 73 .…”

Section: Genetic Tools For Leptospiramentioning

confidence: 99%

Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen

2009

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Leptospirosis is a zoonotic disease which has emerged as a major cause of morbidity and mortality among impoverished populations. One centenary after the discovery of the causative spirochaetal agent, little is understood of Leptospira pathogenesis, which in turn has hampered the identification of new intervention strategies to address this neglected disease. However the recent availability of complete genome sequences for Leptospira and discovery of genetic tools to transform the pathogen has led to major insights into the biology and pathogenesis of this pathogen. We discuss the life cycle of the bacterium and the new advances that have been made and their implications for the future prevention of this disease.

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“…Alternatively, hybrid derivatives of pTD1 containing inserts of foreign (e.g., from E. coli) DNA should yield readily observable high-molecular-weight tandem multimers (5). The presence of a plasmid with ssDNA features suggests that expression of spirochete genes, previously reported to be poor in E. coli (1,12,17,22), may 220…”

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confidence: 99%

Homology of a plasmid from the spirochete Treponema denticola with the single-stranded DNA plasmids

1992

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The 2,647-bp nucleotide sequence of cryptic plasmid pTD1, isolated from the oral spirochete Treponema denticola, was determined. The sequence revealed two open reading frames, A and B, which encode polypeptides of 335 and 235 amino acids, respectively. Open reading frame A shows sequence similarity to genes that encode replication proteins from a group of plasmids common in gram-positive bacteria, which replicate via a single-stranded intermediate.The order Spirochaetales comprises two families of morphologically similar but otherwise diverse bacteria (9, 16); these include species that are pathogenic for both humans and animals. Despite their medical significance, genetic manipulation of these organisms is still at a preliminary level, in part because of the lack of any known system of genetic transfer. The identification of a small, circular 2.6-kb plasmid, pTD1, in the cultivable oral spirochete Treponema denticola raised the possibility of a plasmid-based genetic transfer system within this group of bacteria (8). As the first step in the development of pTD1 as a shuttle vector, we determined its nucleotide sequence to distinguish between essential regions required for replication and nonessential areas that would be suitable as cloning sites. This is the first reported sequence of an extrachromosomal element from within this genus.Identification of proteins encoded by pTDI. Gene expression in pTD1 was demonstrated in a cell-free prokaryotic DNA-directed in vitro transcription-translation assay, based on a 30S ribosomal Escherichia coli extract (Amersham, France SA). The gene products were identified by autoradiography of incorporated L-[35S]methionine (37 TBq/mmol; Amersham, France SA), after conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 10% discontinuous system (11), and this revealed the presence of four major protein bands. In comparison to molecular size markers, these corresponded to 32, 28, 25, and 21 kDa (Fig. 1). These size estimations suggest total coding regions in the plasmid of approximately 3,300 bp. Since the plasmid is only 2,647 bp long, the amino acid sequence of each of the four protein products cannot be unique. This could be a consequence, for example, of the initiation of translation at alternate sites within a coding region. pTD1 structure and DNA sequence analysis. Plasmid pTD1 was obtained from T. denticola ATCC 33520 which was maintained anaerobically as previously described (8). The entire nucleotide sequence, comprising 2,647 bp (Fig. 2), was determined for both strands. Figure 2 indicates the restriction endonuclease sites utilized to subclone fragments of pTD1 into pTZ18R (Pharmacia) for sequencing by the dideoxy method of Sanger et al. (19). The methods used for ligation, transformation, preparation of single-stranded DNA (ssDNA), running of agarose gels, and restriction endonuclease reactions were as described by Sambrook et * Corresponding author. al. (18).The relative ease with which clones could be made, and also their stability, varied consider...

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Cloning of dapD, aroD and asd of Leptospira interrogans serovar icterohaemorrhagiae, and nucleotide sequence of the asd gene

Cited by 21 publications

References 26 publications

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

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

Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen

Homology of a plasmid from the spirochete Treponema denticola with the single-stranded DNA plasmids

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