Genetic Manipulation of <i>Leptospira biflexa</i>

Louvel, Hélène; Picardeau, Mathieu

doi:10.1002/9780471729259.mc12e04s05

Cited by 30 publications

(35 citation statements)

References 16 publications

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“…We have applied the method previously used with the saprophyte L. biflexa (14,15) for use with pathogenic Leptospira spp. Initially, the plasmid vector pSC189 (4), containing both the hyperactive transposase C9 and transposon terminal inverted repeats flanking a kanamycin resistance gene, was used to deliver Himar1 into the L. interrogans genome (2).…”

Section: Resultsmentioning

confidence: 99%

“…The plasmid pSC189ColE1 was constructed by amplifying the ColE1 origin of replication from pBluescript II (using primers 5Ј-A AAATACGTAAGCAAAAGGCCAGGAAC-3Ј and 5Ј-AAAACTGCAGGAT CAAAGGATCTTCTTG-3Ј), and the product was digested with SnaBI and PstI then ligated into similarly digested pSC189, replacing OriR6k. The plasmids pSHT, pKMars (14), and pSC189ColE1 were used to perform random transposon mutagenesis in L. interrogans strains as described previously (2). Briefly, L. interrogans was grown to exponential phase and then washed and concentrated in water.…”

Section: Methodsmentioning

confidence: 99%

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Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species

et al. 2009

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Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.Leptospira interrogans is a spirochete that is the main causative agent of leptospirosis. This zoonosis has emerged as a major public health problem in much of the developing world, with more than 500,000 cases of severe leptospirosis reported each year, for which the mortality rate is more than 10% (17).The genus Leptospira is composed of both saprophytic and pathogenic species. The genome sequences of two epidemic strains of L. interrogans serovars Lai and Copenhageni have been determined (20,25). More recently a human and an animal L. borgpetersenii isolate were sequenced (3), and this year, we determined the genome sequence of the saprophyte L. biflexa (22). The resulting sequences provide an invaluable source of information for identification of genetic determinants involved in the pathogenicity and environmental biology of the organism. For example, the host-adapted L. borgpetersenii genome is 16% smaller and has many more pseudogenes than the L. interrogans genome. These findings suggest that genome reduction has resulted in a reduced environmental transmission potential (3). L. interrogans has 627 genes that are absent in the L. biflexa genome, and more than 500 of these genes have unknown functions, suggesting the presence of novel virulence mechanisms (22). However, the lack of tools for L. interrogans genetics has hindered elucidation of the role of these genes in pathogenesis.Pathogenic leptospires are difficult...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species

et al. 2009

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“…Directed mutagenesis was carried out in L. biflexa serovar Patoc strain Patoc1 as previously described (Louvel & Picardeau, 2007). Briefly, a pGEM7Z-f + (Promega) derivative plasmid was used for the construction of plasmids containing the genes LEPBIa2006 and LEPBIa2008.…”

Section: Methodsmentioning

confidence: 99%

Biofilm formation by saprophytic and pathogenic leptospires

et al. 2008

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“…Significant advances in genetics of Leptospira spp. have been made over the last few years (8,11). For generating antibiotic resistance genetic markers, our group focused on antibiotics other than those used therapeutically.…”

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“…(1). Further studies have used Spc and Kan markers to screen for transformants resulting from plasmid replication or chromosomal integration in leptospires (8,11). As the proportion of allelic-exchange mutants is low and as chromosomal integration generally occurs through a single recombination event, a plasmid containing the rpsL wild-type gene as a counterselectable marker in a streptomycin (Str)-resistant strain of L. biflexa (due to a mutation in rpsL) was also used to eliminate clones harboring the plasmid and/or clones that have integrated the plasmid through a single-crossover event (9,17,20).…”

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Antibiotic Resistance Markers for Genetic Manipulations of Leptospira spp

Giuseppe

Picardeau

2010

Appl Environ Microbiol

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We measured the frequency of appearance of spontaneous mutants resistant to gentamicin, kanamycin, streptomycin, and spectinomycin in saprophytic and pathogenic Leptospira strains. The mutations responsible for the spontaneous resistance to streptomycin and spectinomycin were identified in the rpsL and rrs genes, respectively. We also generated a gentamicin resistance cassette that allows the use of a third selectable marker in leptospires. These results may facilitate further advances in gene transfer systems in Leptospira spp.

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Genetic Manipulation of Leptospira biflexa

Cited by 30 publications

References 16 publications

Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species

Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species

Biofilm formation by saprophytic and pathogenic leptospires

Antibiotic Resistance Markers for Genetic Manipulations of Leptospira spp

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