Nucleotide sequence analysis of the Leptospira biflexa serovar patoc rpsL and rpsG genes

Zuerner, Richard L.; Charon, Nyles W.

doi:10.1128/jb.172.10.6165-6168.1990

Cited by 6 publications

(8 citation statements)

References 33 publications

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“…The rpsL + gene was amplified from L. biflexa (Zuerner and Charon, 1990) with primers LpR1 and LpR2, using Pfu polymerase (Stratagene), and cloned into the Sma I site of pGKLep4 (Saint Girons et al ., 2000) to produce pGKLrL. Str r L. biflexa cells were electroporated with pGLKrL as described above; individual kanamycin‐resistant colonies were then inoculated into kanamycin‐containing EMJH liquid medium and incubated at 32°C to stationary phase.…”

Section: Methodsmentioning

confidence: 99%

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non‐motile mutants deficient in endoflagella

Picardeau¹,

Brenot

Girons

2001

Molecular Microbiology

138

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SummaryLeptospira spp. offer many advantages as model bacteria for the study of spirochaetes. However, homologous recombination between introduced DNA and the corresponding chromosomal loci has never been demonstrated. A unique feature of spirochaetes is the presence of endoflagella between the outer membrane sheath and the cell cylinder. We chose the flaB flagellin gene, constituting the flagellar core, as a target for gene inactivation in the saprophyte Leptospira biflexa. The amino acid sequence of the FlaB protein of L. biflexa was most similar to those of spirochaetes Brachyspira hyodysenteriae (agent of swine dysentery), Leptospira interrogans (agent of leptospirosis) and Treponema pallidum (agent of syphilis). A suicide vector containing the L. biflexa flaB gene disrupted by a kanamycin marker was UV irradiated or alkali denatured before electroporation. This methodology allowed the selection of many kanamycin-resistant colonies resulting from single and double cross-over events at the flaB locus. The double recombinant mutants are nonmotile, as visualized in both liquid and semi-solid media. In addition, a flaB mutant selected for further analysis was shown to be deficient in endoflagella by electron microscopy. However, most of the transformants had resulted from a single homologous recombination event, giving rise to the integration of the suicide vector. We evaluated the effect of the sacB and rpsL genes in L. biflexa as potential counterselectable markers for allelic exchange, and then used the rpsL system for the positive selection of flaB double recombinants in a streptomycinresistant strain. Like the flaB mutant studied above, the Str r double cross-over mutant was non-motile and deficient in endoflagella. Our results demonstrate that FlaB is involved in flagella assembly and motility. They also show the feasibility of performing allelic replacement in Leptospira spp. by homologous recombination.

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Section: Methodsmentioning

confidence: 99%

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non‐motile mutants deficient in endoflagella

Picardeau¹,

Brenot

Girons

2001

Molecular Microbiology

138

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“…A possible insertion may also be present between the serovar icterohaemorrhagiae rpoC and rpsLG genes. These genes map together in both serovar pomona and L. biflexa serovar patoc (50,51) but are separated by at least 130 kb in serovar icterohaemorrhagiae.…”

Section: Discussionmentioning

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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“…A further interest for rpsL resided in its recent efficient use as a counter‐selectable marker to promote allelic exchange in the PFRA strain [17]. The comparison of the RpsL sequences from PFRA and PITAL strains to the RpsL sequence from the PUSA strain [11] was surprising since the amino acid sequences were identical. However, 30 silent differences, all located in the third letters of the codons, were found between the rpsL genes from PFRA and PUSA strains (Fig.…”

Section: Resultsmentioning

confidence: 99%

“… Comparison of the nucleotide sequences of the rpsL gene from PFRA (AJ310919) and PITAL (AJ310918) strains to that of PUSA (M5776) [11]. Asterisks indicate differences.…”

Section: Resultsmentioning

confidence: 99%

“…DNA sequencing was performed on the PCR products using primers FD3 and FD3Rev (5′‐AGA TTC CAG ACT AAC AGT TT‐3′). In order to amplify the rpsL gene, the following primers were used: rpA (5′‐TGA AGA TGA GTT GGA AGA AG‐3′) 176 bp upstream of the ATG and LpR2 (5′‐TGT ATT TAG GAT CGC CTT CG‐3′) 52 bp downstream of the stop codon [11]. Sequencing was then performed.…”

Section: Methodsmentioning

confidence: 99%

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First evidence for a restrictionâmodification system in Leptospira sp.1

Brenot

Werts

Ottone

et al. 2001

FEMS Microbiology Letters

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The LE1 leptophage exhibited a host range restricted to the saprophytic Leptospira biflexa [Saint Girons et al., Res. Microbiol. 141 (1990) 1131-1133] and mainly to the Patoc 1 strain (hereafter called PFRA) kept in the Paris, France collection. Results of titration of LE1 lysates indicated the presence of a host-controlled modification and restriction system within PUSA (Patoc 1 strain maintained in the Morgantown, WV, USA collection) that was absent in PFRA. Because genomic DNA of PITAL (Patoc 1 strain maintained in Trieste, Italy) appeared smeared in pulsed field gel electrophoresis (PFGE), this strain is likely to contain nucleases that are activated upon DNA isolation. Moreover, comparative NotI digestions of PUSA and PFRA DNAs, as visualized by PFGE, indicated that PUSA belonged to a different serovar than PFRA. Finally, 16S ribosomal sequence analysis indicated that PUSA belonged to the saprophytic Leptospira meyeri species, while PITAL and PFRA appertained to L. biflexa. The evolutionary significance and the importance of the restriction and modification enzymes or non-specific nucleases within strains for genetic experiments are discussed.

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Nucleotide sequence analysis of the Leptospira biflexa serovar patoc rpsL and rpsG genes

Cited by 6 publications

References 33 publications

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non‐motile mutants deficient in endoflagella

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non‐motile mutants deficient in endoflagella

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

First evidence for a restrictionâmodification system in Leptospira sp.1

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Nucleotide sequence analysis of the Leptospira biflexa serovar patoc rpsL and rpsG genes

Cited by 6 publications

References 33 publications

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non‐motile mutants deficient in endoflagella

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non‐motile mutants deficient in endoflagella

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

First evidence for a restrictionâmodification system in Leptospira sp.1

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First evidence for a restrictionâmodification system in Leptospira sp.1