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

Abstract

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.

Asymmetrical flagellar rotation in Borrelia burgdorferi nonchemotactic mutants

“…Several lines of evidence indicate that the PFs are not only involved in motility, but also influence the shape of that part of the cell in the domain where they reside. For example, in the Leptospiraceae, the relatively short PFs cause the ends of the cell to form hook or spiral shaped ends (4)(5)(6). In Treponema phagedenis, the PFs are associated with the bent-shaped ends of the cell.…”

A Genomic Island of the Pathogen Leptospira interrogans Serovar Lai Can Excise from Its Chromosome

“…Genomic DNA of Leptospira was isolated using the phenol-chloroform method as previously described (17). For pulsed-field gel electrophoresis (PFGE), cells were embedded in agarose plugs as previously described (5).…”

“…The DNA was amplified using Taq polymerase (Amersham Biosciences, Little Chalfont, England) under the following conditions: 2 min at 95°C, followed by 35 cycles of 10 s at 95°C, 15 s at 55°C, and 5 min at 72°C, and then one cycle of 10 min at 72°C. For Southern blot analysis, genomic DNA was digested, subjected to electrophoresis in a 1% agarose gel, and transferred onto nylon membranes as previously described (17). Probes were generated by PCR and radiolabeled with [␣-32 P]dATP using a commercial kit (Megaprime; Amersham Biosciences).…”

“…Probes were generated by PCR and radiolabeled with [␣-32 P]dATP using a commercial kit (Megaprime; Amersham Biosciences). Membranes were hybridized overnight at 55 or 60°C in rapid hybridization buffer (Amersham Biosciences) and then washed as previously described (17).…”

“…a Genomic DNA from a collection of strains was digested with EcoRI, separated on an agarose gel, transferred to a nylon membrane, and hybridized with two probes of LaiGI I (LA1773 and LA1839) at 55°C as previously described (17). ϩ, presence; Ϫ, absence.…”