Development of a system for genetic manipulation of Bartonella bacilliformis

Abstract

Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of all Bartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes ClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kan(r) clones of B. bacilliformis. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and fla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by fla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG). These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.

FIG. 1

Illustration of suicide plasmid and schematic representation of site-specific fla disruption. (A) The B. bacilliformis suicide plasmid pUB1 harbors a multiple cloning site, the kanamycin resistance cassette neomycin phosphotransferase I (nptI), and the pMB1 replicon. (B) The flagellin gene-targeting suicide plasmid, derived from pUB1, is shown with the 508-bp BglII-KpnI internal fragment of fla (fla′) The transformable strain, JB584 (Km^s, fla⁺), containing the wild-type 1,127-bp fla ORF, is shown. Homologous recombination resulted in site-specific insertion of pUB508 at the fla locus, generating strain JB585 (Km^r, fla). Note the position of the flagellin (FLA5′ and FLA3′) and kanamycin (NPTI5′ and NPTI3′) amplimers, indicated by the small arrows. (Figure not drawn to scale.)

FIG. 2

Electrophoretic analysis of PCR products derived from B. bacilliformis fla mutant and trans-complemented strains. PCR products generated by amplification of chromosomal DNA obtained from parent and recombinant strains by using three amplimer sets, nptI (NPTI5′ and NPTI3′), fla (FLA5′ and FLA3′), and junction (jct) (NPTI5′ and FLA3′) were respectively used to detect the kanamycin cassette, the flagellin gene, and the junction between fla and the inserted pUB508. Bars below the gel indicate the amplimer set used in each reaction. Amplimer sets and the respective DNA templates used in this analysis are as follows: lane 1 (NPTI5′ and NPTI3′, Fla5′ and Fla3′; no template), lane 2 (NPTI5′ and NPTI3′; JB584), lane 3 (NPTI5′ and NPTI3′; JB585), lane 4 (FLA5′ and FLA3′; JB584), lane 5 (FLA5′ and FLA3′; JB585), lane 6 (NPTI5′ and FLA3′; JB584), lane 7 (NPTI5′ and FLA3′; pUB508), lane 8 (NPTI5′ and FLA3′; pUB508 + JB584), lane 9 (NPTI5′ and FLA3′; JB585), lane 10 (FLA5′ and FLA3′ JB686), lane 11 (NPTI5′ and FLA3′ JB686). PCR products were resolved by agarose gel electrophoresis (1% agarose) and visualized by ethidium bromide staining. DNA size markers (lane M) are shown to the right.

FIG. 3

Detection of the flagellin gene in wild-type, mutant, and trans-complemented strains of B. bacilliformis by DNA hybridization. (A) Ethidium bromide-stained agarose gel (1% [wt/vol] agarose) containing λ HindIII size standards (lane 1), ClaI-digested chromosomal DNA from KC584 (lane 2), JB584 (lane 3), JB585 (lane 4), and JB686 (lane 5), and ClaI-digested pBBRFLAG (lane 6). (B) Corresponding Southern blot following hybridization with the fla probe. Note the distinct two-band hybridization pattern in strains containing a disrupted fla gene (strains JB585 and JB686) and the presence of pBBRFLAG in the trans-complemented strain, JB686.

FIG. 4

Analysis of flagellin synthesis in the B. bacilliformis fla mutant and complemented mutant by SDS-PAGE and immunoblotting. (A) Whole-cell extracts of parent, flagellin mutant, and trans-complemented mutant strains were separated by SDS-PAGE (12.5% polyacrylamide) and stained with Coomassie brilliant blue. The 42-kDa flagellin polypeptide is present in the parent strain JB584 (lane 1) and is absent from the kanamycin-resistant flagellin mutant strain JB585 (lane 2). Flagellin synthesis is detected in trans from the complementation plasmid pBBRFLAG in strain JB686 (lane 3). (B) Corresponding immunoblot reacted with rabbit anti-flagellin polyclonal antiserum, indicating that flagellin synthesis is restored in the trans-complemented strain. Molecular mass standards are indicated to the left (in kilodaltons), and the 42-kDa flagellin polypeptide is marked by the arrow.

FIG. 5

Ultrastructure of generated strains as shown by TEM. Transmission electron micrographs were prepared by staining 5-day-old cultures of B. bacilliformis with 2% uranyl acetate. Flagella are observed the wild-type strain (KC584) (A) and the transformable strain (JB584) (B) but are absent from the fla mutant strain (JB585) (C) Polypeptide synthesis, secretion, and assembly of the flagella is restored in the trans-complemented strain (JB686) (D). Bars, 0.5 μm.