Horizontal transfer of iturin A operon, itu, to Bacillus subtilis 168 and conversion into an iturin A producer

Abstract

Iturin A and its derivatives are lipopeptide antibiotics produced by Bacillus subtilis and several closely related bacteria. Three iturin group operons (i.e., iturin A, mycosubtilin, and bacillomycin D) of those antibiotic-producing strains have been cloned and sequenced thus far, strongly implying the horizontal transfer of these operons. To examine the nature of such horizontal transfer in terms of antibiotic production, a 42-kb region of the B. subtilis RB14 genome, which contains a complete 38-kb iturin A operon, was transferred via competent cell transformation to the genome of a non-iturin A producer, B. subtilis 168, using a method based on double-crossover homologous recombination with two short landing pad sequences (LPSs) in the genome. The recombinant was positively selected by confirming the elimination of the cI repressor gene, which was localized between the two LPSs and substituted by the transferred segment. The iturin A operon-transferred strain 168 was then converted into an iturin A producer by the introduction of an sfp gene, which encodes 4'-phosphopantetheinyl transferase and is mutated in strain 168. By inserting the pleiotropic regulator degQ, the productivity of iturin A increased sevenfold and was restored to about half that of the donor strain RB14, without the transfer of additional genes, such as regulatory or self-resistance genes.

FIG. 1.

(A) Structure and flanking regions of the iturin A, mycosubtilin, and bacillomycin D operons. Shaded bridges between different strains connect regions homologous with the iturin A operon of RB14, while dotted bridges connect regions homologous with the plipastatin operon of 168. Solid black regions in the iturin A and bacillomycin D operons indicate regions homologous with the xynD gene of 168. Vertical lines, horizontal lines, and areas not linedrepresent B. subtilis RB14 DNA, B. amyloliquefaciens FZB42 DNA (19), and B. subtilis ATCC 6633 DNA (3), respectively. Fengycin is another name for plipastatin (19). (B) Whole-genome map of strain 168 (diagonal lines). The counterparts of the relevant genes in panel A are mapped. (C) Strain development. The positive-selection system is also indicated. The CI repressor in 6234/cI represses Pr-neo expression, resulting in neomycin susceptibility. However, once substitution of the cI gene by transformed DNA takes place, the transformant becomes neomycin resistant. Solid black regions show the tetracycline (labeled pBR) and ampicillin (labeled 322) resistance gene sides of the split pBR322 sequence. The genes cat and spc represent the chloramphenicol and spectinomycin resistance genes, respectively. The genes cI and Pr-neo indicate the CI repressor gene and the Pr promoter-driven neomycin resistance gene, respectively. BEST6234, 6234/cI, and 6234/itu also have a Pr-neo cassette in their genome; however, this cassette is absent in the RM/iS2 and RM/iSd series. The NeoR/S labels at the far right indicate neomycin resistance (R) or sensitivity (S). LPS, landing pad sequence; LPA, LPSs array.

FIG. 2.

Confirmation of iturin A operon transfer. (A) Southern hybridization analysis. SfiI and NotI digestion of BEST6234 and 6234/itu genomic DNA prepared in an agarose gel block were fractionated by CHEF pulsed-field gel electrophoresis and subjected to Southern hybridization analysis using pBRE4H8 as the probe. Letters correspond to labeled portions of the map in panel B. EtBr, ethidium bromide. (B) Physical map around leuB of iturin A operon-transferred strain. Lettered fragments on the map were actually observed as bands on the photographs in panel A. This figure is drawn on the basis of the whole-genome sequence of strain 168 (DDBJ/EMBL/GenBank accession no. AL009126). Diagonal and vertical lines represent B. subtilis 168 DNA and B. subtilis RB14 DNA, respectively.

FIG. 3.

HPLC peak patterns of three lipopeptides (iturin A [IT], surfactin [SF], and plipastatin [PL]) (top) and those focused on iturin A (bottom) produced by RM125 (A), 6234/itu (B), RM/Sp6 (C), RM/iS2 (D), RM/iSd12 as represented by four RM/iSd strains (E), and RB14 (F) in no. 3S medium at 30°C for 120 h. Two distinct HPLC conditions were used to analyze one sample. The top chromatographs were obtained with a two-eluent gradient, while bottom chromatographs were obtained with one eluent. Peaks 1, 2, 3, and 4 correspond to iturin A whose β-amino acids are n-C₁₄-β-amino acid, anteiso-C₁₅-β-amino acid, iso-C₁₅-β-amino acid, and n-C₁₆-β-amino acid, respectively.