Integrated physical and genetic mapping of Bacillus cereus and other gram-positive bacteria based on IS231A transposition vectors

Abstract

The genome structure of Bacillus cereus is relatively complex, its DNA being modulated between a size-varying chromosome and large plasmids. To study the genetic organization of the B. cereus type strain ATCC 14579, thermosensitive transposition vectors were designed on the basis of IS231A-derived cassettes containing uncommon restriction sites. A highly preferred insertion site for IS231A was detected in the chromosome by Southern blotting and pulsed-field gel electrophoresis (PFGE) analyses of independent insertion mutants. However, once this insertional hot spot was occupied, secondary IS231A insertions occurred randomly, as demonstrated by isolation of independent B. cereus auxotrophs at a frequency of approximately 0.6%. The hot-spot site, as well as several auxotrophic mutations, were mapped by using NotI, SfiI, and AscI PFGE restriction profiles. It was confirmed by sequencing that one of the insertions, generating an Ade- phenotype, had disrupted a gene of the purine synthesis pathway. These results showed that combined PFGE and sequencing analyses of mini-IS231A insertions enable the construction of integrated physical and genetic maps of B. cereus type strain. Moreover, the presence of the ultrarare I-SceI restriction site in the mini-IS231A allowed the isolation, in double-insertion mutants, of contiguous and nonoverlapping large chromosomal fragments, convenient for direct sequencing. The system detailed in this report is therefore a powerful tool for comparative genetic studies among members of the B. cereus group (i.e., B. cereus, B. thuringiensis, B. mycoides, and B. anthracis) and could also be applied to more distantly related gram-positive bacteria.

FIG. 1

IS231A-based transposition vectors pGIC055 and pGIC057. ERY, Ery^r gene; G+ ORI, replicative origin for gram-positive host; P_deg, strong gram-positive promoter; TnpA, transposase; RCP, rare-cutting polylinker; black triangles represent the IRs of IS231A. The mini-IS resistance gene is Sp^r (pGIC055) or Kan^r (pGIC057).

FIG. 2

PFGE profiles of total DNA from B. cereus strains restricted by NotI (A), AscI (B), SfiI (C), and I-SceI (D). Y, yeast chromosome marker; λ, lambda ladder marker (New England Biolabs); HS, hot-spot candidate with one single mini-IS Kan^r insertion; TS, B. cereus ATCC 14579 type strain; A1 and A2, ade mutants; G1, gua mutant; H1 and H2, his mutants; M1 and M2, met mutants; U1, ura mutant; Aph., aphenotypic double-insertion mutant.

FIG. 3

B. cereus chromosomal map. Based on the position of the dnaA gene and by comparison of the locations of other genes with those of B. subtilis, the restriction map recently published by Carlson et al. (7) has been reoriented to place the potential replicative origin site at the 0°/360° point. Several relevant loci have been retained; their positions were arbitrarily set in the middle of the chromosomal fragment to which they hybridize (7). plc, phospholipase C; inA, inhibitor A; pdh, pyruvate dehydrogenase. Nx and Ax correspond to NotI and AscI restriction fragments, respectively. The hot-spot insertion site as well as eight other insertions generating auxotrophies are indicated. Aph represents an aphenotypic secondary transposition event.

FIG. 4

IS231A target DNA sequence of the hot-spot insertion site (A) and Ade⁻ insertion site (B) and representation of their respective EcoRI fragments cloned in pGIC102 (A) and pGIC105 (B). The 11 bp boxed on the sequence represent the duplicated target site (DR). IR_L and IR_R refer to IR left and right, respectively, by reference to the transposase gene orientation in IS231A; small black arrows indicate the cleavage generated by the transposase on each strand of the DNA. The size of the EcoRI fragment cloned in pGIC102 (A) is 7.3 kb, including 1.9 kb for the mini-IS Kan^r. This fragment is 3.2 kb in the case of pGIC105 (B), from which 1.6 kb pertains to the mini-IS Sp^r. As for B. subtilis, the putative B. cereus purL and purF genes share a 25-bp overlap.

FIG. 5

Hybridization of the total DNA of B. cereus auxotrophs restricted by EcoRI with a probe corresponding to the Sp^r gene. HS, hot-spot candidate with one single mini-IS Kan^r insertion (negative control); A1, A2, A3, and A4, ade mutants; C1, cys mutant; G1, gua mutant; H1 and H2, his mutants; M1 and M2, met mutants; U1, ura mutant.