Abstract
We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica, which is required for O antigen biosynthesis, into the chromosome of B.pertussis, which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B.pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti-B.bronchiseptica antibody but not the anti-B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B.bronchiseptica. O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B.pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B.pertussis, which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant mutant strain of B.pertussis. The present results demonstrate that this system successfully accomplished the above-described purpose. We consider this system to be applicable to a number of bacteria other than Bordetella.
Highlights
Introduction of largeB. bronchiseptica genomic DNA (gDNA) into the B. pertussis chromosome
We constructed the vector for the BPI system by combining the advantages of the bacterial artificial chromosome (BAC) vector to carry large DNA fragments [14] and the phiC31 phage to integrate these fragments into a bacterial chromosome in a site-specific manner [15] (Fig. 1A and B)
We initially introduced a kanamycin (Km) resistance gene into pBeloBAC11 in place of the chloramphenicol resistance gene, because resistance to chloramphenicol is often unreproducible in Bordetella grown on Bordet-Gengou (BG) agar plates
Summary
Introduction of largeB. bronchiseptica gDNAs into the B. pertussis chromosome. The BAC system is capable of maintaining high-molecular-weight DNA in E. coli [14]. The attL and attR regions were amplified, the B. bronchiseptica gDNA region was not (data not shown), indicating undesirable homologous recombination, possibly because of large homologous sequences between the 49.6-kbp fragment and the chromosome of B. pertussis To overcome this issue, we generated a recA deletion mutant of BpattP (BpattP⌬recA::gfp), because RecA is mainly involved in recombination in bacteria [16, 17]. Fragments I and VIII, containing attL and attR, were only amplified when the gDNA of BpattP⌬recA:: gfp after transformation (BpattP⌬recA::gfp-L1) was used as the template These results indicated that full-length pBPI-L1 containing the 49.6-kbp fragment of B. bronchiseptica gDNA was successfully integrated into the chromosome of B. pertussis. If the genes inserted by the BPI system are functional, O antigen should be expressed in B. pertussis msphere.asm.org 4
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