Insertion of a second copy of the prn genes into the Bp-WWD strain Due to the low level of PRN expression, a second copy of the prn structural gene (under control of the 246 bp fha promoter and its own terminator) was introduced into the Bp-WWD chromosome (posn. 1345693) between the two pseudogenes of putative exported dehydrogenase (posn. 1344710-1345685) and a putative aspartate racemase (posn. 1345693-1346049) (Figure 5A). The pSKPD2Cm3 E. coli vector was constructed where the Cm R gene was inserted between

the upstream and downstream regions flanking the selected insertion site. Another vector was constructed using the same flanking regions and the prn gene under control of Selleckchem Omipalisib the fha promoter (Figure 5B). After insertion of the Cm R marker in the desired location, the Cm R gene was replaced by the prn functional block using the usual allelic-exchange selection and ISRIB mw screening procedures. Figure 5 Vectors for the insertion of a second copy of the prn gene into the B. pertussis chromosome. A: The insertion site for a second copy of the prn gene was selected between two abandoned genes carrying frameshift mutations and a deletion. B: Schematic structure of the prn gene under control of fha promoter and flanking with target integration site. C: Schematic structure of the prn gene under control of its

own promoter and flanking with target TPCA-1 chemical structure integration site. The B. pertussis strains isolated from this construction exercise did not express PRN and the expression level of the other (FHA, PT and hemolysin) antigens was not detectable (data not shown). It was tentatively concluded that the PRN product is toxic if overproduced under control of the stronger fha promoter and only escape mutants having lost the capacity to produce PRN or all virulence factors were viable. It was, therefore, decided to introduce the natural prn promoter in place of the fha promoter. The plasmid pSKPD25FpPRN3 was used to replace

the fha promoter by the original prn promoter to generate a functional cassette with its own natural promoter and terminator (Figure 5C). This functional cassette was inserted at the selected site by the usual allelic-exchange procedure to obtain PRKACG a strain with a second non-tandemly-repeated copy of the prn gene under control of its own promoter. The expected insertion was confirmed by PCR amplification with primers binding to the flanking regions internally in the prn gene. This strain was normally viable and was designated as Bp-WWE. Genetic stability of PT and PRN constructs in Bp-WWE The strain Bp-WWE was cultured and serially sub-cultured in Modified Stainer-Scholte (MSS) medium to reach approximately 50 generations. The last culture was diluted and plated onto MSS agar. Thirty isolated colonies were randomly picked, and analyzed for their S1 and prn genes by PCR (data not shown). The results showed that all colonies contained two copies of S1 and prn genes at the expected positions.