A: 800 ng PT (strain Bp-WWC). B: Control, no PT added. C: 2.6 pg wt PT (strain Tohama) corresponding to the limit of detection. D. 43 pg wt PT (strain

Tohama) Discussion Unmarked gene insertion and replacement were successful, using pSS4245 as vector in B. pertussis. After a second homologous recombination to excise the plasmid, no antibiotic gene marker nor any scars were left in the chromosome when compared with the cre-lox system [29] or earlier allelic-exchange procedures used in Bordetella [22]. Overproduction of genetically-deactivated PT toxin was reported in 1992 [20] by using tandem repeats of ptx genes or another copy inserted into the fha gene. The resulting recombinant B. pertussis strain overproduced PT up to 80

mg/L. Tandemly-repeated LEE011 ic50 genes are a known potential cause of genetic instability. For this reason, the genome sequence of B. pertussis was scanned to look for suitable integration sites. The DNA position between two terminators of pseudo-genes (putative ammonium transporter and putative auto-transporter genes) was selected as integration sites for the ptx cluster. The copy number for the PT structural cluster was limited to two, as overproduction of these virulence factors places a burden on cell metabolism, resulting in slower growth and potentially genetic instability, as shown by preliminary results. Over-expression of prn gene by the fha promoter to drive DNA Damage inhibitor higher expression was apparently toxic to growth of B. pertussis, possibly in resulting from higher PT expression. Our results showed that replacement of the prn promoter with a stronger

one did not provide increased prn expression [21]. Therefore, increasing the gene copy number under the control of the native prn promoter was the approach selected. The fha promoter of the second gene copy was replaced by the native prn promoter to generate a strain with a second copy of the prn gene and its native promoter inserted into another triclocarban location on the chromosome. The toxicity of PRN to the host cell was also reported in E. coli [30]. The fha promoter was then replaced by the native prn promoter, then the resulting strain exhibited normal growth in shake flasks and expressed twice the amount of PRN. The distribution of PRN between culture supernatant and cell extract was modified – a larger fraction of total PRN was found in the supernatant although in shake flasks, the quantities of PRN spontaneously released into the supernatant were minimal. The presence of either two copies of mutated PT gene alone or together with two copies of prn in WWC, WWD or WWE did not show any genetic instability as evidenced by serial-subculture experiment. All recombinant strains showed the presence of two copies of corresponding genes and corresponding amount of PT and PRN. Hence, homologous recombination among the homologous copies was not so far found in these strains.