The resulting mutant strain was designated 81–176cj0596. To confirm that mutation of the cj0596 gene did AMN-107 order not alter the expression of the putative co-transcribed cj0597 gene, quantitative real-time RT-PCR was AZD1152 supplier performed on RNA samples harvested from 81–176 and 81–176cj0596. These studies confirmed that mRNA levels downstream of the mutation in 81–176cj0596 were equivalent to those in 81–176, and that the mutation was non-polar (data not shown). However, to ensure that any phenotypes of the cj0596 mutant were specific for cj0596, we subsequently isolated a reversion of the mutation by replacing the rpsl HP /cat cassette with a wild-type cj0596 gene. Analogous to the counterselection system of Dailidiene

et al. [49], use of the H. pylori rpsL gene decreased background gene conversion events and facilitated the recovery of cells in which the mutagenized cj0596 allele was replaced with the wild-type cj0596 gene. This process was extremely efficient, as ~70% of the recovered streptomycin-resistant colonies contained the desired reversions of the cj0596 mutation. Mutation of cj0596 causes a moderate growth defect of C. jejuni in MH broth The role of Cj0596 in Campylobacter growth was studied by comparing the growth rates of C. jejuni 81–176, 81–176cj0596, and 81–176cj0596 + in MH broth (Figure 5). When inoculated at OD600 ~ 0.06 and growth rate was measured by OD600 (Figure 5A), the mutant initially appeared to have a significant

growth defect illustrated both by a slower increase in OD600 and a lower maximum OD600. However, we also found that a ICG-001 research buy similar starting OD600 resulted in a significantly lower starting CFU for the mutant, indicating that Teicoplanin the OD600 did not accurately reflect

the number of cj0596 mutant CFU. We then grew the mutant under two starting conditions: one had the same starting OD600 as the wild-type and revertant (OD600 ~ 0.06) and the other had approximately the same starting CFU (OD600 ~ 0.2) as the wild-type and the revertant. The mutant inoculated at OD600 ~ 0.2 showed a faster initial increase in OD600, but reached a similar maximum OD600 as the mutant inoculated at OD600 ~ 0.06. However, when growth rate was monitored by plating for viable counts (Figure 5B), the mutant had an initial growth rate more similar to that of wild-type, although both mutant cultures yielded final viable counts lower than wild-type. Wild-type growth characteristics were restored in the revertant strain. Together, these data suggest that mutation of cj0596 resulted in a moderate growth defect, especially later in the growth curve, and that the cj0596 mutant had apparent changes in cell characteristics such that the OD600 had poor concordance with CFU measurements. Figure 5 Growth of C. jejuni strains at 37°C in MH broth. Strains 81–176 (black diamonds), 81–176cj0596 (“”low”" inoculum, red triangles) and 81–176cj0596 + (blue squares) were inoculated at an OD600 of ~0.