Characterization of cj1169c-cj1170c operon The microarray and qRT-PCR results demonstrated that cj1169c and cj1170c were up-regulated in both inhibitory and sub-inhibitory treatments with Ery (Tables 3 and 4). cj1169c and cj1170c

encode a putative periplasmic protein and a 50 kDa outer membrane protein precursor, respectively [23]. Recently, cj1170c was characterized as an outer-membrane tyrosine kinase, phosphorylating a number of membrane proteins [24]. To identify the role of the two genes in adaptation to Ery treatment, both genes were deleted to produce the mutant strain KOp50Q. The mutation did not affect the transcript abundance of the downstream gene, cj1168c, as determined by qRT-PCR (data not CB-839 manufacturer shown). The mutant was complemented to produce strain Comp50Q. The wild-type and mutant strains demonstrated comparable growth rates in MH broth without

or with sub-inhibitory (1/2, 1/4, 1/8, and 1/16× MIC) concentrations of Ery (data not shown). Additionally, no significant BVD-523 difference in motility was observed between the mutant and wild-type strains. Furthermore, the MIC test revealed no significant differences between the wild type strain and KOp50Q in susceptibility to a number of antimicrobials including ampicillin, erythromycin, tylosin, ciprofloxacin, tetracycline, phosphonomycin, cetylpyridinium chloride, chloramphenicol, nalidixic acid, novobiocin, ethidium bromide and crystal violet (results not shown). Likewise, as shown by the disk diffusion assay, no significant differences were revealed between the mutant and wild-type strains in sensitivity to oxidative stress agents including H2O2 and cumene hydroperoxide (data not shown). However, the aerobic stress experiments HSP90 indicated that the mutant was more susceptible than the wild-type strain to higher levels of oxygen, although they showed comparable growth under microaerobic conditions (Figure 2C). Complementation of

the mutant (Comp50Q) partially restored the phenotype to the wild-type level (Figure 2C). To determine the role of cj1169c-cj1170c in colonization of and horizontal transmission between birds, a Z-VAD-FMK order co-mingling chicken experiment was performed with wild-type, mutant (KOp50Q) and complement strains (Comp50Q). All 3 seeder birds in each group became Campylobacter-positive for the respectively inoculated strain at 3 days after inoculation (DAI) as determined by cloacal swabbing and culturing on selective plates. The three KOp50Q-inoculated seeder birds showed attenuated colonization levels compared with those inoculated with the wild-type strain (p = 0.02), while the complement strain resulted in comparable colonization level to that of the wild-type strain (p = 0.32) as determined by culturing cecal contents collected at necropsy on 9 or 12 DAI (Figure 4A).