Care was taken to ensure that this replacement would not produce polar effects by preserving the cj0597 ribosome binding site and by leaving

only 23 bp between the stop codon of cat and the start codon of cj0597. The mutagenized cj0596 allele was introduced into a spontaneous StrepR derivative of C. jejuni 81–176 by electroporation. Several CmR/StrepS transformants were verified as cj0596 mutants by PCR with primers cj0596-F1 and cj0596-R1 (Table 2) and DNA sequencing (data not shown), a representative of which was designated 81–176cj0596 (Table 1) and used for further analysis. Reversion of the cj0596 mutation A revertant of C. jejuni 81–176cj0596 was isolated by replacing the mutated cj0596 allele in 81–176cj0596 with a wild-type cj0596 gene using streptomycin counterselection. Sapanisertib solubility dmso C. jejuni strain 81–176cj0596 + was created by using electroporation to introduce pKR001 into 81–176cj0596 cells, selecting for colonies on plates PD173074 order containing streptomycin (30 μg/ml). Putative revertants were identified by

screening StrR colonies for sensitivity to chloramphenicol (30 μg/ml) to ensure loss of the rpsL HP /cat cassette. Chromosomal DNA was isolated from these transformants and proper replacement of the rpsL HP /cat cassette with wild-type cj0596 was confirmed by PCR using primers cj0596-F1 and cj0596-R1 (Table 2) and by DNA sequencing of the region. Quantitative real-time reverse transcription Alvocidib PCR cDNA was prepared from RNA samples of C. jejuni grown 81–176 and 81–176cj0596 using a GeneAmp RNA PCR kit (Applied Biosystems). An ICycler IQ real-time

PCR detection system (Bio-Rad Laboratories, Hercules, CA) was used to run qRT-PCR with IQ Sybr Green Super pheromone Mix, and primers cj0597RT-F and cj0597RT-R (Table 2). Data were analyzed using Bio-Rad ICycler data analysis software. Control reactions used primers specific for 16S rDNA (16S-RT-F and 16S-RT-R, Table 2), which allows amplification of a non-regulated RNA [52]. Differences in transcript levels among samples were calculated from amplification profiles using the comparative threshold cycle (ΔΔCT) method, as previously described [53]. Growth experiments The growth rates of C. jejuni wild-type 81–176, mutant 81–176cj0596, and revertant 81–176cj0596 + were assessed by growing cells overnight in MH broth, then diluting the following morning in MH Broth to OD600 ~ 0.06 (the cj0596 mutant was additionally inoculated at OD600 ~ 0.2 due to poor correlation between OD600 and CFU for this strain; see Results) and shaking at 37°C under microaerobic conditions. Growth was monitored by measuring OD600 and numbers of viable bacteria were determined by plating serial dilutions of the bacterial suspensions on MH agar and counting the resultant colonies. Motility The motility of C. jejuni 81–176, 81–176cj0596, and 81–176cj0596 + was determined as previously described [54].