Primers cj0596-F1 and cj0596-R1 (Table 2) were designed based on the published NCTC 11168 genome sequence and used in PCR reactions with template genomic DNA prepared using a MasterPure DNA purification kit (Epicentre, Madison, WI) to amplify the region encompassing cj0596. Thermocycler parameters PD0325901 datasheet were 35 cycles of: 94°C for 30 seconds, 51°C for 30 seconds, and 72°C for 4 minutes. PCR products were purified using QIAquick PCR purification kits (Qiagen, Valencia, CA) and were then sequenced directly (both strands), using an ABI 3730xl sequencer (Applied Biosystems). VectorNTI (version 7, Invitrogen, Carlsbad, CA) was used
to analyze DNA sequences. The protein sequences were analyzed for motifs using the ExPASy Prosite server http://au.expasy.org/prosite/[46, 47], and potential signal peptides were evaluated using SignalP 3.0 http://www.cbs.dtu.dk/services/SignalP/[48]. Isolation of a C. jejuni cj0596 mutant A cj0596 mutant of C. jejuni strain 81–176 was constructed using a streptomycin counterselection system find more similar to the heterologous H. pylori-C. jejuni method described by Dailidiene et al. [49] in which an rpsL
gene from C. jejuni was used for counterselection in H. pylori, to decrease background gene conversion events. In the heterologous rpsL HP system reported here, cj0596 was exactly replaced by the
rpsL HP (StrS) gene from H. pylori strain 84–183 (Table 1; [50]) linked to a chloramphenicol acetyltransferase (cat) cassette (CmR). This strategy allows for selection of a mutant (StrS/CmR) based on chloramphenicol resistance and then allows for selection of a revertant strain (StrR/CmS) based on streptomycin resistance. First, a PCR-amplified cj0596 gene was amplified using primers cj0596-F1 and cj0596-R1 designed based on the published C. jejuni NCTC 11168 genome sequence (Table 2) and the resulting product was cloned into pCR II-TOPO creating pKR001 (Table 3). The plasmid pKR001 was subjected to inverse PCR (primers Interleukin-3 receptor cj0596-inv1 and cj0596-inv2) to remove the cj0596 gene and add restriction sites. The inverse PCR product was self-ligated to form plasmid pKR002. The cat cassette was amplified from pRY111 [51] and Age I, Mfe I, and Nhe I sites were added using primers cat-F1 and cat-R1. The resulting PCR product was cloned into pCR II-TOPO to create plasmid pKR018. The rpsL HP gene was amplified and Age I and Mfe I sites were added using primers rpsL HP -F1 and rpsLHP-R1 and the PCR product was cloned into pCR II-TOPO creating plasmid pKR019. Age I and Nhe I were used to excise the cat cassette from pKR018 and linearize pKR002. The cat cassette was ligated into pKR002 creating plasmid pKR020.