The results of this assay suggested that the antibodies against OmpC or OmpF were effective for mediating opsonophagocytosis of ExPEC. This this website may to some extent account for the high protection against challenge with highly virulent ExPEC in the immunized mice. To gain more insight into the mechanisms of immunogenicity and protective efficacy, the

roles of OmpC and OmpF in macrophage adherence and cytokine production should be evaluated. Based on further phylogenetic analysis, the ompC gene was found to be present in all E. coli strains, but ompF was mutated in certain strains. In addition, we found significant recombination signals in both alignments of ompC and ompF. Furthermore, the porin gene ompC showed significant evidence

for positive selection in seven sites, whereas no positively selected sites were detected in ompF. The previous study on the genome-wide positive selection has reported that the E. coli ompC gene shows evidence for selective pressures exerted by phage infectivity (Petersen et al., 2007). Based on more publicly available sequences, we confirmed that E. coli ompC is undergoing strongly positive selection with an enlarged spectrum of positively selected sites identified. This might provide a genetic basis for further uncovering the interactions of the important outer membrane antigen OmpC with phage binding and/or with the host immune system. In conclusion,

PD-0332991 research buy we characterized the immunogenicity of OmpC and OmpF from porcine ExPEC. Our results indicated that surface-exposed outer membrane protein OmpC could be a promising candidate for vaccine development against ExPEC infection. Phylogenetic analysis further showed genetic evidence for positive selection acting on the porin gene ompC under host immunological pressure. This study was supported by Grants from the National Natural Science Foundation of China (NSFC no. 31030065), and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (31121004). “
“Multidrug efflux systems not only Non-specific serine/threonine protein kinase cause resistance against antibiotics and toxic compounds but also mediate successful host colonization by certain plant-associated bacteria. The genome of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum encodes 24 members of the family of resistance/nodulation/cell division (RND) multidrug efflux systems, of which BdeAB is genetically controlled by the RegSR two-component regulatory system. Phylogenetic analysis of the membrane components of these 24 RND-type transporters revealed that BdeB is more closely related to functionally characterized orthologs in other bacteria, including those associated with plants, than to any of the other 23 paralogs in B. japonicum.

Homologous recombination with linear DNA for deletion of the original find more target gene was performed according to the procedure previously reported with modifications (Datsenko & Wanner, 2000). In brief, PCR products containing the kan gene from pKD4 or pKD13 were electroporated into the E. coli strain harboring pKD46r and grown in LB agar containing KM (either 5 or 25 μg mL−1) and/or 3-β-indoleacrylic acid (IAA, inhibitor of TrpR) (25 μg mL−1). Deletion of the target gene was examined by PCR. Colonies grown in LB agar containing KM (5 μg mL−1) and IAA (25 μg mL−1) were suspended with sterile saline. Suspensions were diluted 10-fold serially with sterile

saline. Then, one hundred microliters of samples was spread onto LB agar without any supplement, LB agar containing IAA (25 μg mL−1), LB agar containing tryptophan (Trp, 1 mg mL−1), or LB agar containing Trp (1 mg mL−1) plus IPTG (10 mM) and then cultured at 37 °C for > 24 h. Finally, the number of colonies grown on the plates was enumerated. Colony-forming capacity was determined check details by the appearance of visible colonies within

48 h of cultivation, and as a positive control, approximately 1000 colony-forming units (CFU) per plate of bacteria were spread on a plate. Colonies grown on LB agar containing KM (5 μg mL−1) and IAA (25 μg mL−1) were suspended with sterile saline and adjusted to OD600 nm = 0.08–0.10. These solutions were diluted 10 000-fold in LB broth and incubated in a shaking incubator at 120 r.p.m. at 37 °C for 2 h. After

incubation, IAA (25 μg mL−1) or IPTG (10 mM) plus Trp (1 mg mL−1) was added to each tube. Aliquots from each tube were removed at −1, 0, 1, 3, and 6 h, and then 10-fold serial dilutions were spread onto LB agar plates containing KM (5 μg mL−1) and IAA (25 μg mL−1). Viable colonies were enumerated after 24–48 h incubation at 37 °C with the limit of detection for the time-kill studies being 10 CFU mL−1. When no viable colony was detected in the undiluted culture, the sample was defined as 10 CFU mL−1. The wild-type lacI promoter is L-gulonolactone oxidase very weak (Calos, 1978). For efficient lacI gene expression, the lacI promoter of E. coli K-12 MG1655 was replaced with lacI-35-10 promoter (Glascock & Weickert, 1998) by homologous recombination, and then the promoter of the clpA gene was replaced with lacUV5 promoter (Lanzer & Bujard, 1988), and the ORF of HA tag was fused in-frame to 3′-end of the clpA gene ORF. Next, the ORF region of the sdaB (Shao & Newman, 1993), a homologue of sdaA that is not essential for bacterial survival, was replaced with CP25e promoter, a constitutive promoter with modification for optimal sequences for E. coli (Jensen & Hammer, 1998), and the ubp1 ORF fused in-frame with FLAG tag ORF at 3′-end. No apparent phenotypic change by deletion of sdaB was observed. The protein expression of ClpA-HA (IPTG supplemented condition) and UBP1-FLAG in this strain was confirmed by Western blotting using anti-HA and anti-FLAG antibody respectively (data not shown).