Region B determines capsule (K-antigen) According to the annotation in GenBank , region B in V. parahaemolyticus encodes four hypothetical proteins that are upstream of gmhD and transcribed in the same direction, followed by an operon-like structure of 19 open reading frames in the opposite direction (Figure 2, Table 2). To LY3039478 mouse investigate if region B is related to either O-antigen/K-antigen biogenesis in V. parahaemolyticus, we deleted the entire 21 kb operon of 19 open frames, VP0219-0237, and replaced it with a Cm cassette (Figure 2). The resulting mutant, ∆CPS, displayed a translucent phenotype consistent
with loss of capsule expression, in contrast to an opaque phenotype in the wild type (Figure 3) . Figure 2 Capsule (K-antigen) genes in V. parahaemolyticus O3:K6. a) Bars with mutant names above indicate regions deleted in each mutant. Bent arrow indicates promoter. Design patterns of open reading frames indicate different classes of genes: vertical lines, pathway genes; diagonal lines, processing and transportation genes; grey box, glycosyltransferase; white box, functions click here not clear. b) GC percentage of the sequence in 120 bp windows, aligned to the genes in a. Table 2 K-antigen/Capsule genes of V. parahaemolyticu
s O3:K6 Gene Symbol https://www.selleckchem.com/products/prn1371.html putative function VP0214 gmhD ADP-L-glycero-D-manoheptose-6-epimerase VP0215 hypothetical protein VP0216 hypothetical protein VP0217 putative regulator protein VP0218 hypothetical protein VP0219 hypothetical protein VP0220 wbfF capsule assembly protein VP0221 wzz polysaccharide chain length determinant VP0222 rmlB dTDP-glucose 4,6 dehydratase VP0223 rmlA D-glucose-1-phosphate Neratinib mw thymidylyltransferase VP0224 rmlD dTDP-4-dehydrorhamnose reductase VP0225 hypothetical protein VP0226 glycosyltranferase VP0227 hypothetical protein VP0228 hypothetical protein VP0229 rmlC dTDP-4-dehydrorhamnose 3,5-epimerase VP0230 glycosyltranferase VP0231 UDP-galactose phosphate transferase VP0232 similar to carbamoyl phosphate synthase VP0233 hypothetical protein VP0234 amino transferase VP0235 putative epimerase
VP0236 UDP-glucose 6-dehydrogenase VP0237 UTP-glucose-1-phosphate uridylyltransferase VP0238 rjg hypothetical protein Figure 3 V. parahaemolyticus mutants ∆CPS and ∆0220 display translucent phenotype. Wild type (WT), ∆CPS and ∆0220 have grown on LB agar at 37°C for 24 hours. We then investigated the immunogenicity of wild type and ∆CPS mutant by immuno-blotting. Whole cell lysate treated with DNase, RNase and pronase was separated on SDS gels, stained with stains-all/silver stain; or blotted to PVDF membrane and probed with O3 or K6 specific antiserum. With the O3:K6 wild type, gels stained with stains-all/silver-stain showed low molecular weight bands circa 17 kDa and high molecular weight bands circa 95 kDa (Figure 4). Immuno-blot developed with O3 antiserum only detected the low molecular weight bands.