Secondary

structure predictions showed no transmembrane segments in the mature protein, suggesting that Cj0596 is likely to be a periplasmic protein. Cj0596 is located in the periplasm of C. jejuni The amino acid sequence of Cj0596 suggested that this protein is located in the periplasm. To test this experimentally, western blots were performed on cytoplasmic, inner membrane, periplasmic, and outer membrane fractions of C. jejuni 81–176 using anti-Cj0596, anti-Cj0355, anti-CetA, and anti-MOMP antibodies (Figure 3). As expected, anti-Cj0355 antibodies reacted with a ~25 kDa protein in the cytoplasmic fraction, anti-CetA antibodies find more reacted with a ~50 kDa protein in the inner membrane fraction, and anti-MOMP antibodies reacted with a ~45 kDa protein in the outer membrane. Anti-Cj0596 antibodies reacted with a ~30 kDa protein present primarily in the periplasmic fraction. Figure 3 Localization of Cj0596. Western blots of cell fractions using Cj0355 as a cytoplasmic control, CetA as an inner membrane control, and MOMP as an outer membrane control show that Cj0596 is located in the periplasm of C. jejuni. Cj0596 has PPIase Activity

Cj0596 has one rotamase domain and is similar to E. coli SurA, suggesting that it is a PPIase. The PPIase activity of purified Cj0596 was determined using a coupled assay in which the cleavage of the trans isomer of N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide by α-chymotrypsin results in the release of p-nitroanilide, causing a colorimetric change over time. https://www.selleckchem.com/products/BIBW2992.html Conversion of the cis to the trans isomers of the substrate occurs spontaneously in solution, allowing chymotrypsin cleavage

(Figure 4, squares). However, addition of Cj0596 accelerates this cis-trans conversion, indicative of PPIase activity (Figure 4, diamonds). By using varying concentrations of Megestrol Acetate purified Cj0596 (data not shown) and plotting calculated kobs vs. [PPIase], the PPIase activity (kcat/km) was calculated to be 22.3 mM-1sec-1, an activity consistent with values published for other PPIases [64–66]. Figure 4 PPIase activity of Cj0596. Enzymatic activity of Cj0596 assayed by cleavage of N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide in a chymotrypsin-coupled assay, in which cleavage of the trans isomer of the substrate by chymotrypsin is accelerated by PPIase activity. A representative plot of Absorbance vs. time using purified Cj0596 protein (red diamonds) and negative control (black squares) is shown. Creation of a non-polar cj0596 mutation To test the role of Cj0596 in C. jejuni physiology or pathogenesis, we created a non-polar cj0596 mutant. To facilitate mutant construction, we developed a modified streptomycin counter selection system based on a similar strategy used in H. pylori [49]. The rpsl HP /cat cassette (Methods) was used to precisely replace cj0596, maintaining the ribosome binding site of the downstream cj0597 gene.