Thus, while our results support the role of CsrA as a major regul

Thus, while our results support the role of CsrA as a major regulator of pgaABCD expression, they also suggest that the current model for pgaABCD post-transcriptional regulation, which is based on data obtained in E. coli K-12, PS-341 molecular weight may not readily apply to E. coli C. The additive effect observed upon combining Δpnp 751 with deletions targeting different sRNAs suggest that PNPase and the sRNAs may act independently on pgaABCD regulation. Figure 5 pgaABCD expression in mutants defective for CsrA-dependent regulation elements and/or PNPase. See Table 1 for the complete

list of strains used in these experiments. A Δpnp ΔcsrA double mutant could not be obtained. A. pgaABCD mRNA expression. RNA was extracted from cultures grown in M9Glu/sup to OD600 = 0.8 and analyzed by quantitative RT-PCR as described in Methods. White bars, pnp + strains; dark grey, Δpnp strains. The “Relative expression” values indicated in the graph are the average of three independent experiments, each performed in duplicate, and standard deviations

are shown. The overall p-value obtained by ANOVA is indicated in the graph. Letters provide the representation for posthoc comparisons. According to posthoc analysis (Tukey’s HSD, p < 0.05), means sharing the same letter are not significantly different from each other. B. PNAG production. Crude extracts from overnight cultures were filtered onto RGFP966 chemical structure a nitrocellulose membrane, and PNAG detection was carried out using polyclonal PNAG specific antibodies as detailed in Materials and Methods. PNAG determination was repeated at least four times on three independent EPS extractions with comparable results; data shown are from a typical experiment. Discussion In this report, we have shown that PNPase negatively regulates the production

of the adhesion factor PNAG, thus maintaining the bacterial cells in a planktonic state (Figures 1 3) when grown at 37°C in supplemented minimal medium. Our results are in line with previous Neratinib in vitro works by other groups connecting PNPase to regulation of outer membrane proteins in E. coli[59] and curli production in Salmonella [60]. Thus, PNPase seems to play a pivotal role in regulating the composition of cell envelope and the production of adhesion surface determinants. PNPase-dependent regulation of PNAG production requires its ribonuclease activity, as suggested by the observation that overexpression of RNase II can compensate for lack of PNPase (Figure 1B). Cell aggregation in the absence of PNPase is suppressed by RNase II, but not by RNase R. This reminds what previously showed for cold sensitivity in pnp mutants, which is also solely suppressed by RNase II [61] and reinforces the notion that, albeit partially redundant, RNA degradation pathways possess a certain degree of specificity and are not fully interchangeable [62].

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