Polyclonal rabbit anti-PNPase, anti-RNase E, and/or anti-RhlB helicase antibodies were used to probe RNase E complexes or whole-cell extracts (at a dilution of 1 : 3000) for 1 h at room temperature. A previously published protocol (Rosenzweig et al., 2005) was used Protein Tyrosine Kinase inhibitor with several modifications. In short, 10-fold serial dilutions of saturated bacterial cultures were spotted in duplicate in ~ 2 μL volumes (using a pronger) on 2 LB agar (Difco) plates containing 100 μg mL−1 ampicillin (Sigma) and 0.02% arabinose (Sigma). One plate was placed at 30 °C, while the other was

placed at 4 °C and monitored for 11-day period. Alternatively, cultures were streaked out on the aforementioned plates and monitored for their growth over 11-day period. Previously published protocols (Wu et al., 2009) were employed. In short, saturated cultures were diluted, and subcultures of OD600 nm ~ 0.2 were established in triplicate 100 μL volumes of LB medium (Difco) in 96-well plates. Following static growth at 30 °C for 1.0 h (with the appropriate antibiotic added and arabinose at 0.02%), a stock 0.88 M H2O2 was added to the various cultures yielding H2O2 concentrations of either 0, 20, 50, or 100 mM, respectively. Growth in the liquid cultures was monitored every 30 min over a 12-h period with

continuous agitation. Growth curves were plotted, and the Student’s t-test was used to determine statistical significance with P values < 0.05 considered significant. For plate-based H2O2 assays, 10-fold serial dilutions of saturated bacterial cultures were spotted in duplicate (using a pronger) in ~ 2 μL volumes find more on 2 LB agar (Difco) plates containing 100 μg mL−1 Ampicillin (Sigma) and 0.02% Arabinose (Sigma). Plate H2O2 concentrations were 0, 0.4, 1, 2, 4, and 100 mM. In an attempt to further identify Y. pseudotuberculosis

degradosome constituents, we employed the B2H assay selleck inhibitor (Karimova et al., 1998) to determine whether RhlB and enolase also associate with the RNase E CTD. In this B2H assay, interaction between two proteins results in transcription of the Lac operon and thus blue color on plates containing X-gal. Our data indicated that the RNase E CTD interacted very strongly with full-length RhlB helicase as evidenced by intensely blue colonies (Fig. 1c). In fact, the intensity of blue mirrored that of the positive control Zip–Zip (compare c to b). Blue colonies also appeared when PNPase interacted with RNase E CTD (d); however, the overall intensity of blue was less than that of an RhlB–RNase E CTD interaction (compare d to b). Little interaction occurred between enolase and the RNase E CTD, as evidenced by weekly blue colonies (e). All experimental colonies observed appeared bluer than the empty vector negative control, pKT25RNE-CTD vs. pUT18Cempty vector (compare all to a). In addition to evaluating degradosome interaction of Y. pseudotuberculosis proteins, we also evaluated degradosome interaction of closely related Y.