enterocolitica RNase E CTD interacted with both the Y. pseudotuberculosis and Y. enterocolitica RhlB degradosome-associated proteins. We chose looking at RhlB because it was the strongest binding partner for the Y. pseudotuberculosis RNase E CTD tested earlier (Fig. 1). Interestingly, the Y. enterocolitica RNase E CTD appeared to bind as well to the Y. enterocolitica RhlB protein as it did to the
Y. pseudotuberculosis RhlB protein (Fig. 2). As was observed earlier with the Y. pseudotuberculosis RNase E CTD vs. Y. pseudotuberculosis enolase (Fig. 1), the Y. enterocolitica-derived RNase E CTD also interacted poorly with the Y. pseudotuberculosis derived enolase (Fig. 2). The positive control Cytoskeletal Signaling inhibitor Zip–Zip appeared blue (as expected), while the two empty vector negative controls were white (as expected), pKT25RNE vs. pUT18Cempty and pKT25empty vs. pUT18CRhlB
(Fig. 2). To validate our B2H findings (Figs 1 and 2), co-immunoprecipitation (Co-IP) assays, utilizing polyclonal anti-RNase E antibodies fused to Protein G agarose beads, were employed. Immunoprecipitated complexes were resolved by SDS-PAGE and probed with polyclonal anti-RhlB or anti-PNPase antibodies. In agreement with our B2H results, RhlB clearly co-immunoprecipitated with RNase E (Fig. 3). PNPase also appeared to co-immunoprecipitate with RNase E (Fig. 3) as was demonstrated in earlier work (Yang et al., NU7441 price 2008). These B2H and co-IP experiments indicate that the RhlB and enolase are conserved subunits of the degradosome in Yersiniae. The degradosome and PNPase have previously been implicated in various stress responses, including macrophage-induced stress, and cold stress
(see ‘Discussion’). To more completely understand their role during stress, we exposed a Δpnp mutant and a strain over-expressing an rne truncation to a variety of stresses. This rne truncation removed the CTD responsible for interaction with the other degradosome subunits, and its over-expression has previously been shown to interfere with degradosome assembly (Briegel et al., 2006; Yang et al., 2008). As the ability of Y. pseudotuberculosis to respond Smoothened to HCIS was previously shown to be dependent upon PNPase (Rosenzweig et al., 2005, 2007) as well as upon degradosome assembly (Yang et al., 2008), we were curious as to whether degradosome assembly was required for growth under oxidative stress which would be experienced during macrophage encounters. To test this directly, H2O2 liquid- and plate-based experiments were carried out. For plate-based assays, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 4, 5, 10, 20, 50 and 100 mM H2O2 plate concentrations were all evaluated. The Δpnp mutant formed smaller colonies on plates, which was exacerbated by 0.1–0.4 mM H2O2 (Fig. 4). In a manner similar to how E. coli did not require degradosome assembly during oxidative stress (Wu et al., 2009), interfering with degradosome assembly did not affect growth on H2O2-containing plates (Fig. 4b).
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