5). Remarkably, the more sensitive liquid-based assay revealed two significant effects. First, as indicated by the change

in the slope of the graphs in Fig. 6, the Δpnp mutant had a longer doubling time in H2O2-containing media, but not in control media. In addition, interfering with degradosome assembly caused a reduced culture density as cultures entered stationary phase. Both of these differences were statistically significant. For reasons not well understood, interfering with degradosome assembly in the Δpnp mutant mirrored the phenotype of the Δpnp mutant strain and suppressed the early stationary phenotype when only degradosome assembly was disrupted (Fig. 5). We also tested growth of these same strains at 4 °C (Fig. 6). Not surprisingly, and in agreement with previously published data (Rosenzweig et al., 2005, 2007), the Δpnp mutant was unable to grow at 4 °C (Fig. 6b) despite PCI32765 relatively normal growth at 28 °C (Fig. 6a). When RNE1-465 was expressed,

there was no effect on the cold-sensitive Acalabrutinib mouse phenotype (Fig. 6). These data strongly suggest that the psychrotropic yersiniae’s ability to grow in the cold depends on PNPase in a degradosome-independent manner. To further evaluate the role that degradosome assembly might be playing in yersiniae stress responses, we challenged the strains with several antibiotics that target protein translation, membrane integrity, and cell wall integrity and found that neither the presence of PNPase nor the ability of the Erythromycin yersiniae degradosome to assemble altered antibiotic susceptibility profiles (data not shown). As we observed that over-expression of RNE1-465 led to a significant reduction in biomass during oxidative stress, but that there was no similar reduction in biomass when expressed in the Δpnp background (Fig. 7), we hypothesized that perhaps PNPase affected expression of the plasmid-encoded RNE1-465. Following a 1.5-h induction

of RNE1-465 in both strains and Western blot analysis, we concluded that the truncated RNE1-465 was expressed similarly in both strains and that PNPase was not modulating RNE1-465 expression levels. More specifically, the Y. pseudotuberculosis + empty vector pBAD24 (WT) and Y. pseudotuberculosis Δpnp + empty vector pBAD24 (pnp) controls did not express RNE1-465 when either induced with 0.02% arabinose or not (lanes 1, 2, 5, and 6). However, the Y. pseudotuberculosis + pBAD-RNE1-465 (RNE) and the Y. pseudotuberculosis Δpnp + pBAD-RNE1-465 (pnp/RNE) both expressed the ~ 52 KDa RNE1-465 when induced with 0.02% arabinose (lanes 3 and 7). Yersinia pseudotuberculosis is a very close relative of the etiological agent of plague, Y. pestis, which diverged from Y. pseudotuberculosis between 15 000–20 000 years ago (Achtman et al., 1999). In fact, their RNase E, PNPase, RhlB and enolase proteins are 97–100% identical. Unlike Y.