In this assay, all the three pnp mutants revealed a comparable retardation in growth

(Fig. 4a). As the expression of nlpI was not diminished in any of the three pnp mutants (Fig. 2a), the impaired cold acclimatization could not originate from nlpI, and so in subsequent experiments, we only used the ∆pnp mutant (SFR228). The ∆nlpI mutant also showed a reduced ability to grow at 15 °C comparable to the pnp mutants (Fig. 4a). As pnp expression was unaffected in this mutant, it infers that pnp and nlpI contribute individually to growth of S. Typhimurium at 15 °C. Further evidence to support this view was the observed slower growth for the pnp–nlpI double mutant (SFR394) (Fig. 4a). Complementation of pnp (pMC109) or nlpI (pSFR04) in the respective single mutants almost buy STA-9090 restored

normal growth at 15 °C (Fig. 4b). However, the introduction of either pMC109 or pSFR04 into the pnp–nlpI double mutant resulted in only a Pexidartinib partial restoration of growth at 15 °C. An almost complete restoration of growth was achieved when the pnp–nlpI double mutant was complemented with both nlpI and pnp (Fig. 4c). A second cold acclimatization assay was performed comparing growth on Luria agar plates incubated at either 15 or 37 °C. The ∆pnp, ∆nlpI and pnp–nlpI double mutants were assayed, and the results were comparable to the broth assay at 15 °C (Figs 4 and 5). The ∆pnp and ∆nlpI mutants both showed a restricted recovery when transferred to 15 °C (Fig. 5). In this assay, the growth defect of the pnp–nlpI double mutant appeared more pronounced in relation to either of the single mutants (Fig. 5). Similar to the broth assay, we also observed a restoration of growth when single mutants were complemented with the plasmids expressing the respective pnp or nlpI genes (Fig. 5). However, to restore any significant growth in the

pnp–nlpI double mutant, plasmids coding for both pnp and nlpI had to be introduced (Fig. 5). The contribution of the deaD gene to the cold acclimation response was also determined by observing the growth of mutant SFR456 (∆deaD) in both the broth and plate assays. The mutant revealed a marked growth defect at 15 °C (Figs 4d and 5), but this growth defect was, however, not complemented Aldehyde dehydrogenase by either pnp or nlpI. Altered folding as well as a controlled degradation and stabilization of ribonucleic acids constitutes important elements of bacterial adaptation to altered temperatures (Hurme & Rhen, 1998; Giuliodori et al., 2010). Such alterations also include helicases, RNA chaperons and ribonucleases (Phadtare & Severinov, 2010). Alterations in RNA folding may furthermore act as an endogenous post-transcriptional control of gene expression (Beran & Simons, 2001; Giuliodori et al., 2010). Expression and post-transcriptional regulation of PNPase have been thoroughly detailed in E. coli and serve as a model for temperature-associated post-translational gene regulation. Transcription of E.