Selective mRNA degradation by polynucleotide phosphorylase in cold shock adaptation in Escherichia coli

Upon cold shock, Escherichia coli cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A pnp mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15 degrees C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.     

The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5°C)

The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent adaptations. To investigate Y . enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5°C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy-defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp . Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp , including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5°C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y . enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6-fold higher at 5°C compared with 30°C. A similarly enhanced level of PNPase at 5°C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold-inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.     

RNase activity of polynucleotide phosphorylase is critical at low temperature in Escherichia coli and is complemented by RNase II

In Escherichia coli, the cold shock response is exerted upon a temperature change from 37°C to 15°C and is characterized by induction of several cold shock proteins, including polynucleotide phosphorylase (PNPase), during acclimation phase. In E. coli, PNPase is essential for growth at low temperatures; however, its exact role in this essential function has not been fully elucidated. PNPase is a 3′-to-5′ exoribonuclease and promotes the processive degradation of RNA. Our screening of an E. coli genomic library for an in vivo counterpart of PNPase that can compensate for its absence at low temperature revealed only one protein, another 3′-to-5′ exonuclease, RNase II. Here we show that the RNase PH domains 1 and 2 of PNPase are important for its cold shock function, suggesting that the RNase activity of PNPase is critical for its essential function at low temperature. We also show that its polymerization activity is dispensable in its cold shock function. Interestingly, the third 3′-to-5′ processing exoribonuclease, RNase R of E. coli, which is cold inducible, cannot complement the cold shock function of PNPase. We further show that this difference is due to the different targets of these enzymes and stabilization of some of the PNPase-sensitive mRNAs, like fis, in the Δpnp cells has consequences, such as accumulation of ribosomal subunits in the Δpnp cells, which may play a role in the cold sensitivity of this strain.     

Genetic analysis of polynucleotide phosphorylase structure and functions

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.     

Cold shock and cold acclimation proteins in the psychrotrophic bacterium Arthrobacter globiformis SI55.

The psychrotrophic bacterium Arthrobacter globiformis SI55 was grown at 4 and 25 degrees C, and the cell protein contents were analyzed by two-dimensional electrophoresis. Cells subjected to cold shocks of increasing magnitude were also analyzed. Correspondence analysis of protein appearance distinguished four groups of physiological significance. Group I contained cold shock proteins (Csps) overexpressed only after a large temperature downshift. Group II contained Csps with optimal expression after mild shocks. Group III contained proteins overexpressed after all cold shocks. These last proteins were also overexpressed in cells growing at 4 degrees C and were considered to be early cold acclimation proteins (Caps). Group IV contained proteins which were present at high concentrations only in 4 degrees C steady-state cells and appeared to be late Caps. A portion of a gene very similar to the Escherichia coli cspA gene (encoding protein CS7.4) was identified. A synthetic peptide was used to produce an antibody which detected a CS7.4-like protein (A9) by immunoblotting two-dimensional electrophoresis gels of A. globiformis SI55 total proteins. Unlike mesophilic microorganisms, this CS7.4-like protein was still produced during prolonged growth at low temperature, and it might have a particular adaptive function needed for balanced growth under harsh conditions. However, A9 was induced at high temperature by chloramphenicol, suggesting that CS7.4-like proteins have a more general role than their sole implication in cold acclimation processes.     

capA, a cspA-like gene that encodes a cold acclimation protein in the psychrotrophic bacterium Arthrobacter globiformis SI55.

By use of Arthrobacter globiformis SI55, a psychrotrophic bacterium capable of growth between -5 and +32 degrees C, we cloned and sequenced capA, a gene homologous to cspA encoding the major cold shock protein in Escherichia coli. The deduced protein sequence has a high level of identity with the sequences of other CspA-related proteins from various sources, and no particular residue or domain that could be specific to cold-adapted microorganisms emerged. We show that CapA was produced very rapidly following cold shock, but unlike its mesophilic counterparts, it was still expressed during prolonged growth at low temperature. Its synthesis is regulated at the translational level, and we showed that growth resumption following a temperature downshift correlated with CapA expression. Transient inhibitions in protein synthesis during the first stages of the cold shock response severely impaired the subsequent acclimation of A. globiformis SI55 to low temperature and delayed CapA expression. The cold shock response in A. globiformis SI55 is an adaptative process in which CapA may play a crucial role. We suggest that low-temperature acclimation is conditioned mainly by the ability of cells to restore an active translational machinery after cold shock in a process that may be different from that present in mesophiles.     

Complementation analysis of the cold-sensitive phenotype of the Escherichia coli csdA deletion strain

The cold shock response of Escherichia coli is elicited by downshift of temperature from 37 degrees C to 15 degrees C and is characterized by induction of several cold shock proteins, including CsdA, during the acclimation phase. CsdA, a DEAD-box protein, has been proposed to participate in a variety of processes, such as ribosome biogenesis, mRNA decay, translation initiation, and gene regulation. It is not clear which of the functions of CsdA play a role in its essential cold shock function or whether all do, and so far no protein has been shown to complement its function in vivo. Our screening of an E. coli genomic library for an in vivo counterpart of CsdA that can compensate for its absence at low temperature revealed only one protein, RhlE, another DEAD-box RNA helicase. We also observed that although not detected in our genetic screening, two cold shock-inducible proteins, namely, CspA, an RNA chaperone, and RNase R, an exonuclease, can also complement the cold shock function of CsdA. Interestingly, the absence of CsdA and RNase R leads to increased sensitivity of the cells to even moderate temperature downshifts. The correlation between the helicase activity of CsdA and the stability of mRNAs of cold-inducible genes was shown using cspA mRNA, which was significantly stabilized in the DeltacsdA cells, an effect counteracted by overexpression of wild-type CsdA or RNase R but not by that of the helicase-deficient mutant of CsdA. These results suggest that the primary role of CsdA in cold acclimation of cells is in mRNA decay and that its helicase activity is pivotal for promoting degradation of mRNAs stabilized at low temperature.