Bacillus subtilis pnpA gene product, polynucleotide phosphorylase (PNPase), is involved in double-strand break (DSB) repair via homologous recombination (HR) or non-homologous end-joining (NHEJ). RecN is among the first responders to localize at the DNA DSBs, with PNPase facilitating the formation of a discrete RecN focus per nucleoid. PNPase, which co-purifies with RecA and RecN, was able to degrade single-stranded (ss) DNA with a 3′ → 5′ polarity in the presence of Mn²⁺ and low inorganic phosphate (Pi) concentration, or to extend a 3′-OH end in the presence dNDP·Mn²⁺. Both PNPase activities were observed in evolutionarily distant bacteria (B. subtilis and Escherichia coli), suggesting conserved functions. The activity of PNPase was directed toward ssDNA degradation or polymerization by manipulating the Pi/dNDPs concentrations or the availability of RecA or RecN. In its dATP-bound form, RecN stimulates PNPase-mediated polymerization. ssDNA phosphorolysis catalyzed by PNPase is stimulated by RecA, but inhibited by SsbA. Our findings suggest that (i) the PNPase degradative and polymerizing activities might play a critical role in the transition from DSB sensing to end resection via HR and (ii) by blunting a 3′-tailed duplex DNA, in the absence of HR, B. subtilis PNPase might also contribute to repair via NHEJ.

The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5'-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5' end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5' double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5' end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5' fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript.

The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover.

Protein Y (PY) is an Escherichia coli cold‐shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA, the gene encoding protein PY, demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNAs and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNAs during cold shock.

Background: Transition from planktonic cells to biofilm is mediated by production of adhesion factors, such as extracellular polysaccharides (EPS), and modulated by complex regulatory networks that, in addition to controlling production of adhesion factors, redirect bacterial cell metabolism to the biofilm mode.Results: Deletion of thepnpgene, encoding polynucleotide phosphorylase, an RNA processing enzyme and a component of the RNA degradosome, results in increased biofilm formation inEscherichia coli. This effect is particularly pronounced in theE.colistrain C-1a, in which deletion of thepnpgene leads to strong cell aggregation in liquid medium. Cell aggregation is dependent on the EPS poly-N-acetylglucosamine (PNAG), thus suggesting negative regulation of the PNAG biosynthetic operonpgaABCDby PNPase. Indeed,pgaABCDtranscript levels are higher in thepnpmutant. Negative control ofpgaABCDexpression by PNPase takes place at mRNA stability level and involves the 5’-untranslated region of thepgaABCDtranscript, which serves as acis-element regulatingpgaABCDtranscript stability and translatability.Conclusions: Our results demonstrate that PNPase is necessary to maintain bacterial cells in the planktonic mode through down-regulation ofpgaABCDexpression and PNAG production.