The transient existence of small RNAs free of binding to the RNA chaperone Hfq is part of the normal dynamic lifecycle of a sRNA. Small RNAs are extremely labile when not associated with Hfq, but the mechanism by which Hfq stabilizes sRNAs has been elusive. In this work we have found that polynucleotide phosphorylase (PNPase) is the major factor involved in the rapid degradation of small RNAs, especially those that are free of binding to Hfq. The levels of MicA, GlmY, RyhB, and SgrS RNAs are drastically increased upon PNPase inactivation in Hfq⁻ cells. In the absence of Hfq, all sRNAs are slightly shorter than their full-length species as result of 3′-end trimming. We show that the turnover of Hfq-free small RNAs is growth-phase regulated, and that PNPase activity is particularly important in stationary phase. Indeed, PNPase makes a greater contribution than RNase E, which is commonly believed to be the main enzyme in the decay of small RNAs. Lack of poly(A) polymerase I (PAP I) is also found to affect the rapid degradation of Hfq-free small RNAs, although to a lesser extent. Our data also suggest that when the sRNA is not associated with Hfq, the degradation occurs mainly in a target-independent pathway in which RNase III has a reduced impact. This work demonstrated that small RNAs free of Hfq binding are preferably degraded by PNPase. Overall, our data highlight the impact of 3′-exonucleolytic RNA decay pathways and re-evaluates the degradation mechanisms of Hfq-free small RNAs.

Summary: Pseudomonas putida is recognized as a very promising strain for industrial application due to its high redox capacity and frequently observed tolerance towards organic solvents. In this research, we studied the metabolic and transcriptional response of P. putida KT2440 exposed to large‐scale heterogeneous mixing conditions in the form of repeated glucose shortage. Cellular responses were mimicked in an experimental setup comprising a stirred tank reactor and a connected plug flow reactor. We deciphered that a stringent response‐like transcriptional regulation programme is frequently induced, which seems to be linked to the intracellular pool of 3‐hydroxyalkanoates (3‐HA) that are known to serve as precursors for polyhydroxyalkanoates (PHA). To be precise, P. putida is endowed with a survival strategy likely to access cellular PHA, amino acids and glycogen in few seconds under glucose starvation to obtain ATP from respiration, thereby replenishing the reduced ATP levels and the adenylate energy charge. Notably, cells only need 0.4% of glucose uptake to build those 3‐HA‐based energy buffers. Concomitantly, genes that are related to amino acid catabolism and β‐oxidation are upregulated during the transient absence of glucose. Furthermore, we provide a detailed list of transcriptional short‐ and long‐term responses that increase the cellular maintenance by about 17% under the industrial‐like conditions tested.

The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome. In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms. In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.