Polynucleotide phosphorylase negatively controls biofilm formation by repressing poly-N-acetylglucosamine (PNAG) production in Escherichia coli C

The exoribonuclease polynucleotide phosphorylase (PNPase) encoded by the pnp gene, is a major player in RNA decay and processing. Inactivation of the pnp gene has pleiotropic effects, which include reduced proficiency in homologous recombination and inability to grow at low temperature. Moreover, PNPase affects stability of several small RNAs, thus impacting their ability to regulate their targets. We observed that pnp deletion results in strong cell aggregation and biofilm formation in Escherichia coli C and found that (i) cell aggregation is dependent on the exopolysaccharide poly-N-acetylglucosamine (PNAG), and (ii) pgaABCD transcript levels are higher in the pnp mutant, thus suggesting negative regulation of the PNAG biosynthetic operon pgaABCD by PNPase. A current model of pgaABCD regulation, developed in E. coli K12, maintains that the translation repressor CsrA negatively regulates pgaABCD by binding at multiple sites within the pgaABCD 5’-UTR. Two small RNAs, CsrB and CsrC, positively regulate pgaABCD by sequestering the CsrA protein. A third sRNA, McaS, has been recently involved in positive pgaABCD regulation. In order to get insights into PNPase-dependent pgaABCD regulation mechanism, we assayed luciferase expression using constructs carrying the luxAB reporter genes under the control of pgaABCD regulatory elements (i.e. promoter and 5’-UTR) in pnp+ and pnp- strains. The constructs were also assayed in mutants devoid of pgaABCD in trans regulators (CsrA, CsrB, CsrC and McaS). Surprisingly, we observed that both PNAG production and luciferase expression were increased in mutants lacking CsrB, CsrC or McaS, suggesting that the current model for pgaABCD post-transcriptional regulation may not readily apply to E. coli C. Our results show that PNPase regulates pgaABCD post-transcriptionally through a still unclear mechanism that involves cis determinants laying in the pgaABCD 5’-UTR.