Lack of functional pnp gene results in increased cell aggregation and 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 processing. In Escherichia coli C the lack of a functional pnp gene results in macroscopic aggregation in glucose-based medium. In order to identify specific adhesion factors involved in cell aggregation, we created a set of isogenic derivatives deficient in the production of curli, cellulose, colanic acid and poly-N-acetylglucosamine (PNAG). Only inactivation of the PNAG biosynthetic genes pgaA and pgaC suppressed cell aggregation phenotype in the pnp mutant, suggesting that PNAG mediated cell aggregation in the absence of PNPase. Real Time PCR experiments showed that in the pnp- strain pgaA gene expression is 12-fold higher than in the wild type. Previous observations suggest that PNAG biosynthetic operon pgaABCD is regulated at post transcriptional level through a mechanism involving the operon 5\u2019-UTR. Thus, in order to test whether PNPase directly regulates pgaABCD transcripts, we constructed plasmids in which the luxAB reporter was placed under the control either of the pgaABCD regulatory elements (promoter and 5\u2019-UTR) or of the promoter region alone (\uf044UTR construct). Our results show that the presence of the 5\u2019-UTR is necessary for PNPase-dependent regulation. We also compared by SDS-PAGE the outer-membrane associated proteins (OMPs) expressed by pnp+ and pnp- E. coli C. Consistent with our previous observations, the PgaA protein, necessary for PNAG secretion, was only found in the outer membrane fraction of the pnp- strain. Finally, PNAG overproduction in the pnp- strain was verified by dot-blot analysis with anti-PNAG specific antibodies. Our results clearly show that lack of PNPase positively affects PNAG production, suggesting that PNPase is a negative regulator of pgaABCD operon expression. The molecular mechanism of pgaABCD regulation by PNPase is currently under investigation