A Subset of Exoribonucleases Serve as Degradative Enzymes for pGpG in c-di-GMP Signaling

Abstract

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that regulates processes, such as biofilm formation and virulence. During degradation, c-di-GMP is first linearized to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG) and subsequently hydrolyzed to two GMPs by a previously unknown enzyme, which was recently identified in <i>Pseudomonas aeruginosa</i> as the 3'-to-5' exoribonuclease oligoribonuclease (Orn). Mutants of <i>orn</i> accumulated pGpG, which inhibited the linearization of c-di-GMP. This product inhibition led to elevated c-di-GMP levels, resulting in increased aggregate and biofilm formation. Thus, the hydrolysis of pGpG is crucial to the maintenance of c-di-GMP homeostasis. How species that utilize c-di-GMP signaling but lack an <i>orn</i> ortholog hydrolyze pGpG remains unknown. Because Orn is an exoribonuclease, we asked whether pGpG hydrolysis can be carried out by genes that encode protein domains found in exoribonucleases. From a screen of these genes from <i>Vibrio cholerae</i> and <i>Bacillus anthracis</i>, we found that only enzymes known to cleave oligoribonucleotides (<i>orn</i> and <i>nrnA</i>) rescued the <i>P. aeruginosa</i> Δ<i>orn</i> mutant phenotypes to the wild type. Thus, we tested additional RNases with demonstrated activity against short oligoribonucleotides. These experiments show that only exoribonucleases previously reported to degrade short RNAs (<i>nrnA</i>, <i>nrnB</i>, <i>nrnC</i>, and <i>orn</i>) can also hydrolyze pGpG. A <i>B. subtilis</i><i>nrnA nrnB</i> mutant had elevated c-di-GMP, suggesting that these two genes serve as the primary enzymes to degrade pGpG. These results indicate that the requirement for pGpG hydrolysis to complete c-di-GMP signaling is conserved across species. The final steps of RNA turnover and c-di-GMP turnover appear to converge at a subset of RNases specific for short oligoribonucleotides.<b>IMPORTANCE</b> The bacterial bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling molecule regulates complex processes, such as biofilm formation. c-di-GMP is degraded in two-steps, linearization into pGpG and subsequent cleavage to two GMPs. The 3'-to-5' exonuclease oligoribonuclease (Orn) serves as the enzyme that degrades pGpG in <i>Pseudomonas aeruginosa</i> Many phyla contain species that utilize c-di-GMP signaling but lack an Orn homolog, and the protein that functions to degrade pGpG remains uncharacterized. Here, systematic screening of genes encoding proteins containing domains found in exoribonucleases revealed a subset of genes encoded within the genomes of <i>Bacillus anthracis</i> and <i>Vibrio cholerae</i> that degrade pGpG to GMP and are functionally analogous to Orn. Feedback inhibition by pGpG is a conserved process, as strains lacking these genes accumulate c-di-GMP.