Antisense RNA that affects Rhodopseudomonas palustris quorum-sensing signal receptor expression

Quorum sensing in the bacterium Rhodopseudomonas palustris involves the RpaI signal synthase, which produces p-coumaroyl-homoserine lactone (pC-HSL) and RpaR, which is a pC-HSL-dependent transcriptional activator. There is also an antisense rpaR transcript (asrpaR) of unknown function. Recent RNAseq studies have revealed that bacterial antisense RNAs are abundant, but little is known about the function of these molecules. Because asrpaR expression is quorum sensing dependent, we sought to characterize its production and function. We show that asrpaR is approximately 300-600 bases and is produced in response to pC-HSL and RpaR. There is an RpaR-binding site centered 51.5 bp from the mapped asrpaR transcript start site. We show that asrpaR overexpression reduces RpaR levels, rpaI expression, and pC-HSL production. We also generated an asrpaR mutant, which shows elevated RpaR levels, and elevated rpaI expression. Thus, asrpaR inhibits rpaR translation, and this inhibition results in suppression of RpaR-dependent rpaI expression and, thus, pC-HSL production. The R. palustris asrpaR represents an antisense RNA for which an activity can be measured and for which a distinct regulatory circuit related to a function is elucidated. It also represents yet another subtle regulatory layer for acyl-homoserine lactone quorum-sensing signal-responsive transcription factors.