Hfq-dependent artificial regulatory small RNAs for targeted gene expression control in the nitrogen-fixing...

Artificial trans-encoded small RNAs (atsRNAs), which repress translation by base pairing with target mRNAs, have proven to be powerful tools for regulation of gene expression for reconstructing cell factories and genetic circuits. Currently, rationally designed atsRNAs use mostly E. coli natural trans-acting sRNAs with an Hfq-binding structure and a Rho-independent terminator as scaffolds. However, owing to the structural differences in Hfq proteins between E. coli and other bacteria, the inhibitory efficacy of atsRNAs may be influenced by heterologous sRNA scaffolds, highlighting the necessity of screening native sRNA scaffolds in various bacteria to develop functional synthetic sRNAs. The root-associated bacterium Pseudomonas stutzeri A1501 is a well-studied strain regarding nitrogen fixation, yet post-transcriptional regulatory platforms for this species remain scarce. In this study, we describe the development of an atsRNA platform by employing the natural Hfq-dependent sRNA CrcZ scaffold from the nitrogen-fixing strain P. stutzeri A1501, which is conserved in the Pseudomonas strain. On the basis of the CrcZ scaffold, two atsRNAs targeting the master nitrogen-fixing regulatory complex encoded by the nifLA operon were developed and found to effectively suppress nifLA gene expression at the posttranscriptional level in P. stutzeri A1501. Further studies demonstrated that the 5' untranslated region of target mRNAs and five Hfq-binding sites are optimal features for designing atsRNAs using CrcZ as a scaffold. The well-established effective design principle for synthetic sRNAs with the CrcZ sRNA scaffold will enable more rational and efficient engineering of synthetic gene networks in Pseudomonas strains.