Spot 42 Small RNA Regulates Arabinose-Inducible araBAD Promoter Activity by Repressing Synthesis of the High-Affinity Low-Capacity Arabinose Transporter

The l -arabinose-inducible araBAD promoter (P BAD ) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where P BAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli , Spot 42 sRNA regulates P BAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF , a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of P BAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the P BAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the P BAD system and has implications for improving its usage for tunable gene expression.