Supplementary Material for: Post-Transcriptional Regulation of the Alginate Biosynthetic Gene algD by the Gac/Rsm System in Azotobacter vinelandii

<i>Azotobacter vinelandii </i>is a soil bacterium that produces the polysaccharide alginate. The two-component system GacS/GacA is required for alginate synthesis since a mutation in <i>gacS </i>or <i>gacA </i>significantly reduced the level of transcripts of <i>algD</i>, the gene encoding GDP-mannose dehydrogenase, a key enzyme of the alginate biosynthetic pathway. In many γ-proteobacteria, GacA homologs control the expression of small regulatory RNAs of the RsmZ/Y/X (CsrB/CsrC) family that interact with RsmA (CsrA) proteins. These proteins bind to their target mRNAs acting as translational repressors. The interaction of Rsm/Csr small RNAs with RsmA/CsrA counteract its repressor activity. In this study, one <i>rsmA</i> gene, seven <i>rsmZ</i> and two <i>rsmY</i> homologs were identified in the <i>A. vinelandii</i> genome. Two of the <i>rsmZ</i> homologs, named <i>rsmZ1</i> and <i>rsmZ2</i>, together with <i>rsmA</i>, were characterized. Northern blot analysis was carried out to show that in <i>A. vinelandii, </i>GacA activates <i>rsmZ1</i> and <i>rsmZ2</i> transcription. We also showed that either overexpression of <i>rsmA</i> or inactivation of <i>rsmZ1</i> or <i>rsmZ2</i> diminished the production of alginate. In addition, interaction of RsmA with RsmZ1, RsmZ2 and the <i>algD </i>mRNA was demonstrated in vitro. These results show that GacS/A regulates alginate biosynthesis by post-transcriptional control of <i>algD </i>expression through the Rsm system.