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

Abstract

Azotobacter vinelandii is a soil bacterium that produces the polysaccharide alginate. The two-component system GacS/GacA is required for alginate synthesis since a mutation in gacS or gacA significantly reduced the level of transcripts of algD, 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 rsmA gene, seven rsmZ and two rsmY homologs were identified in the A. vinelandii genome. Two of the rsmZ homologs, named rsmZ1 and rsmZ2, together with rsmA, were characterized. Northern blot analysis was carried out to show that in A. vinelandii, GacA activates rsmZ1 and rsmZ2 transcription. We also showed that either overexpression of rsmA or inactivation of rsmZ1 or rsmZ2 diminished the production of alginate. In addition, interaction of RsmA with RsmZ1, RsmZ2 and the algD mRNA was demonstrated in vitro. These results show that GacS/A regulates alginate biosynthesis by post-transcriptional control of algD expression through the Rsm system.

In Silico Identification and Experimental Characterization of Regulatory Elements Controlling the Expression of the Salmonella csrB and csrC Genes

“…A similar IR sequence has been found upstream from the promoters of csrB and csrC and of rsmB, rsmX, rsmY, and rsmZ genes from different gammaproteobacteria, which are regulated by BarA/SirA orthologous (31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Moreover, a computational sequence analysis, including a wide number of characterized and predicted csrB, csrC, rsmB, rsmX, rsmY, and rsmZ genes revealed some differences between the IR sequences of csrB (TGTGAGAG ATCTCTTACA), csrC (TGTGAGNCATNGNCCATN), and rsmX, rsmY, and rsmZ (TGTAAGNNATNGCTTACA) genes (36).…”

“…The reactions were analyzed in a polyacrylamide-urea gel in parallel with sequencing reactions (indicated by GATC) which were generated with the same template. The black triangle above the gel represents increasing amounts (40,60,80, and 100 nM) of IHF. Reaction without DNase I treatment and reaction treated with DNase I but lacking IHF are indicated as "-" and "ϩ", respectively.…”

RsmA post-transcriptionally controls PhbR expression and polyhydroxybutyrate biosynthesis in Azotobacter vinelandii

“…Except for rsmY2, the GacA-binding sequence TGTAAG-N 6 -CTTACA is present in the promoter regions of all rsm sRNA genes, and transcription of rsmZ1 and rsmZ2 has been confirmed to be dependent on GacA (Manzo et al, 2011). The A. vinelandii Gac-Rsm system has been shown to control expression of algD, encoding the key enzyme of the alginate biosynthetic pathway in strain AEIV.…”

“…A single rsmA gene, seven rsmZ (rsmZ1-rsmZ7) and two rsmY (rsmY1-rsmY2) homologues were previously identified in the A. vinelandii genomes of alginate-producing (mucoid) strain AEIV and the non-mucoid DJ strain (Manzo et al, 2011). Except for rsmY2, the GacA-binding sequence TGTAAG-N 6 -CTTACA is present in the promoter regions of all rsm sRNA genes, and transcription of rsmZ1 and rsmZ2 has been confirmed to be dependent on GacA (Manzo et al, 2011).…”

Roles of Cyclic Di-GMP and the Gac System in Transcriptional Control of the Genes Coding for the Pseudomonas putida Adhesins LapA and LapF

“…This results in a regulatory cascade that involves derepression of the translation of target genes. In Azotobacter vinelandii this system regulates the synthesis of alginate (16), an exopolysaccharide that is part of the extracellular matrix of biofilms in different bacteria. In P. fluorescens, it participates in the regulatory network controlling flagellar motility (17).…”