Transcription of the Azotobacter vinelandii algD gene, which encodes GDP-mannose dehydrogenase (the rate-limiting enzyme of alginate synthesis), starts from three sites: p1, p2, and p3. The sensor kinase GacS, a member of the two-component regulatory system, is required for transcription of algD from its three sites during the stationary phase. Here we show that algD is expressed constitutively throughout the growth cycle from the p2 and p3 sites and that transcription from p1 started at the transition between the exponential growth phase and stationary phase. We constructed A. vinelandii strains that carried mutations in gacA encoding the cognate response regulator of GacS and in rpoS coding for the stationary-phase S factor. The gacA mutation impaired alginate production and transcription of algD from its three promoters. Transcription of rpoS was also abolished by the gacA mutation. The rpoS mutation impaired transcription of algD from the p1 promoter and increased it from the p2 E promoter. The results of this study provide evidence for the predominant role of GacA in a regulatory cascade controlling alginate production and gene expression during the stationary phase in A. vinelandii.

Azotobacter vinelandii produces two polymers: the extracellular polysaccharide alginate and the intracellular polyester poly-␤-hydroxybutyrate (PHB). A cosmid clone (pSMU588) from an A. vinelandii gene library diminished alginate production by A. vinelandii mucoid strain ATCC 9046. The nucleotide sequence and predicted amino acid sequence of the locus responsible for the mucoidy suppression revealed 65% identity to Pseudomonas GacS, a transmembrane sensor kinase of the two-component regulators, whose cognate response regulator, GacA, is a global activator regulating several products and virulence factors. Plasmid pMC15, harboring gacS, and a strain carrying a gacS nonpolar mutation were constructed. Either pMC15 or the gacS mutation significantly reduced alginate production and transcription of algD, the gene coding for the key enzyme GDP-mannose dehydrogenase of the alginate biosynthetic pathway. We found that the gacS mutation also reduced PHB accumulation and impaired encystment. Taken together, these data indicate that in A. vinelandii the gacSA global system regulates polymer synthesis.Azotobacter vinelandii is a nitrogen-fixing soil bacterium that undergoes differentiation to form desiccation-resistant cysts and produces two polymers of industrial importance: alginate and poly-␤-hydroxybutyrate (PHB). Both polymers are involved in the encystment process; alginate is a component of the cyst capsule (28), and PHB accumulation correlates with the frequency of cyst formation (30).A. vinelandii has been shown to possess an alginate biosynthetic gene cluster (2,16,20,25,32), organized in three operons, one of which transcribes algD encoding a GDP-mannose dehydrogenase, which converts GDP-mannose to GDP-mannuronic acid, the substrate for alginate polymerization. The algUmucABCD cluster controls alginate production. AlgU is a E homolog (19). The mucA and mucB genes code for negative regulators of AlgU activity. In strain ATCC 9046, transcription of the algD gene is initiated at three sites, one of which is AlgU dependent (2). AlgU activity was shown to be involved in the encystment process independent of its role in alginate synthesis (23).Three enzymes are involved in PHB biosynthesis in A. vinelandii: a ␤-ketothiolase, an acetoacetyl-coenzyme A reductase and a PHB synthetase (18). PHB synthesis in A. vinelandii was shown to be regulated at the level of the ␤-ketothiolase activity (18). The genes encoding the enzymes participating in PHB synthesis in A. vinelandii have not been reported.Members of our group have previously reported the identification of cosmid pSMU588 from a gene bank of nonmucoid strain UW136, which reduced alginate production in A. vinelandii ATCC 9046 (19). The characterization of pSMU588 reported here allowed us to identify a regulatory gene homologous to Pseudomonas gacS, coding for a sensor kinase of the two-component regulatory systems. This study also provides evidence for GacS playing a role as a regulator of alginate and PHB synthesis in A. vinelandii. a Alginate production was determin...

In Azotobacter vinelandii the two-component GacS/GacA system is required for synthesis of polyhydroxybutyrate (PHB) and of the exopolysaccharide alginate. The RsmA protein was shown to interact with the alginate biosynthetic algD mRNA, acting as a translational repressor, and GacA was found to activate transcription of the rsmZ1 and rsmZ2 genes that encode small RNAs interacting with RsmA to counteract its repressor activity. The phbBAC operon encodes the enzymes of PHB synthesis and is activated by the transcriptional regulator PhbR. This study shows that GacA is required for transcription of one rsmY and seven rsmZ1-rsmZ7 genes present in the A. vinelandii genome, and that inactivation of rsmA results in increased PHB production. Transcriptional and translational phbR-gusA gene fusions were used to show that the gacA mutation negatively affected the expression of the phbR gene at the translational level. We also demonstrated an in vitro interaction of RsmA with RNAs corresponding to phbB and phbR mRNA leaders, and showed that the stability of phbR and phbB mRNAs is increased in the rsmA mutant. Taken together these results indicate that in A. vinelandii, RsmA post-transcriptionally represses the expression of PhbR.

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.

During encystment of Azotobacter vinelandii, a family of alkylresorcinols (ARs) and alkylpyrones (APs) are synthesized. In the mature cyst, these lipids replace the membrane phospholipids and are also components of the layers covering the cyst. In this study, A. vinelandii strains unable to synthesize ARs were isolated after mini-Tn5 mutagenesis. Cloning and nucleotide sequencing of the affected loci revealed the presence of the transposons within the arsA gene of the previously reported arsABCD gene cluster, which encodes a type I fatty acid synthase. A mutant strain (SW-A) carrying an arsA mutation allowing transcription of arsBCD was constructed and shown to be unable to produce ARs, indicating that the ArsA protein is essential for the synthesis of these phenolic lipids. Transcription of arsA was induced 200-fold in cells undergoing encystment, but only 14-fold in aged cultures of A. vinelandii, in accordance with AR synthesis and cyst formation percentages under the two conditions. Although it was previously reported that the inactivation of arsB abolishes AR synthesis and results in a failure in encystment, the arsA mutants were able to form cysts resistant to desiccation. These data indicate that ARs play a structural role in the exine layer of the cysts, but they are not essential for either cyst formation or for desiccation resistance.