The study of alginate biosynthesis, the exopolysaccharide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, might lead to different biotechnological applications. Here we report the cloning of A. vinelandii algA, the gene coding for the bifunctional enzyme phosphomannose isomerase-guanosine diphospho-o-mannose pyrophosphorylase (PMI-GMP). This gene was selected by the complementation for xanthan gum production of Xanthomonas campestris pv. campestris xanB-mutants, which lack this enzymatic activity. The complementing cosmid clones selected, besides containing algA, presented a gene coding for an alginate lyase activity (algL), and some of them also contained algD which codes for GDP-mannose dehydrogenase. We present here the characterization of the A. vinelandii chromosomal region comprising algD and its promoter region, algA and algL, showing that, as previously reported for P. aeruginosa, A. vinelandii has a cluster of the biosynthetic alginate genes. We provide evidence for the presence of an algD-independent promoter in this region which transcribes at least algL and algA, and which is regulated in a manner that differs from that of the algD promoter.

Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor E regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the E factor (AlgU) is essential for alginate production. In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes. In this work, we studied the transcriptional regulation of the A. vinelandii algU-mucABCD gene cluster, as well as the role of the mucA and mucC gene products in alginate production. Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production.

Azotobacter vinelandii is a nitrogen-fixing soil bacterium that undergoes differentiation to form cysts resistant to desiccation. Upon encystment, this bacterium becomes non-motile. As in enteric bacteria, motility in A. vinelandii occurs through the use of peritrichous flagella. Pseudomonas aeruginosa, a phylogenetically close relative of A. vinelandii, possesses a single polar flagellum. The FlhDC proteins are the master regulators of flagella and motility in enterobacteria, whereas FleQ is the master regulator in P. aeruginosa, and it is under AlgU (sigmaE) negative control. At present, nothing is known about the organization and expression of flagella genes in A. vinelandii. Here, we identified the flagella gene cluster of this bacterium. Homologues of the master regulatory genes flhDC and fleQ are present in A. vinelandii. Inactivation of flhDC, but not fleQ, impaired flagella biogenesis and motility. We present evidence indicating that a negative effect of the AlgU sigma factor on flhDC expression causes loss of motility in A. vinelandii, and that CydR (a homologue of Fnr) is under AlgU control and has a negative effect on flhDC expression. Taken together, these results suggest the existence of a cascade consisting of AlgU and CydR that negatively controls expression of flhDC; the results also suggest that the block in flagella synthesis under encystment conditions centres on flhDC repression by the AlgU–CydR cascade.

Random Tn5 mutagenesis over Azotobacter vinelandii mucoid strain ATCC 9046 produced strain LA21, a non-mucoid, non-encysting mutant, carrying the Tn5 insertion within a gene homologous to algK from Pseudomonas aeruginosa encoding a periplasmic protein. algK, algJ and algG were shown to be transcribed as part of the palg8-alg44-algK-algJ-algG operon. A non-polar algK mutant was constructed and showed a non-mucoid phenotype, indicating that algK is essential for alginate production.

Random Tn5 mutagenesis over Azotobacter vinelandii mucoid strain ATCC 9046 produced strain LA21, a non-mucoid, non-encysting mutant, carrying the Tn5 insertion within a gene homologous to algK from Pseudomonas aeruginosa encoding a periplasmic protein. algK, algJ and algG were shown to be transcribed as part of the palg8-alg44-algK-algJ-algG operon. A non-polar algK mutant was constructed and showed a non-mucoid phenotype, indicating that algK is essential for alginate production.