Josefina Guzmán

Expression of the Azotobacter vinelandii Poly-β-Hydroxybutyrate Biosynthetic phbBAC Operon Is Driven by Two Overlapping Promoters and Is Dependent on the Transcriptional Activator PhbR

The Azotobacter vinelandii phbBAC genes encode the enzymes for poly-beta-hydroxybutyrate (PHB) synthesis. The phbR gene, which is located upstream of and in the opposite direction of phbBAC, encodes PhbR, a transcriptional activator which is a member of the AraC family of activators. Here we report that a mutation in phbR reduced PHB accumulation and transcription of a phbB-lacZ fusion. We also report that phbB is transcribed from two overlapping promoters, p(B)1 and p(B)2. The region corresponding to the -35 region of p(B)1 overlaps the p(B)2 -10 region. In the phbR mutant, expression of phbB from the p(B)1 promoter is significantly reduced, whereas expression from the p(B)2 promoter is slightly increased. Two phbR promoters, p(R)1 and p(R)2, were also identified. Transcription from p(R)2 was shown to be dependent on sigma(S). Six conserved 18-bp sites, designated R1 to R6, are present within the phbR-phbB intergenic region and are proposed to be putative binding targets for PhbR. R1 overlaps the -35 region of the p(B)1 promoter. A model for the regulation of phbB transcription by PhbR is proposed.

The GacS Sensor Kinase Regulates Alginate and Poly-β-Hydroxybutyrate Production in Azotobacter vinelandii

Azotobacter vinelandii produces two polymers: the extracellular polysaccharide alginate and the intracellular polyester poly-beta-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.

The Azotobacter vinelandii alg8 and alg44 genes are essential for alginate synthesis and can be transcribed from an algD-independent promoter

A 2.8-kb DNA region, located immediately downstream of algD, contains the A. vinelandii alg8 and alg44 genes, whose sequences are highly homologous to those of the corresponding Pseudomonas aeruginosa genes. These genes occur on a transcript that does not include algD, and are transcribed from a promoter different from that transcribing algD; this is the fourth promoter described within the alginate biosynthetic gene cluster. alg8 and alg44 mutants were constructed and shown to be completely impaired in alginate production. Alg8 shares 28.20% identity and 38.09% similarity to Azorhizobium caulinodans NodC, a glycosyl transferase catalyzing the formation of beta-1,4 linkages. A topological model is predicted, which supports the idea of Alg8 being the polymerase responsible for alginate synthesis.

Azotobacter vinelandii mutants that overproduce poly-β-hydroxybutyrate or alginate

Azotobacter vinelandii produces two polymers of industrial importance, i.e. alginate and poly-β-hydroxybutyrate (PHB). Alginate synthesis constitutes a waste of substrate when seeking to optimize PHB production and, conversely, synthesis of PHB is undesirable when optimizing alginate production. In this study we evaluated the effect of a mutation in algA, the gene encoding the enzyme that catalyzes the first step of the alginate biosynthetic pathway in the production of PHB. We also evaluated production of alginate in strain AT6 carrying a phbB mutation that impairs PHB synthesis. The algA mutation prevented alginate production and increased PHB accumulation up to 5-fold, determined in milligrams per milligram of protein. Similarly, the phbB mutation increased alginate production up to 4-fold.

Transcriptional organization of the Azotobacter vinelandii algGXLVIFA genes: characterization of algF mutants

Azotobacter vinelandii forms desiccation-resistant cysts which contain a high proportion of the exopolysaccharide alginate in their envelope. We have previously shown that the A. vinelandii alginate biosynthetic genes algA and algL are transcribed from a promoter located somewhere upstream of algL. In this study we sequenced the A. vinelandii algX, algL, algV, algI and algF genes located between algG and algA. We carried out primer extension analysis of the algG, algX and algL genes and detected transcription start sites upstream algG but not upstream algX or algL, implying that algG and algX form part of the previously identified algL-A operon. A promoter upstream algA was also detected; however, transcription of algA exclusively from this promoter is not sufficient for the AlgA levels required for alginate production. An algF mutant (AJ34) was constructed by insertion of the Omega-tetracycline cassette in the non-polar orientation. As expected, AJ34 produced unacetylated alginate. Viability of 35day old cysts formed by strain AJ34, but not of those formed by the wild type, was reduced, indicating that acetylation of alginate plays a role in cyst resistance to desiccation.

Role of Azotobacter vinelandii mucA and mucC gene products in alginate production.

Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor sigma(E) regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the sigma(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.

Characterization of the Azotobacter vinelandii algC gene involved in alginate and lipopolysaccharide production

Azotobacter vinelandii is a soil gamma-proteobacteria that fixes nitrogen and forms desiccation-resistant cysts. The exopolysaccharide alginate is an integral part of the layers surrounding the cysts. Here, we reported the cloning of A. vinelandii algC, encoding the enzyme catalyzing the second step of alginate pathway. We showed that AlgC is involved not only in alginate production, but also in lipopolysaccharide (LPS) synthesis and that it seems to have both phosphomannomutase and phosphoglucomutase activities. The transcriptional analysis of the A. vinelandii algC gene showed that it contained two start sites, one of which was dependent on the alternative sigma factor AlgU/AlgT. This finding explains why alginate biosynthesis is dependent on AlgU activity, since all other alginate biosynthetic genes have been characterized previously and algC is the only alginate structural gene that is directly transcribed by this sigma factor.

Relation between structure and function of Neurospora crassa glutamine synthetase

Abstract Two distinct monomers, α and β participate in the structures of diffe r ent oligomers of Neurospora crassa glutamine synthetase (EC 6. 3. 1. 2). In ammonium-limited cultures a tetrameric form composed mainly of α monomers was found. In excess of nitrogen an octameric form composed mainly from β monomers is the predominant oligomeric state. The presence of both monomers was observed in intermediate oligomeric forms.

Sigma Factor RpoS Controls Alkylresorcinol Synthesis through ArpR, a LysR-Type Regulatory Protein, during Encystment of Azotobacter vinelandii

Azotobacter vinelandii is a bacterium which undergoes a differentiation process leading to the formation of metabolically dormant cysts. During the encystment process, A. vinelandii produces alkylresorcinol lipids (ARs) that replace the membrane phospholipids and are also components of the layers covering the cyst. The synthesis of ARs in A. vinelandii has been shown to occur by the activity of enzymes encoded by the arsABCD operon, which is expressed only during the differentiation process. Also, the production of ARs has been shown to be dependent on the stationary-phase sigma factor RpoS, which is also implicated in the control of the synthesis of other cyst components (i.e., alginate and poly-β-hydroxybutyrate). In this study, we identified ArpR, a LysR-type transcriptional regulator expressed only during encystment that positively regulates arsABCD transcription. We show that this activation is dependent on acetoacetyl-coenzyme A (acetoacetyl-CoA), which might provide a metabolic signal for encystment. We also show that RpoS regulates arsABCD expression through the control of arpR transcription.

Molecular Genetics of the Glutamine Synthetases in Rhizobium Species

Soil bacteria of the genus Rhizobium and Bradyrhizobium establish symbiotic interactions with leguminous plants that result in the formation of specialized structures, the nodules, in which the bacteria differentiate into bacteroids and fix nitrogen. Rhizobial glutamine synthetase (GS) activity is very low in the nodule. The ammonia produced by the bacteroids is exported to the plant cell, where it is assimilated by the GS from the plant, whereas in the free-living state, Rhizobium and Bradyrhizobium species assimilate ammonia for growth. Another characteristic of these species is that they possess two glutamine synthetase isozymes, known as GSI and GSII. A third glutamine synthetase isozyme, called GSIII, has been found in R. meliloti and R. etli.