Daniel Segura

Genome Sequence of Azotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes

Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.

Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii

Several aspects of alginate and PHB synthesis in Azotobacter vinelandii at a molecular level have been elucidated in articles published during the last ten years. It is now clear that alginate and PHB synthesis are under a very complex genetic control. Genetic modification of A. vinelandii has produced a number of very interesting mutants which have particular traits for alginate production. One of these mutants has been shown to produce the alginate with the highest mean molecular mass so far reported. Recent work has also shed light on the factors determining molecular mass distribution; the most important of these being identified as; dissolved oxygen tension and specific growth rate. The use of specific mutants has been very useful for the correct analysis and interpretation of the factors affecting polymerization. Recent scale-up/down work on alginate production has shown that oxygen limitation is crucial for producing alginate of high molecular mass, a condition which is optimized in shake flasks and which can now be reproduced in stirred fermenters. It is clear that the phenotypes of mutants grown on plates are not necessarily reproducible when the strains are tested in lab or bench scale fermenters. In the case of PHB, A. vinelandii has shown itself able to produce relatively large amounts of this polymer of high molecular weight on cheap substrates, even allowing for simple extraction processes. The development of fermentation strategies has also shown promising results in terms of improving productivity. The understanding of the regulatory mechanisms involved in the control of PHB synthesis, and of its metabolic relationships, has increased considerably, making way for new potential strategies for the further improvement of PHB production. Overall, the use of a multidisciplinary approach, integrating molecular and bioengineering aspects is a necessity for optimizing alginate and PHB production in A. vinelandii.

Computational and experimental analysis of redundancy in the central metabolism of Geobacter sulfurreducens.

Previous model-based analysis of the metabolic network of Geobacter sulfurreducens suggested the existence of several redundant pathways. Here, we identified eight sets of redundant pathways that included redundancy for the assimilation of acetate, and for the conversion of pyruvate into acetyl-CoA. These equivalent pathways and two other sub-optimal pathways were studied using 5 single-gene deletion mutants in those pathways for the evaluation of the predictive capacity of the model. The growth phenotypes of these mutants were studied under 12 different conditions of electron donor and acceptor availability. The comparison of the model predictions with the resulting experimental phenotypes indicated that pyruvate ferredoxin oxidoreductase is the only activity able to convert pyruvate into acetyl-CoA. However, the results and the modeling showed that the two acetate activation pathways present are not only active, but needed due to the additional role of the acetyl-CoA transferase in the TCA cycle, probably reflecting the adaptation of these bacteria to acetate utilization. In other cases, the data reconciliation suggested additional capacity constraints that were confirmed with biochemical assays. The results demonstrate the need to experimentally verify the activity of key enzymes when developing in silico models of microbial physiology based on sequence-based reconstruction of metabolic networks.

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.

Enzyme INtr, NPr and IIANtr Are Involved in Regulation of the Poly-β-Hydroxybutyrate Biosynthetic Genes in Azotobacter vinelandii

The ptsP, ptsO, and ptsN genes encode Enzyme INtr, NPr, and enzyme IIANtr (IIANtr) proteins of the nitrogen-related phosphotransferase system. These proteins participate in a phosphoryl transfer chain in several bacteria, where IIANtr appears to be the terminal phosphoryl acceptor. Inactivation of the ptsP gene in Azotobacter vinelandii was previously shown to reduce poly-β-hydroxybutyrate (PHB) production. Therefore, the question of a role of the ptsO and ptsN gene products in PHB synthesis was raised. In this work we constructed strains carrying mutations in the ptsO and ptsN genes and tested their effects on PHB accumulation. In the ptsO mutant, PHB accumulation diminished as in the ptsP mutant, while the ptsN mutant accumulated more PHB than the wild-type strain. The negative effects of the ptsP and ptsO mutations on PHB accumulation was suppressed by the ptsN mutation, and a H68A mutation in the phosphorylatable site of IIANtr, impaired PHB accumulation similar to the ptsP mutation. The ptsP and ptsO mutations negatively affected transcription of the phbBAC biosynthetic operon and of the phbR gene coding for a transcriptional activator of phbBAC, whereas the ptsN mutation increased expression of this operon. Taken together our data provide genetic evidence suggesting that the non-phosphorylated form of IIANtr is involved in negative regulation of phbR and phbBAC expression in A. vinelandii.

Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis

The lipids poly-β-hydroxybutyrate (PHB) and alkylresorcinols are the major metabolic products of Azotobacter vinelandii cysts. Cysts are formed in less than 0.01% of late stationary phase cells grown on sucrose. Culturing vegetative cells in n-butanol or β-hydroxybutyrate induces encystment. After induction of encystment, PHB rapidly accumulates in large granules. Then, the cells begin the synthesis of alkylresorcinols that replace the phospholipids in the membranes and are components of the exine, the outer layer of the cyst envelope. Vegetative cells do not synthesize alkylresorcinols. We report here the effect of mutations in the phbBAC operon, coding for the enzymes of the PHB biosynthetic pathway, on the synthesis of alkylresorcinols and cyst formation. The phb mutations did not impair the capacity to form mature cysts. However, the cysts formed by these strains posses a thicker exine layer and a higher content of alkylresorcinols than the cysts formed by the wild-type strain. A blockage of PHB synthesis caused by phb mutations resulted in the synthesis of alkylresorcinols and encystment even under non-inducing conditions. We propose that, as a consequence of the blockage in the PHB biosynthetic pathway, the acetyl-CoA and reducing power pools are increased causing the shift to lipid metabolism required for the synthesis of alkylresorcinols and cyst formation.

Biotechnological strategies to improve production of microbial poly-(3-hydroxybutyrate): a review of recent research work

Poly‐(3‐hydroxybutyrate) [P(3HB)] is a polyester synthesized as a carbon and energy reserve material by a wide number of bacteria. This polymer is characterized by its thermo‐plastic properties similar to plastics derived from petrochemical industry, such as polyethylene and polypropylene. Furthermore, P(3HB) is an inert, biocompatible and biodegradable material which has been proposed for several uses in medical and biomedical areas. Currently, only few bacterial species such as Cupriavidus necator, Azohydromonas lata and recombinant Escherichia coli have been successfully used for P(3HB) production at industrial level. Nevertheless, in recent years, several fermentation strategies using other microbial models such as Azotobacter vinelandii, A. chroococcum, as well as some methane‐utilizing species, have been developed in order to improve the P(3HB) production and also its mean molecular weight.

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.

Isolation and Characterization of Azotobacter vinelandii Mutants Impaired in Alkylresorcinol Synthesis: Alkylresorcinols Are Not Essential for Cyst Desiccation Resistance

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.

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

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.