Defining the regulatory mechanisms of sigma factor RpoS degradation in Azotobacter vinelandii and Pseudomonas aeruginosa.

In several Gram-negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB-like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti-sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three-hybrid system. We propose that both RssB and RssC are necessary for the ClpXP-dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family.

Evaluation of a bacterial group 1 LEA protein as an enzyme protectant from stress-induced inactivation.

Late embryogenesis abundant (LEA) proteins are hydrophilic proteins that lack a well-ordered tertiary structure and accumulate to high levels in response to water deficit, in organisms such as plants, fungi, and bacteria. The mechanisms proposed to protect cellular structures and enzymes are water replacement, ion sequestering, and membrane stabilization. The activity of some proteins has a limited shelf-life due to instability that can be caused by their structure or the presence of a stress condition that limits their activity; several LEA proteins have been shown to behave as cryoprotectants in vitro. Here, we report a group1 LEA from Azotobacter vinelandii AvLEA1, capable of conferring protection to lactate dehydrogenase, catechol dioxygenase, and Baylase peroxidase against freeze-thaw treatments, desiccation, and oxidative damage, making AvLEA a promising biological stabilizer reagent. This is the first evidence of protection provided by this LEA on enzymes with biotechnological potential, such as dioxygenase and peroxidase under in vitro stress conditions. Our results suggest that AvLEA could act as a molecular chaperone, or a "molecular shield," preventing either dissociation or antiaggregation, or as a radical scavenger, thus preventing damage to these target enzymes during induced stress. KEY POINTS: • This work expands the basic knowledge of the less-known bacterial LEA proteins and their in vitro protection potential. • AvLEA is a bacterial protein that confers in vitro protection to three enzymes with different characteristics and oligomeric arrangement. • The use of AvLEA as a stabilizer agent could be further explored using dioxygenase and peroxidase in bioremediation treatments. AvLEA1 protects against freeze-thaw treatments, desiccation, and oxidative damage on three different enzymes with biotechnological potential.

Increased c-di-GMP Levels Lead to the Production of Alginates of High Molecular Mass in Azotobacter vinelandii.

Azotobacter vinelandii produces the linear exopolysaccharide alginate, a compound of significant biotechnological importance. The biosynthesis of alginate in A. vinelandii and Pseudomonas aeruginosa has several similarities but is regulated somewhat differently in the two microbes. Here, we show that the second messenger cyclic dimeric GMP (c-di-GMP) regulates the production and the molecular mass of alginate in A. vinelandii The hybrid protein MucG, containing conserved GGDEF and EAL domains and N-terminal HAMP and PAS domains, behaved as a c-di-GMP phosphodiesterase (PDE). This activity was found to negatively affect the amount and molecular mass of the polysaccharide formed. On the other hand, among the diguanylate cyclases (DGCs) present in A. vinelandii, AvGReg, a globin-coupled sensor (GCS) DGC that directly binds to oxygen, was identified as the main c-di-GMP-synthesizing contributor to alginate production. Overproduction of AvGReg in the parental strain phenocopied a ΔmucG strain with regard to alginate production and the molecular mass of the polymer. MucG was previously shown to prevent the synthesis of high-molecular-mass alginates in response to reduced oxygen transfer rates (OTRs). In this work, we show that cultures exposed to reduced OTRs accumulated higher levels of c-di-GMP; this finding strongly suggests that at least one of the molecular mechanisms involved in modulation of alginate production and molecular mass by oxygen depends on a c-di-GMP signaling module that includes the PAS domain-containing PDE MucG and the GCS DGC AvGReg.IMPORTANCE c-di-GMP has been widely recognized for its essential role in the production of exopolysaccharides in bacteria, such as alginate produced by Pseudomonas and Azotobacter spp. This study reveals that the levels of c-di-GMP also affect the physical properties of alginate, favoring the production of high-molecular-mass alginates in response to lower OTRs. This finding opens up new alternatives for the design of tailor-made alginates for biotechnological applications.

Cyclic di-GMP-Mediated Regulation of Extracellular Mannuronan C-5 Epimerases Is Essential for Cyst Formation in Azotobacter vinelandii.

The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa, MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR.IMPORTANCEA. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell.

Improving glucose and xylose assimilation in Azotobacter vinelandii by adaptive laboratory evolution.

Azotobacter vinelandii is a microorganism with biotechnological potential because its ability to produce alginate and polyhydroxybutyrate. Large-scale biotechnological processes are oriented to sustainable production by using biomass hydrolysates that are mainly composed by glucose and xylose. In the present study, it was observed that A. vinelandii was unable to consume xylose as the sole carbon source and that glucose assimilation in the presence of xylose was negatively affected. Adaptive Laboratory Evolution (ALE) was used as a metabolic engineering tool in A. vinelandii, to improve both carbohydrate assimilation. As a result of ALE process, the CT387 strain was obtained. The evolved strain (CT387) grown in shaken flask cultivations with xylose (8 g L-1) and glucose (2 g L-1), showed an increase of its specific growth rate (µ), as well as of its glucose and xylose uptake rates of 2, 6.45 and 3.57-fold, respectively, as compared with the parental strain. At bioreactor level, the µ, the glucose consumption rate and the relative expression of gluP that codes for the glucose permease in the evolved strain were also higher than in the native strain (1.53, 1.29 and 18-fold, respectively). Therefore, in the present study, we demonstrated the potential of ALE as a metabolic engineering tool for improving glucose and xylose consumption in A. vinelandii.

Outer membrane protein I is associated with poly-ß-hydroxybutyrate granules and is necessary for optimal polymer accumulation in Azotobacter vinelandii on solid medium.

Azotobacter vinelandii is a soil bacterium that is able to synthesize poly-ß-hydroxybutyrate (PHB), a polymer used to produce biodegradable plastic. PHB is stored in the cytoplasm as granules surrounded by several proteins such as the major phasin PhbP, PHB synthase and PHB depolymerase, among others. Many studies have reported the presence of membrane proteins on PHB granules due to contamination during the polymer extraction procedures. Previously, the outer membrane protein I (OprI) was detected on the polymer granules in A. vinelandii. In this study, by using random transposon mutagenesis, we identified that a mutation in the oprI gene diminished PHB accumulation in A. vinelandii on solid medium. Electron microscopy confirmed the low polymer production by the oprI mutant. Analysis of PHB granules by Tricine-SDS-PAGE revealed that the absence of OprI affected the protein profile of the granules, suggesting that OprI could have a structural role in A. vinelandii. Thus, some membrane proteins on PHB granules may not be artefacts as previously described.

Unphosphorylated EIIANtr induces ClpAP-mediated degradation of RpoS in Azotobacter vinelandii.

The nitrogen-related phosphotransferase system (PTSNtr ) is composed of the EINtr , NPr and EIIANtr proteins that form a phosphorylation cascade from phosphoenolpyruvate. PTSNtr is a global regulatory system present in most Gram-negative bacteria that controls some pivotal processes such as potassium and phosphate homeostasis, virulence, nitrogen fixation and ABC transport activation. In the soil bacterium Azotobacter vinelandii, unphosphorylated EIIANtr negatively regulates the expression of genes related to the synthesis of the bioplastic polyester poly-ß-hydroxybutyrate (PHB) and cyst-specific lipids alkylresorcinols (ARs). The mechanism by which EIIANtr controls gene expression in A. vinelandii is not known. Here, we show that, in presence of unphosphorylated EIIANtr , the stability of the stationary phase sigma factor RpoS, which is necessary for transcriptional activation of PHB and ARs synthesis related genes, is reduced, and that the inactivation of genes coding for ClpAP protease complex in strains that carry unphosphorylated EIIANtr , restored the levels and in vivo stability of RpoS, as well as the synthesis of PHB and ARs. Taken together, our results reveal a novel mechanism, by which EIIANtr globally controls gene expression in A. vinelandii, where the unphosphorylated EIIANtr induces the degradation of RpoS by the proteolytic complex ClpAP.

Inactivation of an intracellular poly-3-hydroxybutyrate depolymerase of Azotobacter vinelandii allows to obtain a polymer of uniform high molecular mass.

A novel poly-3-hydroxybutyrate depolymerase was identified in Azotobacter vinelandii. This enzyme, now designated PhbZ1, is associated to the poly-3-hydroxybutyrate (PHB) granules and when expressed in Escherichia coli, it showed in vitro PHB depolymerizing activity on native or artificial PHB granules, but not on crystalline PHB. Native PHB (nPHB) granules isolated from a PhbZ1 mutant had a diminished endogenous in vitro hydrolysis of the polyester, when compared to the granules of the wild-type strain. This in vitro degradation was also tested in the presence of free coenzyme A. Thiolytic degradation of the polymer was observed in the nPHB granules of the wild type, resulting in the formation of 3-hydroxybutyryl-CoA, but was absent in the granules of the mutant. It was previously reported that cultures of A. vinelandii OP grown in a bioreactor showed a decrease in the weight average molecular weight (Mw) of the PHB after 20 h of culture, with an increase in the fraction of polymers of lower molecular weight. This decrease was correlated with an increase in the PHB depolymerase activity during the culture. Here, we show that in the phbZ1 mutant, neither the decrease in the Mw nor the appearance of a low molecular weight polymers occurred. In addition, a higher PHB accumulation was observed in the cultures of the phbZ1 mutant. These results suggest that PhbZ1 has a role in the degradation of PHB in cultures in bioreactors and its inactivation allows the production of a polymer of a uniform high molecular weight.

Two-component system CbrA/CbrB controls alginate production in Azotobacter vinelandii.

Azotobacter vinelandii, belonging to the Pseudomonadaceae family, is a free-living bacterium that has been considered to be a good source for the production of bacterial polymers such as alginate. In A. vinelandii the synthesis of this polymer is regulated by the Gac/Rsm post-transcriptional regulatory system, in which the RsmA protein binds to the mRNA of the biosynthetic algD gene, inhibiting translation. In several Pseudomonas spp. the two-component system CbrA/CbrB has been described to control a variety of metabolic and behavioural traits needed for adaptation to changing environmental conditions. In this work, we show that the A. vinelandii CbrA/CbrB two-component system negatively affects alginate synthesis, a function that has not been described in Pseudomonas aeruginosa or any other Pseudomonas species. CbrA/CbrB was found to control the expression of some alginate biosynthetic genes, mainly algD translation. In agreement with this result, the CbrA/CbrB system was necessary for optimal rsmA expression levels. CbrA/CbrB was also required for maximum accumulation of the sigma factor RpoS. This last effect could explain the positive effect of CbrA/CbrB on rsmA expression, as we also showed that one of the promoters driving rsmA transcription was RpoS-dependent. However, although inactivation of rpoS increased alginate production by almost 100 %, a cbrA mutation increased the synthesis of this polymer by up to 500 %, implying the existence of additional CbrA/CbrB regulatory pathways for the control of alginate production. The control exerted by CbrA/CbrB on the expression of the RsmA protein indicates the central role of this system in regulating carbon metabolism in A. vinelandii.

The signaling protein MucG negatively affects the production and the molecular mass of alginate in Azotobacter vinelandii.

Azotobacter vinelandii is a soil bacterium that produces the polysaccharide alginate. In this work, we identified a miniTn5 mutant, named GG9, which showed increased alginate production of higher molecular mass, and increased expression of the alginate biosynthetic genes algD and alg8 when compared to its parental strain. The miniTn5 was inserted within ORF Avin07920 encoding a hypothetical protein. Avin07910, located immediately downstream and predicted to form an operon with Avin07920, encodes an inner membrane multi-domain signaling protein here named mucG. Insertional inactivation of mucG resulted in a phenotype of increased alginate production of higher molecular mass similar to that of mutant GG9. The MucG protein contains a periplasmic and putative HAMP and PAS domains, which are linked to GGDEF and EAL domains. The last two domains are potentially involved in the synthesis and degradation, respectively, of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), a secondary messenger that has been reported to be essential for alginate production. Therefore, we hypothesized that the negative effect of MucG on the production of this polymer could be explained by the putative phosphodiesterase activity of the EAL domain. Indeed, we found that alanine replacement mutagenesis of the MucG EAL motif or deletion of the entire EAL domain resulted in increased alginate production of higher molecular mass similar to the GG9 and mucG mutants. To our knowledge, this is the first reported protein that simultaneous affects the production of alginate and its molecular mass.

A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii.

In Azotobacter vinelandii, a cyst-forming bacterium, the alternative sigma factor RpoS is essential to the formation of cysts resistant to desiccation and to synthesis of the cyst-specific lipids, alkylresorcinols. In this study, we carried out a proteome analysis of vegetative cells and cysts of A. vinelandii strain AEIV and its rpoS mutant derivative AErpoS. This analysis allowed us to identify a small heat-shock protein, Hsp20, as one of the most abundant proteins of cysts regulated by RpoS. Inactivation of hsp20 did not affect the synthesis of alkylresorcinols or the formation of cysts with WT morphology; however, the cysts formed by the hsp20 mutant strain were unable to resist desiccation. We also demonstrated that expression of hsp20 from an RpoS-independent promoter in the AErpoS mutant strain is not enough to restore the phenotype of resistance to desiccation. These results indicate that Hsp20 is essential for the resistance to desiccation of A. vinelandii cysts, probably by preventing the aggregation of proteins caused by the lack of water. To our knowledge, this is the first report of a small heat-shock protein that is essential for desiccation resistance in bacteria.

Strong seed-bank effects in bacterial evolution.

Bacterial genomes are mosaics with fragments showing distinct phylogenetic origins or even being unrelated to any other genetic information (ORFan genes). Thus the analysis of bacterial population genetics is in large part a collection of explanations for anomalies in relation to classical population genetic models such as the Wright-Fisher model and the Kingman coalescent that do not adequately describe bacterial population genetics, genomics or evolution. The concept of "species" as an evolutionary coherent biological group that is genetically isolated and shares genetic information through recombination among its members cannot be applied to any bacterial group. Recently, a simple probabilistic model considering the role of strong seed-bank effects in population genetics has been proposed by Blath et al. This model suggests the existence of a genetic pool with high diversity that is not subject to classical selection and extinction. We reason that certain bacterial population genetics anomalies could be explained by the prevalence of strong seed-bank effects among bacteria. To address this possibility we analyzed the genome of the bacterium Azotobacter vinelandii and show that genes that code for functions that are essential for the bacterium biology do not have a relation of ancestry with closely related bacteria, or are ORFan genes. The existence of essential genes that are not inherited from the most recent ancestor cannot be explained by classical population genetics models and is irreconcilable with the current view of genes acquired by horizontal transfer as being accessory or adaptive.

Posttranscriptional regulation of PhbR, the transcriptional activator of polyhydroxybutyrate synthesis, by iron and the sRNA ArrF in Azotobacter vinelandii.

Azotobacter vinelandii is a Gram-negative bacterium able to synthesize poly-ß-hydroxybutyrate (PHB), a biodegradable plastic of industrial interest. The phbBAC operon encodes the enzymes of PHB synthesis and is activated by the transcriptional regulator PhbR and the sigma factor RpoS. Iron limitation has been previously reported to increase PHB accumulation in A. vinelandii; however, the mechanism by which iron controls PHB synthesis is unknown. Under iron starvation in Escherichia coli, the RyhB sRNA modulates the translation of genes involved in iron homeostasis. ArrF is the RyhB analogue in A. vinelandii and similarly increases in quantity during Fe(2+) depletion. In this study, we evaluate the effect of iron and ArrF on PHB accumulation, and on phbR and phbBAC expression in A. vinelandii strain UW136. Using transcriptional and translational fusions of phbR and phbB with gusA reporter gene, we found that iron limitation increased the expression of phbBAC at the transcriptional level and posttranscriptionally increased the expression of phbR. We also found that the ArrF sRNA is a positive regulator of phbR expression at the posttranscriptional level. Collectively, these data suggest that iron limitation increases the translation of phbR through ArrF.

The stringent response regulates the poly-ß-hydroxybutyrate (PHB) synthesis in Azotobacter vinelandii.

The stringent response exerted by (p)ppGpp and RNA-polymerase binding protein DksA regulates gene expression in diverse bacterial species. To control gene expression (p)ppGpp, synthesized by enzymes RelA and SpoT, interacts with two sites within the RNA polymerase; site 1, located in the interphase between subunits ß' and ω (rpoZ), and site 2 located in the secondary channel that is dependent on DksA protein. In Escherichia coli, inactivation of dksA results in a reduced sigma factor RpoS expression. In Azotobacter vinelandii the synthesis of polyhydroxybutyrate (PHB) is under RpoS regulation. In this study, we found that the inactivation of relA or dksA, but not rpoZ, resulted in a negative effect on PHB synthesis. We also found that the dksA, but not the relA mutation reduced both rpoS transcription and RpoS protein levels, implying that (p)ppGpp and DksA control PHB synthesis through different mechanisms. Interestingly, despite expressing rpoS from a constitutive promoter in the dksA mutant, PHB synthesis was not restored to wild type levels. A transcriptomic analysis in the dksA mutant, revealed downregulation of genes encoding enzymes needed for the synthesis of acetyl-CoA, the precursor substrate for PHB synthesis. Together, these data indicate that DksA is required for optimal expression of RpoS which in turn activates transcription of genes for PHB synthesis. Additionally, DksA is required for optimal transcription of genes responsible for the synthesis of precursors for PHB synthesis.

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-ß-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.

Alginate synthesis in Azotobacter vinelandii is increased by reducing the intracellular production of ubiquinone.

Azotobacter vinelandii, a soil nitrogen fixing bacterium, produces alginate a polysaccharide with industrial and medical relevant applications. In this work, we characterized a miniTn5 mutant, named GG101, that showed a 14-fold increase in the specific production of alginate when grown diazotrophically on solid minimal medium comparing to the parental E strain (also named AEIV). Quantitative real-time reverse transcription PCR analysis indicated that this increased alginate production was due to higher expression levels of several biosynthetic alg genes such as algD. Sequencing of the locus interrupted in GG101 indicated that the miniTn5 was inserted in the positive strand, and 10 bp upstream the start codon of the gene ubiA, encoding the enzyme for the second step in the biosynthesis of ubiquinone (Q8). Both the transcription of ubiA and the content of Q8 are decreased in the mutant GG101 when compared to the wild-type strain E. Genetic complementation of mutant GG101 with a wild-type copy of the ubiCA genes restored the content of Q8 and reduced the production of alginate to levels similar to those of the parental E strain. Furthermore, respirometric analysis showed a reproducible decrease of about 8 % in the respiratory capacity of mutant GG101, at exponential phase of growth in liquid minimal medium. Collectively, our data show that a decreased content in Q8 results in higher levels of alginate in A. vinelandii.

The Azotobacter vinelandii AlgU regulon during vegetative growth and encysting conditions: A proteomic approach.

In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of Pseudomonas aeruginosa, has been a model for cellular differentiation in Gram-negative bacteria since it forms desiccation-resistant cysts. Previous work demonstrated the essential role of AlgU to withstand oxidative stress and on A. vinelandii differentiation, particularly for the positive control of alginate production. In this study, the AlgU regulon was dissected by a proteomic approach under vegetative growing conditions and upon encystment induction. Our results revealed several molecular targets that explained the requirement of this sigma factor during oxidative stress and extended its role in alginate production. Furthermore, we demonstrate that AlgU was necessary to produce alkyl resorcinols, a type of aromatic lipids that conform the cell membrane of the differentiated cell. AlgU was also found to positively regulate stress resistance proteins such as OsmC, LEA-1, or proteins involved in trehalose synthesis. A position-specific scoring-matrix (PSSM) was generated based on the consensus sequence recognized by AlgU in P. aeruginosa, which allowed the identification of direct AlgU targets in the A. vinelandii genome. This work further expands our knowledge about the function of the ECF sigma factor AlgU in A. vinelandii and contributes to explains its key regulatory role under adverse conditions.

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

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 lacking the Na(+)-NQR activity: a potential source for producing alginates with improved properties and at high yield.

The mutant ATCN4 strain of Azotobacter vinelandii, which lacks the Na(+)-NQR activity and results in an alginate overproduction (highly mucoid phenotype), was cultured in shake flasks in minimal and rich medium, and the chemical composition and rheological properties of the alginate were determined. Mutant ATCN4 exhibited a high efficiency for sucrose conversion to alginate and PHB accumulation, reaching yields that were 3.6- and 1.6-fold higher than those obtained from the wildtype cultures in minimal medium (Burk's sucrose, BS). The alginate produced by ATCN4 in the minimal medium had a high degree of acetylation (≥4 %) and a low G/M ratio (=2) with respect to the polymer synthesised in the rich medium (BS with yeast extract) (degree of acetylation = 0 % and G/M ratio of 4.5). The alginate produced in the minimal medium exhibited a pronounced pseudoplastic behaviour and a higher G* module in comparison to that observed in the alginate obtained in the cultures using a rich medium. The ATCN4 mutant culture in the minimal medium promoted the synthesis of a polymer of improved rheological quality in terms of its mechanical properties. These characteristics make this mutant a valuable source for producing alginates with improved or special properties.

The ribosome rescue pathways SsrA-SmpB, ArfA, and ArfB mediate tolerance to heat and antibiotic stresses in Azotobacter vinelandii.

Bacteria have a mechanism to rescue stalled ribosomes known as trans-translation consisting of SsrA, a transfer-messenger RNA (tmRNA), and the small protein SmpB. Other alternative rescue mechanisms mediated by ArfA and ArfB proteins are present only in some species. Ribosome rescue mechanisms also play a role in tolerance to antibiotics and various stresses such as heat. This study shows that the genome of the soil bacterium A. vinelandii harbours genes encoding for tmRNA, SmpB, two paralogs of ArfA (arfA1 and arfA2), and ArfB. A number of mutant strains carrying mutations in the ssrA, arfA1, arfA2, and arfB genes were constructed and tested for their growth and susceptibility to heat and the antibiotic tetracycline. We found that the inactivation of both ssrA and one or the two arfA genes was detrimental to growth and caused a higher susceptibility to heat and to the antibiotic tetracycline. Interestingly, the arfB mutant strain was unable to grow after 2 h of incubation at 45°C. Inactivation of arfB in the ssrA-arfA1-arfA2 strain caused a lethal phenotype since the quadruple mutant could not be isolated. Taken together, our data suggest that both arfA1 and arfA2, as well as arfB, are functional as back up mechanisms, and that the ArfB pathway has an essential role that confers A. vinelandii resistance to high temperatures.