RESUMO
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.
Assuntos
Azotobacter vinelandii , Proteínas de Escherichia coli , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Escherichia coli/metabolismo , Proteínas de Ligação a DNA/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Escherichia coli/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
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.
Assuntos
Proteínas de Bactérias , Dioxigenases , Desenvolvimento Embrionário , Peroxidases , Proteínas de Plantas , ÁguaRESUMO
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.
Assuntos
Alginatos/metabolismo , Azotobacter vinelandii/metabolismo , GMP Cíclico/análogos & derivados , Polissacarídeos Bacterianos/biossíntese , Alginatos/química , Azotobacter vinelandii/enzimologia , Azotobacter vinelandii/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Peso Molecular , Oxigênio/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Polissacarídeos Bacterianos/químicaRESUMO
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.
Assuntos
Azotobacter vinelandii/enzimologia , Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/metabolismo , GMP Cíclico/análogos & derivados , Alginatos/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Carboidratos Epimerases/genética , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMO
Poly-(3-hydroxyalkanoates) (PHAs) are bacterial carbon and energy storage compounds. These polymers are synthesized under conditions of nutritional imbalance, where a nutrient is growth-limiting while there is still enough carbon source in the medium. On the other side, the accumulated polymer is mobilized under conditions of nutrient accessibility or by limitation of the carbon source. Thus, it is well known that the accumulation of PHAs is affected by the availability of nutritional resources and this knowledge has been used to establish culture conditions favoring high productivities. In addition to this effect of the metabolic status on PHAs accumulation, several genetic regulatory networks have been shown to drive PHAs metabolism, so the expression of the PHAs genes is under the influence of global or specific regulators. These regulators are thought to coordinate PHAs synthesis and mobilization with the rest of bacterial physiology. While the metabolic and biochemical knowledge related to the biosynthesis of these polymers has led to the development of processes in bioreactors for high-level production and also to the establishment of strategies for metabolic engineering for the synthesis of modified biopolymers, the use of knowledge related to the regulatory circuits controlling PHAs metabolism for strain improvement is scarce. A better understanding of the genetic control systems involved could serve as the foundation for new strategies for strain modification in order to increase PHAs production or to adjust the chemical structure of these biopolymers. In this review, the regulatory systems involved in the control of PHAs metabolism are examined, with emphasis on those acting at the level of expression of the enzymes involved and their potential modification for strain improvement, both for higher titers, or manipulation of polymer properties. The case of the PHAs producer Azotobacter vinelandii is taken as an example of the complexity and variety of systems controlling the accumulation of these interesting polymers in response to diverse situations, many of which could be engineered to improve PHAs production.
RESUMO
In bacteria, the 5'-end-dependent RNA degradation is triggered by the RNA pyrophosphohydrolase RppH converting tri/diphosphate to monophosphate transcripts. This study shows that in the soil bacterium Azotobacter vinelandii, inactivation of rppH gene negatively affected the production of bioplastic poly-ß-hydroxybutyrate (PHB) by reducing the expression at the translational level of PhbR, the specific transcriptional activator of the phbBAC biosynthetic operon. The effect of RppH on the translation of phbR seemed to be exerted through the translational repressor RsmA, as the inactivation of rsmA in the rppH mutant restored the phbR expression. Interestingly, in Escherichia coli inactivation of rppH also affected the expression of CsrA, the RsmA homolog. The level of the csrA transcript was higher and more stable in the E. coli rppH mutant than in the wild type strain. Additionally, and in contrast to the csrA mutants that are known to have a defective swimming phenotype, the E. coli rppH mutant showed a hyper-swimming phenotype that was suppressed by a csrA mutation, and the AvRppH restored to wild type level the swimming phenotype to the E. coli rppH mutant. We propose that in both A. vinelandii and E. coli, RppH activity plays a role in the expression of the translational regulator protein RsmA/CsrA.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Ligação a RNA/biossíntese , Proteínas Repressoras/biossíntese , Deleção de Genes , Biossíntese de ProteínasRESUMO
Late embryogenesis abundant (LEA) proteins constitute a large protein family that is closely associated with resistance to abiotic stresses in multiple organisms and protect cells against drought and other stresses. Azotobacter vinelandii is a soil bacterium that forms desiccation-resistant cysts. This bacterium possesses two genes, here named lea1 and lea2, coding for avLEA1 and avLEA2 proteins, both containing 20-mer motifs characteristic of eukaryotic plant LEA proteins. In this study, we found that disruption of the lea1 gene caused a loss of the cysts' viability after 3 months of desiccation, whereas at 6 months, wild-type or lea2 mutant strain cysts remained viable. Vegetative cells of the lea1 mutant were more sensitive to osmotic stress; cysts developed by this mutant were also more sensitive to high temperatures than cysts or vegetative cells of the wild type or of the lea2 mutant. Expression of lea1 was induced several fold during encystment. In addition, the protective effects of these proteins were assessed in Escherichia coli cells. We found that E. coli cells overexpressing avLEA1 were more tolerant to salt stress than control cells; finally, in vitro analysis showed that avLEA1 protein was able to prevent the freeze thaw-induced inactivation of lactate dehydrogenase. In conclusion, avLEA1 is essential for the survival of A. vinelandii in dry conditions and for protection against hyper-osmolarity, two major stress factors that bacteria must cope with for survival in the environment. This is the first report on the role of bacterial LEA proteins on the resistance of cysts to desiccation.
Assuntos
Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Bases de Dados Genéticas , Escherichia coli/metabolismo , L-Lactato Desidrogenase/metabolismo , Mutagênese , Pressão Osmótica , Proteínas de Plantas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Estresse Fisiológico , Temperatura , TermotolerânciaRESUMO
Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases.
Assuntos
Aciltransferases/genética , Proteínas de Bactérias/genética , Transferência Genética Horizontal , Genes Bacterianos , Hidroxibutiratos/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismoRESUMO
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.
Assuntos
Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico HSP20/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Sequência de Bases , Dessecação , Inativação Gênica , Proteínas de Choque Térmico HSP20/química , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Proteoma , Proteômica , Processamento Pós-Transcricional do RNA , Transcrição GênicaRESUMO
The transcription of genes involved in alginate polymerization and depolymerization, as well as the alginase activity (extracellular and intracellular) under oxygen-limited and non oxygen-limited conditions in cultures of A. vinelandii, was studied. Two levels of dissolved oxygen tension (DOT) (1% and 5%, oxygen-limited and non-oxygen-limited, respectively) strictly controlled by gas blending, were evaluated in a wild type strain. In cultures at low DOT (1%), in which a high molecular weight alginate (1200 kDa) was synthesized, the transcription levels of alg8 and alg44 (genes encoding alginate polymerase complex), and algX (encoding a protein involved in polymer transport through periplasmic space) were considerably higher as compared to cultures conducted at 5% DOT, under which an alginate with a low MW (42 kDa) was produced. In the case of genes encoding for intracellular and extracellular alginases, the levels of these transcripts were higher at 1% DOT. However, intracellular and extracellular alginase activity were lower (0.017 and 0.01 U/mg protein, respectively) in cultures at 1% DOT, as compared with the activities measured at 5% DOT (0.027 and 0.052 U/mg protein for intracellular and extracellular maximum activity, respectively). The low alginase activity measured in cultures at 1% DOT and the high level of transcription of genes constituting alginate polymerase complex might be mechanisms by which oxygen regulates the production of alginates with a high MW.
Assuntos
Alginatos/metabolismo , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Consumo de Oxigênio , Polissacarídeo-Liases/metabolismo , Alginatos/química , Azotobacter vinelandii/efeitos dos fármacos , Azotobacter vinelandii/genética , Azotobacter vinelandii/crescimento & desenvolvimento , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Biomassa , Microbiologia Industrial , Peso Molecular , Oxigênio/farmacologia , Polimerização , Polissacarídeo-Liases/efeitos dos fármacos , Polissacarídeo-Liases/genética , Transcrição GênicaRESUMO
We previously showed that in Azotobacter vinelandii, accumulation of polyhydroxybutyrate (PHB) occurs mainly during the stationary phase, and that a mutation in phbR, encoding a transcriptional regulator of the AraC family, reduces PHB accumulation. In this study, we characterized the roles of PhbR and RpoS, a central regulator during stationary phase in bacteria, in the regulation of expression of the PHB biosynthetic operon phbBAC and phbR. We showed that inactivation of rpoS reduced PHB accumulation, similar to the phbR mutation, and inactivation of both rpoS and phbR resulted in an inability to produce PHB. We carried out expression studies with the wild-type, and the rpoS, phbR and double rpoS-phbR mutant strains, using quantitative RT-PCR, as well as phbBâ:â:âgusA and phbRâ:â:âgusA gene fusions. These studies showed that both PhbR and RpoS act as activators of phbB and phbR, and revealed a role for PhbR as an autoactivator. We also demonstrated that PhbR binds specifically to two almost identical 18 bp sites, TGTCACCAA-N(4)-CACTA and TGTCACCAA-N(4)-CAGTA, present in the phbB promoter region. The activation of phbB and phbR transcription by RpoS reported here is in agreement with the observation that accumulation of PHB in A. vinelandii occurs mainly during the stationary phase.
Assuntos
Proteínas de Bactérias/metabolismo , Hidroxibutiratos/metabolismo , Fator sigma/metabolismo , Ativação Transcricional , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Pegada de DNA , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Mutação , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Fator sigma/genéticaRESUMO
Azotobacter vinelandii is a soil bacterium that undergoes differentiation to form cysts that are resistant to desiccation. Upon induction of cyst formation, the bacterium synthesizes alkylresorcinols that are present in cysts but not in vegetative cells. Alternative sigma factors play important roles in differentiation. In A. vinelandii, AlgU (sigma E) is involved in controlling the loss of flagella upon induction of encystment. We investigated the involvement of the sigma factor RpoS in cyst formation in A. vinelandii. We analysed the transcriptional regulation of the rpoS gene by PsrA, the main regulator of rpoS in Pseudomonas species, which are closely related to A. vinelandii. Inactivation of rpoS resulted in the inability to form cysts resistant to desiccation and to produce cyst-specific alkylresorcinols, whereas inactivation of psrA reduced by 50â% both production of alkylresorcinols and formation of cysts resistant to desiccation. Electrophoretic mobility shift assays revealed specific binding of PsrA to the rpoS promoter region and that inactivation of psrA reduced rpoS transcription by 60â%. These results indicate that RpoS and PsrA are involved in regulation of encystment and alkylresorcinol synthesis in A. vinelandii.
Assuntos
Azotobacter vinelandii/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Resorcinóis/metabolismo , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Dessecação , Ensaio de Desvio de Mobilidade Eletroforética , Fator sigma/genética , Fatores de Transcrição/genéticaRESUMO
Azotobacter vinelandii is a nitrogen-fixing soil bacterium that produces the exopolysaccharide alginate. In this report we describe the isolation and characterization of A. vinelandii strain GG4, which carries an nqrE : : Tn5 mutation resulting in alginate overproduction. The nqrE gene encodes a subunit of the Na+-translocating NADH : ubiquinone oxidoreductase (Na+-NQR). As expected, Na+-NQR activity was abolished in mutant GG4. When this strain was complemented with the nqrEF genes this activity was restored and alginate production was reduced to wild-type levels. Na+-NQR may be the main sodium pump of A. vinelandii under the conditions tested ( approximately 2 mM Na+) since no Na+/H+-antiporter activity was detected. Collectively our results indicate that in A. vinelandii the lack of Na+-NQR activity caused the absence of a transmembrane Na+ gradient and an increase in alginate production.
Assuntos
Alginatos/metabolismo , Azotobacter vinelandii/enzimologia , Regulação Bacteriana da Expressão Gênica , Quinona Redutases/metabolismo , Sódio/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/crescimento & desenvolvimento , Elementos de DNA Transponíveis , Mutação , Quinona Redutases/genética , UbiquinonaRESUMO
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.
Assuntos
Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Flagelos/genética , Inativação Gênica , Genes Bacterianos/genética , Teste de Complementação Genética , Genoma Bacteriano/genética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
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.
Assuntos
Azotobacter vinelandii/enzimologia , Azotobacter vinelandii/genética , Genes Bacterianos , Ácido Glucurônico/biossíntese , Lipopolissacarídeos/biossíntese , Fosfoglucomutase/metabolismo , Fosfotransferases (Fosfomutases)/metabolismo , Alginatos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Ácidos Hexurônicos , Dados de Sequência Molecular , Fosfoglucomutase/genética , Fosfoglucomutase/isolamento & purificação , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
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.