RESUMEN
Nitrogen regulation in bacteria involves the capacity to sense the availability of fixed nitrogen and to translate a signal indicating nitrogen deficiency or nitrogen excess into a cellular response. One of the key enzymes in this complex regulation process, the uridylyltransferase/uridylyl-removing (UTase) enzyme, encoded by the glnD gene, was characterised in the diazotroph Azospirillum brasilense, which promotes plant growth. The glnD gene product is responsible for the uridylylation of both P(II)-like nitrogen regulatory proteins, P(II) and P(Z), depending on the nitrogen status of the cell. The nitrogen-regulated activity of the main ammonium-assimilating enzyme, glutamine synthetase, is not altered in a glnD-Tn 5-B30 insertion mutant. UTase influences processes that are regulated by the NtrB-NtrC two-component histidine protein kinase system, such as ammonium uptake and nitrate assimilation. Moreover, the glnD gene product is indispensable for the activation of nitrogen fixation. Transcription of glnD is up-regulated under nitrogen-fixing conditions. This regulation is only partially dependent on the global nitrogen regulation (Ntr) system.
Asunto(s)
Azospirillum brasilense/enzimología , Azospirillum brasilense/genética , Genes Bacterianos , Mutación , Nucleotidiltransferasas/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Bacteriano/genética , Expresión Génica , Datos de Secuencia Molecular , Fijación del Nitrógeno/genética , Proteínas PII Reguladoras del Nitrógeno , Fenotipo , Plantas/microbiologíaRESUMEN
The Azospirillum brasilense Sp245 napABC genes, encoding nitrate reductase activity, were isolated and sequenced. The derived protein sequences are very similar throughout the whole Nap segment to the NapABC protein sequences of Escherichia coli, Pseudomonas sp. G-179, Ralstonia eutropha, Rhodobacter sphaeroides, and Paracoccus denitrificans. Based on whole-cell nitrate reductase assays with the artificial electron donors benzyl viologen and methyl viologen, and assays with periplasmic cell-free extracts, it was concluded that the napABC-encoded enzyme activity in Azospirillum brasilense Sp245 corresponds to a periplasmic dissimilatory nitrate reductase, which was expressed under anoxic conditions and oxic conditions. A kanamycin-resistant Azospirillum brasilense Sp245 napA insertion mutant was constructed. The mutant still expressed assimilatory nitrate reductase activity, but was devoid of its periplasmic dissimilatory nitrate reductase activity.
Asunto(s)
Azospirillum brasilense/enzimología , Nitrato Reductasas/genética , Nitrato Reductasas/metabolismo , Periplasma/enzimología , Aerobiosis , Anaerobiosis , Azospirillum brasilense/citología , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Clonación Molecular , Genes Bacterianos/genética , Datos de Secuencia Molecular , Mutación/genética , Esferoplastos/enzimologíaRESUMEN
The salAB genes of Azospirillum irakense KBC1, which encode two aryl-beta-glucosidases, are required for growth on salicin. In the 4-kb region upstream of the salAB genes, two additional genes, salC and salR, were identified. SalC shows characteristics of the outer membrane receptors in the FepA/FhuA family. The salC AB genes are transcribed as a polycistronic mRNA. The salR gene encodes a protein homologous to the LacI/GalR family of transcriptional repressors. Expression of the sal operon, measured by means of a salC-gusA translational fusion in A. irkense KBC1, requires the presence of aryl-beta-glucosides such as arbutin and salicin. Expression is markedly enhanced when a simple carbon source, like glucose, cellobiose or malate, is added to the medium. In a salR mutant, expression of the salC-gusA fusion does not require an aryl-beta-glucoside inducer. Expression of a salR-gusA fusion is constitutive. The product of arbutin hydrolysis (hydroquinone) partly inhibits the expression of a salC-gusA fusion in arbutin- or salicin-containing minimal medium. This effect is independent of SalR. Salicylalcohol, the hydrolysis product of salicin, also partly inhibits salC expression in a SalR-independent fashion, but only in salicin-containing minimal medium.
Asunto(s)
Azospirillum/genética , Proteínas Bacterianas , Alcoholes Bencílicos/metabolismo , Proteínas Portadoras/genética , Operón , Receptores de Superficie Celular/genética , Proteínas Represoras/genética , beta-Glucosidasa/genética , Secuencia de Aminoácidos , Arbutina/metabolismo , Alcoholes Bencílicos/farmacología , Clonación Molecular , Inducción Enzimática , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Glucósidos/farmacología , Hidrólisis , Hidroquinonas/farmacología , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.
Asunto(s)
Alcaligenes/clasificación , Pseudomonas/clasificación , Alcaligenes/genética , Secuencia de Bases , ADN Bacteriano/genética , ADN Ribosómico/genética , Ácidos Grasos/análisis , Genes Bacterianos/genética , Datos de Secuencia Molecular , Fijación del Nitrógeno/genética , Hibridación de Ácido Nucleico , Sistemas de Lectura Abierta/genética , Oryza/microbiología , Fenotipo , Filogenia , Pseudomonas/química , Pseudomonas/genética , ARN Ribosómico 16S/análisisRESUMEN
Our approach to the isolation of plant-inducible bacterial genes of Azospirillum brasilense, based on the analysis of protein patterns of bacteria grown in the presence and in the absence of plant root exudates, led to the identification of an acidic 40 kDa protein. Cloning and sequencing analysis of the corresponding coding DNA region revealed the presence of two open reading frames transcribed in the same orientation. The deduced ORF1 protein, which corresponds to the 40 kDa protein, is very similar to the periplasmic ChvE protein, identified in Agrobacterium tumefaciens and involved in enhanced virulence. The deduced ORF2 protein shows homology to members of the LysR family of transcriptional regulators. The function of the ChvE-like protein in A. brasilense was investigated further. The protein, designated as SbpA (sugar binding protein A), is involved in the uptake of D-galactose and functions in the chemotaxis of A. brasilense towards several sugars, including D-galactose, L-arabinose and D-fucose. Expression of the sbpA gene requires the presence of the same sugars in the growth medium and is enhanced further in combination with carbon starvation of A. brasilense cells.
Asunto(s)
Azospirillum brasilense/genética , Metabolismo de los Hidratos de Carbono , Proteínas de Transporte de Membrana , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Unión Periplasmáticas , Agrobacterium tumefaciens/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Quimiotaxis/genética , Clonación Molecular , Galactosa/metabolismo , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/metabolismo , Sistemas de Lectura Abierta , Raíces de Plantas/microbiología , Unión Proteica , Alineación de Secuencia , Triticum/metabolismoRESUMEN
The rhizosphere nitrogen-fixing bacterium Azospirillum irakense KBC1 is able to grow on pectin and beta-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes, salA and salB, conferring beta-glucosidase activity to Escherichia coli, have been identified in a cosmid library of A. irakense DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl beta-glucosides. A Delta(salA-salB) A. irakense mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either salA or salB, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB' subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this beta-glucosidase family in the utilization of beta-glucosides for microbial growth.
Asunto(s)
Azospirillum/metabolismo , Alcoholes Bencílicos/metabolismo , Glucósidos/metabolismo , beta-Glucosidasa/genética , Secuencia de Aminoácidos , Arbutina/metabolismo , Azospirillum/enzimología , Azospirillum/genética , Azospirillum/crecimiento & desarrollo , Celobiosa/metabolismo , Clonación Molecular , Eliminación de Gen , Prueba de Complementación Genética , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Cinética , Datos de Secuencia Molecular , Filogenia , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Temperatura , beta-Glucosidasa/química , beta-Glucosidasa/aislamiento & purificación , beta-Glucosidasa/metabolismoRESUMEN
The pelA gene from the N2-fixing plant-associated bacterium Azospirillum irakense, encoding a pectate lyase, was isolated by heterologous expression in Escherichia coli. Nucleotide sequence analysis of the region containing pelA indicated an open reading frame of 1,296 bp, coding for a preprotein of 432 amino acids with a typical amino-terminal signal peptide of 24 amino acids. N-terminal amino acid sequencing confirmed the processing of the protein in E. coli at the signal peptidase cleavage site predicted by nucleotide sequence analysis. Analysis of the amino acid sequence of PelA revealed no homology to other known pectinases, indicating that PelA belongs to a new pectate lyase family. PelA macerates potato tuber tissue, has an alkaline pH optimum, and requires Ca2+ for its activity. Of several divalent cations tested, none could substitute for Ca2+. Methyl-esterified pectin (with a degree of esterification up to 93%) and polygalacturonate can be used as substrates. Characterization of the degradation products formed upon incubation with polygalacturonate indicated that PelA is an endo-pectate lyase generating unsaturated digalacturonide as the major end product. Regulation of pelA expression was studied by means of a translational pelA-gusA fusion. Transcription of this fusion is low under all growth conditions tested and is dependent on the growth phase. In addition, pelA expression was found to be induced by pectin. An A. irakense pelA::Tn5 mutant still displayed pectate lyase activity, suggesting the presence of multiple pectate lyase genes in A. irakense.
Asunto(s)
Azospirillum/genética , Genes Bacterianos , Pectinas/metabolismo , Polisacárido Liasas/genética , Azospirillum/enzimología , Azospirillum/patogenicidad , Azospirillum brasilense/enzimología , Azospirillum brasilense/genética , Cromosomas Bacterianos , Clonación Molecular , Datos de Secuencia Molecular , Enfermedades de las Plantas/microbiología , Polisacárido Liasas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Spectral analysis indicated the presence of a cytochrome cbb3 oxidase under microaerobic conditions in Azospirillum brasilense Sp7 cells. The corresponding genes (cytNOQP) were isolated by using PCR. These genes are organized in an operon, preceded by a putative anaerobox. The phenotype of an A. brasilense cytN mutant was analyzed. Under aerobic conditions, the specific growth rate during exponential phase (mu(e)) of the A. brasilense cytN mutant was comparable to the wild-type specific growth rate (m(e) of approximately 0.2 h-1). In microaerobic NH4+-supplemented conditions, the low respiration of the A. brasilense cytN mutant affected its specific growth rate (mu(e) of approximately 0.02 h-1) compared to the wild-type specific growth rate (mu(e) of approximately 0.2 h-1). Under nitrogen-fixing conditions, both the growth rates and respiration of the wild type were significantly diminished in comparison to those under NH4+-supplemented conditions. Differences in growth rates and respiration between the wild type and the A. brasilense cytN mutant were less pronounced under these nitrogen-fixing conditions (mu(e) of approximately 0.03 h-1 for the wild type and 0.02 h-1 for the A. brasilense cytN mutant). The nitrogen-fixing capacity of the A. brasilense cytN mutant was still approximately 80% of that determined for the wild-type strain. This leads to the conclusion that the A. brasilense cytochrome cbb3 oxidase is required under microaerobic conditions, when a high respiration rate is needed, but that under nitrogen-fixing conditions the respiration rate does not seem to be a growth-limiting factor.
Asunto(s)
Azospirillum brasilense/enzimología , Azospirillum brasilense/crecimiento & desarrollo , Complejo IV de Transporte de Electrones/fisiología , Oxidorreductasas/fisiología , Aerobiosis , Azospirillum brasilense/genética , Secuencia de Bases , Membrana Celular/enzimología , ADN Bacteriano , Complejo IV de Transporte de Electrones/genética , Espectrometría de Masas , Datos de Secuencia Molecular , Mutagénesis , Fijación del Nitrógeno , Oxidorreductasas/genética , Fenotipo , Análisis de Secuencia de ADNRESUMEN
An ammonium transporter of Azospirillum brasilense was characterized. In contrast to most previously reported putative prokaryotic NH4+ transporter genes, A. brasilense amtB is not part of an operon with glnB or glnZ which, in A. brasilense, encode nitrogen regulatory proteins PII and PZ, respectively. Sequence analysis predicts the presence of 12 transmembrane domains in the deduced AmtB protein and classifies AmtB as an integral membrane protein. Nitrogen regulates the transcription of the amtB gene in A. brasilense by the Ntr system. amtB is the first gene identified in A. brasilense whose expression is regulated by NtrC. The observation that ammonium uptake is still possible in mutants lacking the AmtB protein suggests the presence of a second NH4+ transport mechanism. Growth of amtB mutants at low ammonium concentrations is reduced compared to that of the wild type. This suggests that AmtB has a role in scavenging ammonium at low concentrations.
Asunto(s)
Azospirillum brasilense/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Proteínas de Escherichia coli , Genes Bacterianos/genética , Compuestos de Amonio Cuaternario/metabolismo , Azospirillum brasilense/genética , Transporte Biológico , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , Filogenia , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The Azospirillum brasilense trpE gene has been isolated by DNA hybridization and by genetic complementation of an Escherichia coli trpE deletion mutant. DNA sequence analysis of a 3.1-kb PstI restriction fragment of A. brasilense revealed the presence of an open reading frame encoding a putative TrpE(G) fusion protein. Previously an A. brasilense clone containing trpGDC was identified (Zimmer et al. Mol Gen Genet 229:41-51, 1991). It can, therefore, be concluded that A. brasilense contains two trpG genes. A putative leader peptide is found upstream of trpE(G), containing three consecutive tryptophan residues. Putative terminator and anti-terminator loops have also been identified. The LLESX10S motif, which is responsible for feedback inhibition by tryptophan in other TrpE proteins, is absent in the A. brasilense TrpE(G) fused protein.
Asunto(s)
Antranilato Sintasa/genética , Azospirillum brasilense/genética , Genes Bacterianos/genética , Transferasas de Grupos Nitrogenados , Secuencia de Aminoácidos , Clonación Molecular , Dosificación de Gen , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Operón/genética , Señales de Clasificación de Proteína/genética , ARN Bacteriano/química , ARN Mensajero/química , Secuencias Reguladoras de Ácidos Nucleicos/genética , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transferasas/genéticaRESUMEN
Azospirillum brasilense can display a single polar flagellum and several lateral flagella. The A. brasilense Sp7 gene laf1, encoding the flagellin of the lateral flagella, was isolated and sequenced. The derived protein sequence is extensively similar to those of the flagellins of Rhizobium meliloti, Agrobacterium tumefaciens, Bartonella bacilliformis, and Caulobacter crescentus. An amino acid alignment shows that the flagellins of these bacteria are clustered and are clearly different from other known flagellins. A laf1 mutant, FAJ0201, was constructed by replacing an internal part of the laf1 gene by a kanamycin resistance-encoding gene cassette. The mutant is devoid of lateral flagella but still forms the polar flagellum. This phenotype is further characterized by the abolishment of the capacities to swarm on a semisolid surface and to spread from a stab inoculation in a semisolid medium. FAJ0201 shows a normal wheat root colonization pattern in the initial stage of plant root interaction.
Asunto(s)
Azospirillum brasilense/genética , Proteínas Bacterianas , Flagelos/genética , Flagelina/genética , Genes Bacterianos/genética , Secuencia de Aminoácidos , Azospirillum brasilense/citología , Secuencia de Bases , Movimiento Celular/genética , Clonación Molecular , Flagelina/química , Datos de Secuencia Molecular , Mutación , Fenotipo , Raíces de Plantas/microbiología , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , TriticumRESUMEN
Sequence analysis of a fragment of Azospirillum brasilense DNA revealed the presence of a ribA homologue, of which the 3' portion encodes a putative GTP cyclohydrolase II. The 5' portion (approx. half of the ORF) does not show homology to any other sequence from the databases.
Asunto(s)
Azospirillum brasilense/genética , Proteínas Bacterianas/genética , GTP Ciclohidrolasa/genética , Genes Bacterianos , Secuencia de Aminoácidos , Azospirillum brasilense/enzimología , Bacillus subtilis/química , Bacillus subtilis/enzimología , Clonación Molecular , Escherichia coli/química , Escherichia coli/enzimología , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The nucleotide sequence of the Azospirillum brasilense exoB gene, located on plasmid pRhico, has been determined. The A. brasilense ExoB protein shows significant homology with other prokaryotic UDP-glucose 4'-epimerases (EC 5.1.3.2).
Asunto(s)
Azospirillum brasilense/genética , Genes Bacterianos , Análisis de Secuencia de ADN , UDPglucosa 4-Epimerasa/genética , Secuencia de Aminoácidos , Azospirillum brasilense/enzimología , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , UDPglucosa 4-Epimerasa/químicaRESUMEN
Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading frame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.