RESUMEN
Genetic complementation of a spontaneous mutant, impaired in flocculation, Congo red binding, and colonization of root surface, led to the identification of a new regulatory gene in Azospirillum brasilense Sp7, designated flcA. The deduced amino acid sequence of flcA shared high similarity with a family of transcriptional activators of the LuxR-UphA family. The most significant match was with the AgmR protein, an activator for glycerol metabolism in Pseudomonas aeruginosa. Derivatives of Sp7 resulting from site-directed Tn5 mutagenesis in the flcA coding sequence were constructed by marker exchange. Characterization of the resulting mutant strains showed that flcA controls the production of capsular polysaccharides, the flocculation process in culture, and the colonization of the root surface of wheat. This study provides new information on the genetic control of the mechanism of plant root colonization by Azospirillum.
Asunto(s)
Azospirillum brasilense/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Triticum/microbiología , Secuencia de Aminoácidos , Azospirillum brasilense/genética , Azospirillum brasilense/ultraestructura , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Genes Fúngicos , Genes Reguladores , Prueba de Complementación Genética , Genotipo , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Fenotipo , Raíces de Plantas , Proteínas Represoras/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transactivadores/química , Factores de Transcripción/químicaRESUMEN
From the nifc mutant plasmid pPC868, previously shown to carry a DNA duplication responsible for the Nifc phenotype, a 10 kb HindIII fragment was cloned into the multicopy vector pBR325. Restriction analysis of the resulting plasmids and in vitro deleted derivatives confirmed that the mutation was a fusion between his and nifLA. The order was hisG-hisD'-'nifL-nifA so that nifA was transcribed under the control of the his promoter and the nifL gene was altered. In addition the cloned fragment contained the adjacent nifBQ operon, and complementation data revealed that the nifA, nifB and hisG genes were expressed. Synthesis of nifA product under the transcription control of the his (or cat [CmR]) promoter enabled complementation of nifA and nifB mutations either in the absence or the presence of ammonia, but did not restore nitrogen fixation in a glnF mutant. Therefore, the nifA gene product requires glnF for its positive control function in a manner analogous to ntrC. Protein content analysis of minicells containing various multicopy nif plasmids confirmed the genetic organization mentioned above. A new polypeptide of 51,500 daltons was found whose synthesis was observed at 30 degrees C but not at 37 degrees C. According to the physical map, this protein could be the nifB gene product. Our results are in agreement with nifB transcription being under the control of a thermolabile nifA product. Moreover we obtained results suggesting that the presence of multiple copies of a functional nifB gene inhibited nitrogen fixation.
Asunto(s)
Clonación Molecular , ADN Bacteriano/análisis , Klebsiella pneumoniae/genética , Operón , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , Peso Molecular , Mutación , Fenotipo , PlásmidosRESUMEN
The expression of nifA-, niH- and nifB-lacZ fusions was examined in different mutants of Azospirillum brasilense. Mutations in nifA, glnA and glnB severely impaired the expression of nifH- and nifB-lacZ fusions. By contrast, a nifA-lacZ fusion was not affected in a nifA or a glnB background and was only partially impaired in glnA mutants. It is proposed that in A. brasilense, the PII protein and glutamine synthetase are involved in a post-translational modification of NifA.
Asunto(s)
Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Expresión Génica , Genes Bacterianos , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Operón Lac , Datos de Secuencia Molecular , Mutagénesis Insercional , Fijación del Nitrógeno/genéticaAsunto(s)
Colesteatoma/etiología , Otitis Media/complicaciones , Membrana Timpánica , Niño , HumanosRESUMEN
The functional organization of the glnB-A cluster of Azospirillum brasilense, which codes for the PII protein and glutamine synthetase, respectively, was studied with the aid of lacZ fusions, deletion mapping, site-directed mutagenesis, and complementation. It was shown previously by mRNA mapping that the cluster contains two tandemly organized promoters, glnBp1 and glnBp2, of the sigma 70 and sigma 54 types, respectively, upstream of glnB and a third unidentified promoter upstream of glnA. Data obtained with lacZ fusions in the wild-type strain confirmed that cotranscription of glnBA and transcription of glnA alone were oppositely regulated by the cell N status. Quantification of promoter activities showed a high level of transcription from glnBp1p2 and a low level from glnAp under conditions of nitrogen limitation. The opposite situation prevails under conditions of nitrogen excess. As a consequence, PII polypeptide synthesis is increased under conditions of nitrogen fixation, which strongly suggests that PII plays an important role under these conditions. Null mutant strains of glnB, ntrB-ntrC, nifA, and point mutant strains in glnA were analyzed. NtrB and NtrC are not involved in the regulation of glnBA expression, in contrast to PII and glutamine synthetase. Glutamine synthetase probably acts by modulating the intracellular N status, and PII acts by modifying the properties of an unidentified regulator which might be a functional homolog of NtrC. In addition, a Nif- null mutant strain of glnB was characterized further. A Nif+ phenotype was restored to the strain by nifA from Klebsiella pneumoniae but not by nifA from A. brasilense. This mutant strain is not impaired in NifA synthesis, which is relatively independent of the growth conditions in A. brasilense. It is therefore most likely that PII is required for NifA activation under conditions of nitrogen fixation. Deletion mapping and site-directed mutagenesis showed glnAp was located within a 45-bp DNA fragment upstream of the mRNA start site, dissimiar to previously described consensus sites for sigma factors.
Asunto(s)
Azospirillum brasilense/genética , Proteínas Bacterianas/genética , Genes Bacterianos/genética , Glutamato-Amoníaco Ligasa/genética , Regiones Promotoras Genéticas/genética , Amoníaco/metabolismo , Secuencia de Bases , Mapeo Cromosómico , Análisis Mutacional de ADN , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes/genética , Mutagénesis Sitio-Dirigida , Fijación del Nitrógeno/genética , Proteínas PII Reguladoras del Nitrógeno , Fenotipo , Mutación Puntual , Proteínas Recombinantes/biosíntesis , Eliminación de Secuencia , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
In Gram-negative bacteria, the TonB-ExbB-ExbD inner membrane multiprotein complex is required for active transport of diverse molecules through the outer membrane. We present evidence that Serratia marcescens, like several other Gram-negative bacteria, has two TonB proteins: the previously characterized TonBSM, and also HasB, a newly identified component of the has operon that encodes a haemophore-dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquires free or haemoprotein-bound haem and presents it to a specific outer membrane haemophore receptor (HasR). TonBSM and HasB are significantly similar and can replace each other for haem acquisition. However, TonBSM, but not HasB, mediates iron acquisition from iron sources other than haem and haemoproteins, showing that HasB and TonBSM only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system demonstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non-functional in E. coli. Nevertheless, a mutation in the HasB transmembrane anchor domain allows it to replace TonBEC for haem acquisition. As the change affects a domain involved in specific TonBEC-ExbBEC interactions, HasB may be unable to interact with ExbBEC, and the HasB mutation may allow this interaction. In E. coli, the HasB mutant protein was functional for haem uptake but could not complement the other TonBEC-dependent functions, such as iron siderophore acquisition, and phage DNA and colicin uptake. Our findings support the emerging hypothesis that TonB homologues are widespread in bacteria, where they may have specific functions in receptor-ligand uptake systems.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Hemo/metabolismo , Proteínas de la Membrana/metabolismo , Serratia marcescens/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transporte Biológico , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Immunoblotting , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Plásmidos , Serratia marcescens/químicaRESUMEN
It is possible through investigation under the electronic microscope to determine the location and nature of lesions with accuracy. These lesions are mainly found on the epithelium of proximal and distal convoluted tubules. However, the extent of lesions together with the deterioration process of the cell-constituents vary from one structure to the other and even from cell to cell. On the other hand, while blood capillary vessels pertaining to tubules may also show moderate lesions, glomerular and interstitial deterioration is rare. Kidney deterioration which may either result directly from one of the poison effects or from related phenomena, is under discussion.
Asunto(s)
Enfermedades Renales/patología , Riñón/ultraestructura , Paraquat/envenenamiento , Biopsia , Capilares/ultraestructura , Células Epiteliales , Epitelio/ultraestructura , Humanos , Glomérulos Renales/ultraestructura , Túbulos Renales Distales/ultraestructura , Túbulos Renales Proximales/ultraestructura , Masculino , Persona de Mediana EdadRESUMEN
A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nifgene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.
Asunto(s)
Azospirillum brasilense/genética , ADN Bacteriano/genética , Genes Bacterianos , Fijación del Nitrógeno/genética , Proteínas Bacterianas/genética , Bacterias Aerobias Gramnegativas/genética , Klebsiella pneumoniae/genética , Nitrogenasa/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.