RESUMO
Seven diazotrophs that grow well under Mo-deficient, N(2)-fixing conditions were isolated from a variety of environments. These isolates fall in the gamma subdivision of the class Proteobacteria and have genes that encode the Mo nitrogenase (nitrogenase 1) and the V nitrogenase (nitrogenase 2). Four of the isolates also harbor genes that encode the iron-only nitrogenase (nitrogenase 3).
Assuntos
Microbiologia Ambiental , Gammaproteobacteria/isolamento & purificação , Genes Bacterianos , Fixação de Nitrogênio/genética , Nitrogenase/genética , Sequência de Aminoácidos , Gammaproteobacteria/genética , Gammaproteobacteria/crescimento & desenvolvimento , Gammaproteobacteria/metabolismo , Ferro/química , Dados de Sequência Molecular , Molibdênio/química , Nitrogenase/química , Nitrogenase/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Microbiologia da ÁguaRESUMO
A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.
Assuntos
Genes Bacterianos , Bactérias Gram-Negativas/enzimologia , Molibdênio/farmacologia , Fixação de Nitrogênio/genética , Nitrogenase/genética , Azotobacter/enzimologia , Azotobacter/genética , Amplificação de Genes , Bactérias Gram-Negativas/genética , Ferro/química , Dados de Sequência Molecular , Nitrogenase/metabolismo , Filogenia , Reação em Cadeia da Polimerase , Pseudomonadaceae/enzimologia , Pseudomonadaceae/genética , Rhodospirillum rubrum/enzimologia , Rhodospirillum rubrum/genética , Vanádio/químicaRESUMO
AnfA, an activator required for transcription of the structural genes encoding nitrogenase 3 (anfHDGK) in Azotobacter vinelandii, has a potential metal-binding site [(S19)H(C21)FTGE(C26)R] in its N terminus. Growth studies and expression of an anfH-lacZ fusion in mutants containing amino acid substitutions in this site indicate that Ser-19 is not required for AnfA activity whereas Cys-21 and Cys-26 are required. Residual expression of the anfH-lacZ fusion in AnfA- mutants was found to be due to activation by VnfA, the activator required for expression of genes encoding nitrogenase 2.
Assuntos
Azotobacter/genética , Proteínas de Bactérias , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Nitrogenase/genética , Regiões Promotoras Genéticas/genética , Transativadores/genética , Azotobacter/enzimologia , Azotobacter/crescimento & desenvolvimento , Sequência de Bases , Análise Mutacional de DNA , Dados de Sequência Molecular , Molibdênio/metabolismo , Nitrogenase/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Transcrição Gênica , Vanádio/metabolismoRESUMO
Five major anfH-hybridizing mRNA species accumulated in Azobacter vinelandii cells derepressed for nitrogenase-3 (an alternative nitrogenase, which appears to lack Mo and V). Using anfH-, anfD-, anfG-, anfK-, and orflorf2-specific probes and mutant strains of A. vinelandii these mRNA species have been identified as encoding anfHDGKorflorf2 (6.0 kb), anfHDGK (4.3 kb), anfHD (2.6 kb), vnfHorfFd (1.3 kb), and vnfH and (or) anfH (1.0 kb). A 0.6-kb mRNA species, which hybridized only to the orflorf2-specific probe, and a 3.5-kb mRNA species, which hybridized to anfD or anfK, also accumulated under these conditions. Northern blot analysis and S1 nuclease mapping indicate that transcription of the anf structural gene cluster initiates at a unique nif consensus promoter situated 127 base pairs upstream from the anfH coding region. Observation of anfH-hybridizing mRNA species that accumulate in strains derepressed for nitrogen fixation demonstrates that transcription of the anfHDGKorflorf2 cluster is normally repressed by Mo, V, and NH4+, whereas transcription of the vnfHorfFd cluster does not require the presence of V and is repressed only by Mo, but not NH4+. Analysis of the accumulation of mRNAs in a tungsten-tolerant strain revealed that Mo and V repression of anf transcription must occur by different mechanisms.
Assuntos
Azotobacter vinelandii/genética , Proteínas de Bactérias , Genes Bacterianos , Nitrogenase/genética , RNA Mensageiro/metabolismo , Amônia/farmacologia , Azotobacter vinelandii/efeitos dos fármacos , Azotobacter vinelandii/enzimologia , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , DNA Bacteriano , Dados de Sequência Molecular , Molibdênio/farmacologia , Família Multigênica , Nitrogenase/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , Transcrição Gênica , Tungstênio/farmacologia , Vanádio/farmacologiaRESUMO
The nucleotide sequence (6,559 base pairs) of the genomic region containing the structural genes for nitrogenase 2 (V nitrogenase) from Azotobacter vinelandii was determined. The open reading frames present in this region are organized into two transcriptional units. One contains vnfH (encoding dinitrogenase reductase 2) and a ferredoxinlike open reading frame (Fd). The second one includes vnfD (encoding the alpha subunit of dinitrogenase 2), vnfG (encoding a product similar to the delta subunit of dinitrogenase 2 from A. chroococcum), and vnfK (encoding the beta subunit of dinitrogenase 2). The 5'-flanking regions of vnfH and vnfD contain sequences similar to ntrA-dependent promoters. This gene arrangement allows independent expression of vnfH-Fd and vnfDGK. Mutant strains (CA80 and CA11.80) carrying an insertion in vnfH are still able to synthesize the alpha and beta subunits of dinitrogenase 2 when grown in N-free, Mo-deficient, V-containing medium. A strain (RP1.11) carrying a deletion-plus-insertion mutation in the vnfDGK region produced only dinitrogenase reductase 2.
