Nitrogen fixation by Klebsiella pneumoniae is inhibited by certain multicopy hybrid nif plasmids.

In our studies of nif gene regulation, we have observed that certain hybrid nif plasmids drastically inhibit the expression of the chromosomal nif genes of Klebsiella pneumonia. Wild-type (Nif+) K. pneumoniae strains that acquire certain hybrid nif plasmids also acquire the Nif- phenotype; these strains lose 90 to 99% of all detectable nitrogen fixation activity and grow poorly (or not at all) on solid media with N2 as the sole nitrogen source. We describe experiments which defined this inhibition of the Nif+ phenotype by hybrid nif plasmids and identify and characterize four nif DNA regions associated with this inhibition. We show that plasmids carrying these nif regions could recombine with, but not complement, nif chromosomal mutations. Our results suggest that inhibition of the Nif+ phenotype will provide a useful bioassay for some of the factors that mediate nif gene expression.     

Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.

Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.     

Identification of three genes encoding P(II)-like proteins in Gluconacetobacter diazotrophicus: studies of their role(s) in the control of nitrogen fixation

In our studies on the regulation of nitrogen metabolism in Gluconacetobacter diazotrophicus, an endophytic diazotroph of sugarcane, three glnB-like genes were identified and their role(s) in the control of nitrogen fixation was studied. Sequence analysis revealed that one P(II) protein-encoding gene, glnB, was adjacent to a glnA gene (encoding glutamine synthetase) and that two other P(II) protein-encoding genes, identified as glnK1 and glnK2, were located upstream of amtB1 and amtB2, respectively, genes which in other organisms encode ammonium (or methylammonium) transporters. Single and double mutants and a triple mutant with respect to the three P(II) protein-encoding genes were constructed, and the effects of the mutations on nitrogenase expression and activity in the presence of either ammonium starvation or ammonium sufficiency were studied. Based on the results presented here, it is suggested that none of the three P(II) homologs is required for nif gene expression, that the GlnK2 protein acts primarily as an inhibitor of nif gene expression, and that GlnB and GlnK1 control the expression of nif genes in response to ammonium availability, both directly and by relieving the inhibition by GlnK2. This model includes novel regulatory features of P(II) proteins.     

Effect of nitrogenous compounds on nitrogenase gene expression in anaerobic cultures of Anabaena variabilis.

The effects of several organic and inorganic nitrogen compounds on nitrogenase mRNA and enzyme activity levels were examined in anaerobic cultures of Anabaena variabilis 29413. Even low concentrations of exogenous ammonia (20 microM) prevented nitrogenase gene expression. Nitrate, in contrast, had little effect, even at very high concentrations. Neither compound had a significant direct effect on existing enzyme activity. The amino acids glutamine and glutamate did not repress nif gene expression. Methionine sulfoximine, but not 7-azatryptophan, was shown to eliminate the repressive effect of ammonia, and this action occurred at the mRNA level. Low concentrations of carbamyl phosphate caused a rapid decrease in nitrogenase mRNA levels. These results are consistent with the ideas that nif gene regulation in Anabaena spp. occurs primarily at the mRNA level and that ammonia, and possibly also glutamine and glutamate, is not the immediate effector of regulation.     

固氮螺菌的固氮分子调控研究进展

本文对巴西固氮螺菌周氨基因的结构和调控进行综述。其固氮基因的调控可分为两种水平：通过DRAT-DRAG系统的翻译后水平和通过NifA蛋白的转录水平。通过NifA活性进行调控的机理目前尚不明了。     

Nitrogenase promoter-lacZ fusion studies of essential nitrogen fixation genes in Bradyrhizobium japonicum I110.

DNA fragments containing either the nifD or nifH promoter and 5' structural gene sequences from Bradyrhizobium japonicum I110 were fused in frame to the lacZ gene. Stable integration of these nif promoter-lacZ fusions by homologous double reciprocal crossover into a symbiotically nonessential region of the B. japonicum chromosome provided an easy assay for the effects of potential nif regulatory mutants. The level of beta-galactosidase activity expressed from these two nif promoter-lacZ fusions was assayed in bacteroids of B. japonicum I110 wild type and Fix mutants generated by transposon Tn5 mutagenesis and identified in the accompanying paper. No nif-positive regulatory mutants were identified from among an array of Fix- mutants in which Tn5 was inserted 9 kilobase pairs upstream of the nifDK operon and within the 18-kilobase-pair region separating the nifDK and nifH operons. This result indicates that there are no genes in these regions involved in the regulation of nitrogenase structural gene expression. Interestingly, the level of beta-galactosidase activity expressed from the nifH promoter was twice that expressed from the nifD promoter, suggesting that the normal cellular level of the nifH gene product in bacteroids is in a 2:1 ratio with the nifD gene product instead of in the 1:1 stoichiometry of the nitrogenase enzyme complex.     

Expression of regulatory nif genes in Rhodobacter capsulatus.

Translational fusions of the Escherichia coli lacZ gene to Rhodobacter capsulatus nif genes were constructed in order to determine the regulatory circuit of nif gene expression in R. capsulatus, a free-living photosynthetic diazotroph. The expression of nifH, nifA (copies I and II), and nifR4 was measured in different regulatory mutant strains under different physiological conditions. The expression of nifH and nifR4 (the analog of ntrA in Klebsiella pneumoniae) depends on the NIFR1/R2 system (the analog of the ntr system in K. pneumoniae), on NIFA, and on NIFR4. The expression of both copies of nifA is regulated by the NIFR1/R2 system and is modulated by the N source of the medium under anaerobic photosynthetic growth conditions. In the presence of ammonia or oxygen, moderate expression of nifA was detectable, whereas nifH and nifR4 were not expressed under these conditions. The implications for the regulatory circuit of nif gene expression in R. capsulatus are discussed and compared with the situation in K. pneumoniae, another free-living diazotroph.     

Glutamine synthetase mutations which affect expression of nitrogen fixation genes in Klebsiella pneumoniae.

Previous studies have implicated glutamine synthetase (L-glutamate:ammonia ligase [adenosine diphosphate for-ing], EC 6.6.1.2) as a major controlling element of the nitrogen fixation (nif) genes in Klebsiella pneumoniae. We report here the isolation of a new class of K. pneumoniae mutants which exhibit altered patterns of nif and hut (histidine utlization) regulation. The expression of nif in these mutants, which were isolated as Gln+ (glutamine nonrequiring) revertants of a particular glnA mutation, is extremely sensitive to ammonia repression. These mutants have a Nif- Hut- phenotype at external ammonia concentrations at which wild-type strains are Nif+ Hut+. On the other hand, these mutants can be fully derepressed for nif at very low ammonia concentrations. We adopted the nomenclature "GlnR- (Nif- Hut-)" to facilitate discussion of the phenotype of these mutant strains. The mutations in these strains which confer the GlnR- phenotype map at or near glnA, the structural gene for glutamine synthetase.     

nifV is contiguous to nifHDK in Frankia strain FaC1

The organization of genes with the capacity to code for four proteins involved in nitrogen fixation in Frankia strain FaC1 was determined by restriction fragment mapping and nucleotide sequence analysis. Analysis of the 44-kb genomic cosmid clone pFAH 1. isolated from a cosmid library made from Frankia strain FaCl, resulted in the identification of a 7.2-kb Pst I fragment to which Klebsiella nif H, nif D and nif K probes hybridized. This nif -hybridizing fragment was subcloned and analyzed by restriction fragment mapping. Further subcloning of the 7.2-kb fragment and subsequent sequence analysis of approximately 6.8 kb revealed the presence of six open reading frames (ORFs). Four of these ORFs have the potential to code for nif V-, nif H-, nif D- and nif K-like gene products and the two others are unidentified ORFs. The organization of the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes, but the positioning of the nif V-like gene relative to the nif HDK cluster differs, A consensus nif -promoter-like sequence, found 5'to nif H. was not detected upstream of the nif V-like gene. Nine copies of a 7-bp direct repeat were found 5'to ORFA.