Nif Expression Research Articles

Identification and cloning of an Erwinia carotovora subsp. carotovora bacteriocin regulator gene by insertional mutagenesis.

Avirulent Erwinia carotovora subsp. carotovora CGE234-M403 produces two types of bacteriocin. For the purpose of cloning the bacteriocin genes of strain CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-rif-11-2, was isolated. By Tn5 insertional mutagenesis using M-rif-11-2, a mutant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of a contiguous 1,280-bp region were determined. One complete open reading frame (ORF), designated ORF2, was identified within the sequenced fragment. The 3' end of another ORF, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. Downstream from ORF2, the 5' end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 amino acids, which showed high homology with Yersinia enterocolitica Yrp, a regulator of enterotoxin (Y-ST) production; Escherichia coli host factor 1, required for Qbeta-replicase; and Azorhizobium caulinodans NrfA, required for the expression of nifA. ORF2 was designated brg, bacteriocin regulator gene. A fragment containing ORF2 and its promoter was amplified and cloned into pBR322 and pHSG415r, and the recombinant plasmids, pBYL1 and pHYL1, were transferred into E. coli DH5. Plasmid pBYL1 was reisolated and transferred into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-weight bacteriocin.

Differential gene expression in Azospirillum brasilense Cd under saline stress

Previous studies in our laboratory demonstrated that the attachment of Azospirillum brasilense Cd to maize and wheat roots was altered when the bacteria were grown under saline stress. The aim of this work was to analyze the interaction when either the plant or both components were exposed to saline conditions. We demonstrated that this stress altered the early stages of development, in this plant model, leading to an inadequate colonization. The effect of saline stress on the expression of A. brasilense N2 fixation gene promoters was also studied. While nifA expression was increased in stressed bacteria, nifH transcription was diminished, suggesting an alteration in the NifA activation process. The induction of the nif gene transcription by root exudates was investigated, and the results suggest that they could be modulating certain bacterial processes, such as nitrogen fixation. However, stressed bacteria did not respond to the exudate's action. This could be related to the modified membrane composition resulting from changes in proteins, glucans, and capsular, exo- and lipopolysaccharides. We conclude that saline stress alters the Azospirillum brasilense Cd - maize interaction, the normal colonization, and the nitrogen fixation process.Key words: Azospirillum brasilense, saline stress, Azospirillum-root interaction, exudates, nif genes.

The control of Azorhizobium caulinodans nifA expression by oxygen, ammonia and by the HF-I-like protein, NrfA.

The control of Azorhizobium caulinodans nifA expression in response to oxygen and ammonia involves FixLJ, FixK, NtrBC, NtrXY and the HF-I-like protein NrfA. The regulation is thus complex and possibly involves post-transcriptional regulation by NrfA. The coding region of nifA was determined using a translational lacZ fusion and by site-directed mutagenesis to identify which of four in frame AUG codons was used. The major NifA protein is translated from the second AUG codon and is predicted to consist of 613 amino acids. Primer extension analysis showed a major transcript starting 34 bp downstream from the anaerobox in wild-type, nifA, rpoN, ntrC and nrfA strains, but not in a fixK mutant. FixK- and oxygen-dependent transcription of nifA was confirmed by the analysis of four transcriptional nifA-lacZ fusions with fusion junctions at positions +1, +47, +110 and +181 with respect to the start site. Regulation by ammonia was independent of FixK and RpoN, NtrC being only partially required. Thus, there may be another type of nitrogen control that does not involve NtrC in A. caulinodans. NrfA is not required for the initiation of nifA transcription but, most probably, has an effect on nifA mRNA stability and/or translation. NrfA also restores the defect in rpoS translation to an Escherichia coli hfq mutant, indicating that HF-I and NrfA have similar activities in both A. caulinodans and E. coli.

Structural organization and regulation of the nif genes of Herbaspirillum seropedicae

The promoter regions of nifA and nifH genes of Herbaspirillom seropedicae have been isolated and investigated, and the nifA promoter has been characterized in detail. Both regions contain a −24 −12 type promoter and NifA-binding sites; the nifA promoter also has an NtrC-binding site. The nifA expression is activated by NtrC and repressed by ammonium ions but not by oxygen. When the NtrC-binding site was deleted nifA expression was NifA-dependent. The native NifA protein of H. seropedicae was unable to activate nifH expression in Escherichia coli whereas a truncated protein, lacking the N-terminal domain was able to activate nifH expression in the absence of oxygen and presence of iron, indicating that the N-terminal domain regulates NifA activity. The truncated protein also activates nifH in Azospirillum brasilense in the presence or absence of ammonium ions. This suggests that the N-terminal domain senses the nitrogen status of the cell.

Transcription termination within the regulatorynifLA operon ofKlebsiella pneumoniae

The effect of premature stop codons in thenifL gene on the expression ofnifA-lacZ operon and protein fusions inKlebsiella pneumoniae was analysed in detail. Our results revealed transcriptional polarity in this operon. By dissecting the operon, intragenic regions containing Rho-dependent transcription terminators have been identified. As shown for other Rhodependent terminators, their cytosine content is much higher than the incidence of guanines. However, other regions of the operon that have this feature did not show termination activity, suggesting that, contrary to previous reports, a correlation between these parameters cannot readily be established. Some of our results also suggested that, in addition to polarity, other mechanisms may prevent expression ofnifA when translation ofnifL is altered. Their importance for efficient regulation of nitrogen fixation genes is discussed.

Transcription termination within the regulatory

The effect of premature stop codons in the nifL gene on the expression of nifA-lacZ operon and protein fusions in Klebsiella pneumoniae was analysed in detail. Our results revealed transcriptional polarity in this operon. By dissecting the operon, intragenic regions containing Rho-dependent transcription terminators have been identified. As shown for other Rho-dependent terminators, their cytosine content is much higher than the incidence of guanines. However, other regions of the operon that have this feature did not show termination activity, suggesting that, contrary to previous reports, a correlation between these parameters cannot readily be established. Some of our results alos suggested that, in addition to polarity, other mechanisms may prevent expression of nifA when translation of nifL is altered. Their importance for efficient regulation of nitrogen fixation genes is discussed.

Yst gene expression in Yersinia enterocolitica is positively regulated by a chromosomal region that is highly homologous to Escherichia coli host factor 1 gene (hfq).

Yersinia enterocolitica produces heat-stable enterotoxin (Y-ST) as one of its virulence factors. The yst gene, however, frequently and spontaneously becomes inactive (silent) during storage, which is accompanied by concurrent changes in some biological properties such as colony morphology, growth rate, carbon fermentation and ornithine decarboxylase activity. Northern blot analysis revealed that the level of mRNA for yst was repressed. To investigate the regulatory region, we transformed a yst-silent strain with a chromosomal gene library of Y-ST producing an isogenic counterpart. Out of 3604 clones, one clone resumed the Y-ST production and concurrently other biological properties. An open reading frame in this clone was designated as yrp, yersinia regulator for pleiotropic phenotype. Deduced from the nucleotide sequence, Yrp was a small protein of I01 amino acids with no similarity with any regulatory factor described, but showed high homology with an Escherichia coli host factor 1 required for Q beta-replicase, and with an Azorhizobium caulinodans NrfA required for the expression of nifA. The yrp gene mutation caused decreased negative supercoiling of plasmids, as did hfq. The yrp gene could similarly complement Y-ST production in two other silent strains of Y. enterocolitica. In all three silent strains examined, we found various mutations in the yrp region.

Overlapping promoters for two different RNA polymerase holoenzymes control Bradyrhizobium japonicum nifA expression

The Bradyrhizobium japonicum NifA protein, the central regulator for nitrogen fixation gene expression, is encoded in the fixRnifA operon. This operon is activated during free-living anaerobic growth and in the symbiotic root nodule bacteroid state. In addition, it is expressed in aerobic conditions, albeit at a low level. Here, we report that this pattern of expression is due to the presence of two overlapping promoters: fixRp1, which is of the -24/-12 class recognized by the RNA polymerase sigma 54, and fixRp2, which shares homology with the -35 and -10 regions found in other putative B. japonicum housekeeping promoters. Primer extension analyses showed that fixRp1 directed the synthesis of a transcript, P1, that starts 12 nucleotides downstream of the -12 region. In addition to sigma 54, P1 was dependent on NifA and low oxygen tension. Transcripts originating from fixRp2 started at two sites: one coincided with P1, while the most abundant, P2 initiated just two nucleotides further downstream of P1. Expression from fixRp2 was dependent on the upstream -68 promoter region, a region known to bind a putative activator protein, but it was independent of sigma 54 and NifA. This promoter was expressed in aerobic and anaerobic conditions but was not expressed in 30-day-old bacteroids. Mutations in the conserved 12 region for the sigma 54 promoter did not show any transcript, because these mutations also disrupted the overlapping -10 region of the fixRp2 promoter. Conversely, mutations at the -24 region only affected the sigma 54-dependent P1 transcript, having no effect on the expression of P2. In the absence of omega(54), anaerobic expression from the fixRp(2) promoter was enhanced threefold, suggesting that in the wild-type strain, the two RNA polymerase holoenzymes must compete for binding to the same promoter region.

Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression.

The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported. However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions. This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field. The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nif regulon and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule. These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass. The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control. Each treatment was replicated eight times and was conducted at four field sites in Wisconsin. Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9% compared with the yield with the wild-type strain RMBPC and 17.9% over that in the uninoculated control plot at the site where soil nitrogen and organic matter content was lowest. These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season. Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site. At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. We conclude that RMBPC -2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient moisture.

Posttranslational regulation of nitrogenase activity in Azospirillum brasilense ntrBC mutants: ammonium and anaerobic switch-off occurs through independent signal transduction pathways

Nitrogenase activity is regulated by reversible ADP-ribosylation in response to NH4+ and anaerobic conditions in Azospirillum brasilense. The effect of mutations in ntrBC on this regulation was examined. While NH4+ addition to ntrBC mutants caused a partial loss of nitrogenase activity, the effect was substantially smaller than that seen in ntr+ strains. In contrast, nitrogenase activity in these mutants was normally regulated in response to anaerobic conditions. The analysis of mutants lacking both the ntrBC gene products and dinitrogenase reductase activating glycohydrolase (DRAG) suggested that the primary effect of the ntrBC mutations was to alter the regulation of DRAG activity. Although nif expression in the ntr mutants appeared normal, as judged by activity, glutamine synthetase activity was significantly lower in ntrBC mutants than in the wild type. We hypothesize that this lower glutamine synthetase activity may delay the transduction of the NH4+ signal necessary for the inactivation of DRAG, resulting in a reduced response of nitrogenase activity to NH4+. Finally, data presented here suggest that different environmental stimuli use independent signal pathways to affect this reversible ADP-ribosylation system.

The expression of nifA in Azorhizobium caulinodans requires a gene product homologous to Escherichia coli HF-I, an RNA-binding protein involved in the replication of phage Q beta RNA.

We report the characterization of a mutant of Azorhizobium caulinodans, isolated after ethyl methanesulfonate mutagenesis. This Nod+ Nif- Fix- mutant is unable to synthesize 10 of 15 polypeptides normally induced under conditions of nitrogen fixation. By using lacZ fusions it was shown that nifA and nifA-regulated genes were not expressed in this strain. The mutation was complemented by a constitutively expressed nifA gene or by a 1.1-kb DNA fragment from the wild-type strain, whose nucleotide sequence revealed a single open reading frame of 255 bp coding for an 85-amino acid polypeptide. The deduced amino acid sequence is similar to that of HF-I, an RNA-binding protein of Escherichia coli, which is required for replication of bacteriophage Q beta RNA. The similarity can be extended to the function since hfq, the structural gene for HF-I, complemented the A. caulinodans mutant. The corresponding gene in A. caulinodans was termed nrfA (for nif regulatory factor). Inactivation of nrfA in the mutant was due to a missense mutation resulting in the replacement of a cysteine residue by arginine. A null mutant, constructed by disruption of nrfA, exhibited the same phenotype as the missense mutant. Thus, an additional factor can be added to the already complex system of nifA regulation in A. caulinodans.

Oxygen sensitivity and metal ion-dependent transcriptional activation by NIFA protein from Rhizobium leguminosarum biovar trifolii.

The NIFA protein from Rhizobium leguminosarum biovar trifolii (R. trifolii) strain ANU843 lacks an N-terminal domain present in homologous NIFA proteins from other diazotrophs. The R. trifolii nifA gene product is unstable when expressed in Escherichia coli under both aerobic and microaerobic conditions. Stability is increased by fusion of additional amino acids to the N-terminus of the protein or by expression of nifA in sno mutant (presumed protease deficient) strains of E. coli. Transcriptional activation in vivo by R. trifolii NIFA decreases under aerobic growth conditions, or when cultures are depleted of metal ions. In sno mutant strains this decrease in activity reflects a loss of specific activity rather than proteolytic degradation, implying that R. trifolii NIFA requires metal ions for activity and is oxygen sensitive. The addition of 30 amino acids to the amino-terminus of R. trifoli NIFA results in an oxygen-tolerant protein, with metal ion-dependent activity. Metal ions are therefore not only required for oxygen sensing by R. trifolii NIFA but may play an additional role in determining NIFA structure or activity.

Characterization of the ntrBC genes of Azospirillum brasilense Sp7: their involvement in the regulation of nitrogenase synthesis and activity.

A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, was cloned. The nucleotide sequence of a 3.8 kb subfragment was established. This led to the identification of two open reading frames, encoding polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the putative Azospirillum ntrC gene was shown to restore nitrogen fixation under free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulinodans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A brasilense. These mutants were prototrophic and Nif+. However, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusion was expressed in the mutants at about 60% of the level in the wild type. In the presence of ammonia, the fusion was similarly expressed (60% of the maximum) both in the wild type and mutants. Addition of ammonia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitrogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixation, although NtrC controls nifA expression to some extent. They are, however, required for the switch-off of nitrogenase activity.

Autophosphorylation and phosphatase activities of the oxygen-sensing protein FixL of Rhizobium meliloti are coordinately regulated by oxygen.

The FixL and FixJ proteins of Rhizobium meliloti control the expression of other nif and fix genes in response to oxygen levels. FixL is a hemoprotein kinase that senses oxygen availability and responds to the absence of oxygen by activation of its autophosphorylating activity followed by transfer of the phosphate to FixJ. FixJ in turn activates the nifA and fixK promoters. In vitro studies reported here with a soluble truncated version of FixL (FixL*) indicate that, while low oxygen tension specifically increases the autophosphorylating activity of FixL*, the ability of phospho-FixL* to act as a phosphate donor to FixJ is not affected by the presence or absence of oxygen. FixL* is also shown to possess a phosphatase activity that is repressed under anaerobic conditions only when the protein is in the phosphorylated form. A fixL mutant that induces a higher level of nifA promoter activity in the presence of fixJ in vivo displayed both an increased autophosphorylating activity and a decreased phosphatase activity in vitro. These data provide evidence for a role for both autophosphorylation and phosphatase activities of FixL in the mechanism by which oxygen tension within the alfalfa nodule induces expression of bacterial nitrogen fixation genes during symbiosis.

Regulation of nitrogen fixation in Azospirillum brasilense Sp7: involvement of nifA, glnA and glnB gene products.

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.

Ammonia regulation of the Rhizobium meliloti nitrogenase structural and regulatory genes under free-living conditions: involvement of the fixL gene product?

The expression under microaerobic conditions of the Rhizobium meliloti nifA and consequently the nifHDK genes was found to be negatively regulated by ammonia and nitrate. Assimilation of the ammonia to glutamate and glutamine is not required for this regulation to occur. This indicates that ammonia itself, and not a product of its metabolism, may be regulating nif expression. Unlike the situation in Klebsiella pneumoniae, NtrC is apparently not involved in mediating the ammonia effect on nifA expression in R. meliloti. Neither does the fixK gene product, which is known to regulate nifA in R. meliloti, appear to be involved in mediating the ammonia effect. The regulation of nifA by ammonia is shown to be mediated through the FixL protein. A truncated fixJ gene, the product of which has been shown to induce nifA expression irrespective of the oxygen status of the cell, also circumvented the repressive effect of ammonia on nifA expression. This suggests that the ammonia effect is mediated through the FixLJ regulatory cascade. Interestingly no effect of ammonia on fixK expression was observed.

Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism.

Azorhizobium caulinodans ORS571 nifA regulation is partially mediated by the nitrogen regulatory gene ntrC. However, the residual nifA expression in ntrC mutant strains is still modulated by the cellular nitrogen and oxygen status. A second ntrC-homologous region, linked to ntrC, was identified and characterized by site-directed insertion mutagenesis and DNA sequencing. Tn5 insertions in this region cause pleiotropic defects in nitrogen metabolism and affect free-living as well as symbiotic nitrogen fixation. DNA sequencing and complementation studies revealed the existence of a bicistronic operon (ntrYX). NtrY is likely to represent the transmembrane 'sensor' protein element in a two-component regulatory system. NtrX shares a high degree of homology with NtrC proteins of other organisms and probably constitutes the regulator protein element. The regulation of the ntrYX and ntrC loci and the effects of ntrYX, ntrY and ntrX mutations on nifA expression were examined using beta-galactosidase gene fusions. NtrY/NtrX were found to modulate nifA expression and ntrYX transcription was shown to be partially under the control of NtrC.

Involvement of GroEL in nif gene regulation and nitrogenase assembly

Several approaches were used to study the role of GroEL, the prototype chaperonin, in the nitrogen fixation (nif) system. An Escherichia coli groEL mutant transformed with the Klebsiella pneumoniae nif gene cluster accumulated very low to nondetectable levels of nitrogenase components compared with the isogenic wild-type strain or the mutant cotransformed with the wild-type groE operon. In K. pneumoniae, overexpression of the E. coli groE operon markedly accelerated the rate of appearance of the MoFe protein and its constituent polypeptides after the start of derepression. The groEL mutation in E. coli decreased NifA-dependent beta-galactosidase expression from the nifH promoter but did not affect the constitutive expression of nifA from the tet promoter of ntr-controlled expression from the nifLA promoter. The possibility that GroEL is required for the correct folding of NifA was supported by coimmunoprecipitation of NifA with anti-GroEL antibodies. Kinetic analyses of nitrogenase assembly in 35S pulse-chased K. pneumoniae pointed to the existence of high-molecular-weight intermediates in MoFe protein assembly and demonstrated the transient binding of newly synthesized NifH and NifDK to GroEL. Overall, these results indicate that GroEL fulfills both regulatory and structural functions in the nif system.

Influence of oxygen on DNA binding, positive control, and stability of the Bradyrhizobium japonicum NifA regulatory protein

Central to the genetic regulatory circuit that controls Bradyrhizobium japonicum nif and fix gene expression is the NifA protein. NifA activates transcription of several nif and fix genes and autoregulates its expression during symbiosis in soybean root nodules or in free-living microaerobic conditions. High O2 tensions result in the lack of nif expression, possibly by inactivation of NifA through oxidation of an essential metal cofactor. Several B. japonicum nif and fix promoters have upstream activator sequences (UAS) required for optimal activation. The UAS are located more than 100 bp from the -24/-12 promoter and have been proposed to be binding sites for NifA. We investigated the interaction of NifA with the nifD promoter region by using in vivo dimethyl sulfate footprinting. NifA-dependent protection from methylation of the two UAS of this promoter was detected. Footprinting experiments in the presence of rifampin showed that UAS-bound NifA led to the formation of an open nifD promoter-RNA polymerase sigma 54 complex. Shift to aerobic growth resulted in a rapid loss of protection of both the UAS and the promoter, indicating that the DNA-binding and the activation functions of NifA were controlled by the O2 status of the cell. After an almost complete inactivation by oxygen, the NifA protein began to degrade. Furthermore, metal deprivation also caused degradation of NifA. In this case, however, the rates of NifA inactivation and NifA degradation were not clearly distinguishable. The results are discussed in the light of a previously proposed model, according to which the oxidation state of a NifA-metal complex influences the conformation of NifA for both DNA-binding and positive control functions.

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.