Identification of rhizobial strains nodulating Egyptian grain legumes

Fifty four bacterial strains were isolated from root nodules of the grain legumes Cicer arietinum, Lens esculentus, Phaseolus vulgaris, Pisum sativum, and Vicia faba grown in cultivated lands of Beni-Suef Governorate (Egypt). Repetitive extragenic palindromic (REP)-polymerase chain reaction (PCR) clustered the strains into 15 REP-PCR groups. The nearly complete sequence of the 16S rRNA gene from a representative strain of each REP-PCR pattern showed that the strains were closely related to members of the family Rhizobiaceae of the Alphaproteobacteria. Pairwise alignments between globally aligned sequences indicated that the strains from V. faba had 99.6% identity with Rhizobium leguminosarum, and those from P. vulgaris 99.76% and 100% with sequences from R. leguminosarum and R. mesosinicum, respectively. Strains from P. sativum had 99.76%, 99.84%, and 99.92% sequence identity with R. leguminosarum, R. etli, and R. pisi, respectively, and those from L. esculentus had 99.61% identity with sequences from R. leguminosarum. Sequences of the strains from C. arietinum had 100% identity with those of Mesorhizobium amorphae and M. robiniae, respectively. Nitrogenase activity, determined as acetylene-dependent ethylene production, was detected in nodules formed after inoculation of the corresponding host plant with the representative rhizobial species (AU)

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Substrate recognition of nitrogenase-like dark operative protochlorophyllide oxidoreductase from Prochlorococcus marinus.

Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ringDbya protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR plays an important role in chlorophyll biosynthesis of gymnosperms, mosses, ferns, algae, and photosynthetic bacteria in the absence of light. Although DPOR shares significant amino acid sequence homologies with nitrogenase, only the initial catalytic steps resemble nitrogenase catalysis. Substrate coordination and subsequent [Fe-S] cluster-dependent catalysis were proposed to be unrelated. Here we characterized the first cyanobacterial DPOR consisting of the homodimeric protein complex ChlL(2) and a heterotetrameric protein complex (ChlNB)(2). The ChlL(2) dimer contains one EPR active [4Fe-4S] cluster, whereas the (ChlNB)(2) complex exhibited EPR signals for two [4Fe-4S] clusters with differences in their g values and temperature-dependent relaxation behavior. These findings indicate variations in the geometry of the individual [4Fe-4S] clusters found in (ChlNB)(2). For the analysis of DPOR substrate recognition, 11 synthetic derivatives with altered substituents on the four pyrrole rings and the isocyclic ring plus eight chlorophyll biosynthetic intermediates were tested as DPOR substrates. Although DPOR tolerated minor modifications of the ring substituents on rings A-C, the catalytic target ring D was apparently found to be coordinated with high specificity. Furthermore, protochlorophyllide a, the corresponding [8-vinyl]-derivative and protochlorophyllide b were equally utilized as substrates. Distinct differences from substrate binding by LPOR were observed. Alternative biosynthetic routes for cyanobacterial chlorophyll biosynthesis with regard to the reduction of the C8-vinyl group and the interconversion of a chlorophyll a/b type C7 methyl/formyl group were deduced.

Expression and association of group IV nitrogenase NifD and NifH homologs in the non-nitrogen-fixing archaeon Methanocaldococcus jannaschii.

Using genomic analysis, researchers previously identified genes coding for proteins homologous to the structural proteins of nitrogenase (J. Raymond, J. L. Siefert, C. R. Staples, and R. E. Blankenship, Mol. Biol. Evol. 21:541-554, 2004). The expression and association of NifD and NifH nitrogenase homologs (named NflD and NflH for "Nif-like" D and H, respectively) have been detected in a non-nitrogen-fixing hyperthermophilic methanogen, Methanocaldococcus jannaschii. These homologs are expressed constitutively and do not appear to be directly involved with nitrogen metabolism or detoxification of compounds such as cyanide or azide. The NflH and NflD proteins were found to interact with each other, as determined by bacterial two-hybrid studies. Upon immunoisolation, NflD and NflH copurified, along with three other proteins whose functions are as yet uncharacterized. The apparent presence of genes coding for NflH and NflD in all known methanogens, their constitutive expression, and their high sequence similarity to the NifH and NifD proteins or the BchL and BchN/BchB proteins suggest that NflH and NflD participate in an indispensable and fundamental function(s) in methanogens.

Functional genomic analysis of three nitrogenase isozymes in the photosynthetic bacterium Rhodopseudomonas palustris.

The photosynthetic bacterium Rhodopseudomonas palustris is one of just a few prokaryotes described so far that has vnf and anf genes for alternative vanadium cofactor (V) and iron cofactor (Fe) nitrogenases in addition to nif genes for a molybdenum cofactor (Mo) nitrogenase. Transcriptome data indicated that the 32 genes in the nif gene cluster, but not the anf or vnf genes, were induced in wild-type and Mo nitrogenase-expressing strains grown under nitrogen-fixing conditions in Mo-containing medium. Strains that were unable to express a functional Mo nitrogenase due to mutations in Mo nitrogenase structural genes synthesized functional V and Fe nitrogenases and expressed vnf and anf genes in nitrogen-fixing growth media that contained Mo and V at concentrations far in excess of those that repress alternative nitrogenase gene expression in other bacteria. Thus, not only does R. palustris have multiple enzymatic options for nitrogen fixation, but in contrast to reports on other nitrogen-fixing bacteria, the expression of its alternative nitrogenases is not repressed by transition metals. Between 95 and 295 genes that are not directly associated with nitrogenase synthesis and assembly were induced under nitrogen-fixing conditions, depending on which nitrogenase was being used by R. palustris. Genes for nitrogen acquisition were expressed at particularly high levels during alternative nitrogenase-dependent growth. This suggests that alternative nitrogenase-expressing cells are relatively starved for nitrogen and raises the possibility that fixed nitrogen availability may be the primary signal that controls the synthesis of the V and Fe nitrogenases.

Identification of two new genes involved in diazotrophic growth via the alternative Fe-only nitrogenase in the phototrophic purple bacterium Rhodobacter capsulatus.

Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed.

The H-NS-like protein HvrA modulates expression of nitrogen fixation genes in the phototrophic purple bacterium Rhodobacter capsulatus by binding to selected nif promoters.

Genetic analyses based on chromosomal lac fusions to nitrogen fixation (nif) genes demonstrated that NifA-dependent transcriptional activation of expression of Rhodobacter capsulatus nifH and nifB1 was negatively modulated by HvrA, whereas regulation of rpoN, nifA1, and nifA2 was independent of HvrA. Expression of hvrA itself was not influenced by a mutation in ntrC, which is absolutely essential for N(2) fixation. Furthermore, HvrA accumulated to comparable levels in the presence and absence of ammonium, suggesting that the amount of HvrA in the cells does not differ under nitrogenase-repressing or -derepressing conditions. In addition, competitive gel retardation studies with HvrA-His(6) purified from R. capsulatus were carried out, demonstrating preferential binding of HvrA to the nifH promoter region.

Regulation of biological nitrogen fixation.

Biological nitrogen fixation, a process found only in some prokaryotes, is catalyzed by the nitrogenase enzyme complex. Bacteria containing nitrogenase occupy an indispensable ecological niche, supplying fixed nitrogen to the global nitrogen cycle. Due to this inceptive role in the nitrogen cycle, diazotrophs are present in virtually all ecosystems, with representatives in environments as varied as aerobic soils (e.g., Azotobacter species), the ocean surface layer (Trichodesmium) and specialized nodules in legume roots (Rhizobium). In any ecosystem, diazotrophs must respond to varied environmental conditions to regulate the tremendously taxing nitrogen fixation process. All characterized diazotrophs regulate nitrogenase at the transcriptional level. A smaller set also possesses a fast-acting post-translational regulation system. Although there is little apparent variation in the sequences and structures of nitrogenases, there appear to be almost as many nitrogenase-regulating schemes as there are nitrogen-fixing species. Herein are described the paradigms of nitrogenase function, transcriptional control and post-translational regulation, as well as the variations on these schemes, described in various nitrogen-fixing bacteria. Regulation is described on a molecular basis, focusing on the functional and structural characteristics of the proteins responsible for control of nitrogen fixation.

The role of nitrogenase in a cyanide-degrading Klebsiella oxytoca strain.

It is well known that the major function of nitrogenase is to fix atmospheric nitrogen. However, cyanide can also serve as a subtrate for nitrogenase and can be reduced to CH4 and NH4+. A cyanide-degrading Klebsiella oxytoca strain was isolated from cyanide contaminated water. This isolate was also found to have a nitrogen-fixation capability. Nitrogenase activities in this organism could be induced by KCN. However, there was no significant difference of the induction effect between 1 mM KCN and 5 mM KCN. It was found that the cyanide-degrading ability of this isolate could be inhibited by multicopy hybrid pGR112 nif-containing plasmids. Comparing the wild type K. oxytoca strain with the pGR112 plasmid transformed strain, a typical diauxic growth of the wild type strain was observed in a medium containing NH4Cl and KCN. Although the nif plasmid transformed strain also exhibited diauxic growth in the same medium, a much longer second lag phase was noted. In addition, methane, the nitrogenase reduction end product of cyanide, could be detected on cyanide-containing growth cultures. Ammonium chloride, a repressor of nitrogenase gene expression, was consumed prior to KCN in both strains. Again, the degradation of KCN in the pGR112 transformed strain occurred only under loose control of the nitrogenase gene. These findings strongly suggest that nitrogenase may be the sole cyanide-degrading enzyme in this organism.

Characterization of the gamma protein and its involvement in the metallocluster assembly and maturation of dinitrogenase from Azotobacter vinelandii.

Dinitrogenase, the enzyme capable of catalyzing the reduction of N2, is a heterotetramer (alpha 2 beta 2) and contains the iron-molybdenum cofactor (FeMo-co) at the active site of the enzyme. Mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase, which is enzymatically inactive but can be activated in vitro by the addition of purified FeMo-co. Apodinitrogenase from certain mutant strains of Azotobacter vinelandii has a subunit composition of alpha 2 beta 2 gamma 2. The gamma subunit has been implicated as necessary for the efficient activation of apodinitrogenase in vitro. Characterization of gamma protein in crude extracts and partially pure fractions has suggested that it is a chaperone-insertase required by apodinitrogenase for the insertion of FeMo-co. These are three major forms of gamma protein detectable by Western analysis of native gels. An apodinitrogenase-associated form is found in extracts of nifB or nifNE strains and dissociates from the apocomplex upon addition of purified FeMo-co. A second form of gamma protein is unassociated with other proteins and exists as a homodimer. Both of these forms of gamma protein can be converted to a third form by the addition of purified FeMo-co. This conversion requires the addition of active FeMo-co and correlates with the incorporation of iron into gamma protein. Crude extracts that contain this form of gamma protein are capable of donating FeMo-co to apodinitrogenase, thereby activating the apodinitrogenase. These data support a model in which gamma protein is able to interact with both FeMo-co and apodinitrogenase, facilitate FeMo-co insertion into apodinitrogenase, and then dissociate from the activated dinitrogenase complex.

nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations.

The expression of nif genes in Rhodobacter capsulatus depends on the two regulatory genes, rpoN and nifA, encoding a nif-specific alternative sigma factor of RNA polymerase and a nif-specific transcriptional activator, respectively. The expression of the rpoN gene itself is also RPON/NIFA dependent. In order to better characterize the regulation of nif gene induction, chromosomal nifH-, rpoN-, nifA1- and nifA2- lacZ fusions were constructed and the expression of these different nif-lacZ fusions was determined under photoheterotrophic conditions at different starting ammonium concentrations. The two nifA genes were found to be induced first, followed by nifH and finally by rpoN upon weak, medium and strong nitrogen starvation, respectively. This induction profile and the correlation between the expression of the different nif genes suggested that nifA1 expression is the limiting factor for nif gene induction. This hypothesis was tested by construction of different nifA1 overexpressing mutants. Contrary to the current model of nif gene expression in R. capsulatus, which predicted constitutive nif gene expression in such mutants, a strong repression of nifH and rpoN was found at high ammonium concentration. The low nifH expression under these conditions is unaffected by nifA2 and is not increased in a ntrC mutant, ruling out any role of NTRC as a mediator of this repression. This finding implies an additional, so far unidentified, regulation by fixed nitrogen in R. capsulatus. Changing the expression level of rpoN indicated that low levels of RPON are already sufficient for full nifH induction. The nifA1 and rpoN expression mutants were also tested for diazotrophic growth. Similar generation times were determined for the mutants and for the wild type, but diazotrophic growth of the nifA1 over-expressing ntrC mutant RCM14 did not start until after a prolonged lag phase of two to three days.

Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the alpha- and beta-subunits.

The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.