Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense.

The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.     

Cross-functionality of nitrogenase components NifH1 and VnfH in Anabaena variabilis

Anabaena variabilis fixes nitrogen under aerobic growth conditions in differentiated cells called heterocysts using either a Mo nitrogenase or a V nitrogenase. The nifH1 gene, which encodes the dinitrogenase reductase of the Mo nitrogenase that is expressed only in heterocysts, is cotranscribed with nifD1 and nifK1, which together encode the Mo dinitrogenase. These genes were expressed in the presence or absence of molybdate or vanadate. The vnfH gene, which encodes the dinitrogenase reductase of the V nitrogenase, was located about 23 kb from vnfDGK, which encodes the V dinitrogenase; however, like vnfDGK, vnfH was expressed only in the absence of molybdate, with or without vanadate. Like nifH1, the vnfH gene was expressed exclusively in heterocysts under either aerobic or anaerobic growth conditions and thus is under the control of developmental factors. The vnfH mutant was able to grow diazotrophically using the V nitrogenase, because NifH1, which was also made in cells starved for molybdate, could substitute for VnfH. Under oxic conditions, the nifH1 mutant grew in the absence of molybdate but not in its presence, using VnfH, while the nifH1 vnfH double mutant did not grow diazotrophically with or without molybdate or vanadate. A nifH1 mutant that expressed nifDK and vnfH but not vnfDGK was able to grow and fix nitrogen normally, indicating that VnfH could substitute for NifH in the Mo nitrogenase and that these dinitrogenase reductases are not involved in determining the metal specificity of the Mo nitrogenase or the V nitrogenase.     

A DNA fragment hybridizing to a nif probe in Rhodobacter capsulatus is homologous to a 16S rRNA gene

We have sequenced the Rhodobacter capsulatus nifH and nifD genes. The nifH gene, which codes for the dinitrogenase reductase protein, is 894 bp long and codes for a polypeptide of predicted Mr 32,412. The nifD gene, which codes for the alpha subunit of dinitrogenase, is 1,500 bp long and codes for a protein of predicted Mr 56,113. A 776-bp BglII-XhoI fragment containing only nif sequences was used as a hybridization probe against R. capsulatus genomic DNA. Two HindIII fragments, 11.8 kb and 4.7 kb in length, hybridize to this probe. Both fragments have been cloned from a cosmid library. The 11.8-kb fragment contains the nifH, D and K genes, as previously demonstrated (Scolnik and Haselkorn, 1984). In this paper we present evidence that suggests that the 4.7-kb HindIII fragment contains a gene coding for 16S rRNA, and that although homology between nif and this fragment can be observed in filter hybridization experiments, a second copy of the nif structural genes seems not to be present in this region.     

Cloning and expression of draTG genes from Azospirillum lipoferum

A genomic library of Azospirillum lipoferum was constructed with phage lambda EMBL4 as vector. From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), draG, were cloned by hybridization with the heterologous probes of Rhodospirillum rubrum. As in R. rubrum, draT is located between draG and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system). In the crude extract of Escherichia coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity of the cloned genes. Southern hybridization with genomic DNA from different Azospirillum spp., demonstrated a correlation between observable draTG hybridization and the biochemical demonstration of this covalent modification system.     

Role of the dinitrogenase reductase arginine 101 residue in dinitrogenase reductase ADP-ribosyltransferase binding, NAD binding, and cleavage

Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-(32)P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-(14)C]NAD individually upon UV irradiation, but most (14)C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-(14)C]NAD suggested that Arg 101 is not absolutely required for NAD binding.     

Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense.

Reversible ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system, has been characterized in both Rhodospirillum rubrum and Azospirillum brasilense. Although the general functions of DRAT and DRAG are very similar in these two organisms, there are a number of interesting differences, e.g., in the timing and extent of the regulatory response to different stimuli. In this work, the basis of these differences has been studied by the heterologous expression of either draTG or nifH from A. brasilense in R. rubrum mutants that lack these genes, as well as the expression of draTG from R. rubrum in an A. brasilense draTG mutant. In general, these hybrid strains respond to stimuli in a manner similar to that of the wild-type parent of the recipient strain rather than the wild-type source of the introduced genes. These results suggest that the differences seen in the regulatory response in these organisms are not primarily a result of different properties of DRAT, DRAG, or dinitrogenase reductase. Instead, the differences are likely the result of different signal pathways that regulate DRAG and DRAT activities in these two organisms. Our results also suggest that draT and draG are cotranscribed in A. brasilense.     

Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase.

Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG), were constructed, and the effect of this overexpression on in vivo DRAT and DRAG regulation was investigated. Broad-host-range plasmid constructs containing a fusion of the R. rubrum nifH promoter and translation initiation sequences to the second codon of draT, the first gene of the dra operon, were constructed. Overexpression plasmid constructs which overexpressed (i) only functional DRAT, (ii) only functional DRAG and presumably the putative downstream open reading frame (ORF)-encoded protein, or (iii) all three proteins were generated and introduced into wild-type R. rubrum. Overexpression of DRAT still allowed proper regulation of nitrogenase activity, with ADP-ribosylation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexpression. However, overexpression of DRAG and the downstream ORF altered nitrogenase regulation such that dinitrogenase reductase did not accumulate in the ADP-ribosylated form under inactivation conditions, suggesting that DRAG was constitutively active and that therefore DRAG regulation is altered upon overexpression. Proper DRAG regulation was observed in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required for proper DRAG regulation.     

Molecular cloning and physical mapping of the nitrogenase structural genes from the filamentous, non-heterocystous cyanobacterium Pseudanabaena sp. PCC7409

Abstract Sequences homologous to the structural genes for dinitrogenase ( nifD and nifK ) and nitrogenase reductase ( nifH ) have been cloned from the filamentous, non-heterocystous cyanobacterium Pseudanabaena PCC7409. The nifHDK ⁻ homologous sequences were shown to reside on a 6.5-kb Eco RI restriction fragment by using a restriction fragment encoding the Klebsiella pneumoniae nifHDK genes as a heterologous hybridization probe. This 6.5-kb restriction fragment was cloned from a λ gt.wes Eco RI library of the Paseudanabaena sp. PCC7409 genome. This fragment was subcloned into the plasmid vector pUC9 to generate plasmid pPSU20. A detailed physical map of the insert in plasmid pPSU20 was determined, and relative positions of the nifH, nifD , and nifK homologous sequences on this fragment were determined by hybridization analysis with gene-specific fragments derived from the corresponding Anabaena sp. PCC7120 genes. The results indicate that these genes are contiguous in Pseudanabaena sp. PCC 7409 and are arranged in the order nifH, nifD , and nifK . This arrangement resembles that observed for other non-heterocystous cyanobacteria but differs from that observed for Anabaena, Calothrix , and Nostoc species.     

Diversity of Nitrogen Fixation Genes in the Symbiotic Intestinal Microflora of the Termite Reticulitermes speratus

The diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of a lower termite, Reticulitermes speratus, was investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut and were clonally isolated. The phylogenetic analysis of the nifH product amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. A large number of the termite nifH sequences were most closely related to those of a firmicute, Clostridium pasteurianum, with a few being most closely related to either the (gamma) subclass of the proteobacteria or a sequence of Desulfovibrio gigas. Some of the others were distantly related to those of the bacteria and were seemingly derived from the domain Archaea. The phylogenetic positions of these nifH sequences corresponded to those of genera found during a previous determination of rRNA-based phylogeny of the termite intestinal microbial community, of which a majority consisted of new, yet-uncultivated species. The results revealed that we have little knowledge of the organisms responsible for nitrogen fixation in termites.     

巴西固氮螺菌Yu62 draTG基因及其下游区域的定位诱变分析

用卡那霉素盒（Ｋｍ－ｃａｓｓｅｔｔｅ）插入法,对巴西固氮螺菌（Ａｚｏｓｐｉｒｉｌｌｕｍｂｒａｓｉｌｅｎｓｅ）Ｙｕ６２的ｄｒａＴＧ基因及其下游区域进行了诱变,并获得相应的突变株,研究表明ｄｒａＴ变突株的固氮酶活性不再受铵抑制,而ｄｒａＧ突变株在有铵时则丧失固氮酶活性,但当铵耗尽后却不能使像野生型菌株那样恢复活性,ｄｒａＴＧ下游区域突变株ＹＺ４（突变位点距ｄｒａＧ约２ｋｂ）在无氮及限铵条件下,其固氮酶     

Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites.

Nitrogen fixation by the microorganisms in the gut of termites is one of the crucial aspects of symbiosis, since termites usually thrive on a nitrogen-poor diet. The phylogenetic diversity of the nitrogen-fixing organisms within the symbiotic community in the guts of various termite species was investigated without culturing the resident microorganisms. A portion of the dinitrogenase reductase gene (nifH) was directly amplified from DNA extracted from the mixed population in the termite gut. Analysis of deduced amino acid sequences of the products of the clonally isolated nifH genes revealed the presence of diverse nifH sequences in most of the individual termite species, and their constituents were considerably different among termite species. A majority of the nifH sequences from six lower termites, which showed significant levels of nitrogen fixation activity, could be assigned to either the anaerobic nif group (consisting of clostridia and sulfur reducers) or the alternative nif methanogen group among the nifH phylogenetic groups. In the case of three higher termites, which showed only low levels of nitrogen fixation activity, a large number of the sequences were assigned to the most divergent nif group, probably functioning in some process other than nitrogen fixation and being derived from methanogenic archaea. The nifH groups detected were similar within each termite family but different among the termite families, suggesting an evolutionary trend reflecting the diazotrophic habitats in the symbiotic community. Within these phylogenetic groups, the sequences from the termites formed lineages distinct from those previously recognized in studies using classical microbiological techniques, and several sequence clusters unique to termites were found. The results indicate the presence of diverse potentially nitrogen-fixing microbial assemblages in the guts of termites, and the majority of them are as yet uncharacterized.     

Correlation of activity regulation and substrate recognition of the ADP-ribosyltransferase that regulates nitrogenase activity in Rhodospirillum rubrum

In Rhodospirillum rubrum, nitrogenase activity is regulated posttranslationally through the ADP-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). Several DRAT variants that are altered both in the posttranslational regulation of DRAT activity and in the ability to recognize variants of dinitrogenase reductase have been found. This correlation suggests that these two properties are biochemically connected.     

ADP-Ribosylation of variants of Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase

In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase. The structure of the dinitrogenase reductase from Azotobacter vinelandii is known. In this study, mutant forms of dinitrogenase reductase from A. vinelandii that are affected in various protein activities were tested for their ability to be ADP-ribosylated or to form a complex with dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum. R140Q dinitrogenase reductase could not be ADP-ribosylated by DRAT, although it still formed a cross-linkable complex with DRAT. Thus, the Arg 140 residue of dinitrogenase reductase plays a critical role in the ADP-ribosylation reaction. Conformational changes in dinitrogenase reductase induced by an F135Y substitution or by removal of the Fe(4)S(4) cluster resulted in dinitrogenase reductase not being a substrate for ADP-ribosylation. Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenase reductase to form a cross-linkable complex with DRAT. Substitution of D129E or deletion of Leu 127, which result in altered nucleotide binding regions of these dinitrogenase reductases, did not significantly change the interaction between dinitrogenase reductase and DRAT. Previous results showed that changing Lys 143 to Gln decreased the binding between dinitrogenase reductase and dinitrogenase (L. C. Seefeldt, Protein Sci. 3:2073-2081, 1994); however, this change did not have a substantial effect on the interaction between dinitrogenase reductase and DRAT.     

Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.

The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent alpha2beta2gamma2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum- or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an alpha2 dimer containing an Fe4S4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H2 very well but reduces N2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane.     

Isolation and gene quantification of heterotrophic N2-fixing bacterioplankton in the Baltic Sea

Cyanobacteria are regarded as the main N(2)-fixing organisms in marine waters. However, recent clone libraries from various oceans show a wide distribution of the dinitrogenase reductase gene (nifH) originating from heterotrophic bacterioplankton. We isolated heterotrophic N(2)-fixing bacteria from Baltic Sea bacterioplankton using low-nitrogen plates and semi-solid diazotroph medium (SSDM) tubes. Isolates were analysed for the nitrogenase (nifH) gene and active N(2) fixation by nested polymerase chain reaction (PCR) and acetylene reduction respectively. A primer-probe set targeting the nifH gene from a gamma-proteobacterial isolate, 97% 16S rDNA similarity to Pseudomonas stutzeri, was designed for measuring in situ dynamics using quantitative real-time PCR. This nifH gene sequence was detected at two of 11 stations in a Baltic Proper transect at abundances of 3 x 10(4) and 0.8 x 10(3) copies per litre seawater respectively. Oxygen requirements of isolates were examined by cultivation in SSDM tubes where oxygen gradients were determined with microelectrodes. Growth, and thereby N(2) fixation, was observed as horizontal bands formed at oxygen levels of 0-6% air saturation. The apparent microaerophilic or facultative anaerobic nature of the isolates explains why the SSDM approach is the most appropriate isolation method. Our study illustrates how combined isolation, functional analyses and in situ quantification yielded insights into the oxygen requirements of heterotrophic N(2)-fixing bacterioplankton isolates, which were confirmed to be present in situ.     

Study of nitrogen fixation in microbial communities of oil-contaminated marine sediment microcosms

Aerobic microbial degradation of pollutant oil (petroleum) in aquatic environments is often severely limited by the availability of combined nitrogen. We therefore studied whether the microbial community enriched in marine sediment microcosms with an added oil layer and exposure to light harboured nitrogenase activity. The acetylene reduction (AR) assay indeed indicated active nitrogenase; however, similar activity was observed in oil-free control microcosms. In both microcosms, the AR rate was significantly reduced upon a dark shift, indicating that enriched cyanobacteria were the dominant diazotrophs. Analysis of structural dinitrogenase reductase genes (nifH) amplified from both microcosms indeed revealed NifH sequences related mostly to those of heterocystous cyanobacteria. NifH sequences typically affiliating with those of heterotrophic bacteria were more frequently retrieved from the oil-containing sediment. Expression analyses showed that mainly nifH genes similar to those of heterocystous cyanobacteria were expressed in the light. Upon a dark shift, nifH genes related to those of non-heterocystous cyanobacteria were expressed. Expression of nifH assignable to heterotrophs was apparently not significant. It is concluded that cyanobacteria are the main contributors of fixed nitrogen to oil-contaminated and pristine sediments if nitrogen is a limiting factor and if light is available. Hence, also the oil-degrading heterotrophic community may thus receive a significant part of combined nitrogen from cyanobacteria, even though oil vice versa apparently does not stimulate an additional nitrogen fixation in the enriched community.     

Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.