Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus

Summary A DNA region showing homology to Klebsiella pneumoniae nifA and nifB is duplicated in Rhodobacter capsulatus. The two copies of this region are called nifA/nifB copy I and nifA/nifB copy II. Deletion mutagenesis demonstrated that either of the two copies is sufficient for growth in nitrogen-free medium. In contrast, a double deletion mutant turned out to be deficient in nitrogen fixation. The complete nucleotide sequence of a 4838 bp fragment containing nifA/nifB copy I was determined. Two open reading frames coding for a 59653 (NifA) and a 49453 (NifB) dalton protein could be detected. Comparison of the amino acid sequences revealed that the R. capsulatus nifA and nifB gene products are more closely related to the NifA and NifB proteins of Rhizobium meliloti and Rhizobium leguminosarum than to those of K. pneumoniae. A rho-independent termination signal and a typical nif promoter region containing a putative NifA binding site and a consensus nif promoter are located within the region between the R. capsulatus nifA and nifB genes. The nifB sequence is followed by an open reading frame (ORF1) coding for a 27721 dalton protein in nifA/nifB copy I. DNA sequence analysis of nifA/nifB copy II showed that both copies differ in the DNA region downstream of nifB and in the noncoding sequence in front of nifA. All other regions compared, i.e. the 5 part of nifA, the intergenic region and the 3 part of nifB, are identical in both copies.     

In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).     

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

Identification and phenotypical characterization of a cluster of fix genes, including a nif regulatory gene, from Rhizobium leguminosarum PRE

Summary A nif regulatory gene in R. leguminosarum PRE was identified by interspecies DNA hybridization and site-directed Tn5 mutagenesis. Significant homology was found with the K. pneumoniae nifA locus, a R. meliloti symbiotic regulatory gene and E. coli ntrC; Tn5 insertions within this nifA gene inhibit the expression of the nifHDK operon, encoding synthesis of the nitrogenase polypeptides.Specific DNA hybridization also was detected between a downstream adjacent part of the PRE sym plasmid and the R. leguminosarum 248 fixZ gene, a homologue of the K. pneumoniae nifB locus. To detect further fix genes we investigated a region of the sym plasmid which is localized within a short distance upstream from the nifA gene and is transcribed selectively at a high rate during symbiosis. This approach revealed the existence of a fix cluster in which Tn5-mutations cause a Fix^- phenotype although wild-type levels of nitrogenase synthesis were detectable. In a sym plasmid fragment, which is immediately upstream adjacent to the nifA locus and only moderately expressed in Rhizobium bacteroids, a second fix gene conferring the same symbiotic phenotype was detected.     

Characterization of the ntrBC genes of Azospirillam 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.     

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3-region of the nifM gene, the nifL and nifA genes and the 5-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.Communicated by H. Böhme     

Cloning and characterization of nifA and ntrC genes of the stem nodulating bacterium ORS571, the nitrogen fixing symbiont of Sesbania rostrata: Regulation of nitrogen fixation (nif) genes in the free living versus symbiotic state

Summary A cosmid bank of ORS571, a diazotrophic bacterium capable of inducing aerial stem and root nodules on Sesbania rostrata, was constructed in the vector pLAFR1. A DNA probe carrying the Klebsiella pneumoniae nifA gene was used to identify nifA-and ntrC-like regions of ORS571 in the cosmid bank by colony hybridization. Cosmids carrying these regions were mapped by restriction endonuclease analysis, Southern blotting and transposon Tn5 mutagenesis. Selected Tn5 insertion mutations in the nifA/ntrC homologous regions were used for gene-replacement experiments and the resulting ORS571 mutants were examined for Nif, Fix and Ntr phenotypes. Two clearly distinct regulatory loci were thus identified and named nifA and ntrC. Plasmids carrying gene fusions of the ORS571 nifH and nifD genes to lacZ were constructed and the regulation of the ORS571 nifHDK promoter, and of the Rhizobium meliloti nifHDK promoter, was studied under varying physiological conditions in ORS571, ORS571 nifA::Tn5 and ORS571 nitrC::Tn5 strains. A model for the role of nifA and ntrC in the regulation of ORS571 nif and other nitrogen assimilation genes is proposed.     

Identification of nifE-, nifN- and nifS-like genes in Bradyrhizobium japonicum

Summary The 17 kb region between the Bradyrhizobium japonicum nitrogenase genes (nifDK and nifH) was investigated for the presence of further nif or fix genes by site-directed insertion or deletion/replacement mutagenesis and interspecies hybridization. Mutant strains were tested for their ability to reduce acetylene in free-living, microaerobic culture (Nif phenotype) and in soybean root nodules (Fix phenotype). The presence of a gene, previously identified by hybridization with the Klebsiella pneumoniae nifB gene, was proved by isolation of a nifB insertion mutant which was completely Nif^- and Fix^-. Three other regions were found to be homologous to the K. pneumoniae genes nifE, nifN, and nifS, NifE and nifN insertion mutants were completely Nif^-/Fix^- whereas nifS mutants were leaky with 30% residual Fix activity. Taken together, the data show that the B. japonicum genome harbours a cluster of closely adjacent genes which are directly concerned with nitrogenase function.     

The gltF gene of Klebsiella pneumoniae: cloning and initial characterization

Summary From a gene bank of Klebsiella pneumoniae M5a1, a 1.7 kb gene fragment was isolated which was able to restore the Ntr⁺ phenotype and ammonium (methylammonium) transport, but not glutamate synthase in an Escherichia coli glt mutant (glutamate synthase deficiency). The fragment strongly hybridized with the gltF regulatory gene from E. coli. After subcloning the fragment into an overexpression vector, a protein with a molecular weight of 27000 dalton was identified as the gene product. The results indicate that the fragment cloned contains the gltF gene from K. pneumoniae.     

Bending of DNA segments with Saccharomyces cerevisiae autonomously replicating sequence activity, isolated from basidiomycete mitochondrial linear plasmids

Summary Previous studies have indicated that DNA bending is a general structural feature of sequences (ARSs) from cellular DNAs of yeasts and nuclear and mitochondrial genomic DNAs of other eukaryotes that are capable of autonomous replication in Saccharomyces cerevisiae. Here we showed that bending activity is also tightly associated with S. cerevisiae ARS function of segments cloned from mitochondrial linear DNA plasmids of the basidiomycetes Pleurotus ostreatus and Lentinus edodes. Two plasmids, designated pLPO2-like (9.4 kb), and pLPO3 (6.6 kb) were isolated from a strain of P. ostreatus. A 1029 by fragment with high-level ARS activity was cloned from pLPO3 and it contained one ARS consensus sequence (A/T)TTTAT(A/G)TTT(A/T) indispensable for activity and seven dispersed ARS consensus-like (10/11 match) sequences. A discrete bent DNA region was found to lie around 500 by upstream from the ARS consensus sequence (T-rich strand). Removal of the bent DNA region impaired ARS function. DNA bending was also implicated in the ARS function associated with a 1430 by fragment containing three consecutive ARS consensus sequences which had been cloned from the L. edodes plasmid pLLE1 (11.0 kb): the three consecutive ARSs responsible for high-level ARS function occurred in, and immediately adjacent to, a bent DNA region. A clear difference exists between the two plasmid-derived ARS fragments with respect to the distance between the bent DNA region and the ARS consensus sequence(s).     

Molecular analysis of the chromosomal region encoding the nifA and nifB genes of Acetobacter diazotrophicus

The Acetobacter diazotrophicus nifA gene was isolated by its ability to restore a Nif⁺ phenotype to a nifA mutant of Azotobacter vinelandii. Sequencing revealed that the nifA gene was upstream and adjacent to the nifB gene and both are transcribed in the same direction but independently from different promoters. The 3′ end of the nifB gene was located approximately 2.5 kb upstream of the nitrogenase structural gene cluster, nifHDK. The deduced amino acid sequences of the A. diazotrophicus nifA and nifB gene products were most similar to the NifA and NifB proteins of Azorhizobium caulinodans and Rhodobacter capsulatus, respectively. A. diazotrophicus nifA expression was repressed in cultures exposed to high levels of ammonium while oxygen apparently had no influence. Both oxygen and ammonium prevented expression of a nifB-reporter strain, consistent with the observation that ammonium repressed nifA expression, and indicating that A. diazotrophicus NifA activity is inhibited by oxygen as in other Proteobacterial α group diazotrophs.     

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Summary An hydrogenase-deficient (Hup^–) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup^– mutant, strain JP91, selected by its inability to grow autotrophically (Aut^– phenotype) together with other Hup^– mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup^– mutants, span 15–20 kb of DNA.     

Isolation and characterisation of a Bam HI element in psam 3 a gene of Pisum sativum L. induced during early stages of arbuscular mycorrhiza development

An Alu-like element was identified in the 5′-UTR of psam 3, a Pisum sativum L. gene which shows enhanced expression during early events of AM formation. Two sets of primers specific for the 5′-UTR of the gene psam 3 and for the Alu-like element, respectively, were designed to study psam 3 gene organisation by targeted Alu PCR carried out on pea genomic DNA. PCR products free of Alu-like sequences were produced. A 1.0 kb DNA fragment showing up to 65 percnt; similarity to a Bam HI repeated sequence of Vicia faba was isolated in both wild-type and mycorrhiza-resistant pea. Southern blot analysis revealed the presence of other Bam HI related sequences in pea. The possibility that Bam HI repeated sequences might constitute complex repeating units together with an Alu-like element in the P. sativum genome is discussed.     

MEC: A transposable element from Chironomus thummi (Diptera)

Summary Two genomic clones, pC1.2 and p20D (containing inserts of 2.0 and 1.6 kb, respectively) were isolated from the A2b region to polytene chromosome IV of Chironomus thummi thummi salivary gland cells. Upon in situ hybridization to polytene chromosomes of C. thummi thummi and C. thummi piger, p20D DNA hybridized mainly over the A2b region of chromosome IV, whereas pC1.2 DNA hybridized to at least 90 sites distributed over all the chromosomes. A partial nucleotide sequence analysis showed that these clones were very similar and allowed the detection of a 596 by insert in the pC1.2 clone. This insert possesses all of the essential features of a Class II transposable element and was called MEC. It carries a nearly perfect 107 by terminal inverted repeat containing one mismatch and is flanked by a 5 by direct repeat. The 372 by central region contains a short open reading frame with a coding capacity of 58 amino acids.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.This Paper is dedicated to the memory of Professor James B. Boyd     

DNA sequence and genetic analysis of the Rhodobacter capsulatus nifENX gene region: homology between NifX and NifB suggests involvement of NifX in processing of the iron-molybdenum cofactor

Summary Rhodobacter capsulatus genes homologous to Klebsiella pneumoniae nifE, nifN and nifX were identified by DNA sequence analysis of a 4282 bp fragment of nif region A. Four open reading frames coding for a 51188 (NifE), a 49459 (NifN), a 17459 (NifX) and a 17472 (ORF4) dalton protein were detected. A typical NifA activated consensus promoter and two imperfect putative NifA binding sites were located in the 377 bp sequence in front of the nifE coding region. Comparison of the deduced amino acid sequences of R. capsulatus NifE and NifN revealed homologies not only to analogous gene products of other organisms but also to the and subunits of the nitrogenase iron-molybdenum protein. In addition, the R. capsulatus nifE and nifN proteins shared considerable homology with each other. The map position of nifX downstream of nifEN corresponded in R. capsulatus and K. pneumoniae and the deduced molecular weights of both proteins were nearly identical. Nevertheless, R. capsulatus NifX was more related to the C-terminal end of NifY from K. pneumoniae than to NifX. A small domain of approximately 33 amino acid residues showing the highest degree of homology between NifY and NifX was also present in all nifB proteins analyzed so far. This homology indicated an evolutionary relationship of nifX, nifY and nifB and also suggested that NifX and NifY might play a role in maturation and/or stability of the iron-molybdenum cofactor. The open reading rame (ORF4) downstream of nifX in R. capsulatus is also present in Azotobacter vinelandii but not in K. pneumoniae. Interposon-induced insertion and deletion mutants proved that nifE and nifN were necessary for nitrogen fixation in R. capsulatus. In contrast, no essential role could be demonstrated for nifX and ORF4 whereas at least one gene downstream of ORF4 appeared to be important for nitrogen fixation.     

Molecular cloning of the o2-m5 allele of Zea mays using transposon marking

Summary The deposition of zein protein in maize endosperm is under the control of several regulatory loci. The isolation of DNA sequences corresponding to Opaque-2 (O2), one of such loci, is described in this paper. The mutable allele, o2-m5 was first induced moving the Ac transposable element present at the wx-m7 allele to the O2 locus. Genetic data suggest that a functional Ac element is responsible for the observed somatic mutability of o2-m5. The isolation of genomic clones containing flanking sequences corresponding to the O2 gene was possible by screening an o2-m5 genomic libary with a probe corresponding to internal Ac sequences usually absent in the defective element Ds. Out of 27 clones isolated with homology to the central part of Ac element, only clones 6IP and 21IP generated a 2.5 kb internal fragment size of an active Ac element when digested with PvuII restriction enzyme. A sequence representing a XhoI fragment of 0.9 kb lying, in the 6IP clone, adjacent to the Ac elements, was subcloned and utilized to prove that it corresponded to a part of the O2 gene. To obtain this information we made use of: (1) DNAs from several reversions originating from the unstable (o2mk-(r) allele, which, when digested with SstI, showed a correct 3.4 kb fragment typical of non-inserted alleles of the O2 locus; and (2) recessive alleles of the O2 locus which were devoid of a 2.0 kb mRNA, present on the contrary in the wild type and in other zein regulating mutants different from O2.This paper is dedicated to the memory of R. Marotta, who actively participated in the realization of this work     

Reduced but accurate translation from a mutant AUA initiation codon in the mitochondrial COX2 mRNA of Saccharomyces cerevisiae

The gene encoding the XorII methyltransferase (M · XorII) was cloned from Xanthomonas oryzae pv. oryzae and characterized in Escherichia coli. The M · XorII activity was localized to a 3.1 kb BamHI-BstXI fragment, which contained two open reading frames (ORFs) of 1272 nucleotides (424 amino acids) and 408 nucleotides (136 amino acids). Ten polypeptide domains conserved in other M⁵ cytosine methyltransferases (MTases) were identified in the deduced amino acid sequence of the 1272 ORF. E. coli Mrr⁺ strains were transformed poorly by plasmids containing the XorII MTase gene, indicating the presence of at least one ^MCG in the recognition sequence for M · XorII (CGATCG). The 408 nucleotide ORF was 36% identical at the amino acid level to sequences of the E. coli dcm-vsr gene, which is required for very short patch repair. X. oryzae pv. oryzae genomic DNA that is resistant to digestion by Pvul and XorII hybridizes with a 7.0 kb fragment containing the XorII MTase gene and vsr homolog, whereas DNA from strains that lack M · XorII activity do not hybridize with the fragment.The sequence presented in this paper has been submitted to NCBI; the accession number is U06424