Molecular cloning of nif DNA from Azotobacter vinelandii.

Two clones which contained nif DNA were isolated from a clone bank of total EcoRI-digested Azotobacter vinelandii DNA. The clones carrying the recombinant plasmids were identified by use of the 32P-labeled 6.2-kilobase (kb) nif insert from pSA30 (which contains the Klebsiella pneumoniae nifK, nifD, and nifH genes) as a hybridization probe. Hybridization analysis with fragments derived from the nif insert of pSA30 showed that the 2.6-kb insert from one of the plasmids (pLB1) contains nifK whereas the 1.4-kb insert from the other plasmid (pLB3) contains nifD. Marker rescue tests using genetic transformation indicated that the 2.6-kb A. vinelandii nif fragment contains the wild-type alleles for the nif-6 and nif-38 mutations carried by Nif- strains UW6 and UW38. The 1.4-kb insert contains the wild-type allele for the nif-10 mutation carried by Nif- strain UW10.     

Heterologous hybridization of Frankia DNA to Rhizobium meliloti and Klebsiella pneumoniae nif genes

A nif gene probe from Rhizobium meliloti was used to isolate a recombinant bacteriophage from a Frankia sp. ArI3 gene bank. There is a large homology between nif D and nif H genes of R. meliloti or Klebsiella pneumoniae and Frankia DNA sequences. Approximately 4.5 kb to the right of nif K, we have localized a DNA region hybridizing to a R. meliloti probe containing nif A and nif B genes. The extent of the homology was greater for nif B than for nif A.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.     

DNA hybridization analysis of the nif region of two methylotrophs and molecular cloning of nif-specific DNA.

DNA isolated from two diazotrophic methylotrophs, the obligate methanotroph Methylosinus sp. strain 6 and the methanol autotroph Xanthobacter sp. H4-14, hybridized to DNA fragments encoding nitrogen fixation (nif) genes from Klebsiella pneumoniae. This interspecific nif homology was limited to DNA fragments encoding the nitrogenase structural proteins (nifH, nifD, and nifK) and specific methylotroph DNA sequences. The hybridization patterns obtained with the two methylotrophs were dissimilar, indicating that the nif region of methylotrophs is not physically conserved. By using the K. pneumoniae nif structural genes as a probe, a fragment of nif DNA from each methylotroph was cloned and characterized. The DNA fragment from Methylosinus sp. 6 encoded two polypeptides of 57,000 and 34,000 molecular weight.     

Cadmium accumulation and DNA homology with metal resistance genes in sulfate-reducing bacteria

Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains Desulfovibrio desulfuricans DSM 1926 and Desulfococcus multivorans DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the smtAB, cadAC, and cadD genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known smtAB genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of Desulfomicrobium norvegicum I1 and Desulfovibrio desulfuricans DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.     

Bacteria in the gular and paracloacal glands of the American alligator (Alligator mississippiensis; Reptila, Crocodilia)

Plasmid DNA of six strains of Rhizobium galegae was blotted onto nitrocellulose and hybridized with the 4.8 kb PstI fragment of pRme4lb, a megaplasmid carrying the nifH and the nifD genes of Rhizobium meliloti. DNA sequences homologous to the nif genes were localized on the megaplasmid or on the large plasmid bands of the R. galegae strains tested. In three of the strains analysed the nif genes were located on the megaplasmids. In the other three strains investigated, which also possessed megaplasmids, the nif genes were located on the smaller plasmids.     

Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum.

By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains. Two EcoRI restriction fragments from a plasmid of fast-growing R. japonicum hybridized with nif structural genes of R. meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R. meliloti. Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R. japonicum. Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment. These results suggest that the nif gene organizations in fast-growing and in slow-growing R. japonicum strains are different.     

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

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

Dinitrogen-fixing endogenous rhythm in Synechococcus RF-1

Abstract Using the Southern hybridization technique, 22 strains of archaebacteria were tested for the presence of DNA sequences homologous to the Anabaena nifH gene, which codes for the Fe protein component of nitrogenase. Total DNA from all the 14 methanogenic strains tested was shown to carry homology to nifH . With total DNA from 3 Halobacterium strains, hybridization was also detected, though unspecific binding cannot completely be excluded. No hybridization was detected with DNA from Thermoplasma acidophilum , nor with the DNA from 4 strains belonging to the sulfur-dependent branch of the archaebacteria. Results strongly support the hypothesis of an ancient origin of nif genes.     

Localization and physical mapping of a plasmid-borne 23-kb nif gene cluster from Enterobacter agglomerans showing homology to the entire nif gene cluster of Klebsiella pneumoniae M5a1

A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.     

Evidence for plasmid- and chromosome-borne multiple nif genes in Rhizobium fredii.

Rhizobium fredii is a fast-growing rhizobium isolated from the primitive Chinese soybean cultivar Peking and from the wild soybean Glycine soja. This rhizobium harbors nif genes on 150- to 200-megadalton plasmids. By passage on acridine orange plates, we obtained a mutant of R. fredii USDA 206 cured of the 197-megadalton plasmid (USDA 206C) which carries both nif and nod genes. This strain, however, has retained its symbiotic effectiveness. Probing EcoRI digests of wild-type and cured plasmid DNA with a 2.2-kilobase nif DH fragment from Rhizobium meliloti has shown four homologous fragments in the wild-type strain (4.2, 4.9, 10, and 11 kilobases) and two fragments in the cured strain (4.2 and 10 kilobases). EcoRI digests of total DNA show four major bands of homology (4.2, 4.9, 5.8, and 13 kilobases) in both the wild-type and cured strains. The presence of major bands of homology in the total DNA not present in the plasmid DNA indicated chromosomal nif genes. Probing of HindIII digests of total and plasmid DNA led to the same conclusion. Hybridization to the smaller plasmids of USDA 206 and USDA 206C showed the presence of nif genes on at least one of these plasmids, explaining the nif homology in the USDA 206C plasmid digests.     

Evidence for plasmid- and chromosome-borne multiple nif genes in Rhizobium fredii

Rhizobium fredii is a fast-growing rhizobium isolated from the primitive Chinese soybean cultivar Peking and from the wild soybean Glycine soja. This rhizobium harbors nif genes on 150- to 200-megadalton plasmids. By passage on acridine orange plates, we obtained a mutant of R. fredii USDA 206 cured of the 197-megadalton plasmid (USDA 206C) which carries both nif and nod genes. This strain, however, has retained its symbiotic effectiveness. Probing EcoRI digests of wild-type and cured plasmid DNA with a 2.2-kilobase nif DH fragment from Rhizobium meliloti has shown four homologous fragments in the wild-type strain (4.2, 4.9, 10, and 11 kilobases) and two fragments in the cured strain (4.2 and 10 kilobases). EcoRI digests of total DNA show four major bands of homology (4.2, 4.9, 5.8, and 13 kilobases) in both the wild-type and cured strains. The presence of major bands of homology in the total DNA not present in the plasmid DNA indicated chromosomal nif genes. Probing of HindIII digests of total and plasmid DNA led to the same conclusion. Hybridization to the smaller plasmids of USDA 206 and USDA 206C showed the presence of nif genes on at least one of these plasmids, explaining the nif homology in the USDA 206C plasmid digests.     

Cadmium Accumulation and DNA Homology with Metal Resistance Genes in Sulfate-Reducing Bacteria

Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains Desulfovibrio desulfuricans DSM 1926 and Desulfococcus multivorans DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the smtAB, cadAC, and cadD genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known smtAB genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of Desulfomicrobium norvegicum I1 and Desulfovibrio desulfuricans DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.     

Identification of a Rhizobium trifolii plasmid coding for nitrogen fixation and nodulation genes and its interaction with pJB5JI, a Rhizobium leguminosarum plasmid

Rhizobium trifolii T37 contains at least three plasmids with sizes of greater than 250 megadaltons. Southern blots of agarose gels of these plasmids probed with Rhizobium meliloti nif DNA indicated that the smallest plasmid, pRtT37a, contains the nif genes. Transfer of the Rhizobium leguminosarum plasmid pJB5JI, which codes for pea nodulation and the nif genes and is genetically marked with Tn5, into R. trifolii T37 generated transconjugants containing a variety of plasmid profiles. The plasmid profiles and symbiotic properties of all of the transconjugants were stably maintained even after reisolation from nodules. The transconjugant strains were placed into three groups based on their plasmid profiles and symbiotic properties. The first group harbored a plasmid similar in size to pJB5JI (130 megadaltons) and lacked a plasmid corresponding to pRtT37a. These strains formed effective nodules on peas but were unable to nodulate clover and lacked the R. trifolii nif genes. This suggests that genes essential for clover nodulation as well as the R. trifolii nif genes are located on pRtT37a and have been deleted. The second group harbored hybrid plasmids formed from pRtT37a and pJB5JI which ranged in size from 140 to ca. 250 megadaltons. These transconjugants had lost the R. leguminosarum nif genes but retained the R. trifolii nif genes. Strains in this group nodulated both peas and clover but formed effective nodules only on clover. The third group of transconjugants contained a hybrid plasmid similar in size to pRtT37b. These strains contained the R. trifolii and R. leguminosarum nif genes and formed N2-fixing nodules on both peas and clover.     

Identification of DNA regions homologous to nitrogen fixation genes nifE, nifUS and fixABC in Azospirillum brasilense Sp7

A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nifgene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.     

Cloning and sequencing of the nifH gene of Desulfovibrio gigas

The Desulfovibrio gigas nifH gene has been cloned and sequenced. It consists of an open-reading frame of 822 base pairs encoding a 274 amino acid polypeptide. A potential ntrA-dependent promotor sequence is present. The gene lacks an upstream activator sequence homologous to those often found in nif genes subject to activation by nifA.     

福建省放线菌结瘤植物共生菌Frankia遗传多样性研究

[目的]了解福建省放线菌结瘤植物共生固氮菌Frankia的遗传多样性.[方法]利用16S-23SrDNA间隔区(rrn)和nifD-K基因间隔区的PCR扩增和RFLP技术,分析了福建省木麻黄、杨梅、桤木、胡颓子等共生Frankia纯培养菌株的遗传差异.[结果]17个菌株获得rrn扩增片段,2个杨梅菌株和1个胡颓子菌株扩增未成功,酶切图谱经聚类分析表明6个地点的细枝木麻黄、短枝木麻黄、粗枝木麻黄12个共生Frankia菌株同源性高,属于一个类群,2个地点的4个杨梅菌株和1个四川桤木菌株亲缘关系近,为另一类群.25个Frankia菌株的,nifD-K基因间隔区PCR-RFLP分析结果显示,7个地点的3种木麻黄14个菌株聚类为一个类群,4个地点的7个杨梅菌株、2个地点的2个四川桤木菌株以及1个台湾桤木菌株聚类为另一个类群,胡颓子菌株则为独立的类群.[结论]研究结果表明福建省共生Frankia遗传多样性丰富.     

Selection of nitrogen-fixing deficient Burkholderia vietnamiensis strains by cystic fibrosis patients: involvement of nif gene deletions and auxotrophic mutations

Burkholderia vietnamiensis is the third most prevalent species of the Burkholderia cepacia complex (Bcc) found in cystic fibrosis (CF) patients. Its ability at fixing nitrogen makes it one of the main Bcc species showing strong filiations with environmental reservoirs. In this study, 83% (29 over 35) of the B. vietnamiensis CF isolates and 100% of the environmental ones (over 29) were found expressing the dinitrogenase complex (encoded by the nif cluster) which is essential in N(2) fixation. Among the deficient strains, two were found growing with ammonium chloride suggesting that they were defective in N(2) fixation, and four with amino acids supplements suggesting that they were harbouring auxotrophic mutations. To get insights about the genetic events that led to the emergence of the N(2)-fixing defective strains, a genetic analysis of B. vietnamiensis nitrogen-fixing property was undertaken. A 40-kb-long nif cluster and nif regulatory genes were identified within the B. vietnamiensis strain G4 genome sequence, and analysed. Transposon mutagenesis and nifH genetic marker exchanges showed the nif cluster and several other genes like gltB (encoding a subunit of the glutamate synthase) to play a key role in B. vietnamiensis ability at growing in nitrogen-free media. nif cluster DNA probings of restricted genomic DNA blots showed a full deletion of the nif cluster for one of the N(2)-fixing defective strain while the other one showed a genetic organization similar to the one of the G4 strain. For 17% of B. vietnamiensis clinical strains, CF lungs appeared to have favoured the selection of mutations or deletions leading to N(2)-fixing deficiencies.