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.     

Use of bacteriophage Mu to isolate deletions in the his-nif region of Klebsiella pneumoniae.

Klebsiella pneumoniae M5a1 is naturally resistant to infection by bacteriophage Mu. Mutants of K. pneumoniae sensitive to Mu infection were isolated and found to support both lytic and lysogenic development of Mu. K. pneumoniae lysogens containing a heat-inducible Mu prophage integrated in his were isolated. Strains carrying deletions extending from his into nif were obtained after heat treatment of these lysogens. Such deletions should be useful for determining the map order and cistronic organization of the nif genes.     

Mutational alteration of a nitrogen-fixing bacterium to sensitivity to infection by bacteriophage Mu: isolation of nif mutations of Klebsiella pneumoniae M5al induced by Mu.

The nitrogen-fixing bacterium Klebsiella pneumoniae M5al is not sensitive to infection by bacteriophage Mu. A mutant of K. pneumoniae that is sensitive to Mu infection was isolated. Several Mu-induced auxotrophic mutations of K. pneumoniae including nif, trp, and rtl were isolated and genetically characterized. Evidence is presented that the Mu-induced mutations of nif arise as the result of insertion of Mu within (or near) the nif operon(s). The rtl locus, which determines the ability to utilize ribitol as a carbon source, was found to be linked to nif loci.     

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.     

nifV is contiguous to nifHDK in Frankia strain FaC1

The organization of genes with the capacity to code for four proteins involved in nitrogen fixation in Frankia strain FaC1 was determined by restriction fragment mapping and nucleotide sequence analysis. Analysis of the 44-kb genomic cosmid clone pFAH 1. isolated from a cosmid library made from Frankia strain FaCl, resulted in the identification of a 7.2-kb Pst I fragment to which Klebsiella nif H, nif D and nif K probes hybridized. This nif -hybridizing fragment was subcloned and analyzed by restriction fragment mapping. Further subcloning of the 7.2-kb fragment and subsequent sequence analysis of approximately 6.8 kb revealed the presence of six open reading frames (ORFs). Four of these ORFs have the potential to code for nif V-, nif H-, nif D- and nif K-like gene products and the two others are unidentified ORFs. The organization of the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes, but the positioning of the nif V-like gene relative to the nif HDK cluster differs, A consensus nif -promoter-like sequence, found 5'to nif H. was not detected upstream of the nif V-like gene. Nine copies of a 7-bp direct repeat were found 5'to ORFA.     

The genetic transfer, heredity and phenotypic expression of plasmid RP4::Mu cts genes in Bacillus cereus

The transcipients were obtained in intrageneric matings of E.coli donor harbouring the plasmid PR4::Mu cts 62 with Bac. cereus GP7 recipient cells with the frequency 10(-9). The transcipient clone Bac. cereus 682 was selected having stably inherited and expressed the hybrid plasmid PR4::Mu cts 62 genes for antibiotic resistance and temperature sensitivity. Production of the bacteriophage Mu cts 62 particles was not registered in the bacillary transcipient cells. The plasmid RP4::Mu cts 62 genes were localized in the chromosome of Bac. cereus 682 transcipient by the blot-hybridization technique with 32P-labelled DNA of the bacteriophage Mu cts 62 and the plasmid PR4. The transcipient of Bac. cereus 682 has the donor properties and transfers the RP4::Mu cts 62 genes to recipient cells of Bac. cereus DSM 318 with the frequency of 10(-6)-10(-7). The expression and transfer of the gram-negative plasmid genes in gram-positive bacterial cells are discussed.     

Phylogeny and characterization of three nifH-homologous genes from Paenibacillus azotofixans

In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.     

Organization of nitrogen fixation genes in a nonheterocystous, filamentous cyanobacterium

Abstract The nonheterocystous, filamentous cyanobacterium, Plectonema boryanum fixes nitrogen only under microaerophilic conditions. The organization of nitrogen fixation genes ( nifH, D, K ) in Plectonema was determined by using cloned fragments from the Anabaena nif genes as probes in Southern hybridizations. Regions of Plectonema DNA were homologous to Anabaena nifH, nifD , and nifK genes, and the resulting pattern of hybridization was used to construct a map of nifH, D, K DNA isolated from Plectonema cells grown under non-nitrogen fixing conditions (combined nitrogen and O₂ present). The nifH and nifD genes are on the same 3 kbp Hin dIII fragment, and nifK is on a 1 kbp Hin dIII fragment. All three nif fragments are adjacent to one another on a 12 kbp Cla I fragment.     

Nucleotide sequence and expression of nifHD from Frankia strain EuIKl, a symbiont of Elaeagnus umbellata

Nucleotide sequences of nif HD from Frankia strain EuIKl were determined and analysed. The 3.2-kb and 5.5-kb Bam HI fragments of a genomic clone were previously shown to contain nif HD-like sequences and to be contiguous, based on hybridization experiments and partial sequencing data. Sequence analysis of about 3.0 kb from these fragments revealed two open reading frames and beginning of a third in the same orientation, each of which showed high degree of sequence homology with nif H, nif D and nif K, respectively. The deduced amino acid sequence of the nif H ORF, consisting of 861 bp, showed sequence similarity of about 91% with those of other Frankia strains, whereas that of nif D ORF, consisting of 1458 bp, showed about 85% similarity. Intergenic sequence between nif H and nif D was 45 bp and between nif D and nif K was 61 bp. The 5'of nif H revealed putative Shine-Dalgarno sequences; however, a sequence resembling a typical nif -promoter or NifA binding site was not found. Northern hybridization of RNA from the nodules showed that nif HDK were transcribed into a polycistronic mRNA in the symbiont of Elaeagnus umbellata .     

Isolation and characterization of lambda specialized transducing bacteriophages carrying Klebsiella pneumoniae nif genes

Seve lambda dnif specialized transducing bacteriophages were isolated from Escherichia coli strains containing plasmids carrying the his-nif region of Klebsiella pneumoniae. These phages collectively carry deoxyribonucleic acid for all of the genes in the nif regulon and adjacent deoxyribonucleic acid of K. pneumoniae. The phages were isolated by using Mu insertions in the nif region to direct the integration of lambda pMu phages in nif via formation of lambda pMu-Mu dilysogens which, upon induction, yielded lambda dnif phages. This procedure should be generally applicable for isolating lambda specialized transducing phages carrying genes from E. coli or other bacteria.     

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.     

Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.

Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.     

Genetic control of morphogenesis of Pseudomonas aeruginosa transposable phage D3112

The influence of ts mutations in the early and late genes of transposable phage D3112 on phage morphogenesis was studied. The mutations in the early genes A, B and C were shown to suppress morphogenesis of D3112. Six genes (D, E, F, G, H and I), located from 14 to 29 kbp of the phage physical map, control morphogenesis of phage head. Five genes (J, K, L, M and N), clustered in the 29-36 kbp region of the map, control morphogenesis of tail. The similarity of genetic organization of the Escherichia coli transposable phage Mu and the Pseudomonas aeruginosa phage D3112 is discussed.     

Genetic and physical map of the structural genes (nifH,D,K) coding for the nitrogenase complex of Rhodopseudomonas capsulata.

Functional genes coding for the structural components of the nitrogenase complex (nifH,D,K) have been cloned on an 11.8-kilobase-pair HindIII fragment of DNA from the photosynthetic bacterium Rhodopseudomonas capsulata. The genes were physically mapped by hybridization of individual cloned nif genes from Klebsiella pneumoniae and Anabaena sp. strain 7120 to Southern blots of HindIII digests of the cloned R. capsulata fragment, after introduction of HindIII sites into the latter at specified locations by insertion of Tn5. Plasmids with the 11.8-kilobase-pair HindIII fragment containing the Tn5 insertions were also used for complementation tests with chromosomal Nif- mutations and for the generation of subfragments to locate those mutations by marker rescue. The R. capsulata nifH,D,K genes comprise a single unit of expression, with the same organization and polarity as found in K. pneumoniae. However, the R. capsulata nifH,D,K fragment did not complement Nif- point mutations in the corresponding Klebsiella genes, and the Klebsiella nif genes did not function in R. capsulata.