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.     

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.     

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.     

Nucleotide sequence of nifD from Frankia alni strain ArI3: phylogenetic inferences.

The complete nucleotide sequence of the nifD gene encoding the alpha subunit of component I of nitrogenase from Frankia alni strain ArI3 was determined. The coding region is 1,458 bp in length and encodes a polypeptide of 486 residues with a predicted molecular weight of 53,500. Phylogenetic inferences with 12 complete published nifD sequences were drawn using a variety of approaches. Frankia nifD clusters with proteobacteria rather than with Clostridium pasteurianum, the other Gram-positive bacterium studied. Extant eubacterial nif genes seem to have at least three distinct evolutionary origins as a result of ancient gene duplications. Within the Gram-positive bacterial phylum, functional nif genes descend from different duplicates.     

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.     

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.     

Isolation and characterization of Rhizobium (IC3342) genes that determine leaf curl induction in pigeon pea.

Nodulation by the Rhizobium strain IC3342 causes a leaf curl syndrome in certain tropical legumes such as pigeon pea (Cajanus cajan) (N.M. Upadhyaya, J.V.D.K. Kumar Rao, D.S. Letham, and P.J. Dart, Physiological and Molecular Plant Pathology 39:357-373, 1991). Transposon (Tn5) mutagenesis of this leaf curl-inducing (Curl+) Rhizobium strain yielded two Curl- Fix- and three Curl- Fix+ mutants. Plasmid visualization and subsequent Southern blot hybridization analyses with Tn5, nif and nod gene probes showed that the Tn5 element had inserted into the symbiotic (Sym) plasmid in three of the mutants. Restriction endonuclease analyses indicated that none of the Tn5 insertions were closely linked. Tn5-containing EcoRI fragments were cloned from each mutant and used as probes to isolate the corresponding wild-type DNA fragments from a cosmid (pLAFR3) genomic library. Fix+ and/or Curl+ phenotypes were restored in each mutant by the introduction of cosmids containing the corresponding wild-type DNA. A closely related but Curl- Rhizobium strain ANU240 was shown, by Southern hybridization, to contain conserved DNA sequences of all but one of the identified genetic regions of the Curl+ Rhizobium strain IC3342. Cosmids containing the genetic region unique to the strain IC3342, designated lcr1, conferred a Curl+ phenotype on the strain ANU240. DNA sequence analysis of the cloned lcr1 region revealed five open reading frames (ORFs). The ORF2 showed homology with the Escherichia coli regulatory gene ompR, and ORF4 showed homology with E. coli and Rhizobium meliloti regulatory genes fnr and fixK, respectively.     

Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

The nucleotide (nt) sequence of a 2.0-kb NheI-XbaI DNA fragment containing a peptidoglycan hydrolase-encoding gene, lytA, tentatively identified as encoding an N-acetylmuramyl-L-alanine amidase, from Staphylococcus aureus, was determined. The nt sequencing revealed an open reading frame (ORF) of 1443 bp with a consensus ribosome-binding site located 7 nt upstream from the ATG start codon. The primary amino acid (aa) sequence deduced from the nt sequence revealed a putative protein of 481 aa residues with an Mr of 53815. Comparison of the aa sequence of the ORF with aa sequences in the GenBank data base (version 63, March 1990) revealed that the C-terminal sequence showed significant homology to the C-terminal sequence of lysostaphin from Staphylococcus simulans biovar staphylolyticus.     

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.     

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.     

Typing method for N2-fixing bacteria based on PCR-F — application to the characterization of Frankia strains

DNA sequences of an intergenic spacer (IGS) and parts of genes in the nif cluster were amplified by the polymerase chain reaction (PCR) using two primers derived from nifD -and nifK -conserved sequences. The PCR products were cleaved by ten 4–base cutting restriction enzymes and the restriction patterns were used as fingerprints to type Frankia strains. The feasability of this PCR-RFLP method for typing Frankia strains was investigated on Frankia reference strains belonging mainly to the Elaeagnaceae infectivity group but also on new Frankia isolates and on other N₂-fixing microorganisms. By modulating the stringency of the amplifications, we showed the method allowed to target either Frankia strains or the whole N₂-fixing microbial community. DNA digestion patterns were used to estimate the sequence divergence between the Frankia nifD-K fragment. The estimated relationships deduced from these genotypic data correlated well with established Frankia taxonomic schemes.     

Molecular cloning and nucleotide sequence analysis of the maltose-inducible porin gene of Aeromonas salmonicida

Abstract The gene for the Aeromonas salmonicida maltose-inducible porin (maltoporin) was cloned into phagemid pTZ18R in two restriction fragments, 0.6-kb Pst I/ Kpn I and 1.7-kb Sph I, of genomic DNA and their nucleotide sequences were determined. Open reading frames of 1329 and 1335 bp translated into sequences of 443 and 445 amino acids, with a 23 or 25 amino acid signal sequence and a 420 amino acid mature protein of molecular mass 46424 Da. Putative ribosome binding sites, AGGA and GGGAA, occurred 9 bp upstream of two possible ATG initiation codons. The A. salmonicida gene product showed a high degree of similarity with Escherichia coli LamB, and codon usage was very similar to that of another A. salmonicida outer membrane protein but markedly different from those of extracellular proteins.