Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium leguminosarum and R. phaseoli

Summary The ability to identify genes that specify nitrogenase (nif genes) in Rhizobium depends on the close homology between then and the corresponding nif genes of Klebsiella pneumoniae (Nuti et al. 1979; Ruvkun and Ausubel 1980). Rhizobium plasmids of high molecular weight (>100 Md) were separated on agarose gels, transferred to nitrocellulose filters and tested for their ability to hybridise with radioactively labelled pSA30, containing the nifKDH region of K. pneumoniae. Five large plasmids, each present in different strains of R. leguminosarum or R. phaseoli, were found to hybridise. Each of these plasmids had previously been shown to determine other symbiotic functions such as nodulation ability. The nif genes on three different plasmids appeared to be in conserved DNA regions since they were within an EcoRI restriction fragment of the same size.     

Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation

Summary The plasmid pattern of Rhizobium meliloti strain GR4 was studied and a gene bank of one of the large plasmids (pRmeGR4) of 140 Mdal, was constructed using the broad host range vector pRK290. A restriction map was established with EcoRI. Two regions of this plasmid involved in the infectivity of GR4 on Medicago sativa were identified. An EcoRI fragment hybridizing with the PstI-nif fragment of pID1 was also identified. However, no homology to the cloned Klebsiella pneumoniae nitrogenase genes (pSA30) was detected.     

Identification of a regulatory nifA type gene and physical mapping of cloned new nif regions of Azospirillum brasilense

Summary Three new Tn5-mutagenized nif genes of Azospirillum brasilense were characterized. The sizes of the restriction fragments and the restriction maps of the cloned nif DNA regions showed that these nif genes are distinct from those reported earlier, e.g. nifHDK, nifE, nifUS, fixABC. The Nif27 mutant was identified as a nifA type regulatory gene of A. brasilense (a) by genetic complementation with nifA of Klebsiella pneumoniae, (b) by the absence of nitrogenase iron protein in western protein blots and (c) by its inability to activate expression of a nijH-lacZ fusion. The growth characteristics of the three mutants showed that none of them is defective in general nitrogen regulatory (ntr) genes. Also, no homology was detected between the three nif DNA regions of the mutants, cloned in pMS188, pMS189 and pMS197, and the K. pneumoniae nif, gInA or ntr genes. In addition, the fixABC genes of Bradyrhizobium japonicum did not show any hybridization with the cloned Azospirillum genes. Unlike the situation in enteric bacteria, the nif genes in A. brasilense are scattered and span a region of about 65 kb.     

Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti

Summary R. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRmc41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod^-) or nitrogen fixation (Fix^-) have been readily obtained. In some Nod^- mutants the deletion of a segment of plasmid pRme41b was found.Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. ³²P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod^- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digests from the wild-type bacterium and from a series of Nod^- mutants revealed that at least a 24 kb DNA fragment including the nif structural genes was missing from most of the Nod^- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.     

A molecular linkage map of nitrogenase and nodulation genes in Rhizobium trifolii

Summary A molecular map was constructed linking the nitrogenase structural genes (nif) and nodulation genes (nod) in the white clover symbiont, Rhizobium trifolii. In R. trifolii strain ANU843 these two genetic regions are located some 16 kilobases (kb) apart on the 180 kb symbiotic (Sym) plasmid. The molecular linkage of nod and nif genetic regions was established by hybridization analysis using recombinant plasmids containing overlapping cloned sequences. Nodulation genes were located by means of a Tn5-induced nodulation-defective mutant that failed to induce clover root hair curling (Hac^- phenotype). A cloned wild-type DNA fragment was shown to phenotypically correct the Hac^- mutation by complementation. The nifHDK genes were cloned by positive hybridization to another R. trifolii nif-specific probe. Location of the nif genes relative to the nod genes was established by analysis of a Sym plasmid deletion derivative.     

Detection of nif Genes in Nitrogen-Fixing Bacterial Isolate from Tomato (Lycopersicon esculentum Mill cv Pusa Ruby)

Nitrogen-fixing bacterial isolate from the intercellular spaces of tomato root cortical cells was studied for the location of nif genes on the chromosomal or plasmid DNA. The bacterial isolate showed two plasmids of approximate molecular sizes of 220 and 120 kb. Klebsiella pneumoniae nif HDK probe hybridized with the chromosomal DNA and not with the plasmid DNA thereby showing that nif genes are localised on the chromosomal DNA.     

DNA sequence homology in Rhizobium meliloti plasmids

Summary Plasmids were recovered by an alkaline procedure from six symbiotically effective strains of Rhizobium meliloti of diverse geographical origin, reported to harbour only one middle-size large plasmid (ranging from 89 to 143 Megadaltons). Each purified plasmid was digested with eight restriction endonucleases; cleavage patterns were very complex: only KpnI and XbaI gave a limited number of bands. Fingerprints were very different, whatever the restriction enzyme or the geographical origin of the strains. However, Southern DNA-DNA hybridizations revealed that the plasmids showed homologous sequences having a high thermal stability. We gave evidence that some of these sequences are common to all the plasmids of R. meliloti. The biological function of these common sequences is unknown. Hybridization with cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae had demonstrated that nif genes were not located on the middle — size plasmids of R. meliloti studied in this paper.     

italic> of the Whole nif Genes of Klebsiella oxytoca,a Nitrogen Fixer in the Rhizosphere of Rice

We cloned in E. coli the whole 17 nif genes (nifQ-J) of Klebsiella oxytoca NG13 using pBR322 as a vector, and constructed a recombinant plasmid, pNOW25 (nif⁺, Ap^r, 42.6 kb). A non nif DNA fragment was deleted from the plasmid with XhoI, and a smaller plasmid, pNOK31 (nif+, Ap^r, 31.1 kb), was reconstructed.

We constructed the restriction map of the cloned nif genes. The map was the same as that of the K. pneumoniae M5a1 nif genes as to the EcoRI, HindIII, BamHI and XhoI sites, but differed considerably in the PstI, SalI and BglII sites.

E. coli KO60 containing pNOW25 or pNOK31 can grow on a N-free medium. The acetylene reduction activities of KO60 (pNOW25) and KO60 (pNOK31) were 280 nmol and 390 nmol/48 hr per 7 ml of N-free liquid medium, whereas the activity of K. oxytoca NG13 was 3800 nmol. Thus, the expressed activity of the nif system of K. oxytoca is rather low in E. coli even if the nif genes are cloned on a multicopy plasmid.     

Diversity ofFrankia strains isolated from a single alder stand

One hundredFrankia strains isolated from variousAlnus species in a single alder stand were tested for plasmid presence. Plasmid DNA was observed in five of the frankiae strains and was analyzed. We found that plasmids with a similar molecular weight exhibited in fact minor divergences in restriction patterns. The genetic diversity among the five isolates which contained plasmids and seven isolates which contained no plasmid DNA were examined by using restriction endonucleas digestions, Southern hybridization ofnifHDK,nifAB genes, andFrankia cryptic DNA fragments determined at random. Results indicate that genomic DNA digestion patterns and Southern hybridizations to anifHDK probe were not able to discriminate between closely related frankiae. On the other hand, plasmid presence, Southern hybridization to anifAB proble or to a crypticFrankia probe allowed us to delineate groupings of these isolates.     

Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis.

Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into Bacillus subtilis. Four of these plasmids (pUB110, pCM194, pSA2100, and pSA0501) have been selected for further study. These plasmids replicate as multicopy autonomous replicons in both Rec+ and Rec- B. subtilis strains. They may be transduced between B. subtilis strains or transformed at a frequency of 10(4) to 10(5) transformants per microgram of DNA. The molecular weights of these plasmids were estimated, and restriction endonuclease cleavage site maps are presented. Evidence is given that pSA2100, an in vivo recombinant of pSA0501 and pCM194 (S. Iord?nescu, J. Bacteriol. 124:597-601, 1975), arose by a fusion of the latter plasmids, possibly by insertion of one element into another as a translocatable element. Genetic information from three other S. aureus plasmids (pK545, pSH2, and pUB101) has also been introduced into B. subtilis, although no covalently closed circular plasmid DNA was recovered.     

Cloning of pEA3, a large plasmid ofEnterobacter agglomerans containing nitrogenase structural genes

Summary A clone bank of an indigenous plasmid ofEnterobacter agglomerans containing structural nitrogen-fixation (nif) genes was established in a non-mobilisable, multicopy derivative of the cosmid vector pHC79. The restriction enzyme Bam HI was used to establish the clone bank and it was found that 96% of the clones contained inserts. The clones containingnif-genes were identified by Southern hybridisation usingKlebsiella pneumoniae nif DNA (KpnifHDKY) as the radioactive probe. Thenif-genes ofE. agglomerans showed extensive homology to those ofK. pneumoniae but the restriction enzyme fragment patterns of thenif-genes ofE. agglomerans were different. The plasmid bornenif-genes ofE. agglomerans are clustered as inK. pneumoniae.     

Restriction mapping of deletions in the nif region of the Klebsiella pneumoniae chromosome

Summary Chromosomal DNA restriction fragments carrying the nitrogen fixation (nif) and his genes of Klebsiella pneumoniae were identified in hybridization experiments using a plasmid derived from pRD1 as a radioactive probe. Restriction mapping of 26 genetically characterized chromosomal nif deletions provided a map showing the physical location of nif genes along the chromosome.     

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros

The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.Non-standard abbreviations bp base pairs - kb kilobase pairs - kd kilodaltons     

Cloning of two protease genes fromRhodocyclus gelatinosa APR 3-2 and their expression inEscherichia coli

Two different protease genes were cloned fromRhodocyclus gelatinosa APR 3-2 inEscherichia coli HB 101/ with pBR329 or its derivatives. The recombinant plasmids designated as pRP100 and pRP300 contained 11.2 and 10.6 kb DNA fragments, respectively. The differences of both plasmids in restriction enzyme maps indicate that these plasmids contained different protease genes. DNA fragments coding for protease, 6.4 kb and 4.5 kb from pRP100 and pRP300, were subcloned into pRP329 and designated as pRP101 and pRP301, respectively. The two cloned proteases were excreted in culture medium ofE. coli, and ß-lactamase ofE. coli, which was originally localized in periplasmic space, was also excreted in the medium.     

Isolation and organization of genes for nitrogen fixation in Rhodopseudomonas capsulata

Summary A library of Rhodopseudomonas capsulata chromosomal DNA was constructed in the broad host range cosmid vector pLAFR1. The library was used to isolate nitrogen fixation genes by complementation of R. capsulata Nif^- mutants. Four complementing regions were localized on different cloned DNA fragments by Tn5 and mini-Mu mutagenesis. Additional nif genes were identified by recombination of transposons from the nif cosmids into the R. capsulata chromosome resulting in the creation of new Nif^- mutations. Most of the newly cloned DNA fragments containing nif genes were found to be unlinked to any other by Southern hybridization of the cloned DNA to chromosomal DNA blots. One of the new fragments was linked to the nifHDK genes. Another cluster spanning 10–12 kilobase pairs contained a number of nif genes, possibly as many as eight.     

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.     

Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus

To identify Rhodobacter capsulatus nif genes necessary for the alternative nitrogenase, strains carrying defined mutations in 32 genes and open reading frames of nif region A, B or C were constructed. The ability of these mutants to grow on nitrogen-free medium with molybdenum (Nif phenotype) or in a nifHDK deletion background on medium without molybdenum (Anf phenotype) was tested. Nine nif genes and nif-associated coding regions are absolutely essential for the alternative nitrogenase. These genes comprise nifV and nifB, the nif-specific ntr system (nifR1, R2, R4) and four open reading frames, which exhibit no homology to known genes. In addition, a significantly reduced activity of both the alternative nitrogenase and the molybdenum-dependent nitrogenase was found for fdxN mutants. By random Tn5 mutagenesis of a nifHDK deletion strain 42 Anf^? mutants were isolated. Southern hybridization experiments demonstrated that 17 of these Tn5 mutants were localized in at least 13 different restriction fragments outside of known nif regions. Ten different Anf^? Tn5 mutations are clustered on a 6 kb DNA fragment of the chromosome designated anf region A. DNA sequence analysis revealed that this region contained the structural genes of the alternative nitrogenase (anfHDGK). The identification of several Tn5 insertions mapping outside of anf region A indicated that at least 10 genes specific for the alternative nitrogenase are present in R. capsulatus.     

Physical Mapping of Recombinant Plasmids Containing Broad Bean Chloroplast Ribosomal RNA Genes

Two BamHl fragments containing broad bean chloroplast rRNA genes were cloned using the bacterial plasmid pBR322 as a vector and Escherichia coli HB101 as host bacterial. Physical maps of the two cloned ct DNA BamHI fragments containing rRNA genes were constructed by cleavage with several restriction endonucleases and Southern blot hybridization with E. coli 16S-23S rRNAs. Recombinant plasmids pVFBI6 and pVFB32 contain a 16S rRNA sequence on the 4.70 kb BamHl fragment, a 23S rRNA sequence and 4.5S/5S rRNA sequences on the 5.65 kb BamHl fragment, respectively.     

Indigenous plasmids and cultural characteristics of rhizobia nodulating chickpeas (Cicer arietinum L.)

We examined 27 strains of chickpea rhizobia from different geographic origins for indigenous plasmids, location and organization of nitrogen fixation (nif) genes, and cultural properties currently used to separate fast- and slow-growing groups of rhizobia. By using an in-well lysis and electrophoresis procedure one to three plasmids of molecular weights ranging from 35 to higher than 380 Mdal were demonstrated in each of 19 strains, whereas no plasmids were detected in the eight remaining strains. Nitrogenase structural genes homologous to Rhizobium meliloti nifHD, were not detected in plasmids of 26 out of the 27 strains tested. Hybridization of EcoRI digested total DNA from these 26 strains to the nif probe from R. meliloti indicated that the organization of nifHD genes was highly conserved in chickpea rhizobia. The only exception was strain IC-72 M which harboured a plasmid of 140 Mdal with homology to the R. meliloti nif DNA and exhibited also a unique organization of nifHD genes. The chickpea rhizobia strains showed a wide variation of growth rates (generation times ranged from 4.0 to 14.5 h) in yeast extract-mannitol medium but appear to be relatively homogeneous in terms of acid production in this medium and acid reaction in litmus milk. Although strains with fast and slow growth rates were identified, DNA/DNA hybridization experiments using a nifHD-specific probe, and the cultural properties examined so far do not support the separation of chickpea rhizobia into two distinct groups of the classical fast- and slow-growing types of rhizobia.