Degradation of the Herbicide Glyphosate by Members of the Family Rhizobiaceae.

Several strains of the family Rhizobiaceae were tested for their ability to degrade the phosphonate herbicide glyphosate (isopropylamine salt of N-phosphonomethylglycine). All organisms tested (seven Rhizobium meliloti strains, Rhizobium leguminosarum, Rhizobium galega, Rhizobium trifolii, Agrobacterium rhizogenes, and Agrobacterium tumefaciens) were able to grow on glyphosate as the sole source of phosphorus in the presence of the aromatic amino acids, although growth on glyphosate was not as fast as on P(i). These results suggest that glyphosate degradation ability is widespread in the family Rhizobiaceae. Uptake and metabolism of glyphosate were studied by using R. meliloti 1021. Sarcosine was found to be the immediate breakdown product, indicating that the initial cleavage of glyphosate was at the C-P bond. Therefore, glyphosate breakdown in R. meliloti 1021 is achieved by a C-P lyase activity.

Expression and functional analysis of the Rhizobium meliloti nifA gene.

The translational initiation point for Rhizobium meliloti nifA, the specific activator for nitrogen fixation (nif) genes, was determined. When expressed in an Escherichia coli linked transcriptional-translational system the DNA coding for the R.meliloti nifA gene produced four polypeptide bands of 71 000, 67 000, 62 000 and 59 000 daltons. There are three in-frame ATG codons at the N-terminus of the gene; by replacing the poor ribosome binding sites of the native DNA with a synthetic consensus ribosome binding site prior to each of ATG codons the polypeptides were produced at enhanced levels and related to each of the initiation codons. The ability of these specifically expressed polypeptides to activate nif promoters fused to lacZ was determined. Only the fulllength polypeptide activated the Klebsiella pneumoniae nifH, R.meliloti nifH and fixA and Bradyrhizobium japonicum nifH and nifD promoters. The R.meliloti fixA promoter, contrary to previous evidence, could be activated in E.coli. Deletion of the putative N-terminal domain of the R.meliloti nifA gene product greatly increased the ability of the protein to activate nif promoters. However, deletions retaining part of this domain were not functional. This shows that the N-terminal domain is not essential for activity and that its presence decreases the full potential function of the protein. Our results are consistent with the suggesting that this domain has a regulatory role. In contrast to K.pneumoniae nifA protein, the function of the full length and domain deleted forms of R.meliloti nifA gene product was sensitive to oxygen in E.coli.

Electron transfer to nitrogenase in Klebsiella pneumoniae. nifF gene cloned and the gene product, a flavodoxin, purified.

The nifF gene of Klebsiella pneumoniae was cloned into a multicopy plasmid in order to construct a strain that synthesizes and retains an elevated concentration of the gene product relative to the wild-type strain. Characterization of the isolated flavodoxin, which serves as an electron donor to nitrogenase, shows unambiguously that it is the product of the nifF gene.

The use of cloned nif (nitrogen fixation) DNA to investigate transcriptional regulation of nif expression in Klebsiella pneumoniae.

Some restriction endonuclease fragments of nif DNA, when carried on small multicopy plasmids, inhibited nif expression in Klebsiella pneumoniae. A study of this inhibitory effect revealed, (1) that overproduction of the nifL gene product inhibited transcription of two nif operons examined, nifJ and nifHDKY and, (2) that when transcription was initiated from the promoter of the nifHDKY operon on multicopy plasmids there was a corresponding decrease in the transcription rates of the chromosomally located nifJ and nifHDKY but not the nifLA operon. Studies of transcription in vivo also showed that the nifA gene product was essential for transcription initiation from the nifHDKY and nifBQ promoters. These results, taken with earlier observations (see Discussion) provide evidence that the nifL and nifA gene products are respectively a repressor and activator of nif transcription initiation from all nif promoters except that of the nifLA operon.

Overlapping sequences of Klebsiella pneumoniae nifDNA cloned and characterized.

A HindIII (17.0 kb) and an EcoR1 restriction fragment (6.9 kb) of Klebsiella pneumoniae nif DNA were cloned on two small amplifiable plasmids, pCM1 and pSA30 respectively. These plasmids between them carry 14 of the 15 known Klebsiella nif genes. The operon for the three structural genes for nitrogenase, nifpHDK, is carried on pSA30: four and five of the remaining six operons are on pCRA37 and pCM1 respectively. All of the nif genes were assigned to endonuclease restriction fragments of DNA using the Southern blotting technique (Southern, 1975) with total DNA of nif insertion mutants and radioactive plasmid DNA which contained cloned nif DNA sequences. Their locations were consistent with the genetic map of nif genes. The estimated size of the nif gene cluster was 24 kb.

Physical map of chromosomal nitrogen fixation (nif) genes of Klebsiella pneumoniae.

We describe a method for the rapid determination of the physical location of mutations caused by insertion of transposable elements. We used this method to construct a detailed physical map of the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae and to correlate it with the genetic map. Total cellular DNA was isolated from individual strains, each carrying an insertion in 1 of 15 different nif genes. The DNA was digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, denatured, and blotted onto nitrocellulose filter paper. The DNA on the filters was hybridized with (32)P-labeled DNA fragments derived from amplifiable plasmids carrying cloned nif DNA fragments from K. pneumoniae. Altered hybridization patterns caused by insertions into nif genes allowed us to map nif mutations with respect to the previously mapped cleavage sites for various restriction endonucleases. We have used the same method to map the end points of nif deletions. Using this procedure, we assigned physical locations on the K. pneumoniae chromosome to 86 nif insertion mutations and 13 nif deletion end points. This mapping procedure provides a convenient alternative to deletion mapping as a definitive method for mapping insertion mutations within a gene or for ordering genes within a gene cluster. This procedure will be especially useful for mapping mutations conferring phenotypes that are difficult to monitor and for mapping mutations in bacterial species in which techniques for conducting deletion mapping have not been devised.

Spontaneous degradation of pRD1 DNA into unique size classes is recA dependent.

The his and nif genes of the P1 type plasmid pRD1 were lost at a high frequency in a recA+ but not in a recA- Escherichia coli host during growth in a non-selective medium. 92% of the His- Nif- segregants after 6 subcultures retained the genetic markers of the precursor plasmid RP4, while the remainder lost all of the pRD1 markers with the concomitant loss of ccc-DNA. Plasmids purified from the His- Nif- segregants resembled RP4 in the physical and genetic properties examined. The contour length of pRD1 DNA molecules from a recA- strain was 49 micrometer corresponding to a molecular weight of 101 Mdals and the buoyant density was 1.715 g/cm3. In contrast, the contour lengths of plasmid molecules isolated from a recA+ host carrying pRD1 fell into 3 size classes of 49, 19 and 2 micrometer corresponding to molecular weights of 101, 39 and 4 Mdals respectively and two DNA species of buoyant density 1.715 and 1.719 g/cm3 were observed.

Recombinant plasmid that carries part of the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae.

We have cloned fragments of the Klebsiella pneumoniae genome that carry part of the his operon and part of the nitrogen fixation (nif) gene cluster on the amplifiable plasmid pMB9. One particular plasmid, pCRA37, complements mutations in the hisD, nifB, and nifF loci. The physical map of pCRA37 as determined by restriction enzyme analysis correlates with the genetic map of the his-nif region as determined previously by phage P1-mediated cotransductional analysis.

Derivation and properties of F-prime factors in Escherichia coli carrying nitrogen fixation genes from Klebsiella pneumoniae.

A His+ Nif+ Escherichia coli K12, Hfr strain (UNF43) was constructed by an intergeneric mating between a Klebsiella pneumoniae donor strain (HF3) and a his-HFR E. coli strain (SBI824) which transfers his as an early marker. An F-prime nif plasmid, FN39, carrying genes which correspond to the E. coli chromosomal region, metG gnd his shiA, but excluding purF and aroD, was isolated from UNF43. Translocation of carbenicillin resistance genes from a P-type R-factor, R68, to FN39 increased the stability of his and nif on the derivative F-prime, FN68. Sedimentation analysis of both F-primes in sucrose gradients revealed our covalently closed circular(CCC) DNA species of molecular weights 279 +/- 9, 136 +/- 3, 90 +/- 1 and 44 +/- 1 megadaltons. It is suggested that the two smallest CCC-DNA species are component replicons of the composite F-primes of molecular weight 136 +/- 3 megadaltons, and that the molecules of 279 +/- 9 megadaltons are CCC-dimers. FN68 was transferable in intergeneric matings to Klebsiella aerogenes, K. pneumoniae and Salmonella typhimurium but not to Proteus mirabilis; only carbenicillin resistance and sex factor activity were transferred to Erwinia herbicola. nif genes on FN68 were expressed in a Nif- mutant of K. pneumoniae and also in S. typhimurium, which in conventional tests is naturally non-nitrogen-fixing; expression of the his determinant of FN68 became temperature-sensitive in S. typhimurium.