Nucleotide sequence of nifH regions from Methanobacterium ivanovii and Methanosarcina barkeri 227 and characterization of glnB-like genes.

This brief note reports the nucleotide sequences of the second nifH region of Methanobacterium ivanovii and of the two nifH regions of Methanosarcina barkeri 227. In the three cases, nifH is followed by two ORF (open reading frames) similar to ORF105 and ORF128 respectively, which were previously found downstream of Methanococcus thermolithotrophicus nifH. These two ORF are followed by nifD in M. ivanovii as well as in the first nifH region of M. barkeri 227. Both types of ORF exhibit a strong homology with the glnB gene.

Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene.

An integration vector for use in Methanococcus voltae was constructed, based on the Escherichia coli vector pUC18. It carries the structural gene for puromycin transacetylase from Streptomyces alboniger, which is flanked by expression signals of M. voltae structural genes and hisA gene sequences of this bacterium. Transformed M. voltae cells are puromycin resistant. Several types of integration of the vector into the chromosome were found. Only one case was due to nonhomologous recombination. The integrated sequences were stable under selective pressure but were slowly lost in some cases in the absence of the selective drug. The vector could be excised from M. voltae chromosomal DNA, recircularized and transformed back into E. coli.

Nucleotide sequence and expression of the glutamine synthetase structural gene, glnA, of the archaebacterium Methanococcus voltae.

The sequence of a 2,746-bp DNA fragment of Methanococcus voltae carrying the glnA gene for glutamine synthetase (GS), was established. A 1,338-bp open reading frame (ORF), encoding a 446-amino-acid polypeptide of 50,142 Da, was defined as glnA on the basis of its similarity to other glnA genes and on the ability of a DNA fragment carrying this ORF to complement an Escherichia coli Gln- mutant. No sequence homology was found between sequences flanking the M. volae glnA gene and other eubacterial glnA genes. In M. voltae, the gene was transcribed as a monocistronic unit and GS synthesis was partially repressed at high ammonia concentrations. At the amino acid sequence level, the highest similarity was found with GS of Bacillus subtilis and Clostridium acetobutylicum.

Primary structure, functional organization and expression of nitrogenase structural genes of the thermophilic archaebacterium Methanococcus thermolithotrophicus.

Two regions of homology to Anabaena nifH (nitrogenase Fe protein) were detected in the total DNA of the thermophilic nitrogen-fixing archaebacterium Methanococcus thermolithotrophicus. A 2.8 kb HindIII fragment carrying one of these regions was previously cloned and shown to contain a nifH gene (Souillard et al., 1988) now referred to as ORFnifH2. A 3.4 kb PstI fragment and an overlapping 3.8 kb BglII fragment, containing the second region of homology, were cloned, and a DNA region of 4073 bp was sequenced. It contained four complete open reading frames (ORFs) (ORF nifH1, ORF105, ORF128, ORFnifD) and two truncated ORFs (ORFnifK and ORF96). Five ORFs were transcribed in the same direction in the order of ORFnifH1-ORF105-ORF128-ORFnifD-ORFnifk. ORFnifH1, ORFnifD and ORFnifK were assigned from their similarity to eubacterial nifH and nifDK (nitrogenase MoFe protein) genes. Transcription studies showed that ORFnifH1 and ORFnifD were expressed only under nitrogen-fixation conditions, whereas no ORFnifH2 mRNA was detected under the same conditions. A DNA probe containing ORFnifH1 hybridized with a 1.8 kb mRNA, as detected by a Northern blotting experiment. A transcriptional start site was localized 87 and 88 bp upstream from the ATG codon of ORFnifH1. This site is preceded, 21 bp upstream, by the sequence 5'-TTTATATA-3' already found at the same position in several archaebacterial promoters. ORFnifH1 mRNA was too small to encode ORFnifDK. This was confirmed by the fact that another transcription start site was localized 85 bp upstream from the ATG codon of ORFnifD.

Cloning of the trp genes from the archaebacterium Methanococcus voltae: nucleotide sequence of the trpBA genes.

A cosmid bank of Methanococcus voltae DNA was obtained in Escherichia coli after ligation of partially HindIII-digested M. voltae DNA in the HindIII site of the transferable cosmid pVK100. The bank was used to perform complementation experiments with E. coli auxotrophic mutants. Five cosmids complementing trpA shared three adjacent HindIII fragments of 2.1, 2.3 and 14 kb. Two of these cosmids also complemented trpD and carried an additional 4.2 kb HindIII fragment. The trpA- and trpD- complementing regions were more precisely localized using Tn5 mutagenesis. A 1.7 kb PstI fragment, cloned into pUC9 in both orientations, was responsible for the trpA complementation. This fragment was sequenced and an open reading frame (ORF) of 852 nucleotides (ORFtrpA) encoding a 284 amino acid polypeptide of mol. wt. 31,938 was found. The amino acid sequence was compared with that of the alpha subunit of tryptophan synthase (trpA gene product) from nine eubacterial species and to the N-terminal part of the tryptophan synthase of Saccharomyces cerevisiae (TRP5 gene product). Similarity varied from 24% (Brevibacterium lactofermentum) to 35% (S. cerevisiae). The nucleotide sequence of the region upstream from M. voltae ORFtrpA was determined and revealed the presence of an ORF of 1227 nucleotides (ORFtrpB) encoding a 409 amino acid polypeptide of mol. wt. 44,634. The polypeptide sequence was similar to the beta subunit of tryptophan synthase (trpB gene product) from six eubacterial species and to the C-terminal part of the tryptophan synthase of S. cerevisiae. Similarity varied from 49% (S. cerevisiae, B. lactofermentum) to 58% (Pseudomonas aeruginosa). This high conservation supports the hypothesis of a common ancestor for the trpA and trpB genes of archaebacteria, eubacteria and eucaryotes. M. voltae ORFtrpA and ORFtrpB, which are transcribed in the same direction, are separated by a 37 bp AT-rich region. Immediately upstream from ORFtrpB, the 3' end of an ORF homologous to E. coli and Bacillus subtilis trpF was found. As the trpD-complementing region was located upstream from the trpFBA sequenced region, the organization of trp genes in the archaebacterium might thus be trpDFBA. Such an organization resembles that of enteric eubacteria, in which the trpEDCFBA genes are grouped in a single operon. However, M. voltae ORFtrpA and ORFtrpB do not overlap, in contrast with what is found in most eubacteria.

Analysis of drug resistance in the archaebacterium Methanococcus voltae with respect to potential use in genetic engineering.

The sensitivity of the methanogenic archaebacterium Methanococcus voltae to 12 inhibitors was tested in liquid medium. Four compounds appeared to be inhibitors of growth. Their MICs were as follows: pseudomonic acid, 0.1 micrograms/ml (0.19 microM); puromycin, 2 micrograms/ml (3.6 microM); methionine sulfoximine, 30 micrograms/ml (170 microM); and fusidic acid, 100 micrograms/ml (170 microM). On solid medium, the MICs were similar and the frequency of spontaneous resistance was found to be 5 X 10(-5) (methionine sulfoximine), 10(-7) (pseudomonic acid), and less than 10(-7) (puromycin and fusidic acid). Pseudomonic acid was found to inhibit isoleucyl-tRNA synthetase activity as measured by the in vitro aminoacylation of M. voltae tRNA with L-[U-14C]isoleucine. Fusidic acid and puromycin were shown to inhibit poly(U)-dependent polyphenylalanine synthesis in S30 extracts. Acetylpuromycin was inhibitory at much higher concentrations both in vivo and in vitro for M. voltae. Thus, the pac gene of Streptomyces alboniger, which is responsible for acetylation of puromycin and which conferred resistance to puromycin when introduced in eubacteria and eucaryotes, is a potential selective marker in gene transfer experiments with M. voltae. The latter was recently shown to be transformable. The same would be true for the cat gene of Tn9, which encodes resistance to fusidic acid in eubacteria in addition to resistance to chloramphenicol.

Coumarin and quinolone action in archaebacteria: evidence for the presence of a DNA gyrase-like enzyme.

The action of novobiocin and coumermycin (two coumarins which interact with the gyrB subunit of eubacterial DNA gyrase) and ciprofloxacin (a fluoroquinolone which interacts with the gyrA subunit of DNA gyrase) was tested on several archaebacteria, including five methanogens, two halobacteria, and a thermoacidophile. Most strains were sensitive to doses of coumarins (0.02 to 10 micrograms/ml) which specifically inhibit DNA gyrase in eubacteria. Ciprofloxacin inhibited growth of the haloalkaliphilic strain Natronobacterium gregoryi and of the methanogen Methanosarcina barkeri. In addition, ciprofloxacin partly relieved the sensitivity to coumarins (and vice versa). Novobiocin inhibited DNA replication in Halobacterium halobium rapidly and specifically. Topological analysis has shown that the 1.7-kilobase plasmid from Halobacterium sp. strain GRB is negatively supercoiled; this plasmid was relaxed after novobiocin treatment. These results support the existence in archaebacteria of a coumarin and quinolone target related to eubacterial DNA gyrase.

Nucleotide sequence of regions homologous to nifH (nitrogenase Fe protein) from the nitrogen-fixing archaebacteria Methanococcus thermolithotrophicus and Methanobacterium ivanovii: evolutionary implications.

DNA fragments bearing sequence similarity to eubacterial nif H probes were cloned from two nitrogen-fixing archaebacteria, a thermophilic methanogen, Methanococcus (Mc.) thermolithotrophicus, and a mesophilic methanogen, Methanobacterium (Mb.) ivanovii. Regions carrying similarities with the probes were sequenced. They contained several open reading frames (ORF), separated by A + T-rich regions. The largest ORFs in both regions, an 876-bp sequence in Mc. thermolithotrophicus and a 789-bp sequence in Mb. ivanovii, were assumed to be ORFsnif H. They code for polypeptides of mol. wt. 32,025 and 28,347, respectively. Both ORFsnifH were preceded by potential ribosome binding sites and followed by potential hairpin structures and by oligo-T sequences, which may act as transcription termination signals. The codon usage was similar in both ORFsnifH and was analogous to that used in the Clostridium pasteurianum nifH gene, with a preference for codons ending with A or U. The ORFnifH deduced polypeptides contained 30% sequence matches with all eubacterial nifH products already sequenced. Four cysteine residues were found at the same position in all sequences, and regions surrounding the cysteine residues are highly conserved. Comparison of all pairs of methanogenic and eubacterial nifH sequences is in agreement with a distant phylogenetic position of archaebacteria and with a very ancient origin of nif genes. However, sequence similarity between Methanobacteriales and Methanococcales is low (around 50%) as compared to that found among eubacteria, suggesting a profound divergence between the two orders of methanogens. From comparison of amino acid sequences, C. pasteurianum groups with the other eubacteria, whereas comparison of nucleotide sequences seems to bring C. pasteurianum closer to methanogens. The latter result may be due to the high A + T content of both C. pasteurianum and methanogens ORFsnif H or may come from an ancient lateral transfer between Clostridium and methanogens.

Cloning and expression in Escherichia coli of the TL-DNA gene 4 of Agrobacterium tumefaciens under the control of the PR promoter of bacteriophage lambda.

A plasmid was constructed that directs expression of the TL-DNA gene 4 protein in E. coli. The different steps of the construction were as follows: i) a region of gene 4 encoding the amino-terminal portion of the protein was fused in frame to DNA encoding an enzymatically active carboxy-terminal fragment of beta-galactosidase. The hybrid gene was poorly expressed from the upstream lambda PL promoter carried by the vector. ii) in order to generate an efficient procaryotic ribosome binding site, a DNA fragment carrying the lambda PR promoter with the nearby Shine-Dalgarno (SD) sequence of gene cro was placed in front of the gene 4-lacZ fusion. A recombinant plasmid, termed pGV793, that expressed efficiently a fused protein 4-beta-galactosidase was identified among the Lac+ clones. DNA sequencing analysis showed that pGV793 carried a hybrid ribosome binding site composed of the cro SD sequence, a five bp sequence and the ATG codon of gene 4. Plasmid pGV793 directed the synthesis of three polypeptides of molecular weight 132 Kd, 126 Kd and 122 Kd that carried beta-galactosidase antigenic determinants. The largest polypeptide had the expected size for the hybrid protein. The fusion proteins which accounted for about 0.5% of the total cellular proteins were purified by immunoadsorption using anti-beta-galactosidase antiserum. iii) the complete gene 4 coding sequence was reconstituted, with the lambda PR promoter in place. The resulting pGV822 plasmid expressed a polypeptide whose molecular weight 27 Kd corresponded to the expected size for the gene 4 product. The pI was about 7.

Cloning and expression of a DNA fragment carrying a his nifA fusion and the nifBQ operon from a nif constitutive mutant of Klebsiella pneumoniae.

From the nifc mutant plasmid pPC868, previously shown to carry a DNA duplication responsible for the Nifc phenotype, a 10 kb HindIII fragment was cloned into the multicopy vector pBR325. Restriction analysis of the resulting plasmids and in vitro deleted derivatives confirmed that the mutation was a fusion between his and nifLA. The order was hisG-hisD'-'nifL-nifA so that nifA was transcribed under the control of the his promoter and the nifL gene was altered. In addition the cloned fragment contained the adjacent nifBQ operon, and complementation data revealed that the nifA, nifB and hisG genes were expressed. Synthesis of nifA product under the transcription control of the his (or cat [CmR]) promoter enabled complementation of nifA and nifB mutations either in the absence or the presence of ammonia, but did not restore nitrogen fixation in a glnF mutant. Therefore, the nifA gene product requires glnF for its positive control function in a manner analogous to ntrC. Protein content analysis of minicells containing various multicopy nif plasmids confirmed the genetic organization mentioned above. A new polypeptide of 51,500 daltons was found whose synthesis was observed at 30 degrees C but not at 37 degrees C. According to the physical map, this protein could be the nifB gene product. Our results are in agreement with nifB transcription being under the control of a thermolabile nifA product. Moreover we obtained results suggesting that the presence of multiple copies of a functional nifB gene inhibited nitrogen fixation.

Constitutive expression of nitrogen fixation (nif) genes of Klebsiella pneumoniae due to a DNA duplication.

A spontaneous mutant of Klebsiella pneumoniae exhibiting nitrogen fixing activity in the presence of ammonia was isolated from a nifL ::Mu mutant. The main features of the nif constitutive mutation, designated nif-8388, were as follows: (i) neither ammonia nor bases repressed, but amino acids partially repressed, nitrogen fixation; (ii) the mutation caused an escape from the regulatory effect of glnA and glnG mutations of K. pneumoniae but not that of a glnF mutation; (iii) it enabled the activation of the nifH -lac fusion in the presence of oxygen with or without ammonia and a nifL -lac fusion in the presence of ammonia without oxygen; (iv) the mutation allowed nitrogen fixation at 37 degrees C when plasmid-borne. Restriction analysis and Southern hybridization using Mu DNA and the 8.1-kb nifQBALF EcoRI fragment as probes demonstrated that the nif-8388 mutation was a tandem duplication of 10 kb in the nifL region in which no Mu DNA was present. This duplication led to an operon fusion between nifLA and his since Nifc expression was shown to be increased with a specific inducer of the his operon. These results provide further evidence that the nifA product is a nif-specific activator, and that the nifL product is involved in oxygen repression and temperature control. In addition, they suggest that there is an autoactivation of nifLA transcription by the nifA product and that glnF could act in nif regulation by a mechanism other than the glnG-mediated control of nifLA transcription.

The polar effect on nifM of mutations in the nifU,-S,-V genes of Klebsiella pneumoniae depends on their plasmid or chromosomal location.

Polar mutations, mostly Mu and Tn insertions, were used to study the transcriptional organization of the nif gene cluster of Klebsiella pneumoniae, but there remained some ambiguity in the operon structure of the nifF, -M, -V, -S, -U genes. Complementation data presented here confirm that nifUSVM is a single operon transcribed from nifU to nifM. However, the polar effect on nifM of point, insertion and deletion mutations in nifU, -S, and -V is stronger when the mutation is on the chromosome rather than on a plasmid.

The use of translocatable genetic elements to construct a fine-structure map of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster.

The transposons Tn5, Tn7 and Tn10 and bacteriophage Mu have been used to derive insertion mutations in the Klebsiella pneumoniae nif gene cluster. A large number of deletion mutants have been derived by imprecise excision of insertion mutations and these deletions have been used to construct a fine-structure map of the nif cluster. Comparison of this genetic map with a physical map of the nif cluster derived by Reidel et al. (1979) showed a very good correlation between genetic and physical mapping methods. A new complementation group, designated nifU, has been identified and mapped between nifN and nifS. Polarity studies on the 14 nif cistrons now identified suggests that they are organized in at least seven transcriptional units and that all the multicistronic units are transcribed in the same direction.

Genetic and biochemical analysis of mutants induced by bacteriophage Mu DNA integration into Klebsiella pneumoniae nitrogen fixation genes.

Polar mutations were obtained by integration of bacteriophage Mu c+ or Mu cts DNA into the Klebsiella pneumoniae nif genes located on plasmid pCE1, a derivative of pRD1. In addition, nif deletions were isolated from nif::Mu cts plasmids. Complementation data allowed the characterization of twelve nif cistrons, nine corresponding to previously identified genes. Polar effect of Mu DNA insertions suggested the existence of at least six transcription units: 1) nif K, nif D and nif H--2)nif A and nif L--3) nif E and a new gene--4) nif B--5) nif F--6) nif J. Nif K, nif D and nif H, which are most probably the structural genes for nitrogenase, seem to belong to the same operon transcribed from nif H to nif K. This was confirmed by SDS gel autoradiography of pulse labelled proteins. Moreover it was possible to identify, on the autoradiograms, a polypeptide which likely is the product of nif J and whose biosynthesis is under the control of nif A.