Lionel Sibold

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

SummaryAn 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.

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.

SummaryPolar 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) nifK, nifD and nifH-2) nifA and nifL-3) nifE and a new gene-4) nifB-5) nifF-6) nifJ. NifK, nifD and nifH, which are most probably the structural genes for nitrogenase, seem to belong to the same operon transcribed from nifH to nifK. 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 nifJ and whose biosynthesis is under the control of nifA.

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

SummaryDNA fragments bearing sequence similarity to eubacterialnifH 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 inMc. thermolithotrophicus and a 789-bp sequence inMb.ivanovii, were assumed to be ORFsnifH. 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 theClostridium pasteurianum nifH gene, with a preference for codons ending with A or U. The ORFnifH deduced polypeptides contained 30% sequence matches with all eubacterialnifH 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 eubacterialnifH sequences is in agreeement with a distant phylogenetic position of archaebacteria and with a very ancient origin ofnif 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 bringC. pasteurianum closer to methanogens. The latter result may be due to the high A+T content of bothC. pasteurianum and methanogens ORFsnifH or may come from an ancient lateral transfer betweenClostridium and methanogens.

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

SummaryUsing the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.

Primary structure and expression of a gene homologous to nifH (nitrogenase Fe protein) from the archaebacterium Methanococcus voltae

SummaryIn Methanococcus voltae, a 3.0 kbp HindIII fragment carrying homology to nifH was recently cloned. In Escherichia coli maxicells, the fragment directed the synthesis of a 30 K polypeptide encoded by the region homologous to nifH. Plasmids carrying the fragment did not complement Klebsiella pneumoniae nifH mutants and did not inhibit the nitrogen fixation of a Nif+ strain. The complete nucleotide sequence of the nifH homologous region was determined. It contained an open reading frame (ORFnifH) of 834 bp encoding 278 amino acid residues (mol. wt. 30,362). The ORFnifH was surrounded by regions of very high A+T content as observed with other mc. voltae genes. The region upstream from ORFnifH contained potential prokaryotic-like promoters and a potential ribosome binding site located 5 bp preceding the translation initiation codon. Using a translational fusion to lacZ of a DNA fragment carrying the putative promoter region and the 5′ end of ORFnifH, it was shown in E. coli that (i) a promoter activity was effectively carried by the cloned fragment and (ii) this activity was not significantly modified by the presence of nifA or ntrC products provided by multicopy plasmids. Though the codon usage was characteristic of Mc. voltae, ORFnifH was very similar to eubacterial nifH genes, in particular the position of the cysteine residues was highly conserved. These data confirmed the high conservation of nifH sequences. SAB values (binary matching coefficients) of 0.5 were found with eubacterial nifH genes at the nucleotide or amino acid level suggesting that the mc. voltae ORFnifH sequence was distantly related to eubacterial nifH sequences.

Nucleotide sequence ofnifH regions fromMethanobacterium ivanovii andMethanosarcina barkeri 227 and characterization ofglnB-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.

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

SummaryA 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. 31938 was found. The amino acid sequence was compared with that of the α 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. 44634. The polypeptide sequence was similar to the β 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.

Genes for tRNA and their putative expression signals in Methanococcus

Summary Genes for tRNA are suitable systems for the search for the consensus signal sequences involved in gene expression in archaebacteria, because they are subject to the same regulatory mechanisms and their products display identical functions. To this end, a number of tRNA genes from Methanococcus vannielii were cloned and sequenced. They belong to a total of six putative transcriptional units comprising 11 tRNA genes. Together with the tRNA gene sequences previously reported, this brings the total of Methanococcus tRNA genes now analyzed to 19, organized in seven putative transcriptional units. In two of the tRNA gene clusters (one comprising two genes, the other six) one of the genes possesses opposite transcriptional polarity and is separated from the remaining gene(s) by a spacer of 146 and 115 nucleotides respectively. Comparision of the region flanking the seven transcriptional units at the 5′ end yielded a consensus sequence between −25 and −50 bases upstream. In the tRNA gene clusters with opposite transcriptional polarity this sequence occurred twice and also in inverse polarity. This observation and the fact that this sequence was the only detectable motif of homologous primary structures in 5′ upstream regions of tRNA genes indicates that it may be involved in transcription initiation. Common motifs at the 3′ flanking regions, which may possibly be involved in transcription termination, are also presented.

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.