Albrecht Klein

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.

Methanococcus voltae harbors four gene clusters potentially encoding two [NiFe] and two [NiFeSe] hydrogenases, each of the cofactor F420-reducing or F420-non-reducing types

SummaryFour gene clusters were identified inMethanococcus voltae which probably all encode hydrogenases of the [NiFe] type. One of these contains four genes, including those for the three subunits of the known [NiFeSe] hydrogenase capable of reducing the natural deazaflavin cofactor F420. In a second homologous cluster, the gene encoding the subunit corresponding to that which contains selenium in the know enzyme has a cysteine codon in the relevant position. In addition, two more gene clusters were detected which are very similar both in gene order and sequence to one which encodes a hydrogenase that reduces viologens inMethanobacterium thermoautotrophicum, but whose natural electron acceptor is as yet unknown. Again, in one of these clusters, one of the structural genes, which codes for a hydrogenase subunit containing the putative Ni-binding site, contains a selenocysteine codon. The homologous gene in the other clusters again shows a cysteine codon in the corresponding location. The four gene clusters are closely linked. Those encoding the two selenium-free enzymes are arranged in opposite polarities with a relatively short intergenic region. This arrangement is discussed in terms of a possible joint transcriptional regulation.

The [NiFe] hydrogenases of Methanococcus voltae: genes, enzymes and regulation.

Methanococcus voltae carries genetic information for four [NiFe] hydrogenases. Two of the hydrogenases are predicted to contain selenocysteine on the basis of in-frame TGA codons, while the genes encoding the two other enzymes contain cysteine codons at homologous positions. Their predicted subunit compositions and their electron acceptor specificities are similar to those of the respective selenium-containing enzymes. The selenium-containing hydrogenases have been purified and characterized. Only one of them reduces the deazaflavin F420. The activity of the F420-nonreducing enzyme is exceptionally high. The selenium atom has been shown by EPR spectroscopy to be a ligand to the Ni atom in the primary reaction centers in both enzymes. The spectroscopic analyses also yielded a description of the electronic configuration around the NiFe center at different oxidation states and in the presence of the competitive inhibitor, CO. The genes encoding the selenium-free hydrogenases are expressed only in the absence of selenium. They are linked by an intergenic region in which regulatory cis elements were defined by employing reporter gene constructs and site-directed mutagenesis.

Isolation and characterization of insertional mutations in flagellin genes in the archaeon Methanococcus voltae

Methanococcus voltae is a flagellated member of the Domain Archaea that has four flagellin genes arranged in two transcriptional units. One transcriptional unit encodes only flaA while the second is a multi‐cistronic unit encoding three flagellin genes (flaB1flaB2, and flaB3 ) as well as at least seven other open reading frames downstream. The polymerase chain reaction was used to amplify an internal fragment of the flaA gene which was subsequently cloned into an insertion vector developed for M. voltae. Transformation of protoplasts with this vector led to the isolation of mutant strains that had insertions in flaA or flaB2. Mutant strains carrying insertions in flaA had flagella that were similar to wild‐type cells in both number and appearance when viewed using the electron microscope. In addition, some of these mutant strains had profiles identical to the wild type in immunoblots developed with antisera raised against the 31 kDa flagellin of M. voltae. All flaA mutant strains and the wild‐type cells showed immuno‐cross‐reactive bands at 33 and 31 kDa (corresponding to purified flagellins) as well as at 18 kDa. Some flaA mutant strains also showed an immuno‐cross‐reactive band at 27 kDa which probably represents a truncated flagellin produced by the insertion vector. However, both types of flaA mutant strains were less motile than the wild type in semi‐swarm plate experiments. The mutant strain with an insertion in flaB2 was non‐flagellated when examined by electron microscopy and it was non‐motile in semi‐swarm plate experiments. It represents the first structural mutant strain isolated in a methanogen. This mutant strain lacked the 33, 31, and 18 kDa immuno‐cross‐reactive bands observed in the wild type and flaA mutant strains, and instead had a novel band at 20 kDa. This band may represent an unmodified flagellin which still has an attached leader peptide. If so, then one of the downstream genes in the multi‐cistronic transcriptional unit may encode a leader peptidase for the flagellin system.

Mutants in flaI and flaJ of the archaeon Methanococcus voltae are deficient in flagellum assembly.

The fla gene locus of Methanococcus voltae encodes the major structural components of the flagellum as well as other flagellar‐related proteins. The flaHIJ genes have been found in all flagellated archaea, suggesting a central role in flagella biogenesis. FlaI shares similarity with the type II and type IV secretion NTPases (such as PilB, VirB11 and TadA), and FlaJ exhibits similarity to putative bacterial integral membrane proteins involved in type IV pilus biogenesis such as TadB. In this study, reverse transcription polymerase chain reaction (RT‐PCR) and Northern blotting data revealed that flaHIJ are co‐transcribed with the upstream structural flagellin genes, thus demonstrating the expression of the entire fla gene cluster in vivo. Non‐polar mutants in flaI and flaJ of M. voltae were isolated using insertional inactivation via a novel mutagenic vector. These mutants were non‐motile and non‐flagellated by microscopy, demonstrating the involvement of FlaI and FlaJ in flagella biogenesis. Interestingly, all the mutants maintained the ability to produce and localize flagellins to the cytoplasmic membrane. Amino‐terminal sequencing of flagellins produced by the flaJ mutant strain revealed that the flagellins did not have their cognate leader peptides, thus indicating that preflagellin processing had occurred in vivo. This result was confirmed using an in vitro processing assay. The fla− phenotype and protein secretion characteristics of the flaI and flaJ mutants therefore implicate these respective genes in archaeal flagellin secretion and assembly. These findings further support a model describing the archaeal flagellum as a novel prokaryotic motility structure.

Comparative analysis of genes encoding methyl coenzyme M reductase in methanogenic bacteria

SummaryThe sequence of the gene cluster encoding the methyl coenzyme M reductase (MCR) in Methanococcus voltae was determined. It contains five open reading frames (ORF), three of which encode the known enzyme subunits. Putative ribosome binding sites were found in front of all ORFs. They differ in their degrees of complementarity to the 3′ end of the 16 S rRNA, which is discussed in terms of different translation efficiencies of the respective genes. The codon usage bias is different in the subunit encoding genes compared with the two other ORFs in the cluster and two other known genes of Mc. voltae. This is interpreted in terms of increased translational accuracy of the highly expressed MCR subunit genes. The derived polypeptide sequences encoded by the five ORFs of the MCR cluster were compared to those of the respective genes in Methanobacterium thermoautotrophicum Marburg and Methanosarcina barkeri. Conserved regions were detected in the enzyme subunits, which are candidates for factor binding domains. Conserved hydrophobic sequences found in the α and β subunits are discussed with respect to the membrane association of the enzyme.

Use of theEscherichia coli uidA gene as a reporter inMethanococcus voltae for the analysis of the regulatory function of the intergenic region between the operons encoding selenium-free hydrogenases

TheEscherichia coli β-glucuronidase geneuidA was linked to a region of theMethanococcus voltae genome containing the putative promoter of a gene for a DNA-binding protein and introduced into theM. voltae chromosome. It was found that the enzyme was expressed in the cells in easily measurable amounts. The reporter gene was then placed under the control of the intergenic region found between two divergently transcribed gene groups encoding selenium-free hydrogenases, which are measurably transcribed only after selenium depletion. This region is supposed not only to contain the divergent promoters governing the transcription of the hydrogenase genes but also cis regulatory elements necessary for the negative transcriptional regulation in which selenium is involved. It was shown that the intergenic region functioned as a promoter region for the reporter gene in either orientation. The additional finding thatβ-glucuronidase expression was dependent on selenium depletion localizes the cis regulatory elements to the intergenic region between the two hydrogenase operons.

Selenium is involved in the negative regulation of the expression of selenium-free [NiFe] hydrogenases in Methanococcus voltae

Competitive polymerase chain reactions (PCR) were used to analyze quantitatively the transcription patterns of the four different gene groups encoding [NiFe] hydrogenases in Methanococcus voltae. In cells growing in the presence of selenium, transcripts of only two of the hydrogenase transcription units could be detected in quantities above background levels. The missing transcripts encode the selenium-free F420-non-reducing and F420-reducing hydrogenases. In cells grown without selenium these transcripts are detectable, indicating the involvement of selenium in their transcriptional regulation.

Influence of illumination on the electronic interaction between 77Se and nickel in active F420-non-reducing hydrogenase from Methanococcus voltae

The selenium‐containing F420‐non‐reducing hydrogenase from Methanococcus voltae was anaerobically purified. The enzyme as isolated showed an EPR spectrum with g x,y,z = 2.21, 2.15 and 2.01. Upon illumination this spectrum disappeared and a new signal with the lowest g value at 2.05 arose. EPR studies were carried out either with the enzyme containing natural selenium or enriched in the nuclear isotope 77Se. The hyperfine splitting caused by 77Se in the ‘dark’ signal is shown to be highly anisotropic. In contrast the splitting is nearly isotropic after illumination. A new model for the nickel site is proposed to explain these observations.

Physical mapping of genes coding for two subunits of methyl CoM reductase component C of Methanococcus voltae

SummaryA genomic library of EcoRI*-digested cellular DNA from Methanococcus voltae was constructed in an expression vector, which allows the induction of fusion proteins of the Mc. voltae gene products with an MS2 RNA polymerase N-terminal fragment. Antibodies were raised against the subunits of the methyl CoM reductase component C complex. They were used to screen clones from the genomic library for the synthesis of polypeptides carrying antigenic determinants of the reductase subunits. Plasmids containing fragments of two of the three subunit genes were isolated. The genes were shown to be adjacent to each other by hybridization against restriction fragments of cellular DNA and Mc. voltae DNA cloned in a bacteriophage lambda replacement vector. The direction of the transcription of one of the genes was established by partial sequence determination and it was shown that the two genes have a common transcript.