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.     

Chemotaxis in the archaebacterium Methanococcus voltae.

The archaebacterium Methanococcus voltae, was shown to be chemotactic. Acetate, isoleucine, and leucine were identified as attractants; whereas histidine was not an attractant. A motile, generally nonchemotactic mutant was isolated.     

Heat shock response of the archaebacterium Methanococcus voltae.

The general properties of the heat shock response of the archaebacterium Methanococcus voltae were characterized. The induction of 11 heat shock proteins, with apparent molecular weights ranging from 18,000 to 90,000, occurred optimally at 40 to 50 degrees C. Some of the heat shock proteins were preferentially enriched in either the soluble (cytoplasm) or particulate (membrane) fraction. Alternative stresses (ethanol, hydrogen peroxide, NaCl) stimulated the synthesis of subsets of the heat shock proteins as well as unique proteins. Western blot (immunoblot) analysis, in which antisera to Escherichia coli heat shock proteins (DnaK and GroEL) were used, did not detect any immunologically cross-reactive proteins. In addition, Southern blot analysis did not reveal any homology between M. voltae and four highly conserved heat shock genes, mopB and dnaK from E. coli and hsp70 genes from Drosophila species and Saccharomyces cerevisiae.     

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

Summary 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.The order of the other authors is not indicative of the relative importance of their experimental contributions which are considered to be equivalentWe mourn the loss of our colleague and friend Lionel Sibold, who died while this work was still in progress     

Characterization of a P-type ATPase of the archaebacterium Methanococcus voltae

The vanadate-sensitive ATPase of Methanococcus voltae has been purified by a procedure which includes, purification of the cytoplasmic membrane by sucrose gradient centrifugation, solubilization with Triton X-100, and DEAE-Sephadex and Sephacryl S-300 chromatography. While the DEAE-Sephadex step provided a preparation consisting of two polypeptides (74 and 52 kDa), the Sephacryl S-300 step yields a product with a subunit of 74 kDa. Incubation of either membranes or purified ATPase with [gamma-32P]ATP followed by acidic (pH 2.4) lithium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the vanadate-sensitive labeling of a 74-kDa acyl phosphate intermediate. These results indicate that the M. voltae ATPase is of the P-type.     

Identification of a vanadate-sensitive, membrane-bound ATPase in the archaebacterium Methanococcus voltae.

Membrane-bound ATPase activity was detected in the methanogen Methanococcus voltae. The ATPase was inhibited by vanadate, a characteristic inhibitor of E1E2 ATPases. The enzyme activity was also inhibited by diethylstilbestrol. However, it was insensitive to N,N'-dicyclohexylcarbodiimide, ouabain, and oligomycin. The enzyme displayed a high preference for ATP as substrate, was dependent on Mg2+, and had a pH optimum of approximately 7.5. The enzyme was completely solubilized with 2% Triton X-100. The enzyme was insensitive to oxygen and was stabilized by ATP. There was no homology with the Escherichia coli F0F1 ATPase at the level of DNA and protein. The membrane-bound M. voltae ATPase showed properties similar to those of E1E2 ATPases.     

Physical and genetic map of the Methanococcus voltae chromosome

A physical map of the Methanococcus voltae chromosome was constructed on the basis of restriction mapping and cross-hybridization experiments, employing total and partial digests obtained with rarely cutting restriction enzymes. On the basis of the sum of the fragment sizes of digests with seven enzymes the chromosome length was calculated to be approximately 1900 kb. The derived map is circular. Hybridization of gene probes to mapped restriction fragments has led to a genetic map of genes for structural RNAs as well as proteins, including enzymes involved in the methanogenic pathway.     

Isolation of flagella from the archaebacterium Methanococcus voltae by phase separation with Triton X-114.

The flagella of Methanococcus voltae were isolated by using three procedures. Initially, cells were sheared to release the filaments, which were purified by differential centrifugation and banding in KBr gradients. Flagella were also prepared by solubilization of cells with 1% (vol/vol) Triton X-100 and purified as described above. Both of these techniques resulted in variable recovery and poor yield of flagellar filaments. Purification of intact flagella (filament, hook, and basal body) was achieved by using phase transition separation with Triton X-114. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella revealed two major proteins, with molecular weights of 33,000 and 31,000. This result indicates the likely presence of two flagellins. The filament had a diameter of 13 nm. The basal structure consisted of a small knob, while a slight thickening of the filament immediately adjacent to this area was the only evidence of a hook region. Flagella from three other Methanococcus species were isolated by this technique and found to have the same ultrastructure as flagella from M. voltae. Isolation of flagella from three eubacteria and another methanogen (Methanospirillum hungatei [M. hungatii]) by the phase separation technique indicated that the detergent treatment did not affect the structure of basal bodies. Intact ring structures and well-differentiated hook regions were apparent in each of these flagellar preparations.     

Purification and characterization of an 8-hydroxy-5-deazaflavin-reducing hydrogenase from the archaebacterium Methanococcus voltae

A methylviologen and 8-hydroxy-5-deazaflavin(F420)-reducing hydrogenase was purified over 800-fold to near homogeneity from the archaebacterium Methanococcus voltae with 10 U mg-1 F420-reducing activity. It is the only hydrogenase in this organism. The enzyme showed Km values of 16 microM for F420 and 1.2 mM for methylviologen. A turnover number of 1050 min-1 was calculated for the minimal active unit. The protein tends to aggregate. The molecular mass of the minimal active unit is 105 kDa. Larger molecules of 745 kDa were regularly observed. The enzyme was resolved into subunits with molecular masses of 55 kDa, 45 kDa, 37 kDa and 27 kDa by SDS/polyacrylamide gel electrophoresis. Reversible conversion of an anionic into an uncharged form was observed by DEAE-cellulose chromatography with concomitant changes in substrate specificities. The methylviologen-reducing activity was heat-resistant up to 65 degrees C and was not affected by antiserum raised against the native enzyme, while F420 reduction was inactivated by both treatments. Nickel and selenium contents were determined as 0.6-0.7 mol each, FAD content as 1 mol and iron as 4.5 mol/mol protein (105 kDa), respectively. Electron micrographs taken from the purified enzyme show ring-shaped molecules of 18 nm diameter, which represent the high-molecular-mass species of the enzyme.     

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

Summary 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. 31938 was found. The amino acid sequence was compared with that of the 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 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.     

Complementation of argG and hisA mutations of Escherichia coli by DNA cloned from the archaebacterium Methanococcus voltae

DNA derived from the methanogenic archaebacterium Methanococcus voltae was digested with PstI restriction endonuclease and cloned into the PstI site of pBR322. The recombinant plasmids generated were used to transform a multiply auxotrophic strain of Escherichia coli with selection for tetracycline resistance. Plasmids complementing the argG(pAW1) or hisA(pAW2) mutations were isolated and characterized. Nick-translated pAW1 and pAW2 hybridized to the predicted M. voltae PstI fragments but not to digested E. coli DNA. A novel 55,000-dalton protein was synthesized in UV-irradiated cells by pAW1, whereas pAW2 synthesized a novel 26,000-dalton protein. Derivatives of pAW1 carrying insertion elements no longer complemented the argG mutation and failed to produce the 55,000-dalton protein. When an AccI fragment was deleted from pAW2, complementation of hisA did not occur and no 26,000-dalton protein was synthesized. The effect of orientation of the cloned DNA within the vector on complementation and polypeptide synthesis was examined.     

Antibiotic resistance caused by permeability changes of the archaebacterium Methanococcus vannielii

Abstract Spontaneous mutants of the methane-producing archaebacterium Methanococcus vannielii have been isolated which are resistant to bromoethane-sulphonate, A2315-A (a virginiamycin), neomycin and thiostrepton. Ultraviolet (UV) irradiation followed by growth in bacitracin led to the isolation of bacitracin-resistant mutants. In vitro translation reactions using ribosomes prepared from neomycin or A2315-A-resistant mutants were sensitive to neomycin or A2315-A indicating that the in vivo resistance of the mutants must reflect changes in the permeability of the M. vannielii proteinaceous cell envelope.     

Transduction-Like Gene Transfer in the Methanogen Methanococcus voltae

Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.     

Genetic transformation in the methanogen Methanococcus voltae PS.

Mutations causing requirements for histidine, purine, and vitamin B12 were obtained in strain PS of Methanococcus voltae (archaebacteria) upon irradiation with UV or gamma rays. The first two mutations were shown to revert at low frequencies and were used to demonstrate the occurrence of transformation with homologous, wild-type DNA. The transformation rates obtained for these presumably chromosomal markers were in the range of 2 to 100 transformants per microgram of DNA. Mutants resistant to 2-bromoethanesulfonate and to 5-methyl-DL-tryptophan were also isolated.     

Identification of the Methanococcus voltae S-layer structural gene.

We have established that the gene which we had previously identified as encoding the Methanococcus voltae P-type ATPase is, in fact, the structural gene for the M. voltae S-layer protein. This conclusion is based on a comparison of the N-terminal sequence of S-layer protein prepared by two independent methods with that derived from the nucleotide sequence of the cloned gene. This conclusion was further supported by immunocytochemical localization of the antigen directed against the antibodies used in the cloning experiments.     

Measuring genetic stability in bacteria of potential use in genetic engineering

Four commonly used conjugation techniques, colony cross streak (CCS), broth mating (BM), combined spread plate (CSP), and membrane filtration (MF), were compared with each other regarding reliability, sensitivity, and complexity in evaluating the transfer of conjugative plasmids. Five plasmids representing several incompatibility groups plus a variety of laboratory and environmental isolates were used as mating pairs. The suitability of each method was evaluated for use in a routine assessment of the genetic stability of genetically engineered microorganisms. By the CSP and MF techniques with laboratory strains such as Escherichia coli and Pseudomonas species as recipients, transconjugants were usually produced in 100% of the mating trials. However, when environmental strains isolated from plants and soil were used as recipients, transconjugants were detected in 100% of some crosses and in as little as 30% in other crosses depending on the plasmid and recipient used. In general, differences in the percentage of successful matings between the CSP and MF techniques compared with the BM and CCS techniques were not statistically significant at the P less than or equal to 0.05 level. Occasionally, certain mating pairs consistently produced transconjugants by CCS or BM but not by CSP or MF. Since any single conjugation mating technique is not completely reliable in detecting transconjugants, we have developed a combined mating technique which integrates the CCS, CSP, BM, and MF methods as a single procedure to assess the mobility of plasmid DNA of genetically engineered microorganisms.     

Measuring genetic stability in bacteria of potential use in genetic engineering

[This corrects the article on p. 105 in vol. 53.].