Analogue-resistant and auxotrophic mutants ofMethanobacterium thermoautotrophicum

Kiener, Andreas; Holliger, Christof; Leisinger, Th.

doi:10.1007/bf00692718

Cited by 29 publications

(19 citation statements)

References 12 publications

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“…These applications of the enzyme were examined with ,the Marburg straiti of Methanobacterium thermoautotrophicum, using a DEAE-cellulose-purified pr-eparation. This organism contains a 4.5-kilobase cryptic plasmid (18), and auxotrophic as well as resistance mutants are available (10,11,21). The partially purified enzyme proved to be remarkably active.…”

Section: Discussionmentioning

confidence: 99%

Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum

Kiener¹,

König²,

Winter³

et al. 1987

J Bacteriol

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The pseudomurein-degrading enzyme from autolysates of Methanobacterium wolfei was purified approximately 500-fold to electrophoretic homogeneity by ion-exchange chromatography under anaerobic conditions. Analysis of the soluble cell wall fragments produced by the pure enzyme from a cell wall preparation of M. thermoautotrophicum indicated that it is a peptidase hydrolyzing the e-Ala-Lys bond of pseudomurein. A partially purified preparation of pseudomurein endopeptidase was free of nuclease activity and thus proved useful for the preparation in high yields of undegraded chromosomal and plasmid DNA from M. thermoautotrophicum. The partially purified enzyme was also used for the preparation of protoplasts, which were stabilized by 0.8 M sucrose. Under growth conditions the protoplasts produced methane and increased up to 100-fold in size, but failed to regenerate a cell wall.Methanogenic bacteria exhibit a striking heterogeneity with respect to the composition of their cell wall sacculus in that four basic types of cell walls are found in this physiological group of archaebacteria (3, 5). Methanosarcina spp. possess a methanochondroitin cell wall (15). Methanococcus spp. have a sodium dodecyl sulfate (SDS)-sensitive cell wall of regularly arranged protein subunits (24). Tubelike sheaths, also consisting of proteins, are isolated from Methanospirillum spp. (9), and the cell wall sacculi of the gram-positive genera Methanobacterium, Methanobrevibacter, Methanothermus, and Methanosphaera consist of pseudomurein, which, in contrast to the murein of eubacteria, contains N-acetyl-L-talosaminuronic acid instead of Nacetyl-D-muramic acid (12, 13). Also, the peptide moieties of pseudomurein consist exclusively of L-amino acids.Several reports describe gentle lysis of methanogen cell walls by either chemical or enzymatic agents. Methanococcus vannielii lysed within minutes in solutions containing 0.001% SDS (7). Methanobacterium formicicum was lysed by proteinase K, by pronase, and by an extracellular serine protease produced by a Streptomyces isolate, two enzymes acting on the cytoplasmic membrane but not on the pseudomurein sacculus (2). Spontaneous spheroplast or protoplast formation has been reported for Methanobacterium bryantii (6) and Methanosarcina barkeri (4). Dithiothreitol treatment at alkaline pH has been used to form spheroplasts of Methanospirillum hungatii (25), and in the case of Methanobacterium thermoautotrophicum protoplasts were obtained by treating cells that were grown in the presence of 50 mM sorbitol with lysozyme (22). The methods described for the lysis of Methanobacterium spp. cell walls have certain disadvantages for practical use in the preparation of lysates or protoplasts. The organisms must be grown under stress conditions or else the enzymes applied, such as proteinase K, may damage cell compounds.We therefore have explored whether the lytic activity that has recently been detected in Methanobacterium wolfei may be of use as an experimental tool. The lytic activity of this organism is formed...

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Section: Discussionmentioning

confidence: 99%

Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum

Kiener¹,

König²,

Winter³

et al. 1987

J Bacteriol

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“…This is compounded by their insensitivity to many of the antibiotics effective against the eubacteria [I 51, and to the difficulty in selecting for auxotrophic mutants [16]. The finding that Mc.…”

Section: Discussionmentioning

confidence: 99%

Amino acid biosynthesis and sodium‐dependent transport in Methanococcus voltae, as revealed by ¹³C NMR

Ekiel

Jarrell

Sprott

1985

European Journal of Biochemistry

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Of several methanogenic bacteria examined only Methanococcus voltae readily incorporated exogenous amino acids into cell protein. This was easily shown, since growth in the presence of exogenous amino acids resulted in a loss of signal intensities from those carbon atoms normally labelled by [13C]acetate during biosynthesis. From 80% to 95% of the Ser, Lys, Pro or Val incorporated into protein could be supplied directly from the growth medium. In contrast, Asp and Glu, if supplied to the medium, accounted for only a small percentage of the total acidic amino acid used in protein synthesis. Constitutive transport systems took up a wide range of amino acids at rates of 0.1–4.1 nmol min−1 mg−1. The transport systems required Na+, with the possible exception of the basic amino acid lysine, and were inhibited by N‐ethylmaleimide or 3,3′,4′,5‐tetrachlorosalicylanilide. No interconversion of Ile to other amino acids was detected when cells were given [13C]Ile during growth, whereas the expected labelling of the Asp and Glu families of amino acids resulted when [13C]Asp was provided to the culture. Mc. voltae synthesized its amino acids from acetate via routes fully consistent with those found in Methanospirillum hungatei [Ekiel, I., Smith, I. C. P. & Sprott, G. D. (1983) J. Bacteriol. 156, 316–326]. Propionate could substitute for an auxotrophic requirement for Ile, resulting in the synthesis of Ile with the β‐carbon originating from the carboxyl of acetate and the α‐carbon from the carboxyl of propionate. No labelling of Ile from [13C]acetate could occur without the fatty acid. These results provide strong evidence for the carboxylation of propionate to form 2‐oxobutyrate as intermediate in Ile biosynthesis, and show that the metabolic defect in Ile biosynthesis occurs prior to 2‐oxobutyrate synthesis. The presence of constitutive amino acid transport systems and multiple routes for ile biosynthesis make Methanococcus voltae an attractive methanogen for genetic studies.

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“…Mutants that are resistant to 2-bromoethanesulfonic acid (BES), an analog of methyl-coenzyme M (CoM) and a cofactor in methanogenesis (3,31), have been reported for several methanogens including Methanosarcina barkeri (26), Methanobacterium thermoautotrophicum (17), Methanococcus vannielii (10), and Methanococcus voltae ( (30), and agar medium was prepared anaerobically as described by Jones et al (16). Complex medium (16) was used for all plating experiments and for maintenance of stock cultures.…”

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Characterization of bromoethanesulfonate-resistant mutants of Methanococcus voltae: evidence of a coenzyme M transport system

Santoro¹,

Konisky²

1987

J Bacteriol

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Mutants of Methanococcus voltae were isolated that were resistant to the coenzyme M (CoM; 2-mercaptoethanesulfonic acid) analog 2-bromoethanesulfonic acid (BES). The mutants displayed a reduced ability to accumulate [35S]BES relative to the sensitive parental strain. BES inhibited methane production from CH3-S-CoM in cell extracts prepared from wild-type sensitive or resistant strains. BES uptake requwred the presence of both CO2 and H2 and was inhibited by N-ethylmaleimide and several reagents that are known to disrupt energy metabolism. The mutants showed normal uptake of isoleucine and were not cross-resistant to either azaserine or 5-methyltryptophan and, thus, were neither defective in general energy-dependent substrate transport nor envelope permeability. Both HS-CoM and CH3-S-CoM prevented the uptake of BES and protected cells from inhibition by it. We propose that M. voltae has an energy-dependent, carrier-mediated uptake system for HS-CoM and CH3-S-CoM which can also mediate uptake of BES.

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Analogue-resistant and auxotrophic mutants ofMethanobacterium thermoautotrophicum

Cited by 29 publications

References 12 publications

Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum

Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum

Amino acid biosynthesis and sodium‐dependent transport in Methanococcus voltae, as revealed by ¹³C NMR

Characterization of bromoethanesulfonate-resistant mutants of Methanococcus voltae: evidence of a coenzyme M transport system

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Analogue-resistant and auxotrophic mutants ofMethanobacterium thermoautotrophicum

Cited by 29 publications

References 12 publications

Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum

Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum

Amino acid biosynthesis and sodium‐dependent transport in Methanococcus voltae, as revealed by 13C NMR

Characterization of bromoethanesulfonate-resistant mutants of Methanococcus voltae: evidence of a coenzyme M transport system

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Amino acid biosynthesis and sodium‐dependent transport in Methanococcus voltae, as revealed by ¹³C NMR