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

Abstract

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.

In vivo transposon mutagenesis of the methanogenic archaeon Methanosarcina acetivorans C2A using a modified version of the insect mariner -family transposable element Himar1

“…The coenzyme M (CoM) analog BES is a potent inhibitor of the enzyme methyl-CoM reductase that catalyzes the final step in methane production. In earlier studies, M. voltae strains incapable of either BES or CoM uptake were isolated as BES R mutants (37). However, the mutated gene(s) in these strains were never identified.…”

Isolation of a coenzyme M-auxotrophic mutant and transformation by electroporation in Methanococcus voltae

“…In the experiments shown, the chromosomal DNA used for electroporation of the his-12 auxotroph was partially digested with the restriction enzyme EcoRI; however, partial digestion with HindIlI gave similar levels of electroporation as did undigested DNA. For electroporation of the pur-J aux- (12). BES, a methyl-CoM analog, is an inhibitor of methyl reductase (5), an enzyme complex responsible for the final steps of methanogenesis.…”

“…We recently reported that this marine methanogen is capable of taking up 2-mercaptoethanesulfonic acid (HS-CoM, coenzyme M) via an energy-dependent, carrier-mediated uptake system (4,12). HS-CoM is an intermediate in methanogenesis and carries a methyl moiety in the form of methyl-S-CoM, the terminal carrier of C1 in the process of methane formation (6,14).…”

Structure and organization of the hisA gene of the thermophilic archaebacterium Methanococcus thermolithotrophicus

“…The methanococci are attractive subjects for molecular biology experiments and as candidate species for developing archaebacterial genetics. They grow relatively quickly, form colonies on agar-solidified defined media (1,8,21), contain plasmids (23), can be transformed (3), give rise to mutants (7,16), and can be lysed for DNA isolation simply by the addition of detergents (12) proCJ 19 tsx purEl galK2 trp-3 hisA4 argG36 rpsL xyl-Imtl-I ilvA6 thr-J met-12 (6; obtained from R. Curtiss III).Transformants of E. coli X760 which grew at 37°C on plates containing supplemented M9-glucose minimal-salts medium lacking histidine but containing 100 ,ug of ampicillin per ml were selected (2). One such transformant contained plasmid pET9301 which was shown by DNA sequencing (14) to contain a 2,155-base-pair (bp) HindIII-BamHI-generated fragment of M. thermolithotrophicus genomic DNA (Fig.…”

“…The methanococci are attractive subjects for molecular biology experiments and as candidate species for developing archaebacterial genetics. They grow relatively quickly, form colonies on agar-solidified defined media (1,8,21), contain plasmids (23), can be transformed (3), give rise to mutants (7,16), and can be lysed for DNA isolation simply by the addition of detergents (12) proCJ 19 tsx purEl galK2 trp-3 hisA4 argG36 rpsL xyl-Imtl-I ilvA6 thr-J met-12 (6; obtained from R. Curtiss III).…”