Distinct regulators control the expression of methanol methyltransferase isozymes in Methanosarcina acetivorans C2A.
ABSTRACT The mtaCB1, mtaCB2 and mtaCB3 operons encode isozymes of methanol methyltransferases in Methanosarcina acetivorans C2A and are among the most highly regulated genes known in Archaea. Here we identify cis and trans acting elements that affect the expression of these operons. In vivo reporter gene constructs expressed from sequentially truncated promoters show that the mRNA transcripts for these operons have large 5' untranslated regions. Regions upstream of the transcription start site (TSS) are important for induction of the mtaCB1 and mtaCB2 operons and for repression of the mtaCB3. Regions downstream of the TSS are important for expression of the mtaCB2 and mtaCB3 operons, but are dispensable for mtaCB1 expression. Proteins encoded by the MA0459 and MA0460 loci are required for induction of the mtaCB1 operon, while those encoded by MA4383, MA4397 and MA4398 are required for induction of mtaCB2. Proteins encoded by MA4397 and MA4398 are also required for methanol-specific repression of the mtaCB3 operon and, thus, are the first archaeal examples of regulatory proteins that simultaneously act in both repression and activation. We refer to these genes as methanol-specific regulators and designate MA0459, MA0460, MA4383, MA4397 and MA4398 as msrA, msrB, msrC, msrD and msrE respectively.
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ABSTRACT: Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in separate polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced' together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques.Gene 05/1989; 77(1):61-8. · 2.20 Impact Factor
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ABSTRACT: The enzyme system catalyzing the formation of methyl-coenzyme M from methanol and coenzyme M in Methanosarcina barkeri is composed of the three different polypeptides MtaA, MtaB and MtaC of which MtaC harbors a corrinoid prosthetic group. The heterologous expression of mtaA and mtaB in Escherichia coli has been described previously. We report here on the overproduction of the apoprotein of MtaC in E. coli, on its reconstitution to the active holoprotein with either cob(II)alamin or methyl-cob(III)alamin, and on the properties of the reconstituted corrinoid protein. Reconstituted MtaC was found to contain 1 mol bound cobamide/mol. EPR spectroscopic evidence is presented for a His residue as an axial ligand to Co2+ of the bound corrinoid. This active-site His was identified by site-directed mutagenesis as His136 in the MtaC sequence that contains four His residues. The reconstituted MtaC, in the cob(I)amide oxidation state, was methylated with methanol in the presence of MtaB and demethylated with coenzyme M in the presence of MtaA. In the presence of both MtaB and MtaA, methyl-coenzyme M was formed from methanol and coenzyme M at specific rates comparable to those determined for the enzyme system purified from M. barkeri. M. barkeri contains an isoenzyme of MtaA designated MtbA. The isoenzyme reacted with MtaC with only 2.5% of the activity of MtaA.European Journal of Biochemistry 06/1998; 253(3):698-705. · 3.58 Impact Factor
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ABSTRACT: The effect of medium osmolarity on the morphology and growth of Methanosarcina barkeri, Methanosarcina thermophila, Methanosarcina mazei, Methanosarcina vacuolata, and Methanosarcina acetivorans was examined. Each strain was adapted for growth in NaCl concentrations ranging from 0.05 to 1.0 M. Methanosarcina spp. isolated from both marine and nonmarine sources exhibited similar growth characteristics at all NaCl concentrations tested, demonstrating that these species are capable of adapting to a similar range of medium osmolarities. Concomitant with the adaptation in 0.4 to 1.0 M NaCl, all strains disaggregated and grew as single cells rather than in the characteristic multicellular aggregates. Aggregated cells had a methanochondroitin outer layer, while disaggregated single cells lacked the outer layer but retained the protein S-layer adjacent to the cell membrane. Synthesis of glucuronic acid, a major component of methanochondroitin, was reduced 20-fold in the single-cell form of M. barkeri when compared with synthesis in aggregated cells. Strains with the methanochondroitin outer cell layer exhibited enhanced stability at low (<0.2 M NaCl) osmolarity and grew at higher temperatures. Disaggregated cells could be converted back to aggregated cells by gradually readapting cultures to lower NaCl (<0.2 M) and Mg (<0.005 M) concentrations. Disaggregated Methanosarcina spp. could also be colonized and replica plated with greater than 95% recovery rates on solidified agar basal medium that contained 0.4 to 0.6 M NaCl and either trimethylamine, methanol, or acetate as the substrate. The ability to disaggregate and grow Methanosarcina spp. as viable, detergent-sensitive, single cells on agar medium makes these species amenable to mutant selection and screening for genetic studies and enables cells to be gently lysed for the isolation of intact genetic material.Applied and Environmental Microbiology 11/1993; 59(11):3832-9. · 3.68 Impact Factor