Bose A, Metcalf WW.. Distinct regulators control the expression of methanol methyltransferase isozymes in Methanosarcina acetivorans C2A. Mol Microbiol 67: 649-661

Department of Microbiology, University of Illinois at Urbana-Champaign, B103 CLSL, 601 S. Goodwin, Urbana, IL 61801, USA.
Molecular Microbiology (Impact Factor: 4.42). 03/2008; 67(3):649-61. DOI: 10.1111/j.1365-2958.2007.06075.x
Source: PubMed


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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    • "It was found that in the case of MsrA and MsrB, both proteins act in concert to upregulate expression of MtaCB1, and knockout of either can prevent expression [73]. Similarly, knockout of either msrD or msrE will prevent expression of MtaCB2 [73]. MsrD and to a lesser extent MsrE also repress MtaCB3 [73]. "
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    ABSTRACT: Progress towards a complete model of the methanogenic archaeum Methanosarcina acetivorans is reported. We characterized size distribution of the cells using differential interference contrast microscopy, finding them to be ellipsoidal with mean length and width of 2.9 μ m and 2.3 μ m, respectively, when grown on methanol and 30% smaller when grown on acetate. We used the single molecule pull down (SiMPull) technique to measure average copy number of the Mcr complex and ribosomes. A kinetic model for the methanogenesis pathways based on biochemical studies and recent metabolic reconstructions for several related methanogens is presented. In this model, 26 reactions in the methanogenesis pathways are coupled to a cell mass production reaction that updates enzyme concentrations. RNA expression data (RNA-seq) measured for cell cultures grown on acetate and methanol is used to estimate relative protein production per mole of ATP consumed. The model captures the experimentally observed methane production rates for cells growing on methanol and is most sensitive to the number of methyl-coenzyme-M reductase (Mcr) and methyl-tetrahydromethanopterin:coenzyme-M methyltransferase (Mtr) proteins. A draft transcriptional regulation network based on known interactions is proposed which we intend to integrate with the kinetic model to allow dynamic regulation.
    Archaea 03/2014; 2014(4):898453. DOI:10.1155/2014/898453 · 2.71 Impact Factor
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    • "A plating procedure for growth of M. acetivorans on an agar-free surface, which involves filtration of cells onto nitrocellulose filters and incubating them on media-soaked filter paper, has been established [19] and would have to be used in conjunction with pMG3 to obtain colonies not producing Bla in the absence of methanol. Presently, five trans-acting factors, MsrA, MsrB, MsrC, MsrD, and MsrE, are known to regulate expression of the genes encoding three methanol-specific MT1 isoforms MtaCB1, MtaCB2, and MtaCB3 on the level of transcription initiation [45]. The methanol-specific MT1 isoforms are also regulated at the posttranscriptional level [46] but the factor(s) involved are unknown. "
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    ABSTRACT: The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β -galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β -lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β -lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.
    Archaea 01/2014; 2014(9):725610. DOI:10.1155/2014/725610 · 2.71 Impact Factor
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    • "by an uncharacterized mechanism that was likely either early transcriptional termination or endoribonuclease activity against the 5′ UTR [61]. In M. acetivorans, 5′ UTRs are also known to be involved in regulating the expression of different isozymes of methanol specific methyltransferases [62]. "
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    ABSTRACT: Methane-producing Archaea are of interest due to their contribution to atmospheric change and for their roles in technological applications including waste treatment and biofuel production. Although restricted to anaerobic environments, methanogens are found in a wide variety of habitats, where they commonly live in syntrophic relationships with bacterial partners. Owing to tight thermodynamic constraints of methanogenesis alone or in syntrophic metabolism, methanogens must carefully regulate their catabolic pathways including the regulation of RNA transcripts. The transcriptome is a dynamic and important control point in microbial systems. This paper assesses the impact of mRNA (transcriptome) studies on the understanding of methanogenesis with special consideration given to how methanogenesis is regulated to cope with nutrient limitation, environmental variability, and interactions with syntrophic partners. In comparison with traditional microarray-based transcriptome analyses, next-generation high-throughput RNA sequencing is greatly advantageous in assessing transcription start sites, the extent of 5' untranslated regions, operonic structure, and the presence of small RNAs. We are still in the early stages of understanding RNA regulation but it is already clear that determinants beyond transcript abundance are highly relevant to the lifestyles of methanogens, requiring further study.
    Archaea 02/2013; 2013(4):586369. DOI:10.1155/2013/586369 · 2.71 Impact Factor
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