Carbon-dependent control of electron transfer and central carbon pathway genes for methane biosynthesis in the Archaean, Methanosarcina acetivorans strain C2A

Abstract

Background: The archaeon, Methanosarcina acetivorans strain C2A forms methane, a potent greenhouse gas, from a variety of one-carbon substrates and acetate. Whereas the biochemical pathways leading to methane formation are well understood, little is known about the expression of the many of the genes that encode proteins needed for carbon flow, electron transfer and/or energy conservation. Quantitative transcript analysis was performed on twenty gene clusters encompassing over one hundred genes in M. acetivorans that encode enzymes/proteins with known or potential roles in substrate conversion to methane.

Results: The expression of many seemingly "redundant" genes/gene clusters establish substrate dependent control of approximately seventy genes for methane production by the pathways for methanol and acetate utilization. These include genes for soluble-type and membrane-type heterodisulfide reductases (hdr), hydrogenases including genes for a vht-type F420 non-reducing hydrogenase, molybdenum-type (fmd) as well as tungsten-type (fwd) formylmethanofuran dehydrogenases, genes for rnf and mrp-type electron transfer complexes, for acetate uptake, plus multiple genes for aha- and atp-type ATP synthesis complexes. Analysis of promoters for seven gene clusters reveal UTR leaders of 51-137 nucleotides in length, raising the possibility of both transcriptional and translational levels of control.

Conclusions: The above findings establish the differential and coordinated expression of two major gene families in M. acetivorans in response to carbon/energy supply. Furthermore, the quantitative mRNA measurements demonstrate the dynamic range for modulating transcript abundance. Since many of these gene clusters in M. acetivorans are also present in other Methanosarcina species including M. mazei, and in M. barkeri, these findings provide a basis for predicting related control in these environmentally significant methanogens.

Figure 1

Differential expression of genes annotated for fmd and fwd in M. acetivorans. Panel A) the six and five gene fmdE1F1A1C1D1B1 and fmdF2A2C2D2B2 clusters encoding the two putative molybdenum-type formylmethanofuran dehydrogenase enzyme complexes. Panel B) the four and three gene fwdD1B1A1C1 and fwdG2B2D2 clusters encoding the two putative tungsten-type formylmethanofuran dehydrogenase enzyme complexes. The Genebank identification number (MA number) is shown below each gene while the individual gene designation is shown above. Panel C) RT-PCR data for the indicated fmd and fwd genes. Values are expressed as copy number (Methods).

Figure 2

Differential expression of genes in M. acetivorans annotated for hdr (hetero-disulfide reductase). Panel A) Genes encoding the putative membrane-type hetero-disulfide reductase subunits, hdrED1 and hdrD2. Panel B) Genes encoding the putative soluble-type hetero-disulfide reductase subunits, hdrA1 pfd, hdrC1B1, and hdrA2C2B2. The Genebank identification number (MA number) is shown below each gene while the individual gene designation is shown above. Panel C) RT-PCR data for the indicated hdr genes.

Figure 3

Differential expression of genes annotated for vht (F420 non-reducing hydrogenase) and frhADGB (F420 reducing hydrogenase) in M. acetivorans. Panel A) The genes encoding the frhADGB F420 reducing hydrogenase subunits. Panel B) The genes encoding the vhtG1A1C1D1 and the vhtG2A2C2 F420 non-reducing hydrogenases. The Genebank identification number (MA number) is shown below each gene while the individual gene designation is shown above. Panel C) RT-PCR data for the indicated genes.

Figure 4

Differential expression of genes related to electron transport in M. acetivorans. The orientation and relative length of each gene is indicated by the open arrows. The Genebank identification number (MA number) is shown below each gene. Panels: A) The eight gene rnf cluster; B) the seven gene mrp cluster; C) the fourteen gene fpo cluster; and D), RT-PCR data for the indicated rnf, mrp, and fpo genes.

Figure 5

Expression of the atpDCIHBEFAG and the ahaHIKECFABD gene clusters encoding the bacterial-type and the archaea-type ATP synthase complexes of M. acetivorans, respectively. The Genebank identification number (MA number), and individual gene designation are shown above or below each gene. Panel C shows RT-PCR data for the indicated atp and aha gene clusters.

Figure 6

Differential expression of genes induced in presence of acetate. Panel A) The indicated genes include ack (acetate kinase), pta (phosphoacetyl transferase), and a gene designated aceP encoding a putative acetate uptake system. The RT-PCR data were determined as described in Materials. Panel B) Transcript abundance for aceP from cells grown in the presence or absence of the methanogenic substrate, methanol with the indicated amounts of acetate present.

Figure 7

Location of the mRNA 5'ends for the hdrE1, hdrA1, mrpA, fpoP, pta, aceP, and ahaA genes. Top panel; Sequence gels for the mrpA, fpoP, ahaA and aceP genes along with the corresponding DNA ladders. RNA prepared from methanol or from acetate-grown cells is indicated by Me and Ac, respectively. Bottom panel: the alignment of the upstream DNA sequences relative to the start of transcription (+1 position). The position of the initiation codon is boxed where the numbering is relative to the start of transcription. The putative TATA-box sequences are double underlined and the BRE-regions are indicated by a solid underline. The mRNA 5' end positions for the pta, hdrA1, and hdrE1 genes were determined with a ubiquitous ladder (data not shown).

Figure 8

Overview of differential gene expression in M. acetivorans in response to methanol versus acetate utilization. A boxed number indicates the fold-increase in mRNA levels seen for the indicated gene(s) during acetate versus methanol growth conditions. A circled number indicates the fold-increase in mRNA levels during methanol versus acetate growth conditions. All data are from this study except for the mcr, mtr, mer, mtd, mch, and ftr gene ratio data derived from a prior microarray study [6]. The genes/enzymes are: ack, acetate kinase; pta, phosphotransacetylase; cdh, carbon monoxide dehydrogenase; MT1, mtaB2 methyl transferase 1; MT2, mtaA, methyltransferase 2; mcr, methylcoenzyme M reductase; mtr, methyl -H4 MPT:HSCoM methyltransferase; mer, methylene -H4 MPT reductase; hmd, methylene -H4 MPT dehydrogenase; mch, methenyl -H4 MPT cyclohydrolyase; ftr, formyl MFR:H4MPT formyl transferase; fmd, formyl methanofuran dehydrogenase Mo-type; fwd, formyl methanofuran dehydrogenase W-type; fpo, F420 H2 dehydrogenase; hdr, heterodisulfide reductase; rnf, Rnf-type complex; mrp, Mrp-type complex. The control gene was MA3998. Methanophenazine is represented by MPH. The proposed acetate transporter protein is indicated by AceP while the unknown transporter(s) for one carbon compounds is indicated by a question mark.

Figure 9

Phylogenic tree of the atp and aha ATP synthase proteolipid subunit c for the methanogens M. acetivorans, M. mazei, and M. barkeri, and for the bacterial homologs indicated in reference [26]. The predicted or experimentally determined ion transferred is indicated from data provided in Additional file 2, Figure S2.