BACKGROUND
methanosarcina acetivorans strain c2a is a mesophilic anaerobic archaean isolated from a kelp-degrading enrichment of marine origin  <cit> .
it is one of the more metabolically versatile methanogens in that it can use acetate as well as one-carbon substrates including mono-methylamine, di-methylamine, tri-methyl amine, methanol, or carbon monoxide as a sole source of carbon and energy.
as a result, it contributes to the formation of two green house gases, methane and carbon dioxide during the natural recycling of organic carbon in anaerobic environments.
the biochemical pathways for carbon flow from the alternative substrates to methane are reasonably well established  <cit> .
however, little is yet known about the expression of the genes encoding the described pathway enzymes or accessory proteins needed for electron and carbon flow.
additionally, the genome contains seemingly redundant copies of many other genes with implied roles in carbon or energy metabolism  <cit> .
for example, m. acetivorans possesses four gene clusters annotated for formylmethanofuran dehydrogenase, three gene sets annotated for hydrogenase, five distinct clusters of genes encoding membrane-bound and/or soluble-type heterodisulfide reductase enzymes, and two gene clusters encoding distinct membrane bound atp sythase complexes.
orthologs of many of these genes are present in other described methanosarcinaceae species including m. acetivorans, m. mazei, and m. barkeri , plus in other methanogenic species.
athe presence/absence of the corresponding genes/enzymes in the three genomes are indicated by y  or n .
for a complete inventory of all m. acetivorans genes and designations listed see figures 1- <dig>
the expression and/or physiological roles of many of these genes are either poorly understood or unknown.
initial genomic and proteomic studies with m. acetivorans and m. mazei have initially addressed this but did not clearly resolve these questions due in part to dna/protein sequence similarities and/or detection limits of the methods used  <cit> .
additionally, these approaches did not quantitatively address how mrna abundance levels vary during the alternative cell growth conditions.
in the present study we address the above questions using m. acetivorans as a model system to examine gene expression in response to substrate availability.
using quantitative pcr and supporting molecular methods, the resulting data establish expression levels of genes for over twenty enzymes/enzyme complexes for carbon flow and/or energy conservation.
the resulting findings define two major substrate-specific gene families for acetate and methanol utilization for this model organism.
these studies also lay a foundation to purse the molecular basis of central catabolic pathway gene regulation in this major class of methanogenic archaea.
RESULTS
gene redundancy in the m. acetivorans genome
the m. acetivorans genome contains many seemingly redundant copies of genes annotated with roles in methanogenesis  <cit> .
these include two sets of genes annotated for a molybdate-type formylmethanofuran dehydrogenase , and two gene sets for a tunsten-type formylmethanofuran dehydrogenase , five heterodisulfide reductase-like hdred and hdrabc gene clusters for reduction of coenzyme m-coenzyme b heterodisulfide, two sets of vht genes for f <dig> non-reducing hydrogenase, and two sets of genes for atp synthesizing complexes  <cit> .
additional genes include frh hydrogenase-like genes, plus additional genes for rnf- and mrp-type membrane associated bacterial electron transfer complexes, plus genes needed for acetate metabolism .
homologous and seemingly "redundant" genes/gene sets are also found in the genomes of m. mazei, and m. barkeri .
the reason for these genome makeups is currently unknown.
m. acetivorans was used as a model microorganism to evaluate expression of over twenty sets of genes using gene specific primer pairs designed to eliminate cross-hybridization when dna sequence similarity exists .
rt-pcr, ppcr, and 5' analysis was then performed using rna isolated from m. acetivorans cells grown with either acetate or methanol as the sole source of carbon and energy.
in this study, a number of new m. acetivorans gene designations were established to distinguish among homologous orfs .
formylmethanofuran dehydrogenase  gene expression
two of the four previously annotated sets of genes for formylmethanofuran dehydrogenasethese were designated as molybdenum-type enzymes and are named here as fmde1f1a1c1d1b <dig> and fmdf2a2c2d2b <dig> .
two additional gene sets were annotated as tungsten-type formylmethanofuran dehydrogenase, and are designated here as fwdd1b1a1c <dig> and fwdg2b2d <dig> .
using qpcr analysis methods , the molybdenum-type operon reporter genes fmde <dig> and fmda <dig>  were shown to be expressed at 14-fold higher levels during methanol growth conditions relative to acetate growth .
the second set of reporter genes  were expressed about 2-fold higher during these conditions, but the maximal level of expression was less than 5% of that seen for the fmde <dig> and fmda1genes.
noteworthy, the fmde <dig> and fmda <dig> gene expression values were within the same range observed for the fpon and fpol genes that encode subunits of the f <dig> h <dig> dehydrogenase needed for central pathway electron transfer functions .
the high transcript abundance of the fmde1f1a1c1d1b <dig> gene cluster implies a major role of this gene set during methanogenesis in contrast to that for the fmd <dig> gene set.
the annotated tungsten containing formylmethanofuran dehydrogenase gene cluster fwdd1b1a1c <dig> reporter genes designated fwdb <dig> and fwda <dig>  were also expressed 15-fold higher levels during methanol growth relative to acetate .
interestingly, this was within the magnitude observed for the fmde1f1a1c1d1b <dig> gene cluster.
however, the second tungsten-type gene cluster , was constitutively expressed and at a level about one-half of that observed for either fwda <dig> or fwdb <dig>  these fmd/fwd transcript abundance measurements clearly demonstrate that two of the four fmd and fwd gene clusters  are highly transcribed in response to substrate availability, and furthermore this suggests that two distinct formylmethanofuran dehydrogenase activities are concurrently utilized during methanol growth conditions .
heterodisulfide reductase gene expression
m. acetivorans genome analysis revealed five genes/gene clusters annotated as heterodisulfide reductase, an enzyme essential for electron transfer from methanogenic electron donors to methyl-com reductase .
these include genes for a membrane-type protein designated here as hdre <dig>  hdrd <dig> and hdrd <dig> similar to those needed for methane formation in m. barkeri  <cit> .
an additional six genes encoding soluble-type heterodisulfide reductase proteins are also present in the genome.
they include the hdra <dig> gene associated with a poly-ferredoxin-like gene , an unlinked set of hdrcb genes called hdrc1and hdrb <dig>  and a third hdr gene cluster designated hdra <dig> hdrc <dig> hdrb <dig> .
quantitative gene expression experiments  revealed that the membrane-type hdrd <dig> gene was most highly expressed during acetate cell growth conditions, and where methanol conditions gave slightly lower transcript abundance .
in contrast, hdrd <dig> gene expression was very low , suggesting a minor or no direct function in methanogenesis.
interestingly, the abundance of the soluble-type hdra1pfd and hdrc1b <dig> gene transcripts were also nearly as high as for the membrane type hdred <dig> genes .
here, acetate growth gave three-fold higher hdra <dig> transcript levels versus methanol growth conditions.
the participation of a soluble-type hdrabc enzyme in m. acetivorans metabolism is currently unknown but must now be considered.
an orf following the hdra <dig> gene is annotated as a polyferredoxin , and this suggests a role for this protein in electron transfer to couple the soluble-type hdr complex with an appropriate electron donor complex.
in contrast, hdra <dig> and hdrb <dig> transcript abundance was about two to twenty-fold lower under the corresponding conditions.
this suggests a minor role for the second set of hdrabc-type genes  in methanogenesis.
the hdra1pfd and hdrc1b1genes for the soluble-type enzyme subunits are located at different chromosomal loci, and are coordinately expressed since their mrna abundance levels are alike .
additionally, the pcr-based gene experiments also demonstrate that the hdra1pfd and the hdred <dig> genes are each expressed as operons .
taken together, these data are consistent with a need for both a membrane-type and a soluble type hdr enzyme for electron transfer/energy conservation under acetate and methanol cell growth conditions.
this suggests that distinct electron transfer pathways are operating to service the alternative hdr enzymes.
the vht and frh gene clusters
the m. acetivorans genome lacks an echabcdef gene cluster encoding an ech-type hydrogenase with described roles in hydrogen uptake and ion translocation in m. mazei  <cit> .
since m. acetivorans cells do not exhibit significant hydrogenase activity  <cit> , some other mechanism must provide a means for electron transfer from cellular donor to hdr.
interestingly, the m. acetivorans genome contains two sets of genes  for f420-nonreducing hydrogenase-types .
it also contains a set of frhadgb genes for a coenzyme f420-type hydrogenase .
quantitative rt-pcr assays  established that the vhtg <dig> and vhtc1genes were each expressed at four- to six-fold higher levels during methanol growth conditions, and this is within the range seen for the fpol and fpon genes needed for methyl group oxidation for methanol and acetate metabolism.
in contrast, expression of the vhtg <dig> and vhtc <dig> genes was low under all conditions examined .
finally, the frha and frhb gene expression levels were low relative to vhtg <dig> or fpol , and this suggests a minor role for the frhadgb and vhtg2a2c <dig> gene clusters in either methanol or acetate-dependent cell growth.
since vhtg <dig> transcript abundance was elevated and about half of that observed for the fpol and fpon genes that encode subunits of the f <dig> h <dig> dehydrogenase , this implies a significant physiological role for the vhtg1a1c1d <dig> gene products during methanol growth.
the biochemical and physiological roles for the vhtg <dig> and vhtc <dig> hydrogenase-type genes in m. acetivorans are presently unknown.
the rnfxcdgeaby gene cluster is abundantly expressed
m. acetivorans contains a set of six genes  annotated as nqr <dig>  <cit>  that are absent in the m. mazei, and m. barkeri genomes .
these genes were subsequently re-designated rnfcdgeab based on sequence comparisons to the rnf and nqr-type genes in other microorganisms,  <cit> .
this gene cluster also contains two additional genes of unknown function that we designate here as rnfx and rnfy  whereby the first  precedes rnfc and the second  follows rnfb.
we propose that these genes may encode unique input/output modules for membrane associated electron transfer since they are absent in other microbial genomes.
during acetate cell growth relative to methanol growth conditions, the rnfx, rnfg, and rnfa reporter genes exhibited elevated transcript abundance .
each gene was also more highly expressed than many reference genes involved in central methanogenesis .
therefore, the rnfxcdgeaby gene expression data support the proposal that the products participate in electron transfer during acetate metabolism as proposed via methanophenazine  <cit> .
in addition, they must also function during methanol culture conditions based on transcript abundance .
other roles can be envisioned including participation in electron transfer to a soluble-type heterodisulfide reductase via a poly-ferredoxin .
the mrpabcdefg gene cluster is acetate induced
the m. acetivorans genome contains a set of seven genes called mrpabcdefg  with similarity to the gene clusters found in a variety of bacterial species but absent in either m. barkeri or m. mazei   <cit> .
the mrp-encoded protein complex in bacillus subtilis was shown to confer a role in multiple drug resistance and/or ph regulation  <cit> .
as revealed by the m. acetivorans transcript analysis studies , the mrpa and mrpf reporter genes were expressed more highly during acetate cell growth conditions  relative to methanol growth.
these levels were above the expression levels observed for the ack, pta, and hdr genes needed for acetate utilization, and within the range seen for the rnf gene cluster.
these findings imply a major role for the six mrp gene products in acetate metabolism versus methanol metabolism.
expression of the atp and aha genes encoding atp synthase complexes
m. acetivorans contains genes for a bacterial-type f0f <dig> synthase encoded by the ma <dig> to ma <dig> genes designated here as atpdcihbefag, plus an archaeal-type a0a <dig> atp synthase encoded by the ahahikecfabd genes  .
although prior dna microarray experiments  <cit>  demonstrated that six of the nine genes in the archaeal-type a0a <dig> atp synthase  encoding the atp-hydrolysing/synthesizing domain  were expressed two-fold higher in acetate grown cells relative to methanol, the other genes were not  <cit> .
it is still unknown how their expression varies quantitatively relative to atpdcihbefag gene cluster expression.
corresponding dna microarray studies with the atpdcihbefag genes that encode a bacterial-like f0f <dig> complex revealed that only two of the nine genes  were expressed significantly higher in acetate by  <dig>  and  <dig>  fold, respectively: the remaining genes were either not detected or did not exhibit changes.
lastly, relative to central pathway genes for acetate and methanol utilization, it was unresolved how the aha and atp gene sets are expressed since the microarray data did not address this.
from the rt-pct transcript abundance studies, three representative aha genes representing the archaeal-type a0a <dig> atp synthase genes were highly expressed relative to the atp reporter genes .
acetate cell growth conditions resulted in two-fold higher aha transcript levels relative to methanol cell growth.
these genes were the most highly expressed in the cell regardless of the growth condition.
in contrast, the bacterial-type f0f <dig> atpd, atpa and atpg genes were expressed at less than 2% of the level seen for the ahai, ahac and ahab genes: this suggests a minor role for the atp genes in methanogenesis in contrast to the aha gene cluster.
acetate-induced genes
one m. acetivorans gene of unknown function  was revealed by our prior dna microarray studies to be more highly expressed during acetate growth conditions relative to methanol cell growth .
inspection of the amino acid sequence revealed six trans-membrane spanning regions reminiscent of a membrane solute uptake system .
to extend these ma <dig> gene expression findings, quantitative pcr experiments were performed .
ma <dig> was expressed at a 125-fold higher level during acetate versus methanol cell growth conditions.
interestingly, when methanol was also present in the culture medium in addition to acetate, ma <dig> expression was suppressed to a level seen when only methanol was present .
this indicates that the ma <dig> gene is expressed only when the energetically superior carbon substrate is absent, consistent with a proposed role in acetate uptake.
the m. acetivorans ma <dig> orf is designated acep for its role in an acetate-dependent membrane function.
two other genes required for acetate utilization are ack  and pta  that encode acetate kinase and phosphoacetyl transferase, respectively .
quantitative pcr experiments  established that both genes were highly expressed and at levels similar to acep when acetate was the sole substrate.
the 11-18-fold differential pta and ack gene expression findings are similar to previous reports in m. acetivorans and m. thermophila  <cit> .
location of the fpop, hdre, hdra <dig>  mrpa, pta, acep, and ahaa promoters
the mrna 5' ends of the fpopabcdhijjklmno, hdred <dig>  hdra1-pfd, mrpabcdefg, pta ack, acep and ahahikecfabd genes/clusters were determined to locate their corresponding promoter elements.
using primer extension methods , all but one of the promoter elements were demonstrated to have long un-translated regions  that range from  <dig> to  <dig> nucleotides in length.
for example, the acep 5' mrna end is located  <dig> nucleotides upstream of the translational start site.
similar findings were seen for the mrpa, fpop, ahah, hdre, and hdra genes.
only the pta gene had a relatively short utr .
we did not detect mrna 5' ends for either rnfx or hdrc <dig>  alignment of all the upstream regions of these promoter elements  revealed the highly conserved sequence present in other archaeal promoters, the tata box  located approximately 20- <dig> nt upstream of the + <dig> mrna start site .
this site is bound by the tbp protein that aids rna polymerase binding  <cit> .
in contrast, the bre box elements were not well conserved.
when the utr elements and the upstream regions were further examined using a suite of bioinformatics tools , no clearly discernable dna sequence elements with either dyad symmetry or direct repeats were found.
similarly, no conserved regions within the rna utr's were seen for the coordinately expressed hdra1pfd and hdrc1b <dig> genes sets.
discussion
prior microarray and proteomic experiments reported transcript/protein ratios for a subset of the m. acetivorans genes addressed in this study  <cit> .
however, by the limitations of the methods used, these studies did not provide expression ratios for many other key methanogenic pathway genes nor did they report information for other genes with potential roles in cell energy generation.
therefore quantitative pcr gene expression studies were undertaken here using m. acetivorans as a model to organism to examine which of the seemingly redundant gene copies in methanosarcina species are utilized during growth on the alternative methanogenic substrates, acetate and methanol.
as a result, we may interpret the resulting data as a readout of cell commitment to make rna.
from these experiments six points are readily apparent.
first, this study establishes the simultaneously high levels of gene expression for both a molybdenum-type  and a tungsten-type  formyl methanofuran dehydrogenase enzyme in m. acetivorans .
in contrast, the fmd <dig> and fwd <dig> gene clusters were not.
the co-expression of the fmd <dig> and fwd <dig> gene clusters during routine cell culture suggest that both tungsten and molybdate oxyanions are limiting during cell growth.
alternatively, the cell may somehow require the two gene sets to catalyze different reactions in methanogenic metabolism.
studies of the methanobacterium wolfei and methanobacterium thermoautotrophicum enzymes indicate that a tungsten-containing isoenzyme was constitutively expressed and that a molydate-containing isoenzyme was induced by molybdate ions  <cit> .
studies are in progress to establish if one or both of these oxyanion-metals modulate expression of the m. acetivorans fwd <dig> and/or fmd <dig> gene clusters.
the m. acetivorans expression findings predict that the homologous fmd <dig> and the fwd1-type gene clusters in m. barkeri and in m. mazei are used to make the major formyl methanofuran dehydrogenase enzymes .
interestingly, the m. barkeri genome lacks the annotated fwd <dig> tungsten-type enzyme.
second, all sequenced methanosarcina genomes contain multiple hdr genes encoding a membrane-type as well as a soluble-type heterodisulfide reductase .
based on the transcript abundance studies in m. acetivorans, the membrane-type hdr complex encoded by the hdred <dig> genes was the most abundantly expressed gene cluster .
this is consistent with the biochemical role for the membrane bound enzyme in m. barkeri  <cit> .
however, given the high transcript levels for the hdra <dig> and hdrb <dig> genes in cells grown with either acetate or methanol, a physiological role is hereby predicted for a soluble-type hdrabc heterodisulfide reductase in m. acetivorans metabolism, and by inference, in m. mazei and m barkeri.
the presence of a poly-ferredoxin-like gene immediately downstream of the hdra <dig> gene  provides one candidate for electron transfer from primary electron donors  to this hdr soluble-type enzyme .
transcript abundance for both the hdred <dig> and hdra1b <dig> genes were within the same magnitude observed for the fpon and fpol genes  that encode subunits of the f <dig> h <dig> dehydrogenase needed for central carbon flow to carbon dioxide.
since genes for both a membrane-type and a soluble-type hdr enzyme are co-expressed, this suggests that multiple pathways exist for electron transfer and/or energy conservation in m. acetivorans.
by inference, the homologous hdra pfd and hdrc1b1gene sets in m. barkeri and m. mazei are also highly expressed and operative.
the energetic implication for having distinct hdr-type enzymes is unknown.
possibilities include adaptation to different substrate levels and/or alternative modes of energy conservation  <cit> .
third, regarding the m. acetivorans sets of frh, vhtg <dig>  and vhtg <dig> genes , plus the two electron transfer complexes encoded by rnfxcdgeaby and mrpabcdefg genes , only the vhtg <dig>  rnf and mrp gene sets were abundantly expressed.
the vhtg1a1c1d1gene cluster encoding a methanophenazine-linked type hydrogenase was expressed at four- to six-fold higher levels during methanol growth conditions, and within the range seen for the fpol and fpon genes needed for methyl group oxidation for methanol and acetate metabolism.
this is also in the range seen for methanol-dependent fmda <dig>  and fwda <dig> expression .
in contrast, no vht gene expression was detected in m. acetivorans when a vht-uida promoter assay system was used  <cit> .
whether the high vhtg <dig> and vhtc <dig> mrna levels detected here  versus the low values by the vht-uida promoter assay is due to strain differences, cell growth, and/or in the analytical methods used is unknown.
the vhtg1a1c <dig> like-hydrogenase genes are conserved among the three methanosarcina strains  where a more complex gene expression pattern is evident across these species  <cit> .
interestingly, the m. acetivorans vht mrna expression pattern was similar to that seen in m. mazei  <cit> , and a physiological role is implied for the m. acetivorans vht genes.
the rnf and mrp gene clusters are unique to the metabolism of m. acetivorans since related gene clusters are absent in either of the m. mazei and m. barkeri genomes .
as noted by li, the rnfxcdgeaby gene products are logical candidates to fulfill the role of the ech-type hydrogenases present in m. mazei and m. barkeri  <cit> .
by this scheme, the rnf complex would accept electrons derived from the carbon monoxide dehydrogenase  complex via an associated ferredoxin encoded by the complex.
the membrane associated rnf-type complex is then proposed to transfer electrons on to the membrane associated methanophenazine cofactor  that in turn is reoxidized by a membrane-type heterodisulfide reductase .
from the hdr transcript studies , this enzyme would be encoded by the hdred <dig> gene set since hdrd <dig> expression was low.
by an alternative model, one might envision a role for the rnf complex in transferring electrons to the soluble heterodisulfide reductase complex encoded by the hdra <dig> pfd and hdrc1b <dig> genes via protein-protein interactions.
the poly-ferredoxin encoded by pfd  from the soluble-type heterodisulfide gene cluster is one candidate to interact with one of the unique rnf complex proteins such as rnfx or rnfy.
either model is compatible with the essentiality for rnf based on the effect of an rnf deletion strain that is unable to grow with acetate as a sole carbon supply.
little biochemical data exist to distinguish among these possibilities.
based on the role of the mrp complex in cytoplasmic ph homeostasis in bacillus halodurans, a similar function was proposed for the m. acetivorans mrp-like complex  <cit> .
both belong to the group i class of proteins and exhibit similar gene compositions and gene order  <cit> .
interestingly, several alternative roles have been suggested for the bacterial mrp genes and include exchange of another type of mono-valent ion, in detoxification, and in interactions with another cellular enzyme to form a membrane complex somehow associated with cellular ion partitioning  <cit> .
a role for the m. acetivorans gene products in cytoplasmic ph homeostasis or the other above roles would make it distinct from other methanosarcina species since related mrp genes are absent in the other sequenced genomes .
in this regard, phenotypic analysis of m. acetivorans mrp mutants will be of special interest.
the high similarity of the m. acetivorans mrp genes relative to those in the bacteria, suggest an origin in the methanogen by lateral gene transfer event from a group i organism.
do the m. acetivorans mrp and rnf transcript abundance data provide additional clues about the roles of either mrp or rnf?
the genes for both are among the most highly expressed in the cell , where the mrp gene expression pattern is similar to levels for the ack and pta genes needed for acetate utilization .
the 8-fold higher mrp expression level relative to methanol growth approximates the 8- <dig> fold seen for the ack and pta genes  in support of a primary role in acetate-dependent metabolism, rather than in detoxification and/or ion homeostasis.
in contrast, a second pattern of gene expression is seen for the central pathway genes involved in one carbon oxidations  that are all more highly expressed by  <dig> to  <dig> fold when methanol is the sole substrate .
a third set of genes required for both acetate and methanol metabolism are differentially expressed at an intermediate level .
the rnf gene expression pattern  falls in this group.
it is interesting to speculate that some of these genes may be controlled in response to electron flow rather than the carbon supply .
forth, the quantitative atpase gene expression studies demonstrate that the archaeal-type a0a <dig> atp synthase encoded by the ahahikecfabd genes are among the most highly expressed genes in the cell .
in contrast, transcript abundance for the bacteria type atpdcihbefag genes was about 175-fold lower than these aha cluster genes during either acetate or methanol growth.
although it is conceivable that the atp-type genes may be significantly expressed under unknown growth conditions, an alternative possibility is that they constitute a "dead-ended" lateral gene transfer event  <cit> .
interestingly, the deletion of the atp gene region of m. acetivorans conferred no phenotype  <cit> .
the atpx gene present in the m. acetivorans and m. barkeri genomes is conserved in some, but not all bacterial-like atp synthase operons.
it is present in the rhodoferax ferrireducens dsm  <dig>  desulfuromonas acetoxidans dsm  <dig> and shewanella frigidimarina ncimb genomes .
since the synteny of atpx in the above operons is conserved, atpx is not due to an isolated insertion event in the m. acetivorans genome.
biochemical studies have identified essential amino acids involved in translocation of sodium ions by the proteolipid c subunit of the ilyobacter tartaricus atpase  <cit> .
to address whether na+ or h+ ions are transported by the m. acetivorans archaeal-type a0a <dig> atp synthase, the ahak gene encoding proteolipid c subunit was aligned with the corresponding subunits of i. tartaricus plus other well studied microorganisms .
four amino acid residues at positions  <dig>   <dig>   <dig>  and  <dig> in the i. tartaricus protein to specify na+ ion movement  <cit> .
these four residues are conserved in m. acetivorans, in contrast to e. coli that is a proton translocating enzyme.
this suggests the archaeal-type a0a <dig> atp synthase also transfers na+ ions rather than protons to form atp, in keeping with the example of pyrococcus furiosus  <cit> .
furthermore, the archaeal type ahak subunit in the three methanosarcina strains form a distinct protein subclass given the presence of an additional three amino acids relative to position  <dig> of the i. tartaricus subunit, and a three amino acid deletion corresponding to position 47- <dig> of i. tartaricus.
amino acid alignments of the a0a <dig> atp synthases subunits from the m. mazei and m. barkeri proteolipids suggest the same conclusion for these highly related archaeal complexes .
interestingly, the alignment of the c proteolipid subunit  of the m. acetivorans bacterial-type f0f <dig> synthase also suggests specificity for na+ ions.
a neighbor-joining tree of the archaeal and bacterial c-type polypeptides  reveals a relatively conserved origin of the archaeal-type a0a <dig> atp synthase in the methanosarcina species.
strikingly, the bacterial-type f0f <dig> synthase genes present in m. acetivorans and m. barkeri are more distantly related to either the archaeal or bacterial type enzymes.
this branch of atp metabolism genes/proteins remains poorly understood and awaits further study.
fifth, a candidate gene encoding a potential acetate uptake system for m. acetivorans was identified .
this gene exhibits the same expression patterns as the ack and pta genes needed for activation of the methanogenic substrate following its entry into the cell.
expression of acep was suppressed by the energetically favorable substrate, methanol .
the acep protein is predicted to have six transmembrane-spanning alpha-helical regions .
noteworthy, acep homologs are present in other methanogens including m. mazei, m. barkeri, m. maripaludis, and m. hungatei, and they constitute a distinct class of archaea transporters.
related genes are also present in many bacterial species , suggesting the possibility of a lateral gene transfer event from a bacterium into the methanosarcina sp.
as was proposed as one explanation for their large genome sizes  <cit> .
experiments are in progress to characterize the membrane function of the m. acetivorans protein since no archaeal or bacterial homologs shown in additional file  <dig>  figure s <dig> have been examined to date.
carbon control in the archaea
considerable information is available concerning carbon control of gene expression in bacterial and eukaryal systems, but little is yet known about related carbon control in the archaea.
few studies have been reported for any archaeal species but include microarray studies in pyrococcus furiosus  <cit> , m. mazei  <cit> , and m. acetivorans  <cit> .
the present experiments extend these studies to address a larger set of genes needed for carbon flow and electron transfer leading to methane formation from two key methanogenic substrates .
it provides a foundation of rna transcript abundance and 5' end data to begin exploring regulatory controls in this organism at the level of regulated mrna synthesis and turnover.
little is known about the relative contributions of archaea transcription factors, translation factors, and/or small rna's in gene regulation in the methanosarcina species to provide the distinct patterns of gene expression observed here.
m. acetivorans clearly maintains a cellular commitment to dynamically control transcript levels in response to methanogenic substrate type where two major gene families are further defined by this study.
CONCLUSIONS
of the twenty m. acetivorans gene clusters examined in this study, all but four were differentially expressed by  <dig> to 200-fold during acetate versus methanol cell growth .
the majority of these queried genes are present all sequenced methanosarcina genomes that include m. acetivorans, m. mazei and m. barkeri  and include the genes for multiple heterodisulfide reductase and hydrogenase-like enzymes.
exceptions are the echabcdef, vhogac, rnfxcdgeaby, and mrpabcdefg genes that encode known or predicted electron transfer complexes for ion movement and/or electron transfer.
a number of the above orfs are assigned new gene designations to aid in their description.
the m. acetivorans gene expression data  provides a foundation to understand how energy-yielding pathways are regulated in this model organism and in related methanogens.
it is unknown if this control occurs by the actions of classical transcription factors like those found in bacteria and eukaryotes, and/or by rna control mechanisms involving attenuation, regulated termination and/or small rnas.

 methanosarcina acetivorans strain c2a is a mesophilic anaerobic archaean isolated from a kelp-degrading enrichment of marine origin. it is one of the more metabolically versatile methanogens in that it can use acetate as well as one-carbon substrates including mono-methylamine, di-methylamine, tri-methyl amine, methanol, or carbon monoxide as a sole source of carbon and energy. as a result, it contributes to the formation of two green house gases, methane and carbon dioxide during the natural recycling of organic carbon in anaerobic environments. the biochemical pathways for carbon flow from the alternative substrates to methane are reasonably well established. however, little is yet known about the expression of the genes encoding the described pathway enzymes or accessory proteins needed for electron and carbon flow. additionally, the genome contains seemingly redundant copies of many other genes with implied roles in carbon or energy metabolism. for example, m. acetivorans possesses four gene clusters annotated for formylmethanofuran dehydrogenase, three gene sets annotated for hydrogenase, five distinct clusters of genes encoding membrane-bound and/or soluble-type heterodisulfide reductase enzymes, and two gene clusters encoding distinct membrane bound atp sythase complexes. orthologs of many of these genes are present in other described methanosarcinaceae species including m. acetivorans, m. mazei, and m. barkeri, plus in other methanogenic species. athe presence/absence of the corresponding genes/enzymes in the three genomes are indicated by y or n. for a complete inventory of all m. acetivorans genes and designations listed see figures 1- the expression and/or physiological roles of many of these genes are either poorly understood or unknown. initial genomic and proteomic studies with m. acetivorans and m. mazei have initially addressed this but did not clearly resolve these questions due in part to dna/protein sequence similarities and/or detection limits of the methods used. additionally, these approaches did not quantitatively address how mrna abundance levels vary during the alternative cell growth conditions. in the present study we address the above questions using m. acetivorans as a model system to examine gene expression in response to substrate availability. using quantitative pcr and supporting molecular methods, the resulting data establish expression levels of genes for over twenty enzymes/enzyme complexes for carbon flow and/or energy conservation. the resulting findings define two major substrate-specific gene families for acetate and methanol utilization for this model organism. these studies also lay a foundation to purse the molecular basis of central catabolic pathway gene regulation in this major class of methanogenic archaea.  gene redundancy in the m. acetivorans genome the m. acetivorans genome contains many seemingly redundant copies of genes annotated with roles in methanogenesis. these include two sets of genes annotated for a molybdate-type formylmethanofuran dehydrogenase, and two gene sets for a tunsten-type formylmethanofuran dehydrogenase, five heterodisulfide reductase-like hdred and hdrabc gene clusters for reduction of coenzyme m-coenzyme b heterodisulfide, two sets of vht genes for f non-reducing hydrogenase, and two sets of genes for atp synthesizing complexes. additional genes include frh hydrogenase-like genes, plus additional genes for rnf- and mrp-type membrane associated bacterial electron transfer complexes, plus genes needed for acetate metabolism. homologous and seemingly "redundant" genes/gene sets are also found in the genomes of m. mazei, and m. barkeri. the reason for these genome makeups is currently unknown. m. acetivorans was used as a model microorganism to evaluate expression of over twenty sets of genes using gene specific primer pairs designed to eliminate cross-hybridization when dna sequence similarity exists. rt-pcr, ppcr, and 5' analysis was then performed using rna isolated from m. acetivorans cells grown with either acetate or methanol as the sole source of carbon and energy. in this study, a number of new m. acetivorans gene designations were established to distinguish among homologous orfs. formylmethanofuran dehydrogenase gene expression two of the four previously annotated sets of genes for formylmethanofuran dehydrogenasethese were designated as molybdenum-type enzymes and are named here as fmde1f1a1c1d1b and fmdf2a2c2d2b. two additional gene sets were annotated as tungsten-type formylmethanofuran dehydrogenase, and are designated here as fwdd1b1a1c and fwdg2b2d. using qpcr analysis methods, the molybdenum-type operon reporter genes fmde and fmda were shown to be expressed at 14-fold higher levels during methanol growth conditions relative to acetate growth. the second set of reporter genes were expressed about 2-fold higher during these conditions, but the maximal level of expression was less than 5% of that seen for the fmde and fmda1genes. noteworthy, the fmde and fmda gene expression values were within the same range observed for the fpon and fpol genes that encode subunits of the f h dehydrogenase needed for central pathway electron transfer functions. the high transcript abundance of the fmde1f1a1c1d1b gene cluster implies a major role of this gene set during methanogenesis in contrast to that for the fmd gene set. the annotated tungsten containing formylmethanofuran dehydrogenase gene cluster fwdd1b1a1c reporter genes designated fwdb and fwda were also expressed 15-fold higher levels during methanol growth relative to acetate. interestingly, this was within the magnitude observed for the fmde1f1a1c1d1b gene cluster. however, the second tungsten-type gene cluster, was constitutively expressed and at a level about one-half of that observed for either fwda or fwdb these fmd/fwd transcript abundance measurements clearly demonstrate that two of the four fmd and fwd gene clusters are highly transcribed in response to substrate availability, and furthermore this suggests that two distinct formylmethanofuran dehydrogenase activities are concurrently utilized during methanol growth conditions. heterodisulfide reductase gene expression m. acetivorans genome analysis revealed five genes/gene clusters annotated as heterodisulfide reductase, an enzyme essential for electron transfer from methanogenic electron donors to methyl-com reductase. these include genes for a membrane-type protein designated here as hdre hdrd and hdrd similar to those needed for methane formation in m. barkeri. an additional six genes encoding soluble-type heterodisulfide reductase proteins are also present in the genome. they include the hdra gene associated with a poly-ferredoxin-like gene, an unlinked set of hdrcb genes called hdrc1and hdrb and a third hdr gene cluster designated hdra hdrc hdrb. quantitative gene expression experiments revealed that the membrane-type hdrd gene was most highly expressed during acetate cell growth conditions, and where methanol conditions gave slightly lower transcript abundance. in contrast, hdrd gene expression was very low, suggesting a minor or no direct function in methanogenesis. interestingly, the abundance of the soluble-type hdra1pfd and hdrc1b gene transcripts were also nearly as high as for the membrane type hdred genes. here, acetate growth gave three-fold higher hdra transcript levels versus methanol growth conditions. the participation of a soluble-type hdrabc enzyme in m. acetivorans metabolism is currently unknown but must now be considered. an orf following the hdra gene is annotated as a polyferredoxin, and this suggests a role for this protein in electron transfer to couple the soluble-type hdr complex with an appropriate electron donor complex. in contrast, hdra and hdrb transcript abundance was about two to twenty-fold lower under the corresponding conditions. this suggests a minor role for the second set of hdrabc-type genes in methanogenesis. the hdra1pfd and hdrc1b1genes for the soluble-type enzyme subunits are located at different chromosomal loci, and are coordinately expressed since their mrna abundance levels are alike. additionally, the pcr-based gene experiments also demonstrate that the hdra1pfd and the hdred genes are each expressed as operons. taken together, these data are consistent with a need for both a membrane-type and a soluble type hdr enzyme for electron transfer/energy conservation under acetate and methanol cell growth conditions. this suggests that distinct electron transfer pathways are operating to service the alternative hdr enzymes. the vht and frh gene clusters the m. acetivorans genome lacks an echabcdef gene cluster encoding an ech-type hydrogenase with described roles in hydrogen uptake and ion translocation in m. mazei. since m. acetivorans cells do not exhibit significant hydrogenase activity, some other mechanism must provide a means for electron transfer from cellular donor to hdr. interestingly, the m. acetivorans genome contains two sets of genes for f420-nonreducing hydrogenase-types. it also contains a set of frhadgb genes for a coenzyme f420-type hydrogenase. quantitative rt-pcr assays established that the vhtg and vhtc1genes were each expressed at four- to six-fold higher levels during methanol growth conditions, and this is within the range seen for the fpol and fpon genes needed for methyl group oxidation for methanol and acetate metabolism. in contrast, expression of the vhtg and vhtc genes was low under all conditions examined. finally, the frha and frhb gene expression levels were low relative to vhtg or fpol, and this suggests a minor role for the frhadgb and vhtg2a2c gene clusters in either methanol or acetate-dependent cell growth. since vhtg transcript abundance was elevated and about half of that observed for the fpol and fpon genes that encode subunits of the f h dehydrogenase, this implies a significant physiological role for the vhtg1a1c1d gene products during methanol growth. the biochemical and physiological roles for the vhtg and vhtc hydrogenase-type genes in m. acetivorans are presently unknown. the rnfxcdgeaby gene cluster is abundantly expressed m. acetivorans contains a set of six genes annotated as nqr that are absent in the m. mazei, and m. barkeri genomes. these genes were subsequently re-designated rnfcdgeab based on sequence comparisons to the rnf and nqr-type genes in other microorganisms,. this gene cluster also contains two additional genes of unknown function that we designate here as rnfx and rnfy whereby the first precedes rnfc and the second follows rnfb. we propose that these genes may encode unique input/output modules for membrane associated electron transfer since they are absent in other microbial genomes. during acetate cell growth relative to methanol growth conditions, the rnfx, rnfg, and rnfa reporter genes exhibited elevated transcript abundance. each gene was also more highly expressed than many reference genes involved in central methanogenesis. therefore, the rnfxcdgeaby gene expression data support the proposal that the products participate in electron transfer during acetate metabolism as proposed via methanophenazine. in addition, they must also function during methanol culture conditions based on transcript abundance. other roles can be envisioned including participation in electron transfer to a soluble-type heterodisulfide reductase via a poly-ferredoxin. the mrpabcdefg gene cluster is acetate induced the m. acetivorans genome contains a set of seven genes called mrpabcdefg with similarity to the gene clusters found in a variety of bacterial species but absent in either m. barkeri or m. mazei. the mrp-encoded protein complex in bacillus subtilis was shown to confer a role in multiple drug resistance and/or ph regulation. as revealed by the m. acetivorans transcript analysis studies, the mrpa and mrpf reporter genes were expressed more highly during acetate cell growth conditions relative to methanol growth. these levels were above the expression levels observed for the ack, pta, and hdr genes needed for acetate utilization, and within the range seen for the rnf gene cluster. these findings imply a major role for the six mrp gene products in acetate metabolism versus methanol metabolism. expression of the atp and aha genes encoding atp synthase complexes m. acetivorans contains genes for a bacterial-type f0f synthase encoded by the ma to ma genes designated here as atpdcihbefag, plus an archaeal-type a0a atp synthase encoded by the ahahikecfabd genes. although prior dna microarray experiments demonstrated that six of the nine genes in the archaeal-type a0a atp synthase encoding the atp-hydrolysing/synthesizing domain were expressed two-fold higher in acetate grown cells relative to methanol, the other genes were not. it is still unknown how their expression varies quantitatively relative to atpdcihbefag gene cluster expression. corresponding dna microarray studies with the atpdcihbefag genes that encode a bacterial-like f0f complex revealed that only two of the nine genes were expressed significantly higher in acetate by and fold, respectively: the remaining genes were either not detected or did not exhibit changes. lastly, relative to central pathway genes for acetate and methanol utilization, it was unresolved how the aha and atp gene sets are expressed since the microarray data did not address this. from the rt-pct transcript abundance studies, three representative aha genes representing the archaeal-type a0a atp synthase genes were highly expressed relative to the atp reporter genes. acetate cell growth conditions resulted in two-fold higher aha transcript levels relative to methanol cell growth. these genes were the most highly expressed in the cell regardless of the growth condition. in contrast, the bacterial-type f0f atpd, atpa and atpg genes were expressed at less than 2% of the level seen for the ahai, ahac and ahab genes: this suggests a minor role for the atp genes in methanogenesis in contrast to the aha gene cluster. acetate-induced genes one m. acetivorans gene of unknown function was revealed by our prior dna microarray studies to be more highly expressed during acetate growth conditions relative to methanol cell growth. inspection of the amino acid sequence revealed six trans-membrane spanning regions reminiscent of a membrane solute uptake system. to extend these ma gene expression findings, quantitative pcr experiments were performed. ma was expressed at a 125-fold higher level during acetate versus methanol cell growth conditions. interestingly, when methanol was also present in the culture medium in addition to acetate, ma expression was suppressed to a level seen when only methanol was present. this indicates that the ma gene is expressed only when the energetically superior carbon substrate is absent, consistent with a proposed role in acetate uptake. the m. acetivorans ma orf is designated acep for its role in an acetate-dependent membrane function. two other genes required for acetate utilization are ack and pta that encode acetate kinase and phosphoacetyl transferase, respectively. quantitative pcr experiments established that both genes were highly expressed and at levels similar to acep when acetate was the sole substrate. the 11-18-fold differential pta and ack gene expression findings are similar to previous reports in m. acetivorans and m. thermophila. location of the fpop, hdre, hdra mrpa, pta, acep, and ahaa promoters the mrna 5' ends of the fpopabcdhijjklmno, hdred hdra1-pfd, mrpabcdefg, pta ack, acep and ahahikecfabd genes/clusters were determined to locate their corresponding promoter elements. using primer extension methods, all but one of the promoter elements were demonstrated to have long un-translated regions that range from to nucleotides in length. for example, the acep 5' mrna end is located nucleotides upstream of the translational start site. similar findings were seen for the mrpa, fpop, ahah, hdre, and hdra genes. only the pta gene had a relatively short utr. we did not detect mrna 5' ends for either rnfx or hdrc alignment of all the upstream regions of these promoter elements revealed the highly conserved sequence present in other archaeal promoters, the tata box located approximately 20- nt upstream of the + mrna start site. this site is bound by the tbp protein that aids rna polymerase binding. in contrast, the bre box elements were not well conserved. when the utr elements and the upstream regions were further examined using a suite of bioinformatics tools, no clearly discernable dna sequence elements with either dyad symmetry or direct repeats were found. similarly, no conserved regions within the rna utr's were seen for the coordinately expressed hdra1pfd and hdrc1b genes sets. discussion prior microarray and proteomic experiments reported transcript/protein ratios for a subset of the m. acetivorans genes addressed in this study. however, by the limitations of the methods used, these studies did not provide expression ratios for many other key methanogenic pathway genes nor did they report information for other genes with potential roles in cell energy generation. therefore quantitative pcr gene expression studies were undertaken here using m. acetivorans as a model to organism to examine which of the seemingly redundant gene copies in methanosarcina species are utilized during growth on the alternative methanogenic substrates, acetate and methanol. as a result, we may interpret the resulting data as a readout of cell commitment to make rna. from these experiments six points are readily apparent. first, this study establishes the simultaneously high levels of gene expression for both a molybdenum-type and a tungsten-type formyl methanofuran dehydrogenase enzyme in m. acetivorans. in contrast, the fmd and fwd gene clusters were not. the co-expression of the fmd and fwd gene clusters during routine cell culture suggest that both tungsten and molybdate oxyanions are limiting during cell growth. alternatively, the cell may somehow require the two gene sets to catalyze different reactions in methanogenic metabolism. studies of the methanobacterium wolfei and methanobacterium thermoautotrophicum enzymes indicate that a tungsten-containing isoenzyme was constitutively expressed and that a molydate-containing isoenzyme was induced by molybdate ions. studies are in progress to establish if one or both of these oxyanion-metals modulate expression of the m. acetivorans fwd and/or fmd gene clusters. the m. acetivorans expression findings predict that the homologous fmd and the fwd1-type gene clusters in m. barkeri and in m. mazei are used to make the major formyl methanofuran dehydrogenase enzymes. interestingly, the m. barkeri genome lacks the annotated fwd tungsten-type enzyme. second, all sequenced methanosarcina genomes contain multiple hdr genes encoding a membrane-type as well as a soluble-type heterodisulfide reductase. based on the transcript abundance studies in m. acetivorans, the membrane-type hdr complex encoded by the hdred genes was the most abundantly expressed gene cluster. this is consistent with the biochemical role for the membrane bound enzyme in m. barkeri. however, given the high transcript levels for the hdra and hdrb genes in cells grown with either acetate or methanol, a physiological role is hereby predicted for a soluble-type hdrabc heterodisulfide reductase in m. acetivorans metabolism, and by inference, in m. mazei and m barkeri. the presence of a poly-ferredoxin-like gene immediately downstream of the hdra gene provides one candidate for electron transfer from primary electron donors to this hdr soluble-type enzyme. transcript abundance for both the hdred and hdra1b genes were within the same magnitude observed for the fpon and fpol genes that encode subunits of the f h dehydrogenase needed for central carbon flow to carbon dioxide. since genes for both a membrane-type and a soluble-type hdr enzyme are co-expressed, this suggests that multiple pathways exist for electron transfer and/or energy conservation in m. acetivorans. by inference, the homologous hdra pfd and hdrc1b1gene sets in m. barkeri and m. mazei are also highly expressed and operative. the energetic implication for having distinct hdr-type enzymes is unknown. possibilities include adaptation to different substrate levels and/or alternative modes of energy conservation. third, regarding the m. acetivorans sets of frh, vhtg and vhtg genes, plus the two electron transfer complexes encoded by rnfxcdgeaby and mrpabcdefg genes, only the vhtg rnf and mrp gene sets were abundantly expressed. the vhtg1a1c1d1gene cluster encoding a methanophenazine-linked type hydrogenase was expressed at four- to six-fold higher levels during methanol growth conditions, and within the range seen for the fpol and fpon genes needed for methyl group oxidation for methanol and acetate metabolism. this is also in the range seen for methanol-dependent fmda and fwda expression. in contrast, no vht gene expression was detected in m. acetivorans when a vht-uida promoter assay system was used. whether the high vhtg and vhtc mrna levels detected here versus the low values by the vht-uida promoter assay is due to strain differences, cell growth, and/or in the analytical methods used is unknown. the vhtg1a1c like-hydrogenase genes are conserved among the three methanosarcina strains where a more complex gene expression pattern is evident across these species. interestingly, the m. acetivorans vht mrna expression pattern was similar to that seen in m. mazei, and a physiological role is implied for the m. acetivorans vht genes. the rnf and mrp gene clusters are unique to the metabolism of m. acetivorans since related gene clusters are absent in either of the m. mazei and m. barkeri genomes. as noted by li, the rnfxcdgeaby gene products are logical candidates to fulfill the role of the ech-type hydrogenases present in m. mazei and m. barkeri. by this scheme, the rnf complex would accept electrons derived from the carbon monoxide dehydrogenase complex via an associated ferredoxin encoded by the complex. the membrane associated rnf-type complex is then proposed to transfer electrons on to the membrane associated methanophenazine cofactor that in turn is reoxidized by a membrane-type heterodisulfide reductase. from the hdr transcript studies, this enzyme would be encoded by the hdred gene set since hdrd expression was low. by an alternative model, one might envision a role for the rnf complex in transferring electrons to the soluble heterodisulfide reductase complex encoded by the hdra pfd and hdrc1b genes via protein-protein interactions. the poly-ferredoxin encoded by pfd from the soluble-type heterodisulfide gene cluster is one candidate to interact with one of the unique rnf complex proteins such as rnfx or rnfy. either model is compatible with the essentiality for rnf based on the effect of an rnf deletion strain that is unable to grow with acetate as a sole carbon supply. little biochemical data exist to distinguish among these possibilities. based on the role of the mrp complex in cytoplasmic ph homeostasis in bacillus halodurans, a similar function was proposed for the m. acetivorans mrp-like complex. both belong to the group i class of proteins and exhibit similar gene compositions and gene order. interestingly, several alternative roles have been suggested for the bacterial mrp genes and include exchange of another type of mono-valent ion, in detoxification, and in interactions with another cellular enzyme to form a membrane complex somehow associated with cellular ion partitioning. a role for the m. acetivorans gene products in cytoplasmic ph homeostasis or the other above roles would make it distinct from other methanosarcina species since related mrp genes are absent in the other sequenced genomes. in this regard, phenotypic analysis of m. acetivorans mrp mutants will be of special interest. the high similarity of the m. acetivorans mrp genes relative to those in the bacteria, suggest an origin in the methanogen by lateral gene transfer event from a group i organism. do the m. acetivorans mrp and rnf transcript abundance data provide additional clues about the roles of either mrp or rnf? the genes for both are among the most highly expressed in the cell, where the mrp gene expression pattern is similar to levels for the ack and pta genes needed for acetate utilization. the 8-fold higher mrp expression level relative to methanol growth approximates the 8- fold seen for the ack and pta genes in support of a primary role in acetate-dependent metabolism, rather than in detoxification and/or ion homeostasis. in contrast, a second pattern of gene expression is seen for the central pathway genes involved in one carbon oxidations that are all more highly expressed by to fold when methanol is the sole substrate. a third set of genes required for both acetate and methanol metabolism are differentially expressed at an intermediate level. the rnf gene expression pattern falls in this group. it is interesting to speculate that some of these genes may be controlled in response to electron flow rather than the carbon supply. forth, the quantitative atpase gene expression studies demonstrate that the archaeal-type a0a atp synthase encoded by the ahahikecfabd genes are among the most highly expressed genes in the cell. in contrast, transcript abundance for the bacteria type atpdcihbefag genes was about 175-fold lower than these aha cluster genes during either acetate or methanol growth. although it is conceivable that the atp-type genes may be significantly expressed under unknown growth conditions, an alternative possibility is that they constitute a "dead-ended" lateral gene transfer event. interestingly, the deletion of the atp gene region of m. acetivorans conferred no phenotype. the atpx gene present in the m. acetivorans and m. barkeri genomes is conserved in some, but not all bacterial-like atp synthase operons. it is present in the rhodoferax ferrireducens dsm desulfuromonas acetoxidans dsm and shewanella frigidimarina ncimb genomes. since the synteny of atpx in the above operons is conserved, atpx is not due to an isolated insertion event in the m. acetivorans genome. biochemical studies have identified essential amino acids involved in translocation of sodium ions by the proteolipid c subunit of the ilyobacter tartaricus atpase. to address whether na+ or h+ ions are transported by the m. acetivorans archaeal-type a0a atp synthase, the ahak gene encoding proteolipid c subunit was aligned with the corresponding subunits of i. tartaricus plus other well studied microorganisms. four amino acid residues at positions and in the i. tartaricus protein to specify na+ ion movement. these four residues are conserved in m. acetivorans, in contrast to e. coli that is a proton translocating enzyme. this suggests the archaeal-type a0a atp synthase also transfers na+ ions rather than protons to form atp, in keeping with the example of pyrococcus furiosus. furthermore, the archaeal type ahak subunit in the three methanosarcina strains form a distinct protein subclass given the presence of an additional three amino acids relative to position of the i. tartaricus subunit, and a three amino acid deletion corresponding to position 47- of i. tartaricus. amino acid alignments of the a0a atp synthases subunits from the m. mazei and m. barkeri proteolipids suggest the same conclusion for these highly related archaeal complexes. interestingly, the alignment of the c proteolipid subunit of the m. acetivorans bacterial-type f0f synthase also suggests specificity for na+ ions. a neighbor-joining tree of the archaeal and bacterial c-type polypeptides reveals a relatively conserved origin of the archaeal-type a0a atp synthase in the methanosarcina species. strikingly, the bacterial-type f0f synthase genes present in m. acetivorans and m. barkeri are more distantly related to either the archaeal or bacterial type enzymes. this branch of atp metabolism genes/proteins remains poorly understood and awaits further study. fifth, a candidate gene encoding a potential acetate uptake system for m. acetivorans was identified. this gene exhibits the same expression patterns as the ack and pta genes needed for activation of the methanogenic substrate following its entry into the cell. expression of acep was suppressed by the energetically favorable substrate, methanol. the acep protein is predicted to have six transmembrane-spanning alpha-helical regions. noteworthy, acep homologs are present in other methanogens including m. mazei, m. barkeri, m. maripaludis, and m. hungatei, and they constitute a distinct class of archaea transporters. related genes are also present in many bacterial species, suggesting the possibility of a lateral gene transfer event from a bacterium into the methanosarcina sp. as was proposed as one explanation for their large genome sizes. experiments are in progress to characterize the membrane function of the m. acetivorans protein since no archaeal or bacterial homologs shown in additional file figure s have been examined to date. carbon control in the archaea considerable information is available concerning carbon control of gene expression in bacterial and eukaryal systems, but little is yet known about related carbon control in the archaea. few studies have been reported for any archaeal species but include microarray studies in pyrococcus furiosus, m. mazei, and m. acetivorans. the present experiments extend these studies to address a larger set of genes needed for carbon flow and electron transfer leading to methane formation from two key methanogenic substrates. it provides a foundation of rna transcript abundance and 5' end data to begin exploring regulatory controls in this organism at the level of regulated mrna synthesis and turnover. little is known about the relative contributions of archaea transcription factors, translation factors, and/or small rna's in gene regulation in the methanosarcina species to provide the distinct patterns of gene expression observed here. m. acetivorans clearly maintains a cellular commitment to dynamically control transcript levels in response to methanogenic substrate type where two major gene families are further defined by this study.  of the twenty m. acetivorans gene clusters examined in this study, all but four were differentially expressed by to 200-fold during acetate versus methanol cell growth. the majority of these queried genes are present all sequenced methanosarcina genomes that include m. acetivorans, m. mazei and m. barkeri and include the genes for multiple heterodisulfide reductase and hydrogenase-like enzymes. exceptions are the echabcdef, vhogac, rnfxcdgeaby, and mrpabcdefg genes that encode known or predicted electron transfer complexes for ion movement and/or electron transfer. a number of the above orfs are assigned new gene designations to aid in their description. the m. acetivorans gene expression data provides a foundation to understand how energy-yielding pathways are regulated in this model organism and in related methanogens. it is unknown if this control occurs by the actions of classical transcription factors like those found in bacteria and eukaryotes, and/or by rna control mechanisms involving attenuation, regulated termination and/or small rnas. 
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. 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. 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. the above findings establish the differential and coordinated expression of two major gene families in m. acetivorans in response to carbon/energy supply. 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. analysis of promoters for seven gene clusters reveal utr leaders of 51- nucleotides in length, raising the possibility of both transcriptional and translational levels of control. furthermore, the quantitative mrna measurements demonstrate the dynamic range for modulating transcript abundance. these include genes for soluble-type and membrane-type heterodisulfide reductases, hydrogenases including genes for a vht-type f non-reducing hydrogenase, molybdenum-type as well as tungsten-type 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.
 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.  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, hydrogenases including genes for a vht-type f non-reducing hydrogenase, molybdenum-type as well as tungsten-type 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- nucleotides in length, raising the possibility of both transcriptional and translational levels of control.  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. 