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J Bacteriol. 2010 March; 192(6): 1511–1517.
Published online 2010 January 14. doi:  10.1128/JB.01165-09
PMCID: PMC2832540

Functional Analysis of the Three TATA Binding Protein Homologs in Methanosarcina acetivorans[down-pointing small open triangle]

Abstract

The roles of three TATA binding protein (TBP) homologs (TBP1, TBP2, and TBP3) in the archaeon Methanosarcina acetivorans were investigated by using genetic and molecular approaches. Although tbp2 and tbp3 deletion mutants were readily obtained, a tbp1 mutant was not obtained, and the growth of a conditional tbp1 expression strain was tetracycline dependent, indicating that TBP1 is essential. Transcripts of tbp1 were 20-fold more abundant than transcripts of tbp2 and 100- to 200-fold more abundant than transcripts of tbp3, suggesting that TBP1 is the primary TBP utilized during growth. Accordingly, tbp1 is strictly conserved in the genomes of Methanosarcina species. Δtbp3 and Δtbp2 strains exhibited an extended lag phase compared with the wild type, although the lag phase for the Δtbp2 strain was less pronounced when this strain was transitioning from growth on methylotrophic substrates to growth on acetate. Acetate-adapted Δtbp3 cells exhibited growth rates, final growth yields, and lag times that were significantly reduced compared with those of the wild type when the organisms were cultured with growth-limiting concentrations of acetate, and the acetate-adapted Δtbp2 strain exhibited a final growth yield that was reduced compared with that of the wild type when the organisms were cultured with growth-limiting acetate concentrations. DNA microarray analyses identified 92 and 77 genes with altered transcription in the Δtbp2 and Δtbp3 strains, respectively, which is consistent with a role for TBP2 and TBP3 in optimizing gene expression. Together, the results suggest that TBP2 and TBP3 are required for efficient growth under conditions similar to the conditions in the native environment of M. acetivorans.

The basal transcription machinery of the Archaea resembles that of the RNA polymerase II system in the Eukarya domain that includes two essential general transcription factors, TATA binding protein (TBP) and transcription factor B (TFB) (3-5, 16, 25, 42). In order to establish promoter-directed transcription, TBP binds a TATA box located ~25 bp upstream of the transcription start site. The binding of TBP to the promoter allows TFB to bind at sites both upstream and downstream of the TATA box. TFB then recruits RNA polymerase to the promoter to establish the preinitiation complex, which is followed by transcription (2).

Genes encoding multiple homologs of TBP and/or TFB have been identified in the genomes of several species in the domain Archaea. It has been proposed that the function of the homologs is to direct gene-specific transcription, analogous to the function of alternative σ factors specific to the domain Bacteria (1). Experimental evidence that supports this hypothesis has been reported for the archaeon Halobacterium sp. NRC-1, which contains six tbp genes and seven tfb genes (9, 12). A comprehensive systems approach provided evidence suggesting that there is global gene regulation by specific TFB-TBP pairs (12). In another study, two different mutants in which there was deletion of either the tbpD or tfbA homolog exhibited coordinated downregulation of 363 genes compared with the parental strain, further supporting the hypothesis that there is global gene regulation by specific TFB-TBP pairs (9). Many of the regulated genes are involved in the heat shock response, and one of the TFB homologs binds specifically to the promoter of the heat shock protein gene hsp5 (31). The functional roles of multiple TFB homologs in various hyperthermophilic archaeal species have been investigated as well (33, 35, 36). In contrast to the reports for Halobacterium, none of these reports presented direct evidence that there is differentially gene regulation in vivo by the different TFB homologs. One factor complicating the study of Halobacterium sp. NRC-1 is the presence of multiple homologs of both TBP and TFB, which gives this organism the opportunity to exploit as many as 42 different TBP-TFB combinations for recruitment of RNA polymerase to specific promoters. However, this is not the case for Methanosarcina acetivorans C2A, a genetically tractable methanogen with genome annotations for three homologs of TBP and only a single TFB (32, 39, 40), which provides an opportunity to study the role of multiple TBPs in the context of a single TFB. M. acetivorans is the only acetotrophic methanogen that has been isolated from a marine environment where it must compete for acetate with acetotrophic sulfate-reducing bacteria (23, 34, 37-39). Here we report the results obtained using molecular and genetic approaches to examine the roles of the three TBP homologs in M. acetivorans. These results show that TBP1 is essential and that TBP2 and TBP3 are not essential, although they have specialized roles during adaptation to acetate and during growth with growth-limiting acetate concentrations.

MATERIALS AND METHODS

Cell growth.

The methods used for growth and harvesting of M. acetivorans C2A (= DSM 800) as single cells (40) in high-salt (HS) broth with acetate, methanol, and trimethylamine (TMA) have been described previously (26). Growth was monitored by measuring the optical density at 600 nm. The acetate concentration was maintained at 75 to 100 mM by periodically supplementing cultures with 10 M glacial acetic acid from a sterile anaerobic stock solution. Acetic acid was added at a molar ratio of acetic acid to the methane produced of 1:1; the amount of methane produced was calculated based on the volume of gas overpressure released from the culture vessel headspace. The pH of cultures grown on acetate fluctuated from 6.5 to 7.5 during growth. Cultures with initial concentrations of sodium acetate of 20 and 50 mM were not supplemented with acetic acid during growth, and the pH at the end of growth did not exceed 7.5.

TaqMan quantitative reverse transcription-PCR (qRT-PCR).

TaqMan assays were performed essentially as described previously (26), using total RNA isolated at mid-exponential phase (A600, approximately 0.6 to 0.7, 0.4 to 0.5, and 0.6 to 0.7 for methanol-, acetate-, and TMA-grown cells, respectively). The TaqMan primers and probes used are listed in Table S2 in the supplemental material. The primers and probes used for 16S rRNA have been described previously (26). To determine the numbers of transcripts of tbp1, tbp2, and tbp3 for wild-type M. acetivorans, in vitro-transcribed tbp1, tbp2, and tbp3 RNA standards were generated using a MEGAscript T7 kit (Ambion, Austin, TX) according to the manufacturer's instructions. Linearized plasmids containing tbp1, tbp2, or tbp3 cloned into the pET21a vector under control of the T7 promoter were used as templates for the in vitro transcription reaction. Completed-reaction mixtures were treated with TURBO DNA-free DNase to remove template DNA and were then purified using an RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Product size and purity were checked by agarose gel electrophoresis. The RNA standards were diluted to obtain 1 × 10−11 copies μl−1. A standard curve was generated using the averages of triplicate measurements for 10-fold serial dilutions of the RNA standards. The cycle threshold (CT) values for triplicate measurements for serially diluted total RNA from methanol-grown cells plotted against the standard curve were used to calculate the number of transcript copies per nanogram for tbp1, tbp2, and tbp3.

Construction of M. acetivorans deletion mutant strains.

Liposome-mediated transformation and homologous recombination-mediated gene replacement were performed as described previously (6, 32) to generate M. acetivorans Δtbp2::pac, Δtbp3::pac, and PmcrB(tetO1)::tbp1 strains [designated the Δtbp2, Δtbp3, and ΔPtbp1::PmcrB(tetO1)-tbp1 strains, respectively]. The Δtbp2 and Δtbp3 strains were generated in an M. acetivorans C2A background, and the ΔPtbp1::PmcrB(tetO1)-tbp1 strain was generated in an M. acetivorans WWM75 background. Cells were transformed with 2 μg pMR18, pMR19, or pMR58 linearized by restriction digestion, and transformants were selected on HS agar media with 0.8% (wt/vol) agar, with either 250 mM methanol or 50 mM TMA, and with 2 μg ml−1 puromycin (Sigma, St. Louis, MO) from a sterile, anaerobic 100× stock solution. For generation of the ΔPtbp1::PmcrB(tetO1)-tbp1 strain, 100 μg ml−1 tetracycline (Sigma, St, Louis, MO) was added to HS agar in addition to the other components.

Southern blot analysis.

Digoxigenin (DIG)-labeled probes were generated using a DIG High Prime I DNA labeling and detection starter kit (Roche) according to the manufacturer's guidelines. Probes 1, 2, and 3 were PCR amplified from M. acetivorans genomic DNA using primers TBP2up-F and TBP2up-R, primers TBP3up-F and TBP3up-R, and primers TBP1in-F and TBP1in-R, respectively (see Table S2 in the supplemental material). The positions of these probes on the M. acetivorans chromosome are indicated in Fig. S1A in the supplemental material. Aliquots of genomic DNA (1 to 5 μg) from the wild-type C2A, WWM75, Δtbp2, Δtbp3, and ΔPtbp1::PmcrB(tetO1)-tbp1 M. acetivorans strains were digested overnight with either NdeI, PvuII, or EcoRI, and fragments were separated on 0.6% (wt/vol) agarose gels, denatured, neutralized, and transferred to positively charged nylon membranes (Boehringer Mannheim). Membranes were hybridized with DIG-labeled probes by following the manufacturer's guidelines.

Adaptation to different methanogenic substrates.

Portions (10 ml) of early-stationary-phase wild-type and Δtbp2 or Δtbp3 cell cultures (A600, approximately 1.2, 0.9, and 1.3 for methanol-, acetate-, and TMA-grown cells, respectively) were pelleted by centrifugation anaerobically for 10 min at 5,000 × g. Cells were washed once with 10 ml medium without substrate and resuspended in 10 ml medium without substrate, and 1-ml portions were inoculated into 100 ml medium containing the new methanogenic substrate.

DNA microarray analysis.

RNA was collected from four replicate cultures each of the wild-type, Δtbp2, and Δtbp3 strains grown on methanol at mid-exponential phase (A600, ~0.6). The purity and concentration of RNA samples were checked using a NanoDrop spectrophotometer (Fisher Scientific, Pittsburg, PA), and RNA quality was assessed using an Agilent 6000 bioanalyzer nano RNA assay. RNA samples were labeled with a Message Amp II bacterial kit (Ambion Inc., Austin, TX) used according to the manufacturer's instructions, using aminoallyl UTP in place of biotin-modified nucleotides. Briefly, 500 ng of total RNA was polyadenylated and subsequently reverse transcribed with primer T7oligo(dT)VN. Second-strand cDNA synthesis was performed, and the resulting double-stranded DNA was used as a template for in vitro transcription. Aminoallyl-modified UTP was incorporated into the resulting complementary RNA. Twenty micrograms of this modified aminoallyl RNA was coupled with Cy3 or Cy5 using a CyDye postlabeling reactive dye pack (GE Healthcare, Buckinghamshire, England) according to the aminoallyl message Amp II kit instructions (Ambion, Austin, TX). Samples were hybridized to NimbleGen 4-plex arrays (catalog number A7240-00-01; design name 080303 MacetC2A EXP X4). Two-color hybridizations were performed, and 2 μg of each sample was hybridized to an array. Array hybridization, washing, scanning, and data acquisition were performed according to the manufacturer's instructions (Roche NimbleGen Inc., Madison, WI). All data analysis was carried out using the ArrayStar (v3) software package (DNAStar, Madison, WI). Robust multichip averaging (RMA) and quantile normalization (7, 21, 22) were applied to the entire data set, which consisted of four biological replicates and two technical replicates (dye swap) for each strain. Statistical analyses were carried out with the normalized data using a moderated t test with false discovery rate (FDR) multiple-test correction (Benjamini-Hochberg) to determine differential transcript abundance. Changes in transcript abundance were considered significant if they met the following criteria: ≥2-fold change in abundance, P value of <0.05, and log2 transcript abundance of >11.0 under the conditions with greater abundance. The Z test with FDR multiple-test correction (Benjamini-Hochberg) was used to assess the statistical significance of the sampling distribution of genes according to their COG functional categories.

RESULTS

Transcription of tbp1, tbp2, and tbp3.

The genome of M. acetivorans is annotated with genes encoding three TBP homologs (15) distinguished by sequence identity (see Table S1 in the supplemental material). In previous proteomics studies, peptides specific to TBP1, TBP2, and TBP3 were detected in cell lysates of methanol-, acetate-, and CO-grown M. acetivorans (26, 30). Transcript abundance was determined for each TBP gene by real-time quantitative reverse transcription-PCR (qRT-PCR) at mid-exponential phase during growth with methanol, acetate, or trimethylamine (TMA). The transcript abundance for tbp1 was ~20-fold greater than the transcript abundance for tbp2 in cells cultured with any of the three growth substrates, ~100-fold greater than the transcript abundance for tbp3 during growth on methanol or TMA, and ~200-fold greater than the transcript abundance for tbp3 during growth on acetate (Table (Table1).1). The transcript abundance for tbp2 was ~3-, 5-, and 10-fold greater than the transcript abundance for tbp3 during growth on methanol, TMA, and acetate, respectively (Table (Table1).1). The transcript abundance values for tbp1, tbp2, and tbp3 were also determined in early log phase and late log phase during growth on methanol. In each case, the ratio of tbp2 and tbp3 transcript abundance to tbp1 abundance remained the same (data not shown). Together, these results are consistent with undetermined physiological functions for each of the three TBPs.

TABLE 1.
Abundance of tbp1, tbp2, and tbp3 transcripts in M. acetivorans cultured with various substrates

Analysis of tbp mutant strains.

To assess the biological significance of each TBP homolog in M. acetivorans, we attempted to delete the three encoding genes individually by homologous recombination and selection for puromycin resistance in the presence of methanol in the growth medium. Mutant strains with either tbp2 or tbp3 deleted (the Δtbp2 and Δtbp3 strains) were obtained and confirmed by Southern hybridization (see Fig. S1 in the supplemental material), indicating that TBP2 and TBP3 are not essential for growth on methanol. The levels of the tbp2 and tbp3 transcripts in the Δtbp2 and Δtbp3 strains, respectively, were below the limits of detection by qRT-PCR, and the transcription of both of the remaining TBP genes in these strains was not affected (data not shown). Attempts to delete tbp1 by double crossover were unsuccessful. Therefore, a conditional tbp1 expression strain was constructed using a previously described approach (18). Instead of deleting the tbp1 coding sequence, we modified the promoter for tbp1 by replacing the native promoter (Ptbp1) with a chimeric version of the mcrB promoter that contains an operator sequence for the Tn10 TetR protein [PmcrB(tetO1)]. The Tn10 TetR protein gene is constitutively expressed from two remote sites on the chromosome. Transcription of PmcrB(tetO1) is repressed by the Tn10 TetR protein in the absence of tetracycline. Thus, in the strain obtained [ΔPtbp1::PmcrB(tetO1)-tbp1], tbp1 can be expressed only in the presence of tetracycline, and its growth was comparable to that of the parental strain in the presence of 100 μg ml−1 of tetracycline (Fig. (Fig.1).1). However, no growth was observed in the absence of tetracycline, indicating that tbp1 is essential for growth (Fig. (Fig.11).

FIG. 1.
Growth of the ΔPtbp1::PmcrB(tetO1)-tbp1 strain (bottom panel) and its parental strain, WWM75 (top panel). Strains were cultured with 50 mM TMA in the presence (filled circles) or absence (open circles) of 100 μg ml−1 of tetracycline. ...

To identify growth phenotypes associated with deletion of either tbp2 or tbp3, substrate-dependent cell growth was investigated by comparing the initial growth rates, the maximum A600 values, and the lag times needed to reach mid-log phase for the Δtbp2 and Δtbp3 strains with those of the wild type when the organisms were cultured with a substrate at initial concentrations of 250 mM for methanol, 100 mM for TMA, and 100 mM for acetate. The acetate concentration was then maintained in the range from 75 to 100 mM by periodic addition of acetic acid. The growth parameters of the Δtbp2 and Δtbp3 strains were indistinguishable from those of the wild-type strain when the organisms were cultured with the methylotrophic substrates methanol and TMA (data not shown). However, on average, the lag time until mid-log phase was reached for acetate-grown cultures of the Δtbp3 strain (25.7 ± 6.1 days; n = 30) inoculated with methanol-grown cells was 12 days longer than that for the wild-type strain (13.2 ± 3.7 days; n = 30) (Fig. (Fig.2A).2A). The Δtbp3 strain also exhibited a comparably longer lag time when it was transitioning from growth on TMA to growth on acetate (data not shown). Once the mutant strains were fully adapted to growth on acetate (after ≥30 generations of growth on acetate), the growth parameters were indistinguishable from those of the wild-type strain during culture with acetate maintained at a concentration of 75 to 100 mM (data not shown). These results suggest that TBP3 has a role in the transition from growth on methylotrophic substrates to growth on acetate maintained at concentrations between 75 and 100 mM. Although its phenotype was not as robust as the phenotype observed for the Δtbp3 strain, the Δtbp2 strain also showed a trend toward a lag phase longer than that of the wild-type strain (18.4 ± 4.2 days; n = 30) when it was transitioning from growth on methanol to growth on acetate (Fig. (Fig.2A2A).

FIG. 2.
Representative growth curves for the wild-type, Δtbp2, and Δtbp3 strains of M. acetivorans cultured with acetate (single replicates). (A) Growth with the acetate concentration maintained between 75 and 100 mM. The inocula were cultured ...

To examine the roles of TBPs under more ecologically relevant growth conditions, growth parameters of the Δtbp2 and Δtbp3 strains (Table (Table2)2) were determined with 20 and 50 mM acetate, concentrations that are nearer the acetate concentrations (~1 to 2 mM) in the native environment of M. acetivorans (23, 38, 39). In this experiment, the inocula were fully adapted to growth on acetate, and the cultures were not supplemented with acetic acid during growth. The growth rates and lag times for the Δtbp2 strain were comparable to those for the wild-type strain. However, the maximum A600 for the Δtbp2 strain was ~20% less than the maximum A600 for the wild-type strain during growth with 50 mM acetate, which is consistent with a role for TBP2 in efficient growth with growth-limiting acetate concentrations (Table (Table22 and Fig. 2B and C). For the Δtbp3 strain cultured with 50 mM acetate, the growth rates and yields were ~40% and ~30% less, respectively, than those for the wild-type strain, and the lag time was 6 days longer than the lag time for the wild-type strain (Table (Table22 and Fig. Fig.2B).2B). The growth defects of the Δtbp3 strain were more profound when it was cultured with 20 mM acetate. Seven replicate cultures of the Δtbp3 strain were prepared, all of which showed little detectable growth over the first 3 weeks of incubation (Fig. (Fig.2C).2C). However, 4 of these cultures eventually began to grow after this period. The growth rates and yields of these 4 cultures were significantly lower than the growth rates and yields of the wild-type cultures, and the lag time was 17 days longer than the lag time of the wild-type cultures (Table (Table2).2). There were no differences in any of the growth parameters between either mutant strain and the wild type when the organisms were cultured with limiting methanol concentrations (25 and 50 mM) (data not shown). These results indicate that TBP3 is important for optimal growth with limiting concentrations of acetate.

TABLE 2.
Growth parameters of M. acetivorans wild-type and mutant strains

Involvement of TBP2 and TBP3 in gene regulation.

It was anticipated that the defects in the growth of the two mutant strains compared with the wild type might be reflected in the gene expression profiles. Indeed, for the Δtbp2 and Δtbp3 strains we identified 92 and 77 genes, respectively, which exhibited a ≥2-fold change in transcript abundance compared with the wild type; several of these genes were present in both strains (Fig. (Fig.3;3; see Tables S3 and S4 in the supplemental material), which is consistent with a role for TBP2 and TBP3 in the regulation of gene expression. Interestingly, a substantial proportion of the genes in the Δtbp3 strain and the majority of the genes in the Δtbp2 strain exhibited increased transcript abundance compared with the transcript abundance in the wild type (Fig. (Fig.3;3; see Tables S3 and S4 in the supplemental material), suggesting that TBP2 and TBP3 may be involved in the negative regulation of numerous genes. Among the genes with increased transcript abundance in the Δtbp2 strain compared with the wild type, the genes encoding proteins in the amino acid transport and metabolism and inorganic ion transport and metabolism functional categories were highly overrepresented (P = 1.57 × 10−13 and P = 8.70 × 10−4, respectively) (see Tables S3 and S4 in the supplemental material). However, the biological significance of this trend is not known. Numerous other genes were identified that exhibited decreased transcript abundance in either the Δtbp2 or Δtbp3 strain compared with the wild type. Although none of these genes had functional annotations clearly linked to acetate metabolism (see Tables S3 and S4 in the supplemental material), genes that may provide added fitness under low-nutrient-availability conditions were identified. Of note were two genes encoding putative Hsp60 proteins (MA0857 and MA4386), both of which exhibited greatly decreased transcript abundance in both the Δtbp2 and Δtbp3 strains compared with the wild type and for which a larger decrease was observed for the Δtbp3 strain (see Tables S3 and S4 in the supplemental material), which is consistent with roles for TBP2 and TBP3 in the regulation of their transcription.

FIG. 3.
Distribution of genes for which there was a difference in transcript abundance between wild-type M. acetivorans and mutant strains. The left side and right sides of the Venn diagram show the numbers of genes whose transcript abundance changed only in ...

DISCUSSION

This study, the first genetic analysis of general transcription factors for a methane-producing archaeon, provided initial insight into the roles of the three TBP homologs in M. acetivorans.

TBP1 has a greater role than TBP2 or TBP3 in M. acetivorans.

We propose that in M. acetivorans TPB1 plays a greater role in gene expression than TBP2 and TBP3. This proposal is supported by the following observations: (i) transcripts of tbp1 were found to be substantially more abundant than transcripts of tbp2 and tbp3 in cells cultured on methanol, acetate, or TMA; (ii) the genes encoding either TBP2 or TBP3 could be knocked out by homologous recombination, and the strains obtained could be cultured with acetate and methylotrophic substrates; (iii) a Δtbp1 strain could not be obtained by homologous recombination-mediated gene replacement; and (iv) the ΔPtbp1::PmcrB(tetO1)-tbp1 strain was unable to grow in the absence of tetracycline, which inhibits the expression of tbp1. Accordingly, genes encoding TBP1 orthologs are strictly conserved among Methanosarcina species (see Table S1 in the supplemental material). The genome of Methanosarcina mazei Gö1 (10) contains two orthologs of tbp1 (MM1028 and MM1027), and the gene products exhibit ≥96% amino acid sequence identity to TBP1 of M. acetivorans. Despite this high level of identity to TBP1, the annotations for the proteins encoded by these genes are TBP1 (MM1028) and TBP2 (MM1027). In the interest of keeping a uniform nomenclature, we suggest here that these proteins be renamed TBP1a (MM1027) and TBP1b (MM1028). The genome of Methanosarcina barkeri Fusaro (available at www.tigr.org) also contains a tbp1 gene, whose product exhibits 97% amino acid sequence identity to TBP1 of M. acetivorans. The TBP1 protein from M. acetivorans exhibits 88 and 80% identity with the only TBPs annotated for the phylogenetically and metabolically related organisms Methanococcoides burtonii and Methanosaeta thermophila (see Table S1 in the supplemental material).

For organisms with multiple TBP homologs, the concept that one TBP is the primary TBP for gene expression is not unique. It has been suggested that TBPe plays a dominant role among the TBP homologs of Halobacterium sp. NRC-1 based on proteomic analyses in which TBPe was the sole TBP detected in Halobacterium sp. NRC-1 cell lysates (12, 17). Furthermore, like TBP1 in the Methanosarcinaceae, TBPe is the most phylogenetically conserved of the TBP homologs in the haloarchaea and is the only TBP annotated for the genomes of several species (12).

Although construction of the Δtbp2 and Δtbp3 strains established that TBP2 and TBP3 are not essential, it is yet to be determined if either gene is essential under any conditions. No phenotypic effects were observed for these strains cultured with methylotrophic substrates, which could indicate either that neither TBP2 nor TBP3 is important during growth on these substrates or that TBP2 can replace TBP3 and vice versa. Nonetheless, the finding that not all TBP homologs are essential for growth of M. acetivorans is consistent with findings for the archaeon Halobacterium sp. NRC-1, from which four of the six tbp homologs were successfully deleted (8, 12). The results for Halobacterium sp. NRC-1 do not rule out the possibility that the nonessential TBP homologs provide added fitness under various growth conditions. Alternatively, the results presented here suggest that TBP2 and TBP3 of M. acetivorans do this.

TBP2 and TBP3 are important for growth with limiting concentrations of acetate.

Previous results (26, 28, 29) showed that all three TBP proteins are present in M. acetivorans during growth. The >20-fold excess of tbp1 transcripts compared to tbp2 and tbp3 transcripts argues against the idea that TBP2 and TBP3 are needed simply to increase intracellular TBP concentrations. Instead, the results presented here indicate that TBP2 and TBP3 are important for optimal growth with limiting acetate concentrations. The results also indicate that TBP3 plays a larger role than TBP2. Consistent with this idea, the genomes of both M. mazei and M. barkeri are annotated with only one additional gene encoding a TBP (MM2184 and MbarA1062), which has the highest level of identity to TBP3 of M. acetivorans (see Table S2 in the supplemental material). In contrast to the growth phenotypes of the Δtbp2 and Δtbp3 strains reported here, tbp2 transcripts were more abundant than tbp3 transcripts during growth on acetate (Table (Table1),1), which is consistent with a more prominent role for tbp2 than for tbp3 during growth on acetate, an anomaly that has not been explained at this juncture.

In marine environments, acetotrophic sulfate-reducing species have a competitive advantage over methanogens for acetate (34, 37), which suggests that TBP3 may be necessary for M. acetivorans to utilize this substrate in its native habitat. The 20 mM acetate used in growth-limiting experiments reported here was the minimum concentration that allowed determination of reliable growth parameters, although the levels of acetate reported for various marine sediments (~1 to 2 mM) are well below this value (23, 38). Thus, growth defects of the mutants are expected to be even more severe in the native habitat of M. acetivorans. Conversely, it has been reported that methanogens compete very well with sulfate reducers for methylotrophic substrates (34) due in part to the greater energy available from conversion of these substrates to methane than from conversion of acetate to methane (41).

Potential involvement of TBP2 and TBP3 in gene regulation.

The Methanosarcina species are the most metabolically diverse species in the methane-producing Archaea (13, 14), and the global gene expression profiles for various Methanosarcina species differ substantially during growth with different substrates (11, 20, 26-30). However, how the pathways are regulated is largely unknown. The growth defects of the Δtbp2 and Δtbp3 strains suggest that TBP2 and TBP3 may be necessary for the regulation of genes when M. acetivorans encounters growth-limiting concentrations of acetate or when it is switching from growth on methylotrophic substrates to growth on acetate. If TBP2 and TBP3 have roles in the regulation of genes specific for diverse metabolic pathways, methanogenic species with less metabolic diversity than Methanosarcina species might be expected to have fewer TBP homologs. Consistent with this idea, most methanogenic species that obtain energy for growth only by reducing carbon dioxide to methane have genomic annotations for only one tbp gene (www.tigr.org). The only exceptions are the genomes of “Candidatus Methanoregula boonei 6A8,” which has three tbp genes, Methanococcus maripaludis C5, which has two tbp genes (the C7 and S2 strains each have one tbp gene), Methanocorpusculum labreanum Z, which has two tbp genes, Methanoculleus marisnigri JR1s which has two tbp genes, and Methanospirillum hungatei JF-1s, which also has two tbp genes. Furthermore, M. burtonii, which utilizes only methylotrophic substrates, and M. thermophila, which utilizes only acetate, have genomes annotated with only one TBP gene (www.tigr.org). Therefore, it is tempting to speculate that during the acquisition of metabolic diversity in Methanosarcina species, multiple TBPs evolved to play key roles in optimizing gene expression. Indeed, DNA microarray analyses have suggested that TBP2 and TBP3 are involved in the regulation of gene expression. The results presented here suggest that TBP3 is important for adaptation to growth on acetate from growth on methylotrophic substrates and that TBP2 and TBP3 are important for optimal growth during culture with growth-limiting acetate concentrations. Increases in transcription of numerous genes in the Δtbp2 and Δtbp3 strains compared with wild-type M. acetivorans could potentially cause protein imbalances that place further energetic burdens on cells when nutrient availability is poor. Conversely, decreases in transcription of other genes could compromise functions necessary for efficient growth under these conditions. Although none of the annotations for genes with decreased transcript abundance identified functions that could be linked specifically to acetate metabolism, it is plausible that some differentially regulated genes have roles necessary for efficient growth on acetate. For example, the proteins encoded by MA0857 and MA4386, which are homologous to group II chaperonins present in the Archaea and Eukarya domains (19, 24), could presumably function in proper folding of nascent peptides or denatured proteins to facilitate the metabolic transition from methanol to acetate. These changes in transcription are consistent with roles for TBP2 and TBP3 in the optimization of gene expression necessary for efficient transition from one-carbon substrates, such as methanol, to the least energetically favorable substrate, acetate, particularly when the acetate concentration is growth limiting, as it is in the native environment.

Conclusion.

In this study, the physiological roles of the three TBP homologs in M. acetivorans were investigated. TBP1 is essential and has a greater role than TBP2 or TBP3. Although not essential for cell growth, TBP3 is important for optimal growth when cells are adapting to growth on acetate from growth on methylotrophic substrates, and both TBP2 and TBP3 are important for optimal growth with ecologically relevant growth-limiting acetate concentrations. The alternative TBPs TBP2 and TBP3 may have a role in optimization of gene expression which helps M. acetivorans in the metabolic transition to growth on acetate and a role in efficient utilization of ecologically relevant concentrations of acetate.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank W. W. Metcalf (University of Illinois, Urbana, IL) for donation of all plasmids and strains used for the genetic manipulation of M. acetivorans, D. Grove (Penn State Genomics Core Facility, University Park, PA) for optimization of TaqMan assays, and C. Praul and H. Xiang (Penn State Genomics Core Facility, University Park, PA) for DNA microarray labeling, hybridization, and scanning.

This work was funded by grants from the Pew Scholar's Program in the Biomedical Sciences and the National Institutes of Health (GM071897) to K.S.M. and by grants from the National Science Foundation and Department of Energy to J.G.F.

Footnotes

[down-pointing small open triangle]Published ahead of print on 14 January 2010.

Supplemental material for this article may be found at http://jb.asm.org/.

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