Proteome of Methanosarcina acetivorans Part II: Comparison of Protein Levels in Acetate- and Methanol-Grown Cells Qingbo Li,† Lingyun Li‡, Tomas Rejtar‡, Barry L. Karger,*,‡ and James G. Ferry*,† Center for Microbial Structural Biology, Department of Biochemistry and Molecular Biology, 205 South Frear Laboratory, Penn State University, University Park, Pennsylvania 16802 and Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115 Received September 21, 2004
Methanosarcina acetivorans is an archaeon isolated from marine sediments which utilizes a diversity of substrates for growth and methanogenesis. Part I of a two-part investigation has profiled proteins of this microorganism cultured with both methanol and acetate as growth substrates, utilizing twodimensional gel electrophoresis and MALDI-TOF-TOF mass spectrometry. In this report, Part II, the analyses were extended to identify 34 proteins found to be present in different amounts between methanol- and acetate-grown M. acetivorans. Among these proteins are enzymes which function in pathways for methanogenesis from either acetate or methanol. Several of the 34 proteins were determined to have redundant functions based on annotations of the genomic sequence. Enzymes which function in ATP synthesis and steps common to both methanogenic pathways were elevated in acetate- versus methanol-grown cells, whereas enzymes that have a more general function in protein synthesis were in greater amounts in methanol- compared to acetate-grown cells. Several group I chaperonins were present in greater amounts in methanol- versus acetate-grown cells, whereas lower amounts of several stress related proteins were found in methanol- versus acetate-grown cells. The potential physiological basis for these novel patterns of protein synthesis are discussed. Keywords: two-dimensional gel electrophoresis • regulation • Methanosarcina acetivorans • mass spectrometry • methanol • acetate • methanogenesis • archaea
Introduction mazei1
The genomes of Methanosarcina and Methanosarcina acetivorans,2 the largest yet sequenced among species belonging to the Archaea domain, reflect the wide range of metabolic capabilities which distinguish these methane-producing microbes from all other Archaea. For example, Methanosarcina species are capable of growth and methanogenesis with acetate and several single-carbon compounds such as methanol.3-5 Not surprisingly, these microbes have evolved mechanisms for regulation of protein synthesis in response to growth conditions. For example, it is reported that Methanosarcina species, when cultured with acetate, down-regulate the synthesis of proteins specific for the conversion of methanol to methane, and when cultured with methanol, down-regulate the synthesis of proteins specific for the metabolism of acetate.6-9 This property can be exploited with proteomic approaches to identify novel proteins essential for specific cellular responses to growth conditions. This global experimental approach is expected to provide a foundation for understanding the systems biology of the M. acetivorans cell. A larger inventory of regulated * To whom correspondence should be addressed. J.G.F.: Tel: (814) 8635721. Fax: (814) 863-6217. E-mail:
[email protected]. B.L.K.: Tel: (617) 3732867. Fax: (617) 373-2867. E-mail:
[email protected]. † Center for Microbial Structural Biology, Department of Biochemistry and Molecular Biology, Penn State University. ‡ Barnett Institute and Department of Chemistry, Northeastern University. 10.1021/pr049831k CCC: $30.25
2005 American Chemical Society
genes also provides greater potential for discovery of fundamentals of gene regulation transferable to the Archaea in general. Remarkably, there are only two reports of a global proteomic investigation of changes in protein levels in response to growth conditions of methane-producing species,10,11 and none reported for the Methanosarcina. In Part I,12 we report an initial characterization of the proteome of M. acetivorans grown with either acetate or methanol as the energy source. Here, we build on this result, identifying 34 proteins that are present in different amounts between acetate- and methanol-grown M. acetivorans, and discuss the implications for further understanding of the biology of the cell.
Materials and Methods Methods for culturing M. acetivorans with acetate or methanol, protein extraction, and protein identifications were as previously described.12 Two-dimensional gel electrophoresis (2DE) was performed as described,12 except 18-cm, pH 4-7, IPG strips (Amersham Pharmacia, Piscataway, NJ) were used for the first dimension separation with 240 µg protein loaded during the rehydration step, and ExcelGel 12.5% homogeneous gels (Amersham Pharmacia) were selected for the second dimension separation. Gel images were generated using a Microtek ScanMaker 5900 scanner (Microtek International, Inc., Redondo Journal of Proteome Research 2005, 4, 129-135
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Li et al.
Figure 1. Comparison of 2-DE gels containing proteins from acetate- or methanol-grown Methanosarcina acetivorans. Panel A, cells grown with acetate (AC). Panel B, cells grown with methanol (ME). The numbers are the loci of the genes encoding the protein identified in the spot (see Table 1). Numbered spots were found to have at least 2-fold greater intensities compared to spots in the equivalent positions of the other gel that are indicated by the circles. Paralogs contained in a single spot are indicated in brackets.
Beach, CA). Images of triplicate gels representing acetate- and methanol- grown cells were analyzed using PDQuest image analysis software (BioRad, Hercules, CA) to quantify the difference in intensity of spots between gels.13 Spot intensities were normalized against the total spot optical density for each gel. For each growth condition, the triplicate gel images were assembled as a replicate group to generate the master gel image. The resulting pair of master gels for the acetate- and methanol-grown cells were compared to generate the mean spot intensity ratios. Student’s t-test (95% confidence interval) was performed to determine the significance of the regulated spot intensity differences. After in-gel tryptic digestion, the extracted peptides were concentrated and desalted using C18 ZipTip (Millipore, Bedford, MA) and analyzed using AB 4700 TOF/TOF Proteomics Analyzer (Applied Biosystems, Framingham, MA) in both the MS and MS/MS modes, as previously described.3
Results and Discussion Two-Dimensional Gel Analysis and Protein Identification. Figure 1 shows one of three pairs of gels containing observable proteins of methanol- or acetate-grown cells. A pH gradient of 4-7 was used for the first dimension, as the bulk of proteins were shown previously to be located in this pH range.12 Two of the three pairs were selected for protein identification by MALDI-TOF-TOF analysis. The following criteria were set to select spots and identify proteins present in different amounts in response to the growth substrate. Spots with at least a 2-fold difference in intensity were selected, which resulted in ap130
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proximately 45 spots from each of the two gel pairs that were submitted for protein identification. Spots with more than one identified protein were excluded except for spots containing paralogs that could be distinguished by MS/MS analysis. Here, paralogs are defined as the products of genes with redundant annotations.14 Proteins were further excluded that were present in multiple spots with different MW’s suggesting degradation or modification. However, proteins in multiple spots with different pI’s and the same MW’s were included. After applying these criteria, 34 proteins were identified in 42 spots (Table 1). The results were identical for the two gel pairs. For each growth condition, triplicate gel images were analyzed with PDQuest software for quantitation of the intensities of the 42 spots. The means for each growth condition were determined for all 42 spots (Table 1). Student’s t-test analysis indicated that the differences in the means were at least 2.0-fold for 33 spots and at least 1.5-fold for the remaining 9 spots with a statistical significance of p < 0.05 (Table 1). The results indicated that 14 proteins were in greater amounts in acetate- versus methanolgrown cells, and 20 were in greater amounts in methanolcompared to acetate-grown cells. Methanogenesis from Methanol. The product of MA0010 was in greater amounts in methanol-grown M. acetivorans, in accord with previous results reported for M. thermophila.8 The amount of the products of MA4430 and MA3733 were also greater in methanol-grown M. acetivorans. All three products are enzymes which catalyze steps (Figure 2) essential in the pathway of methanogenesis from methanol and are nonessential for methanogenesis from acetate.15 The results suggest
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Proteome of Methanosarcina acetivorans Part II
Table 1. Proteins with Differential Abundance in Acetate versus Methanol-Grown Cells of Methanosarcina acetivorans mascot scoreb loci
gene annotationa
spot intensityc (ppm)
pI
AC
ME
AC
31,783
4.83
ND
251
805 ( 166
4841 ( 520
0.17
19,594 59,032
6.62 4.90
ND 145
42 304
small heat shock protein methanol-5hydroxybenzimidazolylcobamide co-methyltransferase, isozyme 1 methanol-5hydroxybenzimidazolylcobamide co-methyltransferase, isozyme 1 carbon-monoxide dehydrogenase, subunit γ ribosomal protein S3p translation elongation factor 1, subunit R translation elongation factor 2
17,683 51,162
4.78 4.82
124 ND
ND 344
(400) 3214 ( 1087 2907 ( 948 4554 ( 1285 3914 ( 1596
1591 ( 447 7689 ( 1021 16093 ( 3981 957 ( 228 40986 ( 14231
(
