Abstract
Phosphotransacetylase and acetate kinase catalyze the activation of acetate to acetyl coenzyme A in the first step of methanogenesis from acetate in Methanosarcina thermophila. The genes encoding these enzymes (pta and ack) have been cloned and sequenced. They are arranged on the chromosome with pta upstream of ack (M.T. Latimer, and J. G. Ferry, J. Bacteriol. 175:6822-6829, 1993). The activities of phosphotransacetylase and acetate kinase are at least 8- to 11-fold higher in acetate-grown cells than in cells grown on methanol, monomethylamine, dimethylamine, or trimethylamine. Northern blot (RNA) analyses demonstrated that pta and ack are transcribed as an approximately 2.4-kb polycistronic message and that the regulation of enzyme synthesis occurs at the mRNA level. Primer extension analyses revealed a transcriptional start site located 27 bp upstream from the translational start of the pta gene and 24 bp downstream from a consensus archaeal boxA promoter sequence. S1 nuclease protection assays detected transcripts with four different 3' ends, each of which mapped to the beginning of four consecutive direct repeats. Northern blot analysis using an ack-specific probe detected both the 2.4-kb polycistronic transcript and a smaller 1.4-kb transcript which is the estimated size of monocistronic ack mRNA. A primer extension product was detected with an ack-specific primer; the 5' end of the product was in the intergenic region between the pta and ack genes but did not follow a consensus archaeal boxA sequence. This result, as well as detection of an additional 1.4-kb mRNA species, suggests processing of the polycistronic 2.4-kb transcript.
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- Aceti D. J., Ferry J. G. Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis. J Biol Chem. 1988 Oct 25;263(30):15444–15448. [PubMed] [Google Scholar]
- Alam J., Whitaker R. A., Krogmann D. W., Curtis S. E. Isolation and sequence of the gene for ferredoxin I from the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol. 1986 Dec;168(3):1265–1271. doi: 10.1128/jb.168.3.1265-1271.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
- Jablonski P. E., DiMarco A. A., Bobik T. A., Cabell M. C., Ferry J. G. Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila. J Bacteriol. 1990 Mar;172(3):1271–1275. doi: 10.1128/jb.172.3.1271-1275.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klionsky D. J., Skalnik D. G., Simoni R. D. Differential translation of the genes encoding the proton-translocating ATPase of Escherichia coli. J Biol Chem. 1986 Jun 25;261(18):8096–8099. [PubMed] [Google Scholar]
- Köpke A. K., Leggatt P. A. Initiation of translation at an AUA codon for an archaebacterial protein gene expressed in E.coli. Nucleic Acids Res. 1991 Oct 11;19(19):5169–5172. doi: 10.1093/nar/19.19.5169. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Latimer M. T., Ferry J. G. Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila. J Bacteriol. 1993 Nov;175(21):6822–6829. doi: 10.1128/jb.175.21.6822-6829.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lundie L. L., Jr, Ferry J. G. Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase. J Biol Chem. 1989 Nov 5;264(31):18392–18396. [PubMed] [Google Scholar]
- Peabody D. S. Translation initiation at non-AUG triplets in mammalian cells. J Biol Chem. 1989 Mar 25;264(9):5031–5035. [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith M. R., Mah R. A. Growth and methanogenesis by Methanosarcina strain 227 on acetate and methanol. Appl Environ Microbiol. 1978 Dec;36(6):870–879. doi: 10.1128/aem.36.6.870-879.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sowers K. R., Thai T. T., Gunsalus R. P. Transcriptional regulation of the carbon monoxide dehydrogenase gene (cdhA) in Methanosarcina thermophila. J Biol Chem. 1993 Nov 5;268(31):23172–23178. [PubMed] [Google Scholar]
- Zinder S. H., Elias A. F. Growth substrate effects on acetate and methanol catabolism in Methanosarcina sp. strain TM-1. J Bacteriol. 1985 Jul;163(1):317–323. doi: 10.1128/jb.163.1.317-323.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]