JOURNAL OF BACTERIOLOGY, Oct. 1991, p. 6383-6389

Vol. 173, No. 20

0021-9193/91/206383-07$02.00/0 Copyright C) 1991, American Society for Microbiology

Cloning, Sequence Analysis, and Functional Expression of the Acetyl Coenzyme A Synthetase Gene from Methanothrix soehngenii in Escherichia coli RIK I. L. EGGEN,* ANS C. M. GEERLING, ALEX B. P. BOSHOVEN, AND WILLEM M. DE VOS Bacterial Genetics Group, Department of Microbiology, Wageningen Agricultural University, P.O. Box 8033, 6700 EJ Wageningen, The Netherlands Received 12 April 1991/Accepted 5 July 1991

In the acetoclastic methanogen Methanothrix soehngenii, acetate is activated to acetyl coenzyme A by acetyl coenzyme A synthetase (Acs). The acs gene, coding for the single Acs subunit, was isolated from a genomic library of M. soehngenii DNA in Escherichia coli by using antiserum raised against the purified Acs. After introduction in E. coli, the acs gene was expressed, resulting in the production of an immunoreactive protein of 68 kDa, which is approximately 5 kDa smaller than the known size of purified Acs. In spite of this difference in size, the Acs enzymes are produced in similar quantities in E. coli and M. soehngenii and show comparable specific activities. Upstream from the acs gene, consensus archaeal expression signals were identified. Immediately downstream from the acs gene there was a putative transcriptional stop signal. The amino acid sequence deduced from the nucleotide sequence of the acs gene showed homology with those of functionally related proteins, i.e., proteins involved in the binding of coenzyme A, ATP, or both. In the fermentative decomposition of organic material, H2,

MATERIALS AND METHODS

CO2, and acetate are the main products that are converted to

Bacterial strains, vectors, and growth conditions. The bacterial strains, phages, and plasmids used in this study are listed in Table 1. M. soehngenii Opfikon (provided by A. J. B. Zehnder [13]) was cultivated on acetate (kindly supplied by M. S. M. Jetten) as the sole carbon and energy source as described previously (15). Escherichia coli strains were cultivated in Luria broth-based medium (28). When appropriate, ampicillin (50 ,g/ml), 5-bromo-4-chloro-3-indolyl-,3-D-galactoside (0.004%), or isopropyl-p-D-thiogalactopyranoside (1 mM) was added to the medium. DNA techniques and sequence analysis. DNA isolations, ligations, and transformations and other DNA manipulations were done by established procedures (28). DNA sequencing was carried out by the dideoxy chain termination method (29). The sequence strategy is shown in Fig. 1. Computer analysis of the sequence was done with the PC/GENE program version 5.01 (Genofit, Geneva, Switzerland) and the GCG package version 6.0 (5). All enzymes were purchased from Life Technologies Inc. (Gaithersburg, Md.), Pharmacia LKB Biotechnologies (Uppsala, Sweden), or Boehringer (Mannheim, Germany). Oligonucleotides were synthesized with a Biosearch Cyclone DNA synthesizer (New Brunswick Scientific Corp.) at the Netherlands Institute for Dairy Research, Ede, The Netherlands. Isolation of RNA and primer extension. RNA was isolated from exponentially growing M. soehngenii cells as described by Aiba et al. (2). Primer extension was carried out as described previously (8) with a synthetic oligonucleotide primer (5' CTTCTCAGTCTTGAACCC 3') that is complementary to residues 439 to 456 in the acs gene (see Fig. 3). Preparation of antiserum against acetyl-CoA synthetase. Antiserum was raised in rabbits against purified Acs (kindly provided by M. S. M. Jetten [14]). The specificity of the antiserum was tested in immunoblot experiments with total extracts from E. coli or M. soehngenii cells or purified Acs from M. soehngenii.

methane in the final step of the anaerobic degradation. It is generally accepted that about 70% of the methane produced in anaerobic ecosystems is derived from acetate (12, 30, 38). Thus acetoclastic methanogens play a pivotal role in the anaerobic treatment of organic wastes. To be able to catabolize acetate to methane, acetate must first be activated to acetyl coenzyme A (acetyl-CoA) (32). For this activation process, different pathways have been described in species of Methanosarcina and Methanothrix, the two genera of the archaeal domain (34) that are able to convert acetate. In Methanosarcina spp., acetyl-CoA is formed activated by acetate kinase and then phosphotransacetylase (1, 9, 19, 22, 31). In Methanothrix spp., acetate is activated in one step by the enzyme acetyl-CoA synthetase (Acs) (14, 17, 25): acetate + ATP + coenzyme A-*acetyl-CoA + AMP + PPi. Then the acetyl-CoA is converted into CO2 and CH4 in a series of reactions, the first of which is catalyzed by carbon monoxide dehydrogenase (Cdh). Acs from Methanothrix soehngenii Opfikon has recently been purified and characterized (14). This enzyme, with a native molecular mass of 148 kDa, is composed of two identical subunits of 73 kDa and constitutes up to 4% of the soluble cell protein. Kinetic data indicate that two binding sites for ATP occur in Acs. The molecular analysis of the acs gene could be of help in elucidating the domains of the enzyme that are involved in coordinate binding of the cofactors coenzyme A (CoA) and ATP. In addition, this knowledge can be used to compare the organization, location, and the expression signals of the acs gene with those of the recently characterized cdh genes from the same organism (8). In this report we describe the cloning, functional expression, and characterization of the acs gene from the acetoclastic methanogen M. soehngenii Opfikon. *

Corresponding author. 6383

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TABLE 1. Bacterial strains, phages, and plasmids Strain, phage, or

Reference

Relevant

properties Rlvnprptisor source

plasmid Bacterial strains Methanothrix soehngenii Opfikon Escherichia coli K803 Escherichia coli DH5a Escherichia coli TG1

Acetoclastic methanogen

13 35 20 11

F- lacZAM15 recAl hsdRJ7 supE44C (lacZYA-argF) F'[traD35 proAB+ lacIq lacZAM15] supE hsdA59 lac-proAB)

Phages XEMBL3 M13mpl8 M13mpl9

10 36 36

Plasmids pUC18 pUC19 pLUW101 pLUW102 pLUW103 pLUW104 pLUW105 a

Apra Apr 3.4-kb Sall fragment of M. soehngenii DNA in pUC19, Apr As pLUW101, fragment in opposite orientation 2.4-kb AspI fragment of pLUW101 in pUC18, Apr 2.9-kb XhoI-Sall fragment of pLUW101 in pUC18, Apr As pLUW101; frameshift mutation created by digesting with ClaI, filling with Klenow fragment, and religating; Apr

36 36 This work This work This work This work This work

Apr, resistant to ampicillin.

Construction and screening of a gene library of M. soehngenii DNA. A previously constructed gene library of M. soehngenii DNA in lambda phage EMBL3 (8) was plated on E. coli K803 and transferred to nitrocellulose filters (28). Recombinant bacteriophages were screened for their capacity to produce (upon infection) antigens that bound the

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Cloning, sequence analysis, and functional expression of the acetyl coenzyme A synthetase gene from Methanothrix soehngenii in Escherichia coli.

In the acetoclastic methanogen Methanothrix soehngenii, acetate is activated to acetyl coenzyme A by acetyl coenzyme A synthetase (Acs). The acs gene,...
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