Vol. 174, No. 20JOURNAL OF BACTERIOLOGY, Oct. 1992, p. 6590-65990021-9193/92/206590-10$02.00/0Copyright C) 1992, American Society for Microbiology

Identification and Molecular Characterization of theAcetyl Coenzyme A Synthetase Gene (acoE)

ofAlcaligenes eutrophusHORST PRIEFERT AND ALEXANDER STEINBUCHEL*

Institut fur Mikrobiologie der Georg-August-Universitat zu Gottingen,Grisebachstrasse 8, W-3400 Gottingen, Germany

Received 1 May 1992/Accepted 30 July 1992

The gene locus acoE, which is involved in the utilization of acetoin in Alcaligenes eutrophus, was identifiedas the structural gene of an acetyl coenzyme A synthetase (acetate:coenzyme A ligase [AMP forming]; EC6.2.1.1). This gene was localized on a 3.8-kbp SmaI-EcoRI subfragment of an 8.1-kbp EcoRI restrictionfragment (fragment E) that was cloned recently (C. Frfind, H. Priefert, A. Steinbuichel, and H. G. Schlegel,J. Bacteriol. 171:6539-6548, 1989). The 1,983 bp acoE gene encoded a protein with a relative molecular weightof 72,519, and it was preceded by a putative Shine-Dalgarno sequence. A comparison analysis of the amino acidsequence deduced from acoE revealed a high degree of homology to primary structures of acetyl coenzyme Asynthetases from other sources (amounting to up to 50.5% identical amino acids). Tn5 insertions in twotransposon-induced mutants ofA. eutrophus, that were impaired in the catabolism of acetoin were mapped 481and 1,159 bp downstream from the translational start codon ofacoE. The expression ofacoE in Escherichia coliled to the formation of an acyl coenzyme A synthetase that accepted acetate as the preferred substrate (100%orelative activity) but also reacted with propionate (46%) and hydroxypropionate (87%); fatty acids consistingof four or more carbon atoms were not accepted. In addition, evidence for the presence of a second acylcoenzyme A synthetase was obtained; this enzyme exhibited a different substrate specificity. The latter enzymeis obviously required for the activation of propionate, e.g., during the formation of the storage compoundpoly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) when propionate is provided as the sole carbon source.An analysis of mutants provided evidence that the expression of the uptake protein for propionate depends onthe presence of alternate sigma factor U54.

Many microorganisms are able to grow on acetate as thesole carbon source. Before it is oxidized to gain energy orused as a precursor for the biosynthesis of lipids, acetatemust be activated to acetyl coenzyme A (acetyl-CoA) (6).For this activation process, two main mechanisms have beenelucidated. The primary route in bacteria, as it occurs, e.g.,in Escherichia coli, is via the sequential reactions of twoenzymes: first, acetate kinase (EC 2.7.2.1) catalyzes theATP-dependent conversion of acetate to acetyl phosphate,and second, phosphotransacetylase (EC 2.3.1.8) catalyzesthe transfer of the acetyl moiety from acetyl phosphate tocoenzyme A, with the concomitant release of Pi (6, 29, 40,49). In other microorganisms, as in some ascomycete fungiand in some acetoclastic methanogens, acetate is activatedby only a single reaction, which is catalyzed by acetyl-CoAsynthetase (acetate:CoA ligase [AMP forming]; EC 6.2.1.1)(2, 13, 23): acetate + ATP + coenzyme A -) acetyl-CoA +AMP + PPi.The aerobic bacterium Alcaligenes eutrophus also utilizes

acetate as a sole carbon source (55). A. eutrophus lacksacetate kinase (47), and during growth on acetate, theformation of acetyl-CoA synthetase and of the key enzymesof the glyoxylate cycle, isocitrate lyase (EC 4.1.3.1) andmalate synthase (EC 4.1.3.2), is induced (53). Acetyl-CoAsynthetase also may be required byA. eutrophus if acetate is

* Corresponding author.

formed endogenously from other carbon sources (47). Thissituation occurs in A. eutrophus and in Pelobacter carbin-olicus during the degradation of acetoin, which is initiated bythe direct cleavage of acetoin into acetyl-CoA and acetalde-hyde (15, 32); the latter compound is oxidized in A. eutro-phus to acetate. The genes essential in A. eutrophus foracetoin catabolism recently were localized on five differentgenomic EcoRI restriction fragments (A, B, C, D, and E)(15). A cluster of the structural genes for the a and ,Bsubunits of acetoin:2,6-dichlorophenolindophenol oxidore-ductase (acoA and acoB, respectively), for a dihydrolipo-amide acetyltransferase (acoC), for a transcriptional activa-tor protein (acoR), and for a protein of as-yet-unknownfunction (acoX) was localized on fragments A and C, whichare directly linked in the genome (25, 35). Fragment Bharbored an rpoN-like gene (15, 39). The structural gene foracetaldehyde dehydrogenase II (acoD), which is responsiblefor the oxidation of acetaldehyde to acetate, was localizedon fragment D (36).The present study was aimed at the molecular character-

ization of the gene locus acoE, which is involved in thecatabolism of acetoin in A. eutrophus and which was iden-tified as the structural gene of an acetyl-CoA synthetase(acetate:CoA ligase [AMP forming]; EC 6.2.1.1). Sinceacetyl-CoA is also a precursor for the synthesis of poly(3-hydroxyalkanoic acids) (PHA), we also investigated therelevance of acetyl-CoA synthetase and of related syn-thetases for the synthesis of PHA from organic acids in A.eutrophus.

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ACETYL-CoA SYNTHETASE GENE OF A. EUTROPHUS 6591

TABLE 1. Bacterial strains, plasmids, and DNA fragments used in this study

Strain, plasmid, or DNA Relevant characteristics Source or referencefragment

StrainsAlcaligenes eutrophusH16 Wild type, autotrophic, prototrophic DSM 428, ATCC 17699H1098 TnS-induced mutant (acoR) impaired in acetoin catabolism 15H1075 TnS-induced mutant (rpoN) impaired in acetoin catabolism 15N1231 TnS-induced mutant (rpoN) impaired in acetoin catabolism 15HF149 TnS-induced mutant (rpoN) impaired in acetoin catabolism 19HFO9 rpoN mutant impaired in acetoin catabolism 14H1053 TnS-induced mutant (acoB) impaired in acetoin catabolism 15H1068 TnS-induced mutant (acoD) impaired in acetoin catabolism 15H1050 TnS-induced mutant (acoE) impaired in acetoin catabolism 15H1094 TnS-induced mutant (acoE) impaired in acetoin catabolism 15

Escherichia coliJM83 ara A(lac-proAB) rpsL (Smr) thi-1 4)80 lacZAl15 54XL1-Blue recAl endA1 gyrA96 thi hsdR17 (rK- MK') supE44 reL1 A- lac [F' proAB 7

laclqZAM15 TnlO(tet)]S17-1 recA; harbors the tra genes of plasmid RP4 in the chromosome; proA thi-1 44

PlasmidspVK1O1::E Tcr Kmr; harbors fragment E in the EcoRI site 15pBluescript KS- Apr lacPOZ'; T7 and T3 promoter Stratagene, San Diego, Calif.pBluescript SK- Apr lacPOZ'; T7 and T3 promoter StratagenepUC9-1 Apr lacPOZ' 17

DNA fragments ETHlO50 Genomic EcoRI restriction fragment E of A. eutrophus mutants affected in 15and ETHl' the catabolism of acetoin, harboring TnS

MATERIALS AND METHODSBacterial strains and plasmids. The strains ofA. eutrophus

and Escherichia coli, the plasmids, and the DNA fragmentsused in this study are listed in Table 1.Growth of bacteria and analysis of PHA content and com-

position. Cells of E. coli were grown at 37°C in Luria-Bertani(LB) medium (41). Cells ofA. eutrophus were grown at 30°Ceither in nutrient broth medium (0.8% [wt/vol]) or in mineralsalts medium (MM) (43) supplemented with filter-sterilizedcarbon sources as indicated in the text. To achieve theextensive accumulation of PHA, we reduced the concentra-tion of NH4Cl in MM to 0.05 or 0.005% (wt/vol). The PHAcontent in lyophilized cells and the PHA composition weredetermined by gas chromatography as described previously(50).

Determination of enzyme activities. Approximately 0.5 to1.0 g (wet weight) of cells was washed and resuspended in 2ml of potassium phosphate buffer (100 mM, pH 7.0). Thecells were disrupted by sonication, and the resulting crudeextracts were centrifuged at 100,000 x g for 1 h to obtainsoluble extracts, which were subjected to enzyme assays.Acetyl-CoA synthetase activity (acetate:CoA ligase [AMPforming]; EC 6.2.1.1) was determined by three differentmethods. (i) In the enzyme assay of Oberlies et al. (31), theformation ofAMP from ATP was monitored by coupling thereaction to the oxidation of NADH via adenylate kinase,pyruvate kinase, and lactate dehydrogenase. (ii) In theenzyme assay described by Brown et al. (6), the formation ofacetyl-CoA from acetate and coenzyme A was measured bycoupling the reaction to the reduction of NAD+ via malatedehydrogenase and citrate synthase. (iii) In the hydroxamatemethod of Berg (4), the formation of acethydroxamate from

acetyl-CoA and hydroxylamine was determined. Blankswithout coenzyme A and ATP were prepared for each set ofsamples. Protein was determined as described by Lowry etal. (28).

Electrophoretic methods. Proteins were separated underdenaturating and nondenaturating conditions in polyacryl-amide gels and stained with Coomassie brilliant blue asdescribed previously (35). To stain gels for acetyl-CoAsynthetase activity, we used the calcium PPi precipitationmethod of Nimmo and Nimmo (30) but with the followingmodifications. After electrophoresis, the gels were soakedfor 20 min at 30°C in a buffer containing 110 mM Tris-HCl,5.5 mM MgCl2, and 50 mM CaCl2 (pH 8.5). Coenzyme A,ATP, and potassium acetate were added to final concentra-tions of 10 mM, 10 mM, and 1 M, respectively. Bands ofprecipitated calcium PPi became visible after 3 to 4 h ofincubation at 30°C.

Isolation and manipulation of DNA. Plasmid DNA andDNA restriction fragments were isolated and analyzed bystandard methods referred to in our previous study (35).

Transfer ofDNA. Competent cells ofE. coli were preparedand transformed by the CaCl2 procedure described by Ha-nahan (16). Conjugations ofA. eutrophus (recipient) and ofE. coli S17-1 (donor) harboring hybrid plasmids were per-formed on solidified nutrient broth medium as described byFriedrich et al. (14). *

Synthesis of oligonucleotides. Synthetic oligonucleotideswere synthesized in 0.2-p,mol portions from deoxynucleo-side phosphoramidites (3) in a Gene Assembler Plus appara-tus as specified by the manufacturer (Pharmacia-LKB, Upp-sala, Sweden). Oligonucleotides were released from thesupport matrix, and protection groups were removed by 15 h

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6592 PRIEFERT AND STEINBUCHEL

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ORF3 1 s !ORF2FIG. 1. Localization of acoE. (a) Physical map of fragment E. (b) Restriction sites and open reading frames (ORF) probably representing

coding DNA and the structural gene of the acetyl-CoA synthetase (acoE). Triangles indicate the positions of TnS::mob insertions in mutantsofA. eutrophus, which were impaired in growth on acetoin. The hairpin-like structure downstream of acoE is indicated.

of incubation at 55°C in 25% (vol/vol) ammonium. Oligonu-cleotides were finally purified by passage through an NAP-5column (Pharmacia-LKB).DNA sequence determination and analysis. The dideoxy

chain termination method of Sanger et al. (42) was used withthe modifications described previously (35). Synthetic oligo-nucleotides were used as primers, and the primer-hoppingstrategy (48) was used to determine the DNA sequence ofboth strands. Nucleotide and amino acid sequences wereanalyzed with the Genetics Computer Group SequenceAnalysis Software Package (GCG Package, version 6.2, June1990) as described by Devereux et al. (11).

Chemicals. Restriction endonucleases, T4 DNA ligase,lambda DNA, and enzymes or substrates used in the enzymeassays were obtained from C. F. Boehringer & Soehne(Mannheim, Germany) or from GIBCO/BRL GmbH (Eggen-stein, Germany). Agarose (type NA) was purchased fromPharmacia-LKB. Radioisotopes were obtained from Amer-sham/Buchler (Braunschweig, Germany), Fluka Chemie(Buchs, Switzerland), Serva Feinbiochemica (Heidelberg,Germany), or Sigma Chemie (Deisenhofen, Germany).

Nucleotide sequence accession number. The nucleotide andamino acid sequence data reported in this paper have beensubmitted to the EMBL, GenBank, and DDBJ nucleotidesequence data bases under accession number M97217.

RESULTS

Nucleotide sequence ofacoE and mapping ofTnS insertions.The TnS::mob insertions in two mutants ofA. eutrophus thatonly grew with a doubling time of 6 h after a lag phase of 20to 25 h when acetoin was provided as the sole carbon sourcealready had been physically mapped at a distance of approx-imately 0.7 kbp on a 3.8-kbp EcoRI-SmaI subfragment(ES38) of fragment E (15). This subfragment was ligated to

EcoRI-SmaI-treated plasmids pBluescript KS- and pBlue-script SK- and transformed into E. coli XL1-Blue. One ofthe resulting hybrid plasmids (pSES38) was used as thetemplate DNA. In combination with universal and syntheticprimers, and by use of the primer-hopping strategy, theentire nucleotide sequence of both strands of the 3.8-kbpEcoRI-SmaI fragment was determined (Fig. 1 and 2). TheG+C content of this fragment was 66.2 mol%, close to thevalue reported for genomic DNA of A. eutrophus (66.3 to66.9 mol%) by Davis et al. (10). An open reading frame of1,983 bp (ORF1), which was referred to as acoE, wasidentified. The ATG codon at position 722 (Fig. 2) waspreceded by a putative Shine-Dalgarno sequence at a dis-tance of 8 nucleotides. The only sequence (ATGACA-17bp-CAGCAG) that exhibited a certain homology to the E.coli consensus sequence (TTGACA-17 bp-TATAAT) for&70-dependent promoters (18) was identified approximately400 bp upstream of this putative start codon ofacoE (Fig. 2).An inverted repeat that was followed by a run of T residuesand that may represent a factor-independent transcriptionalterminator was identified 30 bp downstream from the trans-lational stop codon of acoE (Fig. 2). The free energy of thisstructure is approximately -91.2 kJ/mol, according to Ti-noco et al. (51).For determination of the exact locations of Tn5 insertions,

SalI restriction fragments of EcoRI fragnents ETHl094 andETH1050, which were cloned from mutants H1094 andH1050, respectively, were ligated to Sall-treated pUC9-1DNA and transformed into E. coli JM83. The resultinghybrid plasmids were used as templates for DNA sequencingin combination with a synthetic oligonucleotide that hybrid-ized at a distance of 63 to 79 bp from the end of ISSOL (24),and the TnS insertions were mapped 481 and 1,159 bpdownstream from the putative translational start codon ofacoE in mutants H1050 and H1094, respectively (Fig. 2).

FIG. 2. Nucleotide sequence of the 3.8-kbp SmaI-EcoRI fragment. Amino acids deduced from the nucleotide sequence are specified bystandard one-letter abbreviations. The putative ribosome-binding sites are indicated by boxes and by "S/D." The putative promoter isoverlined and indicated by "-10" and "-35." The position of the hairpin-like structure downstream of acoE is indicated by inverted arrows.Triangles indicate the positions of TnS::mob insertions in mutants ofA. eutrophus.

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1CCCGGGCGATATTCAGCACGGCGATGCCGGTCATCGCCAGCAGCACCGATGCCACCG TGCGCCGCGACAGCCGTTCGCGC AGCAGCAGCCACGACAGGAT

10o1 CGCCACCGTGGCCGGGATGGTGC TGGTGATCACGCCGGCCGCCATGGCGCTGGTCAGCCGCACGCCCATTGAGCATCAGC AGCGTAAACAGGAAGGTGCC"-35"

2 01 GAAGAACGCCTGCAGGAACAGGTTCAGCC ATTCCCCGCGCGAGACCCGGCGCATGCGCGCGGGCCGGTACCAGGGCGCCAGGCAGACGATGGCAATGACA"-10"

301 AAACGCAGCAGCGCGAACAGCAGCACCGGCACATGCGCGATGATCGATTTGC CGAGGCCCACGTTGCTGCCGACCAGTGCCATCGAGGCAACCAGGAAGA

4 01 GGGCGTAGACCGGGGAGAATGCTGAAGGG TTGGCGGGCACGGGTACGGCCTTGGTCTTCCGGCGGGCGCCGCGGCGGGTGCTGAGC CGGCAGGAGCGGCA

s0o AGGGTAAGCGCCGCGATCAGGTATGGAAC TCGCGGCATTATAGCTGTGCATGCCTAATGGAGAAGGCTGCACTTTCGCGGCACGTGCGTGGATTTTTCCC

601 GCTGCATTGCAACAAGTTGCAGGCACGcCCCGGCCCACCG TCAGGTCTTACCGTCAGGTTTAAGTAAGCCGTCGGGCGTAACGTGGCAGCCAAAGCC AGT

M S A I E S V M Q E H R V F N P P E G F A S Q A A I P701 CCAAAAaTCG AGCAAACCTATGTCCGCCATCGAATCGGTGATGC AAGAGCATCGCGTGTTCAACCCGCCCGAAGGCTTCGCCAGCCAGGCCGCGATCC

rS/D- acoES M E a Y Q a L C D E A E R D Y E G F W a R H A R E L L H W T K P

801 CCAGCATGGAGGCCTACCAGGCGCTGTGCGACGAAGCCGAGCGTGACTATGAAGGTTTCTGGGCGCGCCACGCGCGCGAGCTGCTGCACTGGACCAAGCC

F T K V L D Q S N A P F Y K W F e D G E L N a S Y N C L D R N L Q90o1 CTTCACCAAGGTGCTGGACCAAAGCAACGCACCGTTCTAC AAGTGGTTCGAAGACGGCGAGCTCAACGCCTCTTACAACTGCCTGG ACCGCAATCTGCAG

N G N A D K V A I V F E A D D G S V T R V T Y R E L H G K V C R F Aoo 1 AACGGCAATGCGGACAAGGTCGCGATCGTGTTCGAGGCCGACGACGGCAGCGTGACGCGCGTC ACCTACCGCGAGCTGCATGGCAAGGTGTGCCGCTTCG

N G L K A L G I R K G D R V V I Y M P M S V E G V V A M Q A C A Ro01 CCAACGGCCTGAAGGCGCTCGGC ATCAGGAAGGGCGACCGCGTGGTGATCTACATGCCGATGTCGGTCGAAGGCGTGGTCGCGATGCAGGCC TGCGCACG

L G A T H S V V F G G F S A K S L Q E R L V D V G A V A L I T A D12 01 CbGZCGCCACGCACTCGGGGTGGTTTCGGCGGCTTCTCGGCCAAGTCGCTGCAGGGAGCGTGCTGGTGACGTGGGCGCGGTGGCGC TGATCACCGCCGAC

30

E Q M R G G K A L P L K A I A D D A L A L G G C E A V R N V I V Y R1301 GAGCAGATGCGCGGCGACAGGTATGAACTCGCGGCATCATCGCCGATGACGCGCTGGCGCTGGGCGGCTGCGAGGCCGTCAGGAACGTGATCGTCTACC

R T G G K V A W T E G R D R W M E D V S A G Q P D T C E A E P V S1401 GCCGCACCGGCGGCAAGGTTGCCTGGACCGAAGGCCGCGACCGCTGGATGGAAGATGTCAGCGCTAGCCGTCGGGCGATACCTGCGAAGCCGAGCCGGTGAG

A E MP L F V L Y T S G S T G K P K G V QN S T G G Y L L W A L M15o CGCCGAGCACCCGCTGTTCGTGCTCTACACCTCCGGCTCCACCGGCAAGCCCAAGGGCGTGCAGCACAGCACCGGCGGCTACCTGCTGTGGGCGCTGATG

T M KE T F D I K P D I G W v T G H T Y I A Y G P L1601 ACAATGAAGTGGACCTTCGACATCAAGCCCGACGACCTGTTCTGGTGTACCGCGGCACTCGGCTGGGTCACCGGCCACACCTATATTGCCTACGGCCCGC

A A G A TD V v F E G V P T Y P N A G R F W D M I A R H K V S I F1701 TGGCCGCGGGCGCCACCCAGGTGGTGTTCGAAGGC GTGCCGACCTACCCCAACGCCGGCCGCTTCTGGGACATGATCGCGCGCCAC AAGGTCAGCATCTT

Y T A P TA I R S L I K A A E A D E K I H P K MY D L S S L R L L1801 CTACACCGCGCCGACCGCGATCCGCTCGC TGATCAAGGCCGCCGAGGCCGACGAGAAGATCCACCCGAAACAGTACGACkTGTCCAGCCTGCGCCTGCTC

G T V G E P I N P E A W S W Y Y K N I G N E R C P I V D T TW Q T E1201 GGCACCGTGGGCGAGCCGATCAATCCCGAAGCCTGGATGTGGTACTACAAGAACATCGGCAACGAGCGCTGCCCGATCGTCGACACCTTCTGGCAGACCG

T G G H AI T P L P G A T P L V P G S C T L P L P G I M A A I V D2001 AGACCGGGCCGGCCACATGATCACGCGCTGCCCGCGGCGCGACGCCGGGTGCCGGGTTCGTGCACGCTGCCGTGCGGCATCAGGAATGGCCGCCATCGTCGA

E T G H D V P N G N G G I L V V K R P W P A M I R T I W G D P E R2101 CGAGACCGGCCATGACGTGCCCAAGGCCAACGGCGGCATCCGTGGAGTCAAGCGTCCGTGGCCGGCCATGATCCGCACCATCTGGGGCGATCCGGAGCGC

A R K S Y F P E E L G G K L Y L A G D G s I R D K D T G Y F T I M G2 201 TTCAGGAAGAGCTACTTCCCCGAAGAGCTCGGCGGCAAGC TCTACCTGGCCGGCGACGGCTCGATCCGCGACAAGGACACCGGCTACTTCACCATCATGG

R I D D V L N V S G H R M G T M E I E S A L V S N P L V A PA V2 301 GCCGCATCGACGACGTGCTGAACGTGTCGGGCCACCGCATGGGGGACGATGGAGATCG AGTCCGCGCTGGTGTCCAACCCGCTGGTGGCTGAAGCCGC CGT

V G R P D D M T G E A I C A F V V L K R S R P T G E E A V K I A T2401 GGTGGGCCGCCCCGACGACATGACCGGCGAGGCCATCTGCGCCTTCGTCGTGCTCAAGCGTTCGCCCGAAACTGGCGAAGAGGCCGTCAAGATCGCGACG

E L R N W V G K E I G P I A K P K D I R F G D N L P K T R S G K I M2101 GAGCTGCGCAACTGGGTCGGCAAGGAGATCGGCCCGATCGCCAAGCCCAAGGACATCCGCTTTGGCGACATCCTGCCCAAGACGCCTCTCGGGCAAGATCA

R R L L R S L A K G E E I TP D T S T L E N P A I L E M L K VA D2601 TGCGCGCCTGCTGCGGTCGCTGGCCAAGGGGGAGTGAATCACGCAGGACACCTTCGACGCTGGAGAATCCGGCCATCCTGGAGCAGCTCAAGCAGGCGCA

2701 GTGATCGTCTGGCTTCGGCCCGATCGAGCCCTTGAACCCGCCGCCGCGTTCCCGCGCGCCGGCGGGTTTGTTACGATAGCCCCCCACCGACCACDCCGS/D

M P E AF S R F R R R L L L Y Y G L F T L G L F G F V G M M G L2801 TCCCCAATGCCTGAAGCCCAGTCGCGCTTTCGCCGACGGTTGCTGCTGTACTACGGCCTGTTCACGCTAGGGTTGTTCGGCTTCGTCGGCATGATGGGGC

0RF2L E K S N a D A L W L G Y V F L F AAI Y A c I G L I C R T S

2 901 TGCTTGAAAAGTCCAATGCCGATGCGCTG TGGCTCGGCTACGTCTTCCTCTTCATCACCATCGCGATCTATGCCTGCATCGGCCTGATCTGCCGCACCTC

D L N E Y Y V A S R R V P A R I A A DR M S A S F I G3001 TGACCTGACGAGTACTACGTTGCCTCGCGGCGCGTGCCGGCGCTGTTCAACGGCATGGCGATTGCCGCCGACTGGATGAGCGCGGCGTCCATTCATCGGC

L a G I L F A S G Y E G L A Y V M G W T G G Y C L V A F L L A P Y L3101 CTGGCCGGCATCCTGTTTGCCTCGGGCTACGAAGGGCATCCCACGATGATGGGCTGGACCGGCGGCTACTGCCTGGTGGCCTTCCTGCTGGCGCCGTACC

R K Y G G Y T I P D F L A A R Y G N G K P G G N L P V R a I A V L3201 TACGCAAGTACGGCGGCTDACCCATTACCGATTTCCTCGCGGCGCGCTACGGCAACGGCAAGCCCGGCGGCCACCTGCCAGTGCGCGCGATCGCGGTGCT

a A S L C S F V Y L V a Q I Q G V G L V V T R F I G V E F A V G I3301 GGCGGCTCDGCTCTGCTCCTTCGTCTACCTGGTTGCGCAGATCCAGGGCGTTCCTGGTGGTGACGCGCTTTATCGGCGTGGAGTTTGCC GTCGGC ATC

F F G L A G I L V C S F L G G M R a V T W T Q V A Q Y I M M I V A F3401 TTCTTCGGGCTGGCCGGCATCCTGGTGTGCTCGTTCCTGGGCGGCATGCGCGCGGTCACATGGACGCAGGTGGCGCAGTACATCATGCTGATCGTCGCCT

L V T V S M I a W K H H H E A L P Q L S Y G T L L S Q L D A R E Q3501 TCCTG-GTCACGGTATCGATGATCGCGTGGAAGCATCACCACGAAGCGTTGCCGCAGCTCAGCTACGGCACGCTGCTGTCGCAACTCGACGCGCGCGAGCA

Q L E R E P A E Q A V R E Y Y R Q Q A I L L Q E R I A R L P D S F3601 GCAGCTGGAGCGCGAGCCGGCCGAGCAGGCCGTGCGCGAGTACTACCGGCAGCAGGCCATCCTGCTGCAGGAGCGCATCGCCCGGC TGCCCGATTCCTTT

A L E R D A L D A R L Q D L R T R N A P L R D I K S L E R E R L E F3701 GCCCAGGAGCGTGACGCGCTCGACGCGCGGCTGCAGGACC TGCGCACGCGCAATGCCCCGCTGCGCGACATCAAGTCGCTGGAGCGCGAACGGCTGGAAaT

3201 IC

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An open reading frame of 996 bp (ORF2) was identified onthe same strand 103 nucleotides downstream of acoE (Fig. 1and 2). It was preceded by a putative Shine-Dalgamo se-quence at a distance of 8 nucleotides from the ATG atposition 2807 (Fig. 2). The 3' end of ORF2 was locatedbeyond the EcoRI restriction site of the fragment. The G+Ccontents of acoE and ORF2 were 66.9 and 65.5 mol%,respectively. The codon usage for acoE and ORF2 obeyedthe rules of Bibb et al. (5) and was very similar to thosereported for other genes ofA. eutrophus, such as acoD (36),acoXABC (35),phbC (34),phbH andphbI (37), adh (22), andhoxFUYH (52). The amino acid sequence deduced from thenucleotide sequence of ORF2 was compared with the pri-mary structures of other proteins available from the NationalBiomedical Research Foundation protein data base. It ex-hibited an identity of 21.8% and a similarity of 51.1% withthe sequence of the Acetobacter aceti pyrroloquinolinequinone-dependent alcohol dehydrogenase (20), which islocated on the outer surface of the cytoplasmic membrane.However, to improve the alignment, we had to introduceextended gaps. Yet to be investigated in the future iswhether ORF2 represents the gene for a cryptic alcoholdehydrogenase that oxidizes ethanol to acetaldehyde inmutants ofA. eutrophus able to grow fast on ethanol (21, 47).An open reading frame of 423 bp (ORF3) was identified

upstream of acoE on the same strand. The translational stopcodon of ORF3 was located 299 bp upstream of the startcodon of acoE, whereas the 5' end was located beyond theSmaI restriction site of the fragment. Three open readingframes (ORF4, 1,407 bp; ORF5, >603 bp; ORF6, >552 bp)were identified on the inverse strand; however, none waspreceded by a reliable Shine-Dalgarno sequence. The codonusages for ORF3 to ORF6 deviated widely from the theoret-ical values calculated as described by Bibb et al. (5).

Properties of the acoE gene product. The relative molecularweight of the gene product, calculated from the amino acidsequence deduced from acoE, was 72,519. A comparison ofthis amino acid sequence with the primary structures ofother proteins available from the EMBL nucleotide se-quence data base revealed extensive homologies with acetyl-CoA synthetases from the anaerobic acetoclastic bacteriumMethanothrix soehngenii (50.5% identical amino acids [12])and the ascomycete fungus Aspergillus nidulans (49.1% [9])or Neurospora crassa (47.2% [9]). The ATG at position 722in the nucleotide sequence shown in Fig. 2 is most probablythe translational initiation codon of acoE. This conclusionwas reached (i) from the alignment of the amino acidsequence deduced from acoE with the primary structures ofother acetyl-CoA synthetases (Fig. 3), (ii) from the presenceof a reliable Shine-Dalgarno sequence upstream of this ATG,and (iii) from the size (Mr = 71,000 ± 1,500) of the acetyl-CoA synthetase protein expressed in the recombinant strainof E. coli XL1-Blue harboring plasmid pSES38, as revealedby sodium dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis (Fig. 4).

Heterologous expression of acetyl-CoA synthetase from A.eutrophus in E. coli. Plasmid pSES38, which harbors the3.8-kbp EcoRI-SmaI fragment with acoE downstream fromand colinear with the lacZ promoter of pBluescript SK-,conferred acetyl-CoA synthetase activity to recombinantstrains of E. coli XL1-Blue (Table 2). When 0.01 or 1 mMisopropyl-13-D-thiogalactopyranoside (IPTG) was added tothe medium, the growth of the recombinant strains wasstrongly retarded (data not shown) and the specific activityof acetyl-CoA synthetase was much lower than in theabsence of IPTG (Table 2). This inhibition may have been

due to the overproduction of an inactive form of the acoEproduct. The specific activity was even higher in the recom-binant strains than in acetate-grown cells of A. eutrophus(Tables 2 and 3). From visual examinations of the electro-pherograms of extracts separated in SDS-polyacrylamidegels, it was estimated that acetyl-CoA synthetase probablycontributed more than 50% of the total protein in these cells(Fig. 4). In recombinant strains of E. coli XL1-Blue thatharbored plasmid pKES38 with acoE antilinear to the lacZpromoter, acetyl-CoA synthetase activity was expressed at amuch lower level. Furthermore, its expression as well as thegrowth of these strains was not affected by IPTG (Table 2).With recombinant strains of E. coli XL1-Blue harboring onlyvector pBluescript KS- or pBluescript SK-, much loweracetyl-CoA synthetase activities were measured (Table 2);these activities may have been due to the endogenousacetyl-CoA synthetase (see below). The results of theseexperiments clearly indicated that with pSES38, acoE wastranscribed from the lacZ promoter of the vector; however,expression, although much weaker, also occurred withpKES38. These results provide evidence that the regionupstream of acoE in ES38 functions as a promoter in E. coli.The acetyl-CoA synthetase protein expressed in E. coli

XL1-Blue(pSES38) exhibited the same electrophoretic mo-bility as that synthesized in A. eutrophus during growth onacetate. This result was revealed by the separation of solubleextracts in native polyacrylamide gels, with subsequentstaining for acetyl-CoA synthetase activity (Fig. 5). Faintprecipitin bands indicating the formation of calcium PPi werevisible at identical positions. These bands were absent inextracts derived from mutants H1050 (Fig. 5) and H1094(data not shown). One additional band of a different size wasvisible in extracts of A. eutrophus and E. coli. In A.eutrophus, this protein probably represents an additionalacyl-CoA synthetase (see below), whereas in E. coli, thisprotein probably represents the endogenous acetyl-CoAsynthetase.

Substrate specificity of acetyl-CoA synthetase. A variety offatty acids of short chain length were tested as substrates foracetyl-CoA synthetase by use of extracts derived from E.coli XL1-Blue(pSES38) expressing the A. eutrophus proteinand three different assay systems. The data listed in Table 2were obtained by the spectrophotometric assay of Oberlieset al. (31) and were confirmed by two other enzyme assays(4, 6; data not shown). The highest specific activities wereobtained with acetate, which is presumably the naturalsubstrate of this enzyme. Propionate was also used as asubstrate, but the rate was only 50% compared with that ofacetate. Interestingly, 3-hydroxypropionate was a muchbetter substrate than propionate, and the specific activitieswere comparable to those obtained with acetate (Table 2).Acetoacetate, butyrate, 3-hydroxybutyrate, and 4-hydroxy-butyrate were not used as substrates. To prove that AMPrather than ADP is formed by the reaction of acetyl- oracyl-CoA synthetase, we added P1,P5-di(adenosine-5)penta-phosphate (Ap5A), a specific inhibitor of adenylate kinase(27), to the reaction mixture. With all substrates tested,except for acetoacetate, ADP formation by the endogenousadenylate kinase was completely inhibited by 50 mM Ap5A;the inhibition disappeared after the addition of an excess ofadenylate kinase.

Expression of acetyl-CoA synthetase in mutants impaired inacetoin metabolism. To analyze whether acoE is required notonly for the catabolism of acetoin but also for the utilizationof fatty acids, we compared the growth ofvarious acoXABC,acoD, acoR, and acoE mutants, impaired in acetoin catab-

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VOL. 174, 1992 ACETYL-CoA SYNTHETASE GENE OF A. EUTROPHUS 6595

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A.e. 98 V FRIMH G K C FtVSVS[i1G VEi 154M.. 116 K.A IW P Z PT Q P JQ KIIi G D1 L Y K EVN IC ....S. SGLI13& -24IPQL PIIA 174A.n. 111 N VI I Y--. A1.EPN......IGI1LRE[IIJIWV1JQR __3TVAIIL?~IIII.jA1 168N.c. 73 N -:::T1IY-- EPVR- E SK LELAI PZ A IY.. 129

A.e. 155 QOG L3THA,_.VVF13$A Q ER L iV3AVALI::...:A Q M L~3A L D.A1D A L 214M..175 IMLA K13S:WVV ..FS SS GM D AA HC13RIIII3jFtaG V1E-A 232

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A.en. 569 S l "R 627N j KfM.s65 I~t--L~ V13N 5P L~j DA -A IIRKIT.J3 I .A A. VH ~ 3 LCfS4I 557 GAVA. SU--KG..TNQVK... ILQVRLjS IIAAI(AVV(:JDLt$.ZI 3N.c.532L~JQL~jVNL ..~LJAV...KEGAQANALRQLcJYPSLN3V.JRRSL FLALLJAYvPDTL.K 41.]8A.ec. 3628SftttSAK1A TQvSTESALjJFKQF6

66045 H .-Y H MVGL-I?PMNEA "P"K .IV 692 R SYFPE G L-An.631 ~~~~~~~ RIIATVH :AS K 670 E..N.c. 5489 D~IJK V ~IQLG.I TLJENELiJSVAK"IDVV .A. 626 VP R 2FIG.3.Homology of the gene product of acoE of~~A. eutrohus wit LAcey-snhtseFrmoteorcsWh'aioaisequncesofte actyl-oA sntheasesfromM sohngeii (.s. 12]) fro A. idulns (.n. 9]),androm..-r--a-N.c.-9])weraligndwihthaofhe actyl-oA sythetse ofA. etrophs deuced rom coE (.e.) Amin acis arespecfied y stndardone-ette

Abbreviations GPs P(ahS) wErE Introucedint theQ.,:seune toimprovethe algnen.Amnoacdreide idnia to ths in theAeutropusactyl.CAsynhetas at apartiular squenc posiion ae boxd andshade. Amin acid thatare pesumalypotionsof.ATbNding40sie (1,12 areinictedb +E (tp A) or x (tp B). A- V AHAserskTndcaeh psiiosofamn acd cnere in al ote acetY-C7Asynthetases but not.....in .he ..eu.opus.cey.-oAsynheas......................

oAism, with thtoMhecrepodnGarnEtais(525,resltswer obaiedfo D-yxpopoy-o syn-835,36)inMMcontainng acetat or propioate as thesole thetse activit, whereas o differeces.occu..d.for.acecaronsorc.llmuans,exep fr iOO ndH194 toceyl o poponl-oAsythtae ctviy.Inerstgrewonacetate (0.5% [wt/vo])....wih. doubling time of igy.btr.Co.ytets.civt a signficntlapproximately 3 h, ~lik thewild tpe. In cotas,muat higher in actt-rw cel of bot acc muat (Tbl 3).0H.30526anIA9a obigtm f prxmtl . hsefc waKvnmrrnucdwe th aoh onacetate.~~~-WhnaeaegonclswrMnlyeo uat eegoni meimcnannO rcoepuacty-ndacl-o sntetse, cey-CA ynheas ceoi a crbn orcs.Whn heeelsweeSareseActivitwa5pro1aey65fl lwRinaIuat tteedo heodgot phseasdecrbe preythaninthewiltype or the oher mutants (Tble 3). Simila ously (15), thyexhibited.30 or.15-fold-hiher.butyryl-.o

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

- 94- 67

- 43

- 30

- 20

- 14.4

FIG. 4. Expression of acoE in E. coli. Cytoplasmic fractions obtained from cells of recombinant strains of E. coli grown in LB mediumor in LB medium supplemented with 1 mM IPTG and cells ofA. eutrophus grown on acetate or on fructose plus acetoin were separated inSDS-polyacrylamide gels as described in Materials and Methods and stained for protein with Coomassie brilliant blue. Lanes: 1,A. eutrophusH1094 (fructose plus acetoin) (162 ,ug of protein); 2, A. eutrophus H16 (fructose plus acetoin) (200 ,ug of protein); 3, A. eutrophus H1094(acetate) (145 ,g of protein); 4,A. eutrophus H16 (acetate) (168 p,g of protein); 6 and 12, E. coli XL1-Blue harboring plasmid pSES38 (withoutIPIG) (252 and 85 p,g of protein, respectively); 7 and 15, E. coli XL1-Blue harboring plasmid pSES38 (with IPTG) (194 and 48 pg of protein,respectively); 8 and 14, E. coli XL1-Blue harboring the vector pBluescript SK- (with IPTG) (133 and 112 ,ug of protein, respectively); 9 and10, E. coli XL1-Blue harboring plasmid pKES38 (without or with IPTG, respectively) (146 and 135 p,g of protein, respectively); 11, E. coliXL1-Blue harboring the vector pBluescript KS- (with IPTG) (122 p,g of protein). The molecular masses of standard proteins (lanes 5 and 13)are given in kilodaltons. Acs, acetyl-CoA synthetase.

propionyl-CoA synthetase activity, respectively, than thewild type or the other mutants (data not shown). This resultindicates that the expression of acyl-CoA synthetase isincreased to compensate for the loss of acetyl-CoA syn-thetase.

Effect of alternate sigma factor o9" on the expression ofacetyl-CoA synthetase and on the utilization of propionate.Growth of wild-type strain H16 and of mutants H1098,

TABLE 2. Substrate specificity of acetyl-CoA synthetase ofA. eutrophus expressed in E. coli XLl-Bluea

Acyl-CoA synthetase activity(U/g of protein) with:

Plasmid IPTGAcetate Propionate 3-Hydroxy-propionate

pBluescript KS- - 64 <2 <2pBluescript KS- + 52 <2 <2pBluescript SK- - 55 <2 <2pBluescript SK- + 49 <2 <2pKES38 - 212 38 192pKES38 + 117 17 165pSES38 - 1,191 549 1,035pSES38 + 347 130 301

a Cells of recombinant strains of E. coli were grown for 12 h at 37C in LBmedium in the presence (+) or absence (-) of 1 mM IPTG. The acyl-CoAsynthetase activities were determined in soluble extracts at 30'C by themethod of Oberlies et al. (31) as described in Materials and Methods. Thecuvette contained 100 mM Tricine N-[tris(hydroxymethyl)methyljglycine-KOH buffer (pH 8.2), 1mM indicated substrate, 0.2mM coenzyme A, 1.5 mMATP, 2 mM glutathione, 2 mM MgCl2, 2 mM phosphoenolpyruvate, 0.4 mMNADH, 0.8 U of pyruvate kinase, 0.7 U of adenylate kinase, and 3 U oflactate dehydrogenase. One unit of enzyme activity is defined as the amountrequired for the conversion of 1 pmol of substrate per min.

H1053, H1068, H1050, and H1094 on propionate was accom-plished with solidified MM containing sodium propionate atconcentrations up to 0.2% (wtlvol); at a higher concentrationof propionate, growth was inhibited. In contrast, mutantsHF149, HF09, H1075, and N1231, which are defective in theipoN-like gene for the alternate sigma factor cr"4, did notutilize propionate as a sole carbon source for growth. Sincethe growth of mutants HF149 and H1075 on acetate was not

TABLE 3. Acyl-CoA synthetase activities in acetate-grown cellsof mutants of A. eutrophus, impaired in acetoin catabolism'

Acyl-CoA synthetase activity(U/g of protein) with:

Strain 3-Hydroxy- Aceto-Acetate onaPie Butyrate propi- ace-

onate tate

H16 (wild type) 337 616 471 283 90H1098 (acoR mutant) 251 535 472 251 101H1075 (rpoN mutant) 337 631 500 272 124HF149 (rpoN mutant) 330 697 514 277 98H1053 (acoC mutant) 286 570 476 323 81H1068 (acoD mutant) 309 624 480 309 127H1050 (acoE mutant) 23 677 743 50 138H1094 (acoE mutant) 17 606 639 60 123

a Cells were grown at 30°C in MM containing acetate as a carbon source tothe late exponential phase and harvested. Acyl-CoA synthetase activitieswere determined in soluble extracts at 30°C by the method of Oberlies et al.(31) as described in Materials and Methods. The cuvette contained 100 mMTricine-KOH buffer (pH 8.2), 1 mM indicated substrate, 0.2mM coenzyme A,1.5 mM ATP, 2 mM glutathione, 2 mM MgCl2, 2 mM phosphoenolpyruvate,0.4 mM NADH, 0.8 U of pyruvate kinase, 0.7 U of adenylate kinase, and 3 Uof lactate dehydrogenase. One unit of enzyme activity is defined as theamount required for the conversion of 1 pmol of substrate per min.

Acs _

Acs

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A B1 2 3 4 5 6 7 1 2 3 4 5 6 7

- Acs

FIG. 5. Functional expression ofacoE in E. coli. Cytoplasmic fractions obtained from recombinant strains of E. coli grown in LB mediumor in LB medium supplemented with 1 mM IPTG and fromA. eutrophus grown on acetate or fructose plus acetoin were separated in a 7.5%(wt/vol) polyacrylamide gel as described in Materials and Methods and stained for acetyl-CoA synthetase (A) and subsequently for proteinwith Coomassie brilliant blue (B). Lanes: 1,A. eutrophus H1050 (fructose plus acetoin) (228 ,g of protein); 2,A. eutrophus H16 (fructose plusacetoin) (203 p1g of protein); 3, A. eutrophus H1050 (acetate) (224 pg of protein); 4, A. eutrophus H16 (acetate) (170 ,g of protein); 5 and 6,E. coli XL1-Blue harboring plasmid pSES38 (without or with IPTG, respectively) (194 and 206 p.g of protein, respectively); 7, E. coliXL1-Blue harboring the vector pBluescript SK- (with IPTG) (153 p.g of protein). Acs, acetyl-CoA synthetase.

restricted and extracts from these cells exhibited acetyl-CoAsynthetase activity at the same level as the wild type, theexpression ofacoE is not dependent on RpoN. Furthermore,these cells exhibited propionyl-CoA synthetase activity atthe wild-type level, indicating that the expression of aprotein either for the uptake of propionate or for the conver-sion of propionyl-CoA to succinyl-CoA seems to be C"4dependent in A. eutrophus.

Effect ofacoE and of the rpoN-like gene on the accumulationof PHA. PHA are of biotechnological interest since they arebiodegradable thermoplastics that can be produced fromrenewable resources (45). Copolyesters of 3-hydroxybutyricacid (3HB) and 3-hydroxyvaleric acid (3HV) [poly(3HB-co-3HV)] are already produced on an industrial scale with A.eutrophus from a mixture of glucose and propionate (8).Since the enzymatic studies described above provided evi-dence that the acoE gene product uses propionate in additionto acetate as a substrate, we investigated the effect of thecopy number of acoE on the incorporation of 3HV intoPHA. When A. eutrophus H16 harboring plasmidpVK1O1::E and thus possessing an increased number ofacoE copies was cultivated under nitrogen-limiting condi-tions in MM with propionate as the sole carbon source, theextent of PHA accumulation as well as the composition ofthe accumulated poly(3HB-co-3HV) was not different fromthat in the parent strain. In addition, mutants H1050 andH1094 were not affected in the accumulation of PHA underthese conditions. These results clearly indicated that acoE isnot relevant for making propionate accessible for conversionto 3-hydroxyvaleryl-CoA and for incorporation as 3HV intopoly(3HB-co-3HV) in A. eutrophus under normal cultureconditions.Mutants defective in the rpoN-like gene, such as H1075,

N1231, HF09, and HF149, did not utilize propionate as asole carbon source for growth (see above). To study theeffect of this gene locus on the accumulation of poly(3HB-co-3HV), we grew the cells in nutrient broth medium,harvested them by centrifugation, and resuspended them ata density of approximately 1 g/liter in MM containing nonitrogen source and 0.5 or 1.0% (wt/vol) sodium propionateas the sole carbon source. The cells were cultivated aerobi-cally at 30°C for 48 h. Whereas strain H16 and mutantsdefective in acoXABC, acoD, or acoE accumulatedpoly(3HB-co-3HV) to approximately 70% (wt/wt) of thecellular dry matter, with a molar fraction of 3HV rangingfrom 50 to 70%, the rpoN mutants mentioned above accu-mulated poly(3HB-co-3HV) only to approximately 10% (wt/wt). However, the constituents occurred at almost the samemolar ratios. This result provides evidence against '54-dependent expression of a protein for the conversion ofpropionyl-CoA to succinyl-CoA in A. eutrophus. In combi-nation with the results of studies of the expression ofacyl-CoA synthetase (see above), these results stronglysuggest that the formation of a transport protein for propi-onate depends on the presence of the RpoN-like sigma factorin A. eutrophus. Evidence for the requirement of the rpoN-like gene for other transport processes has been alreadyobtained for A. eutrophus (38). Therefore, an additionalmarker for the rpoN-like gene different from those describedby Romermann et al. (39) was identified.

DISCUSSION

The acoE locus was identified as the structural gene of anacetyl-CoA synthetase ofA. eutrophus. The positions of TnSinsertions in two mutants of A. eutrophus impaired in the

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catabolism of acetoin were mapped within acoE. The mo-lecular data obtained for acoE and the amino acid sequencededuced from acoE were consistent with the enzyme data.acoE is not clustered with genes for other proteins that areessential for the utilization of acetoin in A. eutrophus, suchas the acoXABC operon or acoD (35, 36). Although asequence that exhibited only weak homology to the entero-bacterial consensus sequence for cr70-dependent promoterswas identified upstream of acoE, acoE is obviously ex-pressed by its own promoter in E. coli. The involvement ofthe alternate RpoN-like sigma factor cr54 in the expression ofacoE was excluded, since rpoN mutants of A. eutrophusexpressed acetyl-CoA synthetase during growth on acetate.The ribosome-binding site (AGGAG) upstream of acoEpermitted a high level of heterologous expression of acetyl-CoA synthetase in E. coli. Similar Shine-Dalgarno se-quences upstream of the translational initiation codons ofseveral A. eutrophus genes had been detected previously(for an overview, see reference 46).The amino acid sequence of the acetyl-CoA synthetase

deduced from acoE provided the second primary structurefor a bacterial acetyl-CoA synthetase. In total, four acetyl-CoA synthetases from prokaryotic or eukaryotic origin nowhave been cloned; they are highly homologous and share upto 50.5% identical amino acids. Certain regions of theseacetyl-CoA synthetases seem to be even more conserved(Fig. 3), such as, e.g., the putative ATP binding sites (1, 12).The extensive homologies among the acetyl-CoA syn-thetases ofA. eutrophus, M. soehngenii, A. nidulans, and N.crassa reflect the specificity of the enzymes. Althoughacetate is the preferred substrate of acetyl-CoA synthetases,propionate is also used, but at a diminished rate comparedwith that of acetate, by the enzymes of A. eutrophus (46%relative activity), yeast (60% [33]), and M. soehngenii (5%[23]). Fatty acids with more than three carbon atoms areusually not accepted as substrates by acetyl-CoA syn-thetases. Interestingly, yeast acetyl-CoA synthetase acceptsas a substrate 3-chloropropionic acid, which is similar in sizeto butyric acid, whereas butyric acid is not accepted. Theauthors postulated some favorable electrostatic interactionof the halogen atom with the substrate binding site of theenzyme (33). Perhaps the hydroxyl group of 3-hydroxypro-pionate has a similar effect and results in a relatively highactivity of the acetyl-CoA synthetase from A. eutrophuswith this substrate (87%).The growth ofA. eutrophus on propionate as a sole carbon

source occurred only at a low concentration of this substrate(0.2% [wt/vol]). This result is probably due to the toxic effectexerted by propionate and also by various other fatty acidsof medium chain length, as shown recently (26). Further-more, on the basis of indirect evidence, it may be assumedthat the presence of the RpoN-like sigma factor is necessaryfor the formation of the uptake protein for propionate in A.eutrophus. Propionate is then activated to propionyl-CoA byan acyl-CoA synthetase, which is different from the acoEgene product. However, overexpression of this acyl-CoAsynthetase may compensate for the loss of acetyl-CoAsynthetase in acoE mutants. Evidence for the presence ofmore than one protein exhibiting acetyl-CoA synthetaseactivity inA. eutrophus was obtained in previous studies (47,53). An investigation of the effect of acoE copy number onthe accumulation of PHA indicated that acoE is not requiredin vivo for the activation of propionate for subsequentincorporation as 3HV into poly(3HB-co-3HV) in A. eutro-phus under normal culture conditions. Further studies willfocus on the isolation and characterization of acyl-CoA

synthetases that are required for the activation of fatty acidsconsisting of three or more carbon atoms. These enzymesseem to be of special interest in order to control theincorporation of hydroxyalkanoic acids other than 3HB intothe storage compound PHA in A. eutrophus.

ACKNOWLEDGMENTSThis study was supported by a grant from the Deutsche Fors-

chungsgemeinschaft (Ste 386/3-2). H.P. received a scholarship fromthe Deutsche Gesellschaft fir Chemisches Apparatewesen, Chemis-che Technik und Biotechnologie e.V. (DECHEMA).

REFERENCES1. Albano, M., R. Breitling, and D. A. Dubnau. 1989. Nucleotide

sequence and genetic organization of the Bacillus subtilis comGoperon. J. Bacteriol. 171:5386-5404.

2. Armitt, S., W. McCullough, and C. F. Roberts. 1976. Analysis ofacetate non-utilizing (acu) mutants in Aspergillus nidulans. J.Gen. Microbiol. 92:263-282.

3. Beaucage, S. L., and M. H. Caruthers. 1981. Deoxynucleosidephosphoramidites-a new class of key intermediates for deoxy-polynucleotide synthesis. Tetrahedron Lett. 22:1859-1862.

4. Berg, P. 1962. Assay and preparation of yeast aceto-CoA-kinase. Methods Enzymol. 5:461-467.

5. Bibb, M. J., P. R. Findlay, and M. W. Johnson. 1984. Therelationship between base composition and codon usage inbacterial genes and its use for the simple and reliable identifi-cation of protein-coding sequences. Gene 30:157-166.

6. Brown, T. D. K., M. C. Jones-Mortimer, and H. L. Kornberg.1977. The enzymic interconversion of acetate and acetyl-coen-zyme A in Escherichia coli. J. Gen. Microbiol. 102:327-336.

7. Bullock, W. O., J. M. Fernandez, and J. M. Stuart. 1987.XL1-Blue: a high efficiency plasmid transforming recA Esche-richia coli strain with beta-galactosidase selection. BioTech-niques 5:376-379.

8. Byrom, D. 1991. Miscellaneous biomaterials, p. 333-359. In D.Byrom (ed.), Biomaterials. Macmillan Publishers Ltd., Basing-stoke, United Kingdom.

9. Connerton, I. F., J. R. S. Fincham, R. A. Sandeman, and M. J.Hynes. 1990. Comparison and cross-species expression of theacetyl-CoA synthetase genes of the ascomycete fungi, Aspergil-lus nidulans and Neurospora crassa. Mol. Microbiol. 4:451-460.

10. Davis, D. H., M. Doudoroff, and R. Y. Stanier. 1969. Proposal toreject the genus Hydrogenomonas: taxonomic implications. Int.J. Syst. Bacteriol. 19:375-390.

11. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehen-sive set of sequence analysis programs for the VAX. NucleicAcids Res. 12:387-395.

12. Eggen, R. I. L., A. C. M. Geerling, A. B. P. Boshoven, andW. M. de Vos. 1991. Cloning, sequence analysis, and functionalexpression of the acetyl coenzyme A synthetase gene fromMethanothrix soehngenii in Escherichia coli. J. Bacteriol. 173:6383-6389.

13. Flavell, R. B., and J. R. S. Fincham. 1968. Acetate-nonutilizingmutants ofNeurospora crassa. I. Mutant isolation, complemen-tation studies, and linkage relationships. J. Bacteriol. 95:1056-1062.

14. Friedrich, B., C. Hogrefe, and H. G. Schlegel. 1981. Naturallyoccurring genetic transfer of hydrogen-oxidizing ability betweenstrains ofAlcaligenes eutrophus. J. Bacteriol. 147:198-205.

15. Frind, C., H. Priefert, A. Steinbuichel, and H. G. Schlegel. 1989.Biochemical and genetic analysis of acetoin catabolism in Al-caligenes eutrophus. J. Bacteriol. 171:6539-6548.

16. Hanahan, D. 1983. Studies on transformation ofEschenchia coliwith plasmids. J. Mol. Biol. 166:557-580.

17. Hanna, Z., C. Fregeau, G. Prefontaine, and R. Brousseau. 1984.Construction of a family of universal expression plasmid vec-tors. Gene 30:247-250.

18. Hawley, D. K., and W. R. McClure. 1983. Compilation andanalysis of Escherichia coli promoter DNA sequences. NucleicAcids Res. 11:2237-2255.

J. BACTERIOL.

on July 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ACETYL-CoA SYNTHETASE GENE OF A. EUTROPHUS 6599

19. Hogrefe, C., D. Romermann, and B. Friedrich. 1984. Alcali-genes eutrophus hydrogenase genes (Hox). J. Bacteriol. 158:43-48.

20. Inoue, T., M. Sunagawa, A. Mori, C. Imai, M. Fukuda, M.Takagi, and K. Yano. 1989. Cloning and sequencing of the geneencoding the 72-kilodalton dehydrogenase subunit of alcoholdehydrogenase from Acetobacter aceti. J. Bacteriol. 171:3115-3122.

21. Jendrossek, D., A. Steinbuichel, and H. G. Schlegel. 1987. Threedifferent proteins exhibiting NAD-dependent acetaldehyde de-hydrogenase activity from Alcaligenes eutrophus. Eur. J. Bio-chem. 167:541-548.

22. Jendrossek, D., A. Steinbfuchel, and H. G. Schlegel. 1988. Alco-hol dehydrogenase gene from Alcaligenes eutrophus: subclon-ing, heterologous expression in Escherichia coli, sequencing,and location of Tn5 insertions. J. Bacteriol. 170:5248-5256.

23. Jetten, M. S. M., A. J. M. Stams, and A. J. B. Zehnder. 1989.Isolation and characterization of acetyl-coenzyme A synthetasefrom Methanothrix soehngenii. J. Bacteriol. 171:5430-5435.

24. Jorgensen, R. A., S. J. Rothstein, and W. S. Reznikoff. 1979. Arestriction enzyme cleavage map of TnS and location of a regionencoding neomycin resistance. Mol. Gen. Genet. 177:65-72.

25. Kriiger, N., and A. Steinbuchel. 1992. Identification of acoR, aregulatory gene for the expression of genes essential for acetoincatabolism in Alcaligenes eutrophus H16. J. Bacteriol. 174:4391-4400.

26. Liebergesell, M., E. Hustede, A. Timm, A. Steinbuchel, R. C.Fuller, R. W. LeAnz, and H. G. Schlegel. 1991. Formation ofpoly(3-hydroxyalkanoates) by phototrophic and chemolithotro-phic bacteria. Arch. Microbiol. 155:415-421.

27. Lienhard, G. E., and I. I. Secemski. 1973. P1,P5-di(adenosine-5)pentaphosphate, a potent multisubstrate inhibitor of adenylatekinase. J. Biol. Chem. 248:1121-1123.

28. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

29. Lundie, L. L., Jr., and J. G. Ferry. 1989. Activation of acetateby Methanosarcina thermophila. J. Biol. Chem. 264:18392-18396.

30. Nimmo, H. G., and G. A. Nimmo. 1982. A general method forthe localization of enzymes that produce phosphate, pyrophos-phate, or CO2 after polyacrylamide gel electrophoresis. Anal.Biochem. 121:17-22.

31. Oberlies, G., G. Fuchs, and R. K. Thauer. 1980. Acetatethiokinase and the assimilation of acetate in Methanobacteriumthermoautotrophicum. Arch. Microbiol. 128:248-252.

32. Oppermann, F. B., B. Schmidt, and A. Steinbuchel. 1991.Purification and characterization of acetoin:2,6-dichlorophe-nolindophenol oxidoreductase, dihydrolipoamide dehydroge-nase, and dihydrolipoamide acetyltransferase of the Pelobactercarbinolicus acetoin dehydrogenase enzyme system. J. Bacte-riol. 173:757-767.

33. Patel, S. S., and D. R. Walt. 1987. Substrate specificity of acetylcoenzyme A synthetase. J. Biol. Chem. 262:7132-7134.

34. Peoples, 0. P., and A. J. Sinskey. 1989. Poly-p-hydroxybutyratebiosynthesis in Alcaligenes eutrophus H16. Characterization ofthe genes encoding 1-ketothiolase and acetoacetyl-CoA reduc-tase. J. Biol. Chem. 264:15293-15297.

35. Priefert, H., S. Hein, N. Kriger, K. Zeh, B. Schmidt, and A.Steinbuchel. 1991. Identification and molecular characterizationof the Alcaligenes eutrophus H16 aco operon genes involved inacetoin catabolism. J. Bacteriol. 173:4056-4071.

36. Priefert, H., N. Kriger, D. Jendrossek, B. Schmidt, and A.Steinbfichel. 1992. Identification and molecular characterizationof the gene coding for acetaldehyde dehydrogenase II (acoD) ofAlcaligenes eutrophus. J. Bacteriol. 174:899-907.

37. Pries, A., H. Priefert, N. Krtiger, and A. Steinbuchel. 1991.Identification and characterization of twoAlcaligenes eutrophus

gene loci relevant to the poly(P-hydroxybutyric acid)-leakyphenotype which exhibit homology to ptsH and ptsI of Esche-richia coli. J. Bacteriol. 173:5843-5853.

38. Romermann, D., M. Lohmeyer, C. G. Friedrich, and B.Friedrich. 1988. Pleiotropic mutants fromAlcaligenes eutrophusdefective in the metabolism of hydrogen, nitrate, urea, andfumarate. Arch. Microbiol. 149:471-475.

39. Romermann, D., J. Warrelmann, R. A. Bender, and B.Friedrich. 1989. An rpoN-like gene of Alcaligenes eutrophusand Pseudomonas facilis controls expression of diverse meta-bolic pathways, including hydrogen oxidation. J. Bacteriol.171:1093-1099.

40. Rose, I. A., M. Grunberg-Manago, S. R. Korey, and S. Ochoa.1954. Enzymatic phosphorylation of acetate. J. Biol. Chem.211:737-756.

41. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

42. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

43. Schlegel, H. G., H. Kaltwasser, and G. Gottschalk. 1961. EinSubmersverfahren zur Kultur wasserstoffoxidierender Bakte-rien: wachstumsphysiologische Untersuchungen. Arch. Mikro-biol. 38:209-222.

44. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host rangemobilization system for in vivo genetic engineering: transposonmutagenesis in gram-negative bacteria. Bio/Technology 1:784-791.

45. Steinbuchel, A. 1991. Polyhydroxyalkanoic acids, p. 123-213. InD. Byrom (ed.), Biomaterials. Macmillan Publishers Ltd., Bas-ingstoke, United Kingdom.

46. Steinbuchel, A. 1991. Recent advances in the knowledge ofbacterial poly(3-hydroxyalkanoic acid) metabolism and poten-tial impacts on the production of biodegradable thermoplastics.Acta Biotechnol. 11:419-427.

47. Steinbuchel, A., C. Frund, D. Jendrossek, and H. G. Schlegel.1987. Isolation of mutants of Alcaligenes eutrophus unable toderepress the fermentative alcohol dehydrogenase. Arch. Mi-crobiol. 148:178-186.

48. Strauss, E. C., J. A. Kobori, G. Siu, and L. E. Hood. 1986.Specific-primer-directed DNA sequencing. Anal. Biochem. 154:353-360.

49. Thaner, R. K., D. Moller-Zinkhan, and A. M. Spormann. 1989.Biochemistry of acetate catabolism in anaerobic chemotrophicbacteria. Annu. Rev. Microbiol. 43:43-67.

50. Timm, A., and A. Steinbuichel. 1990. Formation of polyestersconsisting of medium-chain-length 3-hydroxyalkanoic acidsfrom gluconate by Pseudomonas aeruginosa and other fluores-cent pseudomonads. Appl. Environ. Microbiol. 56:3360-3367.

51. Tinoco, I., P. N. Borer, B. Dengler, M. D. Levine, 0. C.Uhlenbeck, D. M. Crothers, and J. Gralla. 1973. Improvedestimation of secondary structure in ribonucleic acids. Nature(London) New Biol. 246:40-41.

52. Tran-Betcke, A., U. Warnecke, C. Bocker, C. Zaborosch, and B.Friedrich. 1990. Cloning and nucleotide sequences of the genesfor the subunits of NAD-reducing hydrogenase of Alcaligeneseutrophus H16. J. Bacteriol. 172:2920-2929.

53. Truiper, H. G. 1965. Tricarboxylic acid cycle and related en-zymes in Hydrogenomonas strain H16G+ grown on variouscarbon sources. Biochim. Biophys. Acta 111:565-568.

54. Vieira, J., and J. Messing. 1982. The pUC plasmids, anM13mp7-derived system for insertion mutagenesis and sequenc-ing with synthetic universal primers. Gene 19:259-268.

55. Wilde, E. 1962. Untersuchungen uber Wachstum und Speicher-stoffsynthese von Hydrogenomonas. Arch. Mikrobiol. 43:109-137.

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