4
ELSEVIER Biochimica et Biophysica Acta 1219 (1994) 559-562 BB Biochi~ic~a et Biophysica A~ta Short Sequence-Paper Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12 Asahi Matsuyama a,,, Hideko Yamamoto-Otake a, Jeff Hewitt b, Ross T.A. MacGillivray Eiichi Nakano a a Kikkoman Corporation Research and Development Division, 399 Noda, Noda-city, Chiba, 278, Japan b Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada Received 20 May 1994 b Abstract The phosphotransacetylase gene (pta) from Escherichia coli strain K-12 1100 was identified in a cloned fragment of chromosomal DNA (Yamamoto-Otake, H., Matsuyama, A. and Nakano, A. (1990) Appl. Microbiol. Biotechnol. 33, 680-682). Overexpression in E. coli confirmed the presence of the pta gene within the cloned fragment. DNA sequence analysis of the cloned pta gene indicates that the predicted phosphotransacetylase polypeptide chain is 713 amino acids in length. The carboxyterminal region of the E. coli phosphotransacetylase shows 42.6% sequence identity with the corresponding enzyme from Methanosarcina thermophila (142 out of 333 residues in corresponding positions are identical). Several short regions of high sequence identity may be structurally or functionally important for enzymic activity. Keywords: Phosphotransacetylase; DNA sequence; (E. coli) Phosphotransacetylase (EC 2.3.1.8) reversibly cat- alyzes the following reaction: acetyl CoA + phosphate. • CoA + acetyl phosphate The acetate kinase-phosphotransacetylase (AckA-PTA) system has been identified in the Enterobacteriaceae [1,2] and is thought to play an amphibolic role in the excretion and activation of acetate [3,4]. Coenzyme A thioesters serve as substrates in a large variety of enzyme-catalyzed reactions with acetyl CoA being cen- tral in biological acetylation reactions. Furthermore, the high energy compound acetyl phosphate may began important signal in cross-regulation of carbon and phosphate metabolism [5,6]. To obtain a further understanding of the biological and physiological roles of the AckA-PTA system, we have initiated structural studies of the genes coding for the proteins within these pathways [7,8]. Previously, we * Corresponding author. The nucleotide sequence data presented in this paper have been submitted to the EMBL/GenBank data base under the accession number D21123. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4781(94)00150-2 used functional complementation of a phospho- transacetylase deficient strain of E. coli to isolate a 3.2 kbp fragment of DNA in pAK222 [7]. Functional assays revealed that E. coli strain 1100 containing pAK222 expressed increased levels of both acetate kinase and phosphotransacetylase suggesting that both the ackA and pta genes were located on the 3.2 kbp fragment of E. coli genomic DNA [7]. The nucleotide sequence of the ackA gene and promoter have been determined [8]. As the pta gene is located immediately down- stream of the ackA gene [7], these two genes may comprise all or part of an operon. We now report the nucleotide sequence of the remaining 2.7 kbp of cloned DNA on pAK222 [7]. To confirm the presence of the pta gene, a 2.7 kbp KpnI-SmaI fragment was isolated from pAK222 [7], and ligated into the plasmids pUCll8 and pUCll9. The resulting plasmids (pPT400 and pPT410, respec- tively) contain the 2.7 kbp fragment cloned in opposite orientations with respect to the lac promoter on the vectors. The plasmids pPT400 and pPT410 were then transformed into E. coli JM109 [9] and cell cultures (10 ml) were grown at 30°C to early stationary phase in LB medium containing ampicillin (25 ~g/ml). Phospho-

Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12

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Page 1: Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12

ELSEVIER Biochimica et Biophysica Acta 1219 (1994) 559-562

BB Biochi~ic~a et Biophysica A~ta

Short Sequence-Paper

Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12

Asahi Matsuyama a,,, Hideko Yamamoto-Otake a, Jeff Hewitt b, Ross T.A. MacGillivray Eiichi Nakano a

a Kikkoman Corporation Research and Development Division, 399 Noda, Noda-city, Chiba, 278, Japan b Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada

Received 20 May 1994

b

Abstract

The phosphotransacetylase gene (pta) from Escherichia coli strain K-12 1100 was identified in a cloned fragment of chromosomal DNA (Yamamoto-Otake, H., Matsuyama, A. and Nakano, A. (1990) Appl. Microbiol. Biotechnol. 33, 680-682). Overexpression in E. coli confirmed the presence of the pta gene within the cloned fragment. DNA sequence analysis of the cloned pta gene indicates that the predicted phosphotransacetylase polypeptide chain is 713 amino acids in length. The carboxyterminal region of the E. coli phosphotransacetylase shows 42.6% sequence identity with the corresponding enzyme from Methanosarcina thermophila (142 out of 333 residues in corresponding positions are identical). Several short regions of high sequence identity may be structurally or functionally important for enzymic activity.

Keywords: Phosphotransacetylase; DNA sequence; (E. coli)

Phosphotransacetylase (EC 2.3.1.8) reversibly cat- alyzes the following reaction:

acetyl CoA + phosphate. • CoA + acetyl phosphate

The acetate kinase-phosphotransacetylase (AckA-PTA) system has been identified in the Enterobacteriaceae [1,2] and is thought to play an amphibolic role in the excretion and activation of acetate [3,4]. Coenzyme A thioesters serve as substrates in a large variety of enzyme-catalyzed reactions with acetyl CoA being cen- tral in biological acetylation reactions. Furthermore, the high energy compound acetyl phosphate may began important signal in cross-regulation of carbon and phosphate metabolism [5,6].

To obtain a further understanding of the biological and physiological roles of the AckA-PTA system, we have initiated structural studies of the genes coding for the proteins within these pathways [7,8]. Previously, we

* Corresponding author. The nucleotide sequence data presented in this paper have been submitted to the EMBL/GenBank data base under the accession number D21123.

0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 4 ) 0 0 1 5 0 - 2

used functional complementation of a phospho- transacetylase deficient strain of E. coli to isolate a 3.2 kbp fragment of DNA in pAK222 [7]. Functional assays revealed that E. coli strain 1100 containing pAK222 expressed increased levels of both acetate kinase and phosphotransacetylase suggesting that both the ackA and pta genes were located on the 3.2 kbp fragment of E. coli genomic DNA [7]. The nucleotide sequence of the a c k A gene and promoter have been determined [8]. As the pta gene is located immediately down- stream of the a c k A gene [7], these two genes may comprise all or part of an operon. We now report the nucleotide sequence of the remaining 2.7 kbp of cloned DNA on pAK222 [7].

To confirm the presence of the pta gene, a 2.7 kbp KpnI-SmaI fragment was isolated from pAK222 [7], and ligated into the plasmids pUCll8 and pUCll9. The resulting plasmids (pPT400 and pPT410, respec- tively) contain the 2.7 kbp fragment cloned in opposite orientations with respect to the lac promoter on the vectors. The plasmids pPT400 and pPT410 were then transformed into E. coli JM109 [9] and cell cultures (10 ml) were grown at 30°C to early stationary phase in LB medium containing ampicillin (25 ~g/ml). Phospho-

Page 2: Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12

560 A. Matsuyama et al. / Biochimica et Biophysica Acta 1219 (1994) 559-562

(M) 1

CTGCCTGATTTCACACGCCAGCTCAGCTGTGTGTTTTGTAACCCGCCAAATCGGCGTTAACGAAAGAGGATAAACCGTGT 80

S R l I M L l P T G T S V G L T S V A W R D P C N G T 28

CCCGTATTATTATGCTGATCCC TACCGGAACCAGCGTCGGTCTGACCAGCGTAGCTTGGCGTGATCCGTGCAATGGAACG 160

Q R R S S E R F Q T Y R S A A Y R W R C A D Q T T T I 55

•AAAGGCGTT•GTCTGAGCGTTTTCAAA•CTATCG•TCAGC•GCGTACCGGTGGCGATGCGCCGATCAGACTACGACTAT 240

V R A N S S T T T A A E P L K M S Y V E G L L S S N 81

CGTG•GTGCGAACTCTTCCACCACGACGGCCGCTGAACCGCTGAAAATGAGCTACGTTGAAGGTCTGCTTTCCAGCAATC 320

Q K D V L M E E I V A N Y H A N T K D A E V V L V E G 108

AGAAAGATGTGCTGATGGAAGAGATCGTCGCAAACTACCACGCTAACACCAAAGACGCTGAAGTCGTTCTGGTTGAAGGT 400

L V P T E K H Q F A Q S L N Y E I A K T L N A E I V F 135

CTGGTCC•GACACGTAAGCA••AGTTTG•CCAGTCTCTGAACTACGAAATCGCTAAAACGCTGAATGCGGAAATCGTCTT 480

V M S Q G T D T P E Q L K E R I E L T R N S F G G A 161

CGTTATGTCTCAGGGCACTGACACCCCGGAACAGCTGAAAGAGCGTATCGAACTGACCCGCAACAGCTTCGGcGGTGCCA 560

K N T N I T G V I V N K L N A P V D E Q G R T R P D L 188

AAAACAC•AACAT•ACCGGCGTTATCGTTAACAAA•TGAACGCA••GGTTGATGAACAGGGTCGTACTCGCCCGGATCTG 640

S E I F D D S S K A K V N N V D P A N V Q E S S P L P 215

TCCGAGAT TTTCGACGACTCTTCCAAAGCTAAAGTAAACAATGTTGATCCGGCGAACGTGCAAGAATCCAGCCCGCTGCC 720

V L G A V P W S F D L I A T R A I D M A R H L N A T 241

GGTTCT•GGCGCTGTGCCGTGGAGCTTTGAC•TGAT•GCGACTCGTGCGATCGATATGGCTCGCCACCTGAATGCGACCA 800 I I N E G D I N T R R V K S V T F C A R Q H S A H A G 268

TCATCAACGAAGG•GA•ATCAATACTCGCCGCGTTAAATCCGT•A•TTTCTGCGCACGCCAGCATTCCGCA•ATGCTGGA 880

A L R A G S L L V T S A D R P D V L V A A C L A A M N 295

GCACTTCGTGCCGGTTCTCTGCTGGTGACTTCCGCAGACCGTCCTGACGTGCTGGTGGCCGC TTGCCTGGCAGCCATGAA 960

G V E I G A L L L T G G Y E M D A R l S K L C E R A 321

CGGCGTAGAAATCGGTGCCCTG•TGCTGACTGGCGGTTACGAAATGGACGCGCG•ATTTCTAAACTGTGCGAACGTG•TT i040

F A T G L P V F M V N T N T W Q T S L S L Q S F N L E 348

TCG•TACCGGCCTGCCGGTATTTATGGTGAACA•CAACAC•TGGCAGACCTCTCTGAGCCTGCAGAGCTTCAACCTGGAA 1120

V P V D D H E R I E K V Q E Y V A N Y I N A D W l E S 375

GTTCCGGTTGACGATCACGAAC GTATCGAGAAAGT TCAGGAATACGTTGCTAACTACATCAACGC TGACTGGATCGAATC 1200

L T A T S E R S R R L S P P A F R Y Q L T E L A R K 401

TCTGACTGCCACTTCTGAGCGCAGCCGTCGTCTGTCTCCGCCTGCGTTCCGTTATCAGCTGA•TGAA•TTGCGCGCAAAG !280

A G K R I N L P E G D E P R T V K A A A I C A E R G I 428

CGGGCAAACGTATCGTACTGCCGGAAGGTGACGAACCGCGTACCGT TAAAGCAGCCGCTATCTGTGC TGAACGTGGTATC 1360

A T C V L L G N P A E I N R V A A S Q G V E L G A G I 455

GCAACTTGCGTACTGCTGGG TAATCCGGCAGAGATCAACCGTGTTGCAGCG TC TCAGGGTGTAGAACTGGGTGCAGGGAT 1440

E I V D P E V V R E S Y V G R L V E L R K N K G M T 481

TGAAATCGTTGATCCAGAAGTGGTTCGCGAAAGCTATGT TGGTCG TCTGGTCGAACTGCGTAAGAACAAAGGCATGACCG 1520

E T V A R E Q L E D N V V L G T L M L E Q D E V D G L 508

AAACCGT TGCCCGCGAACAGCTGGAAGACAACGTGGTGCTCGGTACGC TGATGCTGGAACAGGATGAAGTTGATGGTCTG 1600

V E G A V H T T A N T I R P P L Q L I K T A P G S S L 535

GTTTC•GGTGCTGTTCACACTA•CGCAAA•A•CATC•GT•CG•CGCTGCAG•TGATCAAAA•TG•AC•GGG•AG•TC•CT 1680

V S S V F F M L L P E Q V Y V Y G D C A I N P D P T 561

GGTATcTT••GTGTTCTTCATGCTGCTGCCGGAA•AGGTTTACGTTTACGGTGA•TGTGCGATCAAc•CGGAT•CGA•CG 1760

A E Q L A E I A I Q S A D S A A A F G I E P R V A M L 588

CTGAACAGCTGGCAGAAATCGCGATTCAGTCCGCTGATTCCGCTGCGGC•TTCGGTATCGAACCGCGCGTTGCTATGCTC 1840

S Y S T G T S G A G S D V E K V R E A T R L A Q E K R 615

TCCTACTCCACCGGTACTTCTGGTGCAGGTAGCGACGTAGAAAAAGTTCGCGAAGCAACTCGT•TGGCGCAGGAAAAACG 1920

P D L M I D G P L Q Y D A A V M A D V A K S K A P N 641

TC•TGACCTGATGATCGACGGTCCGCTGCAGTACGACGCTGCGGTAATGGCTGACGTTGCGAAATC•AAAG•GCCGAACT 2000

S P V A G R A T V F I F P D L N T G N T T Y K A V Q R 668

CTCCGGTTGCAGGTCGCGCTACCGTGTTCATCTTCCCGGATCTGAACACCGGTAACACCACCTACAAAGCGGTACAGCGT 2080

S A D L I S I G P M L Q G M R K P V N D L S R G A L V 695

TCTGCCGACCTGATC TCCATCGGGCCGATGCTGCAGGGTATGCGCAAGCCGGTTAACGACCTGTCCCGTGGCGCACTGGT 2160

D D I V Y T I A L T A I Q S A Q Q Q * 713

TGACGATATCGTCTA•ACCATCGCG•TGA•TGCGATTCAGTCTGCACAGCAGCAGTAATCTCGTCAT•ATCGCAGCTTTG 2240

CGCTG~GGATATCTGAACCGGA~u~TAATCAC~TTT~GGTTTTTTATTCTCTTAATTTGCATTAATCCTTTCTGATTAT 2320

CTTGCTTAACTGCGC TGCATCAATGAATTGCGCCATCCCACTTTGCATACTTACCACTTTGT TTTGTGCAAGGGAATATT 2400

TGCGCTATGTCCGCAATCACTGAATCCAAACCAACAAGAAGATGGGCAATGCCCGATACGTTGGTGAT TATCT TTTTTGT 2480

TGCTATTTTAACCAGCCTTGCCACCTGGGTAGTTCCGGTGGGGATGTTTGACAGTCAGGAAGTGCAGTATCAGGTTGATG 2560

GTCAAACAAAAACACGCAAAGTCGTAGATCCAAAACCCATTTCCCGGCATTCTGACACCTAACCGAAACAGGCCCGAACC 2640

CTGAAGTATCAACCCGCCGATCAGCTGTTCACGACGGCCGATGAACGCCCCGGG 2694

Page 3: Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12

A. Matsuyama et al. / Biochimica et Biophysica Acta 1219 (1994) 559-562 561

388 400 440

E. coli p PAFRYQLTALARKAGKRIVLPE GDE PRTVKAAAI CAE RG IATCVLLGNPAE I NRVAASQGVE LGAG I E IVD PEVVRE SYVGRLV * * * * * *** ** *** ***** ** ** * * * * *

M. therm. MVTFLEKI SERAKKLNKTIALPE TED I RTLQAAAKI LE RGIAD IVLVGNEAD I KALAGDLDLSKAKIVD PKTYE KK-DEY INAFY 1 40 80

480 520

E. coli E LRKNKGMTE TVARE QLE DNVVLGTI/4LE QD EVDGLVS GAVH TTAN T I RPPLQL I KTAPGS SLVS SVFFMLLPE QVY ...... VY

M. therffg E LRKHKG I TLE NAAE IMSDYVY FAVI4MAKLGEVDGVVSGAAHS S SD TLRPAVQ IVKTAKGAALASAFFI I SVPDCE YGSDGT FLF 120 160

560 600

E. coli GDCAIN pD PTAE QLAE IAI QSAD SAAAFG I E - PRVAMLSY S TGT S GAGSDVE KVREATRLAQE KRPD I/4I DGPLQYDAAVMADVA

M. therm. AD SGIvlVE MPSVE DVAN IAVI SAKTFE LLVQDVPKVAMLSY S TKGSAKS KLTEAT IAS TKLAQE LAPD IAI DGE LQVDAAIVPKVA

200 240

640 680 713

E. coil KSKAPNS PVAGRATVFI FPDLN TGNT TYKAVQRSAD L I S I G ~ D LSRGALVDD IVY T IAL TAI QSAQQQ

M. therr, L AS KAPGS PVAGKANVF I FPD LNCGN IAYK IAQRLAKAEAYGP I TQGLAKP INDLSRGC SD E D IVGAVAI TCVQAAAQDK 280 320 333

Fig. 2. Alignment of the amino acid sequences of the phosphotransacetylase genes from E. coli and M. thermophila. Residues 388-713 of the E. coli enzyme are aligned with residues 1-333 of the M. thermophila enzyme. To maximize the sequence identity, gaps ( - ) have been inserted into the sequences. Identical residues in corresponding positions are denoted by an asterisk (*).

transacetylase activity was assayed by measuring acetyl CoA formation from acetyl phosphate and CoA as described previously [4,7]. The results are shown in Table 1. E. coli containing the vector alone had negli- gible phosphotransacetylase activity whereas E. coli containing pAK222 had 41.1-fold higher activity. E. coli containing pPT400 had a 5-fold higher phospho- transacetylase activity than the vector alone; the activ- ity was inducible with isopropyl-D-thiogalactopyranso- side (IPTG) suggesting that the expression of the pta gene in pPT400 was driven by the lac promoter of pUC. In contrast, phosphotransacetylase activity in E. coli containing pPT410 was low and unchanged by induction with IPTG. From these experiments, it was concluded that pPT400 contained the pta gene in the correct orientation for expression with the lac pro- moter. In addition, the low amount of phosphotrans- ferase activity in pPT400 compared to pAK222 sug- gests that the pta gene in pPT400 does not contain an endogenous promoter. This is consistent with the ackA and pta genes in pAK222 being co-transcribed from the ackA promoter.

To determine the complete nucleotide sequence of the 2.7 kbp KpnI-SmaI fragment in pPT400/410, sev- eral synthetic oligodeoxyribonucleotides were used as primers. The complete nucleotide sequence was deter- mined on both strands and is shown in Fig. 1. To identify the correct reading frame for the pta gene, a sample of phosphotransacetylase was purified from a cell extract of E. coli strain 1100 containing pAK222,

and subjected to amino-terminal sequence analysis in a liquid phase protein Sequenator. The N-terminal se- quence was: X-X-I-I-M-L-I-P. Although no residues could be assigned to the first two positions, the remain- ing sequence is consistent with the GTG codon at position 77 (Fig. 1) being used as the initiator methion- ine codon. In that case, the phophotransacetylase polypeptide consists of 713 amino acids with a pre- dicted molecular weight of 77 160. This is in good agreement with the observed molecular weight of phos- photransacetylase (81000) determined by SDS-gel elec- trophoresis (data not shown).

Recently, the nucleotide sequences of the pta and ack genes from Methanosarcina thermophila have been reported [10]. In this methane-producing acetotroph, the two genes are also adjacent to each other, with the pta gene upstream of the ack gene. The predicted acetate kinase polypeptides from M. thermophila [10] and E. coli [8] are comprised of 408 and 400 amino acids, respectively; these polypeptides share 44% se- quence identity [10]. In contrast, the phospho- transacetylase polypeptide from M. thermophila is comprised of 333 amino acids which is about half of the size of the E. coli enzyme (713 amino acids, see Fig. 1). However, alignment of the carboxy-terminal region of the E. coli phosphotransacetylase with the M. thermophila phosphotransferase [10] reveals exten- sive sequence identity (Fig. 2). After the insertion of seven gaps into the E. coli sequence and a single gap into the M. thermophila sequence, the two poly-

Fig. 1. Nucleotide sequence of the pta gene from E. coli. The nucleotide sequence is numbered from the nucleotide immediately 3' to the end of the Ack,4 sequence reported earlier [8]. The deduced amino acid sequence of phosphotransacetylase is shown above the nucleotide sequence using the one letter code. The proposed Shine-Dalgarno sequence is underlined, and an asterisk denotes the putative stop codon. Following the stop codon, a palindromic sequence is indicated by double arrows; this sequence may function as a transcriptional terminator signal.

Page 4: Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12

562 A. Matsuyama et al. /Biochimica et Biophysica Acta 1219 (1994) 559-562

Table 1 Phosphotransacetylase activity in Escherichia coil K12 JM109 cells containing different plasmids

Plasmid Addition a Specific activity b (units/mg protein)

pUC118 none 2.0 pAK222 none 88.1 pPT400 none 10.2

IPTG 206.2 pPT410 none 5.4

IPTG 5.0

a IPTG was added to the medium at an initial concentration of 1 mM. b All values are the average of two experiments.

p e p t i d e s sha re 42.6% sequence iden t i ty (142 out of 333 res idues in co r r e spond ing pos i t ions are ident ical) . Wi th in these homologous sequences are severa l shor t reg ions of very high sequence iden t i ty such as r e s idues 415-440 ( 1 6 / 2 6 res idues a re ident ical ) , r e s idues 5 8 3 - 595 (10 /13 ) , r es idues 610-629 (14 /20) , res idues 635 - 671 ( 2 7 / 3 7 ) and res idues 676-692 (12 /17) . This se- quence conserva t ion suggests tha t these regions may be i m p o r t a n t for the s t ruc ture a n d / o r funct ion of the p h o p h o t r a n s a c e t y l a s e po lypep t ide . The s ignif icance of the N- t e rmina l ha l f of the E. coli p h o s p h o t r a n s a c e t y - lase is unc l ea r at p resen t .

In E. coli, the reg ion be tween the a c k A gene and the pta gene is 76 bp in length (Fig. 1). Wi th in this in t e rgen ic region, no consensus p r o m o t e r sequences (such as T T G A C A at pos i t ion - 3 5 and T A T A A T at - 10 [11]) were found. Similari ly, no sequences s imilar to the ca tabo l ic r ep re s so r p ro t e in -b ind ing si te ( A A N T -

G T G A N 2 T N 4 C A [12] were found. The close proximi ty of the a c k A and pta genes t o g e t h e r with the conce r t ed ac t ions of the i r gene p roduc t s in the p roduc t i on and excre t ion of ace ta t e suggests tha t the a c k A and pta

genes may compr i se all or pa r t of an ope ron ; however , t r ansc r ip t m a p p i n g would be r equ i r ed to conf i rm this. Lastly, a pe r fec t 11 bp p a l i n d r o m i c sequence is found fol lowing the s top codon (Fig. 1); this s equence p roba - bly compr i ses a t r ansc r ip t ion t e rmina to r .

The au thors wish to thank Hung Vo for helpful t echnica l suggest ions.

R e f e r e n c e s

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(1954) J. Biol. Chem. 211,737-756. [3] Sanwal, B.D. (1970) Bacteriol. Rev. 34, 20-39. [4] Brown, T.D.K., Jones-Mortimer, M.C. and Kornberg, H.C.

(1977) J. Gen. Microbiol. 102, 327-336. [5] Wanner, B.L. (1992) J. Bacteriol. 174, 2053-2058. [6] Feng, J., Atkinson, M.R., McCleary, W., Stock, J.B., Wanner,

B.L. and Ninfa, A.J. (1992) J. Bacteriol. 174, 6061-6070. [7] Yamamoto-Otake, H., Matsuyama, A. and Nakano, E. (1990)

Appl. Microbiol. Biotechnol. 33, 680-682. [8] Matsuyama, A., Yamamoto, H. and Nakano, E. (1989) J. Bacte-

riol. 171,577-580. [9] Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene 33,

103-119. [10] Latmer, M.T. and Ferry, J.G. (1993) J. Bacteriol. 175, 6822-6829. [11] Rosenberg, M. and Court, D. (1979) Annu. Rev. Genet. 13,

319-353. [12] Ebright, R.H. (1982) in Molecular Structure and Biological

Activity (Griffin, J.P. and Duax, W.L., eds.), pp. 91-99, Elsevier, New York.