Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B

FEMS MicrobiologyLetters 70 (1990) 7-14 Published by Elsevier

7

FEMSLE 04033

Cloning and nucleotide sequences of the rndh and sucD genes from Thermus aquaticus B D a v i d J. Nicholls, T r i c h u r K. S u n d a r a m l, T o n y A t k i n s o n and N i g e l P. M i n t o n Division of Biotechnology, Centrefor Applied Microbiologyand Research, Porton. Salisburyand t Departmentof Biochemistry and Applied MolecularBtology, Universityof ManchesterInstitute of Scienceand Technology, Manchester, U.K.

Received22 February 1990 Accepted6 March 1990 Key words: Malate dehydrogenase; SuccinyI-CoA synthetase; Thermus; Nucleotide sequence; Amino acid sequence; Enzyme isolation

1. S U M M A R Y A 3 kb D N A fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coil and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus ]?avus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same o!~eron in T. aquaticus B. Expression of the 7: aquati¢us B mdh gene in E. coil was found to be at a relatively low level. A simple method for purification of thermostable M D H from the E. coil clone containing the T. aquaticus B mdh gene is presented.

Correspm;dence to: David J. Nicholls, Divisionof Biotechnology, Centre for Applied Microbiologyand Research, Porton, Salisbury, U.K.

2. I N T R O D U C T I O N Malate dehydrogenase ( M D H ; L-malate: N A D + oxidoreductase, EC 1.1.1.37), an enzyme of the tricarboxylic acid (TCA) cycle, catalyses the oxidation of malate to oxaloacetate with the generation of N A D H . Several enzymes involved in metabolic pathways closely related to the T C A cycle have been demonstrated in Theemus spp. [1,2]. However, there is no information regarding the genetic organisation or regulation of the T C A cycle enzymes in these organisms. M D H occurs as either a dimer or a tetramer of identical subunits in various organisms [3]. Nucleotide and amino acid sequences for d':meric bacterial M D H s have only been determined in E. coli [4] and T. flavus [5]. A comparison of the nucleotide sequences of the mdh genes from T. flavus [5] and T. aquaticus B should provide useful information regarding the classification of these two species. Moreover, Thermus spp. are extremely thermophilic eubacteria and M D H s isolated from this source are intrinsically more resistant, both to thermal and chemical denaturation, than the homologous mesophilic M D H s [3,6]. As an initial step towards

0378:1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties

understanding the mechanism conferring increased thermostability on the MDH from T. aquaticus B we have determined the nucleotide sequence of the mdh gene and present the predicted amino acid sequence of the protein. We have also isolated in pure state the T. aquaticus B MDH produced from the cloned gene.

3. MATERIALS AND METHODS

E. coil K12 TG-2 ((lac/q AM15-pro), supE, thi, hsdD~/F', traD36 proA+B +, recA) was used as the host strain for all recombinant plasmids and grown routinely in 2 × Y T medium [4] at 37°C. T. aquaticus B NCIB 11247 [7] was grown at 70 °C in the medium of Ramaiey and Hixson [8]. Standard recombinant DNA procedures were employed [9]. Plasm~d pMTL22P [10] and p~age M13mp8 were used as cloning vectors. Nucleotide sequencing was carried out by a random sonication procedure to generate templates [11] and using deoxy-7-deazaguanosine in the dideoxy-chain termination reaction at 50°C [12]. DNA fragments were transferred to a Zeta-probe hybridisation membrane (Bio-Rad, Watford, UK) and hybridised with an oligonucleotide probe as described in [9]. Colonies carrying recombinant plasmids were prepared for h~ situ hybridisation [13] on the membranes which were then washed in 6 × SSC (NaCI 900 mM, Na citrate 90 raM, pH 7.0) containing 0.1% sodium dodecyl sulphate (SDS) at 60°C. The oligonucleotide probe (5'GTG AAG GCA CCC GTA CGC GTG GCG GTI" ACC G G A GCC GCG G-3') was synthesised using an Applied Biosystems 380A DNA synthesiser. MDH from T. aquatieus B cells was purified as described previously [14]. Purification of T. aquaticus B MDH from E. coil TG-2 containing the cloned gene was achieved as follows. A cell extract, prepared from 25 g of cells [15], was heated for 90 min at 70 °C prior to centrifugation (10000 × g, 20 rain). The supernatant fraction was applied to a column (5 ml bed volume) of Procion Red HE-3B-Sepharose equilibrated in 10 mM potassium phosphate buffer, pH 7.2. The column was then exhaustively washed with phosphate

buffer containing 20 mM KCI (200 ml), buffer containing 10 mM L-malate (100 ml), buffer containing 20 mM KCI (50 ml) and buffer containing 20 mM KCI and 0.2 mM NAD. This was followed by a wash with 50 ml of phosphate buffer. MDH was elnted from the column with buffer containing 10 mM L-malate and 0.2 mM NAD. The active fractions were pooled, dialysed against phosphate buffer containing 50% glycerol and stored at - 2 0 ° C . Enzyme, protein assays and SDS-polyacrylamide gel electrophoresis (PAGE) were performed as described previously [14]. NH2-terminal amino acid sequence data were obtained using an Applied Biosystems protein sequencer 470A (Warrington, UK).

4. RESULTS AND DISCUSSION

4.1. Cloning and expression of the T. aquaticus B mdh gene The first 30 NH2-terminal amino acid residues of purified T. aquaticus MDH were found to be identical with the corresponding NH2-terminal amino acid sequence of T. flavus MDH predicted from the nucleotide sequence [5]. On this basis an ofigonucleotide probe was made corresponding to the first 40 nucleotides in the coding region of the T. flavus mdh gene. This probe was found to hybridise with a fragment of about 3 kb in both T. aquaticus B and 7". flavus chromosomal Hindlll digests. Fragr~,ents of about 3 kb were isolated from a HindIII digest of T. aquaticus B chromosomal DNA, ligated with HindIII cleaved pMTL22P and transfo:rmed into E. coil TG-2. Following in situ colony hybridisation using the 40-mer oligonucleotide as a probe, five recombinant clones were identified. Cell extracts prepared from these were subjected to heat treatment (70 ° C, 10 rain) and assayed for MDH. Three of the extracts had significant thermostable MDH activity. The levels of thennostable MDH activity in the other two extracts were about 20-fold lower. Plasmid DNA prepared from the recombinant clones which yielded high ~md low thermostable MDH activities was designated pDN10 and p D N l l , respectively. Restriction analysis revealed both pDN10 and

9 Table l Purification of T. aqualicus B MDH from E. coli TG-2 containing the plasmid pDNI0 Activity (U)

Step

Cell extract 1490 Heat treatment 1420 Elution from

Protein Specific Yield (mg) Activity ($) (U/mg) 1690 215

0.88 6.6

100 95

108.0

60

Procion red

column

847

7.8

p D N l l to contain the same insert D N A but in reverse orientations with respect to the vector lacZ gene promoter. This suggesteo that expression of mdh from pDN10 was due to transcriptional read-through from the vector lacZ gene promoter and that expression from p D N l l was due to transcriptional initiation signals which were weakly active in E. coli and resided within the cioned insert 5' to the mdh gene. Thermostable M D H purified from E. coli TG-2 containing pDN10 migrated as a single band when subjected to S D S - P A G E . From the specific activities of the cell extract and the purified preparation, the M D H was estimated to constitute about

0.8% of the soluble cell protein (Table 1). Expresston of the cloned 7". aquaticus mdh gene in E. coil was very inefficient in comparison with expression of the E. coil mdh gene (51% of soluble cell protein) cloned on a similar plasmid vector [4]. The NH2-terminal amino acid sequence of the thermostable M D H purified from E. coli containing pDN10 was found to be identical to the sequence determined for M D H purified from 7". aquaticus B. 4.2. Nucleotide sequence of the T. aquaticus B mdh gene The nucleotide sequence of the 3 kb HindIIl fragment insert of pDN10 was determined on both strands, employing random M13 templates generated by the sonication procedure [11]. The coding region for M D H in this sequence was identified on the basis of the NH2-terminal sequence of the enzyme. The molecular weight calculated from this sequence (35.397 kDa) is similar to the value previously determined by S D S PAGE of the pure enzyme [3]. A putative ribosome binding site was identified 5' to the mdh gene with complementarity to the 3' end of T. thermophilus 16S r R N A [16]. The high G + C content of the T. aquaticus mdh gene resulted in a

Table 2 Codon utilisation of the T. aquaticus B mdh and sucD genes codon UUU

aa Phe

UUC UUA UUG

s u c D codon aa

0

0

UCU

l0

7

UCC Scr

mdh

s u c D codon aa

0

0

UAU

5

6

UAC

0 0

0 0

UAA UAG

0 13 0

0 10 0

CAU CAC CAA

0 2

0 0

UCA UCG

4 11 0

1 10 1

CCU CCC CCA

CUG

13

8

CCG

3

5

CAG

AUU

0

0

ACU

0

0

AAU

16

27

ACC

10

17

AAC

1

0

ACA

0

0

AAA

CUU CUC CUA

AUC

Leu

mdh

Leu

lie

AUA AUG

Met

GUU GUC

GUA GUG

Val

If

Pro

Thr

6

ACG

4

4

AAG

2

0

GCU

0

l

GAU

6

12

GCC

38

22

GAC

2 18

0 17

GCA GCG

1 9

1 9

GAA GAG

Ala

mdh

s u c D codon aa

0

0

UGU

7

3

UGC

Ter

0 0

1 0

UGA UGG

His

0 4 1

0 7 0

CGU CGC CGA

II

4

CGG

0

0

AGU

11

4

AGC

0

1

AGA

18

15

0

0

17

12

GGC

1 23

1 22

GGA GGG

Tvr

Gin

Asn Lys Asp Glu

Cys

mdh 0

sucD 0

1

1

Tar Trp

1 4

0 1

Arg

0 10 0

0 6 0

8

5

Set

0

0

4

3

Arg

0

0

AGG

I

0

GGU

0

0

16

21

2 9

3 15

Gly

A ~o 6o 9o 120 ~GCTTTTGG GGGG~U.G~CCATCTACATGTA~CC~C~T~CATTGAC~CGGC~.GGC~AT~GTGG~ATGGTGoC~GG~C~ATC~T~G~GAGAC~GCGTC~

K

L L E G K P I

0 .°-o

Y M Y P T S I E A A K A ~ V & N V

A

A

-"'

~ ~ ,~ ,

. . . .

150 180 210 240 TCCAGGGCA~ ' CACCGOCCGSGACGGOCAGTTCOACACCAACCACATOCTCO ACTAOC~CACC/IAGATCGTCCCOGO~,GTOACCOCGOGCA/d&GGGGGAAC ~AGOTCCTAGGGGTCOCCO V q G i TG R EGq F ~ T K O M L D Y G T K % V A G V T P G K G G T E V L O V p

270 300 3~0 360 TCTA:GACACGGTGAA~0 AGQCGOTGGCCCACCACGAGGTGGACGCCTCCATCATCTTCGTG COOr :~CCCGGCCGCC,GCGGACGCCOCCCTG,.~A.~,GCF~CCCACGCCGGGATCCCCOTCA V Y D T V K E A V A H H E V D A S Z I P V p A P A A A D A A L E A A H A G I P L 390 42O 4~0 S 48O TCGTCCTCATCACCGAGGGCATCCCCACCCTGGACATGGTGCGGGCGGTOGAGGAGATCAAGGCCCTGGGAAGOCCCCTCATCGGGGGGAACTGCCCCGGGATCATC&GCGCCG&GGAG & I V L I T E G I P T L D M V R A V E E I K A L G S R L I G G . C P G I I S A E E 510 540 570 6OO CCAAGATCGSGATCATGCCCCOCCACGTCTTCAAGCGGG8CGGGGT6GGGATCATCAGCCGCTCCGG CACOCTCAOCTACOA~COGCAGOCUCCC~COCAC.G C~GgQTCC.~CACCA ";' ~ Z C l M P ; . V F X '~ C R V C I I S R S C T L ' t Y e A A A A L S Q A ~ I,¢ T 6~0 T T T V ~

I

660

G G D P V

690

720

I G T T F K D L L P L F N E D P E T E A V V L I G E I G G

?50

760

~ 810 840 J CGACGA,); A~GA 3~,cGGCGGCTTGGGTGAAGGAC CAoATG ~AGAA G CCGOTGGTGGGCTTCATCGGAGGCCGCTOCGCCCCCAA~G CAA¢CGCA"~CO;CCAC;C~C.~GI~O~ATCATCA 870 900 t 9~0 960 ': $ ; Gc AAC,3TGGGCACCCCGOAGT CC~ GCT C0GGGCCT '~'~OCCOACGCGGGCATC CCOGT COCOGACACCA~ OAC0A ¢ J ~ GTC~AO~T~,GTCJU~G.~~ COCTG~ CTA~AAO~A0 y, ;

~

V

;

T

p

E

$

K

L

R

A

F

A

E

A

G

I

99O --

M K A P V ~ V A V T G A A G q

L

LE

I

p~

V

A

D

T

Z

D

E

l V

1140

AMKA

L E G V

E

L V

K

K

A

L

0

*

--'ST~T.D.

~0~0

~oso

I G Y S L L Y R I A A G E R L G K D Q P V I

1~o ~

P ~0~o

~

V M E L ~ D C

L

1~o

~2oo

A F P L L A G L E A T 6 U ¢ * ¥

A~"

~`AA33A~:;~C~`A~TACGc~TC~T~GTGGGG~G~CCCCG~GGCO0~GATG~A~GG~ACCTTT~GcAGG~AA~GG~A~GAT~TTCA~CGAG~GGG~CGGGccC~GGcCG~ K DA D Y A L L V G A A P R K A G M E R R D L L Q V N G K I F T E Q G R A L A ~ s350 I}BO 1410 t440 :;; ?30 CC,L~; AAG; ACOTOAAOG~CTGCTOGTGGOCAACCCCOCOAACACCAACGCCCTCATCOCCTAC/~G AACGCCCCOGGC~CAACCCCCGGAAO'gTCACCGCCATGACCC~GCT

' ; A R ~ 3 V K V L V V G N P A N T N ~ L I A Y K N A P G L N P R N F T A R T R ~ .~,3A.'CACAA: : ~,3GCCAAO,3CCCAGOTCGCCAA0.t~GACCOGGACGGGCGTGGACCGOATCCGCOGCAT(; AOGGTG'IX~GC0OAAOOA~OOACCATO~'?COCOC*CCTC'i~OOAOGC 3 H N R A K A Q L A K K T G T G V DR I R R M T V W G N H ~ S T N ~ P D L F H A t590 t620 1650 ~680 C; ~.G;T;GACGOCAGGCCCGCCOT~AOC~OTC~ACATC'OAO'~GOTACG.'-G ~G~TC¢'~C~.~CCCACOO¢~CCC~.OC(=OO=C~CC,¢OC.'nC.'-¢CC*C~CCCGG;C.OaCCAOC~C~C

g V D O R P A L E L V D N S W Y ~ K V ~ I P T V A Q ~ G A A %

I Q A ~ A S S A

17tO 1740 c 1770 e 1800 ~GC~A~GC~G,~`~AA~Q~A~AGA~ACATC~G~A~GG~T~CA~GG&~G~GGACTG~TTT~AT~c~T~C~CCM~G~GGAGTA~6G~T~C~GAC'GG~T

A

S

A

A

.

A

A

I

EH

l

R

D

W

A

10

T

P

EO

DWV

S M A V

P

SQG

I:~(O.%

pEO

%

c G~,CTA C? CCT TCCCGG~GACOGCCAAOGACGGGGCGTA CCGGGTCGT(, GAGG6CCTGOAGAI c AACGAGTTCOCOCGCAACCGOATGOACATCACC,~CCCA~ AJ~CTTCTGGACGAGAT

v

Y s

F

PV

T

,**K

DO

A Y R

V V

EO

L

g

I

N E

~

k

R

K BME

I

T

AQ

E L L D B M

~950 c ~geo t 2010 ~e~ ~ 2O4o 3 A~¢A3~T;/J,33CCCTGGO CCTCATCTGAGAGG CTGGCCTCAGAGCCCCCACCGCG CCCTGCGGTGGGGGCTTTACACCACOCCAT~CTGG CTT~COCCAGCATGGOGGCCCCGGCAA ~ ~ v K A L O L I ~* • • I= q

2070 2~ O0 ¢ ~ o c T c c c Tc GGGA; CTTCCCC ; ; ; CCC~'Z'C~ ~ C~ ^A GOc ^ ~ CCGGA^~,~ , T ^ ' ~ C CC.'Z'~ ~ O~CC^ ~ CC ~

^~

~13O ~1 CCC~ ;~O~AO ~,CCACCCXC~ CCOCO¢'~C~CC~ ~ C CT*

~,AAG?COGGGCGGTCCGGCACCCGG'J~CCG(CCCGGC'PTGAAGTGGAGOGTGGCCACC&CAGGCACCCCGCCCGCCCGGCGCACCCGC~CCT'~CACGGCAAAC, GCCG~CGCGCCGAG~ 2~10 2~0 ~370 Z400 ccAAACGTOGTCCACCACCAGGACCCGCTTGCCG AAAAOGAGGACATC~GGGGGAA&CTGCAGGAATACCGOCTC GGGCAAAGCCTCCTCCCCCTCGT~GAACATGACGGCC~CGGT~AO ~o

2460

2490

~%0 ~SSO ~6tO cc~; AGOA~O~CC~CCCA~O~A~O~OO ~OOCOCTOO^~OAO~OO~OTOC~OC?CA~O~CO%QTCOAC ~ O O C C ~ O C O COOC C ~ C O T ~ C ~ C Q ~

2120

Z640

k~ ~TO~C~G~

~6"t.

27OO

L~}O

~6o

2?90

~a~o

Z~50

~aSO

~910

2940

2970

3OOO

TGG CCGG;ACCCCCTGGATG&TO&TGTCCAOC~CGATGOOCCGC~C OA~OOCTQO~GA¢CT'~O~GCOA~COOGGC*~GTOCGCGA~X~CCO ATAAGCCCGaT~CGTOGT CCAGOTCC~ GOCOACe¢ CCO ?C ACCOO C ~ TOC ATOTOOAT00OO A CC TCOTTOAOCAGGOT & ~ GGGTI'GTAGGAG~ G OT&GaGCGC A¢G T ~ A ~ ACC&CC~ O TAGOGC ~ 3150

3180

11

TSCS ESCS mSCSR

20 40 60 MI LVNRETRVLVQG I TGREGQFHTKQMLDYGTK IVAGVTPGKGGTEVLGVPVYDTVKEAVAH MS:LIDKN:K: I C : :F: : S Q : T : : S E : A I A : : : : M : G : V : : : : : : T T H : :L: : F N : :R: : V A A GSYTASRKN:YIDKN:K: I C : :F: : K Q : T : : S Q : A L E : : : : L : G : T : : : : : : K K H : :L: : F N : :K: : K E K

TSCS ESCS mSCSR

80 100 120 140 HEVDAS I I~VPAPAAADAALEAAHAG I PL IVL I TEG I P TLDMVRAVEE I KALG- SRL I GGNCPGI I SAEE TGAT: :V:Y: :A:FCKDSIL: : I D : G : K : I I T : : : : : : T L : :L T V K V K L D E A : - V : M : :P: : : : V : T P G : T G A T : : V : Y : :P : F A A A A I N : : ID:E:P:VVC: : : : : :QQ: :LRVKHKLTRQ:KT:L: :P: : : :I : N P G : ° ° °°

TSCS ESCS mSCSR

160 180 200 TKI GIMPGHVFKI~GRVGI I SRSGTLTYEAAAALSQAGLGTTTTVGI GGDPVIGTTFKDLLPLFNEDPETE :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: C : : : :M: : : I H : K : R I : :V: : : : : : : : : : V H Q T T D V : L : Q S L C I : : : : : :FN:TN: I :C:DV:LK: ..

:A: :

.

TSCS ESCS mSCSR

220 240 * 280 AVVL I GE I GGSDEEEAAAWVKDHMK .... KPVVGF I GGRSAP KGKFdvIGHAGAI I MGNVGTPE ~ KLRAFAE ========================= .... ::::::::::::::::::::::::::::::::::::::::: G I : M : : : : : : H A : :N: : E F L : E : N S G P K A : : : :SF:A: IT: :P:R: : : : : : : : :A:GK:GAKE: IS:LQS .. .

TSCS ESCS mSCSR

3O0 AG I PVADT I DE IVELVKKALG : :V K T V R S L A D I GEALKTVLK : :VIVSMSPAQLGTCMYKEFEKRKML

Fig. 2. Amino acid sequence alignment of succinyI-CoA synthetase a-subunits from Z aquaticus B (TSCS: this study). E. coil [22l (F.F~S) and rat mitochondria [23] (mSCSR). Arbitrary gaps (dashes) were placed in the alignment to maximise sequence homology. The degree of homology between TSCS and ESCS is 56.3% and between TSCS and MSCSR 51.8%. Amino acids conserved in all three sequences are indicated by a colon C), conservative replacement is i-dicateP by .~ ~ngle dot below tLe ~"~gnment and the asterisk indicates the active site phosphohistidine residue.

markedly n o n - r a n d o m c o d o n usage pattern (Table 2) with a s t r o n g preference (95.7%) for G or C in the third positions of degenerate codons. Structural genes f r o m T. thermophilus [17], 7". caldophi/us [18], T. flavus [5] and T. aquaticus B (this study) have a similar c o d o n bias, which is different to that f o u n d in structural genes f r o m 7". acuaticus YT-1 [19,20].

4.3. Comparison with the nucleotide sequence for the T. flavus mdh gene C o m p a r a t i v e alignment of the T. flavus and 7". aquaticus B sequences d e m o n s t r a t e d 20 nucleotide differences, of which n o n e gave r~se to a change in the predicted a m i n o acid sequence. T h e two Thermus mdh genes exhibit a different distribution o f rare c o d o n s (A + T rich), but with no overall

Fig. 1. Nucleotide sequence and predicted amino acid sequences for the T. aquaticus B sucD (87-953) and mdh (968-1951) genes. The predicted amino at,~d sequence corresponding to the 3' terminal part (1-90) of a putative open reading frame (possibly the sucC gene) is also indicated. Nucleetide differences with respect to the T. flavus sequence are shown above the T. aquaticus B DNA sequence in lower case letters. A singie letter denotes a substitution, a vertical arrow denotes an insertion of the indicated nuclcotide. Horizontal arrows indicate regions of dyad symmetry. Putative ribosome binding sites are underlined and denoted as S.D. The experimentally determined amino acid sequence is underlined. The asterisks under the nucleotide sequence indicate termination ¢odons.

change in the total number. The four contiguous differences in positions 2015-2018 (5'-GCAA-3) may have been formed by an inversion of the loop sequence of a putative stem-loop structure. There was a marked avoidance of the TaqI restriction endonuclease recognition sequence " T C G A " , consistent with other Thermus D N A sequences [5,17,18]. 7". aquaticus B and T. flavus appear in the same taxonomic cluster on the basis of several similar biochemical and physiological criteria [21]. The high degree of nucleotide sequence homology between T. aquaticus B and 7". flavus mdh genes (98.9%) supports these observations and is comparable to the degree of homology between the mdh genes from two different E. coil strains, K-12 W3899 and K-12 CS520 (about 99.0~, [4]). This calls into question previous assumptions that 7". aquaticus B and T. flavus are two independent species.

C-terminal sequences of T. aquaticus B and E. coil succinyl-CoA synthetase a-subunits. This suggests that the second O R F is fikely to be the sucC gene of 7'. aquaticua B and that the sucC, sucD and mdh genes may be parts of an operon in T. aquaticus B. Such a genetic arrangement is different from the known arrangement of these genes in

4.4. Identification of open reading frames 5' to the mdh gene

REFERENCES

An open reading frame (ORF) was identified between positions 87 and 951 which was preceded by a putative ribosome binding site (Fig. 1) and had a similar codon usage to the mdh gene. The predicted hypothetical protein (29.794 kDa) was identified as the alpha-subunit of succinyl-CoA synthetase (sucD gene product) of T. aquaticus B by the high degree of amino acid sequence homology (Fig. 2) to the E. coil [22] and rat mitochondrial [23] alpha-subunits. Succinyl-CoA synthetase is also an enzyme of the T C A cycle. A second O R F is apparent originating beyond the 5' limit of the determined sequence and terminating at position 88 (Fig. 1). The part (encoding 29 amino acids) of this putative O R F that is present in the cloned insert has 86~ G + C in the third positions o f degenerate codons. The overlap of the termination codon of this second O R F with the initiation codon of the putative sued gene in 7". aquaticus B is analogous to the genetic organisation of the sucC (beta-subunit of succinyl-CoA synthetase) and sueD genes in E. coil The extent of identity between the 29 amino acid sequence and the C-terminal sequence of the E. coil succinyl-CoA synthetase fl-subunit (29~) is comparable to the degree of identity between the

E. coll.

ACKNOWLEDGEMENTS We thank Kevin Bown for Procion Red HE-3B Sepharose and Roy Hartwell for oligonucleotide synthesis and amino acid sequence analysis. D.J.N. gratefully acknowledges the receipt of an SERC CASE studentship.

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