JOURNAL OF FERMENTATION AND BIOENGINEERING
Vol. 70, No. 2, 71-74. 1990
Cloning and Expression of the Thiobacillus ferrooxidans 3-Isopropylmalate Dehydrogenase Gene in Escherichia colt K E N J I I N A G A K I , * H I R O S H I K A W A G U C H I , YASUYUKI K U W A T A , T S U Y O S H I SUGIO, H I D E H I K O T A N A K A , AND T A T S U O T A N O
Division of Biological Function and Genetic Resources Science, Faculty of Agriculture, Okayama University, Okayama 700, Japan Received 14 February 1990/Accepted 13 May 1990 The 3-isopropyimalate (3-IPM) dehydrogenase [EC 22.214.171.1241 gene leuB of an acidophilic autotroph Thiobacillus ferrooxidans was cloned and expressed in Escherichia colt. Recombinant plasmids pTFL1 and pTFL2, carrying a 6.7-kb PstI fragmen! in lhe opposite orientation, conferred the same level of 3-IPM dehydrogenase activity on an E. colt leuB mutant. Restriction endonuclease mapping and deletion analysis indicated that 3-IPM dehydrogenase gene was on a 3.1-kb PstI-NruI fragment. The expression of the 3-IPM dehydrogenase gene of T. ferrooxidans was repressed by the addition of ieucine in the culture medium of E. coil carrying the plasmid pTFL1. These results indicate that the 6.7-kb fragment contains the leuB structural gene and its regulatory region.
Thiobacillus ferrooxidans is an acidophilic chemolithoautotrophic bacterium that can obtain its carbon by fixing carbon dioxide from the atmosphere and derives its energy by oxidizing either ferrous iron to ferric iron or sulfur compounds to sulfuric acid. This bacterium is one of the most important microorganisms for the bacterial leaching of sulfide ores and for the cycling of inorganic sulfur compounds and iron in natural environments. Previously we reported that T. ferrooxidans could use amino acids, other than glycine, methionine, and phenylalanine, as a sole source of nitrogen (18). However, both the growth rate and growth yield were lower than those in Fe 2 ~ - N H 4 ~ -salts medium, suggesting that the ammonium ion was a superior nitrogen source for the strain compared to amino acids. The biosynthesis of amino acids by the obligate autotroph T. ferrooxidans is poorly understood. Therefore, it is interesting to investigate the enzymes involved in amino acid biosynthesis. Recently, Barros et al. (1, 2) reported the cloning and expression of the glutamine synthetase gene glnA of T. ferrooxidans in
MATERIALS AND METHODS Bacterial strains and media T. ferrooxidans AP19-3 (17-20) was used as a donor strain of the wanted gene. E. colt HB101 (pro, leuB, thi, hsdR, hsdM, recA) (7) and E. colt C600 (leuB, thr, thi) (13) lacking 3-IPM dehydrogenase were used as an E. colt host. T. ferrooxidans was grown on an iron-based medium (17). E. colt strains were grown in Lucia medium; when required, ampicillin (50/~g/ ml) was added. Construction of recombinant plasmids Chromosomal D N A of T. ferrooxidans AP19-3 was isolated by the method of Saito & Miura (14). Plasmid D N A was prepared by a modification of the method of Oka (10). DNAs were digested with PstI endonuclease at 37°C for 2 h (plasmid DNA) or for 3 h (chromosomal DNA). After the digestion, 1 pg of pUC19 and 2/zg of T. ferrooxidans AP19-3 chromosomal D N A were mixed and ligated with T4 D N A ligase overnight at 16°C. This ligation mixture was used to transform E. colt HB101. L e u transformants were selected on a plate of Davis' minimal medium (4) with 5 0 p g / m l ampicillin, 20 p g / m l proline, 5 p g / m l thiamine, and agar (1.5%o). Minipreparations of recombinant plasraids were obtained by the alkaline-SDS lysis procedure (3), and general D N A cloning techniques were as described in Maniatis et al. (12). Southern blot hybridization D N A fragments of T. ferrooxidans API9-3 and E. colt HB101 obtained with Pstl were separated by agarose gel electrophoresis and then transferred to a nylon membrane (Hybond-N +, Amersham) by the method of Southern (16). The 6.7-kb fragment of p T F L I described in Results and Discussion was labeled with [a-3zp]dCTP ( ~ 110 T B q / m m o l , Amersham) using a R a n d o m Primer D N A Lebelling Kit (Takara Shuzo Co., Ltd.), and then used as a D N A probe (5). After the hybridization at 68°C for 18 h, the nylon membrane was washed with l x S S C and 0.1 x S S C ( I x S S C is 0.15M NaC1 plus 0.015 M sodium citrate) to avoid a high back-
E. colt. 3-Isopropylmalate (3-IPM) dehydrogenase [EC 1.1.1. 85] is a key enzyme in leucine biosynthesis and the enzyme has been found in a wide variety of heterotrophic bacteria (9, 15, 21, 22). However, no studies have been made of 3-IPM dehydrogenase in strains of T. ferrooxidans. Investigations on intracellular enzymes in T. ferrooxidans are hampered, as it is an obligate autotroph, and a high yield of cells is difficult to obtain. To overcome this problem we cloned the T. ferrooxidans 3-IPM dehydrogenase gene leuB. This paper describes the cloning and expression of the 3IPM dehydrogenase gene from T. ferrooxidans in E. colt, and the analysis o f the cloned fragment.
* Corresponding author. 71
J. FERMENT. BIOENG.,
INAGAKI ET AL.
9.4 6.6 4.4
FIG. 1. Southern hybridization of [32p] labelled 6.7-kb fragment of pTFLI to total chromosomal digests and plasmid. (A) Photograph of gel containing (in lanes): 1, 2 DNA HindIII digest (marker); 2, 7". ferrooxidans AP19-3 chromosomal DNA Pstl digest; 3, plasmid pTFL1 Pstl digest; 4, E. coli HB101 chromosomal DNA PstI digest. (B) Hybridization analysis of the Southern transfer of the DNAs from gel (A), using 32p_ labelled 6.7-kb fragment of pTFLI as a probe.
ground. The filter was a u t o r a d i o g r a p h e d by exposure to X-ray film (Kodak X - O M A T AR) with an intensifying screen at - 80°C. Assay of 3-IPM dehydrogenase activity Cells o f E. ¢oli and T. ferrooxidans, harvested by centrifugation, were washed three times with 0.1 M sodium phosphate buffer (pH 7.5) and then suspended in the same buffer. The cell suspension was sonicated at 0°C for 15 min. After centrifugation at 105,000 × g for 60 min at 4°C (Hitachi 70P72), the supernatant solution was used as the cell-free extract. The activity o f 3-IPM dehydrogenase in the cell-free extract was assayed by the method o f Parsons et al. (11). The reaction mixture contained, in a total volume o f 3.0 ml: 0 . 1 M Tris-HC1 ( p H 8 . 0 ) , 0 . 5 m M MgC12, 5 0 m M KC1, 0.67 m M N A D +, and 0.67 m M 3-IPM ( W a k o Pure Chemical Industries, Ltd.). Enzyme activity was measured by m o n i t o r i n g the p r o d u c t i o n o f N A D H at 3 4 0 n m on a Beckman DU-65 spectrophotometer. One unit o f the enzyme represents the a m o u n t that catalyzes the f o r m a t i o n o f 1/~mol o f p r o d u c t / r a i n at p H 8.0. Protein concentration was measured by the biuret method (8), with bovine serum a l b u m i n as a standard. RESULTS AND DISCUSSION
Isolation of recombinant plasmid carrying the 3-IPM dehydrogenase gene of T. ferrooxidans AP19-3 E. coli HBI01 (leuB; 3-IPM dehydrogenase-less mutation) was t r a n s f o r m e d with the ligation mixture described in Materials and Methods. A n E. ¢oli HB101 leuB' transf o r m a n t was isolated on Davis' minimal agar medium. The t r a n s f o r m a n t contained a p U C I 9 recombinant plasmid designated p T F L I (9.4 kb). The presence o f the leuB structural gene on pTFL1 was confirmed by r e t r a n s f o r m a t i o n
of the leuB deletion strains E. coli HB101 and E. coli C600. After retransformation, approximately equal numbers of leuB t and ampicillin resistant transformants were obtained on minimal agar plates and Luria agar plates containing ampicillin. Digestion of p T F L 1 with PstI indicated that a 6.7-kb D N A fragment was inserted into p U C I 9 (Fig. 1A). To test for a p r o m o t e r sequence in the 6.7-kb fragment, the fragment was reintegrated into pUC19 in the opposite orientation, generating the plasmid pTFL2. The p T F L 2 complemented the leuB m u t a t i o n of E. coli. This suggested that the cloned fragment contained a p r o m o t e r sequence of T. ferrooxidans functioning in E. coli. Hybridization of cloned fragment and chromosomal DNA The origin o f the 6.7-kb insert in p T F L I was identified by Southern blotting and D N A hybridization (Fig. 1). Plasmid pTFL1 D N A and c h r o m o s o m a l DNAs from T. ferrooxidans AP19-3 and E. coli HB101 were digested with PstI. The digested D N A fragments were fractionated by electrophoresis in 0.7%o agarose gel and transferred to a nylon membrane. Labeled 6.7-kb fragment was used as hybridization probe. The 6.7-kb fragment was hybridized to both T. ferrooxidans AP19-3 c h r o m o s o m a l D N A and pTFL1 digested with PstI (Fig. 1B, lanes 2 and 3). No hybridization was detected between the insert and PstI-digested c h r o m o s o m a l D N A from E. coli HB101 (Fig. 1B, lane 4). These results indicated that the 6.7-kb insert o f hybrid plasmid p T F L I were derived from T. ferrooxidans. The cloning o f the leuB gene from T. ferrooxidans is apparently the first report o f the presence or isolation of a leuB gene from an autotrophic chemolithotrophic bacterium. Expression of 3-IPM dehydrogenase gene in E. coli To examine the extent o f expression of the cloned 3-IPM dehydrogenase gene o f T. ferrooxidans in E. coli, we meas-
CLONING OF T. FERROOXIDANS LeuB GENE
Vor. 70, 1990
3-IPM dehydrogenase activity in cell-free extracts of T. ferrooxidans and E. colt clonesa
Strain (Plasmid) T. ferrooxidans A P 19-3 E. colt HB101 E. colt HB101 (pTFL1) E. colt HB101 (pTFL1) E. colt HBI01 (pTFL2)
Addition of leucine (20 ~g/ml) + --
Specific activityb (units/mg) 0.016 0 0.018 0.085 0.101
a Davis' minimal medium was used for E. colt clones. b Specific activity is defined as/.tmol of products formed per mg of protein per min.
ured the activity o f 3 - I P M d e h y d r o g e n a s e in the cell-free extract. T h e E. coli H B I 0 1 s h o w e d no 3 - I P M d e h y d r o g e n ase activity. As s h o w n in T a b l e 1, the specific activity o f 3 - I P M d e h y d r o g e n a s e o f T. ferrooxidans c o n f e r r e d by p T F L 1 was repressed by the a d d i t i o n o f leucine. In the absence o f leucine, high levels o f the specific activity were o b t a i n e d in the t r a n s f o r m a n t s c o n t a i n i n g the T. ferrooxidans 3 - I P M d e h y d r o g e n a s e gene. F u r t h e r m o r e , the enz y m e p r o d u c t i o n o f the p T F L I t r a n s f o r m a n t was a b o u t 5 times that o f the D N A d o n o r strain T. ferrooxidans; thus c l o n i n g o f a gene into E. coli is a p o w e r f u l m e a n s for purifying the gene p r o d u c t o f the o b l i g a t e a u t o t r o p h T. ferrooxidans. T h e t r a n s f o r m a n t s c a r r y i n g p T F L 1 or p T F L 2 s h o w e d a l m o s t the s a m e activity. T h e results also suggested that the c l o n e d T. ferrooxidans 3 - I P M d e h y d r o g e n a s e w o u l d be expressed in E. coli f r o m its o w n p r o m o t e r . As s h o w n in T a b l e 1, the e x p r e s s i o n o f 3 - I P M d e h y d r o g e n a s e gene o f T. ferrooxidans was repressed by the a d d i t i o n o f leucine in the culture m e d i u m o f E . coli c a r r y i n g the p l a s m i d p T F L 1 . T h e s e results indicate that the c l o n e d 6.7-kb f r a g m e n t contains the leuB s t r u c t u r a l gene and its r e g u l a t o r y region. D N A s e q u e n c i n g e x p e r i m e n t s will give us m o r e i n f o r m a tion. L o c a t i o n o f the 3 - I P M d e h y d r o g e n a s e g e n e in p T F L 1 T o i d e n t i f y the c o d i n g r e g i o n for the 3 - I P M d e h y d r o g e n ase gene in the c l o n e d 6.7-kb insert in p T F L 1 , we constructed v a r i o u s deletion plasmids, and e x a m i n e d their ability to c o m p l e m e n t the leuB m u t a t i o n o f E. colt HB101. T h e results are s u m m a r i z e d in Fig. 2. A m o n g t h e m , p T F L I 0 2 , which has the smallest insert, was sufficient to c o m p l e m e n t the leuB m u t a t i o n . This results indicate that the 3 - I P M d e h y d r o g e n a s e gene is b e t w e e n the Pstl and N r u l sites in the f r a g m e n t . In E. colt (15) and Salmonella typhimurium (22), the biosynthesis o f leucine is c o n t r o l l e d by a cluster o f f o u r structural genes a n d an a d j a c e n t o p e r a t o r r e g i o n with the gene order--leuD leuC leuB leuA leuO--reading in a clockwise d i r e c t i o n on their c h r o m o s o m a l maps. T h e leuA codes for 2 - I P M synthetase gene a n d leuC, leuD codes for 2 - I P M i s o m e r a s e . T h e i r e x p r e s s i o n is c o n t r o l l e d by an a t t e n u a t i o n m e c h a n i s m (6, 23). It w o u l d be very interesting to c o m p a r e the leucine genes o f a u t o t r o p h i c T. ferrooxidans with those o f o t h e r h e t e r o t r o p h i c bacteria. T h e analysis o f the leucine genes o f T. ferrooxidans is in progress. F r o m this w o r k it is c o n c l u d e d that the c l o n e d 6.7-kb f r a g m e n t f r o m T. ferrooxidans c o n t a i n s the leuB structural gene and its r e g u l a t o r y region.
0 i 2 3 4 5 6 7 8 9 Phenotype (kb) -- pUCI9~-T, ferrooxidans- fr agrr~nEl i I Leu+ ANrul + I I I. . . . . f I Leu
I-~-N--~--I-'-/-s-m--~!--Leu+ I.... ~-A-c-c-I-I-I---I
FIG. 2. Restriction and deletion map of pTFL1. Deletion plasmids were constructed by digestion of pTFL 1 with the restriction endonucleases indicated. Sites for restriction endonucleases are: AcclIl (A), BanlI (B), NruI (N), Pstl (P), Sinai (S).
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