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The atp2 operon of the green bacterium Chlorobium limicola
267
Biochimica et Biophysica Acta, 1172 (1993) 267-273 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00
BBAEXP 92472
The atp2 operon of the green bacterium Chlorobium limicola D i a n - L i n Xie a, Holger Lill b Gfinter H a u s k a c, M a s a t o m o M a e d a d Masamitsu Futai d and N a t h a n N e l s o n a " Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ (USA), t, Abteilung Biophysik, Fb 5 Biologic / Chemic, Unicersitdt Osnabriick, Osnabriick (Germany), ' Unirersitdt Regensburg, Lehrstuhl fiir Zellbiologie und Pflanzenphysiologie, Regensburg (Germany) and a Department of Organic Chemistry and Biochemisto', The hlstitue of Scienttfic and Industrial Research, Osaka Unicersity, Osaka (Japan) (Received 23 October 1992)
Key words: Chlorobium; F-ATPase; Operon; Hybrid enzyme; (E. coli)
The operon (atp2) encoding the/3 and E subunits of F-ATPase from Chlorobium limicola was cloned and sequenced. In contrast with purple bacteria these genes are arranged in a separate operon similar to the cyanobacteria. The operon terminates with a pronounced stem-loop structure. About 0.8 kb upstream of the /3 subunit a gcnc encoding the enzyme phosphoenolpyruvate carboxykinase was identified. This gene is transcribed in the opposite direction of the atp2 operon and also ends with a stem-loop structure. These genes of green bacteria are among the first to be sequenced, and thcrcfore the genetic distance between these genes and corresponding genes from other bacteria and eukaryotes was studied. Even though the operon structure resembles that of cyanobacteria, the evolutionary tree compiled from these data places the chlorobium gene close to purple bacteria. Chlorobium limicola/3 and e subunits complemented Escherichia co6 mutants defective in the corresponding subunits, indicating that the hybrid enzyme formed from subunits of the two bacteria is active in ATP synthesis.
Introduction The protonmotive force generated by photosynthesis and respiration can be utilized for ATP formation by the enzyme F-ATPase [1,2]. This enzyme is present in mitochondria, chloroplasts and all eubacteria studied so far [3-5]. It is a multisubunit enzyme containing two distinct sectors, a membrane sector that functions in proton conduction and a catalytic sector that functions in ATP-synthesis and hydrolysis. The genes encoding subunits of the enzyme in bacteria are organized in operons [4-9]. The structure of the various operons is conserved through evolution, but some variations in the number and content of each operon took place. In E. coli all the genes encoding F-ATPase subunits are clustered in a single operon [6]. In R. rubrum there are two separate operons, one contains genes encoding the membrane sector and the second contains genes encoding the catalytic sector [10,11]. In cyanobacteria and chloroplasts the genes encoding /3 and • subunits are clustered in a separate operon (atp2), and the rest of the genes are present in another operon (atpl) or have been exported into the nucleus, respectively [7-9]. It is
not clear what governs the operon structure and the way by which the split operons evolved in the various bacteria and chloroplasts. However, the sequence of the genes within the operons is strictly conserved, and their order is genes encoding a,c,b,6,c~,y,/3 and E. Photosynthetic bacteria utilize two different mechanisms for their light-driven electron transport systems. While purple bacteria contain photosynthetic reaction centers homologous to the chloroplasts Photosystem II, the green bacteria operate with a reaction center homologous to Photosystem I [12-19]. Cyanobacteria contain a photosynthetic machinery resembling that of chloroplasts [20]. Regardless of the kind of reaction centers, the protonmotive force generated by them is utilized by F-ATPases for ATP formation. In this communication we report on the cloning and sequencing of the genes encoding the /3 and • subunits from the green bacterium Chlorobium limicola. We observed that the genes are clustered in an operon which contains only these genes and thereby resembles the atp2 operons of cyanobacteria and chloroplasts.
Experimental procedures Genomic library and screening
Correspondence to: N. Nelson, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110, USA.
Two
genomic
libraries
were
constructed
from
Chlorobium limicola DNA. The first was constructed by
268 partial SaulIIA digestion followed by ligation into the BamHI site of the plasmid Bluescript II SK(+). This library was satisfactory for initial screening, but due to substantial rearrangements it could not be used for cloning the full size atp2 operon. For this purpose, a second library of BarnHl DNA fragments were cloned into the BarnHl site of the plasmid pGEM-4Z. A partial sequence of the gene encoding the subunit was kindly provided by Dr. Lincoln Taiz. An oligonucleotide ATG CCG AGC GCC G T G GGC TAC CAG CCG ACC CTC AGC A was synthesized on an Applied Biosystems 381A DNA Synthesizer. The SaulIIA library was then screened by 32p end-labeled oligonucleotide as previously described [21]. The plasmids isolated from the positive colonies were subjected to dot blot analysis, and the DNA fragments were then analyzed by Southern blots following digestion by SauIIIA. The hybridized DNA fragments were isolated, subcloned into the BarnHI site of pBluescript, and used for screening the second library. Published procedures were used for recombinant DNA methods, purification of genomic DNA, screening of libraries [21], overlapping deletions using exonuclease III digestion [22], and sequencing by the dideoxy chain termination method [23].
HindllI restriction enzyme to construct pKK/3. They were introduced into E. coli strains KY7230 (asn, thi, thy) [28], JP17 (AuncD (deletion of the /3 gene), argH, pyrE, entA, recA::TnlO) [29], KF20 (uncD20 (bQ397--+ end), thi, thy) [30], KF95 (uncD95 (BQ7-~ end), thi, thy) (this study) and KF148 (uncC148 (mutation at Shine-Dalgarno sequence of the • gene), thi, thy) [31]. Expression plasmids for E. coli /3 and • subunits were also used; pTN1670 for /3 subunit [31] and pTN1665 (constructed in this study) for /3 and e subunits. pTN1665 was a derivative of pTN1661 [31]. A rich medium (L broth) with or without ampicillin (50 / , g / m l ) and minimal medium containing thiamine (2 / z g / / m l ) , thymine (50 # g / m l ) , asparagine (50 # g / m l ) , arginine (100 /~g/ml), uridine (100 p,g/ml) and a carbon source (0.2% glucose or 0.4% succinatc) were used [30]. Membranes prepared by a French press were subjected to electrophoresis and immunoblotting using antibodies specific for the E. coli [3 subunit [32,33]. ATPase activity of the membranes were measured according tot he standard assay condition for E. coil F~ ATPase [32]. Protein amounts were estimated by the published method [34] using bovine serum albumin as standard. Results
Computer analysis Sequences of DNA fragments were assembled using DNAstar software. Multiple alignment and comparisons of sequence data with databases were performed by GCG software package [24]. Evolutionary relations and genetic distances were calculated using the PHYLIP package under VAX VMS [25]. For proper alignment we used the GCG GAP program the following way: in the first run, the amino acid sequences were aligned. Next, these alignments were copied to the corresponding DNA sequences and obvious mismatches were corrected in the protein alignments. Such cycles were repeated several times, and finally the alignments of DNA sequences were used in the calculation, employing the D N A M L program under the maximum likelihood model for the evaluation of evolutionary trees. Complementation of Escherichia coli uncD and uncC mutants with Chlorobium limicola /3 and • subunits Cloned C. limicola genes for the /3 and • subunits were ligated into the Pstl site of the pKK233 Escherichia coli expression vector [26]. The introduction of the Pst I site by PCR [27] at the 5' terminus of the/3 genc caused amino acid substitution / 3 G l n - G l u ~ /3Ala-Ala at positions 2 and 3. Another PstI site was introduced by substituting nucleotide residues 54575477 downstream of the termination codon of the e gene. Most of the • gene was eliminated from the resulting plasmid (pKK/3•) by digestion with the
A plasmid isolated from the positive colony obtained by screening the chlorobium library contained a BarnHI DNA fragment of about 4.5 kb. As shown in Fig. 1, the DNA fragment contained three complete and one truncated open reading frame. Fig. 2 shows the DNA sequence of the fragment and the deduced amino acid sequences of the open reading frames encoding the fi and e subunits of F-ATPase. They are located at the 3' end of the DNA fragment. No other genes encoding F-ATPase subunits could be identified in the cloned DNA fragment. Upstream of the gene encoding the /3 subunit, a gene encoding PEP-carboxykinase was identified. This gene is transcribed from the opposite DNA strand to the /3 subunit gene. The open reading frame starts with the ATG at position 2731 and ends with TGA at position 792 (see Fig. 2). Another reading frame encoding an unidentified protein starts at position 609 and is transcribed in the same direction. This arrangement suggests that the genes encoding the /3 and e subunits are transcribed
SphI
0RF
HindH[Sail XmalII
Sinai
KpnI
BamHIHindIll
PEP-C.K. I
1 kb
I
Fig. 1. S c h e m a t i c p r e s e n t a t i o n of t h e open reading frames of the D N A f r a g m e n t c o n t a i n i n g the atp2 operon. Useful restriction sites are i n d i c a t e d and the s t e m - l o o p s are shown by the symbol ~.
269 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1821 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721
CCTTAGCGAACATCGGCGAGTCGTTGCCGTAGTCGGTGCCCATGCGCCCTGGGCGAGCAT GCGACGCCCCAGGTGATCCGTCCGTCGCGGCGCATGTAGGACATCGAGGGCATGAAGAAC GGTCGCGTCCGACTTGTCGGTAATGCCGTTGTTGTCCAGCGAGATGTCGGGATGCAGACC CCGGATGCCGAAGCCTATCTCCGATGTTCCCTCCTTCATGAAGCCGAGGGTTGCCGGGTT GTTCATGACGGCGGAGTTACCGGTGTCGTAGGCTGAGCCGGTGCCGCCCATGGCCATCGA
TTTGGCGCCGTACCCTTCGAGGTTCATACCGTTGGTGGCGAAAGCGGGCGTGGCTCCGAT G A G C A C A A G A A G A G C C A C A G C CGAACATGC GGTTTTTC GAATCATGAT GTCAGTGAGT TG AGGTTTGATGCTTGCGGCCAACCCAATGCCCGTGACCTCGGCAGCCCAATGAAGGCGGGC AAGC G C C G G T C G A T C A T G C A G G A T A T GAGGAAGT GC C G G G T T G A T A A T C C G G A T CAGC GA GGCTCGCTGCACGGCCTGCCATGAAACTTCTTTTCCGTAACCGCCGACTGTCAGACTCGA A A C G C G C A T G G G G C A A C A T G A A T A A A C A A C AGCAATCAGAC C GGAATGCGATATAT GACT ATATAACTGATT GAATATAT GGCAATAT TATCGATT CCGCAACACTTG CC G G G C A A A C A A AACGCTATACCATAA~GCGGACAT GCTGCC CGCTTT TTTTCGTGTATGACTGATGC TCCGATGAAC TCAAGACT C G G C G G A G T A G A G T T C G G G C G C A A G T T C CC AATGCTCC GGAG ACAT CCAGAGCGTCGAAAGCAACAGC TCGCGGATGTGCGT GAACTCTT TGGGAAGC CTGT C G T A A A G C T T T T C A A G C A G C T C C T CGTGTGAAAAAAGC TC CTGCTT CC CTTC GCGATCCA C C G A C A T G A G C T T T G A A A A C T T G T TGCTGGTGAATCCG TC CAGACC CC GCCAGTCGAGCG ATTCATATCTCGGCATCCAGCCAAGCGGGCTTTCCACCGCGCCGGAACGTCCGTGCACAC
GCCCCATCATCCACTTGAGCACCCGCATGTTGTCGCCGAATCCGGGCCAGAGGAACTTGC CGGTTCTC GTCT TGCGGAACCAGT TCACGATGAAAATC CTTGGC GGATCGGAGACGGTTC GC CC G A C A T G G A G C C A G T G G T T GAAGTAGT CGCCCATGTGGTAG CC GC A A A A T G G C A G C A TGGCGAAGGGGTCGACGCACGTCGCCGACCTTGCCGCGGCGGCGGTCTTTTCCGAACCCA TC GTGGCCGCCATGTAAACGCC GTAATACCAGTT C G A G G A C T G A T A G A C C A G C G G A A T G G TGTCGC CGCGCCGCCC G G C G A A A A T G A A A G C C G A A A T C G G C A A C A C C C T C G G A T T T T C C C A A G C G G G A T C G A T C A C GGGG CACTGACT G G C C G G A G C G G T G A A G C G G G A G T T C G G G T G G G CGGCCGGGCGGTCGCATCCT GGAACCCAGGGCTT GC C C T G C C A G T C G A T G A G G T A T T C C G GGGGCGTGTCGGTCATATCCTCCCACCAGACGTCGCCGTCGGGGGTGAGCGCCACGTTGG T G A A G A T G C A G T T G G C G T G A A G G G T G G C C A T C GC GTTCGGAT TGC TCTTGTCC GAGGTGC CCGGAGCCACGC CAAAAAAGCCATAC TCCGGATT GATGGC GT GCAGGCGT CCAT CCTT GC CCTGCTGATCCAGGCGAATGTCGTCGCCGACGGTGGTGATCTTCCACCCCTCCATCTCGC C C G G C G G A A T G A T C A T G G C G A A G T T G G T C T T G C C GCAGGC GC T G G G G A A G G C C G C C G C A A CATAATCTTTTTCACCTGCCGGCGACTCGACGCCGAGGATGAGCATGTGCTCGGCGAGCC AC CCCTCGTC GCGCGC CATCGAGGAGGCGATACGCAGGGC GAAGCACT TTTTGC CAAGCA GT GTATTGCC G T T G T A G C C G C T G C T G T A G G A G A C G A T G G C GT G C T C T T C C G G G A A G T G G A CGATGTACTT GGTGTC GTTGCAGGGCCACGGC ACATCCGC CTGGCCCGGTTC GAGCGGCG C G C C G A C C G A G T G C A G G C A G G G C A C G A A C T CCGCCTCTTC GTCGAGCAGTTC GAGCAC CT G G C G A C C C A T G C G C G T CATGAT GCGCATGT TGGTGACGAC GT AGGGCGAATC GGTGAT CT C G A C C C C G A T G T G C G C G A T G T G C G A G C C GAGC GGCCCCAT GC TGAACGGAAT GACATACA TGGTGCGC C C C T T C A T G C A G C C G G T G A A G A G C T T G T T C A G C G T C G C C T T C A T CTCTTT TG
GAGCGACCCAGTTGTTGGTCGGCCCCGCATCCTGGCGGCGAATGCTGCAAATAAAGGTTC
5401
GATCCTCCACCCTGGC CACATCACTCGGAT CGGAACGGCAGAGGTAGCTGTT CGGACGCT T C T G T T C C G A C A G T T T G A T G A A G G T G C C G C TGTTGACCAT CTCCTCGCACAGGCGGTC GT ACTCCTCGGTCGATCCGTCACACCAGCAAACCGAATCGGGCTGGCACAGCGCAACGGT CT CTTTGACC CACTGGAGAAGTTTCAGGTT CT TGACATAATC TGGCGCATTGAT TGGGAT CG G T T C C A T A A T T G C T G A A A A T C G A T T A T T GAGAGTGTTGCT GATTGAAAGGCC TGAAACAA CGCGCCCCGGCAGGAAATTCCCCCGGGGCATCTGGAAT CAGGCATGTTGACTATCTAAGT GTGCAAAAATATCT AAGCGGACAAAAAAGC CTGGTCATTT TTTAGCCGTCTTTCGCTGAG C G C C G T T C T G A C G A T C T G G T C G A A C T CGCCTTGCTTCT CGGCCCGACAATCACGCCGGTC A A C T T G C G G A G G C A T C G A T G A C A A A G G T GGTCGGAATGCC GGTGATGCCGCCATCGAT GT GGCCGTTGAAGGCCCGGATCAGCTCAGGAGTGGCCATGAGCACCGGCCAGGT CATCCC GT T C T G T C T C A T G A A A T T C T T C A C G T T C GGAACCTTCTCGTTGACCGCAATGCC GACAAAGG T G A A G C C T T T A G C G C C C A G G C T C T C T GAACCTGTACCATATCGGGAATCTCC GACCGGCA G G G C G G G C A C C A G G T G G C G A A G A A A T T G A C G A T A T A G G C C T T G C C C T T G A G G G A G G A C GA GGAGAAGGGCTTGCCGTCAACGGTCACGCCCGAAAAAGAGGGCGCGGGCCAC GGAGCAGC GCTGGCCTGCCCTGAAAAGGTGAAAGAAGCTGCAAAAACCATGAGCGCTGCCATAATGGC G G C A A A A C G G G A G G G A A C T C T T T T CATAGTGATTCTTT A G A T G A C G G T A A A T A G A C G G T A A A T A G A C G G T A A A T C C T G A C A T G C TT A A T G A T C A C A A A C G A T A A A T A G T T C T G A T T T A T A A T A T A A G A A T C C C G G C G C G A T T C C CC TTGGTTCG TC CCTGATATTTCTTTGC GTTTTGAG AACAAATAAGCTATATTTCGTCTCGAATTTTTCGCT TAAGAGCGGGAAAGGCCGATTCAA GCCACCTTAGCTCAGTTGGTAGAGCAACTGTTTCGTAAATAGTAGGTCGTGGGTTCAAGT CCCTCAGGTGGCTCGCTTATGCCGACAACCGAGT CGGGTAATCGGAACAGAACCTGAGAA CGGAGTCGCCGTCGCCGCCAAACGTTTGATTGCAAC CAAAATCCCATATTGAAGAATACC A T G C A A G A A G G T A A G A T T T C C C A G A T C A T C G G A C CT G T C G T T G A C G T T G A C T T T G C T G A A M Q E G K I S Q I I G P V V D V D F A E G G A C A G C T T C C G T C T A T C C T CGATGCCCTCAC GGTCACCCGC CAGGAT GGCTCGAAACTG G O L P S I L D A L T V T R Q D G S K L G T T C T C G A ~ A C G C A G C A G C A C C TCGGCGAAGAGC GCGTTCGCCGAATCGC CATGGAGGGA V L E T Q Q H L G E E R V R R I A M E G ACAGATGGCCTGGTCAGGGGCATGAGCGCCA~CACCGGCA~ACCGATCCAGGTACCG T D G L V R G M S A K N T G K P I Q V P GTAGGCGAAGAGGTGCTCGGCAGAATGCTGAACGTTGTCGGCGATCCCATTGATGGC;tJLA V G E E V L G R M L N V V G D P I D G K GGCCCTGTACTTTCAAAGA~TCCTACTCCAT CCATCGCACCGCTCCCA~TTTGATGAG G P V L S K K S Y S I H R T A P K F D E C T T T C G A C C A A G G C C G A G A T G T T C G A ~ C C GGCATCAAGGTTATCGAC CTTCTCGAACCG L S T K A E M F E T G I K V I D L L E P TACTCGCGCGGCGGCAA~CCGGCTTGTTCGGCGGCGCAGGCGTCGGCAAGACCGTGCTT ¥ S R G G K T G L F G G A G V G K T V L ATCATGGAC~TTATCAACAATATCGC CAAAGAACAGTCCGGTTTCTCGGTATTTGCCGGC I M E L I N N I A K E Q S G F S V F A G GTCGGCGAGCGCACCCGC GAAGGAAACGACCTCTGGCACGAGATGATGGAGTCTGGCGTT V G E R T R E G N D t W H E M M E S G V ATCGACA~CCGCACTCGTGTTC GGCCAGATGAACGAGCCTCCGGGAGCACGTGCGCGC I D K T A L V F G Q M N E P P G A R A R G T C G C C C T G A C C G G C C T G A G C A T C GC C G A A T A C T T C C G T G A T G A A G A G ~ T C G T G A C G T G V A L T G L S I A E ¥ F R D E E G R D V C T T C T G T T C A T C G A C A A C ATCTTC CGCTTCACCCAGGCAGGCTC CGAAGTGTCCGCGCTT L L F I D N I F R F T Q A G S E V S A L C T C G G A C G T A T G C C G A G C GCCGTGGGCTACCAGC C G A C C C T C A G C A C C G A G A T G G G T G A G L G R M P S A V G ¥ Q P T L S T E M G E CTTCAGGACAGAATCACCTCCACCAAGAAAGGTTCGGTCACC TC GGTGCAGGCCATCTAC L Q D R I T S T K K G S V T S V Q A I ¥ GTCCCTGCCGATGACCTTACCGACCCTGCCCCGGCCACCGCGTTCACCCACCTCGACGCC V P A D D L T D P A P A T A F T H L D A ACGACCGTGCTTTCGCGTCAGATCGCCGAGCTGGGCATCTACCCCGCCGTGGATCCGCTC T T V L S R Q I A E L G I ¥ P A V D P L GATTCGACCTCGCGAATCCTCGATCCA~CGTCATCGGCGACGATCACTACGACACCGCC D S T S R I L D P N V I G D D H Y D T A CAGGCCGT CAAGCAGATTCTCCAG CGCTACAAGGACTTGCAGGACATCAT CGCCATTCTC Q A V K Q I L Q R ¥ K D L Q D I I A I L G G T A T G G A C G A G C T G A G C G A T G A C GACAAGCTCGTGGTCGCC C G C G C C C G C A A G G T G C A G G M D E L S D D D K L V V A R A R K V Q CGCTTCCTGTCGCAGCCCTTCTTCGTGGCCGAGGCGTTCACCGGTCTGGCCGGCAAGTAC R F L S Q P F F V A E A F T G L A G K ¥ GTCAAGCT CGAAGACACC ATCAAG GGCTTCAAGGAGATCATC GC AGGCCGTCACGACAAC V K L E D T I K G F K E I i A G R 8 D N CTTCCGGAAGCAGCTTTCTACCTGGTCGGCACTATCGAAGAGGCGGTCGCCAAAGTTAAA L P E A A F ¥ L V G T I E E A V A K V K AC GCTC TAAACCAACGGC AAGACA TG GC AAGTTCAGACAAAG CC TTTAAG CT CGATA.TTG T L * M A S S D K A F K L D I V TAACGCCGCAGAAGCTTTTCTTTTCGGGAGAGGTGACCAGCGTCATCGCTCCGGGCCTGG T P Q K L F F 8 G E V T S V I A P G L D ACGGATTGTTTCAGATTATGAAAGGTCACGCCCCCCTGCTCGCCGCGCTCA~GCGGCA G L F Q I M K G H A P L L A A L K S Q K AGGTGCGGCTCTCTCTTTCAGACAAGTCTGAAGATTCGTTCCAGATTGAAGGGGGATTCT v R L S L S D K S E D S F Q I E G G F F TCGAGGTGAGCGGCAACAAGGCAATTCTGCTTACCGAGGACGTCTCCTGACCCACGGAAG E V S G N K A I L L T E D V S * CATAACCGCATGAAAAAAACATCAGCAGCGCCTCGGAGTGTTGCTGATGTTTTTTTGCAT
5461
TCATGAAAATTCTGATC
3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341
5477
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from an operon separated from the rest of the FATPase subunits. Moreover, at the end of the gene encoding the e subunit there is a pronounced stem-loop structure (Fig. 2) that may serve as a transcriptional termination signal. As shown in Fig. 2, a similar stemloop structure is present at the 3' end of the gene encoding PEP-carboxykinase and in the operon encoding the chlorobium photosynthetic reaction center [19]. The predicted reading frame for PEP-carboxykinase encodes a hydrophilic protein of 646 amino acids with a calculated molecular mass of 72539 Da. The amino acid sequence of this protein shows a 58% identity over a 564 amino acid overlap with a recently reported sequence of PEP-carboxykinase from N. frontalis [35]. It also has a 51% identity with chicken mitochondrial PEP-carboxykinase [36], a 47% identity with the cytosolic enzyme [37], and a 48% identity with the enzyme from Drosophila melanogaster [38]. These observations suggest that the open reading frame upstream of the gene encoding the /3 subunit encodes the chlorobium PEP-carboxykina~e. Fig. 3 depicts the aligned amino acid sequences of PEP-carboxykinases from chlorobium and N. frontalis. An operon encoding the /3 and • subunits of FATPase was identified downstream of the PEP-carboxykinase gene. The operon encodes only these two proteins and terminates by a pronounced stem-loop structure typical of all the chlorobium genes sequences so far [19]. The reading frame of the/3 subunit encodes a protein of 462 amino acids with a calculated molecular mass of 50220 Da. Fig. 4 shows the amino acid alignment of the/3 subunits of F-ATPases from chlorobium and E. coli. As was shown for /3 subunits from various sources, the sequences of the E. coli and chlorobium proteins are highly homologous exhibiting a 72% identity. The chlorobium subunit was about 70% identical to/3 subunits from other sources including chloroplast CF v On the other hand, the chlorobium • subunit underwent a much faster evolution as shown by its relatively small size and low percent identity with corresponding subunits from other sources. The reading frame of the • subunit encodes a protein of 88 amino acids with a calculated molecular mass of 9376 Da. It has a 33% identity in the amino acid sequence with the • subunit of E. coli (Fig. 4) and very poor homology with the corresponding subunits of mammalian mitochondria and plant chloroplasts (not shown).
5040 5100 5160 5220 5280 5340 5400 5460
Fig. 2. The complete D N A sequence of the chlorobium D N A fragment and amino acid sequences of the two reading frames in the atp2 operon. The D N A sequence and the open reading frames are schematically depicted in Fig. 1.
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MPRGNFLPGRvvSGLS••NTLNNRFSA•MEPIP•NAPDYVKNLKLLQWVKET•ALCQPDSVCWCDGSTEEYDRLCEEMVNSGTFIKLSEQKRPNSYLCRS * * :**:::**: :* *:::: ****:***::*** :*: * : * : * * : *
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MAHSVSSVVNKQLLAYIKESSELMTPKDIYVCDGSAEEYHNLCELLVKQGIFTKLNETKRPNCYLARS
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120
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DPSDVARvEDRTF•CSIRRQDAGPTNNWvAPKEMKATLNKLFTGCMKGRTMYVIPFSMGPLGSHIAHIGVEITDSPYvvTNMRIMTRMGRQVLELLDEEA :,:******: *:*** : :********:** ****:** ****************************************** **:::*::**:**:
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NPADVARVEKCTYICSEKEEDAGPTNNWMAPAEMKAKLNGLMKGCMKGRTMYVIPFSMGPIGGPISRVGVEITDSPYvvVNMCIMAKVGKKVLDLLGvDG
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EFVPCLHSVGA•LEPC•QADvPWPCN-DTKY•VHFPEEHA••SYSSGYNGNTLLGKK•FALR•ASSMARDEG-WLAEHMLILGVESPAGEKDYVAAAFPSACG . * * * * ** * * * * ** * . * * * * , : : * * * : * * * * * *** ********************* ::*::* *********:::*:*:*
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TFVPCTHSCLAPLEEGQKDSTWPCNIDNRYIvQFPEEHRIVSVGSGYGGNALLGKKCYALRIATvLSREAGDSLAEHMLILGITNPQGKKYYITAAFPSACG
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KTNFAMIIPPGEMEGWKITTVGDDIRLDQQGKDGRLHAINPEYGFFG•APGTSDKSNPNAMATLHANCIFTN•ALTPDGDVWWEDMT-DTPPEYLIDWQGK ***:**: ::: * * * * : * * * * * : ****** ***** ********** ******* : : : ******** *******:**
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KTNLAML--NATIPGWKIECvGDDIAWLKIGKDGRLWAINPESGFFGvAPGTSYKSNPNAMKSCEKDTIFTNvALTEDGDVWWEGMTKEVPKGKIITWLGK
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Pwv~C~CDRPA---AHPNSRFT/~ASQCPVIDPA~NPRVLPISAFIFAGRRGDTIPLVYQSSNJe/XYGVYMAATMGSEKTAAAARSATCV--DPFAMLPFCGYHMG * .... * ********* .... ** ** . . . . ************************ .** . . . . . . * * . * * * * . .*. . ******~****.** EWSADSGEPKPNLAHPNSRFTARVENVPVGDPGYYALEGVPVSAMIFGGRRENTVPLVFQSRSWKHGVLLGSSVASETTAAAEAAAGQLRFDPFAMLPFCGYNMG
510
520
530
540
550
560
570
580
590
600
I
I
I
[
I
I
I
I
I
I
DYFNHWLHvGRIvSDP--PRIFIVNWFRKTRTGKFL~PGFGDNMRvLK~GRVHGRSG-AvESPLGWMPRYESLDWRGLDGFTSNKFSKIAMSVDR~GKQELF **,1:** : : ::: *:** *****,1 :*:*****:*:* *****::1,,1,::* * *:,1,::* : : * * :*** ::1:::,1:1,* DYFGYWLSFADKYDEAKLPKIFHVNWFRKD-NGRFLWPGYGENSRVLKWIIERVEGKEGIAKETPIGYLPAKGALDLSGLD-vPEADMEKILTVDCKAYLSEv
610
620
630
640
I
I
I
I
SHEELLEKLYDRLPKEFTH
IRELLLSTLWMSPEHWELAPELYSAES
EK IRQYH S KFGS LLP KAL IAE LDALEQRLKAAL
o f the d e d u c e d u s i n g the A L I G N p r o g r a m
Fig. 3. C o m p a r i s o n
aligned
a m i n o acid s e q u e n c e s o f the Chlorobium a n d N. frontalis P E P - c a r b o x y k i n a s e e n z y m e s . T h e s e q u e n c e s w e r e from DNASTAR. I d e n t i c a l a m i n o a c i d s are i n d i c a t e d by a s t e r i s k s a n d c o n s e r v a t i v e a m i n o acid r e p l a c e m e n t s are i n d i c a t e d by c o l o n s .
The different evolutionary rates of two genes encoding by the same operon make them interesting for evolutionary dating [9]. Fig. 5 shows, that in spite of the TABLE
I
('omplementation o[ Escherichia coli [3 and • subunit mutants with ('hlorohiun~ limicola [3 and E subunits i n d r o d u c e d into the E. coli s t r a i n s listed. resistant transformants was checked on a m i n i m a l a g a r p l a t e c o n t a i n i n g s u c c i n a t e as the s o l e c a r b o n s o u r c e . All t r a n s f o r m a n t s g r e w o n g l u c o s e p l a t e s . P l a t e s w e r e i n c u b a t e d at 37°C for 3 days (+. positive growth; -, no growth). Strain KF148 (mutant of Shine-Dalgarno s e q u e n c e ) l a c k i n g t r a n s c r i p t i o n o f the • Expression
Growth
plasmids
were
o f the a m p i c i l l i n
subunit. Strain (mutation)
Growth
on succinate
plasmid introduced none pTN1665 pTNI670 KY7230 (wild) JPI7 (J[3) KF20 ([3Q397 ~ end) KF95 ([3Q7 ~ end) KFI48(Ae)
+ -
pKK[3E
pKK/3
+
+
+
+
+
+
+
+
+ +
+
+ +
+ -
+
-
+
significant different evolutionary rates, the calculated evolutionary trees o f / 3 and • subunits are quite similar. Assuming that at least all the /3-e operons evolved from a single ancestral operon, the above evolutionary trees give credence to the calculation methods used in this work [25]. During the last decade it was demonstrated that the /3 subunit of E. coli can be substituted with /3 subunits from various sources [39,40]. Chlorobium is a strictly anaerobic bacterium and several of its biochemical systems are highly sensitive to oxygen [16,18]. Therefore, it is of interest whether the proteins encoded by its atp2 operon complement E. coli uncD (/3) and uncC (e) subunits to support oxidative phosphorylation in the presence of oxygen. Thus we cloned the C. limicola atp2 operon into the E. coli pKK233 expression vector. Constructed plasmids for the /3 subunit (pKK/3) and /3 and • (pKK/3•) were introduced into E. coli /3 and • subunit mutants. As shown in Table I, pKK/3 similar to pTN1670 (coding the E. coli /3 subunit) complemented JP17, a deletion strain of the /3 subunit gene. The same plasmids complemented a non-
271 i0 20 30 40 50 60 70 80 90 i00 I I I I I I I I I I MQEGKISQ••GPvvDVDFAEC-QLPS•LDALTvTRQDGSKL•LETQQHLGEERVRRIAMEGTDGLVRGMSAKNTGKPIQvP•GEE•LGRMLNvvGDPIDGK *..*** .1.*:****:'1::::*:: ***:* ::::::****:**:**:: ** ***:::*** **::1": ************************* * MATGKIVQVIGAVVDVEFPQDAVPRVYDALEv-QNGNERLVLEvQQQLGGGI~RTIAMGSSDGLRRGLDVKDLEHPIEVPvGKATLGRIMNVLGEPVDMK
A Chlorobium
~.
coli
ii0 120 130 140 150 160 170 180 190 200 I I l I I I I I I [ GPvLSKKSYSIHRTAPKFDELSTKAEMFETGIKvIDLLEPYSRGGKTGLFGGAGVGKTvLIMELINNIAKEQSGFSVFAGVGERTREGNDLWHEMMESGV * : ********************************** *:::***:************ :****:*** ************************* :*:* GEIGEEERWAIHRAAPSYEELSNSQELLETGIKvIDLMCPFAKGGKVGLFGGAGvGKTVNMMELIRNIAIEHSGYSvFAGvGERTREGNDFYHEMTDSNV
210 220 230 240 250 260 270 280 290 300 I I I I I I I I I I •DKTALVFC•QMNEP•GARAR•ALTGLS•AEYFRDEEGRD•LLFIDNIFRFTQAGSE•SALLGRMPSA•GYQ•TLSTEMGELQDRITSTKKGSvTS•QAIY ***..**.********:* *******::** *** ********:***:*:* **************************** **:******:**:*****:* IDKvSL~GQMNEPPGNRLRvALTGLTMAEKFRD-EGRDvLLFvDNIYRYTLAGTEVSALLGRMPSAvGYQPTLAEEMGvLQERITsTKTGSITSVQAVY
310 320 330 340 350 360 370 380 390 400 I I I I I I I I I I VPADDLTDPAPATAFTHLDATT•LSRQIAELGIY•AVDPLDSTSRILD•NVIGDDHYDTAQAvKQILQRYKDLQDIIAILGMDELSDDDKLwARARKvQ ********************************************* *** *:*::*****::*: *********************************** VPADDLTDPsPATTFAHLDATwLSRQIAsLGIYPAvDPLDSTSRQLDPLVVC-QEHYDTARGVQSILQRYQELKDIIAILGMDELSEEDKLwARARKIQ
410 420 430 440 450 I I I I I RFLSQPFFVAEAFTGLAGKYVKLEDTIKGFKEIIAGRHDNLPEAAFYLVGTIEEAVAKVKTL ***********:*** :****:*:***:***:*::* ************************* RFLSQPFFVAEVFTGSPGKYVSLKDTIRGFKGIMEGEYDHLPEQAFYMVGSIEEAVEKAKKL
B Chloribium ~. eoli
i0 20 30 40 50 60 70 80 I I I I I I I I MASSDKAFKLDIvTPQKLFFSGEvTS•IAPGLDGLFQIMKGHAPLLAALKSGKvRLSLSDKSEDSFQIEGGFFE•SGNKAILLTED•S :::**:*:::: :*** *::: ::* :* : * ******:*:*:*::*: : *: : ::**::** :::::*:: MAMTYHLDwSAEQQMFSGLVEKIQVTGSEGELGIYPGHAPLLTAIKPGMIRIvKQHGHEEFIYLsGGILEvQPGNVTvLADTAIRC-QDLD
RAMEAKRKAEEH I S S S HGDVDYAQASAE LAKAI AQLRV IE LTKKAM Fig. 4. Comparison of the amino acid sequences of Chlorobium /3 and • subunits with the corresponding proteins from E. coli. The alignment was performed as in Fig. 3. (A)/3 subunit. (B) • subunit.
~
blastica
/ • RR rubrum
/ / / ~ " A
J
B
~
~ Chlorobium
/
"~Ps 3
J
/
J J
Megaterium
~'Megaterium
~
Chlorobium
R
rubrum
j
.....
/
~ A n a b
....
j ~ ~ / / " / S p a ~
~
.
nach Maize
Fig. 5. Evolutionary trees calculated with the sequences of the genes encoding /3 and • subunits. For the sake of compatibility, trees have been rooted with E. coli as an outgroup. Full species names are: Megaterium, Bacillus megaterium; Chlorobium, Chlorobium limicola; R. rubrum, Rhodospicillum rubrum; R. blastica, Rodopsendomonas blastica; Coccus, Synechococcus spec. PCC 6103; Anabaena, Anabaena spcc. PCC 7120; cystis, Synechocystis spec. PCC 6803; Marchantia, Marchantia polymorpha; pea, Pisum saticum; spinach, Spinacea oleracea; maize, Zea maize; wheat, Triticum aesticum. (A)/3 subunits. (B) • subunits.
272
~C
[Y I
I
I
[
~
Whole Cell
~c
Membrane
Fig. 6. Identification of the Chlorobium limicola [3 subunit synthesized in Escherichia coil [3 subunit tess strain. JPl7 cells with pKK[3 and pTN1670 were grown al 37°C in L broth in the presence of 50 ~g ampicillin. Whole cells (left panel, 200 ~g protein) and membranes (right panel, 80 and 120 #g protein) were analyzed by immunoblotting after SDS-polyacrylamide gel (12.5%) electrophoresis. -, JPI7 cells without plasmid; [3~, with pKK[3; [31-, with pTN1670. The positions of E. coil [3 ([3E) and C. limicola [3 (tic) were indicated by arrows.
sense mutant KF20 but not KF95. However, KF95 was complemented by pKK/3e and pTN1665 (coding E. co#/3 and E subunits), confirming that the mutation of this strain is polar and expression of the downstream uncC (e) gene may be extremely low. Further studies demonstrated that E less mutation of KF148 was complemented by pKK/3E but not by pKK/3 similar to pTN1665 (E. coil /3 and e) and pTN1670 (E. coli /3). The introduction of these plasmids did not affect growth of the wild-type strain. We confirmed that the C. limicola /3 subunit is assembled on the J P I 7 membrane, although the amounts were lower compared with the E. coil /3 subunit (Fig. 6). Amounts of the C. limicola /3 subunit were essentially the same in the absence (pKK/3, Fig. 6) or presence (pKK/3E, now shown) of the C. limicola • subunit. The m e m b r a n e ATPase activity with the C. limicola /3 subunit was about 5% of that of the E. coli [3 subunit. These results suggest that C. limicola /3 and E subunits are functional under aerobic condition when assembled with other subunits of E. coli ATPase. Discussion The green sulfur bacterium Chlorobium limicola is a m e m b e r of a family of photosynthetic bacteria that utilizes a reaction center related to Photosystem I of cyanobacteria and chloroplasts [16-19]. This bacterium grows under strict anaerobic conditions and several of its biochemical systems are sensitive to oxygen [32]. The utilization of light energy involves the reduction of N A D by the reaction center, generation of protonmotive force (pmf) by electron transport through the cytochrome b / c I complex, and the utilization of pmf for ATP-synthesis by the enzyme F-ATPase. This enzyme is universal and functions in respiration, photosynthesis and several other processes that generate pmf. F-ATPases are highly conserved in their structure, subunit composition and amino acid sequences of some of their subunits [3-5]. In all the bacteria studied so far the genes encoding F-ATPase subunits are organized in operons. The order of the different genes in the operons is conserved [4-11]. Even though in differ-
ent bacteria the operon was split in different places, the /3 and E subunits are always transcribed on the same mRNA. While in purple bacteria the genes encoding the /3 and E subunits are present in a large operon encoding other subunits of the catalytic sector [4,5], in cyanobacteria and chloroplasts these genes are present in a separate operon [7-9]. In this work it was shown that the genes encoding /3 and • subunits are present in a separate operon. It is interesting to note that all the organisms containing Photosystem I-like reaction center have the genes encoding the /3 and e subunits of F-ATPase arranged in a separate operon [41]. There is no apparent evolutionary explanation for this correlation. As expected, the amino acid sequence of the chlorobium /3 subunit was highly homologous to the corresponding subunits from various sources. Even though chlorobium is a strictly anaerobic bacterium, the /3 subunit contains no cysteine and only one tryptophan. We proposed that these residues were eliminated from F-ATPases when the oxygen concentration increased in the atmosphere [42]. If this is correct, the early evolution of F-ATPase generated a structure that does not allow the presence of these residues even in anaerobic organisms. In contrast to the /3 subunit, the • subunit underwent major changes in its size and amino acid composition. The chlorobium subunit is considerably smaller than the other known • subunits and its amino acid sequence is not conserved as well as the /3 subunit. Therefore, the /3-e operon is one of the best examples of different rates of evolution in an operon in which its antiquity had not been disputed [41]. Evolutionary dating with two such genes in a single operon can put to rest the argument over the effect of various evolutionary rates on the calculation of genetic distances [43]. The observed placing of both the /3 and • subunits together with the corresponding subunits from R. blastica and R. rubrum suggests that the method used in this study for calculating evolutionary trees is not as sensitive for this property. While the /3 subunit of F-ATPase from C. limicola is highly homologous to/3 subunits from other sources, the • subunit is very different. It is the smallest •
273 subunit reported so far and shows homology only to the N-terminal part of corresponding subunits from bacterial sources. Therefore, it is of interest that such a small C. limicola • subunit could complement the E. coli less mutant. It is also demonstrated that the amino terminal half of the E . coli • subunit whose size is similar to the C. lira!cola • subunit is the functional part of this subunit [31]. The C-terminal part of the E. coli • subunit was implicated in its inhibitory effect on the ATPase activity of the enzyme. Further studies on the hybrid enzyme may shed light on the function of the • subunit in the catalytic activity of F-ATPases.
Acknowledgment We are grateful to Dr. Takato Noumi for constructing pTN1665.
References 1 Mitchell, P. (1961) Nature 191, 144-148. 2 Racker, E. (1976) A New Look at Mechanisms in Bioenergetics, Academic Press, New York. 3 Nelson, N. and Taiz, L. (1989) Trends Biocbem. Sci. 14, 113-116. 4 Futai, M., Noumi, T. and Maeda, M. (1989) Annu. Rev. Biochem. 58, 111-136. 5 Senior, A.E. (1990) Annu. Rev. Biochem. 19, 7-41. 6 Walker, J.E., Saraste, M. and Gay, N.J. (1984) Biochim. Biophys. Acta 768, 164-200. 7 Cozens, A.L. and Walker, J.E. (1987) J. Mol. Biol. 194, 359-383. 8 Curtis, S.E. (1988) Photosynth. Res. 18, 223-244. 9 Lill, H. and Nelson, N. (1991) Plant Mol. Biol. 17, 641-652. 10 Falk, G., Hampe, A. and Walker, J.E. (1985) Biochem. J. 228, 391-407. 11 Falk, G. and Walker, J.E. (1988) Biochem. J. 254, 109-122. 12 Michel, H., Weyer, K.A., Gruenberg, H., Dunger, I., Oesterhelt, D. and Lottspeich, F. (1986) EMBO J. 5, 1149-1158. 13 Trebst, A.Z. (1986) Z. Naturforsch. 41c, 240-245. 14 Komiya, H., Yeates, T.O, Rees, D.C., Allen, J.P. and Feher, G. (1988) Proc. Natl. Acad. Sci. USA 85, 9012-9016. 15 Deisenhofer, J. and Michel, H. (1989) EMBO J. 8, 2149-2170. 16 Hurt, E. and Hauska, G. (1984) FEBS Lett. 168, 149-154. 17 Blankenship, R.E. (1985) Photosynth. Res. 6, 317-333. 18 Nitschke, W. and Rutherford, A.W. (1991) Trends Biochem. Sci. 16, 241-245.
19 Biittner, M., Xie, D.-L., Nelson, H., Pinther, W., Hauska, G. and Nelson, N. (1992) Biochim. Biophys, Acta 1101, 154-156. 20 Chitnis, P.R. and Nelson, N. (1991) in Cell Culture and Somatic Cell Genetics of Plants (Bogorad, L. et al., eds.), Vol. 7B, pp. 177-224, Academic Press, New York. 21 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edn., Cold Spring Harbor Laboratory, Cold Spring Harbor. 22 Henikoff, S. (1984) Gene 28, 351-359. 23 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 24 Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395. 25 Felsenstein, J. (1989) PHYLIP 3.2 Manual, University of California Herbarium, Berkeley. 26 Amann, E. and Brosius, J. (1985) Gene 40, 183. 27 Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Science 239, 487-491. 28 Kanazawa, H., Tamura, F., Mabuchi, K., Miki, T. and Futai, M. (1983) Proc. Natl. Acad. Sci. USA 77, 7005-7009. 29 Lee, R.S.-F., Pagan, J., Wilke-Mounts, S. and Senior, A.E. (1991) Biochemistry 30, 6842-6847. 30 Noumi, T., Oka, N., Kanazawa, H. and Futai, M. (1986) J. Biol. Chem. 261, 7070-7075. 31 Kuki, M., Noumi, T., Maeda, M., Amemura, A. and Futai, M. (1988) J. Biol. Chem. 263, 17437-17442. 32 Iwamoto, A., Omote, H., Tomioka, N., Itai, A., Maeda, M. and Futai, M. (1991) J. Biol. Chem. 266, 16350-16355. 33 Moriyama, Y., Iwamoto, A., Hanada, H., Maeda, M. and Futai, M. (1991) J. Biol. Chem. 266, 22141-22146. 34 Bradford, M.M. (1976) Anal. Chem. 72, 248-254. 35 Reymond, P., Geourijon, C., Roux, B., Durand, R. and Fevre, M. (1992) Gene 110, 57-63. 36 Weldon, S.L., Rando, A., Matathias, A.S., Hod, Y., Kalonick, P.A., Savon, S., Cook, J.S. and Hanson, R.W. (1990) J. Biol. Chem. 265, 7308-7317. 37 Cook, J.S., Weldon, S.L., Garcia-Ruiz, J.P., Hod, Y. and Hanson, R.W. (1986) Proc. Natl. Acad. Sci. USA 83, 7583-7587. 38 Gundelfinger, E.D., Hermanns-Borgmeyer, J., Grenningloh, G. and Zopf, D. (1987) Nucleic Acids Res. 15, 6745-6745. 39 Gromet-Elhanan, Z., Khananshvili, D., Weiss, S., Kanazawa, H. and Futai, M. (1985) J. Biol. Chem. 260, 12635-12640. 40 Richter, M.L., Gromet-Elhanan, Z. and McCarty, R.E. (1986) J. Biol. Chem. 261, 12109-12113. 41 Nelson, N. (1992) Biochim. Biophys. Acta 1100, 109-124. 42 Nelson, N. (1989) J. Bioenerg. Biomembr. 21,553-571. 43 Lake, J.A. (1991) Trends Biochem. Sci. 16, 46-50.
Biochimica et Biophysica Acta, 1172 (1993) 267-273 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00
BBAEXP 92472
The atp2 operon of the green bacterium Chlorobium limicola D i a n - L i n Xie a, Holger Lill b Gfinter H a u s k a c, M a s a t o m o M a e d a d Masamitsu Futai d and N a t h a n N e l s o n a " Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ (USA), t, Abteilung Biophysik, Fb 5 Biologic / Chemic, Unicersitdt Osnabriick, Osnabriick (Germany), ' Unirersitdt Regensburg, Lehrstuhl fiir Zellbiologie und Pflanzenphysiologie, Regensburg (Germany) and a Department of Organic Chemistry and Biochemisto', The hlstitue of Scienttfic and Industrial Research, Osaka Unicersity, Osaka (Japan) (Received 23 October 1992)
Key words: Chlorobium; F-ATPase; Operon; Hybrid enzyme; (E. coli)
The operon (atp2) encoding the/3 and E subunits of F-ATPase from Chlorobium limicola was cloned and sequenced. In contrast with purple bacteria these genes are arranged in a separate operon similar to the cyanobacteria. The operon terminates with a pronounced stem-loop structure. About 0.8 kb upstream of the /3 subunit a gcnc encoding the enzyme phosphoenolpyruvate carboxykinase was identified. This gene is transcribed in the opposite direction of the atp2 operon and also ends with a stem-loop structure. These genes of green bacteria are among the first to be sequenced, and thcrcfore the genetic distance between these genes and corresponding genes from other bacteria and eukaryotes was studied. Even though the operon structure resembles that of cyanobacteria, the evolutionary tree compiled from these data places the chlorobium gene close to purple bacteria. Chlorobium limicola/3 and e subunits complemented Escherichia co6 mutants defective in the corresponding subunits, indicating that the hybrid enzyme formed from subunits of the two bacteria is active in ATP synthesis.
Introduction The protonmotive force generated by photosynthesis and respiration can be utilized for ATP formation by the enzyme F-ATPase [1,2]. This enzyme is present in mitochondria, chloroplasts and all eubacteria studied so far [3-5]. It is a multisubunit enzyme containing two distinct sectors, a membrane sector that functions in proton conduction and a catalytic sector that functions in ATP-synthesis and hydrolysis. The genes encoding subunits of the enzyme in bacteria are organized in operons [4-9]. The structure of the various operons is conserved through evolution, but some variations in the number and content of each operon took place. In E. coli all the genes encoding F-ATPase subunits are clustered in a single operon [6]. In R. rubrum there are two separate operons, one contains genes encoding the membrane sector and the second contains genes encoding the catalytic sector [10,11]. In cyanobacteria and chloroplasts the genes encoding /3 and • subunits are clustered in a separate operon (atp2), and the rest of the genes are present in another operon (atpl) or have been exported into the nucleus, respectively [7-9]. It is
not clear what governs the operon structure and the way by which the split operons evolved in the various bacteria and chloroplasts. However, the sequence of the genes within the operons is strictly conserved, and their order is genes encoding a,c,b,6,c~,y,/3 and E. Photosynthetic bacteria utilize two different mechanisms for their light-driven electron transport systems. While purple bacteria contain photosynthetic reaction centers homologous to the chloroplasts Photosystem II, the green bacteria operate with a reaction center homologous to Photosystem I [12-19]. Cyanobacteria contain a photosynthetic machinery resembling that of chloroplasts [20]. Regardless of the kind of reaction centers, the protonmotive force generated by them is utilized by F-ATPases for ATP formation. In this communication we report on the cloning and sequencing of the genes encoding the /3 and • subunits from the green bacterium Chlorobium limicola. We observed that the genes are clustered in an operon which contains only these genes and thereby resembles the atp2 operons of cyanobacteria and chloroplasts.
Experimental procedures Genomic library and screening
Correspondence to: N. Nelson, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110, USA.
Two
genomic
libraries
were
constructed
from
Chlorobium limicola DNA. The first was constructed by
268 partial SaulIIA digestion followed by ligation into the BamHI site of the plasmid Bluescript II SK(+). This library was satisfactory for initial screening, but due to substantial rearrangements it could not be used for cloning the full size atp2 operon. For this purpose, a second library of BarnHl DNA fragments were cloned into the BarnHl site of the plasmid pGEM-4Z. A partial sequence of the gene encoding the subunit was kindly provided by Dr. Lincoln Taiz. An oligonucleotide ATG CCG AGC GCC G T G GGC TAC CAG CCG ACC CTC AGC A was synthesized on an Applied Biosystems 381A DNA Synthesizer. The SaulIIA library was then screened by 32p end-labeled oligonucleotide as previously described [21]. The plasmids isolated from the positive colonies were subjected to dot blot analysis, and the DNA fragments were then analyzed by Southern blots following digestion by SauIIIA. The hybridized DNA fragments were isolated, subcloned into the BarnHI site of pBluescript, and used for screening the second library. Published procedures were used for recombinant DNA methods, purification of genomic DNA, screening of libraries [21], overlapping deletions using exonuclease III digestion [22], and sequencing by the dideoxy chain termination method [23].
HindllI restriction enzyme to construct pKK/3. They were introduced into E. coli strains KY7230 (asn, thi, thy) [28], JP17 (AuncD (deletion of the /3 gene), argH, pyrE, entA, recA::TnlO) [29], KF20 (uncD20 (bQ397--+ end), thi, thy) [30], KF95 (uncD95 (BQ7-~ end), thi, thy) (this study) and KF148 (uncC148 (mutation at Shine-Dalgarno sequence of the • gene), thi, thy) [31]. Expression plasmids for E. coli /3 and • subunits were also used; pTN1670 for /3 subunit [31] and pTN1665 (constructed in this study) for /3 and e subunits. pTN1665 was a derivative of pTN1661 [31]. A rich medium (L broth) with or without ampicillin (50 / , g / m l ) and minimal medium containing thiamine (2 / z g / / m l ) , thymine (50 # g / m l ) , asparagine (50 # g / m l ) , arginine (100 /~g/ml), uridine (100 p,g/ml) and a carbon source (0.2% glucose or 0.4% succinatc) were used [30]. Membranes prepared by a French press were subjected to electrophoresis and immunoblotting using antibodies specific for the E. coli [3 subunit [32,33]. ATPase activity of the membranes were measured according tot he standard assay condition for E. coil F~ ATPase [32]. Protein amounts were estimated by the published method [34] using bovine serum albumin as standard. Results
Computer analysis Sequences of DNA fragments were assembled using DNAstar software. Multiple alignment and comparisons of sequence data with databases were performed by GCG software package [24]. Evolutionary relations and genetic distances were calculated using the PHYLIP package under VAX VMS [25]. For proper alignment we used the GCG GAP program the following way: in the first run, the amino acid sequences were aligned. Next, these alignments were copied to the corresponding DNA sequences and obvious mismatches were corrected in the protein alignments. Such cycles were repeated several times, and finally the alignments of DNA sequences were used in the calculation, employing the D N A M L program under the maximum likelihood model for the evaluation of evolutionary trees. Complementation of Escherichia coli uncD and uncC mutants with Chlorobium limicola /3 and • subunits Cloned C. limicola genes for the /3 and • subunits were ligated into the Pstl site of the pKK233 Escherichia coli expression vector [26]. The introduction of the Pst I site by PCR [27] at the 5' terminus of the/3 genc caused amino acid substitution / 3 G l n - G l u ~ /3Ala-Ala at positions 2 and 3. Another PstI site was introduced by substituting nucleotide residues 54575477 downstream of the termination codon of the e gene. Most of the • gene was eliminated from the resulting plasmid (pKK/3•) by digestion with the
A plasmid isolated from the positive colony obtained by screening the chlorobium library contained a BarnHI DNA fragment of about 4.5 kb. As shown in Fig. 1, the DNA fragment contained three complete and one truncated open reading frame. Fig. 2 shows the DNA sequence of the fragment and the deduced amino acid sequences of the open reading frames encoding the fi and e subunits of F-ATPase. They are located at the 3' end of the DNA fragment. No other genes encoding F-ATPase subunits could be identified in the cloned DNA fragment. Upstream of the gene encoding the /3 subunit, a gene encoding PEP-carboxykinase was identified. This gene is transcribed from the opposite DNA strand to the /3 subunit gene. The open reading frame starts with the ATG at position 2731 and ends with TGA at position 792 (see Fig. 2). Another reading frame encoding an unidentified protein starts at position 609 and is transcribed in the same direction. This arrangement suggests that the genes encoding the /3 and e subunits are transcribed
SphI
0RF
HindH[Sail XmalII
Sinai
KpnI
BamHIHindIll
PEP-C.K. I
1 kb
I
Fig. 1. S c h e m a t i c p r e s e n t a t i o n of t h e open reading frames of the D N A f r a g m e n t c o n t a i n i n g the atp2 operon. Useful restriction sites are i n d i c a t e d and the s t e m - l o o p s are shown by the symbol ~.
269 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1821 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721
CCTTAGCGAACATCGGCGAGTCGTTGCCGTAGTCGGTGCCCATGCGCCCTGGGCGAGCAT GCGACGCCCCAGGTGATCCGTCCGTCGCGGCGCATGTAGGACATCGAGGGCATGAAGAAC GGTCGCGTCCGACTTGTCGGTAATGCCGTTGTTGTCCAGCGAGATGTCGGGATGCAGACC CCGGATGCCGAAGCCTATCTCCGATGTTCCCTCCTTCATGAAGCCGAGGGTTGCCGGGTT GTTCATGACGGCGGAGTTACCGGTGTCGTAGGCTGAGCCGGTGCCGCCCATGGCCATCGA
TTTGGCGCCGTACCCTTCGAGGTTCATACCGTTGGTGGCGAAAGCGGGCGTGGCTCCGAT G A G C A C A A G A A G A G C C A C A G C CGAACATGC GGTTTTTC GAATCATGAT GTCAGTGAGT TG AGGTTTGATGCTTGCGGCCAACCCAATGCCCGTGACCTCGGCAGCCCAATGAAGGCGGGC AAGC G C C G G T C G A T C A T G C A G G A T A T GAGGAAGT GC C G G G T T G A T A A T C C G G A T CAGC GA GGCTCGCTGCACGGCCTGCCATGAAACTTCTTTTCCGTAACCGCCGACTGTCAGACTCGA A A C G C G C A T G G G G C A A C A T G A A T A A A C A A C AGCAATCAGAC C GGAATGCGATATAT GACT ATATAACTGATT GAATATAT GGCAATAT TATCGATT CCGCAACACTTG CC G G G C A A A C A A AACGCTATACCATAA~GCGGACAT GCTGCC CGCTTT TTTTCGTGTATGACTGATGC TCCGATGAAC TCAAGACT C G G C G G A G T A G A G T T C G G G C G C A A G T T C CC AATGCTCC GGAG ACAT CCAGAGCGTCGAAAGCAACAGC TCGCGGATGTGCGT GAACTCTT TGGGAAGC CTGT C G T A A A G C T T T T C A A G C A G C T C C T CGTGTGAAAAAAGC TC CTGCTT CC CTTC GCGATCCA C C G A C A T G A G C T T T G A A A A C T T G T TGCTGGTGAATCCG TC CAGACC CC GCCAGTCGAGCG ATTCATATCTCGGCATCCAGCCAAGCGGGCTTTCCACCGCGCCGGAACGTCCGTGCACAC
GCCCCATCATCCACTTGAGCACCCGCATGTTGTCGCCGAATCCGGGCCAGAGGAACTTGC CGGTTCTC GTCT TGCGGAACCAGT TCACGATGAAAATC CTTGGC GGATCGGAGACGGTTC GC CC G A C A T G G A G C C A G T G G T T GAAGTAGT CGCCCATGTGGTAG CC GC A A A A T G G C A G C A TGGCGAAGGGGTCGACGCACGTCGCCGACCTTGCCGCGGCGGCGGTCTTTTCCGAACCCA TC GTGGCCGCCATGTAAACGCC GTAATACCAGTT C G A G G A C T G A T A G A C C A G C G G A A T G G TGTCGC CGCGCCGCCC G G C G A A A A T G A A A G C C G A A A T C G G C A A C A C C C T C G G A T T T T C C C A A G C G G G A T C G A T C A C GGGG CACTGACT G G C C G G A G C G G T G A A G C G G G A G T T C G G G T G G G CGGCCGGGCGGTCGCATCCT GGAACCCAGGGCTT GC C C T G C C A G T C G A T G A G G T A T T C C G GGGGCGTGTCGGTCATATCCTCCCACCAGACGTCGCCGTCGGGGGTGAGCGCCACGTTGG T G A A G A T G C A G T T G G C G T G A A G G G T G G C C A T C GC GTTCGGAT TGC TCTTGTCC GAGGTGC CCGGAGCCACGC CAAAAAAGCCATAC TCCGGATT GATGGC GT GCAGGCGT CCAT CCTT GC CCTGCTGATCCAGGCGAATGTCGTCGCCGACGGTGGTGATCTTCCACCCCTCCATCTCGC C C G G C G G A A T G A T C A T G G C G A A G T T G G T C T T G C C GCAGGC GC T G G G G A A G G C C G C C G C A A CATAATCTTTTTCACCTGCCGGCGACTCGACGCCGAGGATGAGCATGTGCTCGGCGAGCC AC CCCTCGTC GCGCGC CATCGAGGAGGCGATACGCAGGGC GAAGCACT TTTTGC CAAGCA GT GTATTGCC G T T G T A G C C G C T G C T G T A G G A G A C G A T G G C GT G C T C T T C C G G G A A G T G G A CGATGTACTT GGTGTC GTTGCAGGGCCACGGC ACATCCGC CTGGCCCGGTTC GAGCGGCG C G C C G A C C G A G T G C A G G C A G G G C A C G A A C T CCGCCTCTTC GTCGAGCAGTTC GAGCAC CT G G C G A C C C A T G C G C G T CATGAT GCGCATGT TGGTGACGAC GT AGGGCGAATC GGTGAT CT C G A C C C C G A T G T G C G C G A T G T G C G A G C C GAGC GGCCCCAT GC TGAACGGAAT GACATACA TGGTGCGC C C C T T C A T G C A G C C G G T G A A G A G C T T G T T C A G C G T C G C C T T C A T CTCTTT TG
GAGCGACCCAGTTGTTGGTCGGCCCCGCATCCTGGCGGCGAATGCTGCAAATAAAGGTTC
5401
GATCCTCCACCCTGGC CACATCACTCGGAT CGGAACGGCAGAGGTAGCTGTT CGGACGCT T C T G T T C C G A C A G T T T G A T G A A G G T G C C G C TGTTGACCAT CTCCTCGCACAGGCGGTC GT ACTCCTCGGTCGATCCGTCACACCAGCAAACCGAATCGGGCTGGCACAGCGCAACGGT CT CTTTGACC CACTGGAGAAGTTTCAGGTT CT TGACATAATC TGGCGCATTGAT TGGGAT CG G T T C C A T A A T T G C T G A A A A T C G A T T A T T GAGAGTGTTGCT GATTGAAAGGCC TGAAACAA CGCGCCCCGGCAGGAAATTCCCCCGGGGCATCTGGAAT CAGGCATGTTGACTATCTAAGT GTGCAAAAATATCT AAGCGGACAAAAAAGC CTGGTCATTT TTTAGCCGTCTTTCGCTGAG C G C C G T T C T G A C G A T C T G G T C G A A C T CGCCTTGCTTCT CGGCCCGACAATCACGCCGGTC A A C T T G C G G A G G C A T C G A T G A C A A A G G T GGTCGGAATGCC GGTGATGCCGCCATCGAT GT GGCCGTTGAAGGCCCGGATCAGCTCAGGAGTGGCCATGAGCACCGGCCAGGT CATCCC GT T C T G T C T C A T G A A A T T C T T C A C G T T C GGAACCTTCTCGTTGACCGCAATGCC GACAAAGG T G A A G C C T T T A G C G C C C A G G C T C T C T GAACCTGTACCATATCGGGAATCTCC GACCGGCA G G G C G G G C A C C A G G T G G C G A A G A A A T T G A C G A T A T A G G C C T T G C C C T T G A G G G A G G A C GA GGAGAAGGGCTTGCCGTCAACGGTCACGCCCGAAAAAGAGGGCGCGGGCCAC GGAGCAGC GCTGGCCTGCCCTGAAAAGGTGAAAGAAGCTGCAAAAACCATGAGCGCTGCCATAATGGC G G C A A A A C G G G A G G G A A C T C T T T T CATAGTGATTCTTT A G A T G A C G G T A A A T A G A C G G T A A A T A G A C G G T A A A T C C T G A C A T G C TT A A T G A T C A C A A A C G A T A A A T A G T T C T G A T T T A T A A T A T A A G A A T C C C G G C G C G A T T C C CC TTGGTTCG TC CCTGATATTTCTTTGC GTTTTGAG AACAAATAAGCTATATTTCGTCTCGAATTTTTCGCT TAAGAGCGGGAAAGGCCGATTCAA GCCACCTTAGCTCAGTTGGTAGAGCAACTGTTTCGTAAATAGTAGGTCGTGGGTTCAAGT CCCTCAGGTGGCTCGCTTATGCCGACAACCGAGT CGGGTAATCGGAACAGAACCTGAGAA CGGAGTCGCCGTCGCCGCCAAACGTTTGATTGCAAC CAAAATCCCATATTGAAGAATACC A T G C A A G A A G G T A A G A T T T C C C A G A T C A T C G G A C CT G T C G T T G A C G T T G A C T T T G C T G A A M Q E G K I S Q I I G P V V D V D F A E G G A C A G C T T C C G T C T A T C C T CGATGCCCTCAC GGTCACCCGC CAGGAT GGCTCGAAACTG G O L P S I L D A L T V T R Q D G S K L G T T C T C G A ~ A C G C A G C A G C A C C TCGGCGAAGAGC GCGTTCGCCGAATCGC CATGGAGGGA V L E T Q Q H L G E E R V R R I A M E G ACAGATGGCCTGGTCAGGGGCATGAGCGCCA~CACCGGCA~ACCGATCCAGGTACCG T D G L V R G M S A K N T G K P I Q V P GTAGGCGAAGAGGTGCTCGGCAGAATGCTGAACGTTGTCGGCGATCCCATTGATGGC;tJLA V G E E V L G R M L N V V G D P I D G K GGCCCTGTACTTTCAAAGA~TCCTACTCCAT CCATCGCACCGCTCCCA~TTTGATGAG G P V L S K K S Y S I H R T A P K F D E C T T T C G A C C A A G G C C G A G A T G T T C G A ~ C C GGCATCAAGGTTATCGAC CTTCTCGAACCG L S T K A E M F E T G I K V I D L L E P TACTCGCGCGGCGGCAA~CCGGCTTGTTCGGCGGCGCAGGCGTCGGCAAGACCGTGCTT ¥ S R G G K T G L F G G A G V G K T V L ATCATGGAC~TTATCAACAATATCGC CAAAGAACAGTCCGGTTTCTCGGTATTTGCCGGC I M E L I N N I A K E Q S G F S V F A G GTCGGCGAGCGCACCCGC GAAGGAAACGACCTCTGGCACGAGATGATGGAGTCTGGCGTT V G E R T R E G N D t W H E M M E S G V ATCGACA~CCGCACTCGTGTTC GGCCAGATGAACGAGCCTCCGGGAGCACGTGCGCGC I D K T A L V F G Q M N E P P G A R A R G T C G C C C T G A C C G G C C T G A G C A T C GC C G A A T A C T T C C G T G A T G A A G A G ~ T C G T G A C G T G V A L T G L S I A E ¥ F R D E E G R D V C T T C T G T T C A T C G A C A A C ATCTTC CGCTTCACCCAGGCAGGCTC CGAAGTGTCCGCGCTT L L F I D N I F R F T Q A G S E V S A L C T C G G A C G T A T G C C G A G C GCCGTGGGCTACCAGC C G A C C C T C A G C A C C G A G A T G G G T G A G L G R M P S A V G ¥ Q P T L S T E M G E CTTCAGGACAGAATCACCTCCACCAAGAAAGGTTCGGTCACC TC GGTGCAGGCCATCTAC L Q D R I T S T K K G S V T S V Q A I ¥ GTCCCTGCCGATGACCTTACCGACCCTGCCCCGGCCACCGCGTTCACCCACCTCGACGCC V P A D D L T D P A P A T A F T H L D A ACGACCGTGCTTTCGCGTCAGATCGCCGAGCTGGGCATCTACCCCGCCGTGGATCCGCTC T T V L S R Q I A E L G I ¥ P A V D P L GATTCGACCTCGCGAATCCTCGATCCA~CGTCATCGGCGACGATCACTACGACACCGCC D S T S R I L D P N V I G D D H Y D T A CAGGCCGT CAAGCAGATTCTCCAG CGCTACAAGGACTTGCAGGACATCAT CGCCATTCTC Q A V K Q I L Q R ¥ K D L Q D I I A I L G G T A T G G A C G A G C T G A G C G A T G A C GACAAGCTCGTGGTCGCC C G C G C C C G C A A G G T G C A G G M D E L S D D D K L V V A R A R K V Q CGCTTCCTGTCGCAGCCCTTCTTCGTGGCCGAGGCGTTCACCGGTCTGGCCGGCAAGTAC R F L S Q P F F V A E A F T G L A G K ¥ GTCAAGCT CGAAGACACC ATCAAG GGCTTCAAGGAGATCATC GC AGGCCGTCACGACAAC V K L E D T I K G F K E I i A G R 8 D N CTTCCGGAAGCAGCTTTCTACCTGGTCGGCACTATCGAAGAGGCGGTCGCCAAAGTTAAA L P E A A F ¥ L V G T I E E A V A K V K AC GCTC TAAACCAACGGC AAGACA TG GC AAGTTCAGACAAAG CC TTTAAG CT CGATA.TTG T L * M A S S D K A F K L D I V TAACGCCGCAGAAGCTTTTCTTTTCGGGAGAGGTGACCAGCGTCATCGCTCCGGGCCTGG T P Q K L F F 8 G E V T S V I A P G L D ACGGATTGTTTCAGATTATGAAAGGTCACGCCCCCCTGCTCGCCGCGCTCA~GCGGCA G L F Q I M K G H A P L L A A L K S Q K AGGTGCGGCTCTCTCTTTCAGACAAGTCTGAAGATTCGTTCCAGATTGAAGGGGGATTCT v R L S L S D K S E D S F Q I E G G F F TCGAGGTGAGCGGCAACAAGGCAATTCTGCTTACCGAGGACGTCTCCTGACCCACGGAAG E V S G N K A I L L T E D V S * CATAACCGCATGAAAAAAACATCAGCAGCGCCTCGGAGTGTTGCTGATGTTTTTTTGCAT
5461
TCATGAAAATTCTGATC
3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341
5477
60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4880 4740 4800 4860 4920 4980
from an operon separated from the rest of the FATPase subunits. Moreover, at the end of the gene encoding the e subunit there is a pronounced stem-loop structure (Fig. 2) that may serve as a transcriptional termination signal. As shown in Fig. 2, a similar stemloop structure is present at the 3' end of the gene encoding PEP-carboxykinase and in the operon encoding the chlorobium photosynthetic reaction center [19]. The predicted reading frame for PEP-carboxykinase encodes a hydrophilic protein of 646 amino acids with a calculated molecular mass of 72539 Da. The amino acid sequence of this protein shows a 58% identity over a 564 amino acid overlap with a recently reported sequence of PEP-carboxykinase from N. frontalis [35]. It also has a 51% identity with chicken mitochondrial PEP-carboxykinase [36], a 47% identity with the cytosolic enzyme [37], and a 48% identity with the enzyme from Drosophila melanogaster [38]. These observations suggest that the open reading frame upstream of the gene encoding the /3 subunit encodes the chlorobium PEP-carboxykina~e. Fig. 3 depicts the aligned amino acid sequences of PEP-carboxykinases from chlorobium and N. frontalis. An operon encoding the /3 and • subunits of FATPase was identified downstream of the PEP-carboxykinase gene. The operon encodes only these two proteins and terminates by a pronounced stem-loop structure typical of all the chlorobium genes sequences so far [19]. The reading frame of the/3 subunit encodes a protein of 462 amino acids with a calculated molecular mass of 50220 Da. Fig. 4 shows the amino acid alignment of the/3 subunits of F-ATPases from chlorobium and E. coli. As was shown for /3 subunits from various sources, the sequences of the E. coli and chlorobium proteins are highly homologous exhibiting a 72% identity. The chlorobium subunit was about 70% identical to/3 subunits from other sources including chloroplast CF v On the other hand, the chlorobium • subunit underwent a much faster evolution as shown by its relatively small size and low percent identity with corresponding subunits from other sources. The reading frame of the • subunit encodes a protein of 88 amino acids with a calculated molecular mass of 9376 Da. It has a 33% identity in the amino acid sequence with the • subunit of E. coli (Fig. 4) and very poor homology with the corresponding subunits of mammalian mitochondria and plant chloroplasts (not shown).
5040 5100 5160 5220 5280 5340 5400 5460
Fig. 2. The complete D N A sequence of the chlorobium D N A fragment and amino acid sequences of the two reading frames in the atp2 operon. The D N A sequence and the open reading frames are schematically depicted in Fig. 1.
270 20
30
40
50
60
70
80
90
I
I
I
I
I
I
I
I
I
MPRGNFLPGRvvSGLS••NTLNNRFSA•MEPIP•NAPDYVKNLKLLQWVKET•ALCQPDSVCWCDGSTEEYDRLCEEMVNSGTFIKLSEQKRPNSYLCRS * * :**:::**: :* *:::: ****:***::*** :*: * : * : * * : *
Chlorobium
~.
10
100 I ****:**
**
MAHSVSSVVNKQLLAYIKESSELMTPKDIYVCDGSAEEYHNLCELLVKQGIFTKLNETKRPNCYLARS
frontalis
Ii0
120
130
140
150
160
170
180
190
I
I
I
I
I
I
I
I
I
200
I
DPSDVARvEDRTF•CSIRRQDAGPTNNWvAPKEMKATLNKLFTGCMKGRTMYVIPFSMGPLGSHIAHIGVEITDSPYvvTNMRIMTRMGRQVLELLDEEA :,:******: *:*** : :********:** ****:** ****************************************** **:::*::**:**:
::
NPADVARVEKCTYICSEKEEDAGPTNNWMAPAEMKAKLNGLMKGCMKGRTMYVIPFSMGPIGGPISRVGVEITDSPYvvVNMCIMAKVGKKVLDLLGvDG
210
220
230
240
250
260
270
280
290
300
I
I
I
I
I
I
I
I
I
I
EFVPCLHSVGA•LEPC•QADvPWPCN-DTKY•VHFPEEHA••SYSSGYNGNTLLGKK•FALR•ASSMARDEG-WLAEHMLILGVESPAGEKDYVAAAFPSACG . * * * * ** * * * * ** * . * * * * , : : * * * : * * * * * *** ********************* ::*::* *********:::*:*:*
*::********
TFVPCTHSCLAPLEEGQKDSTWPCNIDNRYIvQFPEEHRIVSVGSGYGGNALLGKKCYALRIATvLSREAGDSLAEHMLILGITNPQGKKYYITAAFPSACG
310
320
330
340
350
I
I
I
I
I
360
I
370
380
390
I
i
I
KTNFAMIIPPGEMEGWKITTVGDDIRLDQQGKDGRLHAINPEYGFFG•APGTSDKSNPNAMATLHANCIFTN•ALTPDGDVWWEDMT-DTPPEYLIDWQGK ***:**: ::: * * * * : * * * * * : ****** ***** ********** ******* : : : ******** *******:**
::*
400
I :
:*:*
**
KTNLAML--NATIPGWKIECvGDDIAWLKIGKDGRLWAINPESGFFGvAPGTSYKSNPNAMKSCEKDTIFTNvALTEDGDVWWEGMTKEVPKGKIITWLGK
410
420
430
440
450
460
470
480
490
500
I
I
[
I
I
I
I
I
I
I
Pwv~C~CDRPA---AHPNSRFT/~ASQCPVIDPA~NPRVLPISAFIFAGRRGDTIPLVYQSSNJe/XYGVYMAATMGSEKTAAAARSATCV--DPFAMLPFCGYHMG * .... * ********* .... ** ** . . . . ************************ .** . . . . . . * * . * * * * . .*. . ******~****.** EWSADSGEPKPNLAHPNSRFTARVENVPVGDPGYYALEGVPVSAMIFGGRRENTVPLVFQSRSWKHGVLLGSSVASETTAAAEAAAGQLRFDPFAMLPFCGYNMG
510
520
530
540
550
560
570
580
590
600
I
I
I
[
I
I
I
I
I
I
DYFNHWLHvGRIvSDP--PRIFIVNWFRKTRTGKFL~PGFGDNMRvLK~GRVHGRSG-AvESPLGWMPRYESLDWRGLDGFTSNKFSKIAMSVDR~GKQELF **,1:** : : ::: *:** *****,1 :*:*****:*:* *****::1,,1,::* * *:,1,::* : : * * :*** ::1:::,1:1,* DYFGYWLSFADKYDEAKLPKIFHVNWFRKD-NGRFLWPGYGENSRVLKWIIERVEGKEGIAKETPIGYLPAKGALDLSGLD-vPEADMEKILTVDCKAYLSEv
610
620
630
640
I
I
I
I
SHEELLEKLYDRLPKEFTH
IRELLLSTLWMSPEHWELAPELYSAES
EK IRQYH S KFGS LLP KAL IAE LDALEQRLKAAL
o f the d e d u c e d u s i n g the A L I G N p r o g r a m
Fig. 3. C o m p a r i s o n
aligned
a m i n o acid s e q u e n c e s o f the Chlorobium a n d N. frontalis P E P - c a r b o x y k i n a s e e n z y m e s . T h e s e q u e n c e s w e r e from DNASTAR. I d e n t i c a l a m i n o a c i d s are i n d i c a t e d by a s t e r i s k s a n d c o n s e r v a t i v e a m i n o acid r e p l a c e m e n t s are i n d i c a t e d by c o l o n s .
The different evolutionary rates of two genes encoding by the same operon make them interesting for evolutionary dating [9]. Fig. 5 shows, that in spite of the TABLE
I
('omplementation o[ Escherichia coli [3 and • subunit mutants with ('hlorohiun~ limicola [3 and E subunits i n d r o d u c e d into the E. coli s t r a i n s listed. resistant transformants was checked on a m i n i m a l a g a r p l a t e c o n t a i n i n g s u c c i n a t e as the s o l e c a r b o n s o u r c e . All t r a n s f o r m a n t s g r e w o n g l u c o s e p l a t e s . P l a t e s w e r e i n c u b a t e d at 37°C for 3 days (+. positive growth; -, no growth). Strain KF148 (mutant of Shine-Dalgarno s e q u e n c e ) l a c k i n g t r a n s c r i p t i o n o f the • Expression
Growth
plasmids
were
o f the a m p i c i l l i n
subunit. Strain (mutation)
Growth
on succinate
plasmid introduced none pTN1665 pTNI670 KY7230 (wild) JPI7 (J[3) KF20 ([3Q397 ~ end) KF95 ([3Q7 ~ end) KFI48(Ae)
+ -
pKK[3E
pKK/3
+
+
+
+
+
+
+
+
+ +
+
+ +
+ -
+
-
+
significant different evolutionary rates, the calculated evolutionary trees o f / 3 and • subunits are quite similar. Assuming that at least all the /3-e operons evolved from a single ancestral operon, the above evolutionary trees give credence to the calculation methods used in this work [25]. During the last decade it was demonstrated that the /3 subunit of E. coli can be substituted with /3 subunits from various sources [39,40]. Chlorobium is a strictly anaerobic bacterium and several of its biochemical systems are highly sensitive to oxygen [16,18]. Therefore, it is of interest whether the proteins encoded by its atp2 operon complement E. coli uncD (/3) and uncC (e) subunits to support oxidative phosphorylation in the presence of oxygen. Thus we cloned the C. limicola atp2 operon into the E. coli pKK233 expression vector. Constructed plasmids for the /3 subunit (pKK/3) and /3 and • (pKK/3•) were introduced into E. coli /3 and • subunit mutants. As shown in Table I, pKK/3 similar to pTN1670 (coding the E. coli /3 subunit) complemented JP17, a deletion strain of the /3 subunit gene. The same plasmids complemented a non-
271 i0 20 30 40 50 60 70 80 90 i00 I I I I I I I I I I MQEGKISQ••GPvvDVDFAEC-QLPS•LDALTvTRQDGSKL•LETQQHLGEERVRRIAMEGTDGLVRGMSAKNTGKPIQvP•GEE•LGRMLNvvGDPIDGK *..*** .1.*:****:'1::::*:: ***:* ::::::****:**:**:: ** ***:::*** **::1": ************************* * MATGKIVQVIGAVVDVEFPQDAVPRVYDALEv-QNGNERLVLEvQQQLGGGI~RTIAMGSSDGLRRGLDVKDLEHPIEVPvGKATLGRIMNVLGEPVDMK
A Chlorobium
~.
coli
ii0 120 130 140 150 160 170 180 190 200 I I l I I I I I I [ GPvLSKKSYSIHRTAPKFDELSTKAEMFETGIKvIDLLEPYSRGGKTGLFGGAGVGKTvLIMELINNIAKEQSGFSVFAGVGERTREGNDLWHEMMESGV * : ********************************** *:::***:************ :****:*** ************************* :*:* GEIGEEERWAIHRAAPSYEELSNSQELLETGIKvIDLMCPFAKGGKVGLFGGAGvGKTVNMMELIRNIAIEHSGYSvFAGvGERTREGNDFYHEMTDSNV
210 220 230 240 250 260 270 280 290 300 I I I I I I I I I I •DKTALVFC•QMNEP•GARAR•ALTGLS•AEYFRDEEGRD•LLFIDNIFRFTQAGSE•SALLGRMPSA•GYQ•TLSTEMGELQDRITSTKKGSvTS•QAIY ***..**.********:* *******::** *** ********:***:*:* **************************** **:******:**:*****:* IDKvSL~GQMNEPPGNRLRvALTGLTMAEKFRD-EGRDvLLFvDNIYRYTLAGTEVSALLGRMPSAvGYQPTLAEEMGvLQERITsTKTGSITSVQAVY
310 320 330 340 350 360 370 380 390 400 I I I I I I I I I I VPADDLTDPAPATAFTHLDATT•LSRQIAELGIY•AVDPLDSTSRILD•NVIGDDHYDTAQAvKQILQRYKDLQDIIAILGMDELSDDDKLwARARKvQ ********************************************* *** *:*::*****::*: *********************************** VPADDLTDPsPATTFAHLDATwLSRQIAsLGIYPAvDPLDSTSRQLDPLVVC-QEHYDTARGVQSILQRYQELKDIIAILGMDELSEEDKLwARARKIQ
410 420 430 440 450 I I I I I RFLSQPFFVAEAFTGLAGKYVKLEDTIKGFKEIIAGRHDNLPEAAFYLVGTIEEAVAKVKTL ***********:*** :****:*:***:***:*::* ************************* RFLSQPFFVAEVFTGSPGKYVSLKDTIRGFKGIMEGEYDHLPEQAFYMVGSIEEAVEKAKKL
B Chloribium ~. eoli
i0 20 30 40 50 60 70 80 I I I I I I I I MASSDKAFKLDIvTPQKLFFSGEvTS•IAPGLDGLFQIMKGHAPLLAALKSGKvRLSLSDKSEDSFQIEGGFFE•SGNKAILLTED•S :::**:*:::: :*** *::: ::* :* : * ******:*:*:*::*: : *: : ::**::** :::::*:: MAMTYHLDwSAEQQMFSGLVEKIQVTGSEGELGIYPGHAPLLTAIKPGMIRIvKQHGHEEFIYLsGGILEvQPGNVTvLADTAIRC-QDLD
RAMEAKRKAEEH I S S S HGDVDYAQASAE LAKAI AQLRV IE LTKKAM Fig. 4. Comparison of the amino acid sequences of Chlorobium /3 and • subunits with the corresponding proteins from E. coli. The alignment was performed as in Fig. 3. (A)/3 subunit. (B) • subunit.
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Fig. 5. Evolutionary trees calculated with the sequences of the genes encoding /3 and • subunits. For the sake of compatibility, trees have been rooted with E. coli as an outgroup. Full species names are: Megaterium, Bacillus megaterium; Chlorobium, Chlorobium limicola; R. rubrum, Rhodospicillum rubrum; R. blastica, Rodopsendomonas blastica; Coccus, Synechococcus spec. PCC 6103; Anabaena, Anabaena spcc. PCC 7120; cystis, Synechocystis spec. PCC 6803; Marchantia, Marchantia polymorpha; pea, Pisum saticum; spinach, Spinacea oleracea; maize, Zea maize; wheat, Triticum aesticum. (A)/3 subunits. (B) • subunits.
272
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Fig. 6. Identification of the Chlorobium limicola [3 subunit synthesized in Escherichia coil [3 subunit tess strain. JPl7 cells with pKK[3 and pTN1670 were grown al 37°C in L broth in the presence of 50 ~g ampicillin. Whole cells (left panel, 200 ~g protein) and membranes (right panel, 80 and 120 #g protein) were analyzed by immunoblotting after SDS-polyacrylamide gel (12.5%) electrophoresis. -, JPI7 cells without plasmid; [3~, with pKK[3; [31-, with pTN1670. The positions of E. coil [3 ([3E) and C. limicola [3 (tic) were indicated by arrows.
sense mutant KF20 but not KF95. However, KF95 was complemented by pKK/3e and pTN1665 (coding E. co#/3 and E subunits), confirming that the mutation of this strain is polar and expression of the downstream uncC (e) gene may be extremely low. Further studies demonstrated that E less mutation of KF148 was complemented by pKK/3E but not by pKK/3 similar to pTN1665 (E. coil /3 and e) and pTN1670 (E. coli /3). The introduction of these plasmids did not affect growth of the wild-type strain. We confirmed that the C. limicola /3 subunit is assembled on the J P I 7 membrane, although the amounts were lower compared with the E. coil /3 subunit (Fig. 6). Amounts of the C. limicola /3 subunit were essentially the same in the absence (pKK/3, Fig. 6) or presence (pKK/3E, now shown) of the C. limicola • subunit. The m e m b r a n e ATPase activity with the C. limicola /3 subunit was about 5% of that of the E. coli [3 subunit. These results suggest that C. limicola /3 and E subunits are functional under aerobic condition when assembled with other subunits of E. coli ATPase. Discussion The green sulfur bacterium Chlorobium limicola is a m e m b e r of a family of photosynthetic bacteria that utilizes a reaction center related to Photosystem I of cyanobacteria and chloroplasts [16-19]. This bacterium grows under strict anaerobic conditions and several of its biochemical systems are sensitive to oxygen [32]. The utilization of light energy involves the reduction of N A D by the reaction center, generation of protonmotive force (pmf) by electron transport through the cytochrome b / c I complex, and the utilization of pmf for ATP-synthesis by the enzyme F-ATPase. This enzyme is universal and functions in respiration, photosynthesis and several other processes that generate pmf. F-ATPases are highly conserved in their structure, subunit composition and amino acid sequences of some of their subunits [3-5]. In all the bacteria studied so far the genes encoding F-ATPase subunits are organized in operons. The order of the different genes in the operons is conserved [4-11]. Even though in differ-
ent bacteria the operon was split in different places, the /3 and E subunits are always transcribed on the same mRNA. While in purple bacteria the genes encoding the /3 and E subunits are present in a large operon encoding other subunits of the catalytic sector [4,5], in cyanobacteria and chloroplasts these genes are present in a separate operon [7-9]. In this work it was shown that the genes encoding /3 and • subunits are present in a separate operon. It is interesting to note that all the organisms containing Photosystem I-like reaction center have the genes encoding the /3 and e subunits of F-ATPase arranged in a separate operon [41]. There is no apparent evolutionary explanation for this correlation. As expected, the amino acid sequence of the chlorobium /3 subunit was highly homologous to the corresponding subunits from various sources. Even though chlorobium is a strictly anaerobic bacterium, the /3 subunit contains no cysteine and only one tryptophan. We proposed that these residues were eliminated from F-ATPases when the oxygen concentration increased in the atmosphere [42]. If this is correct, the early evolution of F-ATPase generated a structure that does not allow the presence of these residues even in anaerobic organisms. In contrast to the /3 subunit, the • subunit underwent major changes in its size and amino acid composition. The chlorobium subunit is considerably smaller than the other known • subunits and its amino acid sequence is not conserved as well as the /3 subunit. Therefore, the /3-e operon is one of the best examples of different rates of evolution in an operon in which its antiquity had not been disputed [41]. Evolutionary dating with two such genes in a single operon can put to rest the argument over the effect of various evolutionary rates on the calculation of genetic distances [43]. The observed placing of both the /3 and • subunits together with the corresponding subunits from R. blastica and R. rubrum suggests that the method used in this study for calculating evolutionary trees is not as sensitive for this property. While the /3 subunit of F-ATPase from C. limicola is highly homologous to/3 subunits from other sources, the • subunit is very different. It is the smallest •
273 subunit reported so far and shows homology only to the N-terminal part of corresponding subunits from bacterial sources. Therefore, it is of interest that such a small C. limicola • subunit could complement the E. coli less mutant. It is also demonstrated that the amino terminal half of the E . coli • subunit whose size is similar to the C. lira!cola • subunit is the functional part of this subunit [31]. The C-terminal part of the E. coli • subunit was implicated in its inhibitory effect on the ATPase activity of the enzyme. Further studies on the hybrid enzyme may shed light on the function of the • subunit in the catalytic activity of F-ATPases.
Acknowledgment We are grateful to Dr. Takato Noumi for constructing pTN1665.
References 1 Mitchell, P. (1961) Nature 191, 144-148. 2 Racker, E. (1976) A New Look at Mechanisms in Bioenergetics, Academic Press, New York. 3 Nelson, N. and Taiz, L. (1989) Trends Biocbem. Sci. 14, 113-116. 4 Futai, M., Noumi, T. and Maeda, M. (1989) Annu. Rev. Biochem. 58, 111-136. 5 Senior, A.E. (1990) Annu. Rev. Biochem. 19, 7-41. 6 Walker, J.E., Saraste, M. and Gay, N.J. (1984) Biochim. Biophys. Acta 768, 164-200. 7 Cozens, A.L. and Walker, J.E. (1987) J. Mol. Biol. 194, 359-383. 8 Curtis, S.E. (1988) Photosynth. Res. 18, 223-244. 9 Lill, H. and Nelson, N. (1991) Plant Mol. Biol. 17, 641-652. 10 Falk, G., Hampe, A. and Walker, J.E. (1985) Biochem. J. 228, 391-407. 11 Falk, G. and Walker, J.E. (1988) Biochem. J. 254, 109-122. 12 Michel, H., Weyer, K.A., Gruenberg, H., Dunger, I., Oesterhelt, D. and Lottspeich, F. (1986) EMBO J. 5, 1149-1158. 13 Trebst, A.Z. (1986) Z. Naturforsch. 41c, 240-245. 14 Komiya, H., Yeates, T.O, Rees, D.C., Allen, J.P. and Feher, G. (1988) Proc. Natl. Acad. Sci. USA 85, 9012-9016. 15 Deisenhofer, J. and Michel, H. (1989) EMBO J. 8, 2149-2170. 16 Hurt, E. and Hauska, G. (1984) FEBS Lett. 168, 149-154. 17 Blankenship, R.E. (1985) Photosynth. Res. 6, 317-333. 18 Nitschke, W. and Rutherford, A.W. (1991) Trends Biochem. Sci. 16, 241-245.
19 Biittner, M., Xie, D.-L., Nelson, H., Pinther, W., Hauska, G. and Nelson, N. (1992) Biochim. Biophys, Acta 1101, 154-156. 20 Chitnis, P.R. and Nelson, N. (1991) in Cell Culture and Somatic Cell Genetics of Plants (Bogorad, L. et al., eds.), Vol. 7B, pp. 177-224, Academic Press, New York. 21 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edn., Cold Spring Harbor Laboratory, Cold Spring Harbor. 22 Henikoff, S. (1984) Gene 28, 351-359. 23 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 24 Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395. 25 Felsenstein, J. (1989) PHYLIP 3.2 Manual, University of California Herbarium, Berkeley. 26 Amann, E. and Brosius, J. (1985) Gene 40, 183. 27 Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Science 239, 487-491. 28 Kanazawa, H., Tamura, F., Mabuchi, K., Miki, T. and Futai, M. (1983) Proc. Natl. Acad. Sci. USA 77, 7005-7009. 29 Lee, R.S.-F., Pagan, J., Wilke-Mounts, S. and Senior, A.E. (1991) Biochemistry 30, 6842-6847. 30 Noumi, T., Oka, N., Kanazawa, H. and Futai, M. (1986) J. Biol. Chem. 261, 7070-7075. 31 Kuki, M., Noumi, T., Maeda, M., Amemura, A. and Futai, M. (1988) J. Biol. Chem. 263, 17437-17442. 32 Iwamoto, A., Omote, H., Tomioka, N., Itai, A., Maeda, M. and Futai, M. (1991) J. Biol. Chem. 266, 16350-16355. 33 Moriyama, Y., Iwamoto, A., Hanada, H., Maeda, M. and Futai, M. (1991) J. Biol. Chem. 266, 22141-22146. 34 Bradford, M.M. (1976) Anal. Chem. 72, 248-254. 35 Reymond, P., Geourijon, C., Roux, B., Durand, R. and Fevre, M. (1992) Gene 110, 57-63. 36 Weldon, S.L., Rando, A., Matathias, A.S., Hod, Y., Kalonick, P.A., Savon, S., Cook, J.S. and Hanson, R.W. (1990) J. Biol. Chem. 265, 7308-7317. 37 Cook, J.S., Weldon, S.L., Garcia-Ruiz, J.P., Hod, Y. and Hanson, R.W. (1986) Proc. Natl. Acad. Sci. USA 83, 7583-7587. 38 Gundelfinger, E.D., Hermanns-Borgmeyer, J., Grenningloh, G. and Zopf, D. (1987) Nucleic Acids Res. 15, 6745-6745. 39 Gromet-Elhanan, Z., Khananshvili, D., Weiss, S., Kanazawa, H. and Futai, M. (1985) J. Biol. Chem. 260, 12635-12640. 40 Richter, M.L., Gromet-Elhanan, Z. and McCarty, R.E. (1986) J. Biol. Chem. 261, 12109-12113. 41 Nelson, N. (1992) Biochim. Biophys. Acta 1100, 109-124. 42 Nelson, N. (1989) J. Bioenerg. Biomembr. 21,553-571. 43 Lake, J.A. (1991) Trends Biochem. Sci. 16, 46-50.