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Cloning and sequencing of the gene coding for topoisomerase I from the extremely thermophilic eubacterium, Thermotoga maritima
BIt
Biochi~ic~a
et Biophysica A~ta
ELSEVIER
Biochimica et Biophysica Acta 1264 (1995) 279-283
Short sequence-paper
Cloning and sequencing of the gene coding for topoisomerase I from the extremely thermophilic eubacterium, Thermotoga maritima Claire Bouthier de la Tour a,,, H. K a l t o u m a C. Portemer a F. Confalonieri a R. Huber b M. D u g u e t a a Laboratoire d'Enzymologie des Acides Nucl~iques, Institut de G~n~tique et Microbiologie, URA 1354, Centre National de la Recherche Scientifique, Universit~ Paris-Sud, 91405 Orsay Cedex, France b lnstitutfiir Biochemie, Genetik und Mikrobiologie, Universitiit Regensburg, 84000 Regensburg, Germam" Received 18 May 1995; revised 7 September 1995; accepted 20 September 1995
Abstract
A 2767 bp fragment containing a gene coding for a topoisomerase I from the extremely thermophilic eubacterium Thermotoga maritima (Tm TopA) has been cloned and sequenced. The protein is composed of 633 amino acids with a calculated molecular mass of 72 695 Da. It shares significant similarity with the topoisomerases I of mesophilic eubacteria. The highest score is obtained with Bacillus subtilis (44% identity); in particular, T. maritima and B. subtilis possess an insertion of 7-8 amino acids in the vicinity of the active site, that is absent in topoisomerases of other organisms. A specific feature of T. maritima topoisomerase I is its low cysteine content compared to its mesophilic homologs. It contains 5 cysteine residues, of which 4 could constitute a zinc finger motif. Finally, analysis of the regions flanking the gene reveals that Tm TopA is surrounded by two other ORFs, suggesting the occurrence of a polycistronic transcriptional unit. Keywords: DNA topoisomerase I; Bacterium; Thermotoga; Thermophile; (T. maritima)
Thermotoga maritima is an anaerobic eubacterium able to grow at temperatures up to 90°C [1]. Together with Aquifex [2], it is o n e of the most thermophilic eubacteria presently known. Our aim is to understand how D N A superhelicity and D N A stability are controlled by the activity of topoisomerases in bacteria living at very high temperature. It is known that in mesophilic bacteria, e.g., Escherichia coli, the level of superhelical density is maintained by the antagonist activities of a type II topoisomerase (DNA gyrase) that produces negative supercoils and a type I topoisomerase (protein to) that removes them. [3 -5]. In 7". maritima, we know that a gyrase-like topoisomerase II is present (O. Guipaud, personal communication). In addition, two type I topoisomerase activities have been detected [6]. One is able to produce positively supercoiled DNA by an ATP dependent process (reverse gyrase activ-
* Corresponding author. Fax: + 33 1 69417296. The sequence data reported in this paper have been submitted to the Genbank library under the accession number U27841. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 I 6 7 - 4 7 8 1
ity): it is found exclusively in thermophilic organisms [7]. The other acts in a way similar to that of protein to by relaxing negatively supercoiled D N A without the need of ATP. This relaxing activity represents the major activity in thermophilic eubacteria [6], whereas it is very weak or absent in thermophilic archaebacteria [7]. Moreover, in Sulfolobus shibatae extracts, a similar ATP-independent activity was attributed to a proteolytic product of reverse gyrase [8]. Thus, the distribution of type I topoisomerases in Thermotoga maritima appears intermediate between that occurring in mesophilic eubacteria (presence of a protein to and absence of reverse gyrase) and that of thermophilic archaebacteria (presence of reverse gyrase and no evidence for the presence of a to-like protein). Here, we present the PCR mediated cloning and sequencing of a T. maritima topoisomerase gene, whose deduced amino acid sequence shows an important similarity to E. coli protein to (product of TopA gene) [9]. By analogy, we have called this new gene Thermotoga maritima TopA gene (Tm TopA). It is the first report on the sequence of a thermophilic eubacterial topoisomerase. The strategy that we used to identify Thermotoga TopA gene is based on the sequence comparison of protein to
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with the topoisomerase domain of S u l f o l o b u s a c i d o c a l d a r ius reverse gyrase [10]. The existence of amino acid conserved blocks allowed us to choose two of these blocks for PCR amplification: L S A G R V Q (block III Ref. [I0]) and IGRPSTY (block VII ref [10]). We obtained a PCR product with a size of about 900 bp (region from nt 984 to 1892 in Fig. 2) consistent with the approximative distance between the two blocks found both in E. coli TopA and reverse gyrase genes. The deduced amino acid sequence of this fragment showed a high degree of similarity (61%) to the corresponding region of E. coli protein to. Therefore, we used it as a probe to isolate the full length DNA topoisomerase I gene from T. m a r i t i m a . The strategy of cloning and sequencing is illustrated in Fig. 1. Finally, a region of 2667 nucleotides centered around the PCR fragment was identified. The corresponding amino acid sequence reveals three ORFs on the upper strand (Fig. 2A): the central ORF exhibits 40% identity with E. coli topoisomerase I, confirming that we have cloned a topoisomerase gene in T h e r m o t o g a m a r i t i m a , equivalent to E. coli TopA. Starting with the first A T G at position 498 (Fig. 2B) and
ending with a T G A stop signal at position 2397, the T. m a r i t i m a TopA ORF codes for a protein of 633 amino
acids with a deduced molecular mass of 72 695 Da. This value is in good agreement with the molecular mass found for the DNA topoisomerase I purified from another thermotogale, F e r v i d o b a c t e r i u m i s l a n d i c u m (molecular mass: 75 kDa), which shared its essential enzymatic properties with E. coli protein to [11]. By analogy with the other topoisomerases I, the putative active site tyrosine is Tyr-288 (nt 1359) (see also Fig. 4). The whole G + C content of the T. m a r i t i m a TopA ORF (45%) reflects that of the genome (46%) [12]. The codon usage frequencies determined with the DNA strider TM 1.2 software (data not shown) exhibit differences with those of E. coli genes [13]: for instance, AGA and A G G are found to be the preferred codons for arginine ( 3 4 / 4 3 ) , and ATA the preferred codon for isoleucine ( 1 7 / 3 9 ) , whilst these codons are very rare in E. coli. In general, these codons are frequently found in hyperthermophilic organisms exhibiting a low G + C content [ 14,15]. Analysis of the DNA sequence upstream TopA ORF reveals a potential Shine-Dalgarno sequence ( G G A G G ) 5
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Fig. 1. Strategy of cloning and sequencingof the DNA fragment containingthe topoisomerase gene. (A) Strategy of cloning, p 0.9 is the PCR fragment cloned into the Sinai site of pGEM 3Z,f(+ ). P1 and P2 represent the primers used for PCR amplification.PI: 5'-YTNWSNGCNGGNMGNGTNCA-3' (corresponding to LSAGRVQ) and P2: 5'-TANGTNSWNGGNCKNCCNAT-3'(correspondingto IGRPSTY). The DNA of p 0.9 was digested with EcoRl and BamHI to give two fragments X and Y of 576 bp and 331 bp, respectively.They were used as radioactiveprobes against an EcoRI partial digestionof T. maritima genomic DNA (MSB8 strain). X was found to hybridize with a fragment of about 4 kbp and Y with a fragment of about 1 kbp. The 4 kbp genomic region was subsequentlycloned into the EcoRI site of pBR322 (a) and the size selected library obtainedwas screened with the radioactive probe X, using E. coli DH5a as the host strain. Out of about 2000 colonies, 6 positive clones were found. Here is presented the restriction map of one recombinantplasmid identicalfor the 6 clones. In the same manner,the 1 kbp genomicregion was cloned into the EcoRI site of pGEM 3Z f(+ ) (b) and approx. 400 colonies were screened with probe Y. 10 positive clones carrying the same insert were obtained. Analysis of their restriction map is shown. The DNA inserts were then sequenced. The restriction enzymes used were: BamHI (B); SacI (S); EcoRl (E); HindlII (H); Pvul (P). The dashed lines represent the vectors arms. The arrows correspond to the regions sequencedwith a series of primers. (B) Restrictionmap of T. maritima genomicDNA in the region of the topoisomerase gene. The map was obtained by Southern blot hybridizationof different restriction digests of genomic DNA with the cloned PCR fragment (data not shown). A perfect correlation was found betweenthis restrictionmap of T. maritima genomicDNA and the maps of a and b inserts. The hatched box indicates the coding region of the topoisomerase gene.
C. Bouthier tie la Tour et a l . / Biochimica et Biophysica Acta 1264 (1995) 279-283
nt before the ATG initiation codon, but no obvious consensus promoter sequence could be observed. The presence of an upstream ORF (ORFI) overlapping the TopA ORF over 5 nt suggests the occurrence of a polycistronic transcriptional unit with mechanisms of translation-reinitiation at the internal ATG start codon. The partial 166 amino acid sequence encoded by the 3' end of ORF 1 does not match any sequence in Genbank protein data base. It might correspond to the carboxy-terminal part of a protein encoded by an yet unknown gene cotranscribed with the TopA gene. The downstream ORF (ORF 2) possesses 113 nt in common with the 3' end of TopA ORF. The sequenced region encodes a polypeptide showing a significant similarity (about 37% identity) to bacterial D-alanylalanine synthetases involved in the peptidoglycan synthesis [16,17]. Thus, T. maritima TopA gene appears to be an internal gene of a multicistronic message. The finding of
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an ORF encoding a membrane synthesis protein asks the question about the possible role of T. maritima topoisomerase I in the chromosome partitioning concomitant with the elongation of the new cell envelope material. The sequence similarities to other prokaryotic-like type I topoisomerases are summarized in the dendrogram of Fig. 3. Two families (A and B) could be distinguished. The first (B) includes the E, coli TopB gene product that is described to be involved in decatenation reactions [27], and the other (A) includes the E. coli TopA gene product that exhibits a prominent DNA relaxation activity [28]. 7". maritima topoisomerase clearly belongs to this last family. It is interesting to note that this protein is closely related to the other topoisomerases from mesophilic bacteria. The highest similarity is obtained with Bacillus subtilis topoisomerase I (44% identity and 63% similarity). They have a comparable size (about 650 aa) whereas the other TopA
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13 GGGGq'I~ A C C C G T G G T T C C A A A G A C G A T A G A G G C T ~ A A T A G T T C A T C A C G T T G A G A A C C T G G A T T C C A A G A T A G C T C G A 400 TTC ATCGAGG~FFATGGAAAGTTC CGAATCGAATC AGGG'F~ACGGAGTACGA C A G G A A T T T G G G A A G A A G A A T C ~'FI'~ 4 8 0 A A G A G G T ~ C TGATGAGTAAGAAA~AAGAAATATATCGTTGTTG AATCTCCTGC CAAGGCGAAGAC GATC ~G 560 M S K K V K K Y I V V E S P A K A K T I K AG TATTC~AATGAGTACGAAGTATTCC, C~'rC G A T G G G G C A C A T C A T A G A C C ~ Y C C C A A G A G C A A G T T C G G T G T G G A 640 S i L G N E Y E V F A S M G H I I D L P K S K F G V D TC T~GAAAAGGATTTCGAACCGGAATTCGCC, GTGATAAAAC~AAAGAGAAGGTAGT'~AAAAGCTG~GGA~ 720 L E K D F E P E F A V I K ~ K E K V V E K L K D L A K AAAAAGGCGAACTGC TC ATCGCTTCTGATATGGATAGAGAAGGGG AAGCCATAGCCTGGCACATAGCC AGGGTAACGAAC 800 K G E L L I A S D M D R E G E A I A W H I A R V T N AC ACTC~AGAAAGAAC AC,G A T C G T G T T C T C T G A G A T C A C T C C T C G T G T C A T A A G A G A A G C CG'F~AAC,A A ~ C ~ ~ A ego T L G R K N R I V F S E I T P R V I R E A V K N P R E CA~F?GAC A T G A A G A A A G T T C G T G C G C A G ~ A A G A A G G A T T T T A G A C AGGATCGT'~ATTCTC TGAGTCCGG~'rC 960 I D M K K V R A Q L A R R I L D R I V G Y S L S P V L TC TC~AGAAACT~AAATC CAATTTGAGCGCT~TAGAGTC C AATC AGC AAC CTTGAAGCTCGTTTGTGACAGAGAGAGA 1060 'X R N F K S N L S A G R V Q S A T L K L V C D R E R G A A A T C C T C A G G T q ' T G T G C C A A A G A A G T A C C A C A G G A T C A C C G T C AATTTCGATC,G T C T C A C A G C A G A A A T A G A T G T G A A 1120 E i L R F V P K K Y H R I T V N F D G L T A E I D V K A G ; ~ A A A G A A G T T C T T C G A C G C T G A A A C G T T G A A A G A A A T C C A G A G C A T A G A C G A A C T C G q ~ I ~ T A G A A G A G A A A A A G G q'l'T 1 2 0 0 E K K F F D A E T L K E I ~ S I D E U V V E E K K V S C T G . ~ G A A A A A G T T T C ~ G C C T C C A G A A C C C T T C A A A A C C A G T A C C C T C C A G C A G G A A C C G T A T T C T A A A C T G G G T T T C T C C 1280 %" K K F A P P E P F K T S T L Q Q E A Y S K L G F S CTT'?CC A A A A C C A ' ~ A T G A T T G C C C A G C A G T ' f G T A T G A A G G C G T G G A A A C A A A A G A T C ,C,C,CATATCC,C G ' I ~ C A T A A C C T A 1 3 G 0 V $ K T M M I" A Q Q 5 Y E G V E T K D G H I A F I T T* TATGAG AACAGA~CAACGCGTGTTTCGGAC TAC GC GAAAGAAGAAGC CAGAAATCTGATAACGGAGGTCTTTGGAGAAG 1440 M R T D S T R V S D Y A K E E A R N L I T E V F G E E AGTACG ~'FCAAAGAG A G A A A G G A G A A A G A G C A A C G C A A A A A T A C AGGATGCC~C A T G A G G C G A T T C G T C C C A C G A A C 1 5 2 0 Y V G S K R E R R K S N A K I Q D A H E A I R P T N G TTTTC ATGACACCTGAAGAAGCGGGAAAATACCTGAATTCTGAC CAGAAAAAACTCTACGAGTTGATAT~GAAGAGATT 1600 V F M T P E E A G K Y L N S D Q K K L Y E L I W K R F T C T S -]CA T C G C A A A T G A A G C C ~FTCT C A A T A C G A G G A A A C T C G T T ~ TCCTGAGAAC AAAC~ATGGGAAGTACAGATTCA 1680 L A S Q M K P S Q Y E E T R F V L R T K D G K Y R F K A G G G A A C G G T G C T G A A G A A G A T T T T C G A T G G G T A C G A A A A G G T G T G G A A G A C G G A A A G A A A C A C G G G A G A G ~'I'FC~ 1760 G T V L K K I F D G Y E K V W K T E R N T G E F P F GAAAAGGGAGAATC TGTGAAAC CC~TGGTTGTAAAAA TAGAGGAACAGGAAAC CAAAC CGAAAC CAAGGTACACAGAGGG 1840 E E O E S V K P V V V K I E E ~ E T K P K P R Y T E G T T C TC T q ~ S T A A A A G A A A T G G A A A G A C T C C ~ T A T A C - G T C G T C C C A G C A C A T A T G C G T C C A C G A T A A A G C T ' ~ T ~ A A C A 1920 S L V K E M E R L G I G R P S T Y A S T I K L L L N R G G G G T T A C A T A A A A A A G A T C A G G G G C T A T C T C T A T C C A A C GATCG~CTGGAAGCG T T G T C A T G G A T T A T C T C . G A G A A A A A G 2 0 0 0 G Y I K K I R G Y L Y P T I V G S V V M D Y L E K K T A C TC T G A T G T T G ~ T A G C G T T T C C T T C A C A G C TGAAATC,GAAAAC-GAC C T C G A T G A A G T C G A A C A G G G T A A G A A A A C A G A 2 0 8 0 Y S D V V S V S F T A E M E K D L D E V E Q G K K T D C A A A A T C G q ' ~ C T G C G G G A G T T T T A T G A A T C Cq~I'TTCC A G T G T G ~ G A C AGGAACGACAGAATTGTGGTT~ATTTTC CGA 21G0 ~! [ V L R E F Y E S F S S V F D R N D R I V V D P P T C ~ ~ ~ ~ C A G A A A T G T T C G T G T C ,G A A A G G A A A T G A G ~ C T C T C T T T T C - G A A A A T A C G G C T T C T A T ' F P G A A A T G C G A G T G T G G T 2 2 4 0 :~ Q K C E C G K E M R L g F G K Y G F Y L K C ~ C G ~.~-~.~. SC A G A A G C G T G A A A A A C G A T G A A A T T G C C - G T C A T A G A C G A q ~ G A A A A A T A T T C C T G G C ~ A G G A A A G A C A G T ~ A G A G 2 3 2 0 =" P S V K N D E I A V I D D G K I F L G R K D S E S ?'5O ?T C T C C TGATC,C,G A G G A G T G T C G A G G G A A A G G G A A A T C T C T C T G A G A A G C GGAGAAAC,C ~ T A A A A A A G G C T C T T G A A 2 ~ 0 0 Z $ P D G R S V E G K G N L S E K R R K G K K G S STOP T ~ = ~ 3 T T A G A G A A G A T T T T C T C A A G A A A G T G G A T C A G T T G A A A A G T T T T G A T G T G G T C T T C A A C G T T C T C C A T G G A A C T T 24S0 U Z L 5 T [~A . ~ G A T G G A A C G C T T C A G G C A A T T C TC,G A T T T T T T G G G G A T C A G A T A C A C C G G G T C C G A T G C G T T T T C A A G C A T 3 ~ 5 6 0 ATA?~.~/q`~GATAAACTCGTGACTTACAGGTCTTTAAAGGGCACCGTCGA~ATACCTGATTT~TGGAGATCAAG~AAT ~. 2 6 2 0
Fig, 2. Structure of T. maritima TopA gene and flanking regions. (A) Organization of the ORFs contained in the sequenced DNA fragment. The dashed lines correspond to the not yet sequenced regions of ORF 1 and ORF 2. (B) Nucleotide sequence (from nt 320 to nt 2620) and deduced amino acid sequence of the Tm TopA ORF. The Shine-Dalgarno sequence is underlined. The putative tyrosine of the active site is indicated by a star and the peptidic sequence encompassing the Zn finger motif is represented in italic letters and underlined.
282
C. Bouthier de la Tour et aL / Biochimica et Biophysica Acta 1264 (1995) 279-283
EcTopB
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TraE
Fig. 4. Sequence comparison of TopA family topoisomerases in the vicinity of the active site. Alignments were performed with the Pileup program described in Fig. 3 legend. The tyrosine of the active site is indicated by a star. The numbers represent the position of amino acids in the sequences. Identical amino acids were blackened when a strict identity was found for at least four organisms out of 6.
SpTop
BaTop
BsTopA
TmTopA
A
SccTopA
Rg620
ScTop3 Fig. 3. Relatedness of T. maritima topoisomerase I with type I topoisomerases from the prokaryotic family. The dendrogram was obtained from the multiple alignment of topoisomerase sequences using the Pileup program GCG package, version 7.2 [18]. The parameters were the followings: gap creation penalty: 1; gap extension penalty: 0.1. The different organisms used for sequence comparison were: Escherichia coli TopB (EcTopB) [19], RP4 plasmid encoded topoisomerase (TraE) [20], Streptococcus pyrogenes pDBI01 plasmid encoded topoisomerase (SpTop) [21], Bacillus anthracis pXOl plasmid encoded topoisomerase (BaTop) [22], Escherichia coli TopA (EcTopA) [9], Haemophilus influenzae TopA (HiTopA) [23], Bacillus subtilis TopA (BsTopA) [24], Synechococcus sp. TopA (SccTopA) [25], topoisomerase domain of Sulfolobus acidocaldarius reverse gyrase (residues 620-1247) (Rg620) [I0], Saccharomyces cerevisiae Top3 (ScTop3) [26]. A and B families are distinguished.
gene products are proteins of approximately 850 amino acids [9,23,25]. Furthermore, T. maritima topoisomerase shares an insertion of 7 - 8 additional amino acids (Fig. 4) with the Bacillus subtilis protein within the highly conserved region of the putative active site. This observation is particularly intriguing, since artificial insertions of tetrapeptides near the active site of protein to lead to a total loss of DNA relaxation activity [29]. The role of this additional octapeptide in the architecture of the active site has to be determined. No thermophilic specific features are evident that distinguish T. maritima topoisomerase I from its mesophilic
counterparts, except for the number of cysteines (5 residues), which is clearly lower than that of the other topoisomerases I (13 to 15 residues). Knowing the susceptibility of the cysteine residues to oxidation at elevated temperatures [30], this finding could constitute an element to explain the thermoadaptation of T. maritima topoisomerase I. Furthermore, 4 out of these 5 cysteines, located at the carboxy-terminus of the protein (region 2171 to 2237 on Fig. 2B), could constitute a Zn finger motif with the structure: Cys-X-Cys-X16-Cys-X-Cys. This motif exhibits a significant similarity with the last of the three Zn fingers of E. coli protein to (amino acids 710 to 736 in Ref. [12]), that was supposed to be involved in the DNA relaxation activity and especially in the strand passage step [29,31,32]. We plan to elucidate the relevance of this Zn finger motif in the enzymatic activity of T maritima topoisomerase I by site-directed mutagenesis. In conclusion, the topoisomerase gene that we have identified in T. maritima clearly belongs to the prokaryotic topoisomerase I family. The finding of a specific gene excludes the possibility that the ATP-independent activity detected in T. maritima extracts is due to a proteolytic product of reverse gyrase. If an equivalent of E. coli TopB is also present, then T. maritima would possess three kinds of type I topoisomerases: such a multiplicity addresses the question of their functional specialization.
Acknowledgements This work was supported by the Centre National de la Recherche Scientifique and the Universit6 Paris Sud (Unit6 de Recherche Associre 1354). The laboratory is supported in part by A.R.C. (Association pour la Recherche contre le Cancer).
References [1] Huber, R., Langworthy, T.A., KiSnig, H., Thomm, M., Woese, C.R., Sleytr, U.B. and Stetter, K.O. (1986) Arch Microbiol. 144, 324-333. [2] Huber, R., Wilharm, T., Huber, D., Trincone, A., Burggraf, S., Ktinig, H., Rachel, R., Rochinger, I., Frick, H. and Stetter, K.O. (1992) Syst. Appl. Microbiol. 15, 340-351. [3] Wang, J.C. (1987) Biochim. Biophys. Acta 909, 1-9. [4] Drlica, K. (1984) Microbiol. Rev. 48, 273-289.
C. Bouthier de la Tour et al. / Biochimica et Biophysica Acta 1264 (1995) 279-283
[5] Drlica, K. (1992)Mol. Microbiol. 6, 425-433. [6] Bouthier de la Tour, C., Portemer, C., Huber, R., Forterre, P. and Duguet M. (1991) J. Bacteriol. 173, 3921-3923. [7] Bouthier de la Tour, C., Ponemer, C., Nadal, M., Stetter, K.O., Forterre P. and Duguet, M. (1990) J. Bacteriol. 172, 6803-6808. [8] Nadal, M., Couderc, E., Duguet, M. and Jaxel, C. (1994) J. Biol. Chem. 269, 5255-5263. [9] Tse-Dinh, Y.C., Wang J.C. (1986). J. Mol. Biol. 191, 321-331. [10] Confalonieri, F., Elie, C., Nadal, M., Bouthier de la Tour, C., Fonerre, F. and Duguet, M., (1993) Proc. Natl. Acad. Sci. USA 90, 4753-4757. [11] Bouthier de la Tour, C., Portemer, C., Forterre, P., Huber, R. and Duguet, M. (1993) Biochim Biophys. Acta 1216, 213-220. [12] Huber, R., Langworthy, T.A., K~Snig, H., Thomm, M., Woese, C.R., Sleytr, U.B. and Stetter, K.O. (1986) Arch. Microbiol. 144, 324-333. [13] Sharp, P.M., Cowe, E., Higgins, D.G., Shields, D.C., Wolfe, K.H. and Wright, F., (1988) Nucleic Acids Res. 8207-8211. [14] Dalgaard, J.Z. and Garrett, R.A. (1993) In New Comprehensive Biochemistry series, Vol. 26 (Kates, M., Kushner, D.J. and Matheson, A.T., eds.), pp. 535-559, Elsevier, Amsterdam. [15] Liao, D. and Dennis, P.P. (1992) J. Biol. Chem. 267, 22787-22797. [16] Zawadzke, L.E., Bugg, T.D. and Walsh, C.T. (1991) Biochemistry 30, 1673-1682. [17] Robinson, A.C, Kenan, D.J., Sweeney, J. and Donachie, W.D. (1986) J. Bacteriol. 167, 809-817.
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[18] Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395. [19] DiGate, R.J. and Marians, K.J. (1989) J. Biol. Chem. 264, 1792417930. [20] Starck, B, and Lanka, E. (1993) Accession number L10329. [21] Ceglowski, P., Boitov, G.. Lueder, G. and Alonso, J.C. (1992) Accession number X66468. [22] Fouet, A., Sirard, J.C. and Mock, M. (1994) Mol. Microbiol. It. 471-479. [23] Chandler, M.S. and Smith, R.A. (1995) Accession number U20964. [24] De Jong, S. (1994) Accession number L27797. [25] Gabay, C., Hassidim, M., Hurwitz-Lieman, J., Marco, E. and Kaplan, A. (1993) Accession number X72391. [26] Wallis, J.W., Chrebet, G., Brodsky, G_ Rolfe, M. and Rothstein, R. (1989) Cell 58, 409-419. [27] DiGate, R.J. and Marians, K.J. (1988) J. Biol. Chem. 263, 1336613373. [28] Wang, J.C. (1971) J. Mol. Biol. 55, 523-533. [29] Zumstein, L. and Wang, J.C. (1986) J. Mol Biol. 191, 333-340. [30] Volkin, D.B. and Klibanov, A.M. (1987) J. Biol. Chem. 262, 2945 -2950. [31] Tse-Dinh, Y.C. (1991) J. Biol. Chem. 266, 14317-14320. [32] Lima, C.D., Wang, J.C. and Mondragon A. (1993) J. Mol. Biol. 232, 1213-1216.
Biochi~ic~a
et Biophysica A~ta
ELSEVIER
Biochimica et Biophysica Acta 1264 (1995) 279-283
Short sequence-paper
Cloning and sequencing of the gene coding for topoisomerase I from the extremely thermophilic eubacterium, Thermotoga maritima Claire Bouthier de la Tour a,,, H. K a l t o u m a C. Portemer a F. Confalonieri a R. Huber b M. D u g u e t a a Laboratoire d'Enzymologie des Acides Nucl~iques, Institut de G~n~tique et Microbiologie, URA 1354, Centre National de la Recherche Scientifique, Universit~ Paris-Sud, 91405 Orsay Cedex, France b lnstitutfiir Biochemie, Genetik und Mikrobiologie, Universitiit Regensburg, 84000 Regensburg, Germam" Received 18 May 1995; revised 7 September 1995; accepted 20 September 1995
Abstract
A 2767 bp fragment containing a gene coding for a topoisomerase I from the extremely thermophilic eubacterium Thermotoga maritima (Tm TopA) has been cloned and sequenced. The protein is composed of 633 amino acids with a calculated molecular mass of 72 695 Da. It shares significant similarity with the topoisomerases I of mesophilic eubacteria. The highest score is obtained with Bacillus subtilis (44% identity); in particular, T. maritima and B. subtilis possess an insertion of 7-8 amino acids in the vicinity of the active site, that is absent in topoisomerases of other organisms. A specific feature of T. maritima topoisomerase I is its low cysteine content compared to its mesophilic homologs. It contains 5 cysteine residues, of which 4 could constitute a zinc finger motif. Finally, analysis of the regions flanking the gene reveals that Tm TopA is surrounded by two other ORFs, suggesting the occurrence of a polycistronic transcriptional unit. Keywords: DNA topoisomerase I; Bacterium; Thermotoga; Thermophile; (T. maritima)
Thermotoga maritima is an anaerobic eubacterium able to grow at temperatures up to 90°C [1]. Together with Aquifex [2], it is o n e of the most thermophilic eubacteria presently known. Our aim is to understand how D N A superhelicity and D N A stability are controlled by the activity of topoisomerases in bacteria living at very high temperature. It is known that in mesophilic bacteria, e.g., Escherichia coli, the level of superhelical density is maintained by the antagonist activities of a type II topoisomerase (DNA gyrase) that produces negative supercoils and a type I topoisomerase (protein to) that removes them. [3 -5]. In 7". maritima, we know that a gyrase-like topoisomerase II is present (O. Guipaud, personal communication). In addition, two type I topoisomerase activities have been detected [6]. One is able to produce positively supercoiled DNA by an ATP dependent process (reverse gyrase activ-
* Corresponding author. Fax: + 33 1 69417296. The sequence data reported in this paper have been submitted to the Genbank library under the accession number U27841. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 I 6 7 - 4 7 8 1
ity): it is found exclusively in thermophilic organisms [7]. The other acts in a way similar to that of protein to by relaxing negatively supercoiled D N A without the need of ATP. This relaxing activity represents the major activity in thermophilic eubacteria [6], whereas it is very weak or absent in thermophilic archaebacteria [7]. Moreover, in Sulfolobus shibatae extracts, a similar ATP-independent activity was attributed to a proteolytic product of reverse gyrase [8]. Thus, the distribution of type I topoisomerases in Thermotoga maritima appears intermediate between that occurring in mesophilic eubacteria (presence of a protein to and absence of reverse gyrase) and that of thermophilic archaebacteria (presence of reverse gyrase and no evidence for the presence of a to-like protein). Here, we present the PCR mediated cloning and sequencing of a T. maritima topoisomerase gene, whose deduced amino acid sequence shows an important similarity to E. coli protein to (product of TopA gene) [9]. By analogy, we have called this new gene Thermotoga maritima TopA gene (Tm TopA). It is the first report on the sequence of a thermophilic eubacterial topoisomerase. The strategy that we used to identify Thermotoga TopA gene is based on the sequence comparison of protein to
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with the topoisomerase domain of S u l f o l o b u s a c i d o c a l d a r ius reverse gyrase [10]. The existence of amino acid conserved blocks allowed us to choose two of these blocks for PCR amplification: L S A G R V Q (block III Ref. [I0]) and IGRPSTY (block VII ref [10]). We obtained a PCR product with a size of about 900 bp (region from nt 984 to 1892 in Fig. 2) consistent with the approximative distance between the two blocks found both in E. coli TopA and reverse gyrase genes. The deduced amino acid sequence of this fragment showed a high degree of similarity (61%) to the corresponding region of E. coli protein to. Therefore, we used it as a probe to isolate the full length DNA topoisomerase I gene from T. m a r i t i m a . The strategy of cloning and sequencing is illustrated in Fig. 1. Finally, a region of 2667 nucleotides centered around the PCR fragment was identified. The corresponding amino acid sequence reveals three ORFs on the upper strand (Fig. 2A): the central ORF exhibits 40% identity with E. coli topoisomerase I, confirming that we have cloned a topoisomerase gene in T h e r m o t o g a m a r i t i m a , equivalent to E. coli TopA. Starting with the first A T G at position 498 (Fig. 2B) and
ending with a T G A stop signal at position 2397, the T. m a r i t i m a TopA ORF codes for a protein of 633 amino
acids with a deduced molecular mass of 72 695 Da. This value is in good agreement with the molecular mass found for the DNA topoisomerase I purified from another thermotogale, F e r v i d o b a c t e r i u m i s l a n d i c u m (molecular mass: 75 kDa), which shared its essential enzymatic properties with E. coli protein to [11]. By analogy with the other topoisomerases I, the putative active site tyrosine is Tyr-288 (nt 1359) (see also Fig. 4). The whole G + C content of the T. m a r i t i m a TopA ORF (45%) reflects that of the genome (46%) [12]. The codon usage frequencies determined with the DNA strider TM 1.2 software (data not shown) exhibit differences with those of E. coli genes [13]: for instance, AGA and A G G are found to be the preferred codons for arginine ( 3 4 / 4 3 ) , and ATA the preferred codon for isoleucine ( 1 7 / 3 9 ) , whilst these codons are very rare in E. coli. In general, these codons are frequently found in hyperthermophilic organisms exhibiting a low G + C content [ 14,15]. Analysis of the DNA sequence upstream TopA ORF reveals a potential Shine-Dalgarno sequence ( G G A G G ) 5
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Fig. 1. Strategy of cloning and sequencingof the DNA fragment containingthe topoisomerase gene. (A) Strategy of cloning, p 0.9 is the PCR fragment cloned into the Sinai site of pGEM 3Z,f(+ ). P1 and P2 represent the primers used for PCR amplification.PI: 5'-YTNWSNGCNGGNMGNGTNCA-3' (corresponding to LSAGRVQ) and P2: 5'-TANGTNSWNGGNCKNCCNAT-3'(correspondingto IGRPSTY). The DNA of p 0.9 was digested with EcoRl and BamHI to give two fragments X and Y of 576 bp and 331 bp, respectively.They were used as radioactiveprobes against an EcoRI partial digestionof T. maritima genomic DNA (MSB8 strain). X was found to hybridize with a fragment of about 4 kbp and Y with a fragment of about 1 kbp. The 4 kbp genomic region was subsequentlycloned into the EcoRI site of pBR322 (a) and the size selected library obtainedwas screened with the radioactive probe X, using E. coli DH5a as the host strain. Out of about 2000 colonies, 6 positive clones were found. Here is presented the restriction map of one recombinantplasmid identicalfor the 6 clones. In the same manner,the 1 kbp genomicregion was cloned into the EcoRI site of pGEM 3Z f(+ ) (b) and approx. 400 colonies were screened with probe Y. 10 positive clones carrying the same insert were obtained. Analysis of their restriction map is shown. The DNA inserts were then sequenced. The restriction enzymes used were: BamHI (B); SacI (S); EcoRl (E); HindlII (H); Pvul (P). The dashed lines represent the vectors arms. The arrows correspond to the regions sequencedwith a series of primers. (B) Restrictionmap of T. maritima genomicDNA in the region of the topoisomerase gene. The map was obtained by Southern blot hybridizationof different restriction digests of genomic DNA with the cloned PCR fragment (data not shown). A perfect correlation was found betweenthis restrictionmap of T. maritima genomicDNA and the maps of a and b inserts. The hatched box indicates the coding region of the topoisomerase gene.
C. Bouthier tie la Tour et a l . / Biochimica et Biophysica Acta 1264 (1995) 279-283
nt before the ATG initiation codon, but no obvious consensus promoter sequence could be observed. The presence of an upstream ORF (ORFI) overlapping the TopA ORF over 5 nt suggests the occurrence of a polycistronic transcriptional unit with mechanisms of translation-reinitiation at the internal ATG start codon. The partial 166 amino acid sequence encoded by the 3' end of ORF 1 does not match any sequence in Genbank protein data base. It might correspond to the carboxy-terminal part of a protein encoded by an yet unknown gene cotranscribed with the TopA gene. The downstream ORF (ORF 2) possesses 113 nt in common with the 3' end of TopA ORF. The sequenced region encodes a polypeptide showing a significant similarity (about 37% identity) to bacterial D-alanylalanine synthetases involved in the peptidoglycan synthesis [16,17]. Thus, T. maritima TopA gene appears to be an internal gene of a multicistronic message. The finding of
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281
an ORF encoding a membrane synthesis protein asks the question about the possible role of T. maritima topoisomerase I in the chromosome partitioning concomitant with the elongation of the new cell envelope material. The sequence similarities to other prokaryotic-like type I topoisomerases are summarized in the dendrogram of Fig. 3. Two families (A and B) could be distinguished. The first (B) includes the E, coli TopB gene product that is described to be involved in decatenation reactions [27], and the other (A) includes the E. coli TopA gene product that exhibits a prominent DNA relaxation activity [28]. 7". maritima topoisomerase clearly belongs to this last family. It is interesting to note that this protein is closely related to the other topoisomerases from mesophilic bacteria. The highest similarity is obtained with Bacillus subtilis topoisomerase I (44% identity and 63% similarity). They have a comparable size (about 650 aa) whereas the other TopA
ORF2 TopA O R F
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13 GGGGq'I~ A C C C G T G G T T C C A A A G A C G A T A G A G G C T ~ A A T A G T T C A T C A C G T T G A G A A C C T G G A T T C C A A G A T A G C T C G A 400 TTC ATCGAGG~FFATGGAAAGTTC CGAATCGAATC AGGG'F~ACGGAGTACGA C A G G A A T T T G G G A A G A A G A A T C ~'FI'~ 4 8 0 A A G A G G T ~ C TGATGAGTAAGAAA~AAGAAATATATCGTTGTTG AATCTCCTGC CAAGGCGAAGAC GATC ~G 560 M S K K V K K Y I V V E S P A K A K T I K AG TATTC~AATGAGTACGAAGTATTCC, C~'rC G A T G G G G C A C A T C A T A G A C C ~ Y C C C A A G A G C A A G T T C G G T G T G G A 640 S i L G N E Y E V F A S M G H I I D L P K S K F G V D TC T~GAAAAGGATTTCGAACCGGAATTCGCC, GTGATAAAAC~AAAGAGAAGGTAGT'~AAAAGCTG~GGA~ 720 L E K D F E P E F A V I K ~ K E K V V E K L K D L A K AAAAAGGCGAACTGC TC ATCGCTTCTGATATGGATAGAGAAGGGG AAGCCATAGCCTGGCACATAGCC AGGGTAACGAAC 800 K G E L L I A S D M D R E G E A I A W H I A R V T N AC ACTC~AGAAAGAAC AC,G A T C G T G T T C T C T G A G A T C A C T C C T C G T G T C A T A A G A G A A G C CG'F~AAC,A A ~ C ~ ~ A ego T L G R K N R I V F S E I T P R V I R E A V K N P R E CA~F?GAC A T G A A G A A A G T T C G T G C G C A G ~ A A G A A G G A T T T T A G A C AGGATCGT'~ATTCTC TGAGTCCGG~'rC 960 I D M K K V R A Q L A R R I L D R I V G Y S L S P V L TC TC~AGAAACT~AAATC CAATTTGAGCGCT~TAGAGTC C AATC AGC AAC CTTGAAGCTCGTTTGTGACAGAGAGAGA 1060 'X R N F K S N L S A G R V Q S A T L K L V C D R E R G A A A T C C T C A G G T q ' T G T G C C A A A G A A G T A C C A C A G G A T C A C C G T C AATTTCGATC,G T C T C A C A G C A G A A A T A G A T G T G A A 1120 E i L R F V P K K Y H R I T V N F D G L T A E I D V K A G ; ~ A A A G A A G T T C T T C G A C G C T G A A A C G T T G A A A G A A A T C C A G A G C A T A G A C G A A C T C G q ~ I ~ T A G A A G A G A A A A A G G q'l'T 1 2 0 0 E K K F F D A E T L K E I ~ S I D E U V V E E K K V S C T G . ~ G A A A A A G T T T C ~ G C C T C C A G A A C C C T T C A A A A C C A G T A C C C T C C A G C A G G A A C C G T A T T C T A A A C T G G G T T T C T C C 1280 %" K K F A P P E P F K T S T L Q Q E A Y S K L G F S CTT'?CC A A A A C C A ' ~ A T G A T T G C C C A G C A G T ' f G T A T G A A G G C G T G G A A A C A A A A G A T C ,C,C,CATATCC,C G ' I ~ C A T A A C C T A 1 3 G 0 V $ K T M M I" A Q Q 5 Y E G V E T K D G H I A F I T T* TATGAG AACAGA~CAACGCGTGTTTCGGAC TAC GC GAAAGAAGAAGC CAGAAATCTGATAACGGAGGTCTTTGGAGAAG 1440 M R T D S T R V S D Y A K E E A R N L I T E V F G E E AGTACG ~'FCAAAGAG A G A A A G G A G A A A G A G C A A C G C A A A A A T A C AGGATGCC~C A T G A G G C G A T T C G T C C C A C G A A C 1 5 2 0 Y V G S K R E R R K S N A K I Q D A H E A I R P T N G TTTTC ATGACACCTGAAGAAGCGGGAAAATACCTGAATTCTGAC CAGAAAAAACTCTACGAGTTGATAT~GAAGAGATT 1600 V F M T P E E A G K Y L N S D Q K K L Y E L I W K R F T C T S -]CA T C G C A A A T G A A G C C ~FTCT C A A T A C G A G G A A A C T C G T T ~ TCCTGAGAAC AAAC~ATGGGAAGTACAGATTCA 1680 L A S Q M K P S Q Y E E T R F V L R T K D G K Y R F K A G G G A A C G G T G C T G A A G A A G A T T T T C G A T G G G T A C G A A A A G G T G T G G A A G A C G G A A A G A A A C A C G G G A G A G ~'I'FC~ 1760 G T V L K K I F D G Y E K V W K T E R N T G E F P F GAAAAGGGAGAATC TGTGAAAC CC~TGGTTGTAAAAA TAGAGGAACAGGAAAC CAAAC CGAAAC CAAGGTACACAGAGGG 1840 E E O E S V K P V V V K I E E ~ E T K P K P R Y T E G T T C TC T q ~ S T A A A A G A A A T G G A A A G A C T C C ~ T A T A C - G T C G T C C C A G C A C A T A T G C G T C C A C G A T A A A G C T ' ~ T ~ A A C A 1920 S L V K E M E R L G I G R P S T Y A S T I K L L L N R G G G G T T A C A T A A A A A A G A T C A G G G G C T A T C T C T A T C C A A C GATCG~CTGGAAGCG T T G T C A T G G A T T A T C T C . G A G A A A A A G 2 0 0 0 G Y I K K I R G Y L Y P T I V G S V V M D Y L E K K T A C TC T G A T G T T G ~ T A G C G T T T C C T T C A C A G C TGAAATC,GAAAAC-GAC C T C G A T G A A G T C G A A C A G G G T A A G A A A A C A G A 2 0 8 0 Y S D V V S V S F T A E M E K D L D E V E Q G K K T D C A A A A T C G q ' ~ C T G C G G G A G T T T T A T G A A T C Cq~I'TTCC A G T G T G ~ G A C AGGAACGACAGAATTGTGGTT~ATTTTC CGA 21G0 ~! [ V L R E F Y E S F S S V F D R N D R I V V D P P T C ~ ~ ~ ~ C A G A A A T G T T C G T G T C ,G A A A G G A A A T G A G ~ C T C T C T T T T C - G A A A A T A C G G C T T C T A T ' F P G A A A T G C G A G T G T G G T 2 2 4 0 :~ Q K C E C G K E M R L g F G K Y G F Y L K C ~ C G ~.~-~.~. SC A G A A G C G T G A A A A A C G A T G A A A T T G C C - G T C A T A G A C G A q ~ G A A A A A T A T T C C T G G C ~ A G G A A A G A C A G T ~ A G A G 2 3 2 0 =" P S V K N D E I A V I D D G K I F L G R K D S E S ?'5O ?T C T C C TGATC,C,G A G G A G T G T C G A G G G A A A G G G A A A T C T C T C T G A G A A G C GGAGAAAC,C ~ T A A A A A A G G C T C T T G A A 2 ~ 0 0 Z $ P D G R S V E G K G N L S E K R R K G K K G S STOP T ~ = ~ 3 T T A G A G A A G A T T T T C T C A A G A A A G T G G A T C A G T T G A A A A G T T T T G A T G T G G T C T T C A A C G T T C T C C A T G G A A C T T 24S0 U Z L 5 T [~A . ~ G A T G G A A C G C T T C A G G C A A T T C TC,G A T T T T T T G G G G A T C A G A T A C A C C G G G T C C G A T G C G T T T T C A A G C A T 3 ~ 5 6 0 ATA?~.~/q`~GATAAACTCGTGACTTACAGGTCTTTAAAGGGCACCGTCGA~ATACCTGATTT~TGGAGATCAAG~AAT ~. 2 6 2 0
Fig, 2. Structure of T. maritima TopA gene and flanking regions. (A) Organization of the ORFs contained in the sequenced DNA fragment. The dashed lines correspond to the not yet sequenced regions of ORF 1 and ORF 2. (B) Nucleotide sequence (from nt 320 to nt 2620) and deduced amino acid sequence of the Tm TopA ORF. The Shine-Dalgarno sequence is underlined. The putative tyrosine of the active site is indicated by a star and the peptidic sequence encompassing the Zn finger motif is represented in italic letters and underlined.
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EcTopB
t
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RG620'
TraE
Fig. 4. Sequence comparison of TopA family topoisomerases in the vicinity of the active site. Alignments were performed with the Pileup program described in Fig. 3 legend. The tyrosine of the active site is indicated by a star. The numbers represent the position of amino acids in the sequences. Identical amino acids were blackened when a strict identity was found for at least four organisms out of 6.
SpTop
BaTop
BsTopA
TmTopA
A
SccTopA
Rg620
ScTop3 Fig. 3. Relatedness of T. maritima topoisomerase I with type I topoisomerases from the prokaryotic family. The dendrogram was obtained from the multiple alignment of topoisomerase sequences using the Pileup program GCG package, version 7.2 [18]. The parameters were the followings: gap creation penalty: 1; gap extension penalty: 0.1. The different organisms used for sequence comparison were: Escherichia coli TopB (EcTopB) [19], RP4 plasmid encoded topoisomerase (TraE) [20], Streptococcus pyrogenes pDBI01 plasmid encoded topoisomerase (SpTop) [21], Bacillus anthracis pXOl plasmid encoded topoisomerase (BaTop) [22], Escherichia coli TopA (EcTopA) [9], Haemophilus influenzae TopA (HiTopA) [23], Bacillus subtilis TopA (BsTopA) [24], Synechococcus sp. TopA (SccTopA) [25], topoisomerase domain of Sulfolobus acidocaldarius reverse gyrase (residues 620-1247) (Rg620) [I0], Saccharomyces cerevisiae Top3 (ScTop3) [26]. A and B families are distinguished.
gene products are proteins of approximately 850 amino acids [9,23,25]. Furthermore, T. maritima topoisomerase shares an insertion of 7 - 8 additional amino acids (Fig. 4) with the Bacillus subtilis protein within the highly conserved region of the putative active site. This observation is particularly intriguing, since artificial insertions of tetrapeptides near the active site of protein to lead to a total loss of DNA relaxation activity [29]. The role of this additional octapeptide in the architecture of the active site has to be determined. No thermophilic specific features are evident that distinguish T. maritima topoisomerase I from its mesophilic
counterparts, except for the number of cysteines (5 residues), which is clearly lower than that of the other topoisomerases I (13 to 15 residues). Knowing the susceptibility of the cysteine residues to oxidation at elevated temperatures [30], this finding could constitute an element to explain the thermoadaptation of T. maritima topoisomerase I. Furthermore, 4 out of these 5 cysteines, located at the carboxy-terminus of the protein (region 2171 to 2237 on Fig. 2B), could constitute a Zn finger motif with the structure: Cys-X-Cys-X16-Cys-X-Cys. This motif exhibits a significant similarity with the last of the three Zn fingers of E. coli protein to (amino acids 710 to 736 in Ref. [12]), that was supposed to be involved in the DNA relaxation activity and especially in the strand passage step [29,31,32]. We plan to elucidate the relevance of this Zn finger motif in the enzymatic activity of T maritima topoisomerase I by site-directed mutagenesis. In conclusion, the topoisomerase gene that we have identified in T. maritima clearly belongs to the prokaryotic topoisomerase I family. The finding of a specific gene excludes the possibility that the ATP-independent activity detected in T. maritima extracts is due to a proteolytic product of reverse gyrase. If an equivalent of E. coli TopB is also present, then T. maritima would possess three kinds of type I topoisomerases: such a multiplicity addresses the question of their functional specialization.
Acknowledgements This work was supported by the Centre National de la Recherche Scientifique and the Universit6 Paris Sud (Unit6 de Recherche Associre 1354). The laboratory is supported in part by A.R.C. (Association pour la Recherche contre le Cancer).
References [1] Huber, R., Langworthy, T.A., KiSnig, H., Thomm, M., Woese, C.R., Sleytr, U.B. and Stetter, K.O. (1986) Arch Microbiol. 144, 324-333. [2] Huber, R., Wilharm, T., Huber, D., Trincone, A., Burggraf, S., Ktinig, H., Rachel, R., Rochinger, I., Frick, H. and Stetter, K.O. (1992) Syst. Appl. Microbiol. 15, 340-351. [3] Wang, J.C. (1987) Biochim. Biophys. Acta 909, 1-9. [4] Drlica, K. (1984) Microbiol. Rev. 48, 273-289.
C. Bouthier de la Tour et al. / Biochimica et Biophysica Acta 1264 (1995) 279-283
[5] Drlica, K. (1992)Mol. Microbiol. 6, 425-433. [6] Bouthier de la Tour, C., Portemer, C., Huber, R., Forterre, P. and Duguet M. (1991) J. Bacteriol. 173, 3921-3923. [7] Bouthier de la Tour, C., Ponemer, C., Nadal, M., Stetter, K.O., Forterre P. and Duguet, M. (1990) J. Bacteriol. 172, 6803-6808. [8] Nadal, M., Couderc, E., Duguet, M. and Jaxel, C. (1994) J. Biol. Chem. 269, 5255-5263. [9] Tse-Dinh, Y.C., Wang J.C. (1986). J. Mol. Biol. 191, 321-331. [10] Confalonieri, F., Elie, C., Nadal, M., Bouthier de la Tour, C., Fonerre, F. and Duguet, M., (1993) Proc. Natl. Acad. Sci. USA 90, 4753-4757. [11] Bouthier de la Tour, C., Portemer, C., Forterre, P., Huber, R. and Duguet, M. (1993) Biochim Biophys. Acta 1216, 213-220. [12] Huber, R., Langworthy, T.A., K~Snig, H., Thomm, M., Woese, C.R., Sleytr, U.B. and Stetter, K.O. (1986) Arch. Microbiol. 144, 324-333. [13] Sharp, P.M., Cowe, E., Higgins, D.G., Shields, D.C., Wolfe, K.H. and Wright, F., (1988) Nucleic Acids Res. 8207-8211. [14] Dalgaard, J.Z. and Garrett, R.A. (1993) In New Comprehensive Biochemistry series, Vol. 26 (Kates, M., Kushner, D.J. and Matheson, A.T., eds.), pp. 535-559, Elsevier, Amsterdam. [15] Liao, D. and Dennis, P.P. (1992) J. Biol. Chem. 267, 22787-22797. [16] Zawadzke, L.E., Bugg, T.D. and Walsh, C.T. (1991) Biochemistry 30, 1673-1682. [17] Robinson, A.C, Kenan, D.J., Sweeney, J. and Donachie, W.D. (1986) J. Bacteriol. 167, 809-817.
283
[18] Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395. [19] DiGate, R.J. and Marians, K.J. (1989) J. Biol. Chem. 264, 1792417930. [20] Starck, B, and Lanka, E. (1993) Accession number L10329. [21] Ceglowski, P., Boitov, G.. Lueder, G. and Alonso, J.C. (1992) Accession number X66468. [22] Fouet, A., Sirard, J.C. and Mock, M. (1994) Mol. Microbiol. It. 471-479. [23] Chandler, M.S. and Smith, R.A. (1995) Accession number U20964. [24] De Jong, S. (1994) Accession number L27797. [25] Gabay, C., Hassidim, M., Hurwitz-Lieman, J., Marco, E. and Kaplan, A. (1993) Accession number X72391. [26] Wallis, J.W., Chrebet, G., Brodsky, G_ Rolfe, M. and Rothstein, R. (1989) Cell 58, 409-419. [27] DiGate, R.J. and Marians, K.J. (1988) J. Biol. Chem. 263, 1336613373. [28] Wang, J.C. (1971) J. Mol. Biol. 55, 523-533. [29] Zumstein, L. and Wang, J.C. (1986) J. Mol Biol. 191, 333-340. [30] Volkin, D.B. and Klibanov, A.M. (1987) J. Biol. Chem. 262, 2945 -2950. [31] Tse-Dinh, Y.C. (1991) J. Biol. Chem. 266, 14317-14320. [32] Lima, C.D., Wang, J.C. and Mondragon A. (1993) J. Mol. Biol. 232, 1213-1216.