Gene, 109 (1991) 63-69 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/91/$03.50
63
GENE 06186
Sequence and linkage analysis of the C o x i e l l a b u r n e t i i citrate synthase-encoding gene (Recombinant DNA; tricarboxylic acid cycle; rickettsia; homology; succinate dehydrogenase)
Robert A. Heinzen*, Marvin E. Frazier b and Louis P. Mallavia" " Department of Microbiology, Washington State University, Pullman, WA 99164.4340 (U.S.A.) and e Office of Health and Environmental Research, Office of Energy Research, U.S. Department of Energy (GTN), Washington, D C (U.S.A.) Tel. (301)376-1914
Received by R.E. Yasbin: ! July 1991 Revised/Accepted: 13 September/17 September 1991 Received at publishers: 23 September 1991
SUMMARY The nucleotide (nt) sequence of the Coxiella burnetii citrate synthase-encoding gene (gilA), previously cloned in Escherichia coil, was ~etermined. The nt sequence analysis revealed an open reading frame (ORF) of 1290 bp capable of coding for a protein of 430 amino acids (aa) with a deduced Mr of 48 633. Preceding an ATG start codon, a possible transcription start point (tsp) with homology to the E. coil promoter consensus was detected. A poly-purine-rieh region occurred immediately upstream from the gltA reading frame and potentially serves as a ribosome-binding site. Additionally, a G + C-rich region of dyad symmetry 3' to the translational stop codon was found that could possibly function as a Rho-independent transcriptional termination signal. A large, nearly perfect, inverted repeat was identified upstream from the gltA tsp and was shown by Southern analysis to be present in multiple copies in the C. burnetii genome. The deduced aa sequence of C. burnetii GltA was optimally aligned with enzymes from various prokaryotic sources and one eukaryotic source (pig heart). Using perfect aa identity, the C. burnetii enzyme demonstrated the greatest homology with GltA from Acinetobacter anitratum (65 ~). Although only 2 6 ~ aa identity was seen with the pig heart enzyme, many of the residues identified in ligand binding appear to be conserved. Sequencing studies of a region centered approx. 5.6 kb upstream from gltA revealed an ORF read with opposite polarity that encodes a peptide highly homologous to the C terminus of the flavoprotein subunit of E. coil succinate dehydrogenase. This report represents the first nt sequence analysis of a gene ofknown function from the obligate intracellular parasite, C. burnetii.
INTRODUCTION The etiologic agent of human Q fever, C. burnetii, is an obligate intracellular bacterium (reviewed in Baca and Paretsky, 1983). As a mild acidophile it replicates in the hostile, low-pH environment of the eukaryotic host-cell phagolysosome (Burton et al., 1978). In axenic media it has Correspondence to: Dr. L.P. Mallavia, Department of Microbiology, Washington State University, Pullman, WA 99164-4233(U.S.A.) Tel. (509)335-3322; Fax (509)335-3517.
Abbreviations: aa. amino acid(s); bp, base pair(s); dlTPo deoxyinosine triphosphate; ExollI, E. coliexonuclease III; GItA,citrate synthase;gltA,
been demonstrated that optimal metabolism of exogenously applied substrates occurs below pH 5 (Hackstadt and Williams, 1981a,b). Of the substrates tested, TCA cycle intermediates, glutamate and proline are preferred as carbon and energy sources (Hackstadt and Williams, 1981b; 1983; Hendrix and MaUavia, 1983). As with other members of the family Rickettsiaceae, tragene encodingGItA; IR, inverted repeat; kb, kilobase(s)or I000bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; RBS, ribosome-bindingsite(s); REP, repetitive extragenic palindrome; SSC, 0.15 M NaCl/0.015M Na3"citrate pH 7.6; SdhA, flavoprotein subunit of E. coilsuccinate dehydrogenase;sdhA,gene encoding SdhA; TCA, tricarboxylic acid; tsp, transcription start point(s).
64 facultative anaerobes also display allosteric inhibition by 2-oxoglutarate (Wright et al., 1967). Kinetic analysis of the cloned C. burnetii enzyme revealed responses to effector molecules similar to those of enzymes found in Gram + bacteria and eukaryotic cells (Heinzen and Mallavia, 1987). C. burnetii is typically considered a Gram- bacterium. A similar finding was determined for citrate synthase of Rickettsia prowazekii (Phibbs and Winkler, 1982; Wood et al., 1983), also in the family Rickettsiaceae. There is a paucity of information on gene structure and function in C. burnetii. To date only two published C. burnetii nt sequences exist: an operon under heat-shock control (Vodkin and Williams, 1988), and a plasmid-located gene encoding a membrane protein of unknown function (Minnick et al., 1990). This report represents the first analysis of an nt sequence of a gene from C. burnetii encoding a protein of known function. Furthermore, evidence is presented that gltA is closely linked to the sdhA gene encoding SdhA.
ditional genetic analysis of C. burnetii has not been possible due to a lack of axenic cultivation methods. Methods of transformation and mutant isolation are currently unavailable. With this in mind studies were initiated to analyze C. burnetii gene structure and function, and to characterize gene products, by cloning nt sequences into E. coll. The gene encoding citrate synthase (GltA; EC 4.1.3.7), an enzyme presumed critical to this agents' biosynthesis and energy production, was the first such gene cloned into E. coil and characterized (Heinzen and Mailavia, 1987). Citrate synthase has been extensively studied from several prokaryotic and eukaryotic sources (reviewed in Weitzman and Danson, 1976; Wiegand and Remington, 1986). Without exception the active enzyme has been found to be a multimer of a single 48-kDa polypeptide. Gram + and eukaryotic cells produce a 'small', dimeric protein (Weitzman and Danson, 1976). In contrast, Grambacteria construct a 'large', primarily hexameric enzyme (Tung and Duckworth, 1975). The two types of enzymes display different responses to effector molecules. In vitro, the small enzyme is isosterically inhibited by ATP (Jangaard et al., 1968), but not NADH or 2-oxoglutarate (Weitzman and Jones, 1968). A reverse effect is seen with the large enzyme where NADH is an allosteric inhibitor (Weitzman and Jones, 1968). Gram- bacteria that are
A
0
1
i
2
i
i
RESULTS AND DISCUSSION
(a) Nucleotide sequence of the gltA gene The cloning, localization and orientation ofthe C. burnetii gitA gene has been previously described (Heinzen and
3
4
___1
5
i
6
i
I
Hindlll Xhol p L P M I O I"~ - " (7.9 kb) ~..~b,
I
I
i Hindlll BamHIEcoRI ~ P'gltA
Hindlll ~ Xhol _
Pstl
pLPM20 I ~ (5.2 kb)
(2,8 kb)
;~
,=
I
~
I
~.
I
",
HIndlll Sstl Hir¢ll Hincll~" ~
I
gltA
,,,,
XhqL,.'°°'"
pLPM41[
Hirdlll BarnHIEcoRI
If .........
............. B
8 kb
7
i r
t~ ~
~
,=
Ba~-II
~
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~
Fig. !. Subcloning and sequencing strategy of C. buraetii gitA. (A) The gltA + plasmids pLPMI0 and pLPM20 have been previously described (Heinzen and Mallavia, 1987). All regions shown are the cloned C. burnetii chromosomal inserts (size of insert in kb shown in parentheses). The E. coli host for all DNA manipulations was DH50¢(Bethesda Research Laboratories, Gaithersburg, MD). The location and direction ofgltA transcription is designated by a bold arrow. The gltA ORF was confirmed by sequencing of both strands of DNA. The location of the 3'-end of the putative sdhA gene is shown by an open arrow on pLPM 10. The orientation and length of thin arrows represent the direction and extent of the derived sequence. Determination of the nt sequence was accomplished by the dideoxy chain-termination method (Sanger et al., 19"/7) using [0¢-s2p]dCTP (3000 Ci/mmol) or [=-3SS]dATP (1000-1500 Ci/mmol)contained in Sequitid@ (New England Nuclear, Boston, MA) as the radiolabel and a Sequenase® kit (United States Biochemical Corp., Cleveland, OH) containing modified bacteriophage T7 DNA polymerase (Tabor and Richardson, 1987). Band compressions resulting from G + C-rich regions were resolved using dITP in the reactions as recommended by the supplier. Piasmid template DNA was purified by either CsCl-gradient centrifugation (Maniatis et aL, 1982)or by cetyl-trimethyl ammonium bromide (CTAB) precipitation (Dei Sal et aL, 1989). Double-stranded plasmid DNA was alkali-denatured prior to use in sequencing reactions (Chen and Seeburg, 1985) and samples were electrophoresed on 6-8% polyacrylamide gels. (B) Sequencing strategy for the gltA gene. One SstI and two HinclI restriction enzyme sites have been added for reference. A 2.8-kb XhoI + B a m H l fragment ofpLPM20 was subcloned into Xhol + Barn Hl-cut pGEM7 (Promega, Madison, WI) to generate pLPM41. Some sequencing reactions employed overlapping subclones created by the ExoIII and Sl nuclease method of Henikoff 0984) and were sequenced using a T7 promoter primer (Promega, Madison, Wl). When necessary, aynthetic oligo primers were synthesized using an Applied Biosystems model 380A DNA Synthesizer (Applied Biosystems Inc., Foster City, CA). Sequence data was compiled and analyzed using the University of Wisconsin Genetics Computer Group sequence analysis programs (Devereux et al., 1984) managed by the VADAMS ® laboratory at Washington State University. The nt sequence data for gltA will appear in the EMBL, GenBank and DDBJ nt sequence databases under accession No. M36338.
65
Mallavia, 1987). Sequencing was aided by the cloning of a 2.8-kb Xhol + BamHl ghA + fragment from pLPM20 into Xhol + BamHl-cut pGEM7, generating pLPM41 (Fig. 1, A and B). Unidirectional overlapping deletion subclones (Xhol --, BamHl) were constructed from the insert DNA of pLPM41 by the method of Henikoff (1984) and were used to determine the majority of sequences of the noncoding strand. The remaining sequence of this strand and the entire coding strand were completed using gltA-specific oligo primers (Fig. 1B). The sequence of gltA (Fig. 2) revealed an ORF from nt 1 61
&CCGACTATCG~TGTC~GGTGCGATT~GTGGTGGTTTTCAACAAGT&GCCCGT&TGC 60 &GCC.~AGCGA~T&CG~CI~AG~TGTCGTCT&TCGTC~CCGTATTCCGCTTCGCTG 120
121
C&TACGGGCT&TT"T&GeT&CTCGGCCCCCG&GGeGCCTT&GG~,¢CT&CACTTTC&AT¢ 100
181
G¢&CCG&TeGATGTG6GC~TT~J~r~TCC¢&CT*~I~TTC&T~T&C&&6¢&
240
241
CTACACCCGCCr~CACG&ATGAGCA&TCGT~GCTJ~J~£CTTTCTTTT~TC&G
300
RetSer&sn&rgLys&$aLysLeuSerPheGlu&snGln
301 TCGGTTC.,AATTTCCTATTT&TTCACCTACCCTTGGCAAGC~TGTGATTC~CGTI~J~GACA 360 SerValGluPheProZleTyrSerProThrLeuGlyLys&spVa111e&spValLyuThr
361
TTGGGCAATC&TGGTGCCT&TGCGCTGGACGTTGGTTTTT&CTCT&C&GCGGCTTGTGAA 420 LeuGly&snHlsGlyAlaTyr&laLeuAspValGlyPheTyrSerThrAlaAlaCysGlu
421
TCAAAN~TT&CCTTTATCGATGGCGAGA~GGT&TTTTGCTGTATCGGGGGT&TCCAATT
481
Se:LyslleThePhelleAspGlyGluLysGl¥IleLeuLeuTyrArgG1yTyrP¢olle
480
GATCAATTGGCTGAC~TCGG&TT&T&TGGA~GTTTGTTACTTACTG&TGTATGGCG~G $40
AspGlnLeuAlaAspLysSerAspTyrMetGluValCysTyrLeuLeuMetTyrGlyGlu
541
CTCCCGAAC~J~GG&~GG*'~,AATTTGTTCGCACGATTAAAGAAC&CACCAGCGTT 600 LeuProAsnLysGlyGluLysGluLysPheVal&~gThrlleLysGluH£$Th¢SerVal
601
TATGAGCAAGTG&C~TTTTTCA&TGGTTTTCACT&TGACGCGCATCCT&TGGCGATG 660
661
GTGCTT&GC&CC&TCGGCGCTCTTTC&GCTTTCT&TCAC~TGCGTT&GAC~TCACT&J~ 720 ValLe~SerThrlleGlyklaLeuSer&laPheTyrHisksp&laLeuAspZleThrLy8
721
¢¢GGCCG&CCGCGAkT~ATCCGCC&TCCG&TTAATAGCC.~,TGCCCACGCTGGCCGCC 780 P¢o&la&|p&¢gGluLeuSe¢&lalle&rgLeulle&laLysMetProThrLeuAlaAla
781
&TGAGTT&TAAAT&TTC~ATTGGTCAACCGTTT&TGCACCCTCGCCGGGCG&TG~TT&T 640 Ne~SerTyrLysTyrSe¢IleGlyGlnProPheHe~HisP~o&¢g&rg&laHetAlnTy¢
841
GCGGAkAACTTTTT&CAT&TGCTCTTTGGCACCCCTT&CG~AGAC'~CAGAACCTGACCCC 900 &laGluAsnPheLeuHisNe~LeuPheGlyTh~ProTy~GluGluThcGluProAspPro
901
GTCTTAGCTCGCGCG&TGG&TCGTATTTTT&TCCTTC^CGCCG&TC&CGKACA~TGCG 980 valLeu~laArg&laMet~sp&rgllePheZleLeuHlskla~spHisGluGlnAsnkla
961
TCAACGACT&CCGTTCGAGTCGCCGGTTCTACGGG&GCAAATCCGTTTGCTTGT&TTTCG 1020 SecThrThrThrValArgvalAlaGlySerThrGly&laksnProPhe^laCys%leSe~
TyrGluGlnValThrLysPhePheAsnGlyPheHisTyrAspAlaHisProMetAlaHe~
1022 GCGGGT&TTAGTGCTCTCTGGGGCCCTGCCCACGGCGGTGCTAATGAAGCTTGTTTARAT &IaGIFIleSerAlaLeuTepGlyP¢O&IaflLIGlyGIFAlaAInGIuAlaCygLeuAIn
1080
1081
1140
ATGTTGAGAAAAATTGGTGATGAGAAAAATATCGGCCAATAT&TTAAAAAAGCCAAAGAC
RetLeUA[gLFsIleGIyABpG~uLyIAInIIeGlyGInTyrlIeLyILyIAIIL¥I&IP
1141 N~ATGACCCTTTTCGTTTGATGGGCTTTGGCCATCGCGTTTACAAAN~TT&CGATCCC LyIASnABpP[oPheAEgLIuHetGlyPheGlyH£S&rgValTyeL~IABnTFeAIpPro
1200
1201 CGTGCCAA~GTCATGCA~CCTGCTATG-a-AGTCCTCGATGCCGTTGGCCGCCACAAT 1260 A~g&laLysValMetGlnLysTh¢CysTy[GluVa1LeuAsp&lavalG1¥&r~H18&sn 1261
GN~CCTTT&TTT~CTAOCGATA~AATTAGAGI~ATTGCGTT&GAGGATG&TT&TTTC 1320 GluP~oLeuPhoL¥sLouAlslleLyeLeuGluLysZle&lsLeuGluAsp&spTYrPhe
1321
ATTGAOAAAAAACTTTATCCGAATGTGGATTTTTATTCCGGTCTGACGCTAAATGCCATT
1380
lleGluLylL¥BLeuTy~P¢o&lnV~l&spPheTy~Se~GlyLeuThrLeuAlr~lalle 1381
GGC&TCCCTTCTA&TATGTTT&CGGTJ~ATTTTCGCCTT&TC&CG/~CGGTGGGCTGC~TT 1440 Gl¥IleP¢oSer&snHe~PheTh~Va111ePheAluLouSer&r~TheValGlyTrpI1e
262-1551 (1290 bp) capable of coding for a polypeptide (GItA) of 430 aa with a deduced Mr of 48 633. This figure agrees with both the value previously calculated from maxicell analysis (Heinzen and Mallavia, 1987) and the M r values described for other citrate synthase monomers (Wiegand and Remington, 1986). Beginning 10 nt t~pstream from an ATG start codon is a poly-purine-rich sequence, AGGAGCA, that is similar to the E. coli RBS consensus sequence (Gold et al., 1981). A putative RNA-polymerasebinding region, TTGAAA-N~7-TATAGT, was found spaced 19 nt proximal to the predicted RB S. The deduced -10 region (TATAGT) displays strong homology with the E. coil promoter consensus (Hawley and McClure, 1983), differing only in an A-to-G transition in the poorly conserved fifth base position. The deduced -35 region (TTGAAA) shares five out of six nt with the E. coli consensus sequence. An interesting feature upstream from gltA is a large, nearly perfect IR present at nt 46-134 (Fig. 2). Within each arm is an additional 7-nt perfect repeat. The 88-nt sequence comprising the gltA IR was searched against the entire GenBank and EMBL nt sequence databases. Remarkably, the only entry showing significant similarity was found distal to the C. burnetii htpAB operon (Vodkin and Williams, 1988) (Fig. 3). The htpAB operon is thermally regulated and produces proteins homologous to E. coli GroES (HtpA) and GroEL (HtpB) heat-shock proteins (Neidhardt and VanBogelen, 1987). The homologous IR begins 188 nt downstream from the htpB stop codon. Only minor differences were apparent between gltA and htpAB IRs. For example, the sequence intervening the left and right arms of each IR showed no similarity and is 23 and 16 nt in length in the gitA and htpAB IRs, respectively. In addition, the arms of the gltA 5' IR were slightly larger (2-8 nt). A consensus sequence was derived using the aligned left and right arms of both IRs and a synthetic oligo was synthesized corresponding to this sequence (Fig. 3). When this 5'
~
~
3'
gltA (right) AAGTAGGCCGTATGCAGCGAAGCGAAATACGGG htpAB (right) GTA GCCCGTA TGCAGCGAAGCGGA A TA CGGA gltA(IC;left) A A A TA GCCCGTA TGCAGCGA A GCGGA A TA CGGG htpAB(IC;lelt) CGT A T GCA- CGA A GCGGA A TA CGA G
1441 TC&C&TTGG&TGGN~q, TGATG&GCAGTCCCG~TCA~CGCCTCGCTCGCCCCAGACAATT& 1500
SeeHlsTrpMetGluHetHetSerSerPro&spBi~Atg~euAla&rgPro&rqGlnLeu
iS01
T&CACAGGCGARACAG&GCGAGAAGTAATCTCGTTGG&TAAACGTCKGGCATA^CAGCAG
aagTAGCCCGTATGCAFCGAAGCGg AATACGg g 1560
TFeTheGIyGluTh~GluArgGluValIleSerLeuAspLyS&r~GlnAIa 1561
AAAAGAGAT/~qJtC, AAAGAR~TCTCTGGCTGCTTGTGCCAGAGACAAGAACGATGCCTATC
1620
1621
GTTATCCAC,AAGTCACRGCGACCTCCATTTTAAACTTTGTGATCCCGCGCAGATGGAACC
1680
1681 CAGGCGCGCTTTAGCGCGCTGCGCATT&CGTGCGCTGGGTC~CCGCCTGCGGGGGCGACG, 1740 1741 ~GGGTTGGGTTTAAGTTTTTAGTGTCT&CCACTTGTGGTCGTCGCGTGGGTTGTGGCCGA 1800 1801 GATTTTTRAAGATTA.qAGTTTACGTTTATGCCCGCGCGGATCGTGTAGGCCCTGATATTC 1860 1861 TTTGCTTCCTGGGTATRAACATCTGGTGGAGCRACAACGGGATCC 1905
Fig. 2. Nucleotide sequence of the noncoding strand ofgltA. The deduced aa sequence is shown under the nt sequence. Putative -35 and -10 promoter regions and a potential RBS are overlined. A large inverted repeat proximal to the g/tA start is shown with facing arrows. A G + C-rich region (near the bottom) with multiple, small dyad symmetries representing a potential Rho-independent termination region is underlined.
Fig. 3. Comparison of the gltA 5' IR with sequences Y of the C. burnetii htpAB operon (Vodkin and Williams, 1988). The IR sequences have been aligned to illustrate their sequence similarity. The sequence of the IC of the left arm of both IRs is aligned with the right arm. Arrows indicate the position of a smaller 7-bp IR within the 1~:~and right arms of the elt.t 5' IR. A dash represents an absence of an nt. The consensty~, seau~:,ce (CONS) is depicted below the alignment with upper-case let:,l's representing positions in the alignment showing perfect homology and lowercase letters representing positions in the alignment showing less than perfect homology or positions with nt not present in all four aligned sequences. The underlined region of the consensus sequence corresponds to the sequence ofthe synthetic oligo synthesized for use in the Southern analysis described in Fig. 4.
66 oligo was used as a hybridization probe it revealed the IR sequence to be present in multiple copies within the rickettsial genome (Fig. 4). At least six bands of hybridization were observed when EcoRl-digested genomic DNAs from four C. bumetii strains (Mallavia, 1991) were probed. The greater intensity of hybridization signals observed with some EcoRl fragments may indicate the presence of more ~han one copy of the IR. A strong signal was also observed from pLPM30 insert DNA containing the cloned C. burnetii gltA gene (Heinzen and Mallavia, 1987) as expected. The function of the C. burnetii repetitive IRs is open to speculation. Perhaps they represent a class of elemeats analogous to REPs found in E. coli (l~r~gins et ai., 1981; Stern et al., 1984; Newbury et al., 1987). These IRs are remarkably homologous a~d are present in nontranslated regions of 25% of all E. coE ~:'~p.scription units. REPs have been shown to enhance g~:ne expression by stabilizing upstream mRNA via slowing the attack of 3' --, 5' exonuclease (Newbury et al., 1987). Transcription of the gltA 5' IR could result in a very stable (stabilization free energy; zig = - 54.9 kcal/mol) RNA stem-loop structure. Higgins etal. (1981; 1988) have suggested that REPs may have
1
2
3
4
5
6
!
Fig. 4. Southern hybridization analysis employing an oligo probe corresponding to the C. burnetii extragenic IR consensus sequence (Fig. 3). Lanes 1-4 show EcoRl-digested (7. burnetii genomic DNA from Hamilton, Biotzere, Corazon, and Dod strains, respectively (Mallavia, 1991). Lane 5 is HindIlI.digested ~ DNA Mr markers, and lane 6 is EcoRI + Pstl.digested pLPM30 (Heinzen and Mallavia, 1987). The DNA was resolved using a 0.8?/o agarose gel. Transfer to nitrocellulose was done by the method of Southern (1975). Hybridization was to the oligo primer 5'-TAGCCCGTATGCAGCGAAGCG (synthesized as described in Fig. 3) that was end-labeled using T4 kinase (Bethesda Research Laboratories, Gaithersburg, MD) and [~,-32P]CTP (NEN). Washes were carried out in 6 x SSC at 60°C.
arisen from insertion-like elements. However, REPs in E. coli lack short direct repeats at both ends that are trademarks of insertion sequences. The C. burnetii gltA 5' IR similarly lacked such repeats. The lack of distinctive polymorphism in the hybridization patterns of chromosomal DNAs obtained from different C. burnetii strains also argues against a transposable element. The location of the gltA 5' IR may indicate the presence of a hitherto unknown transcriptional unit in the region immediately upstream from, and reading toward, gltA. Few sequence data are currently available for this region (Fig. 2). The 3'-end of the gltA gene is determined by a TAA stop codon at nt 1552, followed by a 56-bp G + C-rich area (nt 1694-1749) of multiple-dyad symmetry. This region, when transcribed, could form a multiple hairpin structure (stabilization free energy; ,4(; = -24 kcal/mol) that might act as a Rho-independent terminator. This sequence is 79?/0 G + C as compared to 44% for the gltA ORF. Band compressions were encountered during sequencing of this region on both strands of DNA that were resolvable only upon incorporation of dITP in the sequencing reactions.
(b) Amino acid sequence homology of GItA A multiple sequence alignment was prepared to compare the deduced aa sequence of C. burnetii GItA with that offive bacterial enzymes and one representative eukaryotic enzyme (Fig. 5). The aa sequence of both E. coli (Bhayama and Duckworth, 1984) and pig heart (Bloxham et al., 1982) GltAs have been determined directly by chemical means while those of Acinetobacter anltratum (Donald and Duckworth, 1987), Pseudomonas aeruginosa (Donald et al., 1989) and Rickettsia prowazekii (Wood et al., 1987) have been deduced from the nt sequence. The pig heart enzyme has been studied further using x-ray crystallography and residues involved in ligand binding have been identified (Remington et al., 1982). When the deduced aa sequence of C. burnetii GltA is optimally aligned with the pig heart sequence only 26% homology (using perfect aa identity) was seen. The greatest divergence occurred toward the N terminus where ligand-binding residues are sparse. However, the C. burnetil sequence displays identical or functionally similar residues at ten of eleven positions identified as involved in substrate binding. The one remaining active site residu~ of pig heart, Arg46, has no obvious homolog in the C. burnetii enzyme. Interestingly enough, when C. burnetii GltA is optimally aligned with the enzymes from R. prowazekii, P. aeruginosa, E. coli and A. anitratum the levels of identity were 56 ~o, 61%, 61% and 64%, respectively. The fact that C. burnetii produces a citrate synthase more homologous to that of bacteria residing in the families Pseudomonadaceae, Enterobacteriaceae and Neisseriaceae raises some interesting questions regarding the evolutionary origin of this agent.
67 1 FNGLTA ~LTA FA|GLTA |COGLTA RFRGLTA CSUGLTA CONJ
AS|IS( LKDZLADLIP U ~ N t I E T I ~ SIUkTGNNA V L E L N . E | I|LPZYSGTL A . O N | AQLXXNGSJUF VELlq~SGTN A.DTN AItLTLNGDTA VllSDVLKGTL TNGNNNNL|~ABLKXMG.KL FKLFZLKASZ SN.RK AitLS~IIN.Q8 V|FPZYSPTL -oIp .... ~..... I---k i-l---g---
Sl
O011~..VOOITVO.gN. G S O ~ D V E I ~ IkI~qG.H~TFD G~DI/VDVMOL TATG.nrT~O G~DYZDZRTL GSKG¥.FTI~D GKDVXOXSRV SAgADY~TYO OEOVXOVI|TL ONBGAYA.LD g-dvtdv ...... g......
*
PHGLTA YGGNItGNKGL VYNTSVLD~D EGXRr.MGY| JULNGLTA FGFI~IkTJ~SCK SKZTF.IDGD KGXLLH|GY~ PAEGLTA PGFMSTASC~ SKXTY.Z~D EGVLLHRGYP ECOGLTA POFTSTASCB S K X T F . Z ~ D EGZLLHMGrP RPROLTA PGFNSTJ~SCQ STZTY.ZDGD EGZLMYRGYD CBUOLTA VGFYSTJUkCE SKXTF.XDG~ KGZLLYRGYP CON8 p G f n s ~ a s c e s k i T - - L D g d kG£11-gGyp
ZPBCQKRL~K AKGG|EPLFE EDQLATQA . . . . . . . . DYLE ZEQLABKS . . . . . . . . DYLE I~LATDN . . . . . . . . NYLE ZKDLA~KN. . . . . . . . 9FLE IDQLADKN . . . . . . . . DYnE g-qlm--s ........ dylE
PHOLTA AJ~qGLTA PAEOLTA ECOGLTA RPMGLTA CBUGLTA C0N8
101 GLFWLLVTG~ IPTEEQVSWL TCYLLL.NGB LPTAEQKV|F TCYLLL.NGB LPTJUkQKEQF VCY~LL.NGB KPTQEQYDB~ VAY.LNIYGE LPSSDQ~CN~ VCYLLN.YOE LPNKGEKEK~ -Cy-GI--GO lP~--q---f
PHGLTA JU~HGLTA FAEGLTA ECOGLTA RPRGLTA CBUGLTA CONS
151 QLSJUkXT.AL NSESNFARAY A|GZN;TKYW SLZYNDCRDL INVGW.GAL SAFYNNNLDI EDXHHBEZTA . . . . . . . ZRL HNL VNCOVX.GAL SAFYHDSLD| TNPKHBQVSA . . . . . . . FRL VNCO.~TGAL JUkFYHDSLDV HNPSNMEZJUk . . . . . . . Zg ZNLAAV.GSL SAFYPDLLNr .NETDY|LTA . . . . . . . ZRL .NVLSTXOAL SArYHDALDI TKPADR|L8A . . . . . . . cl - S - - ~ L - g a ~ safyhd-ld- . . . . h r e - - 8 . . . . . . . .
SKEWAKPJUt~ .DAEVRAHTlt V.GTZKmlTN .KTTVTRHTM TK.KVJUIHSL Vlq.TXKENTS ....... h~-
PS~LON .rP1~qLneNS VH~VSRFrN OFRRDAHPNA VHEQLKTFFN GFRBDAHPIqA XH|QXTRLFO AFMMDSHPFL~ VN~MLBYLFQ TFCSSSHPH~ VYEQVTE~N GFHYDAHPRA v-eq .... fn -F--d-HPN8
201
PHGLTA IYRNLYREGS SZOAZDSKLD WSHNFTHNLG AAHOLTA SYKYTV..OQ PrIYPRNDLN YAENrLHHRr PAEGLTA V~g¥SK..GE PHMYPRHO~ YA|NrLHHMf ECOOLTA CYKYSZ..GQ PFVYPRNDL8 YAGNrLNHMF RPROLTA SYKYSZ..GQ PFXYPDNSLD FT|NrLHRRF CBUOLTA SYKYSZ..GQ PFNNPRRAHN YAENrLNNLF CONS - Y k y s - - - G q p f - y p c n - l PHGLTA AJUqOLTA FAEOLTA ECOOLTA BPRGLTA CNUGLTA CONS PHGLTA JUUqGLTA PAEGLTA ECOGLTA RPROLTA CSUOLTA CONS
251 * YLTZHSDHEG ONVSAHTSHL ZFTLHADHE. QNASTSTVRL ZFILHADHE. QNASTSTVRL ILZLHAOHE. QNASTSTVRT IFXLHADHE. QNASTSTVRZ ZFZLHADHB. QNASTTTVRV Lf~INaDHK- q N s S t s T v c -
IAKLPCVAAg IAKXPTLAAN ZASNPTIAAN LSRNPXHJu~q XAKXPTIAAH XAKNFTLAAN LaS-P~-AAs
YTDAQFT|L . . . . . . . . MRL ATFJUDRDYKV NPVLARAHDR NT~CE.TKPZ SPVLAKJuqDg STFCE.PYEV NPZLERANOR ATPCT.KYK¥ NPZXKNALNK GTPYE.ET|P OPVLARNqOg -Tp. ado
SrAJUU~GLA GFL;GLANQE CZSAGZSALH GFAHGGANEA CZASGZAALH GPANGGANEA CIJUkGZASLW GPAHGGANEA CXSTGZASLW GPAHGGAHEA CXSAGZSAL~ GPAHGGAHEA aOS-glnPfa c L - a g ~ - - L w OPSHGqANea
VGSALSDPYL AGSTGANPYA AGSSGANPFA AOSSGANFrA AGSSGAHPFA AGSTGANFrA
]01 VLVWr,TQLQK EVGKDVSOKK LRDYXNNTLN VLKNLDEZGS VENVAEFNER VKRK. . . . . . VLRHLOEZGD VSNXDKI'VEK AgOg . . . . . N ALKRLEEZSS VKHXPKrFRR AgOg . . . . . N VZNRLKBXGS SENZPKYVAK AKDK. . . . . N CLNNLMnXGD EKNX~YXKK AKDK. . . . . N v l o m L - e t g - - - n t . . . . . k akdk . . . . . .
8GRV~TPGYGI! AVL STOP Y I~.VKLNGIFGH RVYKNFDPRA DIPlPKLNGLPGB RVYKNFDFRA OSrMLHGFGH BV~FKNYDPRA DIPFRLRGFGH RVlrKSYOFRA DPFRLNGFGH RVYKNYDPRA d - f-18GfOH cVykn-OPga
351 PHOLTA TCQREF...A L K H L r . . H . D KVNKQTCDEV LEALG.I.ND PAEGLTA KVMKQTCDEV LQELO.Z.ND ECOGLTA TVHRBTCHEV LKELOT.KDD
PNFKLVAOLY KZVPNVLLEQ GKA;NPWPNV PQLALAMELE RZALNDPYFV EB.K.LYPNV PQLELAHKLE |IARHOPYFV E..RNLYPNV .GLEVAHELE NXALNOPYFZ EK.K.LYPNV RPROLTA AVLKETCKEV LNELGQLDNN PLLQZAXELB ALALKDEYFI EB.K.LYPNV CBUGLTA KVNQKTCYEV LDAVOR.HHE PLFKLAZRLE.KXALEDDYFZ ER.R.LYPNV -£al-d-yfe--k-lyPNV CONS - v m - - t - - - v L--1 ..... d --l-la--Le
AANGLTA
.401 PHGLTA DAHSGVLLQY YG~TENNYYT A~IGLTA OFYSGIILKA ZGZPTEN.FT PAEGLTA ~FYSGIXLKA ZG~PTSN.FT
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.,A,,SZN,s DON.~,*g,.
oerQ.,s.,g DFYSGXZYKA NGZPSQN.FT VLrA,A,,V, . M . n z . . , DFYSOLTLNA IGXPSNN.FT VIFALSRTVG WZSHHIqENNS SPDHRLARPR CONS O f y s G t i l k 8 - O i p - - m - f T V £ F a - e R t v G w i - h w - e m - s - p - - k i - g P [
RPRGLTA
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ECOOLTA QLYZGYEKRD FKS.DZKR RPRGLTA QLYTGYVHRS YKC][VERK CBUGLTA QLYTGSTERS VXSLD.KRQA
CONS
ql¥tg---r-
Fig. 5. Multipleaa sequence alignmentof selected citrate synthases with C. burnetiiGltA. Gaps have been introducedto maximizehomology.The sequences from top to bottom are derived from pig heart (PHGLTA; Bioxham etal., 1982), A.a,itratum (AANGLTA; Donald and Duckworth, 1987), P. aeruginosa (PAEGLTA; Donald et al., 1989), E. coli (ECOGLTA; Bhayama and Duckworth, 1984), R.prowazekii (RPRGLTA; Wood et al., 1987) and C. burnetii(CBUGLTA). Met start codons are not included. The consensus aa sequence is designated CONS; upper-case letters indicatingresidues shared by every sequence a,d lower-caseletters indicatingresiduessharedby fouror five sequences in the alignment.Residues implicated in iigand binding of the pig heart enzyme (Remingtonet al., 1982) are marked by asterisks. Numbering indicates position in alignment.
Indeed, this relationship is in keeping with the results of a recent study by Weisberg etal. (1989) that demonstrated using 16S rRNA sequence homologies that C. burnetii is more related to members of the ~, subdivision of the purple bacteria that includes the families Enterobacteriaceae and Pseudomonadaceae. Other members of the Rickettsiaceae are placed within the ~ subdivision. (c) Linkage with sdh The proximity to gltA in E. coli of TCA cycle genes encoding succinate dehydrogenase complex (sdhCDAB), 2-oxoglutarate dehydrogenase complex (sucAB), and succinyl-CoA synthetase (sucCD) (Bachmann, 1987) prompted a search of both distal and proximal regions of C. burnetii gltA to determine Whether similar linkages are present. Nucleotide sequence information flanking gltA was derived from regions centered approx. 5.6 kb upstream using pLPM10, 3.0 kb upstream and 0.5 kb downstream using pLPM20, and 1.0 and 0.5kb upstream using pLPM41 (Fig. 1). The six possible reading frames for each region were translated and predicted peptides of significant size were searched for homologous sequences in the database. In addition, these peptides were compared individually with the predicted aa sequences of the protein subunits comprising the three respective protein complexes produced by sdhCDAB and sucABCD operons. A peptide of 90 aa derived from a region centered 5.6 kb proximal to thegltA ORF on clone pLPMI0 (Fig. 1A) and transcribed with opposite polarity displayed significant homology (60~o aa identity) to the C-terminal 92 aa of the SdhA (Wood et al., 1984) (Fig. 6). The database search revealed additional homologies to the flavoprotein subunits of E. coli fumarate reductase (Cole, 1982) and Bacillus subtills succinate dehydrogenase (Phillips et al., 1987) of 41 and 27% aa identity, respectively (data not shown). The C. burnetii SdhA peptide coding region terminates with tandem stop codons situated 8 nt from the left EcoRI site of insert D N A of pLPMI0 (Fig. 1)(data not shown). (d) Conclusions (1) A 1290-bp ORF has been identified as coding for C. burnetii GltA with a deduced Mr of 48 633. Consensus E. coli transcriptional and translational initiation signals precede gitA. Following the ORF is a G + C-rich region CBU ECO CBU ECO
AKLTDTSRTFNNARIEALELDNLNEVSYATAVSAQQRTESRGAHSRYDYKERDD I:1 I l l II I:1 I l l l l l i l IIIIlli Illllfllil:l: :Ill ARLDDTSSEFNTQRVECLELDNLMETAYATAVSANFRTESRGAHSRFDFPDRDD
SS0
ANHLKHTVYFRDGH.IAYRPVNHKPKGHDPFPPKSRD ill I :l: :: : I I l l II :llll I ENHLCHSLYLPESESNTRP~TNNEPKLRPAFPPKIRTY589
Fig. 6. Comparisonofthe deduced aa sequenceofa peptide encoded by an ORF centered approx.5.6 kb upstreamfromthe C. burnetiiglt,4translational start with the C-terminalportionof SdhA orE. coli(Wood et ai., 1984). Numberingindicates the appropriate residues of E. coli SdhA. Identical and functionallyequivalent residues are signified by lines and colons, respectively. Abbreviations: CBU, C. burnetii;ECO, E. coll.
68
with properties of a Rho-independent termination site. A large region of dyad symmetry centered 172 bp upstream from the gltA start codon is found in multiple copies throughout the C. burnetii genome and may mediate some unspecified regulatory function. (2) Comparison of the deduced aa sequence of C. burnetii GltA with other citrate synthase sequences shows that the enzyme is more closely related to the 'large' type enzyme produced by free-living Gram- bacteria. This contrasts with data that previously demonstrated the enzyme to have regulatory properties typical of the 'small' enzyme associated with Gram + bacteria and eukaryotic cells. Of the bacterial enzymes, the one produced by R. prowazekii is the least similar despite exhibiting similar responses to enzyme modulators. Although the pig heart and C. burnetii GltA proteins are similar in their responses to effectors, they have only 26 % aa identity. Nonetheless, there is conservation of identical or functionally similar residues at most sites identiqed in pig heart GltA ligand binding. (3) The identification of an ORF centered approx. 5.6 kb upstream from gltA that encodes a peptide with a high degree of homology to SdhA has led us to the conclusion that the gene encoding the C. burnetii homolog of this protein, and indeed, possibly the entire sdh operon, is linked to gltA in a similar fashion as that demonstrated in E. coll. If C. burnetii has an operon coding for a 4-subunit Sdh complex analogous to that described in E. coli with the genes for two hydrophobic subunits (sdhC and sdhD) preceding sdhA, the calculated intergenic region between the translational starts ofgltA and sdh would be approx. 3 kb. This is significantly larger than the approx. 700-bp spacing in E. coli and would presumably preclude a shared gltA-sdh regulatory region as demonstrated for this bacterium (Wilde and Guest, 1986). We are currently investigating regions flanking this peptide by sequencing and complementation of sdh-deficient E. coli mutants to confirm the presence of a gltA-linked sdh operon in C. burnetii.
ACKNOWLEDGEMENTS
We express appreciation to Shirley Schmitt for her excellent technical assistance. This work was supported by grant A120190 from the National Institutes of Health (NIAID), by the Northwest College and University Association for Science (University of Washington) under contract DE-AM06-76-RL02225 with the US Department of Energy and by Battelle, Pacific Northwest Laboratories.
REFERENCES Baca, O.G. and Paretsky, D.: Q-fever and Coxiella burnetii: a model for host-parasite interactions. Microbiol. Rev. 47 (1983) 127-149.
Bachmann, B.J.: Linkage map of Escherichia coil K-12, ed. 7. In: Neidhardt, F.C. (Ed.), Escherichia coil and Salmonella typhimurium: Cellular and Molecular Biology, Vol. 2. American Society for Microbiology, Washington, DC, 1987, pp. 807-876. Bhayama, V. and Duckworth, H.: Amino acid sequence ofEscherichia coli citrate synthase. Biochemistry 23 (1984) 2900-2905. Bloxham, D., Parmelee, D.C., Kumar, S., Walsh, K.A. and Titani, K.: Complete amino acid sequence of porcine heart citrate synthase. Biochemistry 21 (1982) 2028-2036. Burton, P.R., Stuckemann, J., Welsh, R.M. and Paretsky, D.: Some u!trastructurai effects on persistent infections by the rickettsia Coxiella burnetii in mouse L-ceils and green monkey kidney (VERO) cells. Infect. Immun. 21 (1978) 556-566. Chen, E.Y. and Seeburg, P.A.: Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4 (1985) 165-170. Cole, S.T.: Nucleotide sequence coding for the flavoprotein subunit ofthe fumarate reductase of Escherichia coll. Eur. J. Biochem. 122 (1982) 479-484. Del Sai, G., Manfiolet~i,G. and Schneider, C.: The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagmids, phages or plasmids suitable for sequencing. Biotechniques 7 (1989) 514-519. Devereux, J., Haeberli, P. and Smithies, O.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Donald, L.J. and Duckworth, H.W.: Expression and base sequence ofthe citrate synthase gene ofAcinetobacter anitratum. Biochem. Cell. Biol. 65 (1987) 930-938. Donald, LJ., Molgat, G.F. and Duckwortb, H.W.: Cloning, sequencing and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa. J. Bacteriol. 171 (1989) 5542-5550. Gold, L., Pribnow, D., Schneider, T., Shinedling, S., Singer, B. and Stromo, G.: Translation in procaryotes. Annu. Rev. Microbioi. 35 (1981) 365-407. Hackstadt, T. and Williams, J.C.: Biochemical strategem for obligate parasitism of eucaryotic cells by CoxieUa burnetii. Prec. Natl. Acad. Sci. USA 78 (1981a) 3240-3244. Hackstadt, T. and Williams, J.C.: Stability of the adenosine 5'-triphosphate pool in Coxiella burnetii: influence of pH and substrate. J. Bacterioi. 148 (1981b) 419-425. Hackstadt, T. and Williams, J.C.: pH dependence of the Coxiella burnetii glutamate transport system. J. Baeteriol. 154 (1983) 598-603. Hawley, D.K. and McClure, W.R.: Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 11 (1983) 2237-2255. Heinzen, R.A. and Mallavia, L.P.: Cloning and functional expression of the Coxiella burnetii citrate synthase gene in Escherichia coll. Infect. Immun. 55 (1987) 848-855. Henikoff, S.: Unidirectional digestion with exonuelease III creates targeted breakpoints for DNA sequencing. Gene 28 (1984) 351-359. Higgins, C.F., Ames, G.F.-L., Barnes, W.M., Clement, J.M. and Hofnung, M.: A novel intercistronic regulatory element ofprokaryotic operons. Nature 298 (1982) 760-762. Higgins, C.F., McLaren, R.S. and Newbury, S.F.: Repetitive extragenic palindromic sequences, mRNA stability and gene expression: evolution by gene conversion? - a review. Gene 72 (1988) 3-14. Jangaard, N.O., Unkless, J. and Atkinson, D.E.: The inhibition of citrate syntbase by ATP. Biocbim. Biophys. Acta 151 (1968) 225-235. Mallavia, L.P.: Genetics of rickettsiae. Eur. J. Epidemiol. 7 (1991) 213-221. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Minnick, M.F., Heinzen, R.A., Frazier, M.E. and Mallavia, L.P.: Charac-
69 terization and expression ofthe cbbE' gene of Coxieila burnetii. J. Gen. Microbiol. 136 (1990) 1099-1107. Neidhardt, F.C. and VanBogelen, R.A.: Heat shock response. In: Neidhart, F.C. (Ed.), Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol. 2. American Society tbr Microbiology, Washington, DC, 1987, pp. 1334-1345. Newbury, S.F., Smith, N.W., Robinson, E.C., Hiles, I.D. and Higgins, C.F.: Stabilization of translationally active mRNA by procaryotic sequences. Cell 48 (1987) 297-310. Phibbs Jr., P.V. and Winkler, H.H.: Regulatory properties of citrate synthase from Rickettsia prowazekii. J. Bacteriol. 149 (1982) 718-725. Phillips, M.K., Hederstedt, L., Hasnain, S., Rutberg, L. and Guest, J.R.: Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis succinate dehydrogenase complex. J. Bacteriol. 169 (1987) 864-873. Remington, S., Wiegand, G. and Huber, R.: Crystallographic refinement and atomic models of two different forms of citrate synthase at 2.7 and 1.7 A resolution. J. Mol. Biol. 158 (1982) 111-152. Sanger, F., Nicklen, S. and Couison, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol, Biol. 98 (1975) 503-517. Stern, M.J., Ames, G.F.-L., Smith, N.H., Robinson, E.C. and Higgins, C.F.: Repetitive extragenic sequences: a major component of the bacterial genome. Cell 37 (1984) 1015-1026. Tabor, S. and Richardson, C.C.: DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84 (1987) 4767-4771. Tung, E.K. and Duckworth, H.W.: The quaternary structure of citrate
synthase from Escherichia coil K-12. Biochemistry 14 (1975) 235-241. Vodkin, M.H. and Williams, J.C.: A heat shock operon in Coxiella burnetii produces a major antigen homologous to a protein in both mycobacteria and Escherichia coll. J. Bacteriol. 170 (1988) 1227-1234. Weisberg, W.G., Dobson, M.E., Samuel, J.E., Dasch, G.A., Mallavia, L.P., Baca, O., Mandelco, L., Sechrest, J.E., Weiss, E. and Woese, C.R.: Phylogenetic diversity of the rickettsia. J. Bacteriol. 171 (1989) 4202-4206. Weitzman, P.D.J. and Danson, M.J.: Citrate synthase. Curr. Top. Cell. Regul. 10 (1976) 161-204. Weitzman, P.D.J. and Jones, D.: Regulation of citrate synthase and microbial taxonomy. Nature 219 (1968) 270-272. Wiegand, G. and Remington, S.J.: Citrate synthase: structure, function and mechanism. Annu. Rev. Biophys. Chem. 15 (1986) 97-117. Wilde, R.J. and Guest, J.T.: Transcript analysis of the citrate synthase and succinate dehydrogenase genes of Escherichia coli K12. J. Gen. Microbiol. 132 (1986) 3239-3251. Wood, D.O., Atkinson, W.H., Sikorski, R.S, and Winkler, H.H.: Expression of the Rickettsia prowazekii citrate synthase gene in Escherichia coll. J. Bacteriol. 155 (1983) 412-416. Wood, D., Darlison, M.G., Wilde, R.J. and Guest, J.R.: Nucleotide sequence encoding the flavoprotein and hydrophobic subunits of th,,• succinate dehydrogenase of Escherichia coil. Biochem. J. 222 (1984) 519-534. Wood, D.O., Williamson, L.R., Winkler, H.H. and Krause, D.C.: Nucleotide sequence of the Rickettsia prowazekii citrate synthase gene. J. Bacterioi. 160 (1987) 3564-3572. Wright, J.A., Maeba, P. and Sanwal, B.D.: Allosteric regulation of the activity of citrate synthase of Escherichia coil by u-ketoglutarate. Biochem. Biophys. Res. Commun. 29 (1967) 34-38.
63
GENE 06186
Sequence and linkage analysis of the C o x i e l l a b u r n e t i i citrate synthase-encoding gene (Recombinant DNA; tricarboxylic acid cycle; rickettsia; homology; succinate dehydrogenase)
Robert A. Heinzen*, Marvin E. Frazier b and Louis P. Mallavia" " Department of Microbiology, Washington State University, Pullman, WA 99164.4340 (U.S.A.) and e Office of Health and Environmental Research, Office of Energy Research, U.S. Department of Energy (GTN), Washington, D C (U.S.A.) Tel. (301)376-1914
Received by R.E. Yasbin: ! July 1991 Revised/Accepted: 13 September/17 September 1991 Received at publishers: 23 September 1991
SUMMARY The nucleotide (nt) sequence of the Coxiella burnetii citrate synthase-encoding gene (gilA), previously cloned in Escherichia coil, was ~etermined. The nt sequence analysis revealed an open reading frame (ORF) of 1290 bp capable of coding for a protein of 430 amino acids (aa) with a deduced Mr of 48 633. Preceding an ATG start codon, a possible transcription start point (tsp) with homology to the E. coil promoter consensus was detected. A poly-purine-rieh region occurred immediately upstream from the gltA reading frame and potentially serves as a ribosome-binding site. Additionally, a G + C-rich region of dyad symmetry 3' to the translational stop codon was found that could possibly function as a Rho-independent transcriptional termination signal. A large, nearly perfect, inverted repeat was identified upstream from the gltA tsp and was shown by Southern analysis to be present in multiple copies in the C. burnetii genome. The deduced aa sequence of C. burnetii GltA was optimally aligned with enzymes from various prokaryotic sources and one eukaryotic source (pig heart). Using perfect aa identity, the C. burnetii enzyme demonstrated the greatest homology with GltA from Acinetobacter anitratum (65 ~). Although only 2 6 ~ aa identity was seen with the pig heart enzyme, many of the residues identified in ligand binding appear to be conserved. Sequencing studies of a region centered approx. 5.6 kb upstream from gltA revealed an ORF read with opposite polarity that encodes a peptide highly homologous to the C terminus of the flavoprotein subunit of E. coil succinate dehydrogenase. This report represents the first nt sequence analysis of a gene ofknown function from the obligate intracellular parasite, C. burnetii.
INTRODUCTION The etiologic agent of human Q fever, C. burnetii, is an obligate intracellular bacterium (reviewed in Baca and Paretsky, 1983). As a mild acidophile it replicates in the hostile, low-pH environment of the eukaryotic host-cell phagolysosome (Burton et al., 1978). In axenic media it has Correspondence to: Dr. L.P. Mallavia, Department of Microbiology, Washington State University, Pullman, WA 99164-4233(U.S.A.) Tel. (509)335-3322; Fax (509)335-3517.
Abbreviations: aa. amino acid(s); bp, base pair(s); dlTPo deoxyinosine triphosphate; ExollI, E. coliexonuclease III; GItA,citrate synthase;gltA,
been demonstrated that optimal metabolism of exogenously applied substrates occurs below pH 5 (Hackstadt and Williams, 1981a,b). Of the substrates tested, TCA cycle intermediates, glutamate and proline are preferred as carbon and energy sources (Hackstadt and Williams, 1981b; 1983; Hendrix and MaUavia, 1983). As with other members of the family Rickettsiaceae, tragene encodingGItA; IR, inverted repeat; kb, kilobase(s)or I000bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; RBS, ribosome-bindingsite(s); REP, repetitive extragenic palindrome; SSC, 0.15 M NaCl/0.015M Na3"citrate pH 7.6; SdhA, flavoprotein subunit of E. coilsuccinate dehydrogenase;sdhA,gene encoding SdhA; TCA, tricarboxylic acid; tsp, transcription start point(s).
64 facultative anaerobes also display allosteric inhibition by 2-oxoglutarate (Wright et al., 1967). Kinetic analysis of the cloned C. burnetii enzyme revealed responses to effector molecules similar to those of enzymes found in Gram + bacteria and eukaryotic cells (Heinzen and Mallavia, 1987). C. burnetii is typically considered a Gram- bacterium. A similar finding was determined for citrate synthase of Rickettsia prowazekii (Phibbs and Winkler, 1982; Wood et al., 1983), also in the family Rickettsiaceae. There is a paucity of information on gene structure and function in C. burnetii. To date only two published C. burnetii nt sequences exist: an operon under heat-shock control (Vodkin and Williams, 1988), and a plasmid-located gene encoding a membrane protein of unknown function (Minnick et al., 1990). This report represents the first analysis of an nt sequence of a gene from C. burnetii encoding a protein of known function. Furthermore, evidence is presented that gltA is closely linked to the sdhA gene encoding SdhA.
ditional genetic analysis of C. burnetii has not been possible due to a lack of axenic cultivation methods. Methods of transformation and mutant isolation are currently unavailable. With this in mind studies were initiated to analyze C. burnetii gene structure and function, and to characterize gene products, by cloning nt sequences into E. coll. The gene encoding citrate synthase (GltA; EC 4.1.3.7), an enzyme presumed critical to this agents' biosynthesis and energy production, was the first such gene cloned into E. coil and characterized (Heinzen and Mailavia, 1987). Citrate synthase has been extensively studied from several prokaryotic and eukaryotic sources (reviewed in Weitzman and Danson, 1976; Wiegand and Remington, 1986). Without exception the active enzyme has been found to be a multimer of a single 48-kDa polypeptide. Gram + and eukaryotic cells produce a 'small', dimeric protein (Weitzman and Danson, 1976). In contrast, Grambacteria construct a 'large', primarily hexameric enzyme (Tung and Duckworth, 1975). The two types of enzymes display different responses to effector molecules. In vitro, the small enzyme is isosterically inhibited by ATP (Jangaard et al., 1968), but not NADH or 2-oxoglutarate (Weitzman and Jones, 1968). A reverse effect is seen with the large enzyme where NADH is an allosteric inhibitor (Weitzman and Jones, 1968). Gram- bacteria that are
A
0
1
i
2
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i
RESULTS AND DISCUSSION
(a) Nucleotide sequence of the gltA gene The cloning, localization and orientation ofthe C. burnetii gitA gene has been previously described (Heinzen and
3
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6
i
I
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Fig. !. Subcloning and sequencing strategy of C. buraetii gitA. (A) The gltA + plasmids pLPMI0 and pLPM20 have been previously described (Heinzen and Mallavia, 1987). All regions shown are the cloned C. burnetii chromosomal inserts (size of insert in kb shown in parentheses). The E. coli host for all DNA manipulations was DH50¢(Bethesda Research Laboratories, Gaithersburg, MD). The location and direction ofgltA transcription is designated by a bold arrow. The gltA ORF was confirmed by sequencing of both strands of DNA. The location of the 3'-end of the putative sdhA gene is shown by an open arrow on pLPM 10. The orientation and length of thin arrows represent the direction and extent of the derived sequence. Determination of the nt sequence was accomplished by the dideoxy chain-termination method (Sanger et al., 19"/7) using [0¢-s2p]dCTP (3000 Ci/mmol) or [=-3SS]dATP (1000-1500 Ci/mmol)contained in Sequitid@ (New England Nuclear, Boston, MA) as the radiolabel and a Sequenase® kit (United States Biochemical Corp., Cleveland, OH) containing modified bacteriophage T7 DNA polymerase (Tabor and Richardson, 1987). Band compressions resulting from G + C-rich regions were resolved using dITP in the reactions as recommended by the supplier. Piasmid template DNA was purified by either CsCl-gradient centrifugation (Maniatis et aL, 1982)or by cetyl-trimethyl ammonium bromide (CTAB) precipitation (Dei Sal et aL, 1989). Double-stranded plasmid DNA was alkali-denatured prior to use in sequencing reactions (Chen and Seeburg, 1985) and samples were electrophoresed on 6-8% polyacrylamide gels. (B) Sequencing strategy for the gltA gene. One SstI and two HinclI restriction enzyme sites have been added for reference. A 2.8-kb XhoI + B a m H l fragment ofpLPM20 was subcloned into Xhol + Barn Hl-cut pGEM7 (Promega, Madison, WI) to generate pLPM41. Some sequencing reactions employed overlapping subclones created by the ExoIII and Sl nuclease method of Henikoff 0984) and were sequenced using a T7 promoter primer (Promega, Madison, Wl). When necessary, aynthetic oligo primers were synthesized using an Applied Biosystems model 380A DNA Synthesizer (Applied Biosystems Inc., Foster City, CA). Sequence data was compiled and analyzed using the University of Wisconsin Genetics Computer Group sequence analysis programs (Devereux et al., 1984) managed by the VADAMS ® laboratory at Washington State University. The nt sequence data for gltA will appear in the EMBL, GenBank and DDBJ nt sequence databases under accession No. M36338.
65
Mallavia, 1987). Sequencing was aided by the cloning of a 2.8-kb Xhol + BamHl ghA + fragment from pLPM20 into Xhol + BamHl-cut pGEM7, generating pLPM41 (Fig. 1, A and B). Unidirectional overlapping deletion subclones (Xhol --, BamHl) were constructed from the insert DNA of pLPM41 by the method of Henikoff (1984) and were used to determine the majority of sequences of the noncoding strand. The remaining sequence of this strand and the entire coding strand were completed using gltA-specific oligo primers (Fig. 1B). The sequence of gltA (Fig. 2) revealed an ORF from nt 1 61
&CCGACTATCG~TGTC~GGTGCGATT~GTGGTGGTTTTCAACAAGT&GCCCGT&TGC 60 &GCC.~AGCGA~T&CG~CI~AG~TGTCGTCT&TCGTC~CCGTATTCCGCTTCGCTG 120
121
C&TACGGGCT&TT"T&GeT&CTCGGCCCCCG&GGeGCCTT&GG~,¢CT&CACTTTC&AT¢ 100
181
G¢&CCG&TeGATGTG6GC~TT~J~r~TCC¢&CT*~I~TTC&T~T&C&&6¢&
240
241
CTACACCCGCCr~CACG&ATGAGCA&TCGT~GCTJ~J~£CTTTCTTTT~TC&G
300
RetSer&sn&rgLys&$aLysLeuSerPheGlu&snGln
301 TCGGTTC.,AATTTCCTATTT&TTCACCTACCCTTGGCAAGC~TGTGATTC~CGTI~J~GACA 360 SerValGluPheProZleTyrSerProThrLeuGlyLys&spVa111e&spValLyuThr
361
TTGGGCAATC&TGGTGCCT&TGCGCTGGACGTTGGTTTTT&CTCT&C&GCGGCTTGTGAA 420 LeuGly&snHlsGlyAlaTyr&laLeuAspValGlyPheTyrSerThrAlaAlaCysGlu
421
TCAAAN~TT&CCTTTATCGATGGCGAGA~GGT&TTTTGCTGTATCGGGGGT&TCCAATT
481
Se:LyslleThePhelleAspGlyGluLysGl¥IleLeuLeuTyrArgG1yTyrP¢olle
480
GATCAATTGGCTGAC~TCGG&TT&T&TGGA~GTTTGTTACTTACTG&TGTATGGCG~G $40
AspGlnLeuAlaAspLysSerAspTyrMetGluValCysTyrLeuLeuMetTyrGlyGlu
541
CTCCCGAAC~J~GG&~GG*'~,AATTTGTTCGCACGATTAAAGAAC&CACCAGCGTT 600 LeuProAsnLysGlyGluLysGluLysPheVal&~gThrlleLysGluH£$Th¢SerVal
601
TATGAGCAAGTG&C~TTTTTCA&TGGTTTTCACT&TGACGCGCATCCT&TGGCGATG 660
661
GTGCTT&GC&CC&TCGGCGCTCTTTC&GCTTTCT&TCAC~TGCGTT&GAC~TCACT&J~ 720 ValLe~SerThrlleGlyklaLeuSer&laPheTyrHisksp&laLeuAspZleThrLy8
721
¢¢GGCCG&CCGCGAkT~ATCCGCC&TCCG&TTAATAGCC.~,TGCCCACGCTGGCCGCC 780 P¢o&la&|p&¢gGluLeuSe¢&lalle&rgLeulle&laLysMetProThrLeuAlaAla
781
&TGAGTT&TAAAT&TTC~ATTGGTCAACCGTTT&TGCACCCTCGCCGGGCG&TG~TT&T 640 Ne~SerTyrLysTyrSe¢IleGlyGlnProPheHe~HisP~o&¢g&rg&laHetAlnTy¢
841
GCGGAkAACTTTTT&CAT&TGCTCTTTGGCACCCCTT&CG~AGAC'~CAGAACCTGACCCC 900 &laGluAsnPheLeuHisNe~LeuPheGlyTh~ProTy~GluGluThcGluProAspPro
901
GTCTTAGCTCGCGCG&TGG&TCGTATTTTT&TCCTTC^CGCCG&TC&CGKACA~TGCG 980 valLeu~laArg&laMet~sp&rgllePheZleLeuHlskla~spHisGluGlnAsnkla
961
TCAACGACT&CCGTTCGAGTCGCCGGTTCTACGGG&GCAAATCCGTTTGCTTGT&TTTCG 1020 SecThrThrThrValArgvalAlaGlySerThrGly&laksnProPhe^laCys%leSe~
TyrGluGlnValThrLysPhePheAsnGlyPheHisTyrAspAlaHisProMetAlaHe~
1022 GCGGGT&TTAGTGCTCTCTGGGGCCCTGCCCACGGCGGTGCTAATGAAGCTTGTTTARAT &IaGIFIleSerAlaLeuTepGlyP¢O&IaflLIGlyGIFAlaAInGIuAlaCygLeuAIn
1080
1081
1140
ATGTTGAGAAAAATTGGTGATGAGAAAAATATCGGCCAATAT&TTAAAAAAGCCAAAGAC
RetLeUA[gLFsIleGIyABpG~uLyIAInIIeGlyGInTyrlIeLyILyIAIIL¥I&IP
1141 N~ATGACCCTTTTCGTTTGATGGGCTTTGGCCATCGCGTTTACAAAN~TT&CGATCCC LyIASnABpP[oPheAEgLIuHetGlyPheGlyH£S&rgValTyeL~IABnTFeAIpPro
1200
1201 CGTGCCAA~GTCATGCA~CCTGCTATG-a-AGTCCTCGATGCCGTTGGCCGCCACAAT 1260 A~g&laLysValMetGlnLysTh¢CysTy[GluVa1LeuAsp&lavalG1¥&r~H18&sn 1261
GN~CCTTT&TTT~CTAOCGATA~AATTAGAGI~ATTGCGTT&GAGGATG&TT&TTTC 1320 GluP~oLeuPhoL¥sLouAlslleLyeLeuGluLysZle&lsLeuGluAsp&spTYrPhe
1321
ATTGAOAAAAAACTTTATCCGAATGTGGATTTTTATTCCGGTCTGACGCTAAATGCCATT
1380
lleGluLylL¥BLeuTy~P¢o&lnV~l&spPheTy~Se~GlyLeuThrLeuAlr~lalle 1381
GGC&TCCCTTCTA&TATGTTT&CGGTJ~ATTTTCGCCTT&TC&CG/~CGGTGGGCTGC~TT 1440 Gl¥IleP¢oSer&snHe~PheTh~Va111ePheAluLouSer&r~TheValGlyTrpI1e
262-1551 (1290 bp) capable of coding for a polypeptide (GItA) of 430 aa with a deduced Mr of 48 633. This figure agrees with both the value previously calculated from maxicell analysis (Heinzen and Mallavia, 1987) and the M r values described for other citrate synthase monomers (Wiegand and Remington, 1986). Beginning 10 nt t~pstream from an ATG start codon is a poly-purine-rich sequence, AGGAGCA, that is similar to the E. coli RBS consensus sequence (Gold et al., 1981). A putative RNA-polymerasebinding region, TTGAAA-N~7-TATAGT, was found spaced 19 nt proximal to the predicted RB S. The deduced -10 region (TATAGT) displays strong homology with the E. coil promoter consensus (Hawley and McClure, 1983), differing only in an A-to-G transition in the poorly conserved fifth base position. The deduced -35 region (TTGAAA) shares five out of six nt with the E. coli consensus sequence. An interesting feature upstream from gltA is a large, nearly perfect IR present at nt 46-134 (Fig. 2). Within each arm is an additional 7-nt perfect repeat. The 88-nt sequence comprising the gltA IR was searched against the entire GenBank and EMBL nt sequence databases. Remarkably, the only entry showing significant similarity was found distal to the C. burnetii htpAB operon (Vodkin and Williams, 1988) (Fig. 3). The htpAB operon is thermally regulated and produces proteins homologous to E. coli GroES (HtpA) and GroEL (HtpB) heat-shock proteins (Neidhardt and VanBogelen, 1987). The homologous IR begins 188 nt downstream from the htpB stop codon. Only minor differences were apparent between gltA and htpAB IRs. For example, the sequence intervening the left and right arms of each IR showed no similarity and is 23 and 16 nt in length in the gitA and htpAB IRs, respectively. In addition, the arms of the gltA 5' IR were slightly larger (2-8 nt). A consensus sequence was derived using the aligned left and right arms of both IRs and a synthetic oligo was synthesized corresponding to this sequence (Fig. 3). When this 5'
~
~
3'
gltA (right) AAGTAGGCCGTATGCAGCGAAGCGAAATACGGG htpAB (right) GTA GCCCGTA TGCAGCGAAGCGGA A TA CGGA gltA(IC;left) A A A TA GCCCGTA TGCAGCGA A GCGGA A TA CGGG htpAB(IC;lelt) CGT A T GCA- CGA A GCGGA A TA CGA G
1441 TC&C&TTGG&TGGN~q, TGATG&GCAGTCCCG~TCA~CGCCTCGCTCGCCCCAGACAATT& 1500
SeeHlsTrpMetGluHetHetSerSerPro&spBi~Atg~euAla&rgPro&rqGlnLeu
iS01
T&CACAGGCGARACAG&GCGAGAAGTAATCTCGTTGG&TAAACGTCKGGCATA^CAGCAG
aagTAGCCCGTATGCAFCGAAGCGg AATACGg g 1560
TFeTheGIyGluTh~GluArgGluValIleSerLeuAspLyS&r~GlnAIa 1561
AAAAGAGAT/~qJtC, AAAGAR~TCTCTGGCTGCTTGTGCCAGAGACAAGAACGATGCCTATC
1620
1621
GTTATCCAC,AAGTCACRGCGACCTCCATTTTAAACTTTGTGATCCCGCGCAGATGGAACC
1680
1681 CAGGCGCGCTTTAGCGCGCTGCGCATT&CGTGCGCTGGGTC~CCGCCTGCGGGGGCGACG, 1740 1741 ~GGGTTGGGTTTAAGTTTTTAGTGTCT&CCACTTGTGGTCGTCGCGTGGGTTGTGGCCGA 1800 1801 GATTTTTRAAGATTA.qAGTTTACGTTTATGCCCGCGCGGATCGTGTAGGCCCTGATATTC 1860 1861 TTTGCTTCCTGGGTATRAACATCTGGTGGAGCRACAACGGGATCC 1905
Fig. 2. Nucleotide sequence of the noncoding strand ofgltA. The deduced aa sequence is shown under the nt sequence. Putative -35 and -10 promoter regions and a potential RBS are overlined. A large inverted repeat proximal to the g/tA start is shown with facing arrows. A G + C-rich region (near the bottom) with multiple, small dyad symmetries representing a potential Rho-independent termination region is underlined.
Fig. 3. Comparison of the gltA 5' IR with sequences Y of the C. burnetii htpAB operon (Vodkin and Williams, 1988). The IR sequences have been aligned to illustrate their sequence similarity. The sequence of the IC of the left arm of both IRs is aligned with the right arm. Arrows indicate the position of a smaller 7-bp IR within the 1~:~and right arms of the elt.t 5' IR. A dash represents an absence of an nt. The consensty~, seau~:,ce (CONS) is depicted below the alignment with upper-case let:,l's representing positions in the alignment showing perfect homology and lowercase letters representing positions in the alignment showing less than perfect homology or positions with nt not present in all four aligned sequences. The underlined region of the consensus sequence corresponds to the sequence ofthe synthetic oligo synthesized for use in the Southern analysis described in Fig. 4.
66 oligo was used as a hybridization probe it revealed the IR sequence to be present in multiple copies within the rickettsial genome (Fig. 4). At least six bands of hybridization were observed when EcoRl-digested genomic DNAs from four C. bumetii strains (Mallavia, 1991) were probed. The greater intensity of hybridization signals observed with some EcoRl fragments may indicate the presence of more ~han one copy of the IR. A strong signal was also observed from pLPM30 insert DNA containing the cloned C. burnetii gltA gene (Heinzen and Mallavia, 1987) as expected. The function of the C. burnetii repetitive IRs is open to speculation. Perhaps they represent a class of elemeats analogous to REPs found in E. coli (l~r~gins et ai., 1981; Stern et al., 1984; Newbury et al., 1987). These IRs are remarkably homologous a~d are present in nontranslated regions of 25% of all E. coE ~:'~p.scription units. REPs have been shown to enhance g~:ne expression by stabilizing upstream mRNA via slowing the attack of 3' --, 5' exonuclease (Newbury et al., 1987). Transcription of the gltA 5' IR could result in a very stable (stabilization free energy; zig = - 54.9 kcal/mol) RNA stem-loop structure. Higgins etal. (1981; 1988) have suggested that REPs may have
1
2
3
4
5
6
!
Fig. 4. Southern hybridization analysis employing an oligo probe corresponding to the C. burnetii extragenic IR consensus sequence (Fig. 3). Lanes 1-4 show EcoRl-digested (7. burnetii genomic DNA from Hamilton, Biotzere, Corazon, and Dod strains, respectively (Mallavia, 1991). Lane 5 is HindIlI.digested ~ DNA Mr markers, and lane 6 is EcoRI + Pstl.digested pLPM30 (Heinzen and Mallavia, 1987). The DNA was resolved using a 0.8?/o agarose gel. Transfer to nitrocellulose was done by the method of Southern (1975). Hybridization was to the oligo primer 5'-TAGCCCGTATGCAGCGAAGCG (synthesized as described in Fig. 3) that was end-labeled using T4 kinase (Bethesda Research Laboratories, Gaithersburg, MD) and [~,-32P]CTP (NEN). Washes were carried out in 6 x SSC at 60°C.
arisen from insertion-like elements. However, REPs in E. coli lack short direct repeats at both ends that are trademarks of insertion sequences. The C. burnetii gltA 5' IR similarly lacked such repeats. The lack of distinctive polymorphism in the hybridization patterns of chromosomal DNAs obtained from different C. burnetii strains also argues against a transposable element. The location of the gltA 5' IR may indicate the presence of a hitherto unknown transcriptional unit in the region immediately upstream from, and reading toward, gltA. Few sequence data are currently available for this region (Fig. 2). The 3'-end of the gltA gene is determined by a TAA stop codon at nt 1552, followed by a 56-bp G + C-rich area (nt 1694-1749) of multiple-dyad symmetry. This region, when transcribed, could form a multiple hairpin structure (stabilization free energy; ,4(; = -24 kcal/mol) that might act as a Rho-independent terminator. This sequence is 79?/0 G + C as compared to 44% for the gltA ORF. Band compressions were encountered during sequencing of this region on both strands of DNA that were resolvable only upon incorporation of dITP in the sequencing reactions.
(b) Amino acid sequence homology of GItA A multiple sequence alignment was prepared to compare the deduced aa sequence of C. burnetii GItA with that offive bacterial enzymes and one representative eukaryotic enzyme (Fig. 5). The aa sequence of both E. coli (Bhayama and Duckworth, 1984) and pig heart (Bloxham et al., 1982) GltAs have been determined directly by chemical means while those of Acinetobacter anltratum (Donald and Duckworth, 1987), Pseudomonas aeruginosa (Donald et al., 1989) and Rickettsia prowazekii (Wood et al., 1987) have been deduced from the nt sequence. The pig heart enzyme has been studied further using x-ray crystallography and residues involved in ligand binding have been identified (Remington et al., 1982). When the deduced aa sequence of C. burnetii GltA is optimally aligned with the pig heart sequence only 26% homology (using perfect aa identity) was seen. The greatest divergence occurred toward the N terminus where ligand-binding residues are sparse. However, the C. burnetil sequence displays identical or functionally similar residues at ten of eleven positions identified as involved in substrate binding. The one remaining active site residu~ of pig heart, Arg46, has no obvious homolog in the C. burnetii enzyme. Interestingly enough, when C. burnetii GltA is optimally aligned with the enzymes from R. prowazekii, P. aeruginosa, E. coli and A. anitratum the levels of identity were 56 ~o, 61%, 61% and 64%, respectively. The fact that C. burnetii produces a citrate synthase more homologous to that of bacteria residing in the families Pseudomonadaceae, Enterobacteriaceae and Neisseriaceae raises some interesting questions regarding the evolutionary origin of this agent.
67 1 FNGLTA ~LTA FA|GLTA |COGLTA RFRGLTA CSUGLTA CONJ
AS|IS( LKDZLADLIP U ~ N t I E T I ~ SIUkTGNNA V L E L N . E | I|LPZYSGTL A . O N | AQLXXNGSJUF VELlq~SGTN A.DTN AItLTLNGDTA VllSDVLKGTL TNGNNNNL|~ABLKXMG.KL FKLFZLKASZ SN.RK AitLS~IIN.Q8 V|FPZYSPTL -oIp .... ~..... I---k i-l---g---
Sl
O011~..VOOITVO.gN. G S O ~ D V E I ~ IkI~qG.H~TFD G~DI/VDVMOL TATG.nrT~O G~DYZDZRTL GSKG¥.FTI~D GKDVXOXSRV SAgADY~TYO OEOVXOVI|TL ONBGAYA.LD g-dvtdv ...... g......
*
PHGLTA YGGNItGNKGL VYNTSVLD~D EGXRr.MGY| JULNGLTA FGFI~IkTJ~SCK SKZTF.IDGD KGXLLH|GY~ PAEGLTA PGFMSTASC~ SKXTY.Z~D EGVLLHRGYP ECOGLTA POFTSTASCB S K X T F . Z ~ D EGZLLHMGrP RPROLTA PGFNSTJ~SCQ STZTY.ZDGD EGZLMYRGYD CBUOLTA VGFYSTJUkCE SKXTF.XDG~ KGZLLYRGYP CON8 p G f n s ~ a s c e s k i T - - L D g d kG£11-gGyp
ZPBCQKRL~K AKGG|EPLFE EDQLATQA . . . . . . . . DYLE ZEQLABKS . . . . . . . . DYLE I~LATDN . . . . . . . . NYLE ZKDLA~KN. . . . . . . . 9FLE IDQLADKN . . . . . . . . DYnE g-qlm--s ........ dylE
PHOLTA AJ~qGLTA PAEOLTA ECOGLTA RPMGLTA CBUGLTA C0N8
101 GLFWLLVTG~ IPTEEQVSWL TCYLLL.NGB LPTAEQKV|F TCYLLL.NGB LPTJUkQKEQF VCY~LL.NGB KPTQEQYDB~ VAY.LNIYGE LPSSDQ~CN~ VCYLLN.YOE LPNKGEKEK~ -Cy-GI--GO lP~--q---f
PHGLTA JU~HGLTA FAEGLTA ECOGLTA RPRGLTA CBUGLTA CONS
151 QLSJUkXT.AL NSESNFARAY A|GZN;TKYW SLZYNDCRDL INVGW.GAL SAFYNNNLDI EDXHHBEZTA . . . . . . . ZRL HNL VNCOVX.GAL SAFYHDSLD| TNPKHBQVSA . . . . . . . FRL VNCO.~TGAL JUkFYHDSLDV HNPSNMEZJUk . . . . . . . Zg ZNLAAV.GSL SAFYPDLLNr .NETDY|LTA . . . . . . . ZRL .NVLSTXOAL SArYHDALDI TKPADR|L8A . . . . . . . cl - S - - ~ L - g a ~ safyhd-ld- . . . . h r e - - 8 . . . . . . . .
SKEWAKPJUt~ .DAEVRAHTlt V.GTZKmlTN .KTTVTRHTM TK.KVJUIHSL Vlq.TXKENTS ....... h~-
PS~LON .rP1~qLneNS VH~VSRFrN OFRRDAHPNA VHEQLKTFFN GFRBDAHPIqA XH|QXTRLFO AFMMDSHPFL~ VN~MLBYLFQ TFCSSSHPH~ VYEQVTE~N GFHYDAHPRA v-eq .... fn -F--d-HPN8
201
PHGLTA IYRNLYREGS SZOAZDSKLD WSHNFTHNLG AAHOLTA SYKYTV..OQ PrIYPRNDLN YAENrLHHRr PAEGLTA V~g¥SK..GE PHMYPRHO~ YA|NrLHHMf ECOOLTA CYKYSZ..GQ PFVYPRNDL8 YAGNrLNHMF RPROLTA SYKYSZ..GQ PFXYPDNSLD FT|NrLHRRF CBUOLTA SYKYSZ..GQ PFNNPRRAHN YAENrLNNLF CONS - Y k y s - - - G q p f - y p c n - l PHGLTA AJUqOLTA FAEOLTA ECOOLTA BPRGLTA CNUGLTA CONS PHGLTA JUUqGLTA PAEGLTA ECOGLTA RPROLTA CSUOLTA CONS
251 * YLTZHSDHEG ONVSAHTSHL ZFTLHADHE. QNASTSTVRL ZFILHADHE. QNASTSTVRL ILZLHAOHE. QNASTSTVRT IFXLHADHE. QNASTSTVRZ ZFZLHADHB. QNASTTTVRV Lf~INaDHK- q N s S t s T v c -
IAKLPCVAAg IAKXPTLAAN ZASNPTIAAN LSRNPXHJu~q XAKXPTIAAH XAKNFTLAAN LaS-P~-AAs
YTDAQFT|L . . . . . . . . MRL ATFJUDRDYKV NPVLARAHDR NT~CE.TKPZ SPVLAKJuqDg STFCE.PYEV NPZLERANOR ATPCT.KYK¥ NPZXKNALNK GTPYE.ET|P OPVLARNqOg -Tp. ado
SrAJUU~GLA GFL;GLANQE CZSAGZSALH GFAHGGANEA CZASGZAALH GPANGGANEA CIJUkGZASLW GPAHGGANEA CXSTGZASLW GPAHGGAHEA CXSAGZSAL~ GPAHGGAHEA aOS-glnPfa c L - a g ~ - - L w OPSHGqANea
VGSALSDPYL AGSTGANPYA AGSSGANPFA AOSSGANFrA AGSSGAHPFA AGSTGANFrA
]01 VLVWr,TQLQK EVGKDVSOKK LRDYXNNTLN VLKNLDEZGS VENVAEFNER VKRK. . . . . . VLRHLOEZGD VSNXDKI'VEK AgOg . . . . . N ALKRLEEZSS VKHXPKrFRR AgOg . . . . . N VZNRLKBXGS SENZPKYVAK AKDK. . . . . N CLNNLMnXGD EKNX~YXKK AKDK. . . . . N v l o m L - e t g - - - n t . . . . . k akdk . . . . . .
8GRV~TPGYGI! AVL STOP Y I~.VKLNGIFGH RVYKNFDPRA DIPlPKLNGLPGB RVYKNFDFRA OSrMLHGFGH BV~FKNYDPRA DIPFRLRGFGH RVlrKSYOFRA DPFRLNGFGH RVYKNYDPRA d - f-18GfOH cVykn-OPga
351 PHOLTA TCQREF...A L K H L r . . H . D KVNKQTCDEV LEALG.I.ND PAEGLTA KVMKQTCDEV LQELO.Z.ND ECOGLTA TVHRBTCHEV LKELOT.KDD
PNFKLVAOLY KZVPNVLLEQ GKA;NPWPNV PQLALAMELE RZALNDPYFV EB.K.LYPNV PQLELAHKLE |IARHOPYFV E..RNLYPNV .GLEVAHELE NXALNOPYFZ EK.K.LYPNV RPROLTA AVLKETCKEV LNELGQLDNN PLLQZAXELB ALALKDEYFI EB.K.LYPNV CBUGLTA KVNQKTCYEV LDAVOR.HHE PLFKLAZRLE.KXALEDDYFZ ER.R.LYPNV -£al-d-yfe--k-lyPNV CONS - v m - - t - - - v L--1 ..... d --l-la--Le
AANGLTA
.401 PHGLTA DAHSGVLLQY YG~TENNYYT A~IGLTA OFYSGIILKA ZGZPTEN.FT PAEGLTA ~FYSGIXLKA ZG~PTSN.FT
ECOGLTA
DF¥SGZXLKANGXPSSN.FT
v.ovs~Lo wAo~:.s~ Lo,.,L~B,g V:rA~n~o . , s . . L I N . S o . ,,~g,g vzrA~.~O . : s . . Q z . . ~,~Kz.,g vzr~o
.,A,,SZN,s DON.~,*g,.
oerQ.,s.,g DFYSGXZYKA NGZPSQN.FT VLrA,A,,V, . M . n z . . , DFYSOLTLNA IGXPSNN.FT VIFALSRTVG WZSHHIqENNS SPDHRLARPR CONS O f y s G t i l k 8 - O i p - - m - f T V £ F a - e R t v G w i - h w - e m - s - p - - k i - g P [
RPRGLTA
CBUGLTA
PHGLTA
AANOLTA PAEGLTA
4S1 SNSTDGLZKL Y . . . D S K QLYTGEVQRD ZKR
0LYTGHTQRD FTALKDRG
ECOOLTA QLYZGYEKRD FKS.DZKR RPRGLTA QLYTGYVHRS YKC][VERK CBUGLTA QLYTGSTERS VXSLD.KRQA
CONS
ql¥tg---r-
Fig. 5. Multipleaa sequence alignmentof selected citrate synthases with C. burnetiiGltA. Gaps have been introducedto maximizehomology.The sequences from top to bottom are derived from pig heart (PHGLTA; Bioxham etal., 1982), A.a,itratum (AANGLTA; Donald and Duckworth, 1987), P. aeruginosa (PAEGLTA; Donald et al., 1989), E. coli (ECOGLTA; Bhayama and Duckworth, 1984), R.prowazekii (RPRGLTA; Wood et al., 1987) and C. burnetii(CBUGLTA). Met start codons are not included. The consensus aa sequence is designated CONS; upper-case letters indicatingresidues shared by every sequence a,d lower-caseletters indicatingresiduessharedby fouror five sequences in the alignment.Residues implicated in iigand binding of the pig heart enzyme (Remingtonet al., 1982) are marked by asterisks. Numbering indicates position in alignment.
Indeed, this relationship is in keeping with the results of a recent study by Weisberg etal. (1989) that demonstrated using 16S rRNA sequence homologies that C. burnetii is more related to members of the ~, subdivision of the purple bacteria that includes the families Enterobacteriaceae and Pseudomonadaceae. Other members of the Rickettsiaceae are placed within the ~ subdivision. (c) Linkage with sdh The proximity to gltA in E. coli of TCA cycle genes encoding succinate dehydrogenase complex (sdhCDAB), 2-oxoglutarate dehydrogenase complex (sucAB), and succinyl-CoA synthetase (sucCD) (Bachmann, 1987) prompted a search of both distal and proximal regions of C. burnetii gltA to determine Whether similar linkages are present. Nucleotide sequence information flanking gltA was derived from regions centered approx. 5.6 kb upstream using pLPM10, 3.0 kb upstream and 0.5 kb downstream using pLPM20, and 1.0 and 0.5kb upstream using pLPM41 (Fig. 1). The six possible reading frames for each region were translated and predicted peptides of significant size were searched for homologous sequences in the database. In addition, these peptides were compared individually with the predicted aa sequences of the protein subunits comprising the three respective protein complexes produced by sdhCDAB and sucABCD operons. A peptide of 90 aa derived from a region centered 5.6 kb proximal to thegltA ORF on clone pLPMI0 (Fig. 1A) and transcribed with opposite polarity displayed significant homology (60~o aa identity) to the C-terminal 92 aa of the SdhA (Wood et al., 1984) (Fig. 6). The database search revealed additional homologies to the flavoprotein subunits of E. coli fumarate reductase (Cole, 1982) and Bacillus subtills succinate dehydrogenase (Phillips et al., 1987) of 41 and 27% aa identity, respectively (data not shown). The C. burnetii SdhA peptide coding region terminates with tandem stop codons situated 8 nt from the left EcoRI site of insert D N A of pLPMI0 (Fig. 1)(data not shown). (d) Conclusions (1) A 1290-bp ORF has been identified as coding for C. burnetii GltA with a deduced Mr of 48 633. Consensus E. coli transcriptional and translational initiation signals precede gitA. Following the ORF is a G + C-rich region CBU ECO CBU ECO
AKLTDTSRTFNNARIEALELDNLNEVSYATAVSAQQRTESRGAHSRYDYKERDD I:1 I l l II I:1 I l l l l l i l IIIIlli Illllfllil:l: :Ill ARLDDTSSEFNTQRVECLELDNLMETAYATAVSANFRTESRGAHSRFDFPDRDD
SS0
ANHLKHTVYFRDGH.IAYRPVNHKPKGHDPFPPKSRD ill I :l: :: : I I l l II :llll I ENHLCHSLYLPESESNTRP~TNNEPKLRPAFPPKIRTY589
Fig. 6. Comparisonofthe deduced aa sequenceofa peptide encoded by an ORF centered approx.5.6 kb upstreamfromthe C. burnetiiglt,4translational start with the C-terminalportionof SdhA orE. coli(Wood et ai., 1984). Numberingindicates the appropriate residues of E. coli SdhA. Identical and functionallyequivalent residues are signified by lines and colons, respectively. Abbreviations: CBU, C. burnetii;ECO, E. coll.
68
with properties of a Rho-independent termination site. A large region of dyad symmetry centered 172 bp upstream from the gltA start codon is found in multiple copies throughout the C. burnetii genome and may mediate some unspecified regulatory function. (2) Comparison of the deduced aa sequence of C. burnetii GltA with other citrate synthase sequences shows that the enzyme is more closely related to the 'large' type enzyme produced by free-living Gram- bacteria. This contrasts with data that previously demonstrated the enzyme to have regulatory properties typical of the 'small' enzyme associated with Gram + bacteria and eukaryotic cells. Of the bacterial enzymes, the one produced by R. prowazekii is the least similar despite exhibiting similar responses to enzyme modulators. Although the pig heart and C. burnetii GltA proteins are similar in their responses to effectors, they have only 26 % aa identity. Nonetheless, there is conservation of identical or functionally similar residues at most sites identiqed in pig heart GltA ligand binding. (3) The identification of an ORF centered approx. 5.6 kb upstream from gltA that encodes a peptide with a high degree of homology to SdhA has led us to the conclusion that the gene encoding the C. burnetii homolog of this protein, and indeed, possibly the entire sdh operon, is linked to gltA in a similar fashion as that demonstrated in E. coll. If C. burnetii has an operon coding for a 4-subunit Sdh complex analogous to that described in E. coli with the genes for two hydrophobic subunits (sdhC and sdhD) preceding sdhA, the calculated intergenic region between the translational starts ofgltA and sdh would be approx. 3 kb. This is significantly larger than the approx. 700-bp spacing in E. coli and would presumably preclude a shared gltA-sdh regulatory region as demonstrated for this bacterium (Wilde and Guest, 1986). We are currently investigating regions flanking this peptide by sequencing and complementation of sdh-deficient E. coli mutants to confirm the presence of a gltA-linked sdh operon in C. burnetii.
ACKNOWLEDGEMENTS
We express appreciation to Shirley Schmitt for her excellent technical assistance. This work was supported by grant A120190 from the National Institutes of Health (NIAID), by the Northwest College and University Association for Science (University of Washington) under contract DE-AM06-76-RL02225 with the US Department of Energy and by Battelle, Pacific Northwest Laboratories.
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