Vol. 173, No. 1
Cloning, Sequencing, and Expression of the Gene Encoding the High-Molecular-Weight Cytochrome c from Desulfovibrio
vulgaris Hildenborough
W. BRENT R. POLLOCK,1 MOHAMED LOUTFI,2MIREILLEBRUSCHI,2 BARBARA J.RAPP-GILES,3 JUDY D. WALL,3ANDGERRIT VOORDOUW1*
DivisionofBiochemistry, Department of Biological Sciences, The University of Calgary, Calgary, Alberta, Canada T2NIN41;Laboratoire de ChimieBacterienne, Centre National de la Recherche Scientifique, 13277 Marseille Cedex9, France2; and Departmcnt of Biochemistry, University of Missouri, Columbia, Missouri3
Received26July 1990/Accepted 16 October 1990
Byusing asyntheticdeoxyoligonucleotide probe designedtorecognizethe structuralgeneforcytochromecc3
from Desulfovibrio vulgarisHildenborough,a3.7-kbXhol genomicDNAfragment containingthecc3gene was
isolated. Thegeneencodesa precursorpolypeptideof 58.9kDa,withanNH2-terminal signalsequenceof31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c(Hmc) isolated from thesamestrain by Higuchi et al. (Y. Higuchi, K.
Inaka, N. Yasuoka, and T. Yagi, Biochim. Biophys. Acta 911:341-348, 1987). Since the data indicatethat cytochromecc3and Hmcarethesameprotein,thegenehas been named hmc. The Hmcpolypeptidecontains 31histidinyl residues, 16 of whichareintegraltohemebindingsites.Thus, only 15 ofthe 16 hemescanhave bis-histidinyl coordination.Acomparisonof the arrangement of hemebindingsites andcoordinated histidines inthe amino acidsequencesofcytochromec3andHmc from D. vulgarisHildenboroughsuggests that the latter contains three cytochrome c3-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-rangevectorpJRD215andsubsequentconjugational transfer of the recombinantplasmidintoD.
desulfuricans G200ledtoexpressionofa periplasmicHmcgeneproductwithcovalentlybound hemes.
c-type cytochromes are distinct from other classes of cytochromeinthattheyarecovalentlyboundtotheirhemes byac-thioetherbonds (7).These bonds arise from the Markov- nikov addition of sulfhydrylgroupstothevinylgroupsof the heme. The sulfhydryl groups are derived from the two cysteinyl residues present in the heme binding sequence
C-X-X-C-H. Thehistidinylresidue in thissequenceoccupies the fifth coordination position of the heme Fe atom, while the sixth coordination position is occupied by either a
histidinyl or a methionyl residue. The nature of the ligand occupying this position has a direct effect on the redox potentialof the heme (21).
Desulfovibrio vulgaris contains at least four different c-type cytochromes: cytochrome C553 (23, 32), cytochrome
C3 (14, 35), cytochromeCC3 (18), andhigh-molecular-weight cytochrome c (Hmc) (13, 38). Cytochrome C553 is the only Desulfovibrio cytochrome reportedtobeamonoheme andto have aHis-Met-coordinated heme Featom; the other three cytochromes have more than one heme, all ofwhich are
thoughttohaveHis-His coordination. Complete amino acid
sequence dataand three-dimensional structure information
arerequiredtoascertaintheexact number and organization of the c-type hemes in each of these cytochromes. A determination of the cellular localization of these cy-
tochromes is also important, since Desulfovibrio spp. con-
tain both a periplasmic and a cytoplasmic chain of redox carriers (16, 27). This information can be obtained by se-
quencing the structural genes for these cytochromes. At present, only theD.vulgaris subsp. vulgaris Hildenborough (hereafter referred to as D. vulgaris Hildenborough) cy-
* Corresponding author.
tochromes C553 (32)and C3 (35) structuralgenes have been sequenced. For the two remaining cytochromes, partial amino acid sequence dataare available forcytochromeCC3 (18), and theisolationandamino acidcompositionfor Hmc have beenreported (13). Thiswork hasnowbeenextended toD. vulgarisHildenborough cytochrome CC3, thegenefor which has been clonedandsequenced.Perhaps surprisingly, it appears that cytochrome CC3 and Hmc are the same
protein,andthereforethis workcompletesthecharacteriza- tion ofgenesfor known periplasmic c-typecytochromes in D. vulgaris.
MATERIALS AND METHODS
Strains, vectors, and media. The bacterial strains and plasmidsusedaredescribedinTable 1.Desulfovibrio strains
wereculturedfor18 hat37°Cinstopperedbottles containing 100 ml of Postgate C medium (27) made anaerobic by flushing with N2. The inoculum consisted of 3 ml of a
PostgateB (27)culture thathadbeengrownunderthesame
conditions. Growth ofEscherichia coliTG2 in TY medium
was carriedout aspreviously described (26). E. coli DH5ao and Desulfovibrio desulfuricans G200, used for conjuga- tional transferofbroad-host-range plasmids, werecultured
asdescribed elsewhere(35a).
Biochemical reagents. All enzymes were obtained from eitherPharmacia, Inc., orBoehringer Mannheim Biochem- icals. Theradiochemicals [a-35S]dATP(400 Ci mmol-1; 10 mCi ml-1), [k-32P]dATP (3,000 Ci mmol-P; 10mCi ml-'), and [y-32P]dATP (3,000 Ci mmol-'; 10 mCi ml-') were
purchasedfrom AmershamCorp.andwereused fordideoxy- nucleotide sequencing, nicktranslation,and 5' endlabeling, respectively. Ficoll 400 was purchased from Pharmacia.
220 JOURNALOFBACTERIOLOGY, Jan. 1991, p.220-228
0021-9193/91/010220-09$02.00/0
Copyright ©D1991, AmericanSociety for Microbiology
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TABLE 1. Bacterialstrains and DNAvectors
Strain orvector Genotype,comments, and reference
D. vulgaris Hildenborough NCIMB 8303;isolatedfrom claysoilnearHildenborough, Kent, United Kingdom (27); source of the hmc gene, encodinghigh-molecular-weightcytochrome c
D.desulfuricans G200 SpontaneousNalr mutantofD. desulfuricansG100A (37)
E.coli TG2a A(lac-pro) supEthihsdMhsdR recA(F' traD36proAB+ lacZAM15P);from T. J. Gibson E.coliDH5ot +80dlacZAM15 endAI recAl hsdRJ7(rK- MK+)supE44 thi-J X- gyrA96 relA1[F'A(lacZA-
argF)U169] (12)
pUC8 33
pP6A Contains the hmcgeneofD. vulgaris Hildenboroughon a3.7-kbXhoI insert inpUC8; thisstudy pJRD215 IncQgroup,broad-host-range cloning vector; Kmr Smr (5)
pBPHMC-1 Containsthe D. vulgaris Hildenborough hmcgene onthe2.2-kbSmaIfragmentofpP6A in pJRD215; this study.
a Constructed from E. coli JM101 byT.J. Gibson and M. D.Biggin at the Laboratory of MolecularBiology, MRCCentre, Cambridge,UnitedKingdom.
Polyvinylpyrrolidone (molecularweight, 40,000), bovine se- rum albumin (fraction V), molecular biology-grade sodium dodecylsulfate(SDS),and salmon sperm DNA(sodiumsalt) were purchased from Sigma Chemical Co. Low- and high- gelling-temperature (LGT and HGT) agarose were obtained from Bethesda Research Laboratories, Inc. Nitrocellulose and Hybond-N hybridization transfer membranes were ac- quired from Schleicher & Schuell, Inc., and Amersham, respectively. Acrylamide andN,N'-methylenebisacrylamide werepurchased from BDH Ltd. Low-molecular-weightpro- teinelectrophoresis markers wereobtainedfromPharmacia.
Dialysis tubing (molecular size cutoff, 6 to 8 kDa) was obtained from Spectrum Medical Industries, Inc. All other reagent-grade chemicalswereobtained from either Sigma or FisherScientific Co.
Purification and peptide sequencing of cytochrome CC3.
Cytochrome CC3 was purified from French-pressed D. vul- garisHildenboroughcellsessentiallyasdescribed by Loutfi etal. (18),whoreferred to thisprotein ascytochromeC3(Mr, 26,000). Inaddition, a newpurification procedure was used (not shown) in which this cytochrome was isolated from the periplasmic fractionof D. vulgaris Hildenborough prepared as described elsewhere(31). The yields of both procedures were approximately 1 mg/100 g of wetcells. These isolated proteins were pure as judged by SDS-polyacrylamide gel electrophoresis (PAGE) (not shown), which indicated mo- lecular sizes of 62 and 56 kDa for the holoprotein and apoprotein, respectively.The 553of thereducedholoprotein wasfoundto be 424mM-'cm-', a value similar to that of Higuchietal. (13), who showed the presence of 16.3 hemes perpolypeptide.
The carboxymethylated apoprotein was prepared as pre- viously described (18) and treated variously with CNBr, Asp-N endopeptidase, Arg-C endopeptidase, or Glu-C en- dopeptidase. Automated amino acid sequencing (18) of iso- latedpeptides yielded47peptides which could be organized into12polypeptidesasindicated in thelegend to Fig. 2. The
l
phmc
H H K
hmc
stretches of Hmc amino acid sequence covered by these peptide groups areindicated inFig. 2.
Gene cloning. DNA manipulations were carried out as described by Maniatis et al. (19). Samples (10 ,ug) of D.
vulgaris Hildenborough genomic DNA were digested with various restriction endonucleases, electrophoresed on a 0.7%(wt/vol) HGTagarosegelasdescribedelsewhere(36), and blotted onto anitrocellulose membrane.Followingpre- hybridizationfor 15 min at68°C in0.2% SDS-10x Denhardt solution (19)and 6x SSC (19), the blot was incubated with the5'-end-labeledprobeP43for16h at68°Cin 6xSSC.The sequence ofthe deoxyoligonucleotide probe P43, a47-mer,
was5'-AAGATCGA(AG)AAGCC(CG)GC(CG)AACAC(CG) GC(CG)TGCGT(CG)GACTGCCACAAGGA. This probe wasdesignedtorecognizethe COOH-terminal region of the structuralgeneencodingthesequenceK-I-E-K-P-A-N-T-A- C-V-D-C-H-K-E. The bias toward G or C in the third position ofeachcodonwaschosen on thebasis oftheknown codon usageinthe nucleotidesequencesofthe hydA,B (34) and cyc (35) genes fromD. vulgaris Hildenborough. After incubation, the blot was washedthricewith 2x SSC,dried, and autoradiographed. Autoradiography (not shown) re- vealed a3.7-kbXhoIfragment ofchromosomal DNA to be the one most suitableforcloning.Thiswasaccomplished by sizefractionating chromosomalDNAdigested withXhoI on 1% (wt/vol) LGT agarose and ligating the 3- to 4.5-kb fractioninto the vectorpUC8, which hadbeendigestedwith calf alkaline phosphatase and the restriction endonuclease Sall.E.coliTG2cells,made competentbyCaCl2treatment, were transformedwith theligation mixtureandspreadonto TYplates containingisopropylindolyl-,-D-thiogalactopyran- oside (IPTG),5-bromo-4-chloro-3-indolyl-,3-D-galactopyran- oside (X-Gal), and ampicillin. Southern blots ofplasmids isolatedfrom white recombinant colonies anddigestedwith bothEcoRI andHindlIl wereprobedwithP43asdescribed above. A restriction map of the 3.7-kb insert of the only positiveplasmid obtained (pP6A) is shown inFig. 1.
1
L I
500bp
FIG. 1. Restrictionmapofthe 3.7-kbXhoI fragmentof D. vulgarisHildenborough chromosomal DNAcontainingthe hmc gene(boxed region). The restriction sites forXhoI (X), HindlIl (H),KpnI (K),and SmaI (S) are noted.The position ofan E. coli-like u70promoter sequenceis shown(Phmc); the arrowindicates thedirectionof transcription.
s x
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222 POLLOCK ET AL.
Shotgun nucleotide sequencing. Theprocedure followed for shotgun nucleotide sequencing was essentially that outlined by Bankier and Barrell (1). Plasmid pP6A (1 pRg RI-') was sonicated,and theresulting fragmentswereend repaired and size fractionated as described above. The 0.3- to 0.6-kb fraction was isolated from the gel and ligated into the replicative form of M13mp8 digested with SmaI and calf alkaline phosphatase. Competent E. coli TG2 cells were transfected with the ligation mixture and spread onto TY plates with top agar containing IPTG and X-Gal. Single- stranded DNA was isolated from white recombinant phage plaques by using a 1.5-ml minipreparation procedure (1).
Purifiedphage DNAs (2Rd)weredot blottedon aHybond-N hybridization transfer membrane and fixed by exposure to UV light. Following digestion with EcoRI and PstI, the 3.7-kb insert ofplasmid pP6A was isolated by 1% (wt/vol) LGTagarose gelelectrophoresis, radiolabeledby nicktrans- lation (19), and denatured by boiling. The filter was prehy- bridized in 6x SSC-lOx Denhardt solution-0.5% (wt/vol) SDS for 1h at68°C. The3.7-kbDNAprobewasthenadded to the prehybridization solution, and hybridization was continued for16 h at68°C.Afterhybridization,thefilterwas washed twice in 2x SSC for 1 min at room temperature, followed by a 1-h wash at 68°C in 6x SSC-0.5% (wt/vol) SDS. The dried filter was autoradiographed, and positive clonesweresequenced bythemethod ofSangeretal.(28)as detailed by Bankier and Barrell (1). The sequencing data were processed and analyzed by using the programs of Staden and McLachlan (29, 30).
Broad-host-rangeplasmid construction and bacterialconju- gation. Plasmid pP6A was digested with SmaI and size fractionated as described above. The 2.2-kb fragment was isolated from the gel and ligated to the vector pJRD215, which had been digested with Stul and calf alkaline phos- phatase. E. coli TG2 was transformed with the ligation mixture and spread onto TY-kanamycin plates. Recombi- nant plasmids were purified and analyzed by restriction digestionwithBamHI,resultinginexcision ofthehmc gene as a2.6-kbBamHIfragment (not shown). The recombinant plasmidand the vector alonewere eachtransformed intoE.
coliDH5aandsubsequentlytransferredtoD. desulfuricans G200asdescribed elsewhere (35a).
Total (chromosomal and plasmid) DNA of the recombi- nant exconjugant was prepared as described by Marmur (20),digested withBamHI, andsubjected toSouthern anal- ysis (not shown). The 2.2-kb SmaI fragment derived from plasmid pP6Awas radiolabeledby nick translationand used as theprobe to verify the presence ofplasmid pBPHMC-1 within the recombinant exconjugant D. desulfuricans G200 (pBPHMC-1).
SDS-PAGE andfluorophotography.Desulfovibrio periplas- mic proteins were isolated essentially as described by van der Westen et al. (31). Cultures of D. desulfuricans G200(pJRD215),D.desulfuricansG200(pBPHMC-1),andD.
vulgaris Hildenborough grown in 100 ml of Postgate C mediumwereharvestedby centrifugation at 10,000 x g for 10min at4°C. Each pellet was resuspended in 2 ml ofH20 and transferred to three separate 15-ml glass centrifuge tubes, to each of which was added 2 ml of 0.1 M Tris hydrochloride-0.1 M EDTA (pH 9.0). These suspensions wereheatedto30°C ina60°Cwaterbathand then shakenfor 15 min at room temperature, after which they were centri- fuged for 10 min at 10,000 x g. The supernatant, which contained the periplasmic proteins, was dialyzed for 20 h againsttwo 1,000-mlvolumes ofcold deionized H20.
For SDS-PAGE, 1-ml aliquots of each dialysate were
placed into microfuge tubes and dried by vacuum centrifu- gation, resuspended in 100 IL of H20, 100 p,l of SDS incubationbuffer(26), and 20 ,ulof2-mercaptoethanol, and placed in aboiling waterbathfor 10 min. SDS-PAGEand fluorophotographywerecarriedout as previouslydescribed (26) exceptthat the acrylamidegradient was 10to 15% and the slabgelwasphotographed byusingPolaroidtype55film andan exposuretime of 10 min.
Nucleotide sequence accessionnumber. The sequence data presentedinthispaperhavebeen submittedtoGenBank and assigned accessionnumber M34607.
RESULTS
Cloning. Deoxyoligonucleotide P43, which was designed
to recognize the COOH-terminal sequence ofcytochrome CC3, was used to screen purified recombinantplasmids for the presence ofthis gene. Screeningofapproximately 140 plasmids yielded onepositive plasmid, pP6A. Arestriction map forthe 3.7-kb XhoIinsert ofpP6Ais shown inFig. 1.
hmccoding region. The sequence ofthe first 2,160nucle- otides(nt)ofthepP6Ainsertisshown in Fig. 2.Theregion containingthe structural gene for thecytochrome (nt472to
2106) wasobtainedon bothstrands and has beentranslated into protein. The NH2-terminal sequence found for cy- tochrome CC3 (18) by protein sequencing (peptide P1:
KALP...)ispresentat nt565. Itis clearfrom the nucleotide sequence (Fig. 2) that this sequence is preceded by an
NH2-terminalsignal sequenceof31 residueswhich initiates at nt 472. The gene-translatedprotein sequence confirmed the directly determined sequences forthe 12 polypeptides,
whichtogetherspan85%of thematureamino acidsequence (Fig. 2).
The region of the structural gene corresponding to the mature cytochrome sequence (nt 565 to 2106) encodes an
apoprotein of 55.7 kDa. This sequence includes 16 C-X-X- C-H hemebindingsites, and the holoprotein,with 16cova-
lentlyboundhemes, hasacalculated molecular size of 65.5 kDa. As discussed in detail below, the properties of the cytochrome definedby thenucleotide sequence depictedin Fig. 2 are reminiscent of those of the Hmc first correctly
describedbyHiguchietal.(13). Therefore,this genewill be referredto as thehmc gene, anditsproductwill be referred
to as Hmc.
The Hmc polypeptide contains 31 His residues, 16 of whichareintegraltothe 16 C-X-X-C-H hemebindingsites.
Thus, only 15 of the 16 hemes can have bis-histidinyl
coordination. The hemebindingsites andhistidinylresidues of the Hmc polypeptide are spatially arranged into three complete cytochrome c3-like domains (II, III, and IV) and oneincomplete domain(I) that lacks both the second heme
binding site and its correspondingsixth-coordinate-position
histidine(Fig. 3).VisiblespectroscopicdataonHmc(13)do notdisallowaMetresidueatthisposition.TheE695valuefor His-Met-coordinated c-type cytochromesistypicallyonthe orderof0.9 mM-1 cm-',as determined forcytochromes c
(9)and C553 (23, 38). Higuchi etal. (13)determined anE553.2
valueof 428 mM-1cm-'for Hmc basedonamolecular size of 75 kDa,whichcorrespondstoanE5532valueof374mM-1
cm-1 when adjusted to the correct molecular size of 65.5 kDa. Applyingthisadjusted E553.2valuetothe visible spec- trumofD. vulgaris Hildenborough Hmc shownby Higuchi
etal.(A5532 =0.14;concentrationofHmc,370nM)givesan
A695 on the order of 0.0003. Thus, if there is a His-Met- coordinated heme in Hmc, it has remained undetected because theA695isonly0.2%of the hemeA5532in Hmc. If
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Xb.QI
CTCGAGCGATCCATACTTAACGGCAAGTTTACGAACATAGCATTTCCCGCGCCACCGGTCGGTGACCATTGAAGGTTGCACAGCAGAGGCCCGCGCCCCTTTCACCGCATCGACGTTCAT
10 20 30 40 50 60 70 80 90 100 110 120
-35 -10
GCCCGCAACCACACGTCCCCAAGGGAAAAGGGGGCAATTGCCACATGCGCTGCAATTGCATCTGCGCGTGTTTTTTCACTTGACGCTCZTTGACACAATAGACTACCCCCGCAGTAGGAlTT
130 140 150 160 170 180 190 200 210 220 230 240
TATTCCATACTATACAGGTAGGGTATGCGATGCTCCCCACACCACCGCCCGCCCGCCTGTCGTATCACGAGGACAGCCACGAAGGAATCAGGTTCCCCTCAGGAGGGGGCGACCGGGAAA
250 260 270 280 290 300 310 320 330 340 350 360
r b s M R N
CCACCGTGGCACGTCCGTCTGGCGACGCGCTCCCCGCGGGGAAACGGGGGCGACGTCCGGCTGGGGATGTCTGAGGATGTGTATCACCCATTCTTCCAAAC-Z-&CTGCCACTATGAGGAAC
370 380 390 400 410 420 430 440 450 460 470 480
G R T L L R W A G V L A A T A I I G V G G F W S Q G T T K A L P E G P G E K R A
GGAAGGACACTGCTGCGATGGGCGGGAGTGTTGGCTGCCACCGCCATCATCGGCGTCGGCGGTTTCTGGTCACAGGGGACGACCAAGGCCCTCCCCGAAGGGCCCGGCGAAAAACGCGCC
490 500 510 520 530 540 550 560 570 580 590 600
HindTIl
D L I E I G A M E R F G K L D L P K V A F R H D Q H T T A V T G M G K D C A A C
GACCTGATCGAGATCGGGGCCATGGAACGCTTCGGGAAGCTTGATCTTCCCAAGGTGGCGTTCCGCCACGACCAGCACACCACCGCCGTGACGGGCATGGGCAAGGACTGCGCCGCCTGT
610 620 630 640 650 660 670 680 690 700 710 720
H K S K D G K M S L K F M R L D D N S A A E L K E I Y H A N C I G C H T D L A K
CACAAGAGCAAGGACGGCAAGATGTCGCTGAAGTTCATGCGGCTTGACGACAACAGCGCCGCTGAACTCAAGGAAATCTACCACGCCAACTGTATCGGCTGTCATACCGACCTCGCCAAG
730 740 750 760 770 780 790 800 810 820 830 840
A G K K T G P Q D A E C R S C H N P K P S A A S S W K E I G F D K S L H Y R H V
GCCGGTAAGAAGACCGGCCCGCAGGACGCCGAATGCCGCTCGTGCCACAACCCGAAGCCCTCTGCCGCCTCGTCGTGGAAGGAGATAGGCTTCGACAAGTCGCTGCACTACCGTCACGTG
850 860 870 880 890 90v 910 920 930 ;40 950 960
IiindIIII
A S K A I K P V G D P Q K N C G A C H H V Y D E A S K K L V W G K N K E D S C R
GCGTCCAAGGCCATCAAGCCCGTGGGCGACCCGCAGAAGAACTGCGGCGCTTGCCACCACGTCTATGACGAAGCTTCCAAGAAGCTCGTATGGGGTAAGAACAAGGAAGACTCGTGCCGC
970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080
A C H G E K P V D K R P A L D T A A H T A C I S C H M D V A K T K A E T G P V N
GCCTGTCACGGCGAGAAGCCCGTGGACAAGCGTCCCGCGCTCGACACCGCCGCCCACACCGCGTGCATCAGCTGCCACATGGACGTCGCCAAGACCAAGGCCGAGACCGGCCCGGTCAAC
1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
C A G C H A P E A Q A K F K V V R E V P R L D R G Q P D A A L I L P V P G K D A
TGCGCCGGCTGCCACGCCCCCGAGGCGCAGGCCAAGTTCAAGGTGGTACGCGAAGTGCCCCGCCTCGACCGCGGTCAGCCCGACGCCGCCCTCATCCTGCCCGTACCCGGCAAGGATGCC
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320
P R E M K G T M K P V A F D H K A H E A K A N D C R T C H H V R I D T C T A C H
CCGCGCGAGATGAAGGGCACCATGAAGCCGGTCGCCTTCGACCACAAGGCCCACGAAGCCAAGGCCAACGACTGCCGCACCTGCCACCATGTGCGCATCGACACCTGCACCGCCTGCCAT
1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 1430 1440
__KpnI
T V N G T A D S K F V Q L E K A M H Q P D S M R S C V G C H N T R V Q Q P T C A
ACCGTGAACGGTACCGCAGACAGCAAGTTCGTCCAGCTTGAAAAGGCCATGCACCAGCCCGACTCCATGCGCAGCTGCGTGGGTTGTCACAACACCCGCGTCCAGCAGCCCACC.TGTGCC
1450 1460 1470 1480 1490 1500 1510 1520 1530 1540 1550 1560
G C H G F I K P T K S D A Q C G V C H V A A P G F D A K Q V E A G A L L N L K A
GGTTGCCACGGCTTCATCAAGCCCACGAAGAGCGATGCCCAGTGCGGCGTGTGCCACGTGGCCGCCCCCGGCTTCGACGCCAAGCAGGTCGAAGCGGGCGCATTGCTGAACCTCAAGGCA
1570 1580 1590 1600 1610 1620 1630 1640 1650 1660 1670 1680
E Q R S Q V A A S M L S A R P Q P K G T F D L N D I P E K V V I G S I A K E Y Q
GAGCAGCGTTCGCAGGTTGCCGCCTCCATGCTGTCGGCCCGTCCGCAGCCCAAGGGCACCTTCGACCTCAATGACATCCCCGAAAAGGTCGTCATCGGCTCCATCGCCAAGGAATACCAG
1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
P S E F P H R K I V K T L I A G I G E D K L A A T F H I E K G T L C Q G C H H N
CCCAGCGAGTTCCCGCACCGCAAGATCGTGAAGACGCTCATCGCCGGTATCGGTGAAGACAAGCTGGCCGCCACCTTCCACATCGAGAAGGGCACGCTGTGCCAGGGCTGCCACCACAAC
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920
S P A S L T P P K C A S C H G K P F D A D R G D R P G L K A A Y H Q Q C M G C H AGCCCCGCCAGCCTCACCCCGCCCAAATGCGCGAGCTGCCACGGCAAGCCCTTCGACGCCGACAGGGGCGACCGTCCCGGTCTCAAGGCCGCCTACCACCAGCAGTGCATGGGCTGCCAC
1930 1940 1950 1960 1970 1980 1990 2000 2010 2020 2030 2040
D R M K I E K P A N T A C V D C H K E R A K
GACCGGATGAAGATCGAAAAGCCGGCGAACACCGCCTGCGTCGATTGCCACAAGGAACGCGCGAAP
2050 2060 2070 2080 2090 2100
ATAGGACGGTGAGGAGTACGACATGGATCGCAGAAGATTCCTGACCCTGCTGGGA
2110 2120 2130 2140 2150 2160
p43
FIG. 2. Nucleic acid sequence of the first 2,160 nt of the 3.7-kbXhoI fragment of D. vulgarisHildenborough chromosomal DNA depicted inFig. 1. The -35 and -10 regions of a putative promoter sequence are indicated, as is the ribosome binding site (rbs). The hmc structural gene spans the region from nt 472 (initiatormethionine residue) to nt 2109(termination codon). A vertical arrow marks the signal peptide cleavage site. The overlined regionsrepresent436 ofthe 514 amino acid residues of the mature Hmc polypeptidewhichhave beensequenced atthe peptide level in polypeptides Plto P12. The corresponding positions of thesepolypeptides in thegene-translated sequenceare Pl, nt 565 to 921;P2, nt 1636 to 1971; P3, nt 1375 to 1530; P4, nt955 to 1026; P5,nt 1162 to 1224; P6, nt 1315 to 1374; P7, nt922 to954; P8,nt1069 to 1104; Pg, nt 1105 to 1119; Plo, nt 1255 to 1293; Pll, nt 1594 to 1635; and P12, nt 2008 to 2106. The region corresponding to the deoxyoligonucleotide probe P43 is underlined, and the asterisk in that region denotes the one nucleotide that is mismatched in the P43 sequence. Restriction sites for enzymesXhoI, HindIll, andKpnI are also noted.
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