Molecular Microbiology (1991) 5(2), 401^07 ADONIS 0950382X91000462

The role of a stress-response protein in Saimonellatyphimurium virulence

K. Johnson,^^ I. Charles,^ G. Dougan,** D. Prckard,^P. O'Gaora,^* G. Costa,^ T. Ali.^ I. Miller^^andC. Hormaeche^'Department of Pathoiogy. University of Cambridge,Tennis Court Road. Cambridge CB2 10P, UK.^Department ot Molecular Bioiogy. Wellcome Biotech,Langley Court, Beckenham, Kent BR3 3BS, UK.^Instituto di Microbiologia. Piazza XX, Settembre 40.Messina, Italy.

Summary

We recently described the use of selective transposonmutagenesis to generate a series of avirulent mutantsof a pathogenic strain of Salmonella typhimurium.Cloning and sequencing of the insertion sites from twoof these mutants reveals that both have identicallocations within an open reading frame that is highlyhomologous to a gene, htrA, encoding a heat-shockprotein in Escherichia coli. DNA sequence analysis ofS. typhimurium htrA reveals the presence of a genecapable of encoding a protein with a calculated Mr of49316 that has 88.7% protein:protein homology withits E. coii counterpart. In E. coli, lesions in this gene,also known as degP, reduce proteolytic degradation ofaberrant periplasmic proteins. Characteristics of the5. typhimurium htrA mutants, 046 and 014, in vivo andin vitro suggested that they are avirulent because ofimpaired ability to survive and/or replicate in hosttissues, tn vitro, the 5. typhimurium htrA mutants 046and 014 are not temperature-sensitive but were foundto be more susceptible to oxidative stress than theparent, suggesting that they may be less able towithstand oxidative killing within macrophages.

Introduction

Salmonella spp. are facultative intracellular parasitesresponsible for a range of syndromes in vertebrates,ranging from acute self-limiting gastroenteritis to typhoid

Received 3 August, 1990; revised 13 September, 1990. Present addresses:'Cambridge Antibody Technology, Daly Research Laboratory, BabrahamHall, Babraham, Cambridge CB2 4AT, UK; ^Department ot MolecularBiology, Scripps Clinic and Research Foundation, tO666 North TorreyPines Road, La Jofia, CalHorr}ia 92037-1093, USA; ^Glaxo Laboratories.Greentord Road, Greenford, Middlesex UB6 OHE, UK. "For correspond-ence. Tel. (081) 658 9966; Fax (081) 656 0890.

(Rubin and Weinstein, 1977). Pathogens such as Sal-monella typhi, the causative agent of human Xy'pho'id,exhibit a restricted host range (Collins, 1974), a factorwhich has hampered studies on these organisms. How-ever, some strains of Salmonella typhimurium can estab-lish infections in mice after oral feeding that closelyresemble typhoid fever in humans, providing a modelsystem in which to study pathogenesis of infection andhost immune responses (O'Brian and Metcalf, 1982; Killarand Eisenstein, 1985). Interest has focussed on genes thataffect pathogenicity of the organism, since identificationof such virulence-associated genes would prove helpful inunderstanding the host-pathogen relationship. Moreover,detailed knowledge of these genes could provide practicalbenefits in the form of live attenuated organisms for use asvaccines. Unlike most conventional killed vaccines, liveorganisms induce strong cellular immunity in addition to ahumoral response, which is one of the many features thatmake attenuated salmonellae spp. attractive as livecarriers of heterologous antigens to the Immune system(Dougan et al\ 1986; 1989).

We have utilized the transposon luphoA to generate S.typhimurium mutants of reduced virulence when givingorally to mice (Miller et at., 1989). 'JuphoA is a Tn5derivative containing an alkaline phosphatase gene minusthe signal sequence (Manoil and Beckwith, 1985). Thissystem provides a rapid screen for transposon insertionsinto genes encoding exported products, since cells platedon indicator media produce a colour reaction only if thetransposon has Inserted downstream of a signal se-quence. In this paper we describe the characterization oftwo TnpAio/\ S. typhimurium mutants and demonstratethat attenuation in these isolates is the consequence oftransposon insertion into a heat-shock gene, htrA (Lip-inskaefa/., 1988; 1989; 1990).

Results

Characterization of TnphoA insertion sites in S,typhimurium 014 and 046

From previous Southern blot experiments (Miller ef al.,1989) it was known that the TnpAio/A-in activated gene(s) ofmutants 014 and 046 were likely to reside in the chromo-some on similar 3.9 kb H/ndlll fragments. In order to clonethese transposon-inactivated genes, samples of chromo-somal DNA from mutants 014 and 046 were first digested

402 K. Johnson et al.

M K K T T L A M S A L A L S L G L A L

S P L S A T A A E T S S S A M T A D O M P S L A P M L E K V

M P S V U S ] N V E G S T T V N T P R M P P N F Q Q F F G D7a7*?

D S P F C O D G S P F O N S P F C a G G G N G G N G G D Q O

K

IGATT

N

t_TCT6

A

F1TC

KAAA

P

GG6C:

A

M

ATE'

VGTA

K

EGAA

1

AGCG

0CAG

N

TACG

NCGCC6C6ATTAAC

GCGGC

GCGGC

GGGC

E:GAf:

N:AAC

LCTE

1:ATC

ei6GG

LCTG'

LCTT

L

VGTG

RCGT

G!6GT

1iATC

GGGC

SABC

T

TACG

6GGT

1ATC

M:ATG

sTCC

DGAT

A

SJCA

NAAC

6:GGC

GiGGG

GGGC

GGGG

I

GGGT

BTCC

FTTC

TiACT

niA'

RCGT

K

IOTC

GGGC

AGCT

EGAG

IftTT

K

AAA

L

VGTT

GGGC

1ATC

L

iCTG

1ATT

FTTC:

A

STCG

AGCG

P

ccc

NlAAT

0RAt

0GAT

D

AGCG

1.CTG

SAGT

STCC

AGCC

AGCT

S

LCTG

VGTG

NAAC

EIGAfl

A

GCG

KAAA

D

G66G

NAAC

MATB

L

iTTE

K

AA13

V

BTG

A

RCGT

1CTG

ViGTC

AIGCC

GCGC

VGTG

L

SA6C

N.AAC

KlAAA

KiA«C

VTAr

GGGG

R

G6GC

GGGT

lAAC

AiGCG

VRTT

K

AAA

V

LCTG

FGAG

LCTG

M.ATG

VCTC

DGAT

G

NAAC

LCTG

TACG

K.AAP

TAcr.

pCCG

D

V6TA

1ATC

STCG

V.GTC

NAAr.

RCGT

V

EGAA

GGGT

0.CAG

DGAC

Nf»AC

STCT

T

NAAT

1ATT

MATG

AGCC

HCAC

DGAT

V

¥TAG

NAAC

VGTG

0CAG

VGin

1ATC

A

EGAG

1ACC

E6AA

H.GGfi

Vr,1T

AGCG

1

NAAC

AGCG

y.TAG

GiGGC

DBAT

LCTG

G

FTTT

IATT

GGGC

A6GG

NAAC

]ATT

N

1ATT

1CTG

QCA6

F.TTC

AGr.r.

aCAA

P

•CA6

A6C6

ViGTG

:GTC

SAGC

1ATT

F

TAGC

PGGG

K.AAA

SAGC

VGT

0CA

G

DGA

DGA

RiGG

•:CA

)38139BIO

168

198199990

228229

1080

25B259

1170

288289

328329

1350

L T D . . Q V N . . . 3 8 8A L R A Q V G T M P V G S K I S L G L L R E G K A I T V N L 3 B 9

GGCGCTGCGCGCTCAGGTCGGCACTOTGGCGGTCGeGAGGAAAATCAGCCTCGOTCTGGTGCeTGAAGGTAAAGGGATTACGGTGAATGT 1 5 3 0

E L O O S S O S O V O S S T

Q . . . . N N . - T G TK G V V V S S V K A N S P A A O I G L K K G D V l I G A N Q

Q P V K N I A E L R K I L D S K P S V L A L N l Q R G D S S

L P V N A V l B L N P F L K T G R 6 S P Y N L «

Rg. 1. Nucleotide and derived amino acid sequence of the MrA gene ot S. typhimurium C5 in comparison wilh its E. coii counterpart. The derived aminoacid sequence is shown in singie-ietter code. Amino acid identity between the two sequences is indicated by a '.' and non-homologous residues arewritten above the Saimoneiia C5 sequence. A '-' denotes a missing residue. The upward arrow at nucleotide 945 shows the JnptioA insertion site. Theinverted arrows between nucleotides 1833 and 1855 delineate the core of a sequence with an imperfect dyad symmetry, and is followed by a run of fourT residues: such a sequence is characteristic of a rho-independent termination site. The putative GXGXXG dinucleotide binding pockets and the RGDSmotif are underlined. These sequence data will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Data Libraries under the accession numberX54548.

HtrA involvement in bacteriai viruience 403

with H/ndlll and electrophoresed through a 0,7% agarosegel, A region of the gel corresponding to DNA fragments inthe 3-5 kb size range was excised for each mutant, andthe DNA purified and cloned into pUC19. Colonies con-taining the expected 3,9kb /-//ndlll fragment were isolatedin colony hybridization experiments (Maniatis etai, 1982)using an [a-^^P]-labelled ECORI-XAJOI fragment of thealkaline phosphatase gene of JnphoA (Manoil and Beck-with, 1985) as a probe. These positive colonies containeda portion of the JnphoA transposon and additionally afragment of the transposon interrupted target gene. Usinga 500bp [a-^^P]-label led EcoRI fragment of the targetgene (isolated from the 3,9kb H/ndlll fragment) as a probein Southern blot experiments against parental C5 chromo-somal DNA resulted in the identification of a 3.0 kb bandthat was likely to contain the entire target gene. This 3.0 kbH/ndlll fragment was isolated and cloned into pUC18 bythe method described above using the 500bp EcoRIfragment as a probe in colony hybridization experiments.The 3,0kb H/ndlll fragment was then subcloned intoM13mp18and M13mp19for DNA sequencing. The com-plete sequence of the target gene, Interrupted by inphoAin the S. typhimurium mutants 014 and 046, is shown inFig. 1. The single open reading frame encodes a polypep-tide of 495 amino acids that is 88.7% homologous to the Ecoli heat-shock protein HtrA (Lipinska et ai, 1988). Thesite of TnphoA insertion in 014 and 046 was found to beidentical and thus the two isolates probably arose from acommon transposition event.

Comparison between the S. typhimurium and E. colihtrA sequences

Comparison of the deduced primary sequence of the S,typhimurium HtrA with its E. co//counterpart (Fig. 1) showsa high degree of homoiogy, with many of the amino aciddifferences being conservative substitutions. The E. coliHtrA has a 26 amino acid signal sequence, typical of thoseproteins that are targeted through a cell membrane(Lipinska ef ai, 1990), S. typhimurium HtrA has a verysimilar sequence, with the same Ala-Xaa-Ala cleavagemotif reported to be recognized by E. coli signal pepti-dases (Perlman and Halvorson, 1983). Both proteins havea Gly-Xaa-Gly-Xaa-Xaa-Gly motif, a sequence that hasbeen identified in some proteins as being involved indinucleotide binding (Wierenga and Hoi, 1983). Towardsthe middle of the deduced protein sequence betweenresidues 235 and 239 both proteins have the sequenceGly-Asn-Ser-Gly-Gly which closely resembles the can-onical core sequence Gly-Asp-Ser-Gly-Gly that usuallysurrounds the active serine residues of the catalyticdomains of known serine proteases (Brenner. 1989).Recently, Lipinksa et ai (1990) have demonstrated thatpurified preparations of E. co// HtrA protein do indeed have

a serine protease activity, although the physiologicalsubstrate(s) for the protease have yet to be Identified. Nearthe carboxy-terminus of both proteins is the sequenceArg-Gly-Asp (RGD). which in the S. typhimurium HtrA isfollowed by a Ser residue. The Arg-Gty-Asp-(Ser) motifhas been characterized as being important for cell bindingin some adhesive molecules such as fibronectin (Ruoslahtiand Pierschbacher. 1987), More recently, the Arg-Gly-Asp motif found in the terminal proteins of both bacterio-phage 029 and M2 have been shown to be involved in aprotein:protein recognition event essential for the protein-priming reaction of phage replication {Kobayashi et ai,1989). It appears that Arg-Gly-Asp motifs are a signal formolecular recognition that can function both inter- andintracellularly. although the role of this sequence, if any, inthe function of HtrA protease specificity is unknown.

The only major difference between the E. coli and S.typhimurium sequences occurs at the 3'-end of the gene.The predicted E. coii protein is longer and extends througha region that, in both genes, has a dyad axis of symmetryand a run of T residues typical of a rho-independenttranscriptional terminator (Rosenberg and Court, 1979),

Two distinct families of stress-response genes havebeen described whose transcription depends on differentRNA polymerase sigma (a) factors. To date, most studieshave focused on stress-response genes that are under thecontrol of tr^^, the product of the rpoH gene. Transcriptionof the second family of stress-response genes, charac-terized so far by only E. coli rpoH and htrA, is under thecontrol of the <J^ factor. Erickson and Gross (1989) haveproposed that, for clarification, members of these twofamilies should be designated as either u^^- or o-^-depen-dent genes. Examination of the 5'-untranslated region ofthe S. typhimurium htrA gene for promoter sequencessimilar to those tr^-dependent elements mapped for the E.coli gene (Lipinska ef ai, 1988) shows an interestingalignment. The -35 element, 5'-GAACTT-3'. and -10element, 5'-TCTGA-3'. found in the 5'-untranslated pro-moter region of E. coii htrA are also found, with the samespacing, from nucleotides 323 to 349 of S. typhimuriumhtrA. The sequence immediately prior to the inferredtranscription initiation site for the S, typhimurium gene is5'-TAGA-3'. while its E. co//counterpart has the sequence5'-AACA-3'. This high degree of homoiogy suggests thatthe promoter elements of the S. typhimurium htrA pro-moter may also be recognized by an RNA polymerasesimilar to the E. co//(r^-RNA polymerase holoenzyme.

Stress response of an S. typhimurium htrA mutant

Because of the high degree of homoiogy between the S.typhimurium htrA and its E. coli counterpart, severalexperiments were performed to investigate temperaturesensitivity of the Salmonella 046 htrA mutant. The mutant

404 K. Johnson et al.

was also found to be as resistant as the parent to thebactericidal effect of serum (data not shown). However,despite the use of several growth regimes and a range oftemperatures from 42-50°C. the Salmonella 046 htrAmutant was found to survive as well as the parent atelevated temperatures (data not shown). This suggeststhat temperature-sensitivity cannot account for the re-duced virulence of the Satmonetta htrA mutant.

Heat-shock proteins are more correctly termed 'stressproteins' in that they are produced in response to a varietyof stressful stimuli other than just heat (Lindquist, 1986).Low recovery of htrA mutants from infected tissues (Milleret at., 1989) could reflect an inability to withstand someform of stress experienced within the host. One possibilityis that the mutants are unable to survive and/or replicatewithin the macrophage phagolysosome, a hostile environ-ment and one in which lack of a stress protein may bepleiotropic. Agents that mimic oxtdative killing mechan-isms of the macrophage. such as hydrogen peroxide andredox cycling agents, are known to induce heat-shockproteins in E co//(Christman etai, 1985; Greenberg andDemple, 1989), suggesting that these proteins are part of aprotective response. Figure 2 shows the result of exper-iments designed to test whether the S. typhimurium htrAmutant has increased sensitivity to oxidative killing mech-anisms. The htrA mutant does appear to be more sensitivethan the C5 parent to H2O2 and to a redox cycling agent,menadione. whilst another attenuated S. typhimuriumTnphoA mutant (060) had the same properties as theparent (Fig. 2c). Sensitivity to oxidative stress couldaccount for low recovery of this mutant trom infectedtissues, and possibly its reduced virulence in vivo,although it could be a secondary effect of the lesion.However, inactivation of S. typhimurium htrA does notrender the cells temperature-sensitive, implying a degreeof specificity.

Discussion

In this paper we describe an account of the involvement ofa stress-response gene, htrA, in bacterial virulence. DNAsequence analysis shows that two attenuated S, typhi-muriumJr\phoA mutants, 014 and 046 (Miller et ai., 1989),harbour transposons located within the same site of thehtrA gene. In E. coii the htrA gene is indispensable forbacterial survival above 42''C and such mutants have atemperature-sensitive phenotype. In contrast, S. typhi-murium htrA mutants are not temperature-sensitive andshow similar growth kinetics at elevated temperatures ascompared with wild-type parental strains.

This finding is quite puzzling as the alignment of the 5'non-coding regions of the S. typhimurium htrA with thepromoter sequences mapped for the E. coli htrA gene

00-

80-

60 ^

40-

20-

a

V

10 mM

\

-• r

hydrogen peroxide

\C5Mr/i

10 20 30 40Minutes aftef challenge

(b) "lOmM menodione

50

C5hfrA

10 20Mjnufes offer challenge

(c ) Solid-media sensitivity tests

Treatment

300 f ig H202

30/ ig H2O21 mq menadione

Diameter of clear areo(mm)

C5

17.0130tO.5

0 6 0

17,513.0105

htrA

25.014.513.0

Fig. 2. Sensitivity to hydrogen peroxide and menadione. Panels A and Bshow percentage survival folfowing addition of H^O? to IQmM andmenadione to 40mM, respectively. The resuits of solid media tests(Panel C) show that lower concentrations of H2O2 and menadione arenecessary to prevent growth of the htrA mutant oompared to the C5parent and an irrelevant S. typhimarium InphoA mutant, 060.

(Lipinksa etai, 1988) shows very high homology. The lackof temperature-sensitivity in S. typhimurium htrA mutantsmay suggest that although the genes are highly homo-logous they are regulated and function differently. A morelikely explanation, however, is that the S, typhimurium C5strain, in which the htrA mutant was generated, carries asecondary gene or mutation that may complement someof the functions of the htrA. A similar situation has beenreported by Strauch and Beckwith (1988) for an E. coli lppdegp (htrA) double mutant that has decreased tem-perature-sensitivity relative to an isogenic/pp^ degP(htrA)mutant. It is believed that the mutant deficient in lpp allowsperiplasmic proteins to escape with greater facility. The

HtrA involvement in bacterial virulence 405

lack of temperature-sensitivity in the S. typhimurium htrAmutant may therefore be due to the leakage of aberrantlyfolded proteins from the periplasm at a sufficiently highrate so that their accumulation does not interfere withgrowth.

Heat shock is only one of a number of environmentalstimuli that can regulate a stress-protein response (Lind-quist, 1986; Dorman etai.. 1989). Although S. typhimuriumhtrA mutants have an unimpaired ability to survive atelevated temperatures the data presented in this papershow that they have a decreased ability to surviveoxidative stress. For S. typhimurium to be virulent thebacterial cell has to be able to survive in macrophages(Fields ef a/., 1986). This survival depends on the ability ofthe bacteria to tolerate a range of toxic killing mechanismsincluding the production of hydrogen peroxide. S. typhi-murium htrA mutants, with a reduced ability to surviveoxidative stress, will be less likely to survive in a macro-phage and thus be attenuated.

The oxidative stress response in bacteria is complex inthat mutations in several genes along with htrA can resultIn reduced tolerance to oxidative stress (Fields et ai,1989); indeed, hydrogen peroxide can induce expressionof several dozen genes (Greenberg and Demple, 1989).Another Salmonella spp. gene, phoP. has been shown tobe necessary for sun/ival inside macrophages. This geneencodes a regulation protein that governs the expressionof several genes responsible for the protection of Sal-monella spp. from the bactericidal action of neutrophil ormacrophage-derived cationic proteins or peptides (Grois-man and Saier, 1990). Like htrA. phoP mutations areattenuating. Whether or not the regulatory phoP geneproduct controls htrA expression (or vice versa) remains tobe tested and the exact mechanism by which either ofthese proteins protects Salmonella spp. in vivo is un-known.

In E. coli, the htrA gene product has been shown to beinvolved in degradation of abnormal periplasmic (but notcytoplasmic) proteins such as heterologous fusion pro-teins or abnormally folded molecules and has beenindependently called degP (Strauch and Beckwith, 1988).Recently Lipinska et ai (1990) have shown the E. coli HtrAto be a serine protease, although the physiologicalsubstrate for the enzyme has not been identified. Becauseof the high degree of homology with the E coii HtrA,including conservation of a canonical serine proteasemotif, it seems likely that the S. typhimurium HtrA alsofunctions as a serine protease. The finding of putativedinucleotide and cell-binding motifs within the deducedprotein sequence of HtrA has not yet been correlated withany biological properties of the molecule. We are,however, currently investigating the possible role of theArg-Gly-Asp sequence in virulence by site-directedmutagenesis.

The importance of stress proteins in the pathogenesis ofinfectious diseases has been highlighted recently by thediscovery that the Immunodominant antigens of f^yco-bacteria spp. are stress proteins capable of interactingwith gamma-delta T-cells of the host, the similaritybetween the mycobacterial and host heat-shock proteinssometimes resulting in autoimmunity. Furthermore,several immunodominant antigens of eukaryotic parasiteshave been shown to be stress proteins (Nene et ai, 1986;Johnson et ai, 1989), indicating that the importance ofstress proteins in pathogenesis is not confined to bacterialinfections. As suggested by the inactivation of the htrAgene, another feature of this important group of proteins istheir role in bacterial virulence. The multiplicity of pheno-types apparent in E. coli and S. typhimurium htrA mutantspresumably arises because of a series of related bio-chemical functions of this protein, as is fairly typical ofprokaryotic heat-shock protein mutants. An immediatepractical application of Saimoneiia hfrA mutats would betheir use as live carrier vaccines (Dougan ef ai, 1986,1989), particularly if the mutant exhibited reduced proteo-lysis of heterologous periplasmic proteins; preliminaryexperiments indicate that vaccination with the htrA mutantconfers protection against lethal challenge with virulentorganisms (S. N. Chatfield, unpublished observation). Asthe majority of protective antigens are surface-located,the correct targeting of heterologous antigens could proveimportant for eliciting optimal immune responses, andhtrA mutants could be particularly suitable for such a use.

Experimental procedures

Bacterial strains, bacteriophages, piasmids and growthconditions

S. typhimurium C5 is a prototrophic rTiouse-virulent strain(Hormaeche, 1979). The JnphoA derivatives of C5. TnphoA 060,014 and 046, were described previously (Miller et al.. 1989). E. coliK12 TGI {A{tac-pro), supE. thi. hsdD5/F'traD36proA ' -B * -iaci"Z :iM15) was as described (Carter ef ai.. 1985). Bacterial strainswere grown in Luria broth (LB) or LB solidified with 1.6% (w/v)agar (Davis et ai.. 1980). E. co//TGI was propagated on minimalmedium (Miffer et al.. 1989) as previously described. The cloningvectors, pUC18. pUC19, M13mp18and Ml3mp19 (Messing andVieira, 1982; Yanisch-Perron et ai. 1985) were from Pharmacia.

Sensitivity fo hydrogen peroxide and menadione

Resistance was determined as described previously (Christmanet al., 1985; Greenberg and Demple, 1989). Cells were grown inminimal medium to an OD of 0.2 prior to the addition of H^O? ormenadione. Cells were removed at intervals and diluted in M9salts before being plated out on nutrient media for viable counts.For resistance determination on solid media, cells were initiallygrown overnight at 37°C in nutrient broth. Approximately 2 x 1 0 ^

406 K. Johnson et al.

of washed cells were added to 3 ml of minimal top agar and platedon 85-mm-diameter plates containing 25 ml of minimal agar. Tenmicrolltres of the inhibitor was applied to BBL discs placed in thecentre of the agar. The diameter of the clear zone around the disc(no cell growth) was measured after 24h at 37°C.

DAW manipulations, sequencing and reagents

Chromosomal DNA was prepared as described previously (Mas-kell et al.. 1988). Piasmid DNA was prepared using the alkalinelysis method (Maniatis et ai., 1982). Restriction endonucleaseswere supplied by BRL Gibco Ltd. T4 DNA ligase was fromBoehringer. Southern blots and colony hybridizations with[a-^^P]-dATP nick-translated probes were carried out as de-scribed by Maniatis etal. (1982). Single-stranded DNA templateswere sequenced using T7 potymerase fTabor and Richardson,1987) using a kit supplied by Cambridge Bioscience. Labelledfragments were separated on buffer gradient polyacrylamide/urea gels (Biggin etal.. 1983). The sequence was assembled fromoverlapping H/ndlll. EcoRI and Pst\ fragments cloned intoM13mp18 and M13mp19. Gaps in the sequence were filled inusing synthetic oligonucleotides as specific sequencing primers(Charles etai., 1988).

Oligonucleotides

Oligonucleotides for use as specific primers were made on anSAM1 oligonucleotide synthesizer (Biolabs).

Acknowledgements

We thank Tina Silva for typing ttie manuscript, Hugh Spence formaking oligonucleotides used in the sequencing strategy, andSteve Nicholls for help witti computer analysis. This work wassupported by the Medical Research Council (UK). K.S.J. is a JohnLucas Walker Senior Student.

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