llecular Mlcroblology ( 1994) 13( 1), 141-151

pr n ·~-- ... -~ to PrfA

I n ula or

nr

Robert Lampldla, Roy Gros , Ze\Jka Soko\ovlc, We ner G bel and Jürgen Kreft Theodor-Boveri-lnstitut für Biowissenschaften ('8io2entrum) der Universität Würzburg, Lehrstuhl Mikrobiologie, Am Hubland 0·97074, Wurzburg.

ermany.

pecl J L lvanovll and .t. rnonocytcg nes c n dlffer ntlated bt hernt· oally and how different ho t r-ange • Vlrulence of L monocytogenes I eiependent on. th lntegr ty of prlA whlch po IOvety and co-o dlt1etely r ula

anscription of veral vlrufence gene Un 'I now, prfA homologue had not been ldentlfled ln

b.. IVIIInovll. W have now clo ed a chromo omal eglon from L. lvsnov/1 comprl lng two genea wHh

filgh , omoJogy to th plcA and ptfA gene from L. monocytoge . Dfstal rom prtA, .an open r acJ.. iog frame hlghly homologaus to a phoephortbosyl Ryropho phate syntheta e gene (pr. ~ was newly d ntJfted. d flnlng the border of the vlrulenc gen

qlu ter Transcrlptlon ot th gene for fvanoly ln 0 a,nd exp e lo of oth r enes of the viru ence gen Iu tet in L.. lvsnov/1 w r de ndent on PrtA. Th attem of ertA~pendent erotelne (PdP ) expras

I L. lvsnovll waa lmllar, but not ldenttcal o thal ot L~ monocytogenes Th PrtA protelns. a . pre-

lcted from nucleotJde • quences of bOth pathogenlc Qtrterla pecle ar very slmilar and alto algnlffcant homology to th · Crp-Fnr famlly of g ob I tran crlptlon r ufators.

he two pathogenic Usterla species (L. ivanoilil and L. monocytogenes) are facultattve lntracellutar bacteria, ~ group that a.lso lncludes the causative agents of twberculosis, leprosy, shigellosrs, typhold fever and

Aeeelve.d 25 O<ltober., 1993~ revlsed' 1 AprU. 1994: accepled 11 April, 994. •For correspondence. E-mail miktobloCvax.rz.uniwuerzw burg.d400.de; Tel. (931) 886 4419; Fax (93'1) 888 4402.

Legionnalres1 disease. L fvanovil (lvanov. 1962~ Sae\iger et al. 1984) very rarely infects humans, rather thls specie is prlmarlly an animal pathogen of sheep and cattle that causes abortlon, neonatal sepsis, etc. (lvanov, 1962; Cooper and Dennis, 1978).1t is different from L monocyto­genas using ths ctiteria of biochem\cal and s.erologlcal tes (tvanov. 1975; Rocourt et al., 1983; Seellger et al., 1984). The most virulent Iisteriai specles, L. monöOyta. genes, can cause severe lllness (listeriosls) in animals and man (GeJiin and Broome, 1989). predominantly menln g tls and septicaem a ln immuncompromis~d or pregnant individuals.

A cholesterol .. bindlng (SH .. aatlvated) cytotysln (Aiouf and G.eoffroy. 1991) t termed Iisteriolysin 0 was ·the fir t virutence factor of L. monocytogenes tor whtch an essen­tial rote in pathogenesis could be unequ vocally demon­strated and the gene ( h/y) sequenced (Mengaud et s/.1 1988; Cossart et al., 1989). Other genas whlch are involved in various steps of the complex lntracellular life cycle of L monocytogenes inC'Iude ,genas for lecithtnase (p/cB). metalloprotease (mpl). actln polymerization (actA} and phQSPhatidyllnositol-speciftc phospholipase C (Pt .. PLC. piCA) (rev1ewed in PortnOy et al .• 1992). These four genas. tagether wittl hly, constltute a chromosomal virutence gene ctuster ccrordtnately and positively regulated at the transcnptionaJ Ievei by prfA, the most proximal gene of the cluster located next to plcA. The preclse mechani m of transcfiptlonal activation by PrfA is not known and t has be~n cJalmed that t has no extensive homology to any known proteln (leimelster .. Wächter et al., 1990; Men­gaud et aJ.1 1991; Chakraborty et al., 1992). A 14 bp palin· dromic ONA sequence, whlch ls present upstream of hly (Mengaud et al., 1989) end in modified form in front of the other transcription units of the virulence cluster, i r&quired for efficient Iisterioiysin synthesis (Freitag et al .• 1992). Thls so·called 'PrfA box' is commonly belleved •o be the target site for PrfA.cjependeht regulation. Recently we have shown that under specific culture conditlons Pr1A-dependent protelns (PdPs) are the main surface pro­tains syntheslzed de novo ln L. monocytogenes (Sokolovic et al., 1993).

lt has been reported earlier that a ~gene probe speclfic for prfA of L monocytogenes dld not hybridize to L lvanovll

142 R. Lampfdls et al.

chromosomal DNA; from thl ft was conoluded that vlru· lence gene regulation ln the latter species fs different from L. monocytogenes (Wemars et al., 1992). However Iecithin e and Pl-PLC in additlon to vanolysln 0. can also readily be detected in L. lvanovil suggesting lhat at least som& of the virulenoe genas in thts IIsteriai pecies mlght be slmllar to those of L monocytogenes. Here we show that lndeed L lvanovii conta ns genes that are hlghly homologaus o both prfA and plcA from L. monocytogenes.

Re ul

Characterfzation of the Tn1545 mutant 816

ln many Instaoces insertionat mutagenesis of Gram­positive bacteria by conjugal transfer of enterococoal trans­posons, e.g. Tn916orTn 1545, has proven tobe a powerful genetlc tool '(Gaillard el al., 1986; Kathariou et al. , 1987}. ln our faboratory. several Tn 7545-d rived mutants of L. lvanovil have prevlousiy been oonstructed and described (Schtesinger~ 1988; Kreff et sl .• 1990). One o·f these, mutant 8/6, exh bits a plelotropic phenotype wlth a concomltant reduction in haemolytic acttvity (Fig. 1 A) and a loss of expresslon of Iecithinase (Fig. 1 B} as weil s of PI .. PLC (Fig. 1 C). ln L monocytogenes, the genes

for Iecithinase and PJ .. PLC (Lelmelster~Wächter et al .• 1991) are under the oontro~ of prfA Culture supematants from ttte mutant L. lvanovll straln had abOut 3% (4 HU) of the Wlfd type (128 HU) haemolytlc activity when measured in a liquid assay. On blood agar pla1es, haemoJysls by thfs mutant was onfy moderately reduced (Fig. 1 A). However, no measurable amounts of ivanoJysfn 0 were: detectabte (see Fig, 6 below) and the restdual haemolytic activlty was attributed to the sphingomyelanase secreted by L. iva· nov/1 (Kraft et al. 1989; Vazquez .. ßoland et al., 1989). ~er, it was shown that mutant 8/6 was unable to multlply an Caco-2 eolon carcinoma cens in vitro (Karunasagar et a/. ~ 1993), and that it ls completety avirulent jn mice and eh cken embryos (J. Kraft et a/., unpublished). Theretore, it had propert~es very slmilar to the known plfA-negattve

1. Phenotype of L. lvanov/lwitd·type (a) and its pdA-mutant 81& {b). A. Haemotys s on blood agar after 24 h at 37 C. B. Lecithinase productlon, visuaUzed by precipltate on egg yolk agar. C. Producdon of PI·PLC, vlsualized by halo formatlon on BHl agar wtth n overlay contalnlng 0.2 "' phasphaUdyllnosltol. 1n (B) and (C} lnc:ubatlon w.a for 48 h at 37 C.

mutants of L. monocytogenes (Mengaud et al. 1991 ; Chakraborty et a/., 1992). suggesting that thls mutant had T n 15451n erted in a gene at least functlonally homo­logous to prfA from L. monocytogenes.

Isolation, cloning, and sequencing of the prtA and ptcA genes

Based on the assumptlon that the chromsomal organiza­tlon o·f L. ivanovilwas slmllar to L monocytogenes. we per· formed a polymerase chaln reaction (PCR) amplificatlon of mutant 8/6 chromosomal DNA with ollgonucleotide ptimer derived from the known ends of the transposon Tn 1545 (Caillaud and Courvallnr 1987) and from a prevlously deter­mlned 1/o-upstream sequence (J. Kraft and M. Web r unpublished) The reaction wrth prtmers M395 (Tn 1545 rtght end) a.nd M397 (1/o-upstream) resutted in the ampiH • cation of a 1.3 kb fragment. Sequence detennlnaüon revealed that thls fragment contalned two contlnuous, yet incomplete, open reading frames wlth signtficant homology to the C.terminal part of plcA and the N-termfnal part of prfA from L. monocytogenes. The complete plcA gene wa PCR ampr fied using primers denvßd from the intergenlc seq.uence upstream of llo (J . Kraft unpubUshed) nd rh N-termmus of prfA. Inverse PCR on Ddet-dlgested and selt-llgated ONA from L. lvanovll wild type yfelded a 0.8 kb fragment containing the ~termlnus of prlA. Using the, sequence Information obtalned so far, the complete prtA witd·typß gene was PCR amplif1ed. The entire double-stranded sequence of prfA as weil as of plcA was verified using different sequencing prlmers and has been deposited in the EMBIJGenBank/OOBJ Nuel otld Sequence Oata Llbrarles under the accession number X72685. F~gure 2A ls a schematJc representation of the chromosomaJ region from L. ivanov/i described here. A computer homology search (TFASTA) in the EMBL data base initially detected only a rather low homology of the PrtA protetn. as deduced from the nucleo de sequence, to NtcA. from Synechococcus. The latter proteln is a mem bar of the Crp-Fnr famlly of global transcriptlon reguta or (Vega~Palas et al., 1992). A manuaJ allgnment of the PrfA proteins from both L. ivanovli and L. monocytogene wlth NtcA and with Crp-Fnr from Escherichla coll. optimized with respect to known functlonal residues and doma ns, then reveaJed an e-xtended and slgnificant homology among these protelns (Ffg. 3). Compared to NtcA, the two PrfA protelns contained lde11tical or simllar amlno acids a 79 positlons; the homology to Crp was 75 am no acids (L ivanov/1) and 73 amino acids (L monocyto­genes). respectivety.

Flgure 3 lncludes a comparison of the deduced amlno acid sequences for the PrfA proteins from both L. lvanov/1 and L. monocytogenes strahl L028. The tatter tra n was chosen because its PrfA is rdentical n length to the

p/cA

321 aa e• (327 bp) "PrfA·boX"

~sta t p~LA • • • • , G'rMAMGT t"'l'AGCAA.AAATATCGGA.MMC'rl'T'I"rAATAGGMTAatUCGCG 1032 Me R.BS

• . • • . -----(+1?) . TCCG'l'GATT~~CGACAACTATC'l'TT't'AGTGCGAT't"rA'l"t'ACI\IJ\AMTG 1.092.

. . . WMl}.I(TATGTTAAAMTT'l' CTAAACGAATCCATAA'l'TT'l'AA 115 2

-10 -35

GGÄCCrCATAGACATCCGATAATAÄAGATCAC~~CGATATcTrrxtccCAAT 1212

• • , . • • encl plcA ~CACC~CTATATGATTQGGMMTA'rCC'l'TTTATA'l'TCAAT 121.3

c • ivanovii TCTT1AACAAATG1T~GA 1loJplc A'AAtTTQT • monocyt. Ca1'TAACMATG't'l'AAcG blyfplcA ~~~g~

L. ivanovii TTTaACAAATG'l'cAAA mpl tm'l'"r.AQ~

L. liiOnocyt • T'l'aACAAATGTaM mp.l ttirM:'Ag~

PrfA of Llsteri 143

Ftg. 2. A, Sehemalle representatlon of the chromosomal reglon from L. lvanovif analysed here. The complete nucleo~de sequenc appears ln tt1 EMBL!GenBankiDDBJ NooleaUde Sequence Oata Ubrarles under the accesslon no. X72685. The orl nta on is ccording to the common scheme for the

Usteria virulence gene cluster. RBS: pu ative ribosome--blndlng sita. The vertrcal bars ln the Intergen c reglons lndicate the posltlon ot putative transcrlptlot"' term nators. Th horizontal bar {'8') lndlcates the posltion of the DNA fragment anaJysed ln Flg. 6. The post on and orlentatlon of the PCR prim used here ls lndlcated (arrow wtth M*numbers) as weil as the tnsert on s t of Tn 1545 ln the mutant 816. B. Nucfeotide sequence of the in rgenlc reglon between prfA and plcA from L lva.novll. Putative .-10/- 35 regrons, the putativ libOsOme·blndlng srte (RBS) nd the stem-Joop (putatiVe. transcripUon term nator) distal from plcA are underlined. The presumptfve transor1ption tart te (+ 1?) h been located by analogy wfth .L. monocyto· genes (Mengaud st .a./., 1991 ). Regularfy paced (dA). trac are indlcated by

broken Une above the sequence, and the paJindrome downstream from the prfA promo­ter s lndlcated by a broken dOUble U beloW the sequence·. C. Comparlson of •prfA boxes . lhe sequences for L monocytogsnss hlylplcA and mpl are from Mengaud et sl (1989) and Domann et 81. (1991); the sequence for L ivsnovil mpl is from our own unpubllsh d results. Non·symmetrlcal nucteodde are g n n lower case.

1:.. ivanovll protein whereas PrfA from straln EGO is less milar at its C.terminus; in partioular lt is two amlno

~lds shortet. Other relevant features of plcA and prfA and of the deduced amino aeid sequences are summar· lzed in Table 1. The homology between the PI·PLCe from the two ,Usteria spec1es was evenly distributed over the entlre length of the protein.

t has been shown that Integration of Tn 1645 occurs VI

homologus recomblnation between the transposon end and the target sequence (Trleu-Cuot et aJ., 1993). Our sequenoe analysls supported this notion. ln the mutant 8/6, insertion of Tn 1545 ocourred in-frame at nucleotlde 623 of prfA (-·TACAAA--) thus creating an aHer c- erm nus {206-FYVOTKYKFLUFL YFLKCS . 225) in

Te le. 1. Comparlson ot the prfAipiCA chromosomal reg1on. from L. lvanovil and L. monocytogenss and of the respectlve g;ene ptoduc (EUJ deduced frorn the nucteotid sequences).

(a). Comparison at the nucfeotld& sequence Je.vel

L. ~~ytogenes(bp) L lvanovil (bp) Per cent identity

prfA

(b) Comparlson at the amlno cld sequence Ievei

PrfA

prlA-plaA

2:12 297 78.2

L monocytogenes, No. aa (kDa) L lvanovll No. aa (kDa) Per cent identlty

2.37 (2.7.31)ll 237 (26.88) n .2

Per cent simiJarff}P 89.9

aa: mlno ac ds. a. Strain L028 (Mengaud eta/,, 1991). b. Straln EGO (Leimes er·Wächter et al., 1991).

plcA

951b 96S 72.8

PI-PLC

plcA - hlylflo

242 2 0 n.9

317 (3&.29)b 321 (36.65) 68.4 80.1

c. Calculated by the program BESTFIT of the UWGCG program package (Deve ux et al. 1984).

144 R. Lampidls et al.

Pr A.L P tA . tm NtcA i"'lr Crp

Pr ,L PrtA.Lm

t Fnr C.rp

~rtA.Li PrfA.Lm

c.A rtlr c p

Pr A.LJ. Pda . ldn N cA t"nr Crp

PrfA.Li Pr A.Lm NtcA Fn Cr

Pr .Ll l?rfA.Lnl

t E'nc Ct

~lp.Jf~~~1fll"' E MIPeKR lR IQSGGCAlHCQOCSISQL

MW..GIKR'"I'll

which amino acids 210-225 were changed and whieh was 12 amino aoids shorter than the wlld·type prote n (compate ·to 1g. 3).

ln the intergenlc region between llo and plcA a 20 bp palindromic equence (Fig. 2C) could be detected which wa centred between the putatiVe - 1 0 reglons of the two genes. Jt contalned the 14 bp palindromlc sequence (' rfA box·. Mengaud et al., 1989) identified in front of prfA·regula ed genes ln L monocytogenes. A simllar palln­drome, 12 bp long~ was found ln front of prfA in this case, however. between the putative promoter and the ribo some-blndlng s1te in. the transcribed part ot the prfA gene (FI . 28).

Th C-terminus of a th rd open reading frame (53 am no aclds, see EMBL accession number X72685) was detacted dlsta1 ot plfA; sequencing of a ONA region further upstream dentnled another part (75 amino acids, data not shown) of the same open readlng frame. A com­puter homology search ln the EMBL database revealed a stgniltcant 66% identity/85% simllarity of these poly~

peptldes to the respective regions at the C-termlnus of the enzyme phosphoribosyl pyrophosphate synthetase (PRS, ATP: o·ribose-5-phosphate pyrophosphotransfer· ase. E.C. 2.7.6.1) from Bacillus subtilis (NIIsson et al. , 1989).

l'1 l? 22 49 21

6 6fl '12 91 69

108 0

U5 42

157 157 162 190 159

204 20 209 2ll 204

237 237 222 250 21~

Flg. 3. Multiple amlno ac d uenc aJ gnment of Pl'fA from L lvanovll (PrfA.U; thls work) end l.. monocytogenes L028 (PrfA,Lm; Mengaud et a/., 1991), NtcA frorn Syn«Jhococcus (Vega·Palas et al., 1. 92), nr and Crp from E. co/1 ·(Spiro and Gue t, 990· Cossart and GicqueJ-Sanzey, 1982). The putative hel x~ um-hell a the N-term nus ot PrfA and the DNA-blod ng hellx-tum-hel x of Crp are ndlcated abov or belowth sequence, respeotfvety. The glyclne Ir\ th tum reglon s marked by tl , nd h ghly oon erv gJyclnes n the N-term nal part ara lndlcated by arrows. Amino ecld$ whlch ar slmifar in PrfA and in at least two of the ·Oth r prote n are bOxed. Groups of slmllar amlno seid were follows: l) A, $, T, P, G· I) N. 0, E, 0 : Ii) H. R, K; lv) M, L, I, V; v) F. Y, W.

Transcriptfon analysls of the ivanolysln gene (llo)

ln order to test tf in L. ivanovii the efficient transcriptlon of the ivanolysln 0 gene (1/o) ls dependent on the integrity of prfA as has repeatedly been demonstrated for hly trom L. monocytogenes (leimeister-Wächter et s/, 1990; Mengaud et al., 1991), primer-extenslon experiments were performed on total RNA isolated from L lvanovll wild type and tts mutant 8/8, respectlvely. ~For com­parison, total RNA from L monocytogenes NCTC7973 was lnvestlgated in .paralfef. Ftgure 4 shows that in the L. lvanovii wild type (lane b) transoription of 110 started at position + 131 from the Initiation codon and a second weak start site was located at positlon + 121. ln the prfA mutant 816, efficlent transcriptlon of Uo was abrog ted (tane c). However, weak secondary transcnpt1on start slte further downstream (positions +87, +83) could be detected).

Electrophoretic mobl/ity of the DNA fragment b tween plcA and prfA

The intergenlc reg on between plcA and prfA (Fg. 2C) contains several oligo-dA and ongo-dT tracts. ln partJc.u­lar, three (dA)• s tracts (indicated by a broken llne abov the sequence) spaced 9-10bp apart are found ln the

a b c standard

Flg, 4. Prlmar--e.xtens on anaJysls of listaliolysin 0 transcr ptlon. Lane a: L. monocytogenes NCTC 7973 (h/y-speciflc pr1mer); lane b: t.~ lvsnov wild type an<~ \ane c: mutant e/6 (bo\h wlth an 1/o-spec ftc ptlmer). Tr:artscrlp lengths were datermlned from standard sequenolng raactlons ( · gtlt panel).

putative promoter reg on of prfA. Such a pattem 1s typlcaJ for DNA wlth an intrinsic curvature (Wo and Crothers, 1984). A recomblna.nt prasmld contaJning the Intergenie

bp

1018

616 _ 506-

394 344

298 -

Flg. 5. ElectrophOretio mobiUty of tne h1tergenlc ONA fragment betw$en prfA an<S plcA from L fvanovil. From a recomb1oant plasmld. a 327 bp DNA fragment ('B' in Flg. 2A), comprlslng the entlre Intergenie sequence, was out out by AvaiVKpnl and electrophoresed on 6% polyacrylam de gels at 4 C or 60 c. respeclive\y {lanes b). Lanes e.: length s\Bndard {~ kb ladder, Bethesda Research Laboratorles); fragment slzes are lndlcated on the left. Stalnlng was wtth ethidfum brom de.

PrfA of Llsterla 145

reglon described above was dlgested with Avaii/Kpnl and electrophoresed on 6% polyacrylamide gels at 4 C and n parallel at 60 C (Mlzuno 1987). Figura 5 shows that the 327 bp fragment ('B' in Fig. 2A) which contalned the three (dA)4-s tracts in its centrat part, had an unusual slow mobllity at 4°C but migrated close to normal at 60 C. Thls behaviour was clearly lnd1catlve of a bent DNA structure.

PrfA~dependent profeins (PdPs) of L. tvanovii

Efficient expresslon from the vlrulence gene cluster ln L. monocytogenes and presumably also ln L. lvanovli, ls strictly dependent on an intact prfA gene. Aecentfy, we have demonstrated thaf under specific nutritional con­ditlons t.e. during cultivation in a minimal cell cutture medium (MEM) and a 5% C02 atmosphereJ primartly PdPs were synthesized de novo ln L. monocytogenes. Many of these [355]-methionine-labelled PdPs were local• ized on tha outer oell surtace and thus could be released by a mild treatment with detergent Protein analyses on SOS-PAGE could detect all the known gene products from the virulence oluster among the PdP plus several additional proteins (SokoJovic et aJ. , 1993). Flgure 6 shows the results ot a simitar experiment with wild•type L. /vanovii, lts prfA mutant 8/6 and a prevfously characterized Isogenie mutant deficient for leoithinase. L. monocytogenes Ncrc 1973 e.nd its prlA-defec ve mutant SLCC53 were lncluded for com­parison (lanes a b). ln the mutant 8/6 of L. ivanovii (\ane d). at least 14 protelns cou\d no \enger be detect&d whfch were Iabelied in tha wild•type strain (tane c), identffy .. rng tham as PdPs. By oomparlson with the isogenlc tran .. poson mutant 34/26 Oane e)1 1eci1hlnase was ldentffied es one o1 the PdPs. As was shown above, transcription of the ivanolysln 0 gene (i/o) was also prfA-dependent. lmmuno .. blotting with a specif c polyclooal rabbtt antiserum con­flrmed the transcriptionaJ results when ivanolysin 0 could not be detected tn the prfA mutant 816 (Fig. 6, tane g). No appropriate mut.ants were available for Pl-PL-C (plcA) or Mpf. However, the 34 kDa and 57 kDa proteins that were missing in mutant 8/6 were tentatively identlfled es Pt-PLC (34 kDa) and Mpl (57 kDa) based on the known molecutar weight of Pl-PLC from L. ivanovil (Table 1) and of Mpl· from L monocytogenes. r'espectiVely (Domann et al. , 1991). lnterestingly. no PdP of 92k0a, which in L. manocytogenes has been identified as the actA gene product (Kocks et al. 1992· Domann et al, 1992), was detactable in L. ivanov/i. One protein of about 70 kDa and of unknown function was detected ln mutant 8/6 at a greater Ievei than in the wild-type L ivanovil Figura 6 also shows that the pattem of PdPs 1s slmllar for both Listeria species but also crearty exhib ts number of differences with regard to PdPs of yet unknown functlon

146 R. Lampidis et al.

pl

PI-

Flg. 6 PrlA~epeodent proteins (PdPs). Surlaoe protelns .speclf eally labelred wlth (35SJ-methlonlne ln MEM Md solublllzed wlth 1% SOS {see the Experlmff!ntaf procsdures) were separated by SOS-PAGE (13% polyaerytamide/1% SOS} and autorad ographed (lanes a-G . Lane a: L monooytogenes wild type (NCTC 7973); lane b: L. monocyrogenes PrfA·defic ent mutant {SLCC53); lane c: L ivanovii wild type; lane d; mutant 816 (PrfA·deflcJent)· lane e: mutant 34fl6 (d&ficient tor lecithlnase). lvanoly n 0 (ILO) was detect d in non·tabelled sample by lmmunoblottlng with antt- vanolys n antlserum. Lane f: L. lvanov/1 w td type, 1· ne g: mutant 816. Positions of morecul'ar welght marker are indlcated on th laft, and dentlfled (.Jen products (arrow ~ on the right (PI .. PLC: plcA g ne produot; Lee: lec:ithin se). The putative Mpl proteln ts marked by an ast ri · , nd PdPs ot unknown function by black do1s.

partfcurany in the molecular mass range between 40-50 kDa and above 80 kOa.

Di eusslon

The chrom omal organ zatJon of the virulence gene cluster in L. monocytogenesls known in detan (e.g. Portnoy et al. 1992). ln the case of L lvanovll, the gene (i/o) for the spec1e · -speciflc Iisterioiysin 0, lvanolysin 0 OLO)~ has prevlously been cloned and sequenced (Haas et al. 1992). However, nothing was known about 1he genetlc organizatlon and the regulation of vlrulenc& genes in L. ivanovii. ln thls study, we have characterized a 24nbp chromosomal reg on from L. lvanov1i upstream of the ilo gene.

The O.terminaf part of an open reading frame which showed 66% ldentlty/85'% Slmilarity to the G-terminus of the enzyme PRS from B. subtllis (Nllsson et al., 1989) was identified at the most distal end. An almost identical open read ng frame can be found dtstal from prfA in the sequences publlshed for L monocytogenes (Leime Ster­Wächter et al., 1990; Mengaud et al. 1991) but was not identified as part of a presumptive prs gene. PRS catalyses the biosynthesis of 5-phospho-o-ribosyl-1-pyrophosphate (PRPP). a key intermediate in the

biosynthesis of pyrimidines, purines, hlstidtne, tryptophan and NAD•. From thls, it ls clear that PAS s Indispensable for normal bacterial growth. Although the sequence is incomplete and the proteln functtonally untested, the signlficant homology of the open readlng frame from patho­g.enlc Listerlas to· PRS must be taken as strong evide ce that this reglon deftnes the prev ously unknown 'left border of the virulence gene cluster. A gene encodlng Iactate dehydrogenase has already been ldentified a compnsing the light' border (Vazquez-Boland et al., 1992). ln the reglon between prs and llo. we dent'fied two genas with signiflcant homotogy to the L. monocyto· gene prfA and plcA genes. Differences in the DNA sequence of the prfA genes from the two Llsteria spectes were malnly found Jn GC base palrs; this could exptain why a prfA gene probe from L monocytogenes dld not hybridlze under stringent condltlons to L. ivanovli chromo · somal DNA (Wemars et al., 1 992). The PrfA proteln from L. lvanovfi, as deduced from 1he coding sequence is very slmilar to PrfA from L monocytogenes (Fig. 3 and Table 1). only the reg on between amlno actds 1Q0-120 being rather heterologous (35% identity/85% s milarity).

Our primer-extenslon experiments clearly have demon­s r,ated that in L. ivanovil an efffcient transcr1ptlon of the IIsteriolysin gene (l/o) ls dependent on an lntact prfA gene. For wild-type L. lvanovl1, wa could show that 1/o ·tran· scr pt on starts from two cJosely spaced sites, primar ly at position +131 from the ilo lnitlat1on codon. Upstream trom there no extensive homology to consensus - 1 0/- 35 promoter boxes could be found; however, a 20 bp palindro· mlc sequence {'PrfA box ) centred around posltlon - 42 was detectable. ln the prlA-negative mutant 8/6, a weak transcriptJon of //o~ startlog at posttion t87/+83, was seen. These results are sim lar to hose recentty desoribed for hly from L monocytogenes (Domann et s.l., 1993). No measurable amounts of lvanolysin 0 were detectab'le in the culture supematant of ttlis mutant. and, in contrast to prlA mutants of L. monocytogenes, no evldence for the escape of ·the L. /Vanovli mutant from the phagosome of lnfected Caro-2 cells has been found (Karun agar et aJ. 1993).

ln L. monocytogenss PdPs can speclfically be Iabeiied and 1Solated (Sokolovlc et al.. 9.93). The pattem of PdP from L ivanovii, when assayed by the same method. was slmllar to that trom L. monocytogenes. lvanolysin 0 , PI-PLC and Iecithine e clearly oould be ldentlfied a PdPs.. The presence of a funotjonal Mpl oould not be directty proven because of the Iack of mutants and speclflc antisera. However. our prelimlnary and unpublished data revealed the presence in L. lvanovii ot .a sequence with high homotogy to mpl from L. monocytogene . n addltion. a protein of a slze appropriate for Mpl (57 kDa, Domann et al.. 1991) was mlssing in the prtA mutant 8/6. No PdP of a size comparable to ActA could be found in

L ivanovii. Recently it has been shown that in lnfeated eukaryotic ceHs, L ivanovil also induces act n-tail forma­tlon\ although it is less efffclent than L. monoe;ytogenes (t<arunasagar et al., 1993). Taken together, we conctude that all the oonstituents of the virulence gene cluster from L monocytogenes with the possible exception of ac.tA, are also present ln L. ivanov/1 and are co-ordlnately regu­lated by prfA. The chromosomal organlzation of the prfA, ploA and 1/o genes was found to be identlcal to that ot L. monocytogenes. lt remalhs to be elucidated whether this Js also tn.Je for plcB and mpl genes. lnterestlngly a number of yet unidentlfied PdPs from L, ivanovii showed differences compared to L monooytOgenes. We cannot rute out the possiblfity that a few of the Jow molecular mass protetns constitute degradation products of larger proteins. Among the ditferrng prote1ns was one of Mr 70 that was obviousty enhanced in tha absence of a tunc­tlonaJ prfA genel simßarty to the effect described for a 64 kDa proteln of L. monocytogenes (Sokolovic et aJ., 1993). lt s not known lf the expresslon of these proteins is directly repressed by PrfA or is under the echtrot of another PrfA·acttvated gene.

There ls good evidence that PrfA specifically Intersets wlth the pallndrome ('PrfA bot) found upstream from PrfA-dependent genas (Freitag et al., 1992; 1993). Flgure 2C shows a comparison of the two 'PrfA boxes' 'for which a sequence is avallable for both L. /vanovil and L. monocytogenes ln thls ftgure we have expanded the h!y/plcA box (L. manocytogenes) up to the GC pair, indicat­ing one mismatch. The sequence for mpl of L. ivanovllis from our own unpubllshed work. ilo/plcA and hly/plcA respeotively, are transcribed in opposite, dlrec1ions but use the same 'PrfA box', wh1ch ls 4 bp longer than the respectlve mpl box. The paJindromes from L /vanovil are 2 bp Ionger than fn L. monocytogenes. ln both Iisteriai species the mpl palindrome is lmperfect. Thls may influ­ence the blnding of PrfA and therefore mlght be related to the observation by others (Freitag et al., 1993) that apparently mpl needs higher amounts of PrfA for its actlvatlon than hly/plcA.

The analysts of the lntsrgenie regions between llo or hly and plcA on the one hand and between plcA and prfA on the other hand revealed several interesüng featun~s e>f these DNA sequences. First. both intergenic reglons are remarkably conserved among the two Usteria species (see Table 1). Figura 28 shows that upstream from prlA rather weil oonserved - 35/- 1 0 boxes can be found I and It has been demonstrated (Mengaud et al., 1991; Freitag et al.. 1993) that in L. monocytogenes a mono­clstronic transcnpt of prfA can be initiated from thls proma­tert in addition to the biclstronic p/cA!prfA transcript. ln this case, no PrfA box' was found ovetlapplng with the - 35 region. However we have .now detected a shorter, but otherwise aJmost ident1cal pallndrome (T AACAA TTGTT A

PrfA of Llsterla 147

in L. ivanovil~ TAACAA11'GTTg in L. monocytogenes) downstream from the transcription start site in the non­translated reglon of prfA. Furthermore. in the case of L. ivanovil we could demonstrate experlmentally that thls ONA region ls lntrinsically bent presumably through the presence of regularty spaced (dA)4-s tracts. This inter­ganie region is 25 bp shorter in L monocytogenes but con­talns simllar (dA)n tracts at comparable positions. No such sequenoe pecullarities can be found upstream of other PrfA .. regulated genes. 1t has been shown by ofhers (Fre1tag et al., 1993) that in L. monooytogenes the mono­cistronic transcript from this promoter is greatty enhanced in the absence of a functional PrfA protein. These tesu ts as weil as our own suggest that in thls case PrfA nega­tively controls transcription ot its own gene. most probably by btnding to the paJindrome mentioned above. ln the absence of PrfA, transcrlption may be stimulated by the lntnnsic curvature of this DNA region.

The symmetry of the blnding site on the DNA suggests that PrfA. like other DNA-binding protelns may bind as a dimer. Our oomputer secondary structure predlctions. uslng the aigor\thms of Chou and Fasman (1978) and Rose (1978), for the PrfA proteins from the two Listeria species identlfied several reglons as potential candl­dates tor DNA·blndlng or dimenzation domains. At the No-terminus~ centred atound G-16, a hellx-turn .. h&lix (HTH) motif was predicted which fulfilled almest all of the stereo­chemical constraints establlshed for true DNA·bindlng HTHs (Dodd and Egan, 1990). At the very G-1erminus a rather long tt-helical domain was predicted. Within thls region, leueine and some simrfar amlno acids were found ,in heptad intervaJs (L-1931 VIE-200, Y-207, L·214, L-221 ). Suoh a hep ad array ls charactertstic of the so-called Jeueine Zipper (Landschulz et al., 1 988), a dlmer­izatJon domain found ln both eukaryotes and prokaryotes. Whether these predicted structures constltute functlonally important domalns is not known at present

More importantly, however, a multiple amino ac1d sequence allgnment rev.ealed a strlklng homology of the entJre PrfA sequence wlth the Crp-Fnr famtly of global transcr\ptlon regulator , wh\ch \ncludes NtcA from Synechacoccus (Vega-Patas et s/., 1, 992) Crp and Fnr from E. co/1 (Cossart and Gicquel-Sanzay 1982· Splro and Guest, 1990) (Fig. 3). This finding fits wen lnto the proven roJe of PrfA as a pJejotropic regulatory protein.

The three-dimensional structure of Crp 'ls known at a 0.25nm resolution (Weber and Steitz, 1987); tha proper­fies of this regulator have been reviewed recently (Kolb et al., 1993). The ONA-bindlng domaln of · Crp has been cnaracterized as a HTH. spanning amino acids 168-191 and centred around G-178. PrfA as weil as the other members of the protein famlly show a particularly high degree of simiranty to this domaln at a comparable position (in the case of PnA 17 amino aoids out 24).

148 R. Lampidis et al.

From 1his we concluda that this region constitutes the ONA-btndlng domaln of PtfA. ln the region between amino acld 133-152 of PrfA, we could not detect any par· ticular homology to Crp nor was a HTH predicted hare. which rs in contrast to previous clalms (Freitag et al., , 992). No homology courd be found for the amlno aclds lnvolved ,ln cAMP bindlog by Crp (E-72, A-82, S-83, R-12~, T-127 and S-128} and for the crltlcal cyst.elne residu (0 .. 16, C-20 C·23 and C-2.9) in Fnr {Spiro and Guest, 1990). fn thls oontext lt is lnterestlng that cetlobiose but not gtucose was shown to influenoe PrfA·regulated IIsteriolysin expression ln L monocytogenes (Park and Krolli 1993; Datta and Kotharyl 1,993). Four ofthe glycines strueturally lmportant ln Crp1 whlch are ht9hly conserved among Crp--like proteins, were found at appropriate positions in PrfA (indicated by atrows in Fig. 3). Further~ more. a high probabiUty for short ß-sttands was predicted fn this region. The predicted et .. helicity for the domain between amino acids 109-130 of PrfA was rather low. This is in some cantrast to Crp where an extended ~-he,lix at this position has been Jdantifi d e~perjmental .y

as the dimer contact area (Weber and Stenz. 1987). Othe,r difference from Crp 'nclude a potential second N-termlnal HTH and a leuclne-containlng a:,..helix at the 0-termlnus, atthough possible 1unctfon remain unclear at present. lt seems premature to conclude 'from these data that the three-dimensionat structure o·f PrfA is, simltar to Crp. lncludlng the equlvatent of a nucleotide-blnding domain wlth ß-ron structure near the N-terminus.

To our knowt~ge PtfA is 1he second example of a Crp/ Fnr .. Uke 'regulatory protein fn Gram ... posltlve bacteria, the f rst one be' ng Flp trom Laotobacillus casel (lrvine and ~Guest1 ,. 993}.

Transoription of prfA as weil as ·prtA-tegulated gene expression are affected by sugars (see above) and a number of olher condltions, e.g~ tempem.ture, culture medium and pH (Lelmeister-Wächter et al, 19,92; Soko­loVie et a/., 1993; Oatta and Kothary, 1993). Future lnvestigations wllt be designed to further elucidate the mechanisms by Whioh transcriptlon of pr/A ls regulated, how PrfA ifseit is active.ted and how lt regulates vlrulenoe gene expcess10n in Lfsteria.

Ex . rlm ntal procedur

Bacterlaf stralns and p/asmlds

L. ivanovii ATCC 19119 (SLCC 2379) and L. monocytogenes NCTC 7973 (SLCC 2371) and SLCC53 were obtajned from the Listerls straln collett1on of tha Institute of Hygiene and Microb ology of the Universtty of Würzburg. The mutants of L. ivanov/1 ATCC 191 19 used ln 1hls study have been obtalned by conjugal transfer of the· streptococcal shuttle transposon Tn1545 frorn L. monocytogene BM4140 klndly provded by P. Courvalin (Pasteur Institute. Parts) and have

been descrlbed prevlously (R. Schleslnger, 1988· Kraft et al .• 1990). The E. co/1 strafn DHS-alpha was used for transtorma­tion and ctoning. The E. coll vector plasmld pTZ18R (Mead et al. , 1986} was purchased from Phamac a.

Media snd antiblotics

L ivsnovfl and L. monocytogsnes were grown in brain- h ert Infusion broth (BHI, Dlfco), whereas E. ooll strains were passaged in turla-Bertani (LB) broth at 37 C. For Tn 1545 mutants of L. ivsnovtl, tetraoycllne wa added at 4 ~g ml- . E. co/1 transformants were grown with 100 ~g ml 1 amplcliUn. Blood ag r conststed of blood agar base (Oxo d No. 2) suppfemented WJth 5% (v/v) sheep blood. Egg yolk agar Wa$

prepared by adding 0.5% (v/v) of fre$h egg yolk to a basal medium (1% bactopeptone, 0.2% beef extract, 0.5% Na Cl 0.03% cysteine-HCI, 2% agar, pH 7.4).

Restrietion en~ymes a:nd llgase were purchased from Pharmacia or Boehringe.r and used as recommended by the manufacturer.

Haemolysin and phospholipase c assays

Ha:emolytlc tnres of cuJture supematants were de1er:mJn d in mlcrotltre pletes s descnbed {Domingue~-Rodriguez t 1., 1986). PI·PLC actlvfty of L. fvsnovii stralns was estlmated after lncubatton t 37 c tor 48 h by halo formatlon round · b ctenal co onies on LB agar plates wlth an 0.7% (WN) ag r overlay oontalning 1% {wlv) L-d-phosphat'dyllnositol (Slgma Chemlcals). Lecithinase productlon was visuatlzed by the formation of a preclpltatlon tone around bactenal streaks on egg yotk agar (see above) after 48 h lncubation at 37 C.

Polymerase chaln reaction

Chromosomal DNA fragments of L. ivanovil wild type and its mutant 8/6 were ampllfied by PC.R a.ccordlng to published procedures (S;alkl et al. 1988; Bubart etsJ: , 1992), us1ng Taq ONA polymerase (Pharmacia). Thermal cycling (30 cycles) consisted af denaturatlon for 1 mln ac 9 llc, primer ann·. anng for 1 mn at 52 0 and prlmer exten ion for 2.5mih at 72. C. The oligonucleot de pt1mers, synthe zed on A373 ONA synthes1zer (Apptiad Biological fnstrumen1s) were MS25: · 5'-GiATGTTCCAGGOCTCTCCITC-3' (H~proximal) ; M395: 5' CATAGMTAAGGCTITACGAGC .. 3' (Tn1545 right end): M396: 5'-GA TAAAGTGTGAT AAGTCCAG·3' (Tn 1545 left end); M397: 5'-CCTACACTAAAAGGCCTCCGCGG .. 3~ (11&­upstream); M449; 5'-TCCGCAAATAGMCCTAGC..S' and M450: 5'-TGTTAGCAGAATTCTiTC .. 3' (fo Inverse PCR): M476: 5'·TITTCCTGTATCAAGGGAAC~3' ( ptfA .. intemal).

Nucleotlde sequence and primer .. extenslon ansJyses

Nucleotide sequences were determlned from both strands of recombinant ptasmids contalnlng different segments of the respectfve L ivanovil genas. Sequencing reactions w · re performed wlth a commetcl t kit (Pharmacia) using super .. colled ternplates a.nd syntheti~ oligonucteot1de prirners (15-18 bases). Computeranalysesand homology searches in the EMBL database were performed with the Universlty of

Wisconsin Genetics Computer Group program package (Devereux et al., 1984) run on a VAXNMS oomputer or with the· PRCSIS program package (Hitachl) on a MS-OOS personal computer.

Total RNA was lsolated from Iog-phase lister al ceUs, kept on ·ce for 15mln in 1M Trls--HCI, 0.1 M EDTA, pH e.o and 1 mg ml 1 Jysozyme. Triton X-100 was added to a ·final ooncentratton of 0.1 %, followed by 15 mln incubation on ice. The lysates were extracted twlce wlth 1 volume o1 phenol/ chloreform (1 :1 v/v) at 65 C Nuclelc acids were precip tated wlth 2.5 volumes of1 ethanol and 0.3 M sodium acetate pH 4 8 .a\ - 70 C then oentrifuged through a CsCI density gradient (rotor TLA100.2, 80000 x g, 15 c. 16h). The RNA peltets were dissolved in waterj reprecipltated with ethanol as above, then treated with ANAse .. free DNAse (0'"25 U pl1- 1 in 20 mM Tris·HCI, 1.5 mM MgSO ... pH 7 5) for 40 min at 25 C. These prepa.rat1ons were then extracted twtce with phenoVohloro .. form. Prfmar .. extension reactions were carried out aoeordlng to a prevlously described protocol (Lelmeister-Wächter et er., 1990), uslng synthetic oligonuoleotides spannlng 20 nucleo-­tldes immediately upstream of the Jnltiadon eodon of i/o. or hly &$ prmörs.

Eleotrophore.sls of ONA fragment at 4 C and 60~ was performed in 6% (wlv) pQtya.crylamlde gel With 40 mM Tris­acetate 5 mM sodlum acetate, 1 mM EDTA. pH 7.5 as buffer.

Lßbelllng, isolatlon and gel electrophoresis of PrfA+dependent protelns from Listena

P:'dPs from L. IVsnovli and L. monocytogenes were labellad wlth ~S]~methlonine in MEM (minimum essential medlurn wit}'l Earle'S salts, Gibco) and anatysed as publlshed (Sokolovio et al., 1993). ln brief~ bacterta were grown a1 87 C ·to 00000 1.0. washed and resuspended ln MEM wtthout L..gJutamine and L·methloni·na. After preincubatron without shaklng for 30 min at 31 C in a 5%" C02 atmosphere 25 pCi ·Of FS]-methtonine were added and the bacteria were lncubated lor an additional 60 min. For the isolatton of nonwradlolabelled PdP's, radloaotive methtonlne was omltted. CeHs were harvested by centrift)gation and washed three times wlth phosphate..buffered saline (PBS). Surfac.Q· protelns were released from the bacterta by shaJ<ing for 15 mln in the presence of 1% SOS at room temperature. All released ,proteins were preoipltated with tr1chloroacetic acid (7% final concentration) at 4 ·c. Gel electrophoresis was periormed on SOS slab gets (13% acrylamide; 1% SOS).

I Other technlques

Transfamtatton of E. aol/ DH5~atpha, all DNA manfputatlons and lmmunoblottin·g of non-labelled PdPs were pertormed accordiog to standard procedures (Sambrook et al., 1989}. Potyclonal rabbit anti-lvanolysin 0 antfserum was obtained by repeated sobcutaneous injection of toxln purrfied as pre­vlously described (Kreft et sl. 1989).

Acknowledgements

We are grateful to K. Brehm for valuable advloe and many helpful disaussions and to W. Schwan for critrcaJ readlng of the manuscript. M. Oumbsky and M. Götz are ·thanked tor

PrfA of Listerla 149

thelr expert technlcal assistance. Thls work was supported by grants from the Bundesministerium fOr Forschung und Technolog1e (BMFT 01K188059), tha Deutsche Forschungs­gem insohaft (SI=B 165) and the Fonds der Chemischen lndus rte.

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