Gene. 118 (1992) 121-125 0 1992 Elsevier Scicncc

GENE

Publishers

B.V. All rights rcscrved.

121

0378-I 119:92:‘SO5.00

06570 (CS)

A gene encoding a superoxide dismutase bacterium List eria monocyt ogenes t DNA;

(Recombinant

enzyme

metalloenzyme;

oxidative

stress;

genetic

complementation;

intracellular

pathogenic

bacteria;

hybrid

formation)

Klaus Brehm, Albert Haas*,

Received

paraquat;

of the facultative

by R.E. Yasbin:

9 December

Werner Goebel and Jiirgen Kreft

1991: Revised/Accepted:

2 March,‘13

March

1992: Reccivcd

at publishers:

23 April 1992

SUMMARY

A gent (Imsotl) encoding superoxide dismutase (SOD; EC 1.15.1.1) of the facultative intracellular pathogen, Listericr was cloned by functional complementation of an SOD-deficient Escherichiu coli mutant. The nucleotide sequence was determined and the deduced amino acid (aa) sequence (202 aa) showed close similarity to manganesecontaining SOD’s from other organisms. Subunits of the recombinant L. monocytogenes SOD (re-SOD) and of both E. coli SODS formed enzymatically active hybrid enzymes in vivo. DNA/DNA-hybridization experiments showed that this type of recombinant re-sod gene is conserved within the genus Listeriu.

monocytogenes,

INTRODCCTION

L. monocytngenes is a Gram+

ing severe is able to cells such ‘facultative

bacterial pathogen causopportunistic infections in man and animals. It survive and to multiply within phagocytic host as macrophages and thus has been named a intracellular bacterium’. The uptake of L.

Corresporldence ro: Dr. K. Brehm. Biorentrum

dcr Universit&.

Lehrstuhl Mikrobiologie. Am Hubland, W-8700 Tel. (49-93 I) 888-4419; Fax (49-931) 888-4402. * Present address: Los Angeles. ’ Dedicated

Molecular

CA 90024-1570 to the memory

Abbreviations:

aa, amine

bp; L., Listeria; limi, Mn-SOD.

(USA).

acids(s):

UCLA, 405 Hilgard

Ave.,

Tel. (3 10) 825-6885.

Ap, ampicillin:

iron-containing

L. imwii

B.. Bncillus; bp, base

SOD; kb, kiloasc(s) or 1000

sod gene; ltnsod, L. monoqmgenes

manganese-containing

open reading frame; PA, polyacrylamidc; K, resistant’resistance;

Wilrzburg, (Germany).

of Dr. J.-P. Lccocq

pair(s); d, deletion; Fe-SOD, gene;

Biology Institute.

Wiirzburg

SOD; PAGE,

RBS. ribosome-binding

SOD, supcroxide dismutase; sod. gene encoding denotes plasmid-carrier state.

nt, nucleotide(s);

sod

ORF.

PA-gel electrophoresis: site(s): re-, recombinant; SOD:

Sv, serovar;

[ 1.

monocqltogenes by phagocytes induces a significant oxidative metabolic burst in these cells. resulting in the release of bactericidal superoxide radicals into the phagosome (McGowan et al., 1983). Superoxide dismutase (SOD; EC 1.15.1.1) converts superoxide into hydrogen peroxide, which then is metabolized by catalases and peroxidases. On the one hand, this enzyme is part of the common defense mechanisms of aerobic bacteria against endogenous oxidative stress. On the other hand, listerial SOD can counteract the oxygen-dependent defense mechanisms which play an important role in the killing of bacteria by phagocytic cells. Therefore, SOD is considered to be a putative virulence factor of Listeria (Chakraborty and Goebel, 1988). Recently the sod gene has been cloned from the animal pathogen L. ivanovii using genetic complementation of an E. coli sodA/sodB double mutant, grown under aerobic conditions on minimal medium containing paraquat (Haas and Goebel, 1992). L. monocytogenes is clearly distinct from L. ivanovii with regard to biochemical and serological characteristics (Rocourt, 1988). Among these two pathogenic, facultative intracellular Listerirr species, L. monocytogenes

122

exhibits a significantly higher virulence than L. irw~o~~i. To address the question kvhether diff’erenccs in the structure and regulation of SOD may contribute to the diff‘crcnt pathogenic potential of thcsc t\vo listerial spccics. we have cloned and characterized the .sod gene from L. /12o/roc:\‘rogc’llp.y. In addition, the isolation of this gcnc Lvill enable us to construct defined mutations in the .sodgenc for in viva virulence tests.

EXPERlhlF-NTAL

AND DISCLISSION

(a) Cloning of the L. monocytogenes sod gene and analysis of Escherichia cofi recomhinants .A gcnomic library of H;jzdIII-digcstcd chromosomal DNA from L. ~llollo~~‘ro)perrr.v Svl!2a EGD (S.H.E. Kaufmann. Ulm, Germany) was established using plasmid pTZ l8R (Ap”, IwZ’: Mead ct al., 1986) as a vector and E. chili DH5, (wA1. kllc,ZdMlj, hsdR; Bethesda Rcs. Labs.) as a host. Plasmid DNA prepared from 4000 recombinant clones of this library was then used to transform E. w/i QC779 (.&,425, .sotlBA2; Carlioz and Touati, 1986). In contrast to E. w/i DHSx. strain QC779 is restriction proficient and thus not suitable as a primary host for hetcrologous DNA. The resulting E. co/i QC779 transformants were plated onto sclcctivc LB agar containing IO0 pg Ap:’ ml and 0.05 mM paraquat (methyl viologen. Sigma Chcmical Co.. St Louis, MO). which induces intracellular superoxide generation (Hassan. 1988). and grown aerobically at 37 C. IJnder these conditions E. co/i QC779 cells Lvhich do not harbour a re-\od gene cannot gro\&. Afccr I5 h of incubation, 18 clones resistant to paraquat-induced superoxidc generation were isolated all containing a re-plasmid \vith an identical 2.17-kb insert of L. Il7orloc:l’togerze.sDNA. Retransformation of E. coli QC779 with thcsc rc-plasmids reproducibly yielded transformants which were resistant to paraquat-induced oxidativc stress. Cell lysates from the positive clones were clectrophoresed on nondenaturing PA gels and stained for SOD aclivitj (Beauchamp and Fridovich. 1971). All clones showed a SOD activill band which comigrated with the authentic L. ,,7o/7o~~‘roRrr7e.r SOD (R, value 0.64 relative to bromophenol blue on 12.5”. 777o/7oc:l’rogcl/rr.~ SOD all five aa residues which can be used as ‘primary candidates’ to diffcrentiatc between Mn-SODS and Fc-SODS (Gly’“, Gly”. Phc”“. Gln’“J , ‘4s~“‘: Fig.1) and IJ out of I7 ‘secondar) cundidatcs’ (Parker and Blake. 1OX8a) correspond to the Mn-SOD type. These findings suggest that L. /?7o,lo~~‘toRene.rSOD most probably is a Mn-containing cnzymc. as has already been sho\vn by cnzkmatic and structural analysis for the closely related L. iww\fi SOD (Haas and Goebcl. 1992). (c) SOD activity of various Escherichia coli sod mutants containing pAHAS It has previously been shown that subunits of the rcL. iwu7orii SOD form cnzymatically active hybrids in viva with subunits of both E. c~)li SODS (Haas and Gocbcl, 1992). Here WC show that such a hybrid formation also occurs in 15’.cwli harbouring the re-lwsod gene on pAHA (Fig.2). As has been mcntioncd above, in cell lysates prcparcd from E. c~li QC779,wd,425,rodBA2[pAHA8] onI> one SOD activity band comigrating tvith L. /17o/roc:l’loge/7e.sSOD could be detected (Fig. 2. lane 5). This protein was overexpressed in E. cwli. amounting to about 15”,, of the total soluble cell proteins (not shown). Howcvcr. in E. w/i DH5xyodA ’ .sodB f [pAHA t\vo activity bands in addition to c‘. co/i Fe-SOD, Mn-SOD and re-L. ,7rorzoc:l‘[c~~~rr7e., SOD could be dctcctcd (Fig. 2, la11c 7). one of thcsc ac-

123 .

.

TCGGGAGCATGGTAGGCTATGGTGTAAGAAGAAACTGTGGTTGATAGTAGTT

60

CTATTGAAATAGGACATGACTTTTGCCTTATACGTCATTTCTTTTCACGT~C~

120

TACAAGGAGGAATTTTTAATGACTTACGAATTACCTACCT~TTACCTTATACTTATGATGCT M T Y E L P K L P Y T Y RBS

D

A

), NcoI TTGGAGCCGAATTTTGATAG~CCATGGAAATTCACTTACT IHYTKHHNT LEPNFDKETME

240 34

I,

1,1,

TATGTAACAAAACTAAATGAGCGGTTGCTGGTCATCCTG~CTTGC~GC~TCTGCG YVTXLNEAV~GH~ELASK~~54

300

GAAGAATTAGTTACTAACCTAGATAGCGTTCCTGAAGATAC

360 74

CACGGTGGCGGTCATGCTAACCATACATACATTGTTCTGGTCTATTCTTAGCCC~TGGTGGC HGGGHANHTLFWS I L s P N

G

G

420 94

F

D

E

480 114

PstI TTTAAAGAAAAA TTCAATGCAGCAGCTGCAGCACGTTTTGGTTCTGGTTGGGCTTGGCTA F K E K FNAAAAARFGS G W A

W

L

540 134

P

L

s

600 154

Y

L

K

F

660 174

W

D

E

A uR

A

AA

EcoRI GGCGCTCCAACTGGCAATTTAAAAGCAGCAGC~TCG~GCG~TTCGGTACTTTTGACG~ GAP T GN LKAA I ES E F GT

GTAGTTAATGATGGCAAATTAGAAATCGTTTCTACAGCTAGC V V N D G I v s TANQDS K L E N n

AA

GATGGCAAAACACCCGTTCTTGGCTTAGATGTTTGGGAACTTC DGKTPVLGLDVWEHAY nE A i CAAAACCGTCGTCCTGAATATATCGAAACATTTTGGAATGGCT QNRRPEYI E T F W N D cl

Fig. 1. Nucleotidc and restriction

180 14

A V

I

N

720 194

AACAAACGCTTTGACGCAGCTAAATAATAATCAT~GACTCACTTCGGTGGGTCTTTTTT NKRFDAAK**

780 202

AATTTCTAATGAATTTCTAA

800

sequence

of the //xrod gene and deduced

sites for EcoRl,

,%‘wI and Pstl are indicated.

differ in L. ircmwii SOD are shown below the aa sequence

aa sequence.

The GenBank/EMBL

A possible

weak. Rho-independent

accession

No. is M80526.

transcription

terminator

of L. monoc~‘togenes SOD (boxed). The aa which are primary

A putative

RBS, stop codons

is undcrlincd. candidates

Those aa \vhich to differentiate

be-

tween Mn-SODS and Fe-SODS (Parker and Blake, 1988a) are indicated bq open triangles bclou the scqucncc. and those which arc involved in subunit contact in B. stenn~thermo~philus Mn-SOD (Parker and Blake, 1988b) bq blackened arrowheads. Methods. Chromosomal DNA from Lisrrritr ~+its isolated according

to Flamm

according

to Maniatis

inserts in pTZ18R were subcloned

et al. (1984). Plasmids et al. (1982)

were identified

into pTZ18R

using a plasmid-sequencing oligodcoqribonuclcotidc

were isolated

by the p-galactosidasc

and the nt sequences protocol

primers

from E. cdi by an alkaline

except for the ligation during construction

(Pharmacia

(approx.

complementation

from both strands DNA

sequencing

110nt) were synthesized

lysis procedure,

assay (Maniatis

were determined

kit, 8”,, PA-14””

et al., 1982). Restriction

and analysis

by the dideoxy

fragments

chain-termination

urea (v&v) gels). M 13 universal

on a 380A DNA synthesizer

tivities (R,.value 0.61) also appeared in E. coli QC78 1 (so&t, Mn-SOD-deficient; Carlioz and Touati, 1986). This band migrated between E. coli Fe-SOD and L. monocytogenes SOD. The other activity (R, value 0.47) correlated with the expression of the E. coli Mn-SOD in strain QC870 (so&, Fe-SOD-deficient; Carlioz and Touati, 1986) and migrated between Mn-SOD and L. monocytogenes SOD (Fig.2, lane 1). From these results we concluded that these activities

DN.4 manipulation

of the library (PeEenka et al., 1988). Recombinant

(Applied

were performed

E. co/i DH5r

with DNA

from the pAHAR-insert

method

(Sangcr

and reverse primers

et al.. 1977). as well as

Biosystcms).

constitute enzymatically active hybrids between one subunit of the L. monoq’togelzes SOD and one of the E. coli Fe-SOD or Mn-SOD, respectively. This assumption is supported by the fact that seven out of the eight aa residues involved in subunit contact of B. stearothermophilus SOD (Parker and Blake, 1988b) are conserved in both E. coli SODS and in the L. monocytogenes enzyme (see also Fig.1).

124

1

2

3

4

5

6

7

8

9

IO

Fim 1 Functional cupreawn of rc-b~~ \ot/ m E. wii nnd 111\i\o formation uf active SOD hybrids. Cleared cell lyaatcs from the strains ~ndlcarcd hclou t’ -’ wcrc clcctrophorcsed on nondcnaturing 12.S”,, PA gels and stained for SOD activity. The positions of E. co/i Mn-SOD (MnSOD) and Fc-SOD (FeSOD).

1.. ,)10,10(‘!‘1,,,~~‘,~‘lle( n-SOD

(rSOD)

as ~~‘113s the hybrid SOD forms (rSOD/MnSOD:

Lanes: 1 and 2. E. UI// QC87O[pr\HAX 7 and 8. E. u~li DH.5r[pAHAX

or pTZ 18R]: Y. L. /~~o/~~~~~‘ro~e/rc.s; 10. purified

from ovcrnipht cultures of E. w/i carrying /rr~,,rl,~:~‘~~,,~~~,ea (BHI tans (Iuncs

I x~d 9) OI-5 pg of purified

-

pAHAX

(containing

I~uwd) or the \cctor

rSOD

(lane 10) L\crc clcctrophoresed.

-.-

-

2.0-

i

abcdefgh of HDrdIIl-digested

chromowmal

DNAs

internal fragment of [rxwd (nt 171-756.

Fig. I ).

The SILC (in kh) of hkhridizing fragmcnta IS indicated on the left margin.

L;~ncs:a-h, f.. ,~lorl~n:rrt,~e,rr.v strain

EGD

c. 1.. iww~,i~ (ATCCl91

or Mackancsa

(SLCC5764),

19); d. L. .tre/igrui (SLCC3379):

c.

ijur~~~~lrtr Svba; f. L. we/.shi~rw,-i;g. L. wrrru~~i: h, 1.. ,yw~,i; i. pAHAS,

dlpcstcd with Hirtdlll.

111 f.~ue,-irr strains were from the Special Llatcrln

C’ulturc’ Collcct~on (SLCC). ct a..

Gcrmany.

I “,,

Vethods

DNA frag-

agarose slab gels (Manintis

The DNA

using the

Ltd.) using ;Lcommercial kit (Pharmacia).

(1 h) and hqhridiution

filter\ wcrc w&cd autot-adiographed Na,,citratc

probe was cluted from a TAE-gel

Kit (Bio 101. Inc.. La Jolla. CA), and randomly labclcd with

]dATP (.,\mersham

11)bridiution

EDT,\

on TAE-

1987). denatured and transfcrrcd onto nitrocelluloae (Schlcichcr &

Gcncclcan

1x-“P

\?‘iirrburg.

\+crc elcctrophorcxd

Schucll. Gcrman\i).

or pTZ 18R]:

Cleared cell lysates were ohtaincd aftcl- sonication of ccII\

pTZ18R.

rcspccti\cly

(LB

broth.

37 C) and of cell\

from 1..

Fat- each Iqsatc. 90 log of wlublc pt-o-

staining for SOD activit> was done by the tlitr~~hlUC-tett-;~Loliti~ll

i

I,ig. 3. DN~~~DNR-h~bri~ation from LI\~L’w to a lab&xl

mcnt\

arc indicated

(d) DNA/DNA hybridization experiments To investigate whether DNA sequences homologous to IIHSO~are also present in other species of the genus Li.srerin. we performed DNA/DNA hybridization experiments with an internal fragment of hnsod (Fig. 1. nt 174-756) as a probe. Under high stringency conditions. related nt sequences could be detected in L. ~uotm~~toge~es Svl/Za strain Muckaness as well as in all other species of the genus (L. ivar~~ii, L. seeligeri. L. imncuu, L. welshimeri, L. ulrrrrtl>j. L. gm~i). indicating the conservation of this type of the .so(l gene within the genus (Fig.3).

-

3.0-

L

Negatwc

FeSOD/MnSOD)

and Ft-idovlch. I97 I ),

5.0-

rcspecti\cl~:

rSOD. Methods.

FeSOD/rSOD;

or pTZ IXR]: 5 and 6. E. co/QC779[p-\Hh~

ht-lath. 37 C: hcforc Ilsis cells \\\crc incuhntcd with 50 kcg;ml of mutunolqsin: Sigma Chcm.).

tmcthod (Bcauchamp

kb

rSOD/FeSOD:

or pTZ 18R. rcspccticcly]: 3 and 4. t. w/t QC78l[phH:\X

(16 h) \%crc in

twice for 30 min in I x SSCO. I ‘I,, SDS at 57-C for 24 h at -20-C.

pH 7.6: T4E

11.01X M NaCI

SSC i!, 0.15

is 0.01 M Tris~HCI:O.O2

pH 8.0.

Prc-

h x SSC at j7- C. and

M NaCl~O.015

M

M Na~acctatc’O.02

M

(e) Conclusions (I) Using an improved protocol for the genetic complcmentation of an E. coli sodAjsodB double mutant, the sotl gene from L. /nonoc:rtogerles was selectively cloned from a plasmid library. The aa sequence of the L. r7lorzoc:l’roRellr.c enzyme is almost identical to the previously characterized SOD from L. iwrzovii and closely related to Mn-SODS from other organisms. (2) L. rlrorzoc’l’toRer?esSOD and both E. co/i SODS form enzymatically active hybrids in vivo, probably due to the high homology of aa in the contact region of the enzyme subunits. The nt sequences homologous to Itmod could bc detected in all species of the genus Listeviu. (3) Survival of L. nwrzoc~vtogetzes in phagocytic cells is a crucial step in the development of listerial infections. SOD may play an important role in this process since it contributes strongly to the bacterial defense against toxic oxygen

125 metabolitcs generated by the phagocyte. Recently it has been shown that SOD contributes to the pathogenicity of facultative intracellular bacteria, Nocardiu mteroides (Beaman and Beaman, 1990) and most probably also Shigell~~,flestzeri (Franzon et al., 1990). The availability of the sod gene from L. monoc~togenes, the most virulent Listrriri species, will enable investigations on the synthesis and function of this enzyme within the infected cell. two

other

Recently. using the cloned gene, we have insertional mutagenesis a sod-deficient ,Izonoc:l’togene.r. Experiments with this progress and will help to clarify the role of infections.

constructed by mutant of L. mutant are in SOD in listerial

Dcvcrcux.

J., Hacbcrli,

quencc analysis 276-287. Flamm, R.K.,

Hinrichs.

into Listcria

bctwcen

natiw

Franzon,

J.and

Sansonctti.

and catalasc

Li,\rericr iwrrwrii by functional characterization

01

and homolog!

( 19x3)

P.J.: Contrlhutlon

to S/@~//rr flc,\wri

complcmentation

of the gent product.

of supcr-

pathqcncsls. dlsmutasc

Infect. gcnc from

in Ew/w~rt~hrtr w/i and

hlol. Gcn.

Gcnct.

231 (lY92)

313-322. Hassan,

H.M.:

Biosynthesis

Manual.

and regulation

Cold Spring

of super-ouldc

dlsmutascs.

J.: hlolccular

Harbor

Cloning.

Laboratory.

A

Cold Spring

NY. 1982. .A.P.. Peterson.

peritoneal

macrophagc

sponsc to osonired

P.K..

Kcanc.

phagocytic

\V. and Quit.

P.G.:

Human

killing and chcmolumincsccnt

rc-

Li.s/erirr ,)1,,,10tl’10~~~‘!~‘)1~.\. Infect. Immun. 40 (1983)

440-443. Mead,

D.A..

Szcrcsna-Skorupa.

DNA ‘blue’ T7 promoter

E. and Kcmpw. plasmida:

a vcrsatilc

B.: Smglc stranded tandem

promoter

q s-

Protein Eng. I ( 1986) 67-74.

tcm for cloning and protein cnginccring.

Naka) ama, K.: The aupcrovide dismutase-encoding gcnc of the obligatcl) anaerobic bacterium Bacteroidr.s ,yit~~iw/i.~. Gcnc 96 ( 1990) l49- 150. Parker.

M.W. and Blake, C.C.F.:

peroxide

dismutaaca

mary structure.

Iron- and mangancac-containing

can bc distinguished

FEBS

bq an analysis

su-

of their pri-

Lctt. 229 (198Xa) 377-3X2.

M.\V. and Blake. C.C.F.: from Bucilh

oxide dismutaae

Crystal

structure

of mangancsc

supcr-

.rretrn,rhernro~/~hihr~ at 2.4 A; resolution.

J. Mol. Biol. 199 (19XXb) 649-661.

Beaman, I..V.and Beaman, B.L.: Monoclonal antibodies demonstrate that supcroxidc dismutasc contributes to the protection of Nocrrrdiu crsreroit/~.cwithin the intact host. Infect. Immun. 58 (1990) 3122-3128. CO.

and Fridovich.

and an assay

applicable

J.: Superoxide

dismutase:

to polyacrylamide

improved

gels. Anal.

Bio-

chcm. 44 (1971) 276-2X7. CarlioL. A. and Touati. E. co/i: is superoxide

D.: Isolation dismutase

of superoxide necessary

dismutasc

for aerobic

mutants

in

lift? EMBO J.

5 (I 986) 623-630. virulcncc

Introduction

Infect. Immun. 44

Immun. 5X (1990) 529-535. Haaa. .A. and Goebel, u’.: Cloning of a supcrouldc

Parker. REFERENCES

M.F.:

b> conlugation

L. n2[),loc~~‘t/!~~‘~~~‘\ plasmids.

V.L.. Arondcl.

oxide dismutasc

Harbor.

We are indebted to D. Touati (Paris) for providing us with the E. coli sod mutants. We thank M. Wuenscher for critical reading of the manuscript and M. Dumbsky as well as M. Kcil for excellent technical assistance. This work was supported by grants from the Bundesministerium fiir Forschung und Technologie (BMFT OlKI88059), the Fonds der Chemischen Industric (to J.K.) and by a fellowship to A.H. from the Boehringer Ingelheim Fonds.

set of sc-

157-161.

McGo\$aan,

Chakrabortq.

monocytogcncs

Free Rad. Biol. Med. 5 (1988) 377-3X5. Maniatis. T.. Fritsch. E.F. and Sambrook,

ACKNOWLEDGEMENTS

assays

0.: 4 comprchcnbivc

for the YAX. Nucleic Acid\ Rch. 12 (1981)

D.J. and Thomasho\+,

pAMPI

Laboratory

Beauchamp,

P. and Smithies.

programs

T. and Goebel.

W.: Recent developments

in the study of

of Lbrrricr mono~~‘f~?~c’ne.v. Curr. Top. Microbial.

138 (1YXX) 41-58.

Immunol.

Pei-enka. V., Dvor?ak.

M. and Travnieek.

for cloning of large DNA fragments vectors. Rocourt,

Nucleic

M.: Simple and cfticlcnt method with identical

ends into plasmid

,Acids Rcs. I6 (1988) 4179.

J.: Taxonomy

of the gcnua L~CIL’TUI. Infection

l6S2 (1988) X9-

91. Sangcr.

F.. Nicklen,

terminating

S. and Coulson,

inhibitors.

.-\.R.: DNA acqucncmg

with chain-

Proc. Natl. Acad. Sci. USA 7J (1977) 5463-

5467. Van Camp, W., Bowler, C.. Villarroel. M. and

Inre,

D.: CharactcriLation

cDNAs

from

planls

obtained

Ec~hrrichic~ c,o/i. Proc. Natl. hcad.

R.. Tsang. of iron by

gcnctic

E.W.T.. Van Montagu. supcroxidc

dismutasc

complcmcntation

Sci. LlS.4 X7 (IWO) 9903-9907.

in