INFECrION AND IMMUNITY, Feb. 1993, p. 457-4630019-9567/93/020457-07$02.00/0Copyright X) 1993, American Society for Microbiology

Comparison of the Alpha-Toxin Genes of Clostridium perfringensType A and C Strains: Evidence for Extragenic

Regulation of TranscriptionSEI-ICHI KATAYAMA, OSAMU MATSUSHITA, JUNZABURO MINAMI, SADAO MIZOBUCHI,

AND AKINOBU OKABE*

Department of Microbiology, Kagawa Medical School, Kagawa 761-07, Japan

Received 26 August 1992/Accepted 20 November 1992

The Clostridium perfringens pk gene encoding phospholipase C (alpha-toxin) was cloned from type C NCIB10662, a strain which produces low levels of phospholipase C activity. The nucleotide sequence of a cloned3.1-kb HindIII fragment was determined. The same fragment was also cloned from type A NCTC 8237, a

phospholipase C-overproducing strain. In this case, an open reading frame (ORF2) truncated in the previouslycloned 2-kb fragment was also sequenced. Comparison of the nucleotide sequence between the 3.1-kbfragments of the two type strains shows some differences both in the pkc gene and in ORF2. However, when the3.1-kb fragment was cloned into plasmid pUC19 and expressed in Escherichia coli, theplc genes from both typestrains were similarly expressed and the toxins produced showed similar levels of activity. Northern blotanalysis revealed that the type A strain produced 16 to 23 times more plc mRNA than the type C strain. Theseresults indicate that in C. perfringens the two plc genes are transcribed at different rates, probably because ofa difference in a locus lying outside of the cloned fragments. Gel retardation analysis showed that the type Astrain possessed two different proteins that bound different regions of the plc gene. However, one of theseproteins, which binds within the plc coding region, was not found in the type C strain, suggesting that it playsa role in the regulation of the pie gene expression.

Clostridiumperfringens, the most widely occurring patho-genic bacterium, is divided into five toxigenic types (Athrough E) based on the ability to produce major lethaltoxins (7, 21). The alpha-toxin, phospholipase C (EC3.1.4.3), produced by this organism exhibits several power-ful toxicities, including necrosis, hemolysis, an increase invascular permeability (27), platelet aggregation (18), andmyocardial dysfunction (26). The alpha-toxin has thereforebeen regarded as the major factor responsible for the patho-genesis of gas gangrene caused by this organism. While thistoxin is produced by all types of C. perfringens, type Astrains, which are most often responsible for C. perfringensinfection in humans, produce especially large amounts (14).While the level of phospholipase C seems to determine the

fate and severity of infections by this organism, no correla-tion between the virulence of different strains of C. perfrin-gens and their production of this enzyme (13, 14) has beenfound. There are several possible explanations for thisdiscrepancy. Two possibilities are as follows. (i) The pro-duction of phospholipase C by cultured C. perfringens isgreatly influenced by the growth conditions (14, 16). Thus,the enzyme activity determined in the laboratory would notnecessarily reflect the production of the enzyme in infectedhosts. (ii) The alpha-toxin is a multifunctional protein com-

posed of a domain responsible for phospholipase C (lecithi-nase) activity and another which is required for the othertoxicities (30). Phospholipase C activity would not correlatewith potency of the alpha-toxin if mutations can occur whichaffect only the function of the toxin domain.One approach to understanding the role of the alpha-toxin

in the pathogenicity of C. perfringens is to analyze thephospholipase C (plc) genes from different type strains of C.

* Corresponding author.

perfringens. This would enable comparisons to be made ofboth their protein structures and gene expression. Thecloning and sequencing of plc genes have been reportedindependently from many laboratories (10, 20, 22, 29, 32).However, these genes have all been cloned from either a

type A reference strain, NCTC 8237, or a mutant derivedfrom the type A strain NCTC 8798. We have chosen toexamine the plc genes from type A (NCTC 8237) and C(NCIB 10662) strains which differed markedly in levels ofphospholipase C activity. We cloned a 3.1-kb HindIII frag-ment containing the plc gene and an open reading frame(ORF) designated ORF2 by Saint-Joanis et al. (22) from bothstrains to compare the nucleotide sequences not only of theplc genes but also of ORF2s truncated in previously clonedfragments (22, 31). We have also determined the levels ofplcmRNA produced by the two strains.We have previously shown that a static DNA curvature

occurs immediately upstream of aplc promoter in our type Astrain (31). In addition, ORF2 has been shown to existupstream of this promoter (22). Since these elements maycontribute to the difference in toxin production, the cloned3.1-kb fragments were expressed in Escherichia coli, andexpression of the two plc genes was compared. Finally, wehave carried out a gel retardation analysis to identify DNA-binding proteins with a potential role in regulating plc geneexpression, and evidence that extragenic regulation is in-volved in plc gene expression is presented in this article.

MATERIALS AND METHODS

Bacterial strains and culture conditions. Strains of C.perfringens used in this study were type A NCTC 8237 andtype C NCIB 10662, gifts from S. Nakamura, Departmentof Microbiology, Kanazawa University Medical School,Kanazawa, Japan. They were precultured at 37°C overnight

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458 KATAYAMA ET AL.

either in Gifu anaerobic medium broth (GAM broth; NissuiCo., Tokyo, Japan) or in TYG broth (2) containing 3%Trypticase peptone, 2% yeast extract, 0.5% glucose, and0.1% sodium thioglycolate (pH 7.4). The last preculture wasdiluted 20-fold with fresh GAM or TYG broth and grownat 37°C. E. coli JM109 (34) was used as a host strain forplasmid pUC19 and derivatives. It was grown in L brothcontaining 50 ,ug of ampicillin per ml with vigorous shakingat 37°C.Assays of enzyme activities. Cultures of C. perfringens

were centrifuged at 4,000 x g for 5 min, and supernatantswere stored at -80°C until used. To prepare sonic extracts ofE. coli cultures, 30-ml cultures were chilled on ice when theyreached an optical density (OD) at 600 nm of 0.8, and thenthey were sonicated as described previously (19). Afterdebris was removed by centrifugation at 27,000 x g at 4°Cfor 20 min, supernatants were stored at -80°C until used.Phospholipase C activity was assayed by the method ofKurioka and Matsuda (9) by usingp-nitrophenylphosphoryl-choline (Sigma Chemical Co., St. Louis, Mo.). P-Lactamaseactivity was assayed by the method of Lupski et al. (12) andused to correct phospholipase C activity for plasmid copynumber. Hemolytic activity was assayed by the method ofTitball et al. (29).

Southern hybridization. Chromosomal DNA was preparedfrom the type A and C strains as described elsewhere (20).The DNA was digested with EcoRI or HindIII, electrophore-sed on an 0.8% agarose gel, and transferred to a nylonmembrane (Biodyne B; Pall BioSupport Division, GlenCove, N.Y.). Hybridization was performed as described bySouthern (25) by using an 88-bp DraI-BamHI fragmentlocated within the coding region of the plc gene. This DNAprobe was labeled with digoxigenin-dUTP by the randomprimer method (4), and hybridized DNA fragments were

detected with the Genius DNA labeling and detection kit(Boehringer GmbH, Mannheim, Germany).

Northern hybridization. Both the type A and type C strainswere grown in GAM broth at 37°C. When cultures reachedan OD of 0.8, total RNA was prepared by the sodiumdodecyl sulfate-phenol method (1). The DNA probe usedwas a 531-bp Sau3AI fragment located within the codingregion of the plc gene. This was labeled with [a-32P]dCTP(-3,000 Ci/mmol; Amersham International plc, Bucking-hamshire, England) by the random primer method (4). Spe-cific activities of the probes prepared from the type A and Cplc genes were 1.8 x 108 and 3.4 x 108 cpm/,ug of DNA,respectively. They were used at 6.9 x 106 and 4.3 x 106 cpmper blot, respectively. Northern hybridization and determi-nation of band intensity were carried out as describedpreviously (8).

Cloning and sequencing of a 3.1-kb HindIII fragment of theplc gene. Hindlll fragments of chromosomal DNA (fromboth the type A and type C strains) were separated on an

0.8% agarose gel. An area containing approximately 3.1-kbDNA fragments was excised, and the DNA was eluted fromthe agarose with the Geneclean II kit (Bio 101, Inc., La Jolla,Calif.). This DNA was ligated into the dephosphorylatedHindIII site of pUC19, and E. coli JM109 was transformedwith the resulting mixture. Transformants were screened forhemolysis by overlaying them with blood agar and forphospholipase C activity by a lecithinase assay using egg

yolk agar as described previously (20). Plasmids containingthe 3.1-kb plc genes of the type A and C strains oriented inthe same direction as the lac promoter were designatedpKMA100 and pKMA300, respectively. Plasmids containingthese genes in the opposite orientation were designated

pKMA101 and pKMA301, respectively. Clones to be se-quenced were generated by exonuclease III deletion, andDNA sequencing was performed by the dideoxy-chain ter-mination method (24) with [a-35S]dATP (>1,000 Ci/mmol,Amersham). Homology searches were performed on the PIRdatabase (National Biomedical Research Foundation).

Preparation of protein extracts for DNA gel retardationassays. Cultures grown in 200 ml of GAM broth wereharvested by centrifugation when they reached an OD of 1.0to 1.1. Cell pellets were washed twice with 0.9% NaCl andonce with 100 mM KCl-1 mM EDTA-1 mM dithiothrei-tol-10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 8.0) and resuspended in 5 ml of thesame buffer. The suspension was passed twice through aFrench pressure cell at 10,000 lb/in2. The cell lysate wascentrifuged at 12,000 x g for 30 min, and glycerol was addedto the supernatants at a concentration of 20% (vol/vol).These supernatants were stored in liquid N2 until used.

Probes and competitor DNAs used for gel retardationassays. A derivative of pUC19 which contained a fragment ofthe type Aplc gene extending from nucleotides 182 to 1619(see Fig. 2), previously constructed by nested deletion ofpKMA20 (31), was digested with FokI and HindIII. A 305-bpHindIII-FokI fragment, a 376-bp FokI fragment, and a263-bp FokI fragment were eluted after electrophoresis on a5% polyacrylamide gel (23). These were designated frag-ments A, B, and C, respectively. Fragment A consisted of a12-bp HindIII-PstI fragment of pUC19 and a fragment of thetype A plc gene which encompasses nucleotides 182 to 474and contains the plc promoter, while fragments B and Ccontained only a part of the plc coding region (see Fig. 4A).All of the fragments were labeled with [-y-32P]ATP by T4polynucleotide kinase to prepare 5'-end-labeled probes (23).A 403-bp FokI fragment (nucleotides 72 through 474) con-taining a plc promoter region was also prepared from thetype Cplc gene and labeled as described above. The specificactivities of the labeled probes were between 5.3 x 105 and3.1 x 106 cpm/pmol.DNA gel retardation assays. Gel retardation assays were

carried out essentially as described by Chodosh (3). Reac-tion mixtures contained 12 mM HEPES (pH 8.0), 4 mMTris-HCl (pH 8.0), 60 mM KCl, 1 mM EDTA, 1 mMdithiothreitol, 12% (vol/vol) glycerol, 300 ,ug of bovine serumalbumin per ml, 0.67 mg of poly(dI-dC) per ml, 0.5 to 10 ,ugof protein, and approximately 0.5 ng of end-labeled probe(7,500 to 1,000 cpm), in a final volume of 30 ,lI. Afterincubation at 30°C for 15 min, samples were electrophoresedon a 4% polyacrylamide gel. Gels were fixed in 10% metha-nol-10% acetic acid for 10 min, dried on sequencing gel filterpaper (Bio-Rad Laboratories, Richmond, Calif.), and ex-posed to X-ray film.

Protein determination. Protein concentrations were deter-mined by the method of Lowry et al. (11), using bovineserum albumin as a standard. For determinations of C.perfringens cellular protein, washed cell pellets were pre-treated with perchloric acid-ethanol and solubilized by heat-ing in 2 N NaOH as described by Waterborg and Matthews(33), and then protein concentration was determined.

Nucleotide sequence accession number. The nucleotidesequence of a 3.1-kb HindIII fragment from the C. perftin-gens type C strain containing theplc gene and the ORF2 wasdetermined in this study and has been deposited in theEMBL database under accession number D10248.

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C. PERFRINGENS TYPE A AND C ALPHA-TOXIN GENES 459

E

0

cocE

ID -

CoE

Time (h)

FIG. 1. Growth curves and phospholipase C activity levels for C.perfringens NCTC 8237 (type A) and NCIB 10662 (type C). Growthin GAM broth of the type A (0) and type C (0) strains wasmonitored by measuring the OD at 600 nm. Phospholipase Cactivities in the culture supernatants of the type A (A) and type C(A) strains were determined by usingp-nitrophenylphosphorylcho-line as a substrate. The data are presented as the means of threedeterminations standard deviations.

RESULTS

Comparison of type A and type C strain phospholipase Cactivities. Growth curves of the type A and C cultures grownin GAM broth are shown in Fig. 1. These cultures exhibitedsimilar growth patterns, although the type A strain grewslightly more rapidly and reached a slightly higher final ODthan the type C strain. Phospholipase C activities in theculture supernatants were also determined. In both cultures,phospholipase C activity was highest in the late logarithmicgrowth phase. The enzyme specific activities of 3-h culturesof the type A and C strains were 42.1 ± 2.1 and 1.3 ± 0.4nmol/min/mg of cell protein, respectively. Thus, the type Astrain produced approximately 30 times as much phospho-lipase C activity as the type C strain. When the two typestrains were grown in TYG broth, phospholipase C activitiesin both cultures were much lower than when the strains weregrown in GAM broth. However, the type A strain stillproduced about 20-fold more phospholipase C activity thanthe type C strain. We therefore conclude that there is amarked difference in phospholipase C activity between thetype A and C strains, although the specific ratio variessomewhat, depending on the culture medium.Comparison of type A and type C strain plc genes. The

difference in phospholipase C activities produced by the twotype strains may result from a difference between the twoplcgenes. To examine whether or not the two type strains differin the copy number of theplc gene, chromosomal DNA fromthe type A and C strains was digested with HindIII or EcoRIand subjected to Southern blot hybridization with a digoxi-genin-labeled 88-bp DraI-BamHI fragment. The HindIII andEcoRI fragments detected in the type C DNA, 3.1 and 4.4kb, respectively, were identical to those found in the type ADNA (data not shown). Furthermore, no significant differ-ences in band intensity were observed between the type Aand C strains, indicating that there is no difference in copynumber of the two plc genes.An open reading frame, ORF2, exists upstream from the

plc gene of strain 8-6 (22). This ORF was truncated by the 5'EcoRI end of a 2-kb EcoRI-HindIII fragment which we hadpreviously cloned from type A NCTC 8237 (31), and itsentire nucleotide sequence has not been determined. To

compare nucleotide sequences not only of the plc gene butalso of the ORF2 between the two type strains, the 3.1-kbHindIII fragments from both strains were cloned intopUC19. The nucleotide sequences of the 3.1-kb fragment ofthe type C strain and a 1.1-kb HindIII-EcoRI fragment of thetype A strain were then determined. Both the nucleotidesequence of the plc gene and the deduced amino acidsequence of the alpha-toxin of the type C strain are com-pared with those of the type A strain (Fig. 2). Both immaturetoxins contain 398 amino acid residues, although they differat six positions, two of which reside in the signal peptide.The nucleotide sequences of the two plc promoters wereidentical except for the final nucleotide in the -35 region,which was C in the type A strain and G in the type C strain.Both ORF2 genes encode polypeptides containing 490 aminoacids, which differ at six positions. Inspection of the aminoacid sequences for the putative ORF2 gene products revealsthat they do not possess sequences characteristic of DNA-binding proteins (28).

Expression of the two pkc genes in E. coli. If the differencesin the nucleotide sequences between the two fragmentsdescribed above are responsible for the difference in phos-pholipase C activity in the two type strains, one might expectthat the unequal levels of activity would remain when thesegenes are expressed in E. coli. To test this hypothesis, theactivities of phospholipase C produced by E. coli JM109cells carrying pKMA100, pKMA101, pKMA300, andpKMA301 were compared (Table 1). While the expression ofthe twoplc genes was affected by their orientation in pUC19relative to the lac promoter, virtually the same level ofphospholipase C activity was produced from clones carryingthe plc genes from the two type strains. Furthermore, nomarked difference in the levels of toxins was observed wheneach pair of cultures was compared by Western blotting(immunoblotting) (data not shown). Thus, both the specificactivities as well as the amounts of the two toxins appear tobe almost the same. Note that the E. coli-produced toxinsare not only equal in phospholipase C activity but also inhemolytic activity (Table 1), suggesting that the two proteinsare functionally identical in spite of the four amino acidsubstitutions.Comparison of type A and type C strain plc mRNAs. The

results from expression of the two plc genes in E. coliindicates that the difference in phospholipase C activitybetween the two type strains is not due to a difference in theenzyme activity itself but rather to a difference in productionof the enzyme. Although the cloned plc genes were equallytranscribed in E. coli, the two plc genes might be unequallytranscribed in C. perfringens. To compare the levels ofplcmRNA of the type A and C cultures, total RNA wasprepared when the cultures grew to an OD of 0.8 and thenwas hybridized by Northern blot with a 32P-labeled 531-bpSau3AI fragment located within the plc gene of either thetype A or C strain. The plc mRNA detected in the type Ccultures was estimated to be approximately 1.4 kb, identicalto that found for the type A strain (Fig. 3). When a DNAprobe prepared from the type Aplc gene was used, the levelof plc mRNA in the type A culture was approximately 23times higher than that in the type C culture. When a probefrom the type Cplc gene was used, these levels differed byabout 16-fold. The discrepancy between these two determi-nations could have resulted from a difference in nucleotidesequence between the two probes. These results indicatethat the greater production of alpha-toxin by the type Astrain is due mainly to an increased level ofplc transcripts.

Gel retardation assays. As shown above, the two type

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460 KATAYAMA ET AL. INFECT. IMMUN.

DdeI1 CTAAGTTAAA ACCTGTTTTT GATTGAAAAT TTTTATTATC CATATTAAAA TCCTTTGCCT TATAATTTAT TTCAAATTTT

E L N F G T K S Q F N K N D M

FokI T81 ATTCCATCCC TTATATTATG CGTAAAAATT CTTATTAAAT TAAAAAACAA GATTTAACTT ATTATAQCAQ TAATAATTGT

-10 -10

A G161 AAATTTTCAI ATTAAAAATA AGTTTAACAA TTTAGAGTGG GTGAGCTTAG ATATGTTTAA TTGAAATTTG AATTGTATTC

orf2 -35 -35

DraI.. .. C241 AAAAATATTT TAAAAAATAT TCAAAAATTT AGTGAQTTA TGGTAATTAT ATGGTATAAT TTCAGTGCAA GTGTTAATCG

-35 plc -10

A G

321 TTATCAAAAA AGGGGAGATT AATACTTGAA AAAAATTAALQ aGGGGATAT- AAAAATGAAAA GAAAGATTT GTAAGGCACTSD M K R K I C K A L

GCC G FokI401 TATTTGTCGT ACGCTAGCAA CTAGCCTATG GGCTGGGGCA TCAACTAAAG TCTACGCTTGG GATGGAAAA ATTGATGGAA

I C R T L A T S L W A G A S T K V Y A W D G K I D G TA A

Sau3AI481 CAGGAACTCA TGCTATGATT GTAACTCAAG GGGTTTCAAT CTTAGAAAAT GATCTGTCCAA AAATGAACC AGAAAGTGTA

G T H A M I V T Q G V S I L E N D L S K N E P E S V

DraI561 AGAAAAAACT TAGAGATTTT AAAAGAGAAC ATGCATGAGC TTCAATTAGG TTCTACTTAT CCAGATTATG ATAAGAATGC

R K N L E I L K E N M H E L Q L G S T Y P D Y D K N A

Sau3AISau3AI Sau3AI BamHI

641 ATATGATCTA TATCAAGATC ATTTCTGGGA TCCTGATACA GATAATAATT TCTCAAAGGA TAATAGTTGG TATTTAGCTTY D L Y Q D H F W D P D T D N N F S K D N S W Y L A Y

721 ATTCTATACC TGACACAGGG GAATCACAAA TAAGAAAATT TTCAGCATTA GCTAGATATG AATGGCAAAG AGGAAACTATS I P D T G E S Q I R K F S A L A R Y E W Q R G N Y

FokI801 AAACAAGCTA CATTCTATCT TGGAGAGGCT ATGCACTATT TTGGAGATAT AGATACTCCA TATCATCCTG CTAATGTTAC

K Q A T F Y L G E A M H Y F G D I D T P Y H P A N V T

G881 TGCCGTTGAT AGCGCAGGAC ATGTTAAGTT TGAGACTTTT GCAGAAGAAA GAAAAGAACA GTATAAAATA AACACAGCAG

A V D S A G H V K F E T F A E E R K E Q Y K I N T A G

C A961 GTTGCAAAAC TAATGAGGAT TTTTATGCTG ATATCTTAAA AAACAAAGAT TTTAATGCAT GGTCAAAAGA ATATGCAAGA

C K T N E D F Y A D I L K N K D F N A W S K E Y A RA T

FokI T1041 GGTTTTGCTA AAACAGGAAA ATCAATATAC TATAGTCATG CTAGCATGAG TCATAGTTGG GATGATTGGG ACTATGCAGC

G F A K T G K S I Y Y S H A S M S H S W D D W D Y A A

G1121 AAAGGTAACT TTAGCTAACT CTCAAAAAGG AACAGCAGGA TATATTTATA GATTCTTACA CGATGTATCA GAGGGTAATG

K V T L A N S Q K G T A G Y I Y R F L H D V S E G N D

Sau3AI G1201 ATCCATCAGT TGGAAAGAAT GTAAAAGAAC TAGTAGCTTA CATATCAACT AGTGGTGAAA AAGATGCTGG AACAGATGAC

P S V G K N V K E L V A Y I S T S G E K D A G T D D

T FokI1281 TACATGTACT TTGGAATCAA AACAAAGGAT GGAAAAACTC AAGAATGGGA AATGGACAAC CCAGGAAATG ATTTTATGAC

Y M Y F G I K T K D G K T Q E W E M D N P G N D F M T

1361 TGGAAGTAAA GATACTTATA CTTTCAAATT AAAAGATGAA AATCTAAAAA TTGATGATAT ACAAAATATG TGGATTAGAAG S E D T Y T F K L K D E N L K I D D I Q N M W I R K

T C A G1441 AAAGAAAATA TACAGCATTC CCAGATGCTT ATAAGCCAGA AAATATAAAG GTAATAGCAA ATGGAAAAGT TGTAGTAGAC

R K Y T A F P D A Y K P E N I K V I A N G K V V V DS I

1521 AAAGATATAA ATGAGTGGAT TTCAGGAAAT TCAACTTATA ATATAAAATA ATAAAAGTAA AAAAATAATT ATTGGTTTTGK D I N E W I S G N S T Y N IK * *

HindIII1601 GTGGTATTTA CAAAATAAAA GCTT

FIG. 2. Nucleotide sequence and deduced amino acid sequence of theplc gene from the type C strain NCIB 10662 and a comparison withthose from the type A strain NCTC 8237. The nucleotide sequence of the type Cplc gene and its upstream region is shown above the aminoacid sequence and is numbered on the left side of the diagram. The bases and amino acids of the type Aplc gene (20, 31) which differ fromthose of the type C strain are given on the top and bottom rows, respectively. Regions corresponding to putative -35 and -10 regions anda ribosome binding site are underlined. The -35 and -10 regions of ORF2 are indicated as reported by Saint-Joanis et al. (22). Direct repeatsare indicated by horizontal arrows. The upward arrow marks the N-terminal amino acid residue of the mature alpha-toxin (20, 29). Theposition of (A. T)n tract of static DNA curvature is denoted by closed circles.

strains differed markedly in their expression of the plc gene, perfringens possesses trans-acting proteins that bind theplcalthough the cloned plc genes from both type strains were gene and whether the two type strains differ in such proteins,expressed at similar levels in E. coli. This suggests that the we carried out gel retardation assays using cell extracts fromexpression of theplc gene in C. perfringens might be affected the two type strains (Fig. 4). Three fragments were preparedby a certain factor(s) which is specifically present in either from the type A strain, radiolabeled, and used as DNAthe type A or type C strain. To examine whether or not C. probes: fragment A is a 305-bp DNA containing the plc

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TABLE 1. Expression in E. coli ofplc genes from the type A and C strains

Phospholipase C Hemolytic activity ,-Lactamase activity Phospholipase C Hemolytic activity/Plasmid" activity (U/min/mI)d(atmolamin/mlase activity/0-lactamase ,B-lactamase

(nmol/min/mlY'.c (Umnm) ±om/lceactivity (1O')" activity (102)epKMA100 0.249 ± 0.004 0.67 0.65 ± 0.07 383.0 103pKMfA300 0.247 ± 0.029 0.67 0.51 ± 0.05 484.3 131pKMA101 0.518 ± 0.112 NDf 0.55 + 0.11 941.8pKMA301 0.474 ± 0.014 ND 0.61 ± 0.01 777.0

a The host cell was E. coi JM109.b Phospholipase C activity was determined with p-nitrophenylphosphorylcholine as a substrate by the method of Kurioka and Matsuda (9).c The values represent the means of triplicate determinations with the maximal deviations.d Hemolytic activity against 1% (vol/vol) mouse erythrocytes was determined by the method of Titball et al. (29), with a modification. The reaction mixture

was incubated for 3 h at 37°C because hemolysis was very weak.I ,-Lactamase activity was determined as described in Materials and Methods.f ND, not determined.

promoter; fragments B and C are 376- and 263-bp DNAs,respectively, within the coding region (Fig. 4A). Figure 4Bshows the results with fragment A prepared from the type Astrain by using protein extracts of both the type A and typeC strains. This shows that fragment A migration was re-tarded in the presence of extracts prepared from either typestrain. Furthermore, the amount of the slower-migratingband, protein-fragnent A complexes, increased with in-creasing amounts of protein extract. On the other hand,when fragment B was used in the binding reaction, only thetype A extract gave a band shift. Neither extract gave a bandshift when fragment C was used (Fig. 4C).To determine whether these band shifts were due to

specific protein-DNA interactions, an excess of the unla-beled fragments was added as competitor DNA and theband-shift assay was carried out as described before (Fig. 5).The slower-migrating band derived from fragment A was stillobserved in the presence of unlabeled fragment B, but it

disappeared in the presence of unlabeled fragment A. Simi-larly, the band shift of fragment B was diminished only in thepresence of the unlabeled fragment B. Nucleotide sequencesof the two plc genes coincide within the region correspond-

A

Hind II182 p

Fokl Fokl Fokl474 850 plC 1 113

Fragment A Fragment B :Fragment Cl305 bo) (376 bp (f263 bpJ

B1 2 3 4 5 6 7

(Hindll lISmal)

I I 1 Ou'j DD

C1 2 3 4 5 6 7 8_- -

1 2 3 4 5

28S >23S >18S16S * "

FIG. 3. Northern blot of pkc mRNAs from the type A and Cstrains of C. perfringens. RNA was extracted from cultures whenthey reached an OD of 0.8. Various amounts of RNA (0.07 to 10 ,ug)were denatured, subjected to electrophoresis on a 1% denaturingagarose gel, transferred to a nylon membrane, and hybridized witha 531-bp Sau3AI fragment prepared from the type Aplc gene. Theplc mRNA (1.4 kb) is indicated by an arrow. Lanes: 1, type A, 2 p.g;2, type A, 0.35 ,ug; 3, type A, 0.07 p,g; 4, type C, 2 p,g; 5, type C, 10p,g. The 28S rRNA (4.4 kb) and 18S rRNA (1.9 kb) of rat liver and23S rRNA (2.9 kb) and 16S rRNA (1.5 kb) of E. coli JM109 were

used as size markers and are shown on the left side of the panel.

FIG. 4. Gel retardation assays for binding of protein extracts toplc DNA. (A) Schematic representation of type A DNA fragmentsused in gel retardation assays. Fragments: A, a 305-bp HindIII-FokIfragment containing a vector arm and theplc DNA from nucleotides182 to 474; B, a 376-bp FokI-FokI fragment (nucleotides 475 through850); C, a 263-bp FokI-FokI fragment (nucleotides 851 through 1113).The darkened region represents the plc coding region. Thin linesrepresent flanking vector arms. The location of the plc promoter isindicated by P. (B) Analysis of DNA-protein complexes betweenfragment A and protein extracts of the type A and C strains. All lanescontained 0.5 ng of labeled fragment A. Lanes 1 to 4 contained 0, 0.2,1, and 5 pLg of protein, respectively, from cell extract of the type Astrain. Lanes S to 7 contained 0.2, 1, and 5 ,ug of protein, respectively,from cell extract of the type C strain. (C) Analysis of DNA-proteincomplexes between fragment B or C and protein extracts of the typeA or C strain. Lanes 1 to 5 contained 0.5 ng of labeled fragment B.Lanes 6 to 8 contained 0.5 ng of labeled fragment C. Lanes 1 to 4contained 0, 1, 3, and 10 pg of protein, respectively, from a cellextract of the type A strain. Lane 5 contained 10 p.g of protein froma cell extract of the type C strain. Lane 6 contained no cell extract.Lanes 7 and 8 contained 10 ,ug of protein from cell extracts of the typeA and C strains, respectively. Free DNA and DNA-protein com-plexes were resolved on a 4% polyacrylamide gel and visualized byautoradiography as described in Materials and Methods.

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FIG. 5. Binding competition assays with fragments A and B.Fragments A and B described in the legend to Fig. 4 were used aslabeled probes as well as unlabeled competitor DNAs. (A) Compe-tition data support the binding specificity of the fragment A. Alllanes contained 0.5 ng of labeled fragment A. Lanes: 1, free DNA;2 to 4, 0.5 ,ug of protein from a cell extract of the type A strain. Lane2 lacked competitor DNA. Lanes 3 and 4 contained 50 ng ofunlabeled fragments A and B, respectively, as competitor DNAs.(B) Competition data support the binding specificity of fragment B.All lanes contained 0.5 ng of labeled fragment B. Lanes: 1, freeDNA; 2 to 4, 10 p.g of protein from a cell extract of the type A strain.Lane 2 lacked competitor DNA. Lanes 3 and 4 contained 50 ng ofunlabeled fragments B and A, respectively, as competitor DNAs.

ing to fragment B, while they differ by eight nucleotides inthe region corresponding to fragment A (Fig. 2). However,the same results were obtained when a 403-bp FokI fragment(nucleotides 72 through 474) containing the plc promoterregion was prepared from the type Cplc gene and used as aDNA probe (data not shown). These results indicate thatboth the type A and C strains contain a DNA-bindingprotein(s) specific for fragment A, while only the type Astrain contains a protein(s) specific for fragment B.

DISCUSSION

These studies have shown that the marked difference inlevels of phospholipase C activity produced by the type Aand C strains results from differences in production of thealpha-toxin rather than differences in toxin activity. We havealso shown that when the genes cloned from either typestrain were expressed in E. coli, the levels of activityproduced were virtually identical. The production of phos-pholipase C has been shown to be greatly affected by variousculture conditions (14, 16), and we indeed found that thephospholipase C activities of the type A and C cultures inGAM broth were different from those in TYG broth. How-ever, the relative differences in enzyme activities betweenthe two type strains were very similar under both conditions.This implies that the difference in toxin production arisesfrom an inherent difference between these strains. The levelsofplc mRNA in the two cultures differed to such an extentthat this difference could be regarded as being a major causeof the difference in toxin production. We did not examinestabilities ofplc mRNAs and cannot rule out a lower stabilityof theplc mRNA in the type C strain. However, no degradedtranscript was detected on the Northern blot of the type Cculture (Fig. 3), and no distinct difference was found in thepredicted secondary structures of the two mRNAs. There-fore, we conclude that the difference in toxin production

arises from the two type strains transcribing the plc gene atdifferent rates.What is responsible for the different rates of plc gene

transcription between the type A and C strains? A staticDNA curvature previously suggested to modulate expres-sion of the type Aplc gene (31) is also present upstream ofthe type C gene. As described above, the ORF2 genes do notappear to be involved in regulating plc gene expression,although their deduced amino acid sequences differ slightlybetween the type A and C strains. The two plc genepromoters differ only in the final nucleotide of the -35 region(G/C transversion). The promoter consensus sequence of C.perfringens has been deduced from a percentage conserva-tion study (21). The consensus sequence of the -35 region isTTGANA, and A and G/C are 57 and 7% of the finalnucleotide, respectively. Furthermore, the two genes wereexpressed at similar levels in E. coli, whose RNA poly-merase can transcribe the plc gene as efficiently as C.perfiingens RNA polymerase (5, 22). Thus, the minor pro-moter alteration would not be expected to alter the transcrip-tional rate. In short, the 3.1-kb HindIII fragment does notcontain any features which would account for the differingexpression ofplc gene between the two type strains. There-fore, it appears that the difference must occur in a locuswhich lies outside of this fragment and which might beinvolved in regulating plc gene expression.The results obtained from the gel retardation assays indi-

cate that two different plc gene-binding proteins are presentin the type A strain. A preliminary gel retardation analysisshowed that a protein(s) specific for fragment A but noprotein(s) specific for fragment B was found in type B, C,and E strains. Each of these type strains also produces lowerlevels ofplc gene transcripts than the type A strain (data notshown). This may imply that the presence or absence of theprotein(s) which binds fragment B is responsible for theobserved differences in the transcriptional rate of the plcgene. Repressors of some bacterial operons have beenshown to bind not only near promoters but also withincoding regions (6, 17). We speculate that interaction of thebinding protein(s) with fragment B plays a role in regulationof the plc gene expression. Another possible explanation isthat the proteins specific for fragment A from the two strainsare quite different and may account for the differences intranscriptional rate of the two plc genes. In a region imme-diately upstream of the plc promoter exists a tandemlyrepeated sequence (TATrCAAAAAT), and a similar se-quence has been observed upstream of genes in othergram-positive bacteria (15, 29). This region overlaps thestatic DNA curvature and hence is the most likely bindingsite within fragment A.

ACKNOWLEDGMENTSWe are grateful to Shin-ichi Nakamura (Department of Microbi-

ology, Kanazawa University Medical School, Kanazawa, Japan) forproviding us with the C. perfringens type strains. We also thank JonD. Stewart (Department of Chemistry, The Pennsylvania StateUniversity, University Park) for invaluable assistance in preparingthe manuscript.

This work was supported in part by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Science and Culture ofJapan.

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