Vol. 173, No. 18

JOURNAL OF BACTERIOLOGY, Sept. 1991, p. 5619-5623 0021-9193/91/185619-05$02.00/0 Copyright ©3 1991, American Society for Microbiology

Cloning, Sequencing, and Expression in Escherichia coli of a Streptococcus faecalis Autolysin CATHERINE BtLIVEAU,l CLAUDE POTVIN,1 JEAN TRUDEL,2 ALAIN ASSELIN,2 AND GUY BELLEMARE1* Departement de Biochimie, Faculte des Sciences et de GMnie,' and Departement de Phytologie, Faculte des Sciences de l'Agriculture et de l'Alimentation, Universite Laval, Quebec, Quebec, Canada GIK 7P4 Received 13 March 1991/Accepted 3 July 1991

A Streptococcusfaecalis genomic bank was obtained by partial digestion with MboI and cloning into the Sall restriction site of pTZ18R. Screening of about 60,000 Escherichia coli transformants for cell wall lysis activity was done by exposing recombinant colonies grown on a medium containing lyophilized Micrococcus lysodeikticus cells to chloroform and toluene vapors in order to release proteins. Because this procedure provoked cell death, colonies could not be used directly for transformant recovery; however, recovery was achieved by partial purification of plasmid DNA from active colonies on the agar plate and transformation of E. coli competent cells. About 60 recombinants were found. One of them (pSH6500) codes for a lytic enzyme active against S. faecalis and M. lysodeikticus cell walls. A shorter clone (pSH4000) was obtained by deleting an EcoRI fragment from the 6.5-kb original insert, leaving a 4-kb EcoRI-MboI insert; this subclone expressed the same lytic activity. Sequencing of a portion of pSH4000 revealed a unique open reading frame of 2,013 nucleotides coding for a 641-amino-acid (74-kDa) polypeptide and containing four 204-nucleotide direct repeats.

Bacteria exhibit several types of enzymes, known as autolysins, that can degrade their own cell walls (31); autolysins are usually active on bacterial-wall peptidoglycans (29) and can be as diverse as N-acetylmuramidases (lysozymes), N-acetylglucosaminidases, N-acetylmuramyl-L-alanine amidases, endopeptidases, and transglycosylases (14). A given bacterial species can possess several autolysins; these can be involved in cell wall turnover (6), cell separation, competence for genetic transformation, formation of flagella, and sporulation (25). Some Streptococcus (Enterococcus) autolysins have been characterized. For example, Streptococcus pneumoniae possesses a well-studied cell wall amidase in addition to an endo-,-1,4-N-acetylglucosaminidase (12). Streptococcus faecium has two autolytic hydrolases: muramidase-1, which is a ,-1,4-N-acetylmuramidase with an 87-kDa active form (5), and muramidase-2, which has a 125-kDa and a 75-kDa active form (4). These two S. faecium autolysins differ in substrate specificity, molecular mass, and probably hydrolysis mechanism. It is interesting that the two active forms of S. faecium muramidase-2 can bind penicillin covalently (4). The lytic enzymes produced by S. faecalis (8) were reported to have molecular masses and substrate specificity similar to those of the two S. faecium peptidoglycan hydrolases just described. In this study, we present the cloning, sequencing, and expression in Escherichia coli of one Streptococcus faecalis autolysin. Although the sequence of an S. pneumoniae amidase has been published elsewhere (11), there are no other sequence data available for Streptococcus autolysins.

from Pharmacia. Most restriction enzymes were from Bethesda Research Laboratories or Pharmacia. T4 DNA ligase, Klenow (large) fragment of E. coli DNA polymerase I (PolIk), Bluo-gal (halogenated indolyl-p-D-galactoside), IPTG (isopropyl-p-D-thiogalactopyranoside), and dithiothreitol were from Bethesda Research Laboratories. Prestained protein molecular mass markers were from Bio-Rad. Radioactive ([a-35S]dATP) was from Amersham. Protease type XI (proteinase K), pancreatic RNase A (type IIA), ampicillin, kanamycin sulfate, agarose (type I; low electroendosmosis), low-gelling-point agarose (type VII), Triton X-100, mannitol, and lyophilized Micrococcus lysodeikticus (ATCC 4678) cells were from Sigma Chemical Co. Growth media were from Difco Laboratories. Sequencing primers (T7 promoter and M13 reverse) were from the Regional DNA Synthesis Laboratory, University of Calgary, Calgary, Alberta, Canada. Chromosomal-DNA preparation. An S. faecalis isolate previously described by Leclerc and Asselin (17) was grown with vigorous shaking in brain heart infusion broth at room temperature to an approximate A450 of 0.1. Chromosomal DNA was prepared according to the method of Rodriguez and Tait (24) except that the cell pellet (about 0.35 g, wet weight) was stored at -20°C overnight and subsequently thawed on ice; typical yields of 170 ,ug/liter of culture medium were obtained. Gene library construction. Phage and single-stranded DNA from pTZ18R and its derivatives were prepared as recommended by the supplier. Double-stranded DNA was isolated according to the method of Birnboim (1). The genomic library was constructed as previously described (22) except that MboI was used instead of Sau3A for partial digestion of chromosomal DNA and that 7- to 10-kb fragments were selected for cloning. Clone selection. The procedure for in situ detection is modified from that of Hogg (16). Transformants from the genomic bank were plated on nutrient agar petri dishes containing 50 ,ug of ampicillin per ml and 0.15% (wt/vol)

MATERIALS AND METHODS Bacteria, phage, plasmid, enzymes, and chemicals. E. coli NM522 and JM109, M13KO7 helper phage, pTZ18R doublestranded DNA, nested deletions, and sequencing kits were *

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FIG. 1. Partial restriction map of S. faecalis genomic insertion of clone pSH6500. Restriction endonucleases are indicated as follows: E, EcoRI; A, AccI; H, HindIII; Hp, HpaI; and SM, SalI-Mbol cloning site. (A) Map of the genomic clone. The bottom map represents the 6.5-kb genomic insert (filled area illustrates the sequenced region); the thin line on each side represents the multiple cloning site of pTZ18R. The top map is an enlargement of the sequenced region. The open arrow locates the coding region and indicates the orientation of the autolysin gene; the four 68-aminoacid direct repeats (I, II, III, and IV) are indicated by small filled arrows. (B) Lengths, orientations, and relative locations in the genomic clone of the inserts of three subclones (pSH4000r, pSH2500, and pSH4000) used for sequencing. Activity (+) and lack of activity (-) are indicated.

autoclaved M. lysodeikticus lyophilized cells. Plates were incubated overnight at 30°C until colonies were visible. Whatman 3MM paper disks soaked in toluene-chloroform (1:1) were inserted into the lids. The inverted plates were incubated for 3 to 4 h at 37°C. New 3MM paper disks impregnated with toluene-chloroform (1:1) were used for another incubation in plastic bags for 36 h at 4°C. After the paper disks were discarded, the plates were left for at least 1 h in a sterile hood with the lids off to eliminate residual organic vapors and finally were incubated for a few days in plastic bags with the lids on until lytic zones were observed around colonies. In order to recover recombinants exhibiting lytic activity, colonies were picked up, suspended in 25 RI of TE buffer (10 mM Tris HCI [pH 8], 1 mM EDTA [pH 8]), and phenol extracted, and the DNA thus obtained was used to transform E. coli JM109 competent cells (15). Detection of cell wall hydrolases by SDS-PAGE. For sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), a 10-ml recombinant bacterial culture was pelleted by centrifugation and suspended in 1 ml of denaturing buffer (2% dithiothreitol, 15% sucrose, 3.8% SDS [all wt/vol]). Samples were boiled for 3 min and then sonicated. A 20-,u aliquot was applied to a 10% polyacrylamide gel containing 0.2% (wt/vol) M. lysodeikticus lyophilized cells. Renaturation of lytic enzymes was done by overnight incubation in 25 mM Tris HCl (pH 8.0) containing 1% (vol/vol) Triton X-100 at 37°C with gentle reciprocal shaking (22). Lytic zones in gels appeared as clear bands within the opaque substrate when viewed with light from the back; however, they were photographed by transparency against a dark background and thus appear as dark bands on photographs. Determination and analysis of the nucleotide sequence. Clones used for sequencing were generated with exonuclease III by using a nested deletion kit. The subclones used for this purpose were pSH4000, obtained by removing a

3324-

16FIG. 2. Electrophoretogram of hydrolytic activities against M. lysodeikticus cells observed on SDS-PAGE. The molecular mass markers indicated on the left are phosphorylase b (110 kDa), bovine serum albumin (84 kDa), ovalbumin (47 kDa), carbonic anhydrase (33 kDa), soybean trypsin inhibitor (24 kDa), and lysozyme (16 kDa). Lane S.f., crude protein extract from S. faecalis; lanes pSH6500, pSH4000, pSH4000r, and pTZ18R, sonicated E. coli extracts carrying the cloned S. faecalis cell wall hydrolases (pSH6500, pSH4000, and pSH4000r) and pTZ18R plasmid vector, respectively.

2,500-bp EcoRI fragment from the 5' end of pSH6500; pSH2800, obtained by removing a 1,286-bp HpaI-HindIII fragment at the 3' end of pSH4000; and pSH4000r (r = inverted insert), obtained by inverting the insert of pSH4000 (Fig. 1). For pSH4000 and pSH2800, the exonuclease III reaction started at the third AccI restriction site from the 5' terminus, and for pSH4000r, it started at the HindIlI restriction site. Sequences of both strands were obtained with the T7 DNA Polymerase kit by the method of Sanger et al. (27), using single-stranded templates. Nucleotide and amino acid sequences were analyzed with the help of the University of Wisconsin Genetics Computer Group sequence analysis package (3) and with DNA Strider (18). Nucleotide sequence accession number. The GenBank accession number for the sequence discussed here is M58002. RESULTS Selection of recombinants with hydrolytic activity. A genomic bank was constructed by inserting S. faecalis DNA fragments obtained by partial digestion with MboI into the SalI restriction site of pTZ18R (32). E. coli NM522 was used for transformation, with an efficiency of 107 CFU/,ug of recombinant plasmid DNA. About 60,000 transformants were plated on autoclaved M. lysodeikticus cell-containing medium as substrate for cell wall lytic activity. Sixty recombinants were detected after treatment with organic vapors as described in Materials and Methods. Detection of lytic activity was not possible without this treatment, since the putative cloned hydrolases were not secreted from the E. coli host cell. A genomic clone (pSH6500) exhibiting lytic activity was selected, and a partial restriction map for localizing the lytic gene in the insert was obtained (Fig. 1). Cell wall lytic activity of clones pSH6500 and pSH4000.

S. FAECALIS AUTOLYSIN CLONE

VOL. 173, 1991 -535 AATTTTTCGTTCCGTCAATTCGATGCCTTGTTCTTTTAAAATTTCTTCTGCGATAACTCGTGGTTTCCC

-467

-466 CAATGATTTTGCCACTTGTTCTTCTGTTTCGCCTTCCGCAACACGAGCATCAAATAACGCCTTGTATTTCGCAAGAATA -388 -387 ATAGCTTGATCTTTTGGATTTAATGCTTTTAAATAAATTTTCAGCTCAATAATAAAATGTTCTTTATTCATTCCGCTCC -309 -308 CTCTTTCTACACGATTCATTTCTCATCTTAAACTATAATTTCTTTTTGGGCGAATAACAAGAGGAATTAAAAAAATCAG -230

-229 ACTAAGGTAAAGAAGCGTCTACAACTAAAGTCGCAGTTTTTTTCCGAGTAGTAACAAGAGAGGAAATCCATGTTAAAAA

-151

-150 ACATTACTTCTTTACAATCTAGTTACAGTAATATGAAATTTTGTTGAAGTGTTTTTAATTCGCCAGAAAAAAAGGTATA

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-71 CTTGTTATAATAACAAAACTAAAAAATTACATTTACATATA-A TTAAAAAaGAAA0TTGGGGACGTATCA ATG AAG 6 Met Lys TATAAT 7

AAA GAA TCA ATG TCA CGT ATC GAA AGA AGG AAA GCA CAA CAA AGA AAG AAA ACG CCA GTA 66 Lys Glu Ser Met Ser Arg Ile Glu Ar6 Arg Lys Ala Gln Gln Arg Lys Lys Thr Pro Val

67 CAA TGG AAG AAG AGC ACT ACT TTA TTC AGC TCG GCG TTA ATT GTT TCA TCT GTA GGA ACG 126 Gln Trp Lys Lys Ser Thr Thr Leu Phe Ser Ser Ala Leu Ile Val Ser Ser Val Gly Thr 127 CCC GTT GCG TTA CTA CCA GTG ACT GCT GAG GCA ACA GAA GAG CAG CCA ACA AAT GCG GAA 186 Pro Val Ala Leu Leu Pro Val Thr Ala Glu Ala Thr Glu Glu Gln Pro Thr Asn Ala Glu 187 GTT GCC CAA GCA CCT ACT ACA GAA ACT GGC TTA GTA GAG ACA CCA ACC ACA GAA ACT ACG 246 Val Ala Gln Ala Pro Thr Thr Glu Thr Gly Leu Val Glu Thr Pro Thr Thr Glu Thr Thr 247 CCA GGA ATT ACG GAA CAA CCG ACA ACG GAT TCG TCA ACA ACG ACT GAA TCG ACA ACT GAA 306 Pro Gly Ile Thr Glu Gln Pro Thr Thr Asp Ser Ser Thr Thr Thr Glu Ser Thr Thr Glu 307 TCA TCA AAA GAA ACA CCA ACA ACA CCA AGT ACC GAG CAA CCA ACA GTT GAT TCA ACT ACA 366 Ser Ser Lys Glu Thr Pro Thr Thr Pro Ser Thr Glu Gln Pro Thr Val Asp Ser Thr Thr

367 CCT GTG GAA TCA GGA ACG ACT GAT TCT TCA GTA GCA GAA ATC ACG CCA GTA GCT CCT TCA 426 Pro Val Glu Ser Gly Thr Thr Asp Ser Ser Val Ala Glu Ile Thr Pro Val Ala Pro Ser 427 ACA ACC GAG TCT GAA GCA GCG CCT GCG GTT ACA CCC GAT GAT GAA GTA AAA GTA CCA GAA 486 Thr Thr Glu Ser Glu Ala Ala Pro Ala Val Thr Pro Asp Asp Glu Val Lys Val Pro Glu 487 GCT AGA GTA GCT TCT GCG CAA ACT TTT TCA GCG TTA TCA CCG ACG CAA AGT CCT TCA GAA 546 Ala Arg Val Ala Ser Ala Gln Thr Phe Ser Ala Leu Ser Pro Thr Gln Ser Pro Ser Glu 547 TTT ATT GCC GAG TTA GCT CGT TGT GCA CAA CCT ATT GCG CAA GCC AAT GAT TTA TAT GCA 606 Phe Ile Ala Glu Leu Ala Arg Cys Ala Gln Pro Ile Ala Gln Ala Asn Asp Leu Tyr Ala 607 TCA GTG ATG ATG GCT CAA GCA ATC GTT GAA AGT GGT TGG GGG GCA AGT ACG TTA TCT AAG 666 Ser Val Met Met Ala Gln Ala Ile Val Glu Ser Gly Trp Gly Ala S0r Thr Leu Ser Lys 667 GCA CCA AAC TAT AAC TTA TTT GGG ATT AAA GGC AGC TAC AAT GGA CAA TCT GTC TAT ATG 726 Ala Pro Asn Tyr Asn Leu Phe Gly Ile Lys Gly Ser Tyr Asn Gly Gln Ser Val Tyr Met 727 GAT ACA TGG GAA TAT TTA AAC GGC AAA TGG TTA GTG AAA AAA GAA CCT TTC CGT AAA TAT 786 Asp Thr Trp Glu Tyr Leu Asn Gly Lys Trp Leu Val Lys Lys Glu Pro Phe Arg Lys Tyr 787 CCT TCT TAT ATG GAA TCA TTC CAA GAT AAT GCG CAC GTG CTA AAA ACA ACT TCT TTC CAA 846 Pro Ser Tyr Met Glu Ser Phe Gln Asp Asn Ala His Val Leu Lys ThO Thr Ser Phe Gln

847 GCG GGC GTT TAC TAT TAT GCT GGG GCT TGG AAA AGC AAT ACA AGC TCG TAT CGC GAT GCA 906 Ala Gly Val Tyr Tyr Tyr Ala Gly Ala Trp Lys Ser Asn Thr Ser Ser Tyr Arg Asp Ala 907 ACT GCT TGG TTA ACA GGT CGT TAT GCG ACA GAT CCT AGC TAC AAT GCT AAA TTA AAT AAT 966 Thr Ala Trp Leu Thr Gly Arg Tyr Ala Thr Asp Pro Ser Tyr Asn Ala Lys Leu Asn Asn 967 GTC ATT ACC GCA TAT AAC TTA ACT CAA TAT GAT ACA CCA TCT TCT GGT GGA AA6 ACT GGG 1026 Val Ile Thr Ala Tyr Asn Leu Thr Gln Tyr Asp Thr Pro Ser Ser Gly Gly Asn Thr Gly 1027 GGC GGA ACA GTT AAT CCA GGA ACA GGC GGT TCT AAC AAT CAA TCA GGA ACG AAC ACG TAC 1086 Gly COy Thr Val Asn Pro Gly Thr Gly COy Ser Asn Asn Gln Ser Gly Thr Asn Thr Tyr

1087 TAT ACT GTA AAA TCA GGA GAT ACC TTG AAT AAA ATT GCC GCG CAA TAT GGT GTG AGC GTT 1146 Tyr Thr Val Lys Ser Gly Asp Thr Leu Asn Lys Ile Ala Ala Gln Tyr Gly Val Ser Val 1147

GCT AAT TTA CGC TCA TGG AAC GGC ATC TCT GGC GAT TTA ATT TTC GTT GGT CAA AAG CTC 1206 Ala Asn Leu Arg Ser Trp Asn Gly Ile Ser Gly Asp Leu Ile Phe Val Gly Gln Lys Leu

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1327 GCC GCG CAA TAT GGC GTG ACG GTT GCT AAT TTA CGC TCA TGG AAT GGC ATC TCT GGC GAT Ala Ala Gln Tyr Gly Val Thr Val Ala Asn Leu Arg Ser Trp Asn Gly Ile Ser Gly Asp 1 387 TTA ATT TTC GTT GGT CAA AAA CTC ATC GTG AAA AAA GGT ACT TCA GGT AAC ACC GGT GGC Leu Ile Phe Val Gly Gln Lys Leu Ile Val Lys Lys Gly Thr Ser Gly Asn Thr Gly Gly TCA AGT AAT GGT GGC TCT AAC AAT AAT CAA TCA GGA ACG AAT ACG TAC TAC ACA ATT AAA 1506 Ser Ser Asn Gly Gly Ser Asn Asn Asn Gln Ser Gly Thr Asn Thr Tyr Tyr Thr Ile Lys 1 507 TCA GGC GAT ACC TTG AAC AAA ATT GCT GCG CAA TAT GGC GTG AGT GTT GCT AAT TTA CGC 1566 Ser Gly Asp Thr Leu Asn Lys Ile Ala Ala Gln Tyr Gly Val Ser Val Ala Asn Leu Arg

1567 TCA TGG AAT GGC ATT TCT GGC GAT TTA ATC TTC GCT GGT CAA AAA ATT ATT GTG AAA AAA 1626 Ser Trp Asn Gly Ile Ser Gly Asp Leu Ile Phe Ala Gly Gln Lys Ile Ile Val Lys Lys

1627 GGT ACT TCA GGT AAC ACC GGT GGC TCA AGT AAT GGT GGC TCT AAC AAT AAT CAA TCA GGA 1686 Gly Thr Ser Gly Asn Thr Gly G1y Ser Ser Asn Gly Gly Ser Asn Asn Asn Gln Ser Gly 1687 ACG AAT ACG TAC TAC ACG ATT AA6 TCA GGC GAT ACC TTG AAC AAA ATT TCT GCA CAA TTC 1746 Thr Asn Thr Tyr Tyr Thr Ile Lys Ser Gly Asp Thr Leu Asn Lys Ile Ser Ala Gln Phe 1747 GGT GTT AGT GTT GCT AAT CTA CGT TCA TGG AAC GGG ATC AAA GGC GAT TTA ATT TTT GCT 1806 Gly Val Ser Val Ala Asn Leu Arg Seo Trp Asn Gly Ile Lys Gly Asp Leu Ile Phe Ala

1807 GGT CAA ACA ATC ATC GTG AAA AAA GGC GCT TCT GCA GGT GGC AAT GCT TCT TCA ACA AAT 1866 Gly Gln Thr Ile Ile Val Lys Lys Gly Ala Ser Ala Gly Gly Asn Ala Ser Ser Thr Asn

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Protein extracts from clones pSH6500 and pSH4000 were subjected to SDS-PAGE in the presence of M. lysodeikticus cells for detection of cell wall hydrolases (17, 22). After renaturation in a Triton X-100-containing buffer, cell wall

hydrolases appeared as clear lysis bands against an opaque background. Various bands ranging from 66 to -130 kDa and exhibiting hydrolytic activity towards M. lysodeikticus cells (Fig. 2, lanes pSH6500 and pSH4000) were observed in pSH6500 and pSH4000 extracts. These bands could also be detected in a crude S. faecalis extract (Fig. 2, lane S.f.). Activity was also observed when S. faecalis cell walls instead of M. lysodeikticus lyophilized cells were used (results not shown). No hydrolytic activity was found in extracts from E. coli containing pSH4000r or pTZ18R (Fig. 2, lanes pSH4000r and pTZ18R). Nucleotide sequence of S. faecalis autolysin. The deletion clones produced by exonuclease III digestions, starting at the 5' end generated by the AccI cut (at the most downstream AccI site, Fig. 1), removed the putative S. faecalis upstream promoter sequences and brought the coding region closer to the pTZ18R P-galactosidase promoter (LacZ). No deletion clone corresponding to the first 10 min of the exonuclease III reaction could be recovered, possibly because the resulting recombinants have a lethal effect on E. coli. Consequently, an inactive truncated clone lacking the 3' end (pSH2800) had to be used to produce a set of nested deleted clones suitable for sequencing this 5' part of the coding region. The nucleotide sequence for 2,752 bp (from -535 to +2217 of the start codon) was determined for both strands, and a 641-amino-acid open reading frame starting at nucleotide + 1 and ending at nucleotide +2013 was found (Fig. 3). The coding sequence corresponds to a polypeptide with a molecular mass of 74 kDa (calculated with DNA Strider [18]) showing four direct repeats of 204 nucleotides each located near the carboxy-terminal end of the protein. The analysis of the direct repeats shows that 50 (73.5%) of 68 amino acids are identical in the four repeats (19 of them correspond to silent mutations), 13 (19.1%) are repeated three times, and 5 (7.4%) are identical when compared two by two. There are two unique amino acids and one deletion in the first repeat. The second repeat has one unique amino acid, and the fourth repeat has four unique amino acids and a 5-amino-acid deletion at the 3' end (Fig. 4). In the case of unique amino acids, the substitution is usually caused by a mutation in the first or second nucleotide of the codon, except that in the fourth repeat, mutations can be found in all three nucleotides. Upstream from the ATG initiation codon, a putative ribosome-binding site (28) and a promoterlike sequence (TATA box) can be found at -15 and -29, respectively (Fig. 3). No significant sequence homology could be found between the S. faecalis autolysin and any published cell wall hydrolase sequence, including Bacillus sp. cell wall hydrolase (22), S. pneumoniae cell wall amidase (11), or various

bactenrophage muram-idases (7, 13, 19-;1, L3, wU).

1867 AGT GCA TCA GGC AAA-CGC CAT ACA GTT AAA AGC GGT GAT TCA CTT TGG GGC TTA TCA ATG 1926

Ser Ala Ser Gly Lys Arg His Thr Val Lys Ser Gly Asp Ser Leu Trp Gly Leu Ser Met 1927 CAA TAC GGA ATC AGC ATC CAA AAA ATC AAA CAA TTA AAT GGC TTA AGC GGG GAT ACA ATT 1986 Gln Tyr Gly Ile Ser Ile Gln Lys Ile Lys Gln Leu Asn Gly Leu Ser Gly Asp Thr Ile 1987 TAT ATT GGT CAA ACT TTA AAA GTT GGT TAA

TTTAAGATTGAAAAAAATAGCTATCTTTGGTAACATGAG 2055

Tyr Ile Gly Gln Thr Leu Lys Val Gly End 2056 TCTTATAACATGAGTCTTATAATAAAAAAAACAATGAAAGGAATAGTAGAAAGAACCCATGTOGATAGAGAGCGTATGG 2134 2135

CTGGTGGAAATACGTACAGAAGCTTTTGAACTCGCCTTTAAGTTACTTTTTTGAACAAACCAAGTAGGAAAAGTCGGTG 2213

2214 ACGA

FIG. 3. Nucleotide sequence of cloned S. faecalis autolysin.1. Only the DNA strand corresponding to the mRNA sequence iss

given. The nucleotide numbers are shown at each end of the lines, +1 being the first nucleotide of the putative initiating methionine; the deduced amino acid sequence is given under the nucleotide of 10 nucleotides. A putative riboa putative TATA box at -29 are underlined. A corresponding consensus sequence for a TATA box is shown below the nucleotide sequence. The four 204-nucleotide direct repeats are boxed.

sequence. Dots indicate series some-binding site at -15 and

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DISCUSSION

A method originally designed to clone a Bacillus autolysin (22) was used to isolate several S. faecalis clones active against M. lysodeikticus cells by including a treatment with organic vapors in order to release proteins into the medium for in situ detection. Although this treatment caused cell death, clones could be easily rescued by transformation with plasmid DNA recovered from dead colonies. To our knowledge, this is the first report of the cloning of an S. faecalis autolysin. Because the cell wall lytic activity increased as the clone was shortened from its 5' end, it was not easy to reduce the size of the genomic insertion to less than 4 kb (including 2,000 bp upstream from the ATG) without affecting the viability of the clones. One possible explanation is that the 5' noncoding region may contain negative cis elements acting on the putative endogenous S. faecalis promoter, so that the promoter would be just strong enough in E. coli to permit the detection of some lytic activity but not strong enough to have a lethal effect on the bacterial host. When this promoter region is removed, the autolysin gene then falls under the control of the stronger P-galactosidase promoter (LacZ) in the vector, resulting in a higher level of autolysin expression. Another explanation could be that the S. faecalis promoter is not active in E. coli and all the lytic activity is produced by the ,-galactosidase promoter in pTZ18R. The large 5' noncoding region serving as a negative modulator permits only a few RNA polymerase molecules to go all the way to the transcription start site, but when this region is removed, only then does the promoter become fully active. This last hypothesis seems to be supported by the behavior of clone pSH4000r (an inverted insert of pSH4000), which shows no activity (Fig. 2, lane pSH4000r). Nevertheless, it must be kept in mind that this construct contains the P-galactosidase promoter at one end of the gene and a putative S. faecalis promoter at the other end, and an antagonistic effect could occur.

The shortest clone exhibiting lytic activity is pSH4000. In

the case of the deletion clones produced by the exonuclease III reaction, the longest viable subclones, lacking approximately 300 bp from the 5' end in the coding region, are also inactive. It is possible that the active site of the autolysin is located in this region. One S. faecalis clone (pSH4000) was sequenced, and its cell wall lytic activity was also analyzed by SDS-PAGE. Polypeptides from clone pSH4000 showed the same electrophoretic pattern as clone pSH6500. Activity against M. lysodeikticus and S. faecalis cell walls indicated autolytic activity. Several lytic bands were observed after SDS-PAGE of the cloned S. faecalis autolysin. Some of these bands show the same migration patterns as active proteins from a crude S. faecalis extract (Fig. 2, lane S.f.). The coding capacity of pSH4000 is for a 641-amino-acid protein with a calculated molecular mass of 74 kDa. This molecular mass could well correspond to the lower band of the clone migrating exactly as the two main bands from the crude S. faecalis extract. The band near 130 kDa (Fig. 2, lanes pSH6500 and pSH4000) could possibly be explained by a covalent dimerization of the monomer, representing a modified form of the autolysin in E. coli. The upper band near -130 kDa could not be altered by using large amounts of reducing agent (up to 10% dithiothreitol). This indicates that it is not the result of disulfide bonds between two monomers. Since covalent dimers are often formed in vitro by exposing proteins to chemicals, it is conceivable that covalent dimerization could also occur in E. coli. Activity bands lower than the -130-kDa band were also observed and could represent partial proteolytic active products. It is interesting that the relative importance of these bands was found to vary with E. coli growth. For instance, when E. coli is grown for approximately 24 h, the extract exhibits less of the upper (-130-kDa) band than an overnight (18-h) culture does (data not shown). Multiple bands of autolysin activity are also observed when several gram-positive bacteria are used in this gel assay (17). The activity patterns from clones pSH6500, pSH4000, and S. faecalis are in agreement with previous results of the same gel assay involving a different S. faecalis isolate (17). The faint activities migrating below 29 kDa in the S. faecalis extract (not present in the extract from clones pSH6500 and pSH4000) correspond to enzymes releasing teichuronic acids from M. lysodeikticus cells (29a). The cloned autolysin shares some similarity with previously described S. faecium muramidase-2 (4), and they both have an estimated molecular mass around 75 kDa (we calculated 74 kDa for a 641-amino-acid polypeptide). It is noteworthy that S. faecium muramidase-2 can bind penicillin in a covalent manner (4) and that penicillin-resistant isolates contain increased levels of a previously described 75-kDa penicillin-binding protein (PBP 5) (2, 9). The sequence analysis of clone pSH4000 reveals four direct repeats at the carboxy end of the protein. Even though no significant homology could be found between the complete S. faecalis autolysin sequence and any other published sequences of cell wall hydrolases, it is possible to find up to 46% similarity in 52 amino acids encoded by gene 15 from Bacillus bacteriophage ¢29 (13), which codes for a lysozyme, when only the repeated sequences are compared. Repeats were also observed in other cases: pneumococcal bacteriophage Cp-1 muramidase and the S. pneumoniae amidase both share a carboxy-end homologous domain that contains six repeats of 20 amino acids each (10). For the S. pneumoniae amidase, it was demonstrated that the carboxy end is not essential for activity but is required for the enzyme to be fully functional (26). Further study of clones shortened

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by one or more repeats will help in determining the functions of these repeats in the protein and finding out if they could be involved in binding the substrate. ACKNOWLEDGMENTS We thank Richard Hogue and Denis Leclerc for their collaboration and Colette Tremblay for discussions and revision of the manuscript. This work was partly supported by grants EQ-1696 from the Ministere de l'Education du Quebec, 2098 from the Conseil des Recherches en Peche et Agro-alimentaire du Quebec, and A6923 from the Natural Sciences and Engineering Research Council of Canada.

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