Proc. Natl. Acad. Sci. USAVol. 92, pp. 285-289, January 1995Microbiology

A Staphylococcus aureus autolysin that has anN-acetylmuramoyl-L-alanine amidase domain and anendo-f8-N-acetylglucosaminidase domain: Cloning, sequenceanalysis, and characterizationTADAHIRO OSHIDA*t, MOTOYUKI SUGAIt, HITOSHI KOMATSUZAWAt, YEONG-MAN HONG§,HIDEKAZU SUGINAKAi, AND ALEXANDER ToMAsz*¶*The Rockefeller University, 1230 York Avenue, New York, NY 10021; tDepartment of Microbiology, Hiroshima University, School of Dentistry, 1-2-3 Kasumi,Minami, Hiroshima, Hiroshima 734, Japan; and §APRO Life Science Institute, 45-56 Kurosaki-Matsushima, Muya, Naruto, Tokushima 772, Japan

Communicated by Norton D. Zinder, The Rockefeller University, New York, NY, September 14, 1994

ABSTRACT The Tn551 insertion site of the autolysis-deficient Staphylococcus aureus mutant RUSAL2 was clonedand used to identify the autolysis gene ati in the parent strain,RN450. The open reading frame for atl was 3768 bp in length,encoding a deduced protein of 1256 amino acids and molecularsize of 137,381 Da. The atl gene product is a bifunctionalprotein that has an amidase domain and an endo-13-N-acetylglucosaminidase domain which must undergo proteo-lytic processing to generate the two extraceliular lytic enzymesfound in the culture broth of S. aureus.

Bacteria contain several types of peptidoglycan hydrolases thatcan break covalent bonds in their own cell walls (1, 2). In spiteof their ubiquitous presence among all bacterial species ex-amined, the true physiological or ecological function(s) ofthese enzymes are not known. On the other hand, the activityof some of these hydrolases is clearly involved in phenomenasuch as bacterial autolysis (induced by antibiotics or adversephysiological conditions), cell wall turnover, and cell separa-tion (2-7). At least some peptidoglycan hydrolase(s) may beinvolved in cell wall enlargement, cell division, and/or mor-phogenetic processes (8). Staphylococcus aureus contains atleast three kinds of catalytically distinct peptidoglycan hydro-lases: an N-acetylmuramoyl-L-alanine amidase (AM) (EC3.5.1.28), an endo-f3-N-acetylglucosaminidase (GL) (EC3.2.1.96), and an endopeptidase (2, 9-11). Several staphylo-coccal AMs and GLs of different molecular mass have beendetected in culture broth and cell extracts by SDS/PAGE(12-14). Other physiological roles proposed for the S. aureushydrolases [for instance, in the mechanism of methicillinresistance and antibiotic tolerance (15-18)] remain matters ofspeculation.

Little is known about staphylococcal peptidoglycan hydro-lase genes except for molecular cloning of a DNA fragmentencoding an S. aureus GL (19) and nucleotide sequenceanalysis of a peptidoglycan hydrolase determinant of a pro-phage in S. aureus NCTC8325 (20). We have previouslyisolated an autolysis-deficient mutant, RUSAL2, after Tn551transposon mutagenesis of S. aureus RN450 (18), and this hasenabled us to clone and sequence an autolysis gene (atl) whichwe describe in this communication."1

MATERIALS AND METHODSBacteria and Plasmids. S. aureus RUSAL2 is an autolysis-

defective mutant isolated after transposon TnS51 mutagenesisof S. aureus RN450 (18). S. aureus RN450 and FDA 209P wereused for purification of extracellular lytic enzymes. Escherichia

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coli JM109, used as a host strain, and plasmids pUC19, pUC18,and pBR322 were from Takara Shuzo (Kyoto). S. aureusstrains and E. coli strains were grown in tryptic soy broth(Difco) and Luria-Bertani medium, respectively, with aerationat 370C.

Cloning and Nucleotide Sequencing. Routine DNA manip-ulations were performed essentially as described (21). DNAsequence was determined by the dideoxy chain-terminationmethod (22) with an automated DNA sequencing system(model 373A; Applied Biosystems). Both DNA strands weresequenced by using vector-derived primers.

Protein Isolation and Amino Acid Analysis. A 60-kDa AMand a 51-kDa GL were isolated as described (M.S., unpub-lished work; ref. 14).

Bacteriolytic Enzyme Profiles After SDS/PAGE. Thesewere analyzed as described (13).

RESULTSCloning of the Autolysis Gene ati. To clone the autolysis

gene from the parent RN450, the chromosomal fragmentcarrying the inactivating Tn551 insert in mutant RUSAL2 wasidentified by probing a Kpn I digest of RUSAL2 chromosomalDNA with a 4.1-kb Pst I-EcoRI internal fragment from Tn551.The probe hybridized to a 3.4-kb DNA fragment and a 9.2-kbDNA fragment. The 3.4-kb fragment was subsequently puri-fied and cloned into the Kpn I site ofpUC19 to generate pAT1.The pAT1 DNA was then used to screen restriction digests ofchromosomal DNA from RN450. A 7.3-kb Kpn I fragment wasidentified. However, attempts to clone the whole 7.3-kb KpnI fragment directly from the chromosomal DNA of RN450have been unsuccessful and the complete atl gene was even-tually cloned in overlapping DNA fragments.The steps in this cloning procedure, summarized in Fig. 1,

were as follows. (Step 1) During the unsuccessful attempts toclone the whole 7.3-kb Kpn I fragment one recombinantplasmid, pAT4, carrying a spontaneously truncated Kpn Ifragment was obtained. The insert ofpAT4 was a 4.3-kb moietywhich contained one of the Kpn I ends of the 7.3-kb fragment;this 4.3-kb insert was cloned into pUC19 at the Kpn I cloningsite. (Step 2) Next, two deletion subclones, pAT20 and pAT22,were made from the 4.3-kb fragment ofpAT4 by using the KpnI cloning site of pUC19. (Step 3) By probing the chromosomalDNA digests with the pAT4 DNA, a 2.3-kb Pvu II fragment

Abbreviations: AM, N-acetylmuramoyl-L-alanine amidase; GL, endo-,3-N-acetylglucosaminidase; ORF, open reading frame.tPresent address: Pharmacological Research Laboratory, TanabeSeiyaku Co., Ltd., 2-2-50 Kawagishi, Toda, Saitama 335, Japan.1To whom reprint requests should be addressed.[The sequence reported in this paper has been deposited in theGenBank data base (accession no. D17366).

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KH H Hc PHcPPsle,

pAT4H.

PH P S Hc Hc KPH Lytic ActivityPH P IS "I Amidase Glucosaminidase

pAT5HHM Activity Activity,K - +

H +

+K = = _ pA~~T38pAT40

pAT20-

pAT8M,H

pAT22 -M -HS1 Kb pAT15

pAT6H$pAT1 2$ H,c

nDc1 nzcenDQ nDcslAsetnt,oi,\ pAT16HL -

WIjrI jHFr UHJn3- _ I

+

+

+

+

+

UHL-4 (AUtolysin)

NH2 Amidase IGlucosaminidasetCOOH2588bp 4300bp 4301bp 5743bp

FIG. 1. Genomic organization and sequencing strategy of the atl gene and phenotypes of recombinant plasmids. Heavy line at top indicates thesequenced part ofDNA and small arrows indicate the direction of sequencing. Thick arrows show the open reading frames (ORFs) and the directionof transcription. The coding region of atl is represented by ORF4. K, Kpn I; H, HindIII; Hc, HinclI; P, Pvu II; Ps, Pst I; S, Sph I. Extracts of E.coli JM109 cells carrying each plasmid were examined for bacteriolytic activity in SDS/polyacrylamide gels containing heat-inactivated bacterialcells (S. aureus to determine AM and Micrococcus luteus to determine GL activity): +, lytic band(s); -, no lytic band.

and a 3.7-kb HindlIl fragment were identified. The 2.3-kb PvuII fragment was cloned in the HinclI site ofpUC19 to generatepAT40, and the 3.7-kb HindlIl fragment (after the recessed 3'termini were filled in) was cloned in the Sca I site of pBR322to generate pAT28. (Step 4) By using pAT28 to probe chro-mosomal digests, a 2.5-kb Kpn I-Pst I fragment was identifiedand cloned into pUC18 digested with Kpn I and Pst I, togenerate pAT37 (not shown in Fig. 1). To facilitate sequencing,the 2.5-kb Kpn I-Pst I fragment of pAT37 was then subclonedin the HinclI site of pUC19 after conversion of the termini toblunt-ended molecules, to generate pAT38. (Step 5) Finally, asecond group of overlapping clones was obtained by using theoriginal plasmid pATl to identify hybridizing fragmentsamong HindIll restriction fragments of the parental (RN450)chromosomal DNA. A 3.4-kb Hindlll fragment was identifiedand cloned in the HindIII site of pUC19, to generate pAT5.Plasmid pAT5 was then used to generate a set of deletionsubclones shown in Fig. 1 (pAT8, pAT1S, pAT6, pAT12, andpAT16).

Expression of the atl Gene in E. coli. All the recombinantplasmids shown in Fig. 1, except pAT28, contained the insertsdownstream of the lac promoter of pUC19 in the direction ofgene transcription. Cell extracts of the E. coli strains carrying

A B C D E F G H I200-116-

66-V.:

FIG. 2. Bacteriolytic enzyme profiles on an SDS/7.5% polyacryl-amide gel containing heat-inactivated M. luteus cells (0.05%) assubstrate. After cell extracts of S. aureus RN450 and E. coli clones andthe purified enzymes were subjected to electrophoresis, the gel waswashed for 30 min in distilled water and then incubated in 0.1 Mphosphate buffer (pH 7.0) at 37°C for 16 hr before detection of enzymeactivity. Bands with lytic activity were observed as clear zones in theopaque gel. The clear zones appeared as dark bands after photographyagainst a dark background. Lanes: A, S. aureus RN450 cell extract; B,S. aureus RN450 AM; C, S. aureus RN450 GL; D-I, E. coli JM109carrying pAT40, pAT4, pAT5, pAT8, pAT12, or pUC19, respectively.Positions of molecular mass standards are shown at left (kDa).

recombinant plasmids showed lytic activity in the gels con-taining heat-inactivated bacterial cells (Figs. 2 and 3), indicat-ing that the cloned DNA fragments encoded (a) protein withcell wall hydrolytic activity. The extract of E. coli straincarrying pAT40 showed a lytic band of 58 kDa on the gelcontaining S. aureus cells (Fig. 3) but no lytic activity on thegel containingM luteus cells as substrate (Fig. 2), suggestingthat the band might represent the pure AM. In contrast, theextract of the E. coli strains carrying pAT4, pAT5, pAT8, orpAT12 exhibited lytic bands of 80, 45, 45, and 24 kDa (notshown in Fig. 2), respectively, but only on the SDS/polyacrylamide gels containing M luteus cells as substrate(Fig. 2), indicating that the hydrolase encoded on theseparticular cloned DNA fragments had substrate specificitysimilar to that of pure GL. The molecular sizes of these lyticbands decreased parallel with the truncation of the clonedDNA fragments, suggesting that an active site of the hydrolasewas encoded on the cloned fragment of pAT12 and that themolecular size of the hydrolase was at least 80 kDa. Thesmallest protein (24 kDa) exhibiting lytic activity was observedin extracts from pAT12-carrying cells, which were tested on a12% acrylamide gel containing M luteus cells (data notshown). This low molecular mass lytic band was not seen on thegel in Fig. 2, because it was eluted from the gel under theconditions employed.The most intriguing finding that emerged from these tests

was that pairs of cloned DNA fragments that clearly did notoverlap on the restriction map (e.g., pAT40 and pAT8) couldnevertheless both encode proteins with cell wall hydrolyticactivity. Further, the lytic activity of pAT40 was detectableonly with the amidase substrate, in contrast to pAT8, whichshowed lytic activity only with the glucosaminidase substrate

A B C D E F G H I200-116-

66--

45-.

FIG. 3. Bacteriolytic enzyme profiles with heat-inactivated S.aureus (0.05% in the gel) as substrate. Samples, SDS/PAGE condi-tions, and detection of lytic bands were as in Fig. 2.

Proc- Natt Acad Sci USA 92 (1995)

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(see Fig. 1). These observations suggested either the presence sequences of the ORFs are shown in Fig. 4. The total nucle-of two different hydrolase genes or a single gene encoding a otide sequence has four ORFs all transcribed in the samehydrolase containing two active sites. direction. The autolysin gene atl (ORF4) is preceded by three

Nucleotide and Deduced Amino Acid Sequence of the Pep- ORFs with unknown function. ORF1, at the beginning of thetidoglycan Hydrolase. The DNA sequencing strategy and the cloned DNA, is N-terminally truncated. There are two ATGlocation of ORFs are shown in Fig. 1, and the nucleotide codons in the same correct reading frame of atl. However, thesequence for 6140 bp of DNA and the deduced amino acid second of these is considered to be the initiation codon,

wVACCJUWUTTAAATGGTGATGAGGCTTTaGCAGTTGCTAGAACTAGACATCATGATTCAGACTTAAAACGTG cGTCaAaAGGAATTAaTTAAGATTTTATTCCAAAAAGCAC 120orf l T Q K L N G D E A L A V A R T R H H D S D L K R G Q R Q H B L I X I L F Q X A Q

AGGAAGTGTCATTGATAAACTTGATAACGTGATTCsAKATTGTAGGTAAAAATGCAAAGCATAATTTAACTAACTC TGAAATTAkAGCTTTASCMAAATGTACTTCAAlATGATG 24 0B V D S I D K L D N V I Q I V G K N A K H N L T N S E I X A L AKx X r L T N D V

TTGAAATTAAAACTGCGCAATTaAAAGGTAAAGATGATATGTTAAATGGTATTTACTATTATCATCCAAGTGTTGAAAGCATTCAAAAATATGCAAACTTACTTCGTaAAhGACTTAGAAT 36011 I K T A Q L K G X D D H L N G I r Y Y H P S V lC S I Q K Y A N L L R K D L E L

TATCACCTATTAATGATAAAAATGATTTCTTAGATCAACGTGTCATTAATCATTATGGTTCATTAATACCATTAACGCCTTTAGATAATAGTT ATTGAACAAAACAAAATGATACGA 480S P I N D X N D F L .D Q R V I N H Y G S L I P L T P L D N S L L R X E Q N D T T

CAGATAAAGATAAG;ACATCTAACGAG;AACAGTGATTCAACTAATAACAGTGATTCCAGCAATCAACAACAACCTGCTACAGATCAAAACTCAAATCA1iAATCAAGGTGGCACAACAAG 600D X D X T S N B N S D S T N N S D S S N Q Q Q P A T D Q N S N Q N Q G G T Q Q A

CGCCACAAGCTTCAAKTARCCAAAATGGTGTTGTaAATTAATATTATATGACGATTAhCATTTACTASTTTGAIGWACATCTACATGAGTAGAAAAACATACGAAaAGATTGCAAA 720P Q A S N N Q N G V v N * rbs orf2 11 5 R K T Y E K I A N

TATaAAATGGCATGTTTAATATGTTAGAACAACAAATCATTCATAGCCaAGATATGGCTCATTTTAGAAGTGAATTTTTTTACGTCAATCATGAGCATCGAGaAAAACTATGAACACTCCT 84 0I N G H F N H L B Q Q I I H S Q D M a H F R S 15 P F r V N H 3 H R E N Y E A L L

AATTTATTACAAAAATAGTATCGACAATCCTATTGTAGATGGTGCATGTTATATTTTAGCCCTACCTGaAAATTTCAATAGTGTTGATGTTTTCGAATCAGAGTTACCATTTTCATGGGT 960I Y Y It N S I D N P I V D G A C Y I L A L P 1 I F N S V D V F E S E L P F S N VAAccGAAAcAasGAAAaTGAACACTTAGCATTCCATTACATTTATTG CAGCAGCTTTAGAAGTaAhCTGATGTGAATATATTTAAGCCTTCAGGATTTACAAT 1080

Y D 2 N G I T E T M X S L S I P L Q Y L V A A A L E V T D V N I r X P S G F T MGGGAATGAATAATTGGAATATTGCTCARATGCGAATCTTTTGGCAATATACAGCAATTATTAGAAAAGACATATAACATTATATATTAATTAGCTATAAAGTGATTCACAACAATC 12200G X N N I N I A Q M R I F W Q Y T A I I R X E A L - IN 4*ATCTTTATAGCTTTTTTATGTCTaATTATTTTTGAGGAAAAaTaAcAaCAATTTCTATGTTATTATTAGTAAATAAATGTAAATGATTTTGTTAGGATGTGGAGGAATTTTATGTTTT 132 0

-35 -lO rbs orf 3 M F SCAAAAGTAAACATCAAAGATGTGAGATTG CTTCTATATAAGAAAGa AAGTGTTTGTAGAAGAACAAGOCGTCCC TGAGGAAAGTGAAATTGATGAATATGAATCTGAATCTATTC 1440K V N N Q X M L E D C F Y I R K K V F V E E Q G V P E E S E I D E Y E 5 13 5 I H

ACCTCATTGGATATGATAATGGACAGCCAGTTGCCACTGCTCGAATACGCCCTATTAATGAAACAACTGTCAAAATAGAACGAGTAGCTGTGATGAAATCACTGTGGACAAGGAATGG 15S60L I G Y D N G Q P V A T A R I R P I N E T T V X I E R V a V M X S H R G Q G H G

GTAGAATGCTTATGCAAGCTGTAGAATCATTAGCTAAAGATGAAGGTTTTTACGTAGCTACTATGAATGCCCAATGTCATGCTATCCCATTTTATGAAAGTTTAAACTTTAAAATGAGAG 1680R M L M Q A V E S L A X D E G F Y V A T M N a Q C H A I P F Y E S L N F X M R G

GTAATATATTTCTTGAGGAAGGCATCGAGCATATTGaAAATGACAAAAAGTTAACCTCGCTTAATTAAAAAAA^ TTGTATCTATTTTAGAAACATTTGTAATAAACCAATTCTTAAATTG 18 00N I F L E E G I E H I E H T K X L T S L N * 44

ACATCAATTCTATTTGATTTGTCACGTCACCATTGAGATTCAAAATTTTTTTCATTTTTTACAGTGAAAATGTaAATA=AGaTATATTACMAATGGTTaATACGCACAGGTATATA 192 0_~~~~~~~~~~~~~~~o . < -35 (Pl) -10(Pl)-35(P2) -lO(P2)

AAAAGGTACTATAATGTTAGTAATAATTAATAAATGTTAGGAGTAATAAATAGAATGGCGAAlAAAATTCAATTACAAhCTACCATCAATGGTTGCATTAACGCTTGTAGGTTCAGCAGT 20401 rbs orf4 M A X K F N Y X L P S }( V A L T L V G S a VCACTGCAAaTCAAGTTCAAGCAGCTGAGACGACACAAGATCAAACTATAKTAATAAAACGTTTTAGATAGTAATAAA GTTAAAGCATCGcAACAAGCAAACTGAGGTAAAAAATCC 2160

23 T A H Q V Q A a E T T Q D Q T T N X N V L D S N X V X A T T E Q A X A E V X N PAACGCAlAAaCATTTCTGGCACTCAAGTATATCAAGACCCTGCTATTGTCCAACCAAACGAAoAGCaG TCAAGTAAGTAAAGTTGATACTGCACAAGTAAA 2280

63 T Q N I S G T Q V Y Q D P a I V Q P X T A N N K T G N A Q V S Q X V D T A Q V NTGGTGACACTCGTGCTAATCAATCAGCGACTACAAATAATACGCAGCCTGTTGCAAAGTCAAAGCACTACAGCACCT^^*AAA TAACACTAATGTTACAAATGCTGGTTATAGTTTAGT 2400

103 G D T R a N Q S a T T N N T Q P V A X S T S T T a P x T N T N V T N A G Y S L VTGATGATGAAGATGATAATTCAGAAAATCAAATTAATCCAGAATTAATTAAATCAGCTGCTAAACCTGCAGCTCTTGAAACGCAATATaAAACCGCAGCACCTAAAGCTGCAACTACATC 2520

143 D D E D D N S E N Q I N P E L I K S A A X P A A L E T Q Y X T A A P X a A T T SAGCACCTAAAGCTAAAACTGAAGCGAaCACTAAAGTAACTACTTTTAGCGCTTCAGcacAACCAAGATCAGTTGCTGCaAACACCAAAAACGaGTTTGCCAAAATATaAAACCACAAGTAAA 2 64 0

183 A P K A K T E A T P K V T T F S A S A Q P R S V A A T P X T 8 L P K Y X P Q V NCTCTTCAATTAACGATTACATTTG TAAAAATAACTTaAAAAGCACCTAAAATTGAAGAAGATTATACATC TTACTTCCCTaAATACGCATACCGTiAACGGCGTAGGTCG TCCTGAAGGTAT 2760

223 S S I N D Y I C K N N L X A P X I E E D Y T S Y F P K Y A Y R N G V G R P E G ICGTAGTTCATGATACAGCTAATGATCGTTCGACGATAAATGGTGAAATTAGTTATATGAAAAATAACTATCAAAACGCATTCGTACATGCATTTGTTGATGGGGATCG TATAATCGAAACB 2880

263 V V H D T A N D R S T I N G E I S Y M K N N Y Q N a F V H A F V D G D R I I E TAGCACCAACGGATTACTTATCTTGGGGTGTCGGTGCAGTCGGTAACCCTAGATTCATCAATGTTGAAATCGTACACACACACGACTATGCTTCATTTGCACGTTCAATGNAATAACTATGC 3000

303 A P T D Y L S W G V G A V G N P R F I N V E I V H T H D Y A s F A R S M N N Y ATGACTATGCAGCTACACAATTACAATATTATGGTTTaAAAACCAGACAGTGC TGAGTATGATGGAAATGGTACAGTATGGACTCACTACGCTGTAAGTAAATATTTAGGTGGTACTGACCA 3120

343 D Y A A T Q L Q Y Y G L X P D S A E Y D G N G T V W T H Y A V s X Y L G G T D HTGCCGATCCACATGGATATTTAAGAAGTCATAATTATAGTTATGATCAATTATATGACTTAATTAATGAAAAATATTTAATAAAAATGGGTAAAGTGGCGCCATGGGGTACGC AATCTAC 32 40

383 A D P H G Y L R S H N Y S Y D Q L Y D L I N E X Y L I K M G X V A P W G T Q S .TAACTACCCCTACTACACCATCAAAACCAACAACACCGTCGAAACCATCAACTGGTAAATTAACAGTTGCTGCAAACAATGGTGTCGCACAAATCAAA,CCAACAAATAGTGGTTTATATAC 3360

423 T T P T T P S X P T T P S K P S T G x L T V A A N N G V A o I X P T N S G L Y TTACTGTATACGACAAAACTGGTAAAGCAACTAATGAAGTTCAAAACATTTGC TGTATCTAAAACAGCTACATTAGGTAATCAAAAATTCTATCTTGTTCAAGATTACAATTCTGGTAA 3480

463 T V Y D K T G X A T N E V O X T F A V s K T A T L G N O X F Y L V Q D Y N S G NTAAATTTGGTTGGGTTAAAGAAGGCGATG TGGTTTACAACACAGCTAAATCACC TGTAAATGTAAATCAATCATATTCAATCAAACCTGGTACGAAACTTTATACAGTACCTTGGGGTAC 3600

503 K F G W V K E G D V V Y N T A X s P V N V N Q S Y S I K P G T K L Y T V P W G TATCTAAACAAGTTGCTGGTAGTGTGTCTGGCTCTGGAAACCAAACATTAAGGCTTCAAAGCAACAACAAATTGATAAATCAATTTATTTATATGGCTC TGTGAATGGTAAATCTGGTTG 372 0

5 43 S K O V A G S V S G S G N O T F X A S x O Q Q I D X S I Y L Y G s V N G K S G WGGTAAGTAAAGCATATTTAGTTGATACTGCTAAACCTACGCCTACACCAACACCTAAGCCATCAACACCTACAACAAATAATAAATTAACAGTTTCATCATTAAACGGTGTTGCTCAAAT 384 0

583 V S X A Y L V D T A X P T P T P T P X P S T P T T N N X L T V S S L N G V A Q ITAATGCTAAAAACAATGGCTTATTCACTACAGTTTATGACAAAACTGGTAAGCCAACGAAAGAAGTTCAAACATTTGCTGTAACAAAAGAAGCAAGTTTAGGTGGAAACAAATTCTA 3960

623 N A K N N G L F T T V Y D K T G K P T K E V O X T F A V T K E A s L G G N K F Y-CTTAGTTAAAGATTACAATAGTCCAAC TTTAATTGGTTGGGTTAAACAAGGTGACGTTATTTATAACAATGCAAAATCACC TGTAAATGTAATGCAAACATATACAGTAAAACCAGGCAC 40o80

663. L V X D Y N S P T L I G W V K Q G D V I T N N A X S P V N V M Q T I T V K P G TTAAATTATATTC;AGTACCTTGGGGCACTTATAAACAAGAAGCTGGTGCAGTTTC TGGTACAGGTAACCAAACTTTTAAAGCGAC TAAGCAACAACAAATTGATAAATC TATCTATTTATT *4200

703 K L Y S V P W G T Y K O E A G A V S G T G N O T F K A T K O O O I D X S I Y L FTGGAACnTGTAAATGGTAAATCTGGTTGGGTAAGTaAAAGCATATTTAGCTGTACCTGCTGCACCTAAAAAAGCAGTAGCACAACCAAAAACAGCTGTAAAAGCTTATACTGTTACTAAACC 4 320

743 G T V N G K S G W V S K a Y L A V P A A P K K A V A Q P X T A V X A T T V T X PACAAACGACTCAAACAGTTAGCAAGATTGCTCAAGTTAAACCAAACAACACTGGTATTCGTGCTTCTGTTTATGAAAAAACAGCGAAAAAGGTGCGAAATATGCAGACCGTACGTTC TA 4 44 0

783 Q T T Q T V S X I a Q V X P N N T G I R A S V Y E K T A K N G A K Y A D R T F YTGTAACAAAAGAGCGTGCTCATGGTAATGAAACGTATGTATTATTAAACAATACAAGCCATAACATCCCATTAGGTTGCTTCAATGTAAAAGACTTAAATGTTC^AAAATTAGGCAAAGA 4560

823 V T K E R -A H G N E T Y V L L N N T S H N T P L G W F N V X n T. N V n] N T. r. l FI460

506V l~ ~ I T N S 0 00 0 0 n a M V nPr 0 r0 0 D rV La T L. V D OL F SAG.TTGAs:CAAAAATATACTGTTAATAAATCAAATAACGGCTTATCAATGGTTCTTGGGGTACTAAAAACCAAGTCATTrTTAACAGGCAATAACATTGCTCAAGGTACATTTAA 4

863 v X T T 0 K Y T v N K S N N G L S M V P W G T K N O V I L T G N N I A O G T F NTGCAACGAAA^̂AAGTATCTGTAGGCAAAGATGTTTATTATACGGTACTATTAATAACCGCACTGGTTGGGTAAATC,CAAAATTTAACTGCACCAAC TGCTGTGAAACCAACTACATC 4800

903 A T K O V S V G K D V Y L Y G T I N N R T G N V N A K D L T A P T A V K P T T SAGCTGCCAAAGATTATAACTACACTTATGTAATTAAAAATGGTAATGGTTATTACTATGTAACACCAAATTCTGATACAGCTAAATACTCATTAAAAGCATTTAATGAACAACCATTCGC 4 920

943 A A K D Y N Y T Y V I K N G N G Y Y Y V T P N S D T A K Y S L K A F N E Q P F AAGTTGTTAAAG-^AACAGTCATTAATGGACAAACTTGGTACTATGGTAAATTATCTAACGGTAAATTAGCATGGATTAAATCAACGATTTAGCTAAAGAATTAATTAAGTATAATCAAAC 50O40O

983 V V K E Q V I N G Q T W Y Y G K L S N G K L A W I K S T D L A K B L I X Y N Q TAGGTATGACATTAAACCAAGTTGCTCAAATAChAAGCTGGTTTACAATATAAACCACAA-GTACAACGTGTACCAGGTAAGTGGACAGATGCTAAATTTAATGATGTTAAGlCATGCIAATGGA 5 160

023 G M T L N Q V a Q I Q A G L Q Y K P Q V Q R V P G K N T D A K F N D V K H A H DTACGAAGCGTTTAGCTCAAGATCCAGCATTAAAATATCAATTCTTACGCTTAGACCAACCACAMAAATTTTCTATTGATAAAATTAATCAATTCTTAAAGGTAAAGGTGTATTAGAAAA 5280

063 T K R L A Q D P A L K Y Q P L R L D Q P Q N I S I D K I N Q F L K G K G V L E NCCAAGGTGCTGCATTTAACAAAGCTGCTCAAATGTATGGCATTAATGAAGTTTATCTTATCTSCACATGCCCTATTAGAAACAGGTAACGGTACTTCTCAATTAGCGAAAGGTGCAGATGT 5400

103 Q G A a F N K A A Q H Y G I N E V Y L I S H A L L E T G N G T S Q L A K G a D VAGTGAACAACAAAGTTGTAACTAACTCAAACACGAAATACCATAACGTATTTGGTATTGCTGCATATGATAACGATCCTTTACGTGAAGGTATTAAATATGCTAAACAAGTGGTTGGGA 5520

143 V N N K V V T N 'S N T K Y H N V F G I A A Y D N D P L R Z G I X Y A X Q A G W DCACAGTATCAAAAGCAATCGTTGGTGGTGCTAAATTCATCGGCAACTSCATATGTAAAAGCTGGTCAAMATACACTTTACMAASTGAGATGGAATCC TGCACATCCAGGAACACACCAATA 5 640O

183 T V S K A I V G G A X F I G N S Y V K A G Q N T L Y K M R W N P A H P G T H Q YTGCTACAGATGTAGATTGGGCTAACATCAATGCTAAAATCATCAAAGGCTACTATGATAAAATTGGCGAAGTCGGCAAATACTTCGACATCCCACAATATAAATAAGCAACATGAACATA 5 760

223 A T D V D W A N I N A K I I K G Y Y D K I G E V G K Y F D I P Q Y K *

GGATCAAAAGTCATCCCCCACTATCAATCATGGGGGATGACCTTTGATCCCTTTTTTATACATACACAAGCAAAAATAGCGGTGATTGTTTACCATCAATTTTAACAATCACCGCTACTT S 8800- - '4o 8

TTGCTTGTAATTCATGATTCATSTTTTGTTGTGTGCACAACGACACTAAATTATGTGTTTGCTATTGTCGTGTTACAACGATATGCGTCGTTGATTTAACTTAVAGPTAATTGATSTAA 6000ATTGTCTAATTCGACTTCCGATAAACATTGACATCTTGCTTCAATCAATTCGCAACGTGCAT ATTTATTTGTGA MATTAATGTACGTGCTTGATC AGTCAAnTAAT=TTACATCT 6120TAAAKTCTTCCTAGATTGTT 614

FIG. 4. Nucleotide sequence and deduced amino acid sequence of the S. aureus atl gene. The coding region of atl is represented by ORF4. Twopossible candidates (Pl and P2) for promoter sequences (-35 and -10 regions) and the putative ribosome binding sites (rbs) are shown. Invertedrepeat sequences are indicated by arrows. The three repeated amino acid sequences are underlined.

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288 Microbiology: Oshida et al.

because a Shine-Dalgarno sequence (AGGAG) is located atthe proper position (12-16 bp upstream of the second ATGcodon). The termination codon TAA occurs 3771 bp from theinitiation codon, so that atl encodes an autolysin of 1256 aa and137,381 Da. A putative p-independent terminator that consistsof two stem-loop structures is present downstream from thestop codon at bases 5761-5888. Two possible candidates forpromoter sequences (P1 and P2) are present upstream of atl.A stem-loop structure that may concern the regulation of atlis present upstream of the putative promoter sequence at bases1849-1877. A putative signal sequence is present in the first 29aa and the cleavage site is located after the sequence Val-Gln-Ala, using the strategy described (23). Three repeated se-quences, each about 150 aa long, are located in the centralregion of the amino acid sequence. Alignment (Fig. 5) ofrepeat 1 (Lys441-Leu588) with repeat 2 (Lys610-Leu757) yields113 identities for 148 aa (76%). The similarity between repeat1 and repeat 2 is much greater than those between repeat 3 andrepeat 1 (34%) or repeat 3 and repeat 2 (35%).

Isolation and Characterization of Peptidoglycan Hydro-lases of S. aureus RN450 and FDA 209P. The amidase (AM)and the glucosaminidase (GL) were purified from culturesupernatants of S. aureus RN450 and FDA 209P, respectively,and the N-terminal amino acid sequences of the enzymes weredetermined experimentally to compare with the deducedsequence of atl. The N-terminal amino acid sequences of theAMs of S. aureus RN450 and FDA 209P were determined asSer-Val-Ala-Ala-Thr-Pro-Lys-Thr-Xaa-Leu and Ala-Ser-Ala-Gln-Pro-Arg-Ser-Val-Ala-Ala-Thr-Pro-Lys-Xaa-Ser-Leu-Xaa-Xaa-Tyr-Xaa-Xaa-Gln-Val-Asn, respectively. The twosequences showed an exact match except for a 6-aa deletion inthe N-terminal end of the RN450 AM. The sequence of theFDA 209P AM was in complete agreement with the deducedamino acid sequence of Atl from Ala'99 to Val221. TheN-terminal amino acid sequences of the GLs of S. aureusRN450 and FDA 209P were determined as Ala-Tyr-Thr-Val-Thr-Lys-Pro-Gln-Thr-Thr-Gln-Thr-Val-Ser-Lys-IIe-Ala andAla-Tyr-Thr-Val-Thr-Lys-Pro-Gln-Thr-Thr-Gln-Xaa-Val-Ser-Lys-Ile-Ala-Gln-Val-Lys, respectively. The two sequencesshowed a complete match with each other and were incomplete agreement with the deduced amino acid sequence ofAtl starting at Ala776. If the Atl polypeptide starting at Ala'99and ending at Lys1256 were hydrolyzed between residues Lys775and Ala776 and processed to two separate hydrolases (AM andGL), the calculated molecular masses of the two deducedpeptides (Ala199-Lys775 and Ala776-Lys1256) would be 62,954Da and 53,580 Da, respectively, which correspond precisely tothe molecular masses of purified staphylococcal peptidoglycanhydrolases AM and GL, respectively. The experimentallydetermined amino acid composition of these two hydrolases,purified from the culture medium of S. aureus FDA 209P, alsocorresponded closely to the compositions predicted from thesequence data (data not shown). Proteolytic processing of thebifunctional enzyme is strongly supported by experiments withprotease inhibitors. When RN450 was grown in the presenceof protease inhibitor (p-hydroxymercuribenzoate or phenyl-methylsulfonyl fluoride), the bacteriolytic profile of the cellextract showed a marked decrease in intensities of 51-kDa and

Ri 422: TTTPTrPSKPTTPSKPSTGKLTVAANNGVAQIKPTNSGLYTTVYDKTGKATNE-VQKTFAR2 591: .AK ..PTPT.KPSTPTTNN .... SSL... NAK.N. ..F. P.K.-.R3 763: PKKAVAQP.TAVKAYTV.KPQ.TQTVSKI. .V. .N.T.IRAS. .E. .A.NGAKYADR. .Y

Ri 481: VSKTATLGNQKFYLVQDYNSGNKFGWVKEGDVVYNTAKSPNVNQSY-SIKPGTKLYTVPR2 650: .T.E.S..GN. K ... PTLI .. Q .. .N. M.T.-TV.... SR3 823: .T.ERAH..ETYV.LNNTSHNIPL. .FNVK.LNVQNLGKE.KTT.K.TVN.SNNG.SM..

Ri 540: WGTSKQVAGSVSGSGNQTFKASKQQQIDKSIYLYGSVNGKSGWVSKAYLVDR2 709: .. Y..E. .A.. .T. T. F.T. AVPAAR3 883: ... KN. .ILTGNNIAQG. .N.T. .VSVG.DV .. TI.NRT...NAKD.TAPTA

FIG. 5. Amino acid sequence alignment of the three repeatedsequences (Rl, R2, and R3). Amino acid residues identical to the Rlsequence are indicated by dots.

60-kDa lytic bands together with a comparable increase in theintensity of a 150-kDa lytic band (Fig. 6).Comparison of Amino Acid Sequences of the S. Aureus

Autolysin and Other Cell Wall Hydrolases. The N-terminalpart of the AM domain of S. aureus autolysin shows significantsequence homology with the N-terminal portion of the Bacillussubtilis cell wall hydrolase (CWLA) and the pneumococcalEJL bacteriophageAM (24, 25) (Fig. 7). The degree of identityis 40% for 83 aa between the AM and CWLA, 24% for 163 aabetween the AM and the EJLAM and 26% for 96 aa betweenCWLA and the EJL AM.

DISCUSSIONThe DNA sequence reported here establishes the primarystructure of a bifunctional autolysin encoded by the atl gene inS. aureus. The AM domain of atl extends from bp 2588 to bp4300, with the corresponding Atl polypeptide extending fromAla'99 to Lys775. The GM domain begins at bp 4301 and endsat bp 5743, with the corresponding Atl polypeptide extendingfrom Ala776 to Lys'256. The accuracy of the junction sequencewas confirmed by sequencing the recombinant plasmid pAT28from the HindlIl site in the direction of the Pvu II site. The65-bp sequence obtained showed a complete match with thecorresponding sequences generated by pAT20 and pAT22(subclones of pAT4) between the nucleotide residues A-3071and C-5154.The extract of the E. coli strain carrying pAT4 showed a lytic

band of 80 kDa. This molecular size was much larger than the

v v v

1o.02 A.U.

B00LO

C 10.02

D 1 AU|~~~~~0Io02 A.U.

U-)

Cocn0ai

FiG. 6. Effect of protease inhibitor on the proportion of autolysinbands in staphylococcal extracts. A culture of S. aureus RN450 (3 ml)received p-hydroxymercuribenzoic acid (3 ,uM) when the OD620reached 0.2. After 4 hr of incubation with the protease inhibitor, thecells of treated and control (untreated) cultures were harvested bycentrifugation and were suspended in 100 ,ul of 4% SDS for 30 min atroom temperature. After centrifugation, supernatants were analyzedby SDS/PAGE with heat-inactivated cells incorporated in the gels assubstrate. Extracts of the control cells (A and C) and the inhibitor-treated cells (B and D) were analyzed with M. luteus cells (GLsubstrate) (A and B) or S. aureus cells (AM substrate) (C and D)incorporated in the gel. Positions of the decreased bands are indicatedby filled triangles and that of the increased band by an open triangle.Positions of the molecular mass standards (kDa) are indicated at thebottom. A.U., absorbance unit.

Proc. Natt Acad ScL USA 92 (1995)

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An-AM 199:ASAQPRSVAATPKTSLPKYKPQVNSSINDYICKNNLKAPKIEEDYTSYFPKYAYR-NGVG

CWLA 1: MAIKVVKNLVSKSKYGLKCPNPM

EJL 1: MDIDTSRLRTDLPQ-VGVQ

ATL-AM 258:RPEGIVVHDTANDRSTINGEISYM-KNNYQNAFVHAFVDGDRIIETAPTDYLSW----GV

CWLA 24:KAEYITIHNTANDASAAN-EISYM-KNNSSSTSFHFAVDDKQVIQGIPTNRNAWHTGDGT

EJL 19:PYRQVHAHSTGNRNSTAQNEADYHWRKDPELGFFSHVVGNGRVMIVGPVNNGSW----D

ATL-AM 313: GAV-GNPRFINVEIVHTHDY-ASFARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTH

CWLA 82:NGT-GNRKSIGVEICYSKSGGVRYKAAEKLAIKFVAQLLKERGWGIDRVRKHQDWNGKYC

EJL 75:GGGWNAETYAAVELIESHSTEEEFMEDYRLYIELLRNLADEADL-PKTLDTDDLAGIKTH

ATL-AM 371: -Y-AVSKYLGGTDHADPHGYLRSHNYSYDQLYDLINEKYLIKMGKVAPWGTQSTTTPTTP

EJL 134: EYCTNNQPNNNSDHWDPYPYLAKWGVSREQFKQDIENGLTIEAGWKKNDTGTWVYSDGS

FIG. 7. Alignment of the deduced N-terminal amino acid se-quences of the S. aureus Atl AM, the B. subtilis cell wall hydrolase(CWLA), and the EJL bacteriophage amidase. Colons indicate iden-tities and dots represent conservative amino acid substitutions bothrelative to the S. aureus AM sequence. Hyphens represent breaksintroduced to maximize homology.

calculated molecular mass deduced for GL (53,580 Da). Sincethe cloned DNA fragment of pAT4 encoded the C-terminalpart of AM and the GL protein in the same polypeptide, theresults indicate that the C-terminal part ofAM and the entireGL protein were translated in a single polypeptide chain in thisclone. Since AM and GL were purified from culture broth asseparate enzymes, the bifunctional autolysin encoded by atlmust be processed to generate the two extracellular cell wallhydrolases, perhaps in a manner described for the peptydogly-can hydrolases of Staphylococcus simulans (26).The AM domain of Atl contains repeats 1 and 2 at the

C-terminal part, while the GL domain contains repeat 3 at theN-terminal part. Other cell wall-associated bacterial proteinsalso carry repeated motifs at the C-terminal region to formligand-binding domains (25, 27, 28). By analogy, it is likely thatthe N-terminal part of the AM domain of the staphylococcalautolysin, which is highly homologous to EJL AM and CWLA(see Fig. 7), contains the active center of the enzyme, whereasthe C-terminal part may be involved in binding the protein tosome residues in the cell wall. The active site of the GL domainappears to be located closer to the C-terminus, since theprotein (from His1059 to the C terminus) encoded on thetruncated subclone pAT12 still possessed lytic activity. There-fore the GL seems to have an organization opposite to that ofthe AM, having the N-terminal part containing repeat 3 andthe C-terminal part containing the active site. The differencesin structure of the repeat domains of these two enzymes mightreflect the differences of the recognition sites on staphylococ-cal cell walls.The atl gene was probably developed through fusion ofAM

and GL genes. The DNA sequences coding for the threerepeats were probably formed by duplication of an ancestralDNA segment. The greater similarity between repeats 1 and 2suggests that they were formed by two rounds of duplication.The staphylococcal autolysin presents an attractive model for

the study of the evolution of multimodular enzymes by genefusion and gene duplication and also for the study of themechanisms of processing and maturation of autolytic pro-teins.

We are grateful to Drs. T. Yamaguchi, G. Ohashi, and T. Matsushitafor advice and encouragement; to Drs. K. Omori and E. Osward forhelp in DNA sequencing and analysis; and to Dr. H. de Lencastre forcritical discussion of the results.

1. Ghuysen, J.-M., Tipper, D. J. & Strominger, J. L. (1966) MethodsEnzymol. 8, 685-699.

2. Perkins, H. R. (1980) in Microbial Cell Walls and Membranes, eds.Rogers, H. J., Perkins, H. R. & Ward, J. B. (Chapman & Hall,London), pp. 437-456.

3. Koyama, T., Yamada, M. & Matsuhashi, M. (1977) J. Bacteriol.129, 1518-1523.

4. Wong, W., Chatterjee, A. N. & Young, F. N. (1978) J. Bacteriol.134, 555-561.

5. Tomasz, A., Albino, A. & Zanati, E. (1970) Nature (London) 227,138-140.

6. Handwerger, S. & Tomasz, A. (1985) Rev. Infect. Dis. 7,368-386.7. Lopez, R., Ronda, C. & Garcia, E. (1990) FEMS Microbiol. Lett.

66,317-322.8. Tomasz, A. (1984) in Microbial Cell Wall Synthesis and Autolysis,

ed. Nombela, C. (Elsevier, Amsterdam), pp. 3-12.9. Huff, E. C., Silverman, C. S., Adams, N. J. & Awkard, W. S.

(1970) J. BacterioL 103, 761-769.10. Singer, H. J., Wise, E. M. J. & Park, J. T. (1972) J. Bacteriol. 112,

932-939.11. Tipper, D. J. (1969) J. Bacteriol. 97, 837-847.12. Leclerc, D. & Asselin, A. (1989) Can. J. Microbiol. 35, 749-753.13. Sugai, M., Akiyama, T., Komatsuzawa, H., Miyake, Y. &

Suginaka, H. (1990) J. Bacteriol. 172, 6494-6498.14. Sugai, M., Koike, H., Hong, Y.-M., Miyake, Y., Nogami, R. &

Suginaka, H. (1989) FEMS Microbiol. Let. 61, 267-272.15. Gustafson, J. E., Berger-Bachi, B., Strassle, A. & Wilkinson, B. J.

(1992) Antimicrob. Agents Chemother. 36, 566-572.16. De Jonge, B. L. M., de Lencastre, H. & Tomasz, A. (1991) J.

Bacteriol. 173, 1105-1110.17. Mani, N., Tobin, P. & Jayaswal, R. K. (1993) J. Bacteriol. 175,

1493-1499.18. Oshida, T. & Tomasz, A. (1992) J. Bacteriol. 174, 4952-4959.19. Biavasco, F., Pruzzo, C. & Thomas, C. (1988) FEMS Microbiol.

Lett. 49, 137-142.20. Wang, X., Mani, N., Pattee, P. A., Wilkinson, B. J. & Jayaswal,

R. K. (1992) J. Bacteriol. 174, 6303-6306.21. Sambrook, J., Maniatis, T. & Fritsch, E. F. (1989) Molecular

Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press,Plainview, NY).

22. Sanger, F., Nicklen, S. & Coulson, A. (1977) Proc. Natl. Acad. Sci.USA 74, 5463-5467.

23. Von Heijne, G. (1988) Biochim. Biophys. Acta. 947, 307-333.24. Kuroda, A. & Sekiguchi, J. (1990) J. Gen. Microbiol. 136,

2209-2216.25. Diaz, E., Lopez, R. & Garcia, J. L. (1992) J. Bacteriol. 174,

5516-5525.26. Neumann, V. C., Heath, H. E., LeBlanc, P. A. & Sloan, G. L.

(1993) FEMS Microbiol. Let. 110, 205-212.27. Joris, B., Englebert, S., Chu, C.-P., Kariyama, R., Daneo-Moore,

L., Shockman, G. D. & Ghuysen, J.-M. (1992) FEMS. Microbiol.Lett. 91, 257-264.

28. Wren, B. W. (1991) Mol. Microbiol. 5, 797-803.

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