Gene, 86 (1990) 81-88 Elsevier

GENE03~8

81

Modular organization of the lytic enzymes of Streptococcus pneumonioc and its bacteriophages

(Autolysins; substrate specificity; sequence repetitions; molecular evolution)

Pedro Gar¢ia, Jos6 L. Garcla, Ernesto Garcia, Jos6 M. Sinehez-Puelles and Rubens L6pez

Centro de Investigaciones Biol6gicas, C.S.I.C., 28006 Madrid (Spain)

Received by M. Salas: 23 May 1989 Revised: 29 June 1989 Accepted: 30 June 1989

SUMMARY

The nucleotide sequences ofgenes cpl7 and cpl9 of the Streptococcuspneumoniae bacteriophages Cp-7 and Cp-9, encoding the muramidases CPL-7 and CPL-9, respectively, have been determined. The N-terminal domains of CPLo7 and CPL-9 were virtually identical to that previously reported for the CPL- 1 muramidase. The C-terminal domain of the CPL-7 muramidase, however, was different from those of the host amidase and the phage Cp- 1 and Cp-9 lysozymes. Whereas all enzymes studied are characterized by repeated sequences at their C termini, the repeat-unit lengths are 20 amino acids (aa) in CPL-I, CPL-9 and in the host amidase, but 48 aa in CPL-7. Six repeated sequences represent the C-terminal domains of CPL-I, CPI.-9 and the host amidase, and 2.8 perfect tandem repetitions that of CPL-7. The peculiar characteristics ofthe structure of CPL-7 muramidase correlate with its biochemical and biological properties. Whereas CPL-1, CPL-9 and the pneumococcal amidase strictly depend on the presence of choline-containing cell walls for activity, CPL-7 is able to degrade ceil walls containing either choline or ethanolamine. These results support the previously postulated role for the C-terminal domain of these lyric enzymes in substrata recognition and provide further experimental evidence supporting the notion that the proteins have evolved by an exchange of modular units.

INTRODUCTION

Lysozymes have provided a useful model system to study protein structure, mechanism of enzyme action and evolu- tion (see Joll~,s and Joil~s, 1984 for review). With this interest, we studied the relatiorship between the lytic enzymes of $.pneumoniae ant) ~:s bacteriophages. We

Conespondence to: Dr. R. Lbpez, Centre. de Investiaaciones Bio168icas, VeI~quez, 144, 28006 Madrid (Spain) ~re|. (I)2611800; Fax (I)2627518.

Abbreviations: aa, amino acid(s); Ap, ampicillin; bp, base pair(s); ¢pl, 8erie (DNA) encoding phage Cp lysozyme; Cden-EA, chemically defined medium containin8 EA; EA, ethanolamine; kb, kilobase(s) or 1000 bp; I.vtA, 8ene encoding the pneumococcai major autolysin (amidase); nt, nucleofide(s); ORF, open reading frame; p, plasmid; Pollk, Klenow (large) fraament of E. con DNA polymerase I; a, resistance/resistant; RBS, ribosome-binding site; To, tetracycline; [ ], denotes plasmid-carrier state.

0378-1119/90/S03..50 © 1990 Elsevier Science Publishm B.V. (Biomedical DivJsiou)

found that all lytic enzymes described so far showed a s~ngent dependence on the presence of choline in the cell-wall substrata for activity. Assuming that the peculiar presence of choline in the pneumococcal cell wall might act as an element of strong selective pressure, it was con- ceivable that the similarities between host and phage lysins in the pneumococcal system would be h~4her than in other species. We have recently cloned and sequenced the cpll gene (formerly designated as cpl) of Cp-I that encodes the CPL-I lysozyme (Garcla et al., 1987a; 1988). The determi- nation of the aa sequence of CPL-1 and its comparison to those of the major pneumococcal amidase (Garcia et al., 1986) or to the muramidase of the fungus Chalaropsis (Fouche and Hash, 1978), indicated that the C.terminal domains ofthe CPL-1 muramidase and ofthe host amidase could be responsible for the specific recognition of the choline-containing cell wall. The active center of the enzymes, on the other hand, was anticipated in their

82

N-terminal domains (Garcia et al., 1988). In addition, our results strongly suggested that the C-terminal domains of CPL-I and of the host amidase share a common ancestry.

To further substantiate our assignments of these enzyme domains, we have cloned and sequenced the genes encoding the lyric enzymes of the pneumococcal phages Cp-7 and Cp-9. These phages are morphologically similar to Cp-1 (Ronda et al., 1981) but their DNAs have different restric- tion patterns (L6pez et al., 1984). We show here that the lyric enzyme coded by Cp-7 differs from all others, in that it can degrade pneumococcal cell walls containing either choline or EA. The comparison of the sequence of CPL-7 to those of the lytic enzymes that only recognize choline- containing cell walls provides new insights into the organi- zation of cell wall lysins.

MATERIALS AND METHODS

(a) Bacterial strains, plasmids, phages and growth condi- tions

The Escherichia coli strains employed were HBI01 (r-m- recA 13) (Boyer and Roulland-Dussoix, 1969) and JM 103 (hsdR4[ F' traD36proABiaclqlacZAM 15 ]). The lat- ter was used as a host for phages Ml3mpl0 and Ml3mpl 1 (Messing, 1983). Plasmid4~CIP50 (ApRTcScpll) is a deriva- tive of pBR325 carrying the structural gene encoding the CPL-I muramidase (Garcia etal., 1987a). Plasmids pGLg0 (Garcla et al., 1986) and pRG2 (Ronda et vl., 1987) have been described elsewhere. The pneumococcal bac- teriophages, Cp-I, Cp-$, Cp-7 and Cp-9, were prepared and purified as described elsewhere (L6pez etal,, 1984). $. pneumontae R6 was grown in C medium (Tomasz and Hotchkiss, 1964) supplemented with yeast extract (0.08Yo, Difco Laboratories)or in Cden-EA as previously described (Tomasz and Westphai, 1971).

(b) Materials

Enzymes including restriction endonucleases, T4 DNA ligase, Pollk, nuclease BAL 31, and proteinase K were pur- chased from commercial suppliers (New England BioLabs; Amersham International; Merck). Radionuclides, Hybond membranes, and kits for DNA sequencing, transcription- translation and nick translation were obtained from Amersham. Mutanolysin, a muramidase from Streptomyces globisporus, was purchased from Sigma.

(c) Preparation of cell walls and assay for enzymatic activ- ity

Cell walls labelled with [methyl.aH]choVme (spec. act. 60 Ci/mmol) or [2-*4C]ethanolamine (spec. act. 44 mCi/

mmol) were prepared by biosynthetic labelling of the bac- teria (Mosser and Tomasz, 1970). Th¢ standard assay con- ditions for the pneumococcal amidase (Holtje and Tomasz, 1975) and for the phage muramidases (Garcia et al., 1987a) have been described.

(d) Preparation of DNA

Proteinase K-treated phage DNA was prepared as previ- ously described (L6pez et al., 1984). Plasmid DNA was prepared by the rapid alkaline method described by Birnboim and Doly (1979). Purification of DNA restriction fragments by low-melting temperature agarose gel electro- phoresis was carried out as previously described (Lbpez et al., 1984).

(e) Nucleotide sequence analysis

Nucleotide sequencing was performed by the dideoxy chain-terminating method (Sanger et al., 1977). Data analy- sis was simplified by computer programs run on a Zenith Z-100 PC. Protein similarity searches were conducted using the Protein Sequence Database of the Protein Identification Resource at the National Biomedical Research Foundation with the computer programs of Lipman and Pearson (1985).

RESULTS AND DISCUSSION

(a) Phages Cp-7 and Cp-9 code for lytlc enzymes

Both in vivo and in vitro tests demonstrated that phages Cp-7 and Cp-9 encode lyric enzymes. (l) The lyric behaviour of M31, a strain ofpneumococcus with a complete deletion ofits lytA gene (S/mchez-Puelles et al., 1986a), infected with Cp-7 or Cp-9 was the same as that observed following Cp- 1 infection (not shown). (ii) In vitro transcription.translation assays with Cp.7 or Cp-9 DNA directly showed the synthesis of lyric enzymes active against 8. pneumoniae cell walls (Table I).

(h) Localization and homology relationships between the genes coding for the lyric enzymes of Cp-7 and Cp.9

Southern blots with the pneuroococcal lytA gene as a probe (plasmid pGL80) had previously shown homology with 7.9-kb Hindlll fragments of DNA from phages cp-I and Cp-5 and with a 6.l-kb Hindlll fragment of Cp-9 DNA. Only tittle, if any, homology was detectable with H/ndlll-degraded Cp-7 DNA (Garcia et al., 1988). This result identified the location of the cpi9 gene. To define the location of the cpi7 gene within tL; Cp-7 genome, we

TABLE 1

In vitro transcription-translation ofproteinase K-treated Cp-7 and Cp-9 DNAs"

Additions Released SH label b of phage DNA (cpm)

None 153 Cp-IDNA 5577 Cp-7DNA 2522 Cp-gDNA 6025

a The reaction (1.5 Fg DNA) was carried out at 37 ° C for 60 rain in a final volume of 30/d, according to the recommendations ofthe suppliers of the transcription-translation system. Afterwards, 10/d of [3H]choline- labelled (total cpm 7500) cell walls and 200/d of 50 mM "Iris. maleate buffer pH 6.5, were added, and the incubation was continued at 37°C for 135 min. The reaction was stopped by adding l0/d of 38% formaldehyde and 10/d of 4% bovine serum albumin. Alter centrifugation (10000 × g, 15 rain) radioactivity in the supematants was determined. b Assay of lysozyme activity; radioactivity released from [3H]choline- labelled pneumococcal cell walls (see last sentence of footnote a).

degraded Cp-7 DNA with AccI.'The digest was separated by electrophoresis, blotted, and hybridized with the 32p. labelled 1.4-kb Accl-$phl fragment of Cp-I DNA. This fragment represents the 5'-flanking region of the cpll gene and encodes the N-terminal half of CPL-I. Fig. I shows that this fragment hybridized with two Accl fragments that map at the right end of the genome of Cp-7. In addition, when plasmid pRG2, containing the lyt,4 gene (Ronda et al., 1987), was labelled and used as a probe against Cp-7 DNA, no hybridization bands were found, indicating that the weak hybridization band previously observed cor- responded to an unspecific one (Garcia et al., 1988). These results also suggested that the gene coding for the lysin of Cp-7 may be located in a position similar to that previously determined for Cp-1 (Garcia et el., 1987a).

(c) Analysis of the cpl7 and cpl9 lysin genes

Appropriate overlapping restriction fragments from Cp-7 and Cp-9 DNAs were cloned into M13mpl0 or Ml3mpl 1 and sequenced. The nt sequences of the cpl7 and cpl9 genes are given in Fig. 2. For comparison, the nt sequence of the cpll gene (Garcia et al., 1988) is also given. Only one ORF of significant length (1017 and 1026 nt for cpl9 and cplT, respectively) was found in either gene. Position 1 was assigned to the first nt of the start codon, ATG. At 10 nt upstream from the ATG codon, a putative RBS is located. Altogether, the cpl9 gene has much greater similarity to the cpll gene than to the cpl7 gene. In cpl9, 52 out of the first 600 nt (8.7~o) differ from the cpll gene (21 transversions), whereas only 20 changes (six transversions) were found in the remaining 417 nt (4.8 ~) . On the other hand, 17.3 ~ of the first 600 nt were different when comparing the cpll and

83

A

pCIP2 4-

Sphl

Cp-7 DNA 4-

• !

am

9.4

0.6

pRG2 4-

HindW

Cp-TOl~ 4-

• Accl I

:"- 6~6 - ' 4 , 4

- 0 . 6

PH , . '11

Sp H S P HP I ' ' ' ' " ~ C p - 7

: ~ : ~ AI At

ABel 31 ~ A BOI 31

Fig. I. Southern-blot hybridization analyses of Cp-7 D N A and se-

quencin$ stratagy of the ¢p~7 gene. (Top) Ethidium bromide-stained 0.7 agarose gels and Southern-blot analyses of Cp-7 DNA digested with ,4¢¢I. Hybridization (Southern, 1975) was performed according to Maniatis et al. (1982). Radioactive probes were synthesized by nick- translation in the presence of [32P]dCTP. Radioactive bands were detected with Kodak X-Omat films using intensi~ng screens (Dupont Cronex Lightning Plus)at -70°C. The autoradiovaphs show the frag- ments blotted to Hybond membranes and hybridized at SO ' C for 4 h with (A) 32P-labelled 1.4-kb Ac¢l.$phl fragment of Cp.I DNA that contains the 5'-flanking region of the ¢pli gene and the sequence coding only for the N-terminal halfoftbe CPL-I (Garcia et al., 1988), and (B) 32P-labelled pRG2, a derivative ofpLS I containing the lyt,4 gene (Ronda et al., 1987). Piasmid pCIP2, a derivative ofpACYCI77 containing the ¢pll gene, has been described by Oarcia et ai. (I 987a). The sizes (in kb) oftbe restriction fragments of Hindlll-digested A DNA are indicated. (Bottom) Partial restriction map of Cp-7 DNA and the strategy for determining the nt sequence of the ¢pl7 gene. Location of the restriction sites used for sequencing are indicated. Arrows below the map indicate the direction and extent of sequence determination. Heavy black bars at the top ofthe map indicate the location of the two A¢¢! fragments of Cp-7 DNA that hybridize in the Southern blot analysis shown above. The hatched bar indicates the location of the ¢pi7 gene. Restriction sites: A, Ac¢I; Al,,41ul; H, Hindlll; HP, H/nPI; P, Pmll; S, $au961; Sp, Sphl. d Ba131 indicates the direction of the deletions generated by BAL 31 nuclease for purposes of sequencing. Wavy lines indicate the length of deletions.

cpl7 genes. Of these 104 changes 41 corresponded to trans- versions. In addition, 98 changes (46 transversions) were found when comparing the first 600 nt of the cpl7 and cp19 genes. The most striking diffelences between the cpll/cpl9

84

-315 -208

-24e -240

-$I -120 -120

TTT~AA66CTT66AAST66&AASTSACCSATASCTSEACASGSTTAAAA86ASTTATC~AACATACCCTAACATT cpl-I C11111111111C11111811i81118111111111118T11111 ¢p1-7

~A~TTTTTAC~ATTT~6TA6c66TATTCTTAACAT&TATTCAc6CTAT6~cA6TC6~TCA6ATTTT6CTT~TTATCATTAAcTT6TACTAT~CAcTTTCAATTAT66AAAATcTT6CT6T cpl-I c 1 8 ~ 8 ~ 8 ~ C ~ 8 8 8 t 8 ~ 1 8 t ~ t 8 ~ 8 C 8 8 ~ 8 8 t 8 8 8 8 1 t t 1 ~ t 1 t 8 8 1 1 ~ 8 8 ~ t ~ 6 1 ~ 8 ~ A 1 1 ~ 1 ~ T T ~ 6 ~ C ~ 1 ~ 1 8 ~ cpl-7

881111111111111111111111111111111111111111111111111 ¢pl-9 T&T6~T~TCT~T&TccCT~&TTT&T~Ac~cTA~A~TACAuAA~A&TT~cA~AAAT&©ACC~CACAAcT&~AC~CA~AAAA~AcCT~cTA~AA~A&TTTAAA~A~AAAA8MATA cpl-I ~8~8~&~8~8~T~C8~G~8~88~&~6~68~&8~18~6~A~1~8~8~&~1~8~~6~1~8811~8~8~11~1~888~8~81~88 ¢p107

I 8 1 ~ 6 8 8 & ~ 8 ~ 1 ~ t ~ t t 8 ~ N ~ t ~ 1 ~ . ~ C 8 8 8 ~ 8 ~ s ~ + 8 ~ i ~ i ~ n h u ~ 8 ~ 1 ~ 8 ~ & ~ T ~ t ~ & ~ 8 ~ cpl-9 1AT6~TTAAAAA~AAT6kTTTATTT8TA6AT6TTTcAA~TCACAAC66TTAC6ATATAACA~6TATcTT6~A6CAAAT~sAAC~ACTAACACCATC&TTAA~ATTTCT6AAMTAC6ACC cpl-t I 111~88~88A18188~t~t t81~c1~t6~C18Tc~A~c8~18c~TTt~A~T1~A~A~1~CA1~6~A~8A~1T1~8~1~B~A~8~8~&~B~ cpl-7

121 11~1~1~1~111188~81~88~8~1~84888~8T8~1~88~81~8~88~81~8~88~8~88~18~88~8~8~8~T881~8~88~11~8~8~88188~ cpl-9 121 TATTTAMCCCTT~cTT6TCT~C~CAA~T~A~C~TCMACccTATT~CTTTTATCACTTc8CAC~cTTT~c~A~A~TABCA6AA6CC~AAA8A~AAsC~CA~TTTTTCCTT8AC ¢pl-t 121 t t8111~81~8~11~8~18~1Hc8~8~8~81~c~8~81~T~T8~TT~1T~A~T~A1~A~8~1A8~K~8~8A~6c~A~8~1~T ©pl-7

241 ~ 8 1 ~ 1 ~ C G ~ 1 ~ 1 ~ 1 1 ~ 1 ~ 1 ~ T ~ & ~ n ~ 8 1 ~ & ~ 1 1 ~ 1 ~ 1 C ~ ~ ~ 1 ~ 8 ~ 8 1 ~ 1 ~ 1 ~ 1 A ~ 1 ~ 8 ~ 1 ~ 1 c~i-9 ~41 AAc~T~cCTAT6CAA~TTA&ATACCTT8TATT~ACTAC~A~AC~AcCC~AsC~A~AC~CACAA~C~AACACTAAC~C~T~cTTAC8CTTTAT~CMAT~ATT~CT~AC~cT~ATAT ¢pl-I ~41 ~ 8 ~ c A ~ 8 ~ ~ c ~ A ~ T ~ i T ~ A ~ T o ~ A a ~ T ~ A ~ A ~ c ~ ~ ~ A ~ A ~ A ~ ~ cpi-7

361 ~ c ~ 1 ~ ~ ~ T ~ c ~ T 1 ~ ~ 8 ~ 1 ~ s ~ T T ~ A ~ ~ ~ c ~ ~ e ~ s ~ T ~ A ~ A c ~ cpl=9 361 AAAcCTATTTATTATA~TA~AAAC~TTT~CACAT~ATAAT~T~6ACTATCA~CAAATCCTT~C~CA6TTCCCTAATTCTCTAT~ATT~CA~CTAT~CTTAA~C6~T~TACA~CT CpI-I 361 ~ C ~ ~ 8 8 c ~ 8 ~ 6 ~ T ~ 8 ~ 8 ~ 6 ~ T T ~ A ~ 1 ~ 1 ~ 1 1 ~ 1 ~ 1 ~ 1 1 ~ ~ 1 1 ~ 1 ~ T ~ 1 ~ 8 ~ cpl-7

481 ~ T ~ T 1 ~ T ~ 1 1 T c T ~ t 1 ~ t ~ 1 ~ 1 ~ 1 1 1 1 1 1 1 ~ n ~ 1 ~ 1 1 ~ 1 ~ 1 ~ 1 1 ~ ~ 8 ~ 1 ~ 1 t ~ 1 ~ A n ~ ~ T ~ 1 1 8 ~ 401 &&CTTTGA~TAcTTCCC&A6C&TG~Ac6GGAT&AsAT66T66CA~TATTCT&~TAAccC8TTT~ACAA~A&TATTGTACTsTT&~AC6AT6A&s&&sAc8Ac~A6CCAA&6&CCsCT~e& 401 11~1t~11111~1tT~1tt81~8tt1~T~1C1~1~1~t1t&~11~11tj~1~1~8j~8n~8~t8~81811~T1~8~A~11~A~8T1tT&T~1tC~AT1AAAAc

- P ! s'~n P2 601JIII1181111|111111TIlIIIClII|IIIIIIIlIIClITIIClIT|111111111111 81111111111111181111111118118111811111|1111111111181118 cp|-9 ~ ~6jU~RACAA6hCA6C~A~6~8Te6T66TTCA~AC6AAACAAT6eCA~TTTCCCTT&TAATAAATG66AAAAA&TC~T86TBTsT~sTACTACTTP~ATA6TAAA~AT&TT6CTT& cp|-I 601 8nTc~A1~1A6~CTT1Ct~C~1TA6Cc~AC~B~TC~TTc1~66ACTTT86~61~AC~81c1A~1~8TT1TB8C~6TTTAnc~ATC1A6B6n1T6~1CCCCA~C68TTc1A6A1&A~ cpl-7

• "11-P3 ~-MJ "lI--P4 7~1 1~18~1~11~1811o11~1111111~tt8111111t8~111811u111t881118T~181T1u1~jt1181n188j8t111111881811~1811111111~;~8T11A111 cpl-9 721AC|A~A~T~cT~AAA~A~A~AA~AAT~A~TAc~T~AA~Ac~cs~AAT6~c|AcT8|TT|~T|CTA6T~6~HA6T66TATTAT~T~c~TT~A~C6cTAT~ cpl-I 721 ~T~Av1~1ATCT~A~cnC1ts6~1~nTT~cA6n~-~TAC~oCA1w6cj~A1~A~CATTCAB1nACTTTo~CAAtj1TCA~1A~1T18n~1C~8TTTA~C~AATC1A~TAT cpl=7

.~? M~ Is-~lP6 84J I I 1 1 . . . . ~I11111111~IIC1111111111111811111111111111111111111111111111111 I11111111A11111111111C111181111111111111111111 cpl'9 041 ~TTACT~T~8TCA~6TATAA~AATAACT~ACTATAT~AC~&AT~AAC~T~TAACAT~TTTcTAAT~AATTTATTAA~T~AAAA9~TT~TATTTCAT~AACACAAA~A cpl-i 041 t~cC~Cc~N~c~TC~A~C~|~T~A~AT~TA~C|~T~t~T~A~A~TA~AcA~T4~AA~|A~AT~6~CT~NCAA8~TC1A~A~TTAT cpl=?

.-~-M3 , -e.-. I I ,I 961 I I I I I I I I I I I I I I I I I I I I C I I T I I ~ I I I I I I I I I I I I I I A I I I I I I I I S l I I I I I . . . . . . . . " ~ I I 8 - 1 1 1 ~ A C l I f l I I I I I I I I I I I I I I I I I T I l I I I A I 1 cp|-~ 961 ~AOCTTOC~O~C~TCCAASTTTC~CO~O~CCAOACO6OCTTAT~CCOT~OCA . . . . . . . . . ~A~A~O~AOCTAOT~|OATTTTCCT~CTAOCIGIITTTAT~OTCT|CTAT cpl=l 96111CAOtTTilCGiIJiOIOiOn~TOACCCCCIJOIOiTTCAAOACiAoOTO4ATI~JlTT~CTTTC t/~iCllOTli l t l j l i l i i tAlAii15itOjtt i i j~tuljj i j lTai lJOjt I cpi-9

~ j ~ t ~ j ~ | j ~ j ~ ~ j ~ ~ | ~ j ~ j ~ j ~ C A 6 ~ A c C | C T ~ T T c A T c T ~ A T T | T A T T T C T ~ T A ~ | T ~ T | A T T T T c T ~ 6 c T ~ T ~ T ~ T T ~ c T ~ A T cpl-9 &&TTTTAT&&GC&TCTTCOTCTGO&TT&TCCAOROCO&TOO&~CRO&T~C& cpi-I | ~ 6 ~ | ~ C ~ 1 ~ ~ i i ~ i ~ | i ~ i i ~ i i ~ ~ i i ~ i ~ ~ i i ~ i ~ A ~ ~ i ~ c T Cp| '~

IOTI 1092 IO|t

l i f t OTOCTTTAYACC

cpl-9 cpl=l cpl-7

cpi-9

Fig, 2, Comparison of' the nt sequences of' the ba~;teriophage genes cpi�, cpll and cplT. Only the DNA strand co~'ruponding to the mRNA sequence is shown, Nt sequence xiumbers of'the genes are indicated on the let~. Identical nt to those of~71 (Garcla etal., 1988) are indicated by colons in the sequence oPcpi7 snd ¢pl9, P I-P6 and M I-M3 represent, in this and the Following figures, the sets oF repeated sequences. The heavy underline represents the RBS. Facing arrows show stem-loop structures and the light segment indicates the mRNA transcription stop sequence. The TAA termination codons sre boxed.

and the cpl'~ genes are found within the 400 bp preceding the stop codon, TAA. Here, the sequences diverge completely. Furthermore, a remarkable characteristic of both types of sequences is the presence of repetition sequences. The num. ber of'repetitions era 144-nt repeat is 2.8 in cpl7 (positions 613-756, 757-900, and 901-1017) a~d six in ¢pll/cpl9 with a repeat length of about 60 nt. Th,, Rho-indevendent termi- nator already reported for cpll (Garcia etal., 1988) also appears in cpl7 and cpl9 do~lstream from the stop codon, although several changes were found. The G =+ A change in the stem is compensated by a complementary C =+ U change that maintains the secondary structure of the termi- nator. The sequencing results obtained fully explain the Southern (1975) blots reported above. These showed that DNA/DNA homology was only found between the 5' regions of the three phage genes (Fig. 1).

(d) Comparison of the aa sequences of the lysins of Cp bacteriophages

Based on the analysis ot'tbe nt sequences reported above, we deduced that ¢pl7 and ¢pi9 encode proteins of 38 461 Da and 39107 Da corresponding to 342 and 339 aa residues, respectively (Fig. 3). Only 10 aa were different between CPL-I and CPL-9. Nine of these changes reside in the N-terminal moiety of the proteins, whereas only one aa differed in the part of the molecule homologous to the host amidase (Garcla etal., 1988). On the other hand, 34 out of the first 204 aa were different between CPL-I and CPL-?. Asp-l0 and Glu-37, that have been suggested to be involved in the catalytic process (Garcla etal., 1988), are conserved in CPL-I, CPL-7 and CPL-9. These results strongly sug- gested that CPL-7 and CPL-9 are muramidases (see below). On the other hand, the C-terminal ends ofthese iysins were

completely different. The set of six modular repetitions present in the C-end of CPL- 1 previously described (Garcia et al., 1988) (Pl to P6 in Fig. 3) were replaced by a set of 2.8 perfect tandem repetitions, each 48 aa long (MI to M3

I 5 0

? . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . v . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C ~ L - 7 . . . . . . . . . . . A , . O . . . . S . . . . EA . . . . . . . . V . . . . S . . . . . . . . . . S . . . . . . . . . . . M . . . N ~ . . . A , . t Y . . .

I O 0 1 5 0

o 0

C1~-9 . . . l r . . . . . . . . . . . . . . . N . . . . , . . . . . . . . . . . . . . . T . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . N.

C[H,-I l i l / E I I G ~ C Y L V L D ~ I N A C L R F M ~ I A D A G Y I C P IWSYKPFTHDlilRYClQI LAGFPNSLMIAGYGUIOGTA

~q. -? . . . . . . . . . . . . . . . HA.ASV.R.,T . . . . . . . i . . E , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . .

2O0

~ P t -,,,,-- P2 CPL-9 O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . . . . , . t . : . . . . . . . . . . . . . . . . . . Jl , . . ,- . . . . . . . . . . . . . . .

CPi.-I NFEYFPSIIDGI RYUGYb"SIIPFDIOIll/LLIDOEEIDOIO~TAG~SKGINIFRRNNGSFPYIII~EKIGGIit/YYIFOSI~YCL

CPi.-T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I(E,M! NNEM • L ,T~LTTVNllEVI(16LMGIIGOERYDSLAMIG.OP(IAVgDi( t - - M 1

25O 300

. . T . p 3 o ..--T--P4 - I t - p e . . . . . . ? . . . - ~ . . P G . . . . . . . . . . . . ~PL'9 , - - , , , . , , , . . , . , , . , , , V a . . , , , . , , . , . , , , . , , . , & , , . , . , , . ,

I ~ . - 1 TSEVU~IIElG/TlrLICDII~LNATG,'VLVGllEInTI~TAN4VTGIMtYIOIE/TT14TlIBt~IMINE F I I~GI~VTRIITII~

~q . -? W . h N N I . llt~LLTTVNIEVIG. LMGII.O .It .D|LANIIIGTOIqlA.I~ .V.E ILMNt. I AD~TTVANEVI~ . ~ R y ,,-m-- M ~ - - K - M 3

339

o

~-9 ..,-J ...............

~ L ' I IILAONPSPTKEPOOLITVA

CPI.- ? O|LA .ItQYDP~VQOIO/NEILL8 ~ ' 0

I l k |

Fig. 3, Comparison of the deduced as sequences of the bacteriophage muramidases, CPL-9, CPL-I, and CPL-7, Identical an are indicated by dots. The number above the sequences describes aa alignment coordi- nates,

85

in Fig. 3). A comparative summary of the relevant molecu- lar characteristics of the pneumococcal phage lysins, the host amidase and the related enzyme from Clmlaropsis is depicted in Fig. 4.

(e) Biochemical properties of the CPL-7 lysln

Alignments of the aa sequences of CPL-I, the pneu- mococcal amidase and the muramidase of' the fungus Chalarops~ sp. have suggested that the C-terminal domains of" the CPL-I and of" the amidase might be responsible for the specific recognition of. choline residues present on the pneumococcal cell wail, whereas the active center of. these enzymes should be located in their N-terminal domains (Garcla et al., 1988). If' this hypothesis were correct, the specificity of the CPL-? lysin might differ from that previ- ously reported for CPL-I (Garc|a et al., 1987a), i.e., CPL-7 might be capable ofdegrading pneumococcal walls contain- ing either choline or EA. To test this supposition, we con- structed plasmid pCP70 by cloning the 1.8-kb HinPI-Pmll fragment of Cp-7 DNA containing the cpl7 gene into pBR325 previously digested with ClaI + EcoRV. E. coil cells harboring pCPT0 were sonicated and the extracts tested in vitro using either [3H]choline- or ['4C]EA- labelled pneumococcal cell walls. The results shown in Table II demonstrated that CPL-7 did degrade both sub- strates, in contrast with the choline-dependent cell-wall. hydrolysing specificity characteristic of both the CPL-I muramidase and the host amidase. Moreover, CPL-7 iysin was not inhibited by high concentrations (100mM) of choline (not shown). The specific recognition of choline- containing cell walls by the lyric enzymes of S. pneumonlae and its bacteriophages is a property that has been success- fully used to purify both CPL-I and the host amidase by

Gene or Type of Choline dependence Mr xtO "3 species lysin for activity

amino acid number

o ao ~o ,so zoo eso 3oo 35o ! ! , ! v ! ! '~

Hz, ' ~ ~ C00 ly t._._AA amidose Yes 36 d

cpl - I mummidase Yes 39. I d

cp1-9 muramldase Yes 39. I d

cpl-7 muramidase No 38.5 d

Chalaropsi._.ss muramidase No 22.4 d s,p.

P l leg lell P4 Plb PII

NI-~ -t~,\ \ \"~\\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \~'E~'J~J~'~.t;~.l ' . '~.~l~,; I - COOH PI pip PS P4 P6 P6

I I I -COOH

NH ~ - t ~ \ \ \ \ \ \ \ ' ~ COOH

Fig. 4. Comparison of the most relevant properties of the iytic muramidases of phages, Ep-l, Cp-7, and Cp-9, and of the 8. pneumoniae amidase. The muramidase of the fungus Chalaropsis is also included for comparison. A blackened bar represents the N-terminal part of the pneumococcal amidase, and hatched bars, the homologous N.terminal regions of CPL-I, CPL-7, CPL-9, and Chalaropsis muramidases. Stippled boxes represent the C.terminal homologous repetitions of the host amidase, CPL-I and CPL-9 muramidases. Open boxes represent the C.terminal modular repetitions of the CPL-7 muramidase. A striped bar indicates the interlinking region between the N- and C-terminal regions of the CPL-7 muramidase. A crosshatched bar represents the C-terminal region of the Chalaropsis muramidase. The aa sequences showing a z value higher than 4.7 according to Lipman and Pearson (1985), are represented by identical shading.

86

TABLE !i

IX.gradation of [~4C]ethanolamine-labclled pneumococcal cell wags by lysins of Sme mococcm .m~eumoniae and its bacteriophages"

Strain Enzyme ~ Substra*.e d

[3H]choline cw [14CIEA cw

(cpm released)

HBI01[pBR325] --- 0 0 HBI01 [pGLg0] amidase 7300 0 HBIOI[pCIPS0] CPL-! 7100 0 HBI01[pCPT0] CPI...7 6900 4400 - - mutanolysin 7200 7100

" Exponentially growing cultures of E. coli HBI01 containing the plas- mids indicated were iysed by sonication and the extracts incubated with 13H]cholinc- or ['4C]ethanolamine-labelled pneumococcal walls, as previously described (Garcta et al., 1986). u pOL80 and pCIPS0 are described in MATERIALS AND METHODS, section a, and pCPT0 in RESULTS AND DISCUSSION, section e. © The enzymes were used as described in MATERIALS AND METHODS, section c. d The standard assay conditions are described in Table I, footnote c, cw, cell walls.

affinity chromatography either on choline-Sepharose 6B (Garcla et al., 1987a,b) or on DEAE-ccllulose columns (Sanz et al., 1988). As expected, attempts to purify CPL-7 Following these procedures failed since the protein was not retained by the matrix but eluted from the column with salt washes (not shown).

Crude sonicated extracts obtained From E. coli HB101[pCP70] were used to biochemically characterize CPL-7. When samples ofCPL-7-treated pneumococcal cell walls were reduced with NaB3H 4, hydrolyzed, and chroma- tographed on a Dowex-50 column (Oarcia et al., 1987a), the radioactivity was Found as [ 3H ]muramitol, demonstrat- ing that CPL-7 is a muramidase (not shown).

(t) Biological characteristics of Cp-?

The pneumococcal cells containing EA instead ofcholine in the cell wall are resistant to the lyric activity of the pneumococcal amidase (HOltje and Tomasz, 1975). More- over, we have found that for cultures of EA-grown cells, in addition to the necessity of being 'minipulsed' with choline to be infected by bacteriophage Dp-I (Lbpez et al., 1982), the continuous presence of choline in the cell wall was required for a successful liberation of the phage progeny (Garcia etal., 1983). In contrast, when EA-grown pneumococci were infected with Cp-7, the culture lysed, liberating productive progeny phages. Control cultures of F,s, cells infected with Cp l did not lyse (Fig. 5).

soot- .,B

,ooF / \

,oot, / , TIME (hours)

Fig. 5. Lyric effect of Cp-7 on cultures of EA-grown S. pneumoniae, Cul, tures ofS. pneumoniae R6 grown in Cden-EA medium were infected with Cp-I (@) or Cp-7 (O) at the time indicated by the arrow (multiplicity of infection = 2) and incubated at 30°C. Growth and lysis were followed by nephelometry (N). ] (10N- 7.5 x l0 T colony-forming units/m]. The growth curve of an uninfected culture is also shown (J).

(g) Conclusions

We have previously suggested that the host pneumo- coccal amidase and the Cp-1 muramidase genes originated from the fusion of two functional modules (Garcia et al., 1988). An overall analysis of the results presented here reveals: (l)that the specificity for binding to the cell-wall substrate resides in the C-terminal domain of' the lytic enzymes and that this domain always contains a repeating motif; (li) that the N-terminal domain determines the type of enzyme activity. Taking into account these generalities, we propose a modular organization in the structure of the lytic enzymes of pneumococcus and its bacteriophages. This modular design allows a number of combinatorial possibilities and could be a widespread model for many types of organisms.

Chimeric constructions between the DNAs of different phages (Susking and Botstein, 1978; Balganesh et al., 1987) and structural and functional relationships between 1"4 and $29 lysozymes (Saedi et al., 1987) have supported the idea of modular organization of the phage genomes (Botstein, 1980). We have suggested that the long repeats present in cpll and lyt,4 genes also represent a repetitive module (Oarcfa et al., 1988). Internally repeating unit sequences also occur in the Bacillus subtiliz phage ~p29 muramidase gene (Oarvey et al., 1986; Saedi et al., 1987). They could represent a common feature of most lytic enzymes of phages infecting Gram + bacteria, although confirmation awaits the elucidation of the sequences of additional phage- encoded lysins. In the case of cpl7 gene, the long (144 nO tandem repeat units are identical (Fig. 2). This suggests that the duplication event occurred very recently (Doolittle, 1986).

Many proteins contain internally repeating unit se- quences. Although it is generally assumed that the internal repeats frequently have structure-function connotations, only few cases of direct experimental evidence have been reported (e.g., the DNA-binding capacity of the zinc fingers of various proteins; Klug and Rhodes, 1987). The presence of choline in the cell wall of S. pneumoniae has been reported to be fundamental, both to obtain a successful lyric cycle with all the phages studied up to now (Garcia et al., 1983) as well as for the activity ofthe host aut~lysins (HOltje and Tomasz, 1975; SAnchez-Puelles et al., 1986b). The analysis ofthe primary structures ofthe lytA and cpll genes and of their deduced aa sequences led us to postulate that the tandem repeat present in the C-terminal region of these proteins would participate in the specific recognition of the choline present in the celt wall substrate (Garcia et al., 1988). In agreement with this assignment of a substrate- binding domain, we have found that the CPL-7 enzyme, a muramidase that degrades both choline- and EA-containing cell wails (Table II and Fig. 5) has a different C-terminus, with the N-terminus conserved.

We have previously found a significant aa similarity ofthe N-terminal domain between CPL-1 and the muramidase of Chalaropsis (Garcia et al., 1988). In addition, the two aa that have been reported to be involved in catalytic activity ofthe Chalaropsis enzyme (Asp-6 and Glu-33; Fouche and Hash, 1978) are also located in CPL-I the same distance apart (Asp- 10 and Glu-37). The analysis ofthe aa sequence of CLP-7 and CPL-9 shows that Asp-10 and Glu-37 were unaltered and that the aa changes found in this region in CPL-7 and CPL-9 do not alter the overall aa similarity between these proteins and the muramidase of Chalaropsis (Fig. 4). These results provide additional support to our previous suggestion that the active center of the lytic enzymes ofS. pneumoniae and its bacteriophages is located in their N-terminal domain (Garcia et al., 1988).

The homologies between the C-terminal domains of bac- terial and phage lytic enzymes invite speculations on the possibility that the phage might have interchanged some regions of its genome with the host cell. Although prelimi- nary Southern blot tests to demonstrate homologies between the 3'-end of the cpl7 gene and the DNAs of several Streptococcus species, as well as computer searches for aa and nt similarities have been unsuccessful so far, our results provide additional support to the notion that most proteins evolved by exchanging modular units (Doolittle, 1985). In spite of the difficulties to envisage the precise mechanism for such an interchange, it is interesting to underline that the complete substitution of the C-terminal module in the 3'-end of the cpl7 gene, with respect to that in the cpll gene (Fig. 2), was achieved in cpi7 using a nonintegral repeat which does not substantially alter the sizes of both genes, since only 9 additional nt ba~,¢ been

87

introduced in c4~,v7. On the other hand, 3'-end deletions of the lytA gene have indicated that the repeats are required for optimal enzymatic activity (Sinchez-Puelles et al., 1987). These results suggest that there is a simultaneous require- ment for the presence of repeating motifs and conservation of a certain molecular size to achieve optimal activity of the enzymes coded by the lytic genes. In addition, module substitution seems to report evolutionary advantages to the parasite since the lyric enzyme encoded by Cp-7 has lost the strict dependence exhibited by the pneumococcal autolysins and by the CPL-I/CPL-9 muramidases for the presence of choline in the cell wall substrate for degradation (HOItje and Tomasz, 1975; Garcia et al., 1987). It is conceivable that the long and conserved repetitions found in CPL-7 should confer to this phage a wider host~range spectrum than that of the other pneumococcal phages allowing a successful infection of species, other than pneumococcus, when mul- tiplying in their natural environments.

ACKNOWLEDGEMENTS

The authors are grateful to Prof. T.A. Trantner for advice and a critical review ofthe manuscript. We thank E. Cano and M. Carrasco for their excellent technical assistance. The art work of A. Hurtado is gratefully acknowledged. This work was supported by a grant from Progrema Sectorial de Promocibn General del Conocimiento (PB87-0214). J.M.S.-P. was supported by a postdoctoral fellowship from the Consejo Superior de Investigaciones Cientificas.

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