Gene, 89 (1990) 69-75 Elsevier

69

GENE 03488

Cloning and expression of gene fragments encoding the choline-binding domain of pneumococcal murein hydrolases (Recombinant DNA; Streptococcus pneumoniae; autolytic enzymes; amidase; lysins; lysozyme; pneumococcal phages; affinity purification; DEAE-cellulose)

Jos(~M. Sinchez-Puelles, Jest~s M. Sanz, Jos(~L. Garcia and Ernesto Garcia Centre de Investigaciones Biol6gicas, C.S.I.C., Vel~zquez, 144, 28006 Madrid (Spain) Received by M. Salas: 11 October 1989 Revised: 19 December 1989 Accepted: 20 December 1989

SUMMARY

The cloning in Escherichia coli of the 3' moieties of the iytA and cpl-I genes is described, coding for the C-terminal regions of the lyric amidase of Streptococcus pneumoniae and the phage Cp-I lysozyme, respectively. The truncated genes were overexpressed in E. coli and the purified polypeptides showed a great alTmityfor choline, although they were devoid of cell wall-degrading activity. Biochemical and circular dichroism analyses indicated that these are the domains responsible for the specific recognition of the choline-containing pneumococcal cell walls by the lyric enzymes. The data presented here suggested that these choline-binding domains can function independently of their catalytic domains.

INTRODUCTION

Bacterial autolysins are enzymes that specificallydegrade some bonds in the peptidoglycan backbone of the cell wall and eventually cause cell lysis. The best known bacterial peptidoglycan-hydrolase is the major autolysin of Streptococcus pneumoniae (an N-acetylmuramoyl-L-alanine amidCorrespondence to: Dr. E. Garcia, Centro de Investigaciones Biol6gicas, C.S.I.C., Vel~zquez, 144, 28006 Madrid (Spain) Tel.(1)2611800; Fax (1) 2627518.

Abbreviations: aa, amino acid(s); amidase, N-acetylmuramoyI-L-alanine amidase of 5. pneumoniae; anti-E, serum against the pneumococcal amidase; Ap, ampicillin; bp, base pair(s); BSA, bovine serum albumin; C-CPLI, C-terminal moiety of CPL-I; CPL-I, lysozyme encoded by phage Cp-l; CD, circular dichroism; C-LYrA, carboxy-terminal moiety ofthe pneumococcal amidase; Cm, chloramphenicol; cpl-l,gene encoding phage Cp-I lysozyme; DEAE, diethylaminoethyl; deg, degree(s); EA, ethanolamine; HEWL, hen egg-white lysozyme; kb, kilobase(s) or 1000bp; LMP, low melting point; lytA, gene encoding the major pneumococcal autolysin (amidase); nt, nucleotide(s); p, plasmid; PAGE, polyacrylamide-gel electrophoresis; Poilk, Klenow (large) fragment of E. cog DNA polymerase I; a resistance/resistant; RBS, ribosomebinding site; RF, replicative form; S., Streptococcus; SDS, sodium dodecyl sulfate; Tc, tetracycline; UV, ultraviolet; [ ], denotes plasmid-carrier state. 0378-11191901503.50 © 1990Elsevier Science Publishers B.V.(BiomedicalDivision)

ase of 36 kDa, referred to here as amidase). This enzyme requires the presence of choline residues in the cell wall teichoic acids for activity since ethanolamine(EA)-containing cell walls are not degraded by the amidase (HOltje and Tomasz, 1975). Moreover, it has been reported that the C-terminal region of the amidase is involved in the process of enzyme activation by choline (S~mchez-Puelles et al., 1987). On the other hand, choline has been shown to act as a noncompetitive inhibitor of the amidase by preventing the attachment of the enzyme to the cell wall when added in sufficient amounts (higher than 10 mM; Giudicelli and Tomasz, 1984; Briese and Hakenbeck, 1985). It is generally accepted that the binding of autolysin to its insoluble substrate (the cell wall) is an essential prerequisite for the hydrolysis of covalent bonds in processes that take place as heterogeneous phase reactions, such as hydrolysis of bacterial and fungal or plant cell walls. In agreement with this model, it has been reported that the inhibitory effect of choline disappears when soluble cell wall fractions, rather than intact cell walls, were used. Moreover, soluble substrates prepared from EA-containing cell walls were also hydrolyzed by the amidase (Garcia-Bustos and Tomasz, 1987).

70 Recently, the cpl-I gene of the pneumococcal bacteriophage Cp-l, that codes for the 39-kDa CPL-I lysozyme, has been cloned and expressed in Escherichia coli. The CPL-I lysozyme showed a choline specificity identical to that of the host amidase (Garcia et al., 1987a). Comparisons between the aa sequences of the amidase (Garcia et al., 1986) and the CPL-I lysozyme (Garcia et al., 1988) suggested that these enzymes resulted from the fusion of two functionally different modules. It was proposed that the N-terminal regions should be responsible for the catalytic activities whereas the C-terminal moiety of these enzymes (C-LYTA used for the C-terminal region of the host amidase and C-CPLI for the corresponding region of the Cp-1 lysozyme, hereafter) should represent the choline-binding domain (Garcia et al., 1988). In agreement with this hypothesis, we have recently found that the CPL-7 lysozyme from the pneumococcal phage Cp-7, that did not require the presence of choline in the cell wall for activity, had a completely different C-terminal part whereas its N-terminal domain was practically identical to that of the CPL-1 (Garcia et al., 1990). The aim of the present study was to ascertain whether the C-terminal domains of the pneumococcal lyric enzymes are responsible for the attachment to the choline residues. Thus we attempted to clone and express the 3' ends of the genes en,~oding the phage Cp-1 lysozyme and the S. pneumoniae amidase.

RESULTS AND D~SCUSSION (a) Constructlm~ of recombinant plasmids containing the 3' moieties of the lytA and e f t . l genes and expression of the

encoded peptldes Recombinant plasmids pCEI7 and pCM 1 containing the 3'regions of the lytA and cpl-I genes, respectively, were constructed by cloning the corresponding DNA fragments into plN-lll(lppP-5)-A3, which provided both the appropriate RBS and the ATG start codon. The vector plasmid contained the lac promoter-operator region downstream from the Ipp promoter and, therefore, the synthesis of C-LYTA and C-CPLI was under the control of lac inducers. Fig. I summarizes the detailed construction of pCEI7 and pCM 1. Restriction mapping and determination of the nt sequence indicated the accuracy of the constructions (data not shown). Sonicated extracts obtained from E. coli RB791 har-

boring either pCE17 or pCMI plasmids, grown in the presence or in the absence of 2~o lactose, were analysed by SDS-PAGE and a prominent band could be observed in lactose-induced cultures (Fig. 2, panel A). The mobilities of the induced proteins in denaturing polyacrylamide gels were in good agreement with those MrS predicted, on the basis of the nt sequences, for C-LYTA (15840) and C-CPL1 (18 758), respectively. The nature of the overproduced prorein from E. coli RB791[pCE17] was ascertained by immunoblot analyses. A crude sonicated extract obtained from a lactose-induced culture prepared as indicated in Fig. 2 (panel A, lane 2), was transferred to nitrocellulose and ' reacted with polyclonal serum made against the purified pneumococcal lyric amidase (anti-E serum) (Garcla et al., 1982). The presence of a unique stained band reactive with the anti-E serum is shown in Fig. 2, (panel B, lane 2). The mobility of this stained band agrees with that expected for C-LYTA.

(b) Specific attachment properties of C-LYTA and C-CPLI The strong attachment to choline residues exhibited by the pneumococcal amidase and the CPL-1 lysozyme has provided an appropriate method for enzyme purification by affinity chromatography on choline-Sepharose columns (Briese and Hakenbeck, 1985; Garcia et al., 1987b). In addition, we have previously reported that DEAE behaved as a choline analogue and that affinity chromatography on DEAE-ceUulose was a rapid, efficient and inexpensive method for the one-step purification of choline-dependent cell wall lyric enzymes (Sanz et al., 1988), When crude sonicated extracts obtained from E. coli RB791 cells harboring either pCEI7 or pCMI were applied on cholineSepharose or DEAE-cellulose columns and washed, first with 1.5 M NaCI and then with 1.5 M NaCI plus 2~o choline, only the bands corresponding to C-LYTA and C-CPL 1 were observed in denaturing polyacrylamide gels (Fig. 2, panel A). Western blot analyses, carried out with the purified C-LYTA, showed a positive reaction with the anti-E serum. In addition, the determination of the N-terminal aa sequence of the first ten residues of each purified protein showed the expected sequences predicted for C-LYTA and C-CPLI from their nt sequences (data not shown). On the other hand, we have recently cloned and expressed (in E. coli) the 5'-moieties of the iytA and the cpl-I genes and observed that the N-terminal parts of the host amidase and the CPL-1 lysozyme did not bind either to choline-Sepharose or to DEAE-cellulose columns

Fig. 1. Constructionand structureofrecombinantplasmidscontainingthe 3' moietieso~'theiytAand cpl-I genes.To constructpCEI7, pGLgl containing the intactiytA 8ene(Garciaet al., 1987a),was preparedby the alkalinemethoddescribedby Bimboimand Doly(1979).This plasmidwas digestedwith SnaB! + BamHI and the DNA fragmentencodingthe C-terminalregion of the pneumococcalamidase was purifiedby electrophoresisin 1% LMP agarose (L6pez et ai., 1984).This fragmentwas ligatedto plN-llI(IppP-5).A3(Inouye and Inouye, 1985)that had been digested with EcoRI, blunted with Pollk, and digestedwithBamHI. The ligationmixturewas used to transformthe E. coUstrain RB791 (Brent and Ptashne, 1981).The pCMI was constructed as follows:a SspI.Pstl fragmentof pCIP51, containingthe 3' end of the cpl.l gene, was cloned into Sinai + Pstl-digestedMl3tgl31 RF

D

K N

I

V

... GAG AAG AAT AAT,GTA... s' $spI 3'

E Sm PBg

E S

P

+Stool ÷Psi I +$sp I ÷Psi l

I

W Y V H S ...TGG TAC GTA,CAT TCA...

S'

Sno B I

H

3'

... c ~

H

E

T4 DNA ligose

I

s' H

P

I

V

T,= A ~ COC ATT GTA...

Eco R l

3'

B

g

+$no BI +BornHT

RBS M K G ATCTAGAGGGTAT TA ATAATAATGA AA GGG...

oil

|

s'

+ E~'~RI

+ Bgl lt

|

X~ I

3' E

H

I ~-E'co RI ÷Pollk

÷£coRI .8ore Hr

/'~Bo m H !

"

~

G

G

T4 DNA ligose

T4 DNA l igose

G

G

I

V

H

S

•.. GGG GGA ATT GTA CAT TCA... 5' 3'

/ocp o X = ~ ~ IPPP"~~

H B

M

K

I

P

I

V

... ATG AAA GGG GGA ATT P.~ ArT GTA... 5' £co RI ~'

loc p o X ~

DNA. The resulting plasmid, pCCI0, was digested with EcoRI + Bglll, ligated to EcoRl + BamHl.digested piN-Ill and used to transform the E. coil RB791 strain° The chimeric plasmids pCEI7 and pCMI were sequenced at the insertion sites. The aa sequence (in one-letter code) is indicated above the corresponding nt codons. Boldface letters indicate the aa sequence corresponding to the C-terminal regions of the pneumococcal amidase (in pCE! 7) or CPL-I lysozyme (in pCMI). The aa encoded by the vector are indicated by upper-case letters. Abbreviations: B, BamHl; Bg, Bglll; E, EcoRl; H,

Hindlll; P, Pstl; S, SspI; Sm, Smal.

"/2

B

A !

kDo 97.4

I

I

14.4

3

4

5

6

7

I

I

2

I

--

31.0--

21.5

2

lt~.

~

-"--

-- -

" ~,:~

I

H~ /

:~.i~!~ ¸~•¸ ,•

Fig. 2. Overexpression and purification of C-LYTA and C-CPLI from E. coli. E. coil RB791 cells, harboring either pCEI7 or pCMI, were grown in the presence or in the absence of lactose (2% final concentration). Sonicated samples were electrophoresed through 0.1~ SDS-15~ polyacryl~nide gels. (Panel A)Gel stained with Coomassie brilliant blue. Lanes 1 and 2 show the uninduced and lactose-induced RB791[pCEIT] samples, respectively; lane 3 shows the purified C-LYTA protein obtained as follows: a crude sonicated extract from a lactoseinduced RB791[pCEIT] culture was applied on a choline-Sepharose 6B column, washed first with six bed volumes of ice-cold 0.05 M Tris-maleate buffer, pH 6.9, containing 1.5 M NaCI, and then eluted with the same buffer plus 2~0 choline (Garcla et ~]., 1987b). Fractions were analyzed for homogeneity by SDS.PAGE. Lane 4, standard Mr markers in kDa on the left margin. Lane 5 shows the purified C-CPLI protein obtained from a crude sonicated extract of a lactose-induced RB791[pCMI] culture, following the procedure described above. Lanes 6 and 7 represent the total cellular proteins from lactose-induced and uninduced E. coli RB791[pCM I] cultures, respectively.The N-terminal aa sequences of the purified proteins were determined by Edmar, degradation in a pulse liquid phase protein sequencer model 477A (Applied Biosystems). (Panel B) lmmunoblot using anti-pneumococcal amidase serum (anti-E). Purified C-LYTA protein (lane 1) and total crude extract (lane 2) from lactoseinduced RB791[pCElT] cells, were electrophoresed on a SDS-polyaerylamide gel and transferred to nitrocellulose membranes (Schleicher & Schueil). After incubation ofthe blots with the primary serum (anti.E), they were incubated with a peroxidase-conjugated Aflinipure goat antirabbit serum ($ackson Immunoresearch) and the protein bands were visualized using 4-chloro-l-naphthol (Sigma).

(unpublished observations). These results taken together strongly suggested that the choline-binding capacity of the host amidase and the Cp-I lysozyme resides in their C-terminal regions. It is worth pointing out that the cholinerecognizing truncated proteins were stable enough to allow: (i) high levels of expression, similar to those previously reported for the overproduction of the complete host amidase and the CPL- 1lysozyme using the same expression vector (Garcia et al., 1987b; Sanz and Garcia, 1990); (ii) the binding to the choline-Sepharose or to the DEAE matrix and (iii)that either C-LYTA or C-CPL1 were retained in the column even after extensive washing with 1.5 M NaCI. No peptidoglycan-hydrolase activity was detected when crude E. coil sonicated extracts or pure

preparations of either C-LYTA or C-CPLI were incubated with [3H]choline-labelled pneumococcal cell walls (data not shown). (c) Circular dichroism studies of C-LYTA and C-CPLI The results presented above indicated that C-LYTA and C-CPLI bind to choline-Sepharose or to a choline analogue (DEAE-cellulose). Therefore, we were interested to know whether C-LYTA and C-CPLI were able to bind free choline residues as well. Far UV CD spectroscopy is exquisitely sensitive to the secondary structure of proteins. In the absence of data from X-rays, one of the uses of CD is for analysis of changes in the secondary structure on figand binding (see Johnson, 1988 for a review). Fig. 3 shows the CD spectra, in the near and far UV, of C-LYTA and C-CPL1 recorded in the presence or in the absence of choline. As expected, due to the aa similarities of both proteins, their spectra presented similar patterns of bands, i.e., two maxima (at 225 and 265 nm), and two minima (at 285 and 295 nm). Since choline did not have any CD spectrum by itself, and NaCI solutions giving an ionic strength equivalent to the choline concentrations used produced a negligibleeffect on the spectra (data not shown), it could be concluded that choline did bind specifically to C-LYTA and C-CPL 1 and that the conformational changes that take place on ligand binding are very similar, if not identical, in both proteins. It was noteworthy that the bands observed at 225 and 295 nm coincided with those observed in the CD spectrum of the CPL-1 lysozyme (Sanz and Garcia, 1990). These results reinforced the hypothesis that the conformational changes induced by choline in the CPL-1 lysozyme were due to the specific interaction of choline with the C-terminal region of the protein. As was previously proposed for CPL-1 (Sanz and Garcia, 1990), the presence of a high number of aromatic residues in the six repeated sequences of C-CPLI and C-LYTA (Garcia et ai., 1988), could explain the presence of the unusual CD band observed at 225 nm (Fig. 3). According to these data, it can also be concluded that the C-terminal pan of CPL-1 represents the major contribution to the unusual CD bands observed in the entire protein. The potential contribution of the aromatic residues to the CD spectra of C-CPLI and C-LYTA proteins might explain the fimitations to carry out an accurate calculation of their secondary structure composition using the conventional reference parameters of Chang et al. (1978; data not shown).

(d) Inhibition of the peptidoglycan hydrolase activity by C-LYTA and C-CYLI We have previously reported the construction of lytA genes modified at the 3' end and presented evidence suggesting that the C terminus of the pneumococcal amidase is not essential for the activity of the e ~ n e , although a

73

,-,, A

B Io

I o

," t

:

?

.~

,

I i

,,!./ |i

:

l,/v~ '

'

o

-s ? ~ o I -15 -20

6

;,'

DI so

; I

l,.....l

._.J 3O 20 lO

~V~ ~

.,o

P il -zo zoo z,o ~zo ~3o 2,o z~o z~o ~o 32o wovelencjth (nrn)

Fig. 3. CD spectra of C-CPLI and C-LYTA. Panels A and B show the C-CPLI spectra in the far and near UV, respectively. Panels C and D show the C-LYTA spectra in the far and near UV, respectively. Spectra (continuous lines) were obtained in 20 mM Na. phosphate buffer at pH 7.0. Broken lines correspond to spectra obtained in the presence of 10 or 20 mM choline for C-CPLI and C.LYTA, respectively. CD spectra were recorded in a RousellJouan Dichrograph II with a sensitivityofl × 10- s or 2 × 10 -e A4/mm. Cells of I or 0.05 cm (optical path) were used for spectra in the near UV (320-240 nm) and the far UV (240-200 nm) regions, respectively. The mean residue ellipticity, [0], was calculated from the relation [0] = 3300 × (As. - An) x c- t × d- t where (As. - An) is the difference dichroic absorption, c is the mean residue concentration when spectra are recorded in the far UV (or the protein concentration in the near UV) and d is the optical path expressed in cm. Protein concentrations of 0.096 and 0.77 mg/ml were used to record the CD spectra of C-LYrA in the far and near UV regions, respectively, whereas 0.3 ! mg/mlof C-CPLI was used in both determinations. Results are expressed in molar ellipticities with the dimensions of deg × dl/dm/(mol of residue) in the far UV or deg × dl/dm/(mol of protein) in the near UV. The 0 values represent the arithmetic mean of at least four independent runs, calculated at l-nm intervals. Protein concentrations were determined either by the method of Bradford (1976) or, spectrophotometrically, in a Shimadzu spectrophotometer UV-260,using the calculated C-LYTA and C-CPLI molar extinction coefficients E28 o = 60037 and 81076 M-t × c m - t respectively, according to the parameters published by Fasman (1976), making use of the known aa composition of the truncated proteins (Garcia et al., 1988).

nonaltered C end is required to achieve full activity as well as enzymatic activation by choline (S~chez-Puelles et al., 1987). The results shown in sections b and e demonstrated

that the purified C-terminal domains of the pneumococcal amidase and the CPL-I lysozyme are able to bind choline or choline analogues (i.e., DEAE). As previously pointed out, the binding of the autolysins to its insoluble substrate (i.e., the cell wall) is an essential prerequisite for activity (Giudicelli and Tomasz, 1984). Therefore, if C-LYTA and C-CPLI were also able to attach to choline-confining pneumococcal cell walls, these polypeptides should inhibit the degradative action of the lyric enzymes by masking the choline residues needed for the binding step. This hypothesis should be tested, with C-LYTA and C-CPLI added to the in vitro enzymatic assays in stoichiometric amounts, i.e., in amounts close to those of the choline residues that are 'accessible' in the purified cell walls. According to Mosser and Tomasz (1970), S. pneumoniae contains about 0.5 nmol of choline per ~tg of cell walls. Unfortunately, the number of choline residues in the cell wall flint are actually accessible is not known, but, because of the multilayered structure of t~te peptidoglycan, it is conceivable that they might represent only a minor fraction of the total choline content. On the other hand, it is also possible that C-LYTA and C-CPLI were able to bind to more than one choline residue at once (Garcia et al., 1988). The results shown in Table I indicated that 0.1 nmol of C-LYTA and C-CPLI reduced the hydrolysis of 10 pg of chofine-containing cell w',dls by about 45 ~o and 80 ~o, respectively, whereas similar amounts ofeither BSA or H E W L were unable to inhibit the enzymatic reaction. This finding suggested that the inhibitory effect of C-LYTA and C-CPLI was a specific one and confirmed that both C-LYTA and C-CPLI were able to bind to the choline residues of the pneumococcal cell walls.

TABLE 1 Inhibition of cell wall-hydrolysingactivities by C-LYTA and C-CPL!" Additions

None BSA HEWL C-LYTA C-CPLI

% Radioactivity releasedb Amidase

CPL-I

100 100 100 54.3 16.3

100 100 100 56.4 23.9

a Each assay mixture contained 10/d of [3H]choline-labelled pneumococcal cell walls (1 mg/ml and 7 x i0s cpm/ml; Mosser and Tomasz, 1970) and 0.1 nmoles of the appropriate additive in a final volume of 250 pl of 50 mM Tris-maleate buffer, pH 6.9. After incubation at 4°C for 5 min, 0.1 pmol of either the pneumococcal amidase or the CPL-I lysozymewas added. The samples were incubated for 10 rain at 37°C and the radioactivity released was determined as previously described (Garcla et al., 1987a). b Each valuerepresents the mean ofthree independent experiments.One hundred per cent represeny, about 3500 cpm.

74

(e) Ceuelesions (1) The 3' moieties of the lytA and the cpl-1 genes encoding the C-terminal regions of the pneumococcal amidase (C-LYTA) and the CPL-1 lysozyme (C-CPLI) have been cloned and overexpressed in E. coll. (2) The truncated proteins specifically bind to cholineSepharose or to DEAE-cellulose from which they can be purified to electrophoretic homogeneity in a single step. (3) CD spectroscopy revealed that choline binds to both C-LYTA and C-CPLI inducing similar conformational ch:mges. (4) C-LYTA and C-CPLI are able to attach to cholinecontaining pneumococcal cell walls. (5) An overall analysis of the results presented above revealed that the specificity for binding to the choline-substrates resides in the C-terminal region and suggested that the choline-recognizing regions of the pneumococcal lytic enzymes would constitute an independent and fully functional domain. We have previously suggested that the lytA and cpl-l, cpl-9, and cpl-7 genes originated from the fusion of different modules (Garcta et al., 1988; 1990). This modular design may allow a number of combination possibilities and could be a widespread model for many types of organisms. They could represent a common feature of most lyric enzymes of phages infecting Gram + bacteria although a definite response awaits the elucidation of the sequences of additional phage-encoded lysins. The idea of modular organization of phage DNAs had been proposed by the partial relationships between E. coil phages (Susskind and Botstein, 1978; Botstein, 1980). It has been suggested that the early modules were of exon size and that the proteins of contemporary phages were built up by rearranging such simple ©xons into new combinations (Reanney and Ackerman, 1982). On the basis ofthe present and previously reported results (Garcia et al., 1988), it is conceivable that the choline.binding domains of the iytic enzymes of the pneumococcal system have diverged from a common ancestor. In agreement with this hypothesis, we have recently found that the Cp-7 lysozyme, a lytic enzyme that does not require choline for activity, has a totally different C terminus with the N terminus conserved (Garcia et al., 1990). The availability ofthe cloned 3' ends of the lytA and cpl-I genes represents an important tool to construct chimeric proteins between the host and the phage lyric enzymes, which should provide valuable information conceming domain organization and function.

ACKNOWLEDGMENTS

We thank R. L6pez and P. Garcfa for tlz.ec~tical reading of the manuscript. We also thank the excellent technical assistance of M. Carrasco and E. Cano. The artwork of A.

Hurtado is gratefully acknowledged. This research was supported by a grant from Programa Sectorial de Promoci6n General del Conocimiento (PB87-0214).

REFERENCES Birnboim, H.C. and Duly, J.: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7 0979) 1513-1523. Botstein, D.: A theory of modular evolutionfor bacteriophages. Ann. NY Acad. Sci. 354 (1980) 451-484. Bradford, M.M.: A rapid and sensitive method for the quantitation of microgramquantities of proteins utilizingthe principle of protein-dye binding. Anal. Biochem. 72 (1976) 248-256. Brent, R. and Ptashne, M.: Mechanism of action of the lex~lgene product. Proc. Natl. Acad. Sci. U.S.A 78 (1981) 4204-4208. Briese, T. and Hakenbeck, R.: Interaction ofthe pneumococcal amidase with lipoteichoic acids and choline. Eur. J. Biochem. 146 0985) 417-427. Chang, C.T., Wu, C.C. and Yang, J.T.: Circular dichroic analysis of protein conformation: inclusion of the p.turns. Anal. Biochem. 91 (1978) 13-31. Fasman, G.: Handbook of Biochemistry and Molecular Biology,Vol. 1. CRC Press, Cleveland, OH, 1976. Garcia-Bustos, J.F. and Tomasz, A.: Teichoic acid-containing muropeptides from Streptococcuspneumonioc as substrates for the pneumococcal autolysin. J. Bacteriol. 169 (1987) 447-453. Garcia, E., Rojo, J.M., Garcia, P., Ronda, C., L6pez, R. and Tomasz, A.: Preparation ofantiserom against the pneumococcal autolysin.Inhibition of autolysin activity and some autolytic processes by the antibody. FEMS Microbiol. Lett. 14 (1982) 133-136. Garcia, E., Garcfa, J.L,, Garcia, P., Arraffm, A., S~chez-Pue'Jes, J.M. and L6pez, R.: Molecular evolution of lyric enzymes of $~ptococcus pneumonlae and its bacteriophages. Proc. Natl. Acad. Sci. USA 8'; (1988) 914-918. Garcla, J.L., Garcla, E,, Arrar/ts, A., Oarcla, P., Ronda, C. and L6pez, R.: Cloning, purification, and biochemical characterization of the pneumococcal bacteriophage Cp-I lysin. J. Virol. 61 (1987a) 2573-2580. Garcla, J.L., Garcia, E. and L6pez, R.: Overproduction and rapid purification of the amidase of Streptococcuspneumonlae. Arch. Microbiol. 149 (1987b) 52-56. Garcla, P., Garcia, J.L., Garcia, E. and L6pez, R.: Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in EscAerlr,Aia coll. Gene 43 (1986) 265-272. Garcia, P., Garcia, J.L, GarcJa, E., S/mchez-PueHes,J.M. and L6pez, R.: Modular organization of the lyric enzymes of Streptococcus paeumonlae and its bacteriophages. Gene 86 (1990) 81-88. Giudicelll,S. and Tomasz, A.: Attachment of pneumocoecal autolysinto wall teichoic acids, an essential step in enzymatic wall degradation. J. Bacteriol. 158 (1984) 1188-1190. H01tje, J.-V. and Tomasz, A.: Specificrecognition of choline residues in the cell wan teichoic acid by the N-aeetylmuramyl-L-alanineamidase of pnemnococctm. J. Biol. Chem. 250 (1975) 6072-6076. lnouye, S. and Inouye, M.: Up-promoter mutations in the ~pp gnne of E s ~ ¢o/L Nucleic Acids Ru. 13 (1985) 3101-3110. Johnson, Jr., W.C.: Semndary structure of proteins throuilh circular dichroism spectroscopy. Ammu. Rev. Biephys. Chem. 17 (1988) 145-166. L6pez, R., Ronda, C., Garcia, P., Escarmis, C. and Gm'cla, E.: Restriction cleavage maps of the DNAs of Streptococaa/mmmum/ae bacterio-

75 phages containing protein covalently bound to their 5' ends. Mol. Gen. Genct. 197 (1984) 67-74. Mosser, J.L. and Tomasz, A.: {..~oline-containing teichoic acid as a structural component ofpneumococcal cell wall and its role in sensitivity of lysis by an antolytic enzyme. J. Biol. Chem. 245 0970) 287-298. Reanney, D.C. and Ackermann, H.W.: Comparative biology and evolution of bacteriophages. Adv. Virus Res. 27 (1982) 205-280. S~nchez-PueHes, J.M., Garcia, J.L., L6pez, R. and Garcia, E.: 3'-End modifications of the $1reptococcuspneumoniae lytA gene: role of the

carboxy terminus of the pneumococcal autolysin in the process of enzymatic activation (conversion~ Gene 61 (1987) 13-19. Sanz, J.M. and Oarcla, J.L: Structura! studies of the lysozyme coded by the pneumococcal phage Cp-I. Conformational changes induced by choline. Eur. J. Biochem. 187 0990) 409-416. Sanz, J.M., l~pez, R. and Garcia, J.L.: Structural requirements for 'conversion' of pneumococcal amidase. A new single-step procedure for purification of this autolysin. FEBS Lett. 232 (1988) 308-312. S~mskind, M.M. and Botstein, D.: Molecular genetics of bacteriophage P22. Micrcb. Rev. 42 (1978) 385-413.