Mol Gen Genet (1985) 201:225-230 © Springer-Verlag 1985

Cloning and expression of the pneumococcal autolysin gene in Escherichia coil

Ernesto Garcia, Jos6-Luis Gareia 2, Concepci6n Ronda l, Pedro Garcia 1, and Rubens L6pez 1 1 Instituto de Inmunologia y Biologia Microbiana, Vel~zquez 144, SP-28006 Madrid 2 Antibi6ticos, S.A. Bravo Murillo 38, SP-28015 Madrid, Spain

Summary. A 7.5 kb Bc/I-fragment of Streptococcus pneu- moniae DNA has been cloned in Escherichia coli HB101 using pBR322 as a vector. The new plasmid (pGL30) of 12.0 kb expresses a protein that has been characterized by biochemical, immunological and genetic methods as the in- active form (E-form) of the pneumococcal N-acetyl-mura- myl-L-alanyl amidase (EC 3.5.1.28). Our results demon- strate that the E-form is the primary product of the lyt gene of S. pneumoniae. The inactive E-form can be con- verted to the active C-form in vitro by incubation of the E-form enzyme with choline-containing pneumococcal cell walls at low temperature in a similar way to enzyme produc- tion in the homologous system. The production of this pro- tein in E. coli HB101 was 500-fold higher than in the homol- ogous host. E. coli CSR603 containing pGL30 and labeled with [3SS]methionine synthesized a 35 kd protein, pGL30 can transform at high frequency an autolysin-defective mu- tant of S. pneumoniae to the lyt + phenotype.

Introduction

Bacterial autolysins have been implicated in a large number of important biological phenomena such as cell lysis at the end of the growth cycle or after treatment with fl-lactam antibiotics, liberation of the phage progeny, competence for genetic transformation, etc. (see Tomasz 1984 for a re- view). Streptococcus pneumoniae contains an N-acetyl-mur- amyl-L-alanyl amidase (EC 3.5.1.28) (Howard and Gooder 1974; H61tje and Tomasz 1976), which is responsible for the detergent-induced lysis characteristic of this species (Mosser and Tomasz 1970). The acti~dty of this murein hydrolase is strongly dependent on the presence of choline residues in the cell wall teichoic acid of this bacterium. Replacement of the choline by its analogue ethanolamine causes a series of dramatic changes in the cell physiology (e.g. growth in long chains, loss of competence for transfor- mation, resistance to lysis, etc.) (Tomasz 1968; Tomasz et al. 1970, 1971). All these alterations have been attributed to the fact that ethanolamine-grown cells contain an inac- tive form (E-form) amidase (Tomasz and Westphal 1971). This inactive autolysin can be "converted" to the active form (C-form) by incubation at low temperature with cho- line-containing cell walls.

Offprint requests to. R. L6pez

In autolysis-prone bacteria the host amidase also plays an important role in the liberation of the progeny of the pneumococcal bacteriophage Dp-I (Ronda etal. 1977). However, another autolytic activity is involved in this pro- cess. When autolytic-defective mutants of S. pneurnoniae are infected with phage Dp-l, a phage-associated lysin (PAL) is synthesized (Garcia etal. 1983a). This PAL has been isolated and purified to electrophoretic homogeneity (Garcia et al. 1983 b) and characterized as an endo N-ace- tyl-muramyl-L-alanyl amidase (Garcla et al. 1984). The host pneumococcal amidase and the PAL share several impor- tant properties (H61tje and Tomasz 1975c; Garcia et al. 1983 c) such as requirement of choline residues for activity, inhibition by choline-containing lipoteichoic acid (LTA), etc. In addition, recent experimental evidence indicates that E enzyme and PAL have a similar but not identical amino acid compositions (L6pez et al. 1984). Furthermore, using antibodies against both enzymes (Garcia et al. 1982, 1983 c) we have suggested recently that E-form and PAL are not closely related in conformation (Garcia et al. 1984). In spite of what we have pointed out above, the physiological role of the pneumococcal autolysin in normal cells remains still an open question (Tomasz 1984). Further biochemical stud- ies would require the availability of higher amounts of both proteins, which are difficult to purify by standard tech- niques. Molecular cloning and over-expression would allow sufficient quantities of the enzymes to be obtained.

The recent development in our laboratory of a powerful technique to distinguish between autolysin-containing strains (lyt +) and autolysin-defective strains (lyt-) (Garcia et al. 1985) has opened the possibility of cloning the genetic determinants of the pneumococcal autolysin.

In the present paper, we report the cloning of a 7.5 kilo- base (kb) Bc/I-fragment of S. pneumoniae DNA in Escherichia coli using pBR322 as plasmid vector. This pneu- mococcal insert is able to transform lyt- pneumococcal receptors to the lyt ÷ phenotype. In addition, the cloned DNA fragment is expressed in E. coli as an E-form pneumo- coccal amidase in amounts 500-fold higher than those pro- duced in the homologous host. To our knowledge, this is the first example of cloning of a bacterial autolysin gene.

Materials and methods

Bacterial strains, plasmid and transformation. Strains of S. pneumoniae are listed in Table 1. Transformation of corn-

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Table 1. Strains used in this study

Species Strain Plasmid Plasmid Relevant genotype Source (reference) size or phenotype (kb)

E. coli

S. pneumoniae

HB10a (r~, raft), recA13 Boyer and Roulland-Dussoix (1969) HB101 (pBR322) pBR322 4.3 Amp r, Tet r Bolivar et al. (1977) CSR603 uvrA6, recA1 Sancar et al. (1979) HB101 (pGL30) pGL30 12.0 Amp r This study ~ CSR603 (pGL30) pGL30 12.0 Amp f This study a

R6 Wild type Rockefeller University Rst7 str Rockefeller University R61y4-4 lyt-4 Fischer and Tomasz (1984) FBa hex +, lyt + Barany and Tomasz (1980) M7 hex + , lyt-4 b 708 hex-, lyt + Stassi et al. (1981) M12 hex-, lyt-4 c

Plasmid pGL30 was constructed by inserting a BclI fragment of pneumococcal DNA into the BamHI site of pBR322. The chromosomal insert codes for the pneumococcal autolysin

b Strain constructed by transformation of FB1 with DNA from R61y4-4 c Strain constructed by transformation of strain 708 with DNA from R61y4-4

petent pneumococcal cells was carried out essentially as previously described (Barany and Tomasz 1980) except that the incubation with transforming DNA was for 40 min at 30 ° C and after addition of DNase the cultures were incu- bated at 37 ° C for 2 h before plating. E. coli strain HBI01 was made competent by the rubidium chloride method (Maniatis et al. 1982). Plasmids were amplified in the pres- ence of chloramphenicol (170 gg/ml). Alkaline lysates were prepared as previously described (Birnboim and Doly 1979).

DNA cleavage and ligation. Restriction endonucleases were purchased either from Bethesda Research Laboratories, Maryland, USA, Boehringer Mannheim, FRG or Amer- sham Searle. Calf intestine alkaline phosphatase and T4 DNA ligase were obtained from Boehringer Mannheim. All these enzymes were used according to the recommenda- tions of the suppliers. Before ligation BamHI-digested pBR322 was treated with intestinal alkaline phosphatase to reduce the number of E. coli transformants due to self- ligation of the vector. Ligation mixtures (about 2 gg DNA) were heated at 65 ° C for 5 min and cooled at room tempera- ture. After addition of 1/10 (vol/vol) ligase buffer (0.2 M Tris-HC1, pH7.6; 0,1 M MgC12; 0.1 M dithiothreitol; 6 mM ATP) and 2 units of ligase, the mixtures were incu- bated at 4 ° C for 17 h,

Agarose gel electrophoresis. Restriction fragments and plas- raids were analyzed by electrophoresis in 0.7% or 1.4% gels as previously described (L6pez et al. 1984b).

Preparation of cell walls and assay for lytic activity. The standard assay conditions for the pneumococcal amidase have been described elsewhere (H61tje and Tomasz 1976).

Identification of the lyt+/lyt - phenotype in S. pneumoniae. Pneumococcal colonies can be grown on the surface of C + Y plates supplemented with 250 units/ml catalase (Boehrin- ger Mannheim) (Garcia et al. 1985). Millipore HATF filters were laid upon agar plates and after 1-2 min at room tem- perature the filters were removed and lain colonies up on

the surface of a velvet placed in a 1% (w/v) sodium deoxy- cholate solution buffered with 50 mM potassium phosphate (pH 8.0). After incubation for 10 rain at 37 ° C, the filters were dried briefly at room temperature and stained in a Coomassie brilliant blue solution (0.25% Coomassie bril- liant blue in 45.4% (v/v) methanol and 9.2% (v/v) acetic acid) (Swank and Munkress 1971). After 1 rain the filters were destained over 15-30 s in a solution containing 7.5% acetic acid and 25% methanol and the visible colonies on the filter were eliminated. The filters were washed with tap water and dried. Autolysin-containing colonies (lyt +) appear as blue spots whereas autolysin-deficient colonies (lyt-) appear as white circles (Garcia et al. 1985).

Detection of proteins coded by cloned pneumococcal DNA in pGL30. Proteins encoded by the pneumococcal insert in pGL30 were detected using the maxicell technique as previously described. (Sancar et al. 1979). [~5S]methionine (1500 Ci/mmol) was from Amersham Searle. SDS-poly- acrylamide gel electrophoresis was carried out as described by Laemmli (1970).

Results

Cloning of chromosomal lyt DATA

Chromosomal DNA from strain Rst7 containing the lyt + gene was digested with several restriction endonucleases and the residual transforming activity was assayed using compe- tent cells of strain M12 (hex- lyt-). Pneumococcal DNA treated with BclI maintained almost full lyt + transforming capacity with a remarkable reduction in the mean size of the fragments. To ligate Bcll fragments to a suitable com- patible vector, we chose pBR322, which has only a single BamHI cutting site (in the TeV gene). The ligated DNA was used to transform E. coli strain HBI01 to ampicillin resistance and the transformants were tested for tetracycline resistance. Following this procedure more than 80% of the ampicillin-resistant clones were tetracycline-sensitive. Alka- line lysates of these transformants were tested for the ability to transform the M12 strain of S. pneumoniae. From about

Table 2. Transforming activity of recombinant plasmid pGL30 in hex + and hex- strains of S. pneumoniae

Recipient strain Marker Transformants per ml obtained with donor DNA

Chromosomal pGL30 DNA from Rst7

FB1 (hex +, lyt +) lyt- -- <300 Str r 2.2 x 10 s -

M7 (hex +, lyt-) lyt + 1.8 x 104 7.5 x 105 Str r 1.4 x 105 -

708 (hex-, lyt +) lyt- - <300 Str r 3.4 x 105 -

M12 (hex-, lyt-) lyt + 2.5 x 105 1.5 x 107 Str ~ 2.7 x 105 -

600 recombinant clones, two transformants were isolated, clones 430 and 444, which contained plasmids with pneu- mococcal lyt + D N A inserts. Since restriction enzyme analy- ses indicated that both plasmids contained an identical in- sert, only one of them (pGL30) was chosen for further study. Alkaline lysates of this E. coli strain (i.e. clone 430) analyzed by agarose gel electrophoresis showed a plasmid which migrated more slowly than tlhe parental plasmid pBR322 (data not shown).

Transfer o f lyt + gene f rom pGL30 to S. pneumoniae

Claverys et al. (1981) and more recently Morrison et al. (1984) have shown that fragments of pneumococcal D N A cloned into heterologous plasmid vectors are able to trans- form competent cells of S. pneumoniae. These pure recom- binant E. coli plasmids can transform with high frequency adequate receptor strains. When we transformed an hex lyt- pneumococcal strain (M12) with an alkaline prepara- tion obtained from pGL30, t ransformation frequencies as high as 60% were obtained (Table 2). These results also demonstrate that the muta t ion lyt-4 present in the receptor strains behaved as a low-efficiency marker and fully confirm previous findings in our laboratory using undigested chro- mosomal D N A from S. pneumoniae as donor material (Garcia et al. 1985).

Physical mapping o f the lyt recombinant plasmid pGL30

Restriction enzyme analyses indicated that pGL30 has a a D N A insert of about 7.5 kb (Fig. 1). This insert is cut once by PstI, AccI and PvuII and twice by EcoRI. Several sites recognized by Sau96I and HindIII have been detected and mapped. The existence of multiple HindIII sites and, conse- quently, the small size of the fragments, explains why this particular restriction endonuclease completely destroyed b the transforming capacity of chromosomal lyt D N A (see above). On the other hand, no cuts were found with several restriction enzymes (e.g. BamHI, SstI, SstII, XbaI).

Expression o f the pneumococcal autolysm gene #2 E. coli

The host pneumococcal amidase (autolysin) has been puri- fied to electrophoretic homogeneity as a low specific activity

227

Eco RV

Fig. 1. Restriction map of plasmid pGL30. Light lines correspond to vector pBR322 DNA and heavy lines to DNA of pneumococcal chromosomal origin. The single cut by EcoRI in the vector DNA has been arbitrarily designated as zero map units

Table 3. Pneumococcal cell wall hydrolysis by lysates of E. coli cultures

Condition ~ % radioactivity released b

E. coli HB101 0

E. coli HB101 (pBR322) 0

E. coli HBa01 (pGL30)

No preincubation at 0 ° C, 5 min E 25.2

Preincubation (0 ° C, 5 rain)c 100 t4C-ethanolamine cell walls 7.5

Preincubation + TEPC-15 5 131 75.2

20 gl 48.3 40 131 23.4 20 131 + 30 lag phosphorylcholine 95.2

Preincubation + LTA 0.5 lag/ml 70.3 1.0 lag/ml 48.7 5.0 gg/ml 21.7

A mid-stationary phase culture of E. coli in LB was centrifuged, washed and lysed with lysozyme as previously described (Birn- boim and Doly 1979). Aliquots of 10 gl (for strains HB101 and HB101(pBR322)) or 10 131 of a 100-fold dilution (for HB101(pGL30)) were assayed for autolysin activity in a final reaction volume of 250 gl as described in Materials and methods 100% represents about 7,500 cpm After addition of all components to the reaction mixture, this was preincubated at 0 ° C for 5 min before incubation at 37 ° C for 15 min (H61tje and Tomasz 1976). l~C-ethanolamine-labeled pneumococcal cell walls were used instead of 3H-choline-labeled ones. Myeloma protein (TEPC-15) (2 mg/ml of protein/ml stock solution) was added to the samples at the indicated amounts. Bacterial lysate was added last. Pneumococcal Forssman antigen (lipoteichoic acid, LTA) (Briles and Tomasz 1973) was added to give the final concentration indicated

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Fig. 3. Ouchterlony double-diffusion plate of the lysates of E. coli containing pGL30. Wells: (1) anti-E serum; (2) extract obtained from E. coli HBI01 (pGL30) previously incubated for 5 min at 0 ° C; (3) extract obtained from E. coli HB101 (pBR322); (4) anti- PAL serum; (5) extract obtained from E. coli HB101 (pGL30)

Fig. 2A, B. Autoradiogram of labeled plasmid-coded proteins in UV-irradiated whole cells (" maxicells "). A E. coli CSR603, con- taining pGL30 (lane 1) or pBR322 (lane 2) grown in LB containing 50 gg/ml ampicillin were subjected to SDS-polyacrylamide gel elec- trophoresis. B Maxicell experiments were carried out as previously described (Sancar et al. 1979). Lane 3: E. coli CSR603 containing pGL30; lane 4: cells containing pBR322; lane 5: cells containing no plasmid. Arrows indicate the position of the E enzyme. Molecu- lar weight markers: Phosphorylase B; Bovine serum albumin; Ovoalbumin; Carbonic anhydrase; Soybean Trypsin inhibitor, and Lysozyme

form (E-form) of 35 kd (H61tje and Tomasz 1976). Upon incubation in vitro with purified cell walls of pneumococci the E-form was converted to the active form (C-form) that had a high molecular weight. Lysates of E. coli HB101 (pGL30) were examined for lytic activity against pneumo- coccal cell walls. Table 3 shows that extracts from this E. coli strain did release radioactivity from [3H]choline-la- beled pneumococcal cell walls. The results also demonstrate that preincubation with pneumococcal cell walls is a prerequi- site for activity, indicating that the autolysin is synthesized as an inactive E-form. Furthermore, lipoteichoic acid (LTA) (i.e. Forssman antigen) showed a strong inhibitory activity as previously reported for the pneumococcal ami- dase (H61tje and Tomasz 1975a, b). The autolysin found in E. coli requires choline residues to degrade cell walls as does that isolated from S. pneumoniae (H61tje and Tomasz 1975c). Therefore, it did not hydrolyze ethanolamine-con- taining cell walls and, moreover, when choline-containing cell walls were treated with a phosphorylcholine-specific mieloma antibody, the lytic activity of the E. coli extracts was drastically blocked; however, the addition of phosphor- yleholine immediately restored the autolytic activity (Ta- ble 3).

Plasmid pGL30 was introduced by transformation into E. coli strain CSR603 and, after UV-irradiation, plasmid- coded proteins were labeled with [35S]methionine. "Maxi- cells" were processed for SDS-polyacrylamide gel electro- phoresis and labeled proteins were visualized by autoradi- ography. As expected, there is no incorporation of label

into CSR603 but protein bands of molecular weights 34 K and 30 K were observed in the strain carrying pBR322 (Fig. 2). The protein of higher molecular weight corre- sponds to the tet gene product whereas the other was the product of the amp gene (Sancar et al. 1979). On the other hand, one heavily labeled band of 35 kd, which corresponds to the molecular weight of the purified E-form amidase (H61tje and Tomasz 1976), was observed in the extracts obtained from the strain containing pGL30 (Fig. 2B). As expected, the extract of this strain lacked the band corre- sponding to the tet protein, since the pneumococcal DNA was inserted at the BamHI site in pBR322 (see above). Fur- thermore, the protein band corresponding to the pneumo- coccal autolysin could be observed in gels stained with Coo- massie blue (Fig. 2A).

An antiserum prepared against pneumococcal E-form amidase has been obtained in our laboratory (Garcia et al. 1982). Figure 3 shows that anti-E serum formed a precipitin band against extracts of E. coli HB101 (pGL30) whereas no band was observed against extracts of E. coli HB101 (pBR322). Extracts obtained from E. coli HBI01 or from another E. eoli strain containing an unspecific pneumococ- cal DNA insert in pBR322 did not form any precipitin band (data not shown). On the other hand, we have recently reported (Garcia et al. 1984) that, upon incubation of the E-form amidase with choline-containing cell walls, the C- form amidase shares immunological determinants with the phage Dp-1 associated lysin (PAL). In a similar way, anti- PAL serum did precipitate the converted C-form, but did not form any band against the inactive E-form of the au- tolysin obtained from E. coli HB101 (pGL30) (Fig. 3).

Discussion

S. pneumoniae contains a potent autolytic enzyme (H61tje and Tomasz 1976) which has been implicated in a high number of important processes (Tomasz 1984). Recently it has been demonstrated that the autolysin (lyt) gene be- haves as a low-efficiency marker in transformation experi- ments using strain R61y4-4 as receptor (Garcia et al. 1985). Successful cloning and expression of the pneumoeoccal lyt gene in a heterologous system (E. coli) is a novel procedure for obtaining high amounts of autolytic enzyme.

229

The recombinant plasmid, pGL30, that contains a 7.5 kb insert (Fig. 1) was shown to transform ly t - pneumo- coccal cells at a high frequency (about 60%) (Table 2). Sim- ilar high transformation levels have been obtained pre- viously (Claverys et al. 1981). The increase in transforma- tion frequency of cloned DNA as compared to undigested chromosomal DNA is due to the fact that each donor- cloned DNA molecule carries the selected gene.

In normal pneumococcal cells (i.e. growing in the pres- ence of choline) the autolytic enzyme is mainly in a high molecular weight form (C-form) (H61tje and Tomasz 1976) that exhibits high specific activity. However, when choline is replaced by its analogue ethanolamine in the culture me- dium, pneumococcal cell contain an inactive, low molecular weight form (E-form) of the autolysin. Some experimental evidence has indicated that E-form represents the primary translation product of the lyt gene whereas C-form autoly- sin resulted from activation at a later stage (Hrltje and Tomasz 1976). Unfortunately the in vivo conversion E ~ C is so rapid that it has not been possible to confirm this hypothesis, When E. coli cells containing pGL30 were lysed and the lysate was tested for its activity against pneumocoe- cal cell walls, we observed that an inactive, E-like autolysin was present (Table 3). Preincubation of the lysate with cho- line cell walls at low temperature (i.e. "conversion") was a prerequisite for activity. This activity did degrade choline- containing pneumococcal cell walls but was unable to at- tack ethanolamine-containing ones. On the other hand, the strong dependence of the choline residues on activity of the cloned autolysin was in agreement with previous results obtained with the amidase in the homologous system (H61tje and Tomasz 1975c; Tomasz et al. 1971). These re- sults taken together indicate that the entire lyt gene was cloned in pGL30 and that this gene is transcribed as a low activity E-form amidase. A single protein band of 35 kd (part of the product of the amp gene of the plasmid vector) was observed in maxicell extracts labeled with [35S]meth- ionine containing the recombinant plasmid pGL30 (Fig. 2). The molecular weight of this protein, which was also ob- served in Coomassie blue-stained extracts, is the same as that reported for the electrophoretically pure E-form of the pneumococcal autolysin (H61tje and Tomasz 1976).

Further evidence of the production of the E-form of the pneumococcal autolysin in E. coli came from immuno- logical experiments. Anti-E serum did recognize the autoly- sin of S. pneumoniae in crude extracts of E. coli containing pGL30 but not in other clones containing different inserts. Furthermore, an antiserum obtained against the PAL did not form any band against nonconverted lysates but, intro- ducing a previous conversion step (i.e. E~C), the resulting C-form amidase was detected by the formation of a precipi- tin band (Fig. 3) which is in agreement with previous results from our laboratory (Garcia et al. 1984).

The precise location of the lyt gene in the insert of pGL30 is still unknown. Preliminary results (not shown) indicate that it probably resides in the EcoRI-C fragment of pGL30 (0-3.9 kb of map position). In fact, a plasmid that lacks the smaller PvuII fragment of pGL30 still main- tains full autolysin coding activity. Another open question is whether the lyt gene is transcribed from its own promoter or from one ofE. coli. The difficulty of cloning pneumococ- cal genes that carry strong promoters have been pointed out recently (Stassi and Lacks 1982). It is conceivable that transcription from the promoter of the tetracycline gene

located upstream from the insert contributes to the expres- sion of the lyt gene. However, functioning of the autolysin promoter cannot be ruled out. This possibility is suggested from the remarkable amount of autolysin synthesized in maxicells containing pGL30 (Fig. 2) in spite of the fact that these maxicells probably have a smaller plasmid copy number than those that contain pBR322, as indicated by the diminished amount of fl-lactamase synthesized by the former with respect to the latter, as shown in Fig. 2 B. Fur- thermore, since the pneumococcal autolysin contains low number of methionine residues (L6pez et al. 1984), the high amount of labeling obtained with [35S]methionine in pGL30-containing maxicells must be due to an increased' production of the enzyme. The amount of autolysin con- tained in E. coli extracts is very high compared to the nor- mal levels in the homologous system (Hrltje and Tomasz 1976). In fact, the cloned E-form autolysin can be obtained from E. coli in amounts 500-fold in excess of those found in S. pneumoniae. Further experiments are in progress to use other plasmid vectors suitable for an increased over- expression of the cloned lyt gene of S. pneumoniae, In addi- tion, the cloned fragment is currently being used to deter- mine the nucleotide sequence of the lyt gene.

Acknowledgements. P.G. received a postdoctoral fellowship from C.S.I.C. The technical assistance of Mrs. N. Fontfirbel is acknowl- edged. This work was supported by a grant of the Spain-USA Joint Committee for Scientific and Technological Cooperation (CDB 840 20, 35) and by a grant from CAICYT.

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Communicated by E.K.F. Bautz

Received June 10, 1985