Cloning of a gene encoding a DNA polymerase-exonuclease of Streptococcus pneumoniae

Gene, 44 (1986) 79-88

79

Elsevier GENE

1657

Cloning of a gene encoding a DNA polymerase-exonuclease of Streptococcus pneumoniae (Recombinant DNA; Gram-positive cloning system; DNase colony assay; nuclease detection in SDS gels; gene expression in Escherichia coli; DNA polymerase I; polA)

Susana Martinez”vb, Paloma Lopezavb, Manuel Espinosab and Sanford A. Lacksa* a Biology Department, Brookhaven National Laboratory, Upton, NY 11973 (U.S.A.) Tel. (516)282-3369, Investigaciones Biolhgicas, C.S.I. C., Velazquez. 144, 28006 Madrid (Spain) Tel. 261-18-00 (Received

March

(Revision

received

(Accepted

and ’ Centro de

17th, 1986) May 2nd, 1986)

May 5th, 1986)

SUMMARY

A procedure was developed for cloning and characterizing genes that encode proteins with nuclease activity in the Streptococcus pneumoniae[pLSl] host/vector system. Clones are screened for nuclease activity by a DNase colony assay and the nucleases that they produce are characterized by detection of enzyme activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The method was used to clone the gene encoding a DNA polymerase (Pal)-exonuclease of S. pneumoniae. The activity of this enzyme, the predominant DNA Pol of S. pneumoniae, is tenfold greater in cells carrying the multicopy recombinant plasmid than in cells without the plasmid. The enzyme corresponds to a lOO-kDa polypeptide, and its properties are similar to PolI of Escherichia coli. A restriction map of the pSM22 plasmid containing the pneumococcal polA gene was obtained. The gene was transferred into Bacillus subtilis and E. coli, and it was expressed in both species. Its direction of transcription was determined by placement of the gene in both orientations in an E. coli hyperexpression plasmid. In one of the orientations the pneumococcal PolI enzyme was produced at a level 50-fold greater than normally found in S. pneumoniae, and it comprised 5% of the total protein.

INTRODUCTION

Bacteria generally contain several different DNA polymerase enzymes. In the best characterized case,

* To whom

correspondence

and

reprint

requests

should

be

addressed. Abbreviations: bromide;

WIG,

lyacrylamide; sodium dodecyl carrier

state.

Ap, ampicillin;

bp, base pair(s);

isopropylthiogalactoside; Pol, polymerase;

R, resistance,

sulfate; Tc, tetracycline;

EtdBr, ethidium

kb, 1000 bp; PA, poresistant;

[ 1,designates

SDS, plasmid-

that of the Gram-negative bacterium E. coli, three enzymes are found: Poll (Lehman et al., 1958), which is present in the highest molar concentration and has been implicated in DNA repair (De Lucia and Cairns, 1969) and in discontinuous DNA synthesis (Lehman and Uyemura, 1976); Poll1 (Gefter et al., 1972), for which no function is known; and PolIII, which is responsible for chromosomal DNA synthesis (Gefter et al., 1971). All three enzymes exhibit 3’ to 5’ exonuclease activity, and Poll and PolIII also have 5’ to 3’ exonuclease activity (Deutscher and Kornberg, 1969; Livingston and Richardson, 1975).

0378-I 119/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

80

Three DNA polymerases are found also in the Gram-positive B. sub&s. Here too, PolI is the predominant activity and is implicated in repair, whereas PolIII functions in chromosomal replication. PolIII of B. subti1i.shas exonuclease activity but only in the 3’ to 5’ direction; unlike the E. coli enzyme, it is inhibited by 6-(p-hydroxyphenylhydrazino)-uracil which inhibits chromosomal DNA replication in Gram-positive bacteria (Low et al., 1976). Neither PolI (Okazaki and Kornberg, 1964) nor PolII (Low et al., 1976) from B. subtilis show exonuclease activity. The predominant DNA polymerase in the Gram-positive S. pneumoniae, however, was associated with exonuclease activity after partial purilication (Lacks, 1970). On the basis of substrate and inhibitor specificity, several different polymerases were detected in S. pneumoniae (Firshein and Gelman, 198 1). In the present work a gene encoding a DNA polymerase-exonuclease of S. pneumoniae was cloned in both S. pneumoniae and E. coli and shown to encode a protein resembling PolI from E. coli.

The polA gene of E. coli, which encodes PolI, was cloned in phage 1 by selecting for resistance of transductants to DNA damage by methyl methanesulfonate (Kelley et al., 1977). Its sequence has been determined and shown to encode a polypeptide of 103 kDa. Cloning of the E. coli gene has provided sufficient PolI protein for structural analysis (Ollis et al., 1985) and allowed investigation of enzyme functions essential for cell viability (Joyce and Grindley, 1984). Analysis along these lines of the corresponding pneumococcal gene and its product should now be possible. In particular, it will be of interest to test for a role of the pneumococcal PolI in the Hex system of DNA base mismatch repair (reviewed in Claverys and Lacks, 1986). A role was postulated for such an enzyme in resynthesis of the lengthy tracts of DNA excised in the repair process (Balganesh and Lacks, 1985). Cloning of the gene from S. pneumoniae was accomplished by an approach that depended on the exonuclease activity of the enzyme and was therefore very different from the procedure used for cloning the E. coli gene. The pneumococcal gene was cloned in a Gram-positive cloning system (Stassi et al., 1981) using the broad-host-range multicopy plasmid vector pLS1 (S.A.L., P. L., B. Greenberg and M. E., manuscript submitted for publication). Unique

features of the approach were the screening of clones for exonuclease activity by a DNase colony assay (Lacks, 1970) and the characterization ofthe product of the cloned gene by a method for detecting nuclease activity in PA gels after electrophoresis in the presence of SDS (Rosenthal and Lacks, 1977). These convenient and rapid techniques may be generally applicable to the cloning of genes for DNA polymerases and other proteins that can hydrolyze DNA.

MATERIALS

AND METHODS

(a) Bacterial strains and plasmids

Strains of S. pneumoniae used were the nonencapsulated, nonpathogenic, ‘wild-type’ strain R6 and derivatives as follows : 577 (end- 14) 593 (end-l), 64 1 (end-l noz-19 exo-2), 642 (exo-5), and 708 (end-l exo-2 trt-1 hex-4 malA4594). The end and noz (for no zone) mutations affect the gene encoding the major endonuclease of S. pneumoniae, and exo mutations affect the major exonuclease (Lacks, 1970; Lacks et al., 1975). Strains of B. subtilis and E. coli used were CU403 (Reeve et al., 1973) and BL2lDE3 (Studier and Moffatt, 1986), respectively. Plasmids used were pLS1 (Stassi et al., 1981) and pAR2192 (A.H. Rosenberg, J.J. Dunn and F.W. Studier, manuscript in preparation). (b) Growth and transformation

of bacteria

Cultures of S. pneumoniae and B. subtilis were grown and transformed as previously described (Lopez et al., 1984). E. coli was grown in L broth and transformed according to Kushner (1978). Cultures were treated with plasmid DNA at 1.0 pg/ml. Transformants were selected in agar medium containing Tc at 1 and 50 pg/ml for S. pneumoniae and B. subtilis, respectively, and Ap at 50 pg/ml for E. coli. (c) DNA and plasmid preparation

Chromosomal DNA was prepared from S. pneumoniae according to Hotchkiss (1957). Purified plasmids were prepared by the procedure of Currier and Nester (1976). Crude plasmid prepara-

81

tions, called alkaline lysates, were prepared from E. coli by the method of Birnboim and Doly (1979) and from S. pneumoniue by a modification of that method (Stassi et al., 1981). Cleared lysates of B. subtilis were prepared as previously described (Espinosa et al., 1982). (d) DNA manipulations

Restriction endonucleases were obtained commercially and used as specified by their suppliers. Analytical gel electrophoresis of plasmids and restriction fragments was carried out in 1y0 agarose or 5 % PA with staining by EtdBr. Ligation was carried out as described (Stassi et al., 1981). (e) Enzyme assays

DNA polymerase and exonuclease activities in extracts containing native proteins were determined in vitro as indicated in the legend to Table I. The DNase colony assay was carried out as previously described (Lacks, 1970). For the assay of nuclease activity in gels after electrophoresis in the presence of SDS, the method of Rosenthal and Lacks (1977) was used. However, in some experiments the PA slab gels contained a linear concentration gradient of PA (Studier, 1973). Thymus DNA (Worthington Biochemical), which had been denatured by heating in solution at 2 mg/ml for 15 min at 100’ C, was cast into the gel at a concentration of 30 pg/ml. Bacterial extracts were prepared by lysis with SDS in the presence of phenyhnethylsulfonyl fluoride and applied to the gel after heating for 3 min at 100 ’ C as previously described (Rosenthal and Lacks, 1977). The electrophoresis buffer contained 0.05% SDS (Matheson, Coleman and Bell, product DX2490, lot 27 [Lacks et al., 19791). Electrophoresis was conducted at 20-25°C with a constant current of 26 mA for approx. 3 h. After removal of SDS the gels were incubated at 30°C in 40 mM Tris . HCl, pH 7.6, 2 mM MnCl,. They were periodically stained with EtdBr and photographed under UV illumination. (f) Analysis of plasmid-encoded

proteins

The BL21DE3[pAR2192] host-vector cloning system allows the specific labeling of protein pro-

ducts encoded by genes in the plasmid (Studier and Moffatt, 1986). The cell harbors a defective 1 prophage that contains the phage T7 RNA polymerase gene under the control of the lac repressor, and the vector contains a T7 RNA polymerase promoter site. So in the presence of IPTG, which induces synthesis of the T7 polymerase, and rifampicin, which inhibits the E. coli RNA polymerase but not the T7 polymerase, only plasmid-encoded proteins, the mRNA for which is transcribed from the T7 promoter, and no host-encoded proteins are synthesized. Even without rifampicin, most of the protein synthesized after induction is plasmid-encoded (Studier and Moffat, 1986). To l-ml cultures of BL21DE3 carrying pSM23 or pSM24 grown at 37°C to/t,,, = 0.5 in M9 medium containing 0.2 mg Ap/ml, 0.5 pmols IPTG and, in some cases, 0.2 mg rifampicin were added; after various periods of incubation, 10 pmol of [ 35S]methionine (1000 Ci/mmol) were added; 5 min later the reaction was terminated by chilling the culture, centrifuging the cells, suspending them in 0.2 ml of sample loading solution (50 mM Tris +HCl, pH 7.6, 2 mM EDTA, 1% SDS, 1% mercaptoethanol, 0.025% bromphenol blue, 10% glycerol) and heating for 3 min at 100°C.

RESULTS

AND DISCUSSION

(a) Cloning of a chromosomal clease activity

gene conferring nu-

Cloning was accomplished in an S. pneumoniae host with the vector pLS 1 (Stassi et al., 198 1). Chromosomal DNA of the wild-type strain, R6, was cut with EcoRI and ligated with pLS1 cut at its single EcoRI site. The ligation mixture was used to transform strain 708, which is deficient in the major nucleases of S.pneumoniue (Lacks, 1970). Tcresistant transformants were screened by the DNase plate assay for the ability of colonies to form colorless zones indicative of elevated nuclease levels in the clone (Fig. 1). However, of 6000 TcR colonies so tested in this initial cloning attempt, none gave zones larger than given by the recipient strain. To increase the proportion of transformant clones with recombinant plasmids, enrichment by chromo-

Fig. 1. DNase colony assay of S. pneumonia strains with and without recombinant plasmid pSM22. (Left) R6 (wild type); (center) 708 (end-l exo-2); (right) 708[pSM22]. Cells were plated in agar medium containing DNA and methyl green, and the plates were incubated at 37°C for 30 h. Colorless zones surrounding colonies (dark points) indicate degradation of DNA by nucleases that leak out of the colonies.

somal facilitation (BaIganesh and Lacks, 1984) was carried out. Inasmuch as homology with the chromosome facilitates plasmid establishment (Lopez et al., 1982), this procedure increases the ratio of recombinant plasmids to reconstituted vector plasmids. A crude plasmid extract of a batch culture of TcR transformants from the original transformation was used to transform the same recipient strain. This time three of 500 TcR colonies tested showed larger zones. The colonies were picked and all three contained plasmids of similar size. One was selected and called pSM22. It contains a 4.3-kb insert of chromosomal DNA. When transferred to other strains, pSM22 produced zones indicative of higher levels of nuclease. An example of the DNase colony assay is shown in Fig. 1, which compares wild-type colonies to those of a strain deficient in the major endonuclease and exonuclease in the presence and absence of pSM22. (b) Identi~eation cloned gene

of the enzyme

encoded by the

Crude extracts of various strains with or without pSM22 were analyzed for nuclease components by the method of Rosenthal and Lacks (1977). This procedure allows detection of enzyme activities after SDS-PA gel electrophoresis and determination of the size of the polypeptide responsible for the activity. Fig. 2 shows the results of such analyses. Strains

of S. p~eu~o~~ae that are wild-type for the major endonuclease give a strong nuclease band at 25 kDa (lanes 7,8 and 9). Strains carrying the leaky end-l mutation give a weak band at this position (lanes 1,2

--Pot-Exo

Fig. 2. Gel DNase assay of extracts from ceils with and without pSM22. Total cell extracts containing approximately 5Opg of protein were subjected to electrophoresis in the presence of SDS in a PA siab gel composed of 10% acrylamide, 0.54% bisacrylamide, and containing DNA at 30 pc&/ml. Lanes contained extracts from strains (of S.pneumoniue unless otherwise indicated) as follows: 1,708 (end-l exo-2); 2,708[pSM22]; 3,641 (end-1 noz-19 exo-2); 4, 641jpSM22j; 5, 577 (end-14); 6, 593 (end-l); 7, 642 (exo-5); 8, R6[pSM22]; 9, R6 (wild-type); 10, B. subtiffs CU403[pSM22]; 11, B. sub&s CU403. The gel was incubated for 62 h before staining. Migration of marker proteins is shown on the left margin [sizes in kDa (kd)]; positions of pneumococcal enzymes are shown on the right margin.

83

and 6). This band is very faint in mutants containing noz-19 (lanes 3 and 4), and it is absent in end-14 mutants (lane 5). Strains containing the wild-type gene for the major exonuclease give a band at 38 kDa (lanes 5,6,8 and 9). In all strains of S. pneumoniue that do not contain pSM22, a faint nuclease band was observed at 100 kDa (lanes 1,3,5-7 and 9). This activity apparently corresponds to the enzyme elicited by the cloned gene because a much stronger band was seen in this position when the strains carried pSM22 (lanes 2,4 and 8). As indicated below, this enzyme is a DNA polymerase-exonuclease. An additional faint band at 95 kDa may correspond to a less prominent polymerase-exonuclease different from the cloned enzyme. That pSM22 contains the structural gene for the polymerase-exonuclease is indicated by expression of the cloned nuclease activity in a foreign host. Inasmuch as the vector can function as a replicon in B. subtilis (Espinosa et al., 1982) it was possible to transfer pSM22 to that species. Only cells of strain CU403 of B. subtih that contained pSM22 showed the nuclease band at 100 kDa (lanes 10 and 11). The band just below the main endonuclease band, seen in lanes 7-9, was previously shown to derive from a proteolytic fragment of the endonuclease (Rosenthal and Lacks, 1980). Several bands, some diffuse, in the range from 40 to 90 kDa were visible when pSM22 was present (lanes 2,4 and 8). They may derive also from proteolytic fragments of the lOO-kDa polypeptide that retain nuclease activity. This is supported by the demonstration of nuclease activity in proteolytic fragments of the purified polymerase protein (not shown). Assays of DNA polymerase and nuclease activities in crude extracts of cells that do or do not carry pSM22 show that both activities are increased proportionally by a factor of approx. 15 in the presence of the plasmid (Table I). This was demonstrated in a strain of S. pneumoniae lacking the major endonuclease and exonuclease, in which the residual nuclease activity was predominantly due to a polymerase-exonuclease (Lacks, 1970). Work on purification of the protein encoded by the cloned gene (not shown) confirmed by comigration in two different fractionation systems the dual function of the enzyme, showed that it was made in tenfold higher amount in the presence of the plasmid, and demonstrated that it possessed both 3’ and 5’ exo-

TABLE

I

DNA polymerase Bacterial

and exonuclease

activity in total cell extracts

strain

Specific enzyme (units/mg

S. pneumoniae 641 [pSM22]

E. coli BL21DE3[pAR2192] E. coli BL21DE3[pSM23]

activity

protein) Polymerase

Exonuclease S. pneumoniae 641

a

5

12

44

218

16

8

212

1127

a Cultures of S.pneumoniae were grown in CH medium to A 650-- 0 8. Cultures ofE. coliwere grown in M9 medium containing 0.2 mg Ap/ml to A,, = 0.5, IPTG was added to 0.5 mM, and incubation was continued for 2 h, to give A,,, = 1.1. Cells from 1 liter were sedimented washed

by centrifugation

by suspension

3 mM mercaptoethanol, pH 7.6, centrifuged buffer.

Total

suspensions extracts,

again,

for 20 min at 20000 15 mg/ml,

samples

containing

10 mM Tris

MnCI,,

(Lacks

of extract

16pg

Labeled

Exonuclease

at 30°C

bovine

Tris

in 40~1 of a mixture

serum

albumin,

activity

were terminated

serum

nicked

with pancreatic

albumin,

4pg

salmon

DNase),

were terminated

DNA

15 PM each of dATP,

disks

in nmol/h

40 pg dGTP,

at 55 Ci/mmol.

and the assay mixtures

(Whatman

DEll),

and dried. Tritium

with an efficiency

system. Units correspond

of

IO mM

(previously

six times with 0.5 M Na2HP0,,

and once with ethanol,

scintillation

samples

containing 5 mM MgCl,,

sperm

by chilling,

to DEAE-paper

the disk was counted incorporated

of 40 ~1 of

by incubating

3 FM TTP, and 20 nCi of [3H]TTP

were then washed water,

of 22000 cpm/pg.

fluid was taken for scintillation

was determined

. HCI, pH 7.6, 3 mM mercaptoethanol,

Reactions

0.2pg

described

by addition

in 0.1 ml of a mixture

bovine

were applied

and

as previously

acid; after 20 min at 0°C the mixture was centri-

at 30°C

and dCTP,

of by

1976) from strain 470 grown with [3H-

reactions

Pol activity

extract

by the method was determined

. HCI, pH 7.6, 3 mM mercaptoethanol,

fuged and 60 ~1 ofthe supematant counting.

them of the

activity

and had a specific

3.5 % perchloric

the cell

Protein content

x g.

DNA was prepared

and Greenberg,

methyllthymidine

by passing

cell and clarifying

was determined

Exonuclease

incubating

[3H]DNA.

pressure

HCI,

in 10 ml of the same

were prepared

a French

et al. (1951).

2 mM

and suspended

cell extracts

approx.

Lowry

0.5 M NaCI,

0.1 mM EDTA, and 10 mM Tris

through

by centrifugation

for 10 min at 5000 x g,

in 40 ml of buffer containing

remaining

of 55%

to nucleotides

which

twice with on

in a liquid released

or

at 30°C.

nuclease activities. The cloned gene thus makes an enzyme similar to E. coli PolI and therefore will be designated polA of S. pneumoniae. The justification for calling the cloned gene product PolI is based on its correspondence with the

84

predominant DNA polymerase observed in extracts of S.pneumoniue (Lacks, 1970). This correspondence is supported by (1) a roughly proportional increase of polymerase and exonuclease activity in crude extracts of cells containing pSM22 (Table I); (2) similar migration in SDS-PA gel electrophoresis of the cloned exonuclease polypeptide and that attributed to the original polymerase-exonuclease (Fig. 2) and (3) identical behaviour in gel filtration and affinity chromatography for both DNA polymerase and exonuclease activities of the original and cloned polymerase-exonuclease proteins in their native form (not shown). Evidence for similarity of the PolI enzymes of S. pneumoniue and E. coli includes (1) roughly identical polypeptide size; (2) 5’ as well as 3’ exonuclease activity, and (3) susceptibility to proteolytic fragmentation to give separate polypeptides with exonuclease and polymerase activity (not shown). Although, as in other Gram-positive bacteria, chromosomal DNA synthesis in S.pneumoniue is sensitive to 6-(p-hydroxyphenylhydrazino)~uracil (Mejean and Claverys, 1984), the predominant polymerase-exonuclease of S. pneumoniae is not (not shown). Thus the cloned gene product cannot be the

2

I I

I

I

I

3 I

I

sole polymerase responsible for chromosomal DNA replication, and another enzyme, perhaps like PolIII of B. subtilis (Low et al., 1976), appears necessary. Unlike PolI of B. subtilis (Okazaki and Komberg, 1964), PolI of S. pneumoniue clearly has intrinsic exonuclease activity. The significance of the species differences in bacterial PolI enzymes remains to be established. (c) Characterization of the pneumococcal polA gene A restriction map of the recombinant plasmid pSM22 is shown in Fig. 3. Deletions were introduced into the plasmid by removing restriction fragments and religation. This showed that both the 3.2-kb HindIII-fragment extending into the left side and the 3.3-kb BstEII fragment extending into the right side of the chromosomal insert were essential for gene function. No increase of nuclease activity over the host-cell background was observed with these deleted plasmids. From the nuclease detection gels, the pneumococcal Poll appeared to have a mass of 100 kDa. This would require a coding capacity of 2.7 kb or 63% of the 4.3-kb chromosomal insert. To further characterize the pneumococcal poL4

4 I

I

5 I

I

6 I

I

I

7 I

8 I

I

I

9 kb I

1

pLSl L

pSM22 PO/A pAR2

h

192

pSM23

Fig. 3. Restriction pSM22,

original

chromosomal PAR2192

DNA segment

in the opposite

To construct

pSM23

line) chromosomal presumed Arrows

maps of recombinant recombinant

and pSM24,

DNA. Genetic direction

plasmids. containing

of pSM22

orientation.

origin of replication indicate

plasmid

inserted

To construct

Maps of circular a segment

into PAR2192 pSM22,5

1 pg each of pSM22 symbols:

in vectors;

of transcription.

polA, encodes amp, encodes

plasmids

of chromosomal as shown.

fig of EcoRI-cut

are drawn

in a linear format

pSM24

contains

the chromosomal

Poll of S. pneumoniue; tet, encodes Pr,,

at an EcoRI site.

into pLS 1. pSM23, segment

inserted

DNA from R6 was ligated with 1 pg of EcoRI-cut

cut with EcoRI + PstI and of PAR2192 /&lactamase;

beginning

DNA from S. pneumoniue inserted

p romoter

cut with EcoRI were ligated.

protein

for transcription

responsible

into pLS 1.

(Heavy

for Tc resistance;

ori,

by phage T7 RNA polymerase.

85

gene and to obtain hyperexpression of its enzyme product, the cloned insert was introduced into a T7 RNA polymerase promoter expression plasmid in E. coli (Studier and Moffatt, 1986) (see MATERIALS AND METHODS, section f). Fig. 3 shows the insertion of the EcoRI-fragment containing the pneumococcal poL4 gene into the EcoRI site of the pAR2 192 vector in one orientation to give pSM23. The pneumococcal fragment was inserted in the opposite orientation to give pSM24. Fig. 4 shows by the SDS-PA gel technique that the poZA nuclease was expressed in the presence of pSM23 but not pSM24. So transcription of the gene must proceed in the direction from the BglII site to the BamHI site. From the intensity of bands on the gel (Fig. 4) it was estimated that the E. coli T7 promoter system produced the pneumococcal PolI en-

I

2

Fig. 4. Nuclease ious plasmids.

3

5

4

activity

in extracts

Cell extracts

6

of bacteria

containing

protein were subjected

to electrophoresis

in a slab gel containing

a linear gradient

containing

indicated

var-

amounts

in the presence

of

of SDS

of 5-15 y0 PA and DNA

at 30 pg/ml. Lanes

1-3, extracts

from strains

lanes 4-6,

from strains

of E. coli. Cells of E. coli were

grown 60 pg;

extracts

with IPTG for 3 h. Lanes: 2,

641[pSM22],

BL21DE3[pAR2192], BL21DE3[pSM23], staining.

1,641 3,

(end-l

nor-19

exe-2),

641[pSM22],

6Oncg;

4,

1Opcg; 5, BL21DE3[pSM24],

1Opg;

6,

10 pg. The gel was incubated

Arrow indicates

exonuclease.

2Opg;

of S. pneumofliae;

position

of pneumococcal

for 64 h before polymerase-

zyme to a specific activity 50-fold greater than a normal strain of S. pneumoniae. This order of hyperexpression was observed also by assay of crude extracts (Table I). Cells of E. coli strain BL21DE3 containing either pSM23 or pSM24 were induced with IPTG for various periods with or without addition of rifampitin, and were then pulse-labeled with [ 35S]methionine. The pattern of labeled protein obtained is shown in Fig. 5. With pSM24 (lanes l-5) in the presence of rifampicin, which blocks transcription by the cellular RNA polymerase, the main protein made is the 29-kDa #&lactamase product of the amp gene in the vector (lane 5). A small polypeptide of 11 kDa is also made. With pSM23 (lanes 6-10) in addition to the 29-kDa /I-lactamase and its 31-kDa precursor, a band at 100 kDa that would correspond to the pneumococcal PolI protein is evident in the presence of rifampicin (lane 10). Just below the lOOkDa band is a more diffuse band that may correspond to incompletely synthesized polypeptides or degradation products of PolI. In the absence of rifampicin, labeled bands corresponding to cellular proteins are evident, but the products under T7 promoter control stand out. After 2 h of induction with IPTG, the rate of synthesis of such products was maximal (lane 8). At this time approx. 25% of the protein synthesized appear to be products of the cloned pneumococcal poL4 gene. The gel depicted as an autoradiogram in Fig. 5 was also stained to reveal protein. This allowed an estimate of the total amounts of various protein components present in E. coli after induction of cultures containing either pSM23 or pSM24. The lOO-kDa PolI polypeptide is clearly visible only with pSM23 (lanes 6-10). The amount observed after 3 h incubation with IPTG (lane 9) corresponds to approx. 5% of the total protein. The 11-kDa polypeptide specified by pSM24 is barely visible in lanes 3-5. It may be the product of a gene, possibly truncated in the insert, which is normally transcribed in the direction opposite to that for pol.4. Cloning of the pneumococcal polA gene will allow its mutagenesis in vitro and the insertion of mutated forms into the chromosome of S. pneumoniae either by ectopic integration (Mannarelli and Lacks, 1984) or by direct transformation. This should help determine whether the gene function is essential for cell viability, as is the case for the E. coli poL4 under

86

t 234567891011

12345678910 ’ \‘“l * - . 0%e ”

kd pal -+

‘W.l

^“” Stained

Autorad iogram Fig. 5. Synthesis

of proteins

BL21DE3[pSM24];

6-10,

albumin,

chicken

in E. coli carrying

extracts

ovalbumin,

bovine carbonic

anhydrase,

follows prior to pulse labeling with [%]methionine: lo,30

min, then 90 min with rifampicin

section f; total cell extracts The gel was stained on the right. Arrows

positions

the pool gene

containing

11, M, standard and chicken

proteins

egg lysozyme).

of S.pneumoniae.

(rabbit

muscle

Culture

samples

Lanes:

l-5,

phosphorylase

extracts

b, bovine

were incubated

of

serum

with IPTG

as

lanes 1 and 6,30 min; 2 and 7,60 min; 3 and 8, 120 min; 4 and 9, 180 min; 5 and

before labeling. Culture samples

were prepared

with Coomassie indicate

plasmids

of BL21DE3[pSM23];

for protein

and subjected

were treated

to electrophoresis

blue, dried, and exposed

as indicated

in the presence

in MATERIALS

of SDS in a 5-15%

to film for 48 h. Autoradiogram

AND METHODS, PA gradient

is on the left; gel stained

slab gel.

for protein

is

of polA and amp gene products.

certain conditions (Joyce and Grindley, 1984) and whether its PolI product is implicated in DNA repair processes. Application of the approach used for cloning the pneumococcal polA gene to genes encoding enzymes with nuclease activity in other species is feasible. For example, the ability of the DNase colony assay to detect the presence of endonuclease I in E. coli (Lacks, 1977) could serve as a basis for cloning the gene encoding that enzyme. Attempts to clone other E. coli genes could be made with strains lacking the endonuclease. Wild-type strains could be used in species, such as Haemophilus influenzae, that have intrinsically low levels of nuclease. With other species, such as B. subtilis, that have highly active nucleases (Rosenthal and Lacks, 1977), the use of mutants lacking those enzymes would be necessary, as was the case for S. pneumoniae.

ACKNOWLEDGEMENTS

We thank J.J. Dunn and F.W. Studier for providing us with the E. coli BL21DE3[pAR2192] expression system and information on its use prior to publication. Research at Brookhaven National Laboratory was under the auspices of the U.S. Department of Energy; research at the Centro de Investigaciones Biologicas was under the auspices of the Consejo Superior de Investigaciones Cientilicas. This work was supported in part by U.S. Public Health Service grants AI14885 and GM29721 to S.A.L., by C.S.I.C. grant 608-501 to M.E., and by acooperative research award CCB-8402-036 to M.E. and S.A.L. from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.

87

Lacks,

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T.S. and Lacks,

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Streptococcus pneumoniae and strategies recombinant Balganesh,

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Birnboim,

dure for screening

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