An in vitro coupled transcription-translation system from Staphylococcus aureus

Gene, 106 (1991) 29-34 ~9 1991 Elsevier Science

GENE

Publishers

B.V. All rights reserved.

29

0378-l 119/91/SO3.50

05098

An in vitro coupled transcription-translation (Cell-free

translation;

plasmids;

ribosomal

RNA methyltransferase;

Riaz Mahmood *, Patricia Compagnone-Post Department

system from StaphyZuc~ccu~ CEUWUS erythromycin

induction)

and Saleem A. Khan

of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 (U.S.A.)

Received by R.E. Yasbin: 16 January 1991 Revised/Accepted: 6 May/l7 May 1991 Received at publishers: 14 June 1991

SUMMARY

We have developed a Stuphylvcoccus aureus cell-free system that is capable of directing DNA-dependent synthesis of proteins. The staphylococcal plasmids pE194 and pSK265 were used to characterize this system. The in vitro system was found to direct the synthesis of the appropriate proteins predicted from the nucleotide sequence of the plasmids. As is the case in vivo, low levels of the inducer, erythromycin, promoted the synthesis of the pE194-encoded ribosomal RNA methyltransferase in the in vitro system.

INTRODUCTION

Cell-free systems have proven to be of great value in the study of gene regulation, DNA replication and recombination. Efficient in vitro translation systems have been developed from both prokaryotic and eukaryotic cells. The prokaryotic systems developed include those from Escherichia colt’and Bacillus subtilis (Chen and Zubay, 1983 ; Narayanan and Dubnau, 1987). A cell-free system from S. aureus describing the incorporation of labeled aa into total cellular proteins has also been reported (Mao, 1967). A number of plasmids of S. aureus have recently been the

subject of intensive investigations as models for DNA replication and gene expression in Gram + bacteria. Many plasmids from S. aureus such as pTl81, pE194 and pC194, have been shown to replicate in B. subtifis (te Riele et al., 1986). These plasmids have been extensively used for the cloning of various genes in these organisms. However, the use of an in vitro system from B. subtilis may not be suitable for studies on the regulation of many S. aureus genes. We have, therefore, developed an efficient cell-free system from S. aureus that carries out transcription and translation of input plasmid DNA.

RESULTS Correspondence

to; Dr. S.A. Khan,

and Biochemistry, Biomedical

University

Science

Tel. (412)648-9025; * Present Sciences,

address: Aligarh

Abbreviations: base pair(s);

of Pittsburgh Pittsburgh,

Department Muslim

of

dodecyl

School of Medicine,

Biochemistry,

University, acid(s);

Aligarh,

E. 1240

Er, erythromycin; nt, nucleotide(s): sulfate;

of

infusion

Life

broth;

Cm, chloramphenicol;

kb, kilobase r, ribosomal;

S., Sraphylococcus.

Faculty

202001 (India).

BHI, brain heart

AND DISCUSSION

Genetics

Fax (412)624-1401.

aa, amino

dithiothreitol;

of Molecular

PA 15261 (U.S.A.)

CAT, Cm acetyltransferase;

methyltransferase; sodium

Tower,

Department

bp,

DTT,

or 1000 bp; MTase, n, resistance;

SDS,

(a) In vitro translation of pE194 DNA and pSK265 DNA The well-characterized S. aureus plasmids pE194 and pSK26.5 (a derivative of pC194) were used to analyze the coupled transcription-translation system. Based on its nt sequence as well as its translation products in an in vitro system from B. subtifis (Horinouchi and Weisblum, 1982a; Narayanan and Dubnau, 1987), the pE 194 plasmid encodes four polypeptides of approx. 50 (El), 33 (E2), 29 (E3) and 25 (E4) kDa. The El polypeptide corresponds to the site-

30

’

A

234567

1

B

2

r.^Ci

kDa kDa 55

-

El-,

U’11

El+

E2+

-

55

-

43

-

36

-

29

-

18

E3-* E4+

Fig. 1. SDS-polyacrylamide-gel pEl94cop6

1982a). Plasmid Plasmid

analysis

is a high-copy-number pSK265

of the translation

derivative

is a derivative

RNase and proteinase

ether, and then precipitated

of pE194 and pSK265

on CsCl/ethidium

K were omitted.

multiple cloning sites (Horinouchi

bromide

gradients

The purified plasmids

with ethanol. The S-30 extracts

DNA. (A) Effect of template

DNA concentration.

and was used as the source of pE194 DNA in these studies (Horinouchi

of the CmR pC194 that carries

DNA was isolated by two centrifugations

with pancreatic

products

of the ErR pEl94

were prepared

and Weisblum,

(Clewell and Helinski,

were extracted

Plasmid

and Weisblum,

1982b; Jones and Khan,

1969). However,

1986).

steps involving treatment

three times with phenol, followed by three extractions

from the restriction-deficient

S. aureus strain RN4220 (Kreiswirth

with

et al., 1983).

Cells were grown at 37°C in 3.7% BHI with vigorous aeration. 10 ml of BHI were inoculated with S. (~ureus RN4220 and grown for 5 h. The cultures were diluted to 250 ml with BHI, grown overnight and then transferred to 4 liters of BHI. The cells were grown until the density reached the value of 410-470

Klett units. The extracts

cells were harvested

were prepared

by centrifugation

and washed

essentially

1 M KC], and then with 250 ml of buffer A containing 4°C and resuspended

in 30-35

added and the cell suspension

was incubated

Tris

as described

The mixture

was incubated

X.0114 mM Mg

acetate/60

pH 8.0/20 mM Mg

acetate

The pellet was recentrifuged

above. The top half of the supernatants mM phosphoenolpyruvate/7

at 37°C for 30 min to translate mM K. acetate/l

by Narayanan

and Dubnau

acetate

(1987) with some modifications.

at -70°C

acetate/50

until further

mRNA

mM DTT for 18 h at 4°C with one change

at 30000 x g

from both centrifugations

and mixed with 0.25 vol. of a solution containing

and then dialyzed

against

670 mM

ng per ml of pyruvate

a buffer containing

of buffer. This S-30 extract

at

(8-9 mg) was

Cell lysates were centrifuged

mM DTT/5.5 mM ATP/70 PM each of the 20 aa/

endogenous

use. The cells were thawed

mM KC]/1 mM DTT). Lysostaphin

at 30000 x g for 30 min. The supernatants was collected

The

pH 8.0/14 mM Mg acetate/ 1 mM DTT) containing

at 37°C for 45 min after which 50 ~1 of 0.5 M DTT was added.

was removed.

pH 8.0/20 mM Mg acetate/7

acetate

previously

50 mM KCI. The cell pellets were stored

ml of buffer B (10 mM Tris

for 30 min at 4°C and the supernatant were pooled and centrifuged

as described

first with 500 ml of buffer A (10 mM Tris

10 mM Tris

was divided into aliquots

kinase.

acetate

pH

and stored

in liquid nitrogen. The protein concentration of the extracts was determined by using a BioRad kit. The extracts can be stored for up to one month with a minimal loss of translation activity. In vitro transcription-translation reactions were carried out essentially as described by Narayanan and Dubnau (1987)

for B. subtilis. The reactions

were carried

K acetate/l.5 mM DTT/320 pg polyethylene CTP and UTP/ZO mM phosphoenolpyruvate/0.6 acid/ 11 nCi [“Slmethionine (added

(1087 Cijmmol)/200

last) were used. The reaction

precipitate

the proteins,

out in a final volume

of 20 pl containing

15 mM Mg. acetate

(unless

otherwise

stated)/45

PM

glycol 8000/5 pg E. cofi tRNA/lOO units of the RNase inhibitor RNasim2.3 mM ATP/0.6 mM each of GTP. pg each of pyridoxine HCl, NADP, flavin adenine dinucleotide and leucovorin/0.2 pg p-amino benzoic

mixtures

VM each of the other 19 aa. Unless otherwise were incubated

After 30 min incubation

stated,

for 60 min at 37°C. The reactions

on ice, the proteins

were pelleted by centrifugation

5 pg of plasmid

were stopped

DNA and 200 pg of S-30 extract

by the addition

of 200 ~1 of acetone

for 30 min at 4°C in a microcentrifuge.

to

The pellets

were washed with 1.2 ml of 80% acetone, dried under vacuum and resuspended in 30 ~1 of SDS-PAGE sample buffer. The samples were boiled and protein standards from Diversified Biotech were used as M, run on 12.55, polyacrylamide gels containing 0.1 y,, SDS (Laemmli, 1970). Prestained standards. The gels were dried and exposed to x-ray films (Kodak) at -70°C with intensifying screens, Lanes: 1, no DNA added; 2-4, I, 5 and 10 peg of pEl94cop6

DNA, respectively;

of incubation.

Transcription-translation

extract.

The reactions

of the protein

markers

lanes 5-7,

were incubated are indicated

1, 2.5 and 5 pg of pSK265

reactions

were carried

DNA, respectively.

out as described

for 0.5 (lane 1) or 1 h (lane 2). The positions in kDa

(B) In vitro translation

above using 5 pg of template of the pE194-encoded

products

obtained

after 0.5 and

Ih

pE194 DNA and 200 pg of the protein

El-E4

proteins

are indicated.

The positions

31 specific

recombinase

which is synthesized

constitutively,

and the E4 polypeptide corresponds to RepF which is the replication initiator protein of pE194 (Villafane et al., 1987). The E3 protein corresponds to the rRNA MTase encoded by the ermC gene. The E2 polypeptide corresponds to a readthrough fusion product of the E3 polypeptide that contains an extra 40 aa at its N-terminal end (Narayanan and Dubnau, 1987). This fusion protein is generated as a result of a frame shift during the translation of a leader peptide. Fig. 1A shows the translation products observed with pE194 DNA using cell-free extracts from S. uureus. Background protein synthesis in the absence of added template DNA is observed in many coupled transcriptiontranslation systems usually due to the presence of chromosomal DNA fragments in the S-30 extracts (Chen and Zubay, 1983; Narayanan and Dubnau, 1987). The level of background synthesis using the RN4220 extracts was very low (Fig. 1A, lane 1). This may be due to the mild enzymatic procedure involving lysostaphin used for the lysis of the S. clureus cells that may minimize degradation of the chromosomal DNA. Significant levels of translation products were obtained with 1 pg of pE194 DNA as template. There was a small increase in the levels of translation products when the amount of template DNA was increased to 5 pg (Fig. lA, lane 3) after which there was no further increase in protein synthesis. The results obtained here were similar to those observed for pE 194 in an in vitro system from B. subtilis (Narayanan and Dubnau, 1987). The levels of the El and E3 (rRNA MTase) polypeptides synthesized in the in vitro system were low, whereas the levels of E2 and E4 polypeptides were high. However, as described later, the synthesis of rRNA MTase was induced in vitro in the presence of low concentrations of the Er inducer. Although very little E4 protein (RepF) is synthesized in vivo in B. subtilis minicells carrying pE194 (Shivakumar et al., 1980a), a major band corresponding to the E4 protein was observed using the S. uureus in vitro system. It is likely that the RepF protein is synthesized constitutively in vitro. Similar results were obtained with the in vitro system from B. subtilis (Narayanan and Dubnau, 1987). The plasmid pSK265 is a derivative of pC194 that carries a CmR marker (Horinouchi and Weisblum, 1982; Jones and Khan, 1986). This plasmid was also used as a template in the in vitro transcriptiontranslation system (Fig. lA, lanes 5-7). A major product of approx. 25 kDa was observed that corresponds to CAT. Furthermore, a few smaller bands were observed that probably correspond to breakdown products or products of internal initiation. Two larger bands of approx. 36 and 33 kDa were also observed. One of these may correspond to the replication initiator protein of pC194, RepH (Dagert et al., 1984). The CmR gene present on pSK265 is induced at the posttranscriptional level in S. aureus (Byeon and Weisblum, 1984; Ambulos et al., 1984). However, very little

or no induction presence

of CAT

synthesis

was observed

of Cm in vitro (data not shown),

in the

suggesting

that

CAT is synthesized constitutively in this system. Protein synthesis was inhibited and totally abolished at a concentration of 50 pg Cm/ml (not shown). Higher levels of pEl94-encoded polypeptides were present after 1 h of incubation as compared to 30 min of incubation (Fig. 1B). Similar results were obtained when pSK265 DNA was used as the template (data not shown). (b) Effect of the addition of various levels of S-30 extract In vitro translation reactions were carried out using 5 pg of pE 194 DNA as the template and various amounts of the protein extract. The optimal level of the protein extract was generally found to be 200 pg (Fig. 2). However, it was variable for some extract preparations and the optimal amount had to be determined for each batch of the extract. (c) Effect of Mg’+ and Ca*+ concentrations Fig. 3 shows the effect of Mg2+ concentration on the synthesis of pE194 and pSK265-encoded polypeptides. The optimal concentration of Mg*+ was found to be between 10 and 15 mM for pE194 and 10 mM for pSK265. Translation was inhibited in the presence of Ca” (Fig. 3, lanes 6 and 11). As observed previously with the B. subtilis

123

4

kDa

El-s

-

43

-

36

-

18

E2+ E3+ E4+

Fig. 2. Translation amounts phoresis presence

of pE194cop6

of S-30 extract.

In vitro

DNA

in the presence

translation

reactions

of various and

electro-

were carried out as described in the legend to Fig. 1A in the of 5 pg of plasmid DNA and 15 mM Mg’ +. Lanes: 1-3, 100,

200 and 300 pg of S-30 extract, respectively; lane 4 contained 200 pg of the extract and no plasmid DNA. The El-E4 proteins are indicated.

32 3

12

4

5

6

7

891011 kDa

by Narayanan and Dubnau (1987), this is likely to be due to the effect of Mg2+ on the efficiency of readthrough during the translation of the leader peptide at a run of six

El-, -

36

A residues immediately leader peptide.

E2+ E3+ E4-, CAT-, -

Fig. 3. Effect pSK265

of Mg’+

(lanes 2-6)

200 pg of S-30 extract

concentration

or pE194cop6

4 and

on translation. (lanes 7-11)

in the presence

Mg’ + Lanes: 1, no DNA control; Mg”;

synthesis of E2 protein at higher Mg* + concentrations was accompanied by a reduction in the levels of rRNA methyltransferase (E3). Conversely, more E3 protein was synthesized at lower Mg2 + concentrations than E2. As postulated

9, 15 mM

Mg*+

Plasmid

was

of specified

16

(5 pg)

translated

with

concentrations

of

2 and 7,s mM Mg’ + ; 3 and 8, 10 mM

; 5 and 10,22.5 mM Mg’+ ; 6 and

11, 15 mM Mg’+ and 7.5 mM Ca”. The El-E4 indicated. Refer to Fig. 1A legend for methods.

proteins

and CAT are

system, the synthesis of E2 protein was more efficient at higher Mg2+ concentrations. As predicted, the increased

12345

preceding

the stop codon

of the

(d) Effect of preincubation of the S-30 extract In several prokaryotic coupled transcription-translation systems, the preparation of S-30 extracts involves a preincubation step (Chen and Zubay, 1983). This is done to remove the endogenous mRNA and also to reduce interference from the endogenous chromosomal DNA fragments that are usually present in small quantities in the S-30 extracts. Although we did not encounter much background synthesis using RN4220 extracts (Fig. l-3), we investigated the effect of preincubation of the extracts on in vitro translation. As shown in Fig. 4, higher levels of protein products were synthesized when the extracts were subjected to the preincubation step. This is probably due to the reduced interference from endogenous mRNA. Similar results were obtained when pSK265 DNA was used as the template (data not shown).

6

7

6

9 10

El-

kDa

-

55

-

43 36

E2+ E3-+ E4+

29

18

Fig. 4. Effect of preincubation

of S-30 extracts

on the efficiency

of in vitro translation.

Extracts

were prepared

as described

in the legend to Fig. IA; a

portion of the extract was preincubated for 30 min at 37°C while the other portion was not preincubated. Reactions and electrophoresis were carried out as described in the legend to Fig. IA using extracts that were either preincubated (lanes l-5) or not (lanes 6-10). Plasmid pE194 template DNA (5 pg) was used in all the samples except in lanes 1 and 6 which contained no DNA. The amounts of S-30 extract added were: 100 pg (lanes 2 and 7); 200 pg (lanes 3 and 8); 300 pg (lanes 4 and 9); 400 pg (lanes 5 and IO).

33 (e) Induction of pE194-encoded Er The Er resistance encoded

rRNA MTase synthesis by

the synthesis

by the pE194

posttranscriptionally in B. subtilis, we assume that this is also the case in S. aureus. The synthesis of El and E4 polypeptides was found to be inhibited at 0.05 and 0.25 pg

plasmid

is

inducible. The synthesis of the rRNA MTase encoded by the ermC gene has been shown to be induced at a posttranscriptional level by low levels of Er both in vivo and in vitro (Shivakumar et al., 1980b; Horinouchi and Weisblum, 1980; Narayanan and Dubnau, 1987). In vitro transcription-translation experiments were carried out in the presence of various concentrations of Er and the products analyzed by SDS-polyacrylamide-gel electrophoresis (Fig. 5). The synthesis of rRNA MTase (E3) was increased severalfold in the presence of 0.05 pg Er/ml. At a higher concentration of Er (0.25 pg/ml), translation of E3 was found to be inhibited. It is interesting to note that, while the synthesis of MTase increased upon treatment with Er, there was a concomitant decrease in the synthesis of the E2 protein (Fig. 5, lanes 3-5). This pattern was also observed earlier with B. subtilis (Narayanan and Dubnau, 1987) and shown to be due to the stalling of ribosomes in the leader region resulting in an increase in the synthesis of MTase and a decrease in the synthesis of E2. These results show that the in vitro system from S. aureuf responds to an inducer and faithfully reflects the results observed in vivo for the induction of pE194-encoded rRNA MTase. Since

12345

kDa

of the MTase has been shown to be regulated

Er/ml (Fig. 5, lanes 4 and 5). This effect has been observed previously with the B. subtilis system and is probably due to the inhibitory effect of Er on protein synthesis. The translation of all polypeptides was inhibited in the presence of rifampicin (Fig. 5, lane 2) demonstrating that the rifampicin-sensitive host RNA polymerase is involved in transcription in the in vitro system. (f) Conclusions We have described an in vitro coupled transcriptiontranslation system from S. oureus. This system was characterized by using two well-studied S. aureus plasmids, pE194 and pSK265 (a derivative of pC194), as templates. This system is efficient and directs the synthesis of polypeptides of appropriate sizes. The synthesis of pE194-encoded rRNA MTase was found to be inducible in vitro. The in vitro system is expected to be of value in studies on staphylococcal gene expression.

ACKNOWLEDGEMENTS

We thank the members of our laboratory for helpful discussions. This work was supported by National Institutes of Health grants AI19783 and GM31685. S.A.K. is the recipient of a National Institutes of Health Research Career Development Award.

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