Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids

153

Gene, 31 (1986) 153-163 Elsevicr GENE

1507

advantage of deletions enhancing chloramphenicol Streptococcus pneumoniae plasmids

Selective

(Recombinant

DNA;

instability;

regulation;

promoters;

acetyltransferase

translational

control;

gene expression

ribosome-binding

in

site)

Sara Ballester a) Paloma Lopez”, Juan C. Alonsob, Manuel Espinosa” and Sanford A. Lacks’* ” Instituto

de Inmunologia

Plonck-Institut

~1 Biologia

,ftir Molekulare

;Vtrtiorttrl Ltrboratory~, (Received

September

(Accepted

October

Microbiana.

Genetik,

Upton. NY I I973

C. S. I. C.,

Ihnestrasse

63:‘73-1000

(U.S.A.)

Velasquez. Be&

144, 28006

33 (Germarg~),

Madrid and

(Spain)

Tel. 2611800:

’ Biology, Deprrrtment.

” May-

Brookhuven

Tel. (5161282-3369

18th. 1985) 15th. 1985)

SUMMARY

A hybrid plasmid, pJS37, was made by combining pLS1, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJ S 140 (6.0 kb), from which pC 194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4 (3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLS 1. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.

* To whom

correspondence

and reprint

requests

should

be

INTRODUCTION

addressed. Abbreviations: gene encoding

bp, base pair(s); CAT, Cm acetyltransferase; CAT; ccc, covalently

forming

units;

ethidium

bromide:

open reading

Cm,

ribosome-binding Tc;

kb, kilobase

frame;

closed circular:

chloramphenicol; pair(s);

PA. polyacrylamide;

site: Tc. tetracvcline:

[ 1. designates

037X-l I19:86:$03.50

d,

plasmid-carrier

0

cfu, colony-

deletion;

EtdBr,

oc, open circular;

ORF,

R, resistance;

RBS,

tet.gene for resistance

state.

1986 Elsevier

cuf,

Science

Publishers

to

Naturally occurring plasmids are generally stable in their normal hosts. Two such examples are pMV158 (originating in Streptococcus agaluctiae; Burdett, 1980), or its shortened derivative pLS1 (Stassi et al., 198 I), which confers Tc resistance, and pC194 (originating in Staphylococcus aureus; Iordanescu, 1975), which confers Cm resistance. Hybrid

B.V. (Biomedical

Division)

154

plasmids,

however,

instability

may be segregational,

of the plasmid resulting Ehrlich

are frequently

unstable.

Such

from the cell, or it may be structural,

in deletion

of the plasmid

et al., 1982). In the present

(reviewed

(a) Bacterial strains and plasmids

work, we con-

of the cat genes that confer Cm resis-

tance in Gram-positive ble by growth results (Shaw,

bacteria

1983). The

induci-

mids. The streptococcal plasmid pLS1 (4.4 kb) contains a tet gene which, in S. pneumoniue, confers constitutive resistance to 1 ,rig Tc/ml. The staphylococcal plasmid pC I94 (2.9 kb), which confers indu-

synthesis

of the CAT enzyme

cible

cut gene

carried

B. subtilis by Ehrhch

pC194 has been extensively sequence

is generally

S. pneumoniue strain 708 (n-t-1 hex-4 end-l exo-2 m&4594) was used as recipient for transfer of plas-

levels of Cm, which

at subinhibitory

in increased

AND METHODS

in

structed hybrid plasmids of pLS 1 and pC 194 and examined their stability in S. pneumnnicle. Expression

MATERIALS

resulting in total loss

of this plasmid

studied.

by plasmid

The nucleotide

has been determined

Cm resistance

was introduced

into

(1977); it was prepared

(Cm”),

from

II. subtilis BD 170(trpC2 thr-5)[pC194].

(Hor-

inouchi and Weisblum, 1982), and its cut gene has been used as a model for studying the regulation of gene expression (Duvall et al., 1983; Ambulos et al., 1984; Schotell et al., 1984). Hybrid plasmids containing the pC194 rephcon have been made previously (Ehrlich et al., 1982). These constructs often were unstable. The structural instability of such hybrid plasmids has been extensively analyzed in Bacillus subtilis, with recent reports by Hahn and Dubnau (1985) and Alonso and Trautner (1985). However, no information about pC194-based instability in S. pneumoniue was available. In the present work, hybrid plasmids containing pC194 were shown to be structurally unstable in S. pneumoniue upon selection for CmR. The generation of plasmid deletions was highly reproducible and only a few deleted forms prevailed. These were isolated and characterized. They all conferred higher CAT activity than did pC194. DNA sequence analysis of the deleted plasmids revealed the molecular basis for the enhanced expression of the cut gene. The limited number of deleted forms that accumulated in this study can be attributed to the positive selective advantage afforded by the deletions. This result is in contrast to the multiplicity of deleted forms produced by negative selection against an undeleted plasmid (Espinosa et al., 1984; Lopez et al., 1984). However, results of both types can be embraced by a common theory of structural instability.

(b) Media for growth and selection To grow B. subtilis BD170[pC194], TY medium (Biswal et al., 1967) supplemented with 5 ng Cm/ml was used. Cultures of S. pneumoniue were grown in a casein hydrolysate-based medium previously described (Lacks, 1966). When specified, Tc was added at 1 klg/ml. Selection for CmK was usually at 2.5 ,LLgCm/ml for plasmids pJS37. pJS140, and pC 194 and at 5 Llg Cm/ml for plasmids pJS3 and pJS5. We determined that 0.1 pg Cm/ml did not alter the growth rate of S. pneumoniue 708, and we used this subinhibitory concentration to induce plasmid-containing cultures. Exponentially growing cultures received 0.1 kig Cm/ml, and after 20 min of incubation at 37°C an inhibitory concentration of the drug was added. (c) DNA preparation Alkaline lysates were prepared by a modification of the method of Birnboim and Doly (1979) as described previously (Stassi et al., 1981). Plasmid pC 194 was purified from B. subtifis BD 170[ pC 1941 by the CsCl-EtdBr centrifugation method described Canosi by et al. ( 1978). Plasmids from S. pneumoniue were purified following the method described by Currier and Nester (1976). To isolate ccc-monomer plasmids, alkaline lysates were fractionated by electrophoresis in 0.8:; agarose gels (Bio-Rad Ultra Pure DNA Grade). After staining with EtdBr, the gel segments containing the cccmonomers were excised and DNA was isolated by electroelution and further purification as described (Maniatis et al.. 1982).

155

(d) Plasmid transfer Competent

RESULTS

pneumococcal

cultures

(a) Construction

were treated

essentially as previously reported (Lopez et al., 1982). In some cases, transformants were induced by the addition completion

of 0.1 pg Cm/ml

sion. Drug-resistant

transformants

plates

containing

and/or

2.5 pg Cm/ml.

selective

To construct

media

with

1 pg Tc/ml

selection, content

analysis and ligation

one sample was treated of

of Cm. independent

with an inducing

We analyzed clones

the plasmid from

induced

(lanes l-9) and noninduced (lanes 10-15) Cm’ transformants by agarose gel electrophoresis (Fig. 1A). Both the hybrid plasmid and various derivatives of smaller size were present in most of the

Restriction enzymes were purchased from New England Biolabs or Boehringer Mannheim. Reactions were carried out according to the suppliers’ specifications. Agarose gel electrophoresis was used to analyze restriction products larger than 1.5 kb. Analysis of smaller restriction fragments was performed by electrophoresis in 5”/, PA gels, and bands were revealed either with EtdBr or with the Bio-Rad Silver Stain. To construct the hybrid plasmid pJS37, 1 pg each of pLS 1 and pC 194 were linearized with Hind111 and

clones tested. Twelve bands corresponding to the expected ccc-monomer position of the hybrid plasmid were excised from the gel. DNA was eluted from these gel segments and the presence of the hybrid plasmids was confirmed by restriction analysis. Only one of the two possible orientations was found. After digestion of this hybrid plasmid with Hind111 and religation, transformation gave only plasmids with the original orientation. Whether this was due to transcription of cat and tet genes in opposite directions or to another reason was not further investi-

ligated together with T4 DNA ligase as described (Espinosa et al., 1982). Plasmid pJS140 was constructed from pJS37 by digestion with EcoRI + AccI. Protruding ends were trimmed off with endonuclease Sl, and the DNA was subjected to bluntend ligation.

gated. The hybrid plasmid was called pJS37 (Fig. 2). The ccc-monomers of pJS37 eluted from the gel were used to transform S. pneumoniae. In this

A

C

B

I 2 3 4 5 6 7 8 9 IO II 12 13 14 I5

16 17 I8 19 20 21 __

Fig.

I, Plasmid content in alkaline lysates ofpneumococcal

and pCl94 strain

ligated together.

with the hybrid

transformation

Lanes

plasmid

1-9, induced;

pJS37.

Lanes

16-18,

as (B). Lane 28, linear DNA standards

for 17 h at 35 V. Bands were revealed

clones. (A) CmR clones obtained

lanes 10-15,

by staining

induced;

(MboI-cut

with EtdBr.

at

The ligation mixture was used

to transform S. pneumoniue and transformants were selected simultaneously for CmR and TcK. Prior to

in

concentration (e) Restriction

pC194 and pLS 1

with Hind111 and ligated together

equal concentrations.

expres-

were scored

a hybrid plasmid,

were linearized

for 20 min before the

of the 2-h period of phenotypic

of the hybrid plasmid pJS37

uninduced. lanes

after transformation

(B) CmR clones obtained 19-21,

uninduced.

For method

of Cm induction

8.3

___

6.0

-

pJS37

of strain 708 with pLS

after transformation

(C) TcK clones

phage T7 DNA), in kb. Electrophoresis

21.4

obtained

in 0.8 “o agarose

see legend to Fig. 3.

of the same from the same was conducted

I

156

remains undetermined.

In the absence of Cm, pC194

showed a rate of loss of about 27; per generation S. pneumoniae. the absence generations.

In contrast,

in

pJS37 was fully stable in

of either Tc or Cm for at least 120 Inasmuch as plasmid pLS1 is fully

stable in S. pneumoniue, the hybrid pJS37 appears to use the replication (b) Structural

Results

mechanism

instability

obtained

(Fig. 1) indicated plasmids

of pLS1.

of pJS37

the transfer

of pJS37

that the accumulation

from

of deleted

was related

to the presence

of Cm and

independent of induction. In addition, because the original hybrid was also observed in some clones, the Fig. 2. Physical maps ofpJS37 open segment respectively.

Arrows

orientations.

Identified

Greenberg

Weisblum.

and ofpJS 140. Solid segment and

of the circle indicate

and

indtcate

functions

M.E.,

unpublished),

Alonso, G. Morelli and H. Leonhard. IS truncated.

ordered

Filled portions

ers. Only relevant

species, monomeric transfer the plasmid Again, transformants

CAT

(Hortnouchi

for replication unpublished). by decreasmg

in ORFs represent

restriction

5’ to 3’

arc: TET (TcR; S.A.L.. P.L., B.

1982) and REP (protein

arc labelled alphabetically,

pLS1 and pC194 moieties,

ORFs and their respective

and

of pC194; J. Other ORFs sizes; ORF-C

putative

promot-

sites arc depicted.

deleted forms must have been generated after the establishment of pJS37. To confirm these suppositions, S. pneumoniae 708[pJS37], which was previously selected in the presence of Tc (Fig. lC, lane 22) was subjected to growth in the presence of Cm. A culture of this strain was grown without antibiotic until the middle of exponential phase. Then, portions of the culture were exposed to a high dose of Cm (10 LLg/ml), without (Fig. 3A) or with (Fig. 3B) prior induction. Viability in the uninduced culture decreased by SO’

i5 the avcrqe

0. I?

+ pJS4

(lY75).

dleterlllitl;ttiotls.

0.06

I

and

folIoned

3s nmol

Now

p1.s

wcrc

b! 10 cyclca

by the method

is ckprcsscd

the HCI,

supernatant

of Shaw

to

steps were

buds

centrifugation

pc: I Y‘l

pI.SI

Tris

Sigma)

Cells uere disrupted

was detcrmlned

ct al. (lY5 I). One

least three indcpcndent

and

50 mM

and kept at -20 ‘C. CAT activity

b> the wtorimetric

contwt

induced

incubated

subsequent

(30 s~cyclc).

allquot$

pressure

One wit\

(2X)-3OO,um,

by tow-speed

at I1000

plasmids

ccntrifugation.

All

buds

I :2 (n/v).

debris wcrc eliminated

divided

b)

?-mercaptocthanot.

added at ;I ratio of

vnrioua sctectivc

were

in 7 ml of chilled

at 0 C. Glass

centrifugatlon

subcultures

harvested

~vcre huspendcd

pcrformcd

708 harboring

at 37 C without

of Cm of at

CAT levels conferred

by the

by pJS5 were indeed

The nucleotide sequence in the vicinity of the deletions of the pJS3 and pJS5 was determined for two reasons: (1) to gain insight into the mechanism that generated the deletions, and (2) to determine the effects of the deletions on control signals that regulate cut gene expression. Sequences from the deleted plasmids were compared to those of the parental plasmids pC194 (Horinouchi and Wcisblum, 1982) and pLS1 (SAL., P.L., B. Greenberg and M.E., unpublished) and the results are summarized in Fig. 8. In the case of pJ S3, the deletion of pJ S37 occurred between directly repeated sequences 6 bp long. The effect of the deletion was to eliminate the tet structural gene and to bring its putative promoter to within 200 bp of the putative cat gene promoter. Transcription from the tet promoter presumably raised the level of cur gene expression without affecting its inducibility. In the case of pJS.5, the deletion of pJS 140 again occurred between directly repeated sequences, which this time were 9 bp long. The deletion replaced the cat promoter with the tet promoter. Inasmuch as the deletion end-point was within the putative ribosome binding sequence of the cut gene (Horinouchi and Weisblum, 1982) it removed the proximal sequence of DNA that is complementary to the ribosome binding sequence, as shown by the hairpin structure of Fig. 8. In the mRNA transcribed from the parental plasmids or pJS3, this sequence presumably sequesters the RBS in the absence of Cm and prevents translation of CAT (Horinouchi and Weisblum, 1982; Duvall et al., 1983). The presence

161

T

A

G

T

A

T A

T C A

A C

T-AA n’=c,

P-CAT V’ (~~16bp~GTTTTAATCAAGJJJAGGAGGAGG---ATCAAG~l6Ybp v-v -35 -10

w

6bp

Fig. 8. Partial DNA sequence

ofhybrid

from pL.Sl and pC194 arc indicated. lie within 9-bp repeats

indicated

-35 and --IO polymerase the cur gene IS underlined, purified intestinal restriction

by treatment phosphatase enzyme,

of this sequence

Deletion

sites boxed.

pJS37 and pJS140 showing deletion end-points end-points

and gel filtration.

and labelled DNA sequences

at their

for pJS3 lie within 6-bp repeats

Putative

Inverted

and the rest of the putative

with RNase

promoters

indicate

with restriction

5’-ends

with [y-“P]ATP

were determined

by the chemical

in pJS3 fits with the inducibility

from the tet promoter,

compared

cat promoter, as well as to the elimination lation controls.

possible

RBS is boxed. For nucleotide

After cleavage

of

to the

of trans-

DISCUSSION

This work describes the positive selection in bacteria of specifically deleted plasmid forms as a consequence of an environmental change, namely, the addition of Cm to the culture medium. In the course of the development of new plasmid vehicles for the pneumococcal cloning system, we introduced into S. pneumoniae plasmid pC194 and the hybrid plasmids pJS37 and pJS140 constructed in vitro from pC194 and pLS 1. All three plasmids conferred Cm resistance to S. pneumoniae. The hybrid plasmid pC194-pE194 (termed pSA5700) was previously introduced into S. pneumoniae by selection for

and regulatory

indicated

signals. Portions

by larger lettering;

transcription sequence

enzymes,

and polynucleotide method

of Maxam

start points. The start codon

determination, DNA fragments

kinase. and Gilbert

derived

those for pJS5

P-TET and P-CAT. with

for the rer and cur genes are designated

solid triangles

CAT expression conferred by that plasmid (Table II). Inasmuch as the control is at the translational level, CAT synthesis specified by the supposedly greater amounts of mRNA transcribed from the ter promoter would still be subject to induction by Cm. The absence of the sequestering sequence in pJ S5 fits with the constitutive CAT expression conferred by that plasmid (Table II). The particularly high level of expression in this case could be due to enhanced transcription

bp-rG-

-10

6bp

Ybp

plasmids

by italic lettering.

binding

,7bpaGAAGAA:cl2 -35

After

plasmids

were treated

subcutting

01

were further with calf

with a second

(1980).

erythromycin resistance (Barany et al., 1982). Both pJS37 and pJS140 were structurally and segregationally stable when selected in Tc or grown in the absence of drug. When grown in the presence of Cm, they gave rise to deleted plasmids with three forms - pJ S3, pJS4 and pJS5 - predominating (Fig. 5). Of these, pJ S4 was not an independent replicon, and it was not fully characterized. Plasmids pJS3 and pJS5, however, conferred much higher levels of Cm resistance than the original plasmids (Fig. 7) by effecting an increased production of CAT (Table II). The deletions that produced pJS3 and pJS5 occurred after establishment of the intact hybrid plasmids. This was evident both from the presence of mixed plasmid populations in transformed clones (Fig. 1A) and from the secondary occurrence of deletions when TcR transformants were grown with Cm (Fig. 5). However, it is possible that some deletions (cf. Fig. lA, lane 4) occurred during plasmid establishment, perhaps giving rise to the greater variety of deletions observed after transformation. DNA sequence determinations showed that the deletions occurred between short, direct repeats (Fig. 8), presumably by a homology-dependent recombinatory process. This mechanism for generating deletions is common in bacteria, and it is in one of two modes for generating plasmid deletions in B. suhtifis (Lopez et al., 1984). Such generation of deletions presumably occurs spontaneously and randomly in the plasmid population but at a low

162

frequency. The deleted forms would remain inconspicuous if they lacked a selective advantage, either of a positive sort, due to enhanced deleted plasmid, a detrimental case

or of a negative

feature of the original plasmid.

of ma/ recombinant

(Lopez

capabilities

plasmids

et al., 1984), selection between

the two cases

in the

a mystery. However, comparison of the levels of CAT elicited by pJS3 and pJS5

(Table II) shows that Cm only partially removes the translational

inhibition

It appears positive

that

One observable

creating

problems

nant plasmids rearrangements

gives rise to a few par-

rial evolution.

whereas

positive

advantage

sort can

of deletions

is that negative

gives rise to a multitude

of CAT synthesis.

selective

or negative

accumulation

of deleted forms

selection

selection

In the

in B. s&tilis

was negative;

present case, selection was positive. difference

of the

sort, due to loss of

remains induced

of either

result

a

in the rapid

in plasmids.

In addition

for the propagation

to

of recombi-

in genetic engineering,

such plasmid

may play an important

role in bacte-

ticular forms. Although selection by Cm was positive, the difference in behavior between pJS37 and pJS 140 suggests that the presence of the pC194 replication functions in the former may have added a small negative component to the selective advantage of pJS3. Thus, deleted forms of pJS37 accumulated at a lower Cm concentration than did deleted forms of pJS140 (Fig. 5), and the deleted product in the former case, pJS3, conferred less uninduced Cm resistance than did pJS5, in the latter case (Fig. 7). The nature of the deletions in pJ S3 and pJ SS and their effect on CAT production gave considerable insight into the transcriptional and translational regulation of cat gene expression. Placement of the tet gene promoter proximal to the cat gene promoter in pJS3 increased uninduced CAT production IOOfold (Table II). This presumably resulted from additional transcription from the tet promoter. Inasmuch as CAT synthesis was further increased by induction, the tet promoter transcripts were still subject to translational control. So the tet promoter must initiate transcripts at a much higher rate than the cut promoter. That the cut promoter is rather weak is shown also by the IO-fold lower induced level of CAT with pJS37 as compared to pJS3 (Table II). It would be desirable to confirm these deductions by direct measurement of transcript RNAs. In pJS5 the cat promoter is replaced entirely by the zet promoter. Furthermore, loss of the sequence that could sequester the ribosome binding site in the normal transcript RNA rendered CAT production constitutive. This supports the translational control mechanism proposed by Horinouchi and Weisblum (1982) and demonstrated, also, by others (Duvall et al., 1983; Ambulos et al., 1984; Schotell et al., 1984). The manner by which Cm causes induction

ACKNOWLEDGEMENTS

This research was supported by Grant 608/501 from the C.S.I.C., Spain. The exchange program between The Max-Planck-Gesellschaft and C.S.I.C. supported J.C.A. in Spain. In this connection WC appreciate the support and encouragement of Dr. Thomas Trautner. Work by S.A.L. was under the of U.S.D.O.E. auspices U.S.P.H.S. Grant AI14885.

and

supported

by

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