Gene, 98 (1991) 45S.52

45

Elsevier

GENE

0383 1

Gene expression in Deinococcus DNA;

(Recombinant

radiodurans

Deinococcaceae;

gene fusions;

promoter

probes;

transformation;

duplication

insertion;

direct in-

sertion ; shuttle plasmids)

Michael

D. Smith, C. Ian Masters,

Department

Eileen Lennon, Leslie B. McNeil

and Kenneth W. Minton

ofPathology, F.E. Hebert School of Medicine, Un$ormed Services University of the Health Sciences, Bethesda, MD 20814-4799

(U.S.A.) Received by R.E. Yasbin: 30 March Revised: 16 July 1990 Accepted: 31 August 1990

1990

SUMMARY

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUEl0 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUEl0 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUEl0 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5’-truncated 1acZ. Three fusions that produced high levels of BGal in D. radiodurans were introduced into E. coli, but /?Gal was produced in only one. The results demonstrate that the E. coligenes cat, tet and IacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.

INTRODlJCTlON

The eubacterial family Deinococcaceae is only distantly related to any other group of bacteria. Deinococcus species are as remote from Bacillus or Streptococcus species as they Correspondence

10: Any one of the authors,

Department

of Pathology,

Services University ofthe Health Sciences, 430 I Jones Bridge MD 20814-4799 (U.S.A.) Tel. (202)295-3476; Bethesda,

Uniformed Road,

Ap, ampicillin;

fiGal, P-galactosidase;

cat, gene encoding

CmR; cfu, colony-forming units; Cm, chloramphenicol; D., Deinococcus; IPTG, isopropyl-fi-n-thiogalactopyranoside; kb, kilobase or 1000 bp;

0378-I

I lY:Y1:$03.50

Km, kanamycin;

61 IYY 1 Elsewer

Science

Publishers

B.V.

(Biomedical

Dlvislon)

lacZ,5’-truncated

MU, methyl-umbelliferone; ranoside; nt, nucleotide(s); a plasmid;

Fax (202)295-1640. Abbreviations:

are from E. coli (Brooks et al., 1980). Two D. radiodurans strains are naturally transformable, Rl and Sark (Tigari and Mosely, 1980), and this property has facilitated efforts at genetic characterization. We reported the introduction and expression of two drug-resistance determinants in /acZ; LB, Luria-Bertani

(medium);

MUG, methyl-umbelliferyl-~-D-galactopy0, denotes an insertion in a chromosome

or

‘, resistance/resistant;

‘, sensitive/sensitivity; Tc, tetracycline; TGY, tryptone/glucose/yeast extract (medium); Tn, transposon; fsp, transcription start point(s); wt, wild type; XGal, 5-bromo-4-chloro-3indolyl-p-D-galactopyranoside;

[ 1. denotes plasmid-carrier

‘, gene is truncated state;

::. novel joint.

at the indicated

side;

46 D. rudioduruns by transformation

using methods that produce duplication insertion (Smith et al., 1988). The resistance determinants (on E. coli plasmids) were covalently linked to D. radioduruns chromosomal fragments prior to transformation. Transformants contained the E. coli plasTABLE

I

Bacterial Strain

mid in the host chromosome flanked by direct repeats of host sequences. The presence of flanking repeats facilitates gene amplification under selective pressure (Peterson and Rownd, 1983; Janniere et al., 1985). Insertion by this means of up/z.4 (KmR) or cut (CmR) always resulted in high

strains

and plasmids Source

Description

or plasmid

(reference)

D. radiodurans n RI

wt[pS16]

Lll4

SarkQpEL

Moseley (Brooks This study

I7

L115

SarkQpELlX

This study

Lllh

SarkQpELlY

This study

Sark

wt[pUElO.

Moseley

pUE1 l]

et al., 1981)

(Brooks

et al., IYXI)

E. coli’ F

DH5,

recA 1 endA 1 hsdR17(r,

(+8OdlurZdMlS)d(krcZYA-argF)U169

Bethesda

,m; )

Research

Laboratories

Plasmids’

pUEl I

45 kb; Sark cryptic

plasmid

(Mackay

et al., 1985)

pUEl0

37 kb; Sark cryptic

plasmid

(Mackay

et al., 1985)

pELl

cur; P15A replicon

pEL2

cat; pELI

pEL17

aphA; pRF1::4.7-kb

BclI/BamHI-A::

12.kb Sark chromosomal

DNA

(Smith

et al., 1988)

(Smith

et al., 1988)

Sark chromosomal

DNA

This study

pELIX

uphA; pRF2:: 4.9-kb Sark chromosomal

DNA

This study

pEL1Y

uphA; pRF2::

pKK175-6 pKK232-8

ApR; promoterless ApR; promoterless

pRFl.

aphA; IucZ gene lacking

pRF2.

reading

and pRF3

12.9-kb Sark chromosomal let gene cat gene promoter

km, promoterless

pBD64

car(pClY4),

pA3

pIJEll

pMK20

uphA; ColEl replicon uphA; fragment of RI chromosomal

::pBD64

site; three plasmids

for three

(Brosius,

1984)

Pharmacia

(Brosius,

1984)

Friedberg

(Robinson

et al., 1986)

Lovett (Ambulos

Helinski DNA inserted

(Smith

in pMK20

I 1 Hind111 : :pEL1 Hind111

pS

pUCl9

pSl6

60 kb; Rl cryptic

ps17

pEL2

ps30”

pKK232-8

EcoRI-A

ps31 (I

pKK232-8

HindIII::pUElO

pS3Y”

pKK232-8

SmaI::pUElO

pS4Y Cl

pKK175-6::pS30

pUClY

1ucI’OPZ’

et al., 1986)

(Gryczan et al., 1980) This study

replicon

TuqI-A

pSl5

(Kahn

et al

1979)

et al., 1988)

This study

I EcoRIiHindIII-A

EcoRIIHindIII-A::pUEI

(Smith

et al., 1989a)

(Smith et al., 1988)

plasmid

:: Tn mini-kan

This study and -B::pUEIO

This study

EcoRI-A

This study

HindIII-A

This study

SmaI-A

This study

EcoRI-A

amp pMB9 replicon

“ D. rudioduruns strains were grown with aeration of a plasmid that contains both chromosomal instance, D. rudioduruns Sark derivative Lll4 previously

start

cut-86 gene; ptiB 110 replicon

km; pUB110

ps13

described

and translation

Pharmacia

frames

pPL703

PSI1

This study

DNA

(Yanisch-Perron

at 32°C in TGY broth, and plated on TGY agar. Several strains may be thought

et al., 1985)

of has having insertions

and heterologous sequences and are so designated according to convention (Novick et al., 1976). For has an insertion of pEL17 and so is described as SarkQpEL17. Transformation of D. rudioduruns was

(Smith et al., 1988). Briefly, 0.1 ml (approx.

5

x

lo6 cells) of competent

D. rudiodurans recipients

were transformed

with 1 pg DNA,

diluted tenfold with TGY and incubated at 32°C for 90 min to permit expression before plating on selective agar. Selective drug concentrations in agar plates were 8 Fg Km/ml, 3 Fg Tc/ml and 3 pg Cm/ml for D. rudioduruns RI. D. radioduruns strain Sark was selected with 5 pg Km/ml. Selective plates were incubated

for four days at 32’C

’ E. co/i was grown with aeration

before final colony counts

in LB broth at 37°C

were 30 pg Km/ml, 25 pg Cm/ml, 30 gg/ml c Plasmids are described in terms ofparental EcoRI-A

and -B ::pUElO

EcoRI-A”

indicates

were recorded.

and plated on LB agar. Transformation

Ap, and 15 pg Tc/‘ml. restriction fragments as suggested that pS30 contains

Thus.

Sark[pS30]

carries

pS30 and pUEI1.

(Novick et al., 1976). For example,

the two largest (A and B) EcoRI fragments

fragment of pUEI0, where symbol :: indicates linkage. pi In Sark. pUEl0 was replaced by the CmR pUElO-derivatives introduced.

was by the CaClz method.

Rl[pS30]

the description

ofpKK232-8

pS30, pS31, or pS39, or the TcR pUElO-derivative carries

pS30 and pS16.

Selective drug concentrations ofpS30

as “pKK232.8

joined to the largest (A) EwRI pS4Y when these plasmids

were

levels of gene amplification, up to 50 tandem chromosomal copies of the E. coli plasmid (which contained the drugresistance originally

determinant) plus the flanking host sequence present on the transforming construct (Smith

et al., 1988). The extensive

gene

amplification

observed

following

duplication insertion of aphA or cat suggests that these genes were poorly expressed in D. radioduruns and that numerous copies were required for the drug-resistant phenotype. Further evidence of inefficient expression of E. coli promoters in D. radiodurans was provided during experiments employing direct insertion, instead of duplication insertion, of the aphA gene into the natural plasmids, pUEl0 and pUE11, of D. radiodurans strain Sark. In the absence of flanking direct repeats, as in the case of direct insertion, amplification does not occur (Anderson and Roth, 1977; Peterson and Rownd, 1983; Janniere et al., 1985). Transformation by direct insertion into these lowcopy-number cryptic plasmids was accomplished only when Deinococcus sequences were ligated in vitro to the uphA-containing E. coliplasmid in such a way that D. radiodurans sequences were immediately upstream from aphA (Smith et al., 1989a). The apparent need for particular upstream D. radiodurans sequences, or, lacking such sequences, dramatic amplification of the heterologous drug-resistance determinant, prompted the studies described here in which we sought to determine if Deinococcus promoters were required for efficient expression of heterologous drug-resistance determinants in Deinococcus, what genes could be so expressed, and whether Deinococcus sequences could promote expression in E. coli.

RESULTS

AND DISCUSSION

(a) The Escherichia coli promoter probes pKK232-8 and pKK175-6 The pBR322 derivative, pKK232-8, contains a promoterless cat gene and a similar plasmid, pKK175-6, contains a promoterless tet gene. In both constructions the promoterless gene is preceded by a multiple cloning site and transcriptional terminators are present immediately upstream from the multiple cloning site, and immediately downstream from the cat or tet gene. Insertion of DNA with promoter activity into the multiple cloning site causes expression of the cut or tet genes in E. coli (Brosius, 1984). We have found that insertion of Deinococcus DNA into these vectors causes expression of cat or tet in Deinococcus, as described in the following paragraphs.

(b) Insertion of promoter probe plasmids into the Deinococcus rudiodurans chromosome We previously demonstrated that duplication insertion of pMK20 and pEL1 (not promoter probe plasmids) resulted in remarkably high levels of gene amplification that were easily detected by gel electrophoresis of restriction-cleaved genomic DNA (Smith et al., 1988). pKK232-8 was cleaved at the multicloning site with BamHI and ligated with a partial Sau3A digest of chromosomal DNA from Rl or Sark. The ligation mixtures were used to transform Rl or Sark recipients to CmR. Genomic DNA from several CmK transformants was analyzed by restriction endonuclease digestion and gel electrophoresis. The results showed that duplication insertion of pKK232-8 resulted in D. radioduruns transformants that were not significantly amplified for the insertion (not shown). Transformation of wt RI with genomic DNA from RlSZpKK232-8 CmR strains yielded duplication insertion transformants at a very high frequency ( 105-10h CmR transformants per 5 x 10’ recipients) compared to transformations with genomic DNA from RlOpMK20 or RlS2pELl strains which produced low yields (lo3 - lo4 KmR or CmR transformants per 5 x lo7 recipients). Several reasons for this can be advanced, but one possibility is that RlQpKK232-8 transformants were easier to select because they did not require extensive amplification for the expression of drug resistance. Since cat was effective when introduced on promoter probe pKK232-8, we inquired whether the cat-86 promoter probe pPL703 from Bacillus would behave similarly. pPL703 is composed of the replicon and kan gene from PUB 110, and a promoterless cat-86 gene from B. pumilus. The cut-86 promoter is replaced with a multicloning site (Harwood et al., 1983; Mongkolsuk et al., 1984; McKenzie et al., 1986; Ambulos et al., 1986). pPL703 was cleaved in the multicloning site with EcoRI, BarnHI, SalI, or PstI, ligated with similarly cleaved Sark chromosomal DNA and the ligation mixtures used to transform Sark. Only KmR, and no CmR transformants were recovered, indicating lack of expression of the cat-86 gene. Southern blotting of chroshowed that mosomal DNA of KmR transformants pPL703 was flanked by direct repeats of D. radiodurans chromosomal DNA, and both pPL703 and the chromosoma1 fragments were highly amplified (not shown). This result suggests that the pPL703 kan gene was not efficiently expressed in D. radiodurans. The failure to recover any CmK transformants by duplication insertion of pP703 is in contrast to the efficient duplication insertion of pKK232-8. This difference may reflect failure of D. radiodurans to translate the unusual cat-86 leader sequence and/or

48 recognize

the uncommon

gent (Mongkolsuk investigated.

UUG

start codon of the cat-86

et al.. 1984). However,

this was not

(c) The lian gene is not expressed in low copy number Although the cat86 gene was not expressed, as noted above, KmK resulted from duplication insertion of the kern gene in pPL703. This observation is novel, as previously we had discovered that only two heterologous drug-resistance determinants, crphA and cat, expressed drug resistance in n. radiodurans. Since the pPL703 kan gene was expressed in Sark when introduced as a duplication insertion and amplified, we investigated whether this gene would be expressed when introduced as a direct insertion and consequently present in low copy number. A direct insertion into Sark plasmid pUEl1 was achieved as follows: pS15 is a clone of the largest EcoRI-Hind111 fragment of pUEl1 in pUC19 (Smith et al., 1989a). The TuqI-A fragment of pBD64, which contains the kan gene as well as the Staph_vloco~‘cus uureus cat(pC194) gene (Gryczan et al.. 1980; Sadaie et al., 1980; Matsumura et al., 1984; Horinouchi and Weisblum, 1982; McKenzie et al., 1986), was ligated with pS 15 that had been partially digested with TuqI, and the ligation mixture used to transform D. radioduruns Sark recipients. No KmR or CmR transformants were detected by plating on selective agar. Transformant colonies plated on nonselective agar were screened by colony blot, and a Sark isolate that contained the pBD64 TuqI-A fragment was identified. It was as sensitive as the parental Sark strain to Km and Cm. The isolate contained pA3 (pUE11 : : pBD64 TaqI-A), which was slightly larger than pUEl1, and hybridized to pBD64. It contained a 4-kb TrrqI fragment which comigrated with the TaqI-A fragment of pBD64. The wt Sark plasmid DNA did not contain this fragment. An attempt was made to select for spontaneous mutants in which the CmR or KmR determinants were expressed (for example, by an upstream insertion sequence in pA3), but Sark[pA3] and wt Sark produced the same frequencies of CmR and KmR mutants when 10’ cfu were plated on selective agar. The foregoing results indicate that the 5’. uureus kan gene is expressed by D. radiodurans Sark when introduced by duplication insertion and amplified, but not expressed when introduced as a direct insertion at a specific site in the low-copy-number plasmid pUEl1. In addition, the c~lb(pC194) gene present on the pBD64 TaqI-A fragment was not expressed. These observations are similar to prior results on the E. coli aphA gene, in which it was found that aphA was expressed as an amplified duplication insertion, but not as a direct insertion in pUEl1, unless it was placed immediately downstream from D. rudiodurans-promothg sequences (Smith et al.. 1988; 1989a).

H

ps31

ps39

ps30

ps49

pEL18

B”yL

__Bg’

pEL17

89 sa pEL19

s H S,ESrn

Xh Srn Xh Fig

Bg sa

1

.-’

d

‘lacz

Fig. 1. Plasmids inserted

constructions.

into pS 1 1 by in vitro ligation, producing

from pEL2

by insertion

by the method

pUEI0,

shown in reduced

1989a). The central additional

fragment

pKK232-8

and

scale, is as previously is expanded

cleavage

were not determined

pruc-transposon

were ligated pKKl75-6,

determined

and mapped portion

greater

fusions

vectors.

See section

hatched

(narrow),

transposon shaded,

producing

pS31,

pS39,

pS30

and

pS49,

that contain

of Sark genes with the ‘IrccZ in the E. Ai f. Segments:

pMK20;

pRF

D. radiodurcms sequences;

blackened,

striped (wide), pEL1; striped (narrow,), pruc-

open,

pRF. B, BarnHI;

pKK232-8;

hatched

(wide),

pKK 175-6;

Ba, BunII; Bc, EC/I: B/Be, BarnHI-Bc(I fusion; E, EcoRI; H, HijldIII; when harvested from dtrnl ’

methylation,

when harvested

but is cleaved

M, A4luI; N, NruI;

S, SrrlI; Sm, SmuI;

(d) Introduction

of the probes

in E. coli and D. rcrdiodurcms strains

Bg, BglII; BiSa, BglII-Sau3A CltrI site that is not cleaved

EcoRV;

portion

in vitro with the E. co/i promoter

of replication

mini-&n;

HpcrI; K, KpnI;

detail. The

of pUEIO. Portlons

Sark and R 1. pEL 18, pEL 17, and pEL 19 are E. coli plasmids translational

map of

(Smith et al.,

sites shown within the expanded

in the remaining

shuttle vectors capable

pEL1 was

pS 13. pS 17 was derived

of Way et al. (1984). The restriction

portion

restriction

plasmid

of the Km R TnlO-derivative

mini-!un

pUElO

Krm”

The CmK-conferring

from D. rcrdiodunm~; Hp,

Ns, YJiI; P, P.T/I: Pv, PwtIl;

Sp, SphI; Ss, SstII;

of plasmid

fusion;

C, CluI; C’. E. coli due to

markers

RV.

St, StrrI; Xh, XhoI.

by duplication

in-

sertion or direct insertion

Plasmids pS 1 1 and pEL2 are readily introduced by duplication insertion into the D. radioduruns strain Rl chro-

49 TABLE

II

Drug resistance

determinants

to Deinococcus rudiodurans

introduced

Gene

Phenotype

Gene product

uphA

KmR

Aminoglycoside-3’phosphotransferase

Source

Reference

Tn903

Oka et al. (1981) Beck et al. (1982)

type I

cat cut-86

CmR

Cm acetyltransferase

Tn9

Alton and Vapnek

CmR

Cm acetyltransferase

B. pumilus b

Harwood

cat(pC194)”

CmR

Cm acetyltransferase

PC194

Horinouchi

km

Kmn

Aminoglycoside

pUBll0

Sadaie

tet

Ten

R6-5

“ The cat gene from the S. aureus plasmid

pC194 is called cat(pC194)

h cat-86 was cloned

of a B. pumilus strain

a

from the chromosome

to distinguish

a

b C

I

PR d

e

e

f

I;

lg abcdefg

abcdef

Fig. 2. Schematic ments.

qThe

for duplication

heterologous

D. rudiodurans sequence additional

heterologous

insertion

vs simple

drug-resistance

bcdef is a duplication drug-resistance

struction

can potentially

transforms

or direct insertion.nHomology forming construct pR.aDirect

crosses

insertion

into the chromosome

sequence

bcdef

qHomologous

and

into

an interruption

construct.

of

Thus, this con-

chromosomal

at regions

et al. (1984)

Cohen et al. (1973)

it from the cut gene from TnY.

mosome (Smith et al., 1988). pS11 consists of a 13-kb fragment of D. rudiodurans Rl chromosomal DNA and the E. co/i plasmid pMK20 that confers KmR by virtue of #A (Fig. 1). pEL2 consists of a 10.4-kb D. rudioduruns Rl chromosomal fragment and the E. coli plasmid pEL 1 which confers CmR by virtue of the cut gene (Fig. 1). We inserted the cut plasmid pEL1 into the D. rudioduruns segment of pSl1, forming pS13, and we also inserted a 1.7-kb transposon that contains the uphA gene into the D. rudioduruns segment of pEL2, forming pS17 (Fig. 1; Table I). Thus, pS 13 could transform D. rudioduruns to KmR or CmRKmR by duplication insertion and to CmRKmS only by direct insertion (Fig. 2). Conversely, pS17 could transform D. rudioduruns to CmR or to CmRKmR by duplication insertion and to KmRCmS alone by direct insertion (Fig. 2). Duplication insertion of pS 13 or pS 17 does not necessarily produce transformants that are KmRCmR, since the duplication insertion marker is always flanked by repeats that allow its amplification exclusive of the direct insertion marker (Fig. 2). The results shown in Table III can be summarized as follows: pS13 transformed D. rudiodurmr for the duplication insertion marker (KmR) as well as the parental pS 11.

of the transforming

construct

allows

on both sides of

of homology

flanking

of the trans-

sequences

chromosome

of DR, Interrupting repeats. The

lacking

pairing

/I”, is inserted

(1982)

D. radiodurans by either duplication

pairing with the recipient

insertion:

An

of the bc and def portions

with the corresponding

for homologous

to

construct.

producing

c and d, i.e., a direct insertion

experi-

c? ligated

insertion

determinant,

the middle of the D. radiodurans sequence bcdef between

insertion

determinant

and Weisblum

et al. (1980)

Matsumura

nucleotidyltransferase

(1979)

et al. (1983)

produce

TABLE

III of Deinococcus radiodurms strain

Transformation

RI “

a direct

the chromosomal c? region is lost.

Drug resistance

with the chromo-

Plasmid pSll

pSl3

pEL2

ps17

at either the bc or def D. radioduruns DNA fragments

some may occur

as a

CmR