Mutagenic analysis of the promoter of the Streptomyces fradiae β-lactamase-encoding gene

Gene. 121 (1992) 87-94 0 1992 Elsevier Science

GENE

Publishers

B.V. All rights reserved.

87

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0674 1

Mutagenic analysis p -lactamase-encoding (Streptomyces expression;

of the gene

lividuns; signal peptide; RNA colony hybridization;

Mats Forsman

promoter

transcriptional enzyme

initiation;

of

the

exonuclease

Streptomyces

III-mediated

deletions;

fradiae

promoter

sequence;

activity)

and Micael Gram&-am

Department ofMicrobiology, Notional Dcfknce Research Establishment, S-901 82 Umed. Sweden Received

by K.F. Chater:

3 March

1992; Accepted:

29 June 1992; Received

at publishers:

16 July 1992

SUMMARY

by promoter probing, primer The Streptomyces fradiue P-lactamase promoter (PhlaF) was sequenced and characterized extension, and exonuclease III-mediated deletions. The transcription start point (tsp) was the same in both S. lividuns and S. fiudiue. Oligodeoxyribonucleotide-directed random mutations and site-specific mutations were introduced in the promoter region. The effects of these mutations on transcription were assayed by an RNA colony hybridization method. This analysis identified c&acting sequence determinants located similarly to the -10 and -35 regions of a typical Escherichia coli promoter. Also, a change in the distance between these regions from 19 to 17 bp drastically reduced promoter activity. PhbL was shown not to be recognized by sigma-whiG or by sigma-hrdA, hrdC, or hrdD. Sequence alignment of PhlUFto sigma factor-classified Streptomwes promoters revealed little homology. Thus, P,,(,, is probably recognized by an as yet unidentified sigma factor.

INTRODUCTION

Transcription initiation plays a crucial role in the complex process of gene regulation. In the Gram’, mycelial soil

Correspondence to: Dr. M. Forsman,

FOA 4, S-901 82 Umei,

Sweden.

Tel. (46-90) 106669; Fax (46-90) 106800. Abbreviations:

aa, amino

acid(s);

B., Bacillus;

Bla, /I-lactamase;

bluF,

gene encoding a Bla from S.,fiadiae; bp, base pair(s); ccc, covalently closed circular; cpm, counts per minute; A, deletion; DOG, 2-deoxyglucase; E., Escherichia; Exo III, exonuclease

coding

sigma factor(s)

kilobase polymerase

homologous

or 1000 bp; PolIk, Klenow (large) fragment of E. coli DNA I; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; P, pro-

moter; PEG, polyethylene SDS, sodium transcription

III of E. co/i; hrd, gene(s) ento rpoD of E. coli and B. subtilis; kb.

dodecyl

Sm, streptomycin;

form; S., Streptomyces; Th; thiostrepton;

tsp,

tsr, gene, encoding Th resistance; whiG, gene sigma (0) factor; wt, wild type; [ 1.denotes plasmid-

start point(s);

encoding sporulation carrier state.

glycol; RF, replicative

sulfate;

bacterium Streptomyces, a tremendous promoter sequence diversity is observed (Strohl, 1992). At least seven forms of RNA polymerase holoenzymes have been discovered so far in Streptomyces. The different forms of RNA polymerase are distinguished from each other by the association of the core enzyme (b/? u2) with different c~factors. Thus far, four different u factors, $a, d5, 049 and aG6have been well characterized biochemically (Westpheling et al., 1985; Buttner et al., 1988; Brown et al., 1992). Another 0 factor, cYhiG, was identified by genetic studies (Chater et al., 1989) and shown to be obligatory for sporulation in S. coelicolor A3(2). In addition, S. coelicolor contains four genes which are highly similar to the Escherichia coli rpoD gene, specifying the principal u factor (Tanaka et al., 1988), and these genes have therefore been named hrdA, hrdB, hrdC, and hrdD (homology to rpoD). The hrdB gene encodes a r~factor, g6, which in association with the core components of the RNA polymerase complex directs transcription from the dagAp4 and Bacillus subtilis

88 veg promoters in vitro (Brown et al., 1992). The d5 factor may also be encoded by one of the hrd genes since the reg promoter of B. s~bt~Z~s,which is recognized by d5 (Westpheling

et al., 1985), conforms

Ph,L,P, and we evaluate the expression mediated by this promoter in various G factor-deficient S. ~oei~~~~~)~strains (Table I).

closely to the E. coli con-

sensus sequence. Moreover, the existence of at least one additional D factor, do, has been inferred from in vitro

RESULTS

transcription studies of the Streptomyces gal operon (Westpheling and Brawner, 1989; Westpheling et al., 1990). However, present information does not exclude that OiehiC and d” are identical. Mutations in each of the component regions constituting a typical E. coli promoter are recognized in S. Iividuns in the same way as in E. coli (Jaurin and Cohen, 1984). However, functional motifs for classes of promoters that are transcriptionally active in Streptornyces but not in E. coli

(a) Characterization of the PblaF region A 544-bp upstream Sau3AI DNA fragment, which contains the blaF start codon (Fig. l), was subcloned into the promoter-probe vector pJAS14 (Forsman and Jaurins, 1987). On plates, this construction mediated a strong expression of the ctnz&’ reporter gene in S. lividun~~as measured by spraying nitrocefin. This fragment therefore carries the blaF promoter. ExoIII-mediated deletion of this

have not been systematically characterized. The bloF gene, encoding a constitutively expressed Blactamase of S.,fiadiae, has previously been cloned in S. ~~~~~~~~~, and the nt sequence encoding the S. f~~~~ffe enzyme has been established (Forsman et al., 1990a). In this study, we describe a mutagenic analysis of the

region from a plasmid carrying bluF (pPF58; Fig. 1) almost completely abolished bluF transcription in S. lividuns as judged by RNA colony hybridization. Deletion of only the first 267 bp (p~F60; Fig. 1) of the upstrean~ promoter rcgion had no effect on transcription (Fig. 2). Thus, the region between the deletion end-points in pflF58 and pPF60

TABLE

I

Relevant

strains

AND DISCUSSION

and plasmids Genotype

Reference

s. .~~fff~jfle

U’t

DSM4~~63

S. lividam 1326

wt

Lomovskaya

whiG71 derivative of A3(2), following mutagenesis srrA1 uraA1 g&Ill9 Pgl- SCPIN” SCP2-

Chatcr (1972) Fisher et al. (1987)

Strains

and plasmids

or source

Strains”

et al. (1972)

S. coelicnh~ A3(2) c71 51668

hisAl

J1957

hisA

J1958

hisA

J1959

hisA 1 s?rA 1 um.4 1 gikAl19

I s3rA 1 urd i gNtAl19 Pgl~ hrdC::ermE SCPIN” SCPZ _ I SPA1 urad 1 glkAl19 Pgl- hrdD:h.vg SCPI”‘; SCP2 Pgl- hrdA::spececistrep SCPI”’

Buttner et al. (1990) M. Buttner, John lnncs Institute M. Buttncr.

SCPZ

John fnnes Institute

Plasmidsh ~13487 containing

p/JFZO 1

EcoRI-Hind111 pIJ487 containing

the S..fiadiae

Bla-encoding

a promoter-less

derivative

Bla-encoding

promoter

region originally

This paper

of the S.,fradiue

Bla-encoding gene pIJ487 containing a 267-bp deletion of the 502-bp (pflF2Ol)

pBF60

This paper

gene on a 1.7-kb

fragment

This paper

S.fradiae

cloned

j’ Details concerning medium, transformation, and growth conditions of Srreptomyces are described in the manual of Hopwood et al. (1985). S. coelicobr A3(2) hrd mutants (hrdA, hrdC, and hrdl)) and the isogenic control were initially selected on NMM medium (Hodgson, 1982) containing the appropriate antibiotic~lo~ mM DOGjO.So/ mannitol as carbon source. Thereafter, these strains were routinely grown on RZYE medium containing 0.5”; mannitol. All other Strepfomyces

strains were grown on RZYE agar plates (Thompson

(Bibb et al., 1977) or YED medium (Daza et al., 1989). All standard et al. (1989). h The Streptomyces

plasmids

pJASOl

(Jaurin

and Cohen,

et al., 1980). Liquid cultures

manipuiations

of Srreptomyces were grown in YEME medium

of DNA were, if not otherwise

1984) and the E. coli plasmid

pACYC184

(Chang

stated, done as described and Cohen,

by Sambrook

1978) have been described

previously. The S.fiudiae Bla-encoding gene residing on a 1680-bp Sal1 fragment was previously cloned into M13mp19 (Forsman et al., 199Oa). This construct was digested with Sac1 + BumHI and treated with ExoIII and SI n&ease (Henikoff, 1984). After Polfk treatment, religation. and transformation into E. co/i JM 103, templates were prepared and sequenced. The promoter deletion clones as well as the original construct were digested with Ec(tRI + Hind111 and ligated into pIJ487 digested with the same enzymes. The promoter region was located proximal to the EcoRl site m p,PFZOt, p{jF%, and pBF60.

89

Sal I 1

5 '-GTCGACGGGCGCGTCCGCGGCTCCCCCCAGGAGGGCGCTCCTCGGCGGGCCCGGCC~CGGGC

61

TCGGTCGAGGCCCGGGGGTCCGCGGGCGCGG~CTGCGCGGACCGCTCGTCGGCTGGCGTGT

121

CGGGCCCGGGCTCGCGGGCGGG~TC~G~GACGGCCCCCGGACGGCC~GACCGGGAGG

181

GCGGGGACTGAGCGGTCCTGCGTTCGCTCATCGAGTCGGCGACCCACTCCGTACGAAAAA

241

460 GCGCAGAGGCGTCTTGGCGTGACATAGACATCATTCCCGCAGTGTGCCACGGCTTG~CA

301

CTGCTGGCAGAAACGTTACTCCCGACCACTGTCAAGCACGGCCTCCGCCCCCCGCACGGC

361

GTGGCCCGGGCCGGTTCGGCTGCGCCGCGGTGCCCCACGC

421

ACGGGACGGCCGGGGGCGGTTTTCGGCCG~

481

r 1 58 AGGCACCTCGTCCTGCCGT3_CGGAGAAGGGGTCCATCG~~C - 10 rsglon

- 35 region

Fig. 1. The nt sequence

600

AlaThrAlaAlaAlaAlaGlyProAlaHisA~aAlaProGlyArgGlyA~a GCCACCGCGGCAGCGGCGGGCCCGCGCACGCCCCTCCGGGCGCC-3'

of the P,,,,, region. The bent arrow marks

hybridization

N-terminal

cession

(nt -232).

region to pJASl4. number

sequencing,

The horizontal

experiments.

ate oligo which was used for generating promoter

_

ThrThrAlaArgProAsnArgArgAlaValLeuAlaThrG~yVa~GlyA~aA~aLeuA~a ACCACCGCACGTCCGAACCGCCGAGCCGTCCTCGCCACACAGG~TGGGGGCCGCGCTGGCG

et al., 1990a), to 40 aa as indicated.

(nt + 11) and ppF60

__

540

rification of the protein and subsequent and RNA colony

ValAspArg __

tsp.The identified

25-m arrow represents

Dashed

the random Suu3AI

the position

- 10 and

-35 regions

promoter

mutants

is underlined.

are indicated

to the S. Downward

of the most downstream

site used was that within the BarnHI

Sau3AI

lividam

16s rRNA.

arrows

with overlining.

from the predicted

of the Fl primer (Table II, footnote

line shows the nt complementary

The boxed nt show the location

The upstream

the

the length of the signal peptide has been corrected

After pu-

length, 34 aa (Forsman

d) used in primer extension

The location

of the 50-nt degener-

show the extent of Exo III deletion

in p/?F58

(cleaves at nt 534) site used in the subcloning

site from pSP64

(outside

the sequence

shown).

of the

Sequence

ac-

M94255.

(nt 268-5 11) presumably contained a &-acting element necessary to mediate transcription of blaF. Consistent with this, the tsp of PhluF was shown by primer extension analysis to be from the same nt (501 in Fig. 1) in both S. lividans and S.fiadiae (Fig. 3). (b) Mutational analysis of Phla, To analyse the promoter region in more detail, a degenerate oligo, including nt -50 to - 1, was used to introduce random mutations in the promoter region of blaF. The resulting mutants were categorized into three groups, high, low, and wt level of expression. Representatives of each

1234

B Fig. 2. RNA colony hybridization

of Exo III-promoted

S. lividuns[pflF58]

see Fig. 1); lane 2, S. lividrms[pfiF60];

(promoterless,

lane 3, S. lividans[pflF201];

lane 4, S. lividans[pIJ487].

deletions.

Lane 1,

(A) Filter hybrid-

ized to the Fl oligo (see Fig. 1). (B) Same filter hybridized to the fsrspecific probe, after a high stringency wash. For details see Table II.

group were chosen for sequence analysis. The effect on transcription of these promoter mutations was assayed by a simple and specific RNA colony hybridization method (Forsman et al., 1990b,c). The results are shown in Table II. Random mutations with no effect on transcription were found at positions -20, -25, and -45. Random mutations which differed most from wt level were found at positions - 10 and - 14. This analysis suggested that the - 10 region was important for transcription. To extend the mutational analysis, site-specific mutagenesis of both the -10 and the -35 region was performed. The effects on transcription by the site-specific mutations are presented in Fig. 4 and Table II. Even though down-mutations were predominant, a few up-mutations were also identified. Two of the up-mutations were located at the same nt position (nt -lo), and two other up-mutations mapped at -36 and -39. Inspection of the positions of the single-base substitutions with the most pronounced effect on transcription suggests a -10 region with the sequence CGTCGT, located between positions - 14 and -9. The second sequence motif identified, the -35 region, is probably located at positions -39 to -34, and here the suggested sequence is ATGAAA. To evaluate the importance of a correct spacing between the - 10 region and the -35 region, 1-bp and 2-bp oligo-aided deletions were made in the interregion. A I-bp deletion (d nt -24) in distance had little effect on the

90 TABLE

II

The effect of the different promoter Random

S. fkzdiue Bla promoter

mutations mutants”.d

C( 14)T, A( 16)T

TT

T(9)A T( 12)G

A G G

A(36)C, C(42)G

C

A(20)T, A(25)T C( I S)G, C(24)A

A

G( lO)T, G(46)T

G T

A

T(45)G

G

activityh

0.5 * 0.2

0.5

0.8 & 0.0

tt.7

0.7 _t 0. I

0.6

1.4* 0.2 I .o * 0.2

I .A I.1 I.5 3.2 I.1 0.x 0.6 I1

1.920.3

C

l.O_tO.l 0.8 f 0.1

C( 14)T, G( 19)T, T(29)C G(40)A S. jhdiae

Relative Icvcl of enzyme activtty’

1.6 2 0.3

T

A(20)C, T(45)A

Site-specific

T

T

Relative level of promoter

T

C

T

0.5_tO.l

A

1.2+0.1

Bla promoter

mutants”,r

5’- -CCGAGTGACAGATGAAACCGGTGGGACGGGAGGGACCGTCGTCGTGGATGT~’ A(39)T

cagTtga

2.2 * 0.3

2.1

A(39)C

cagCtga

I. I 5 0.4

0.8

0.4 + 0.1

0.3 0.9

T(38)G G(37)T

agaGgaa ga

A(36)G

0.7 * 0.1

tTaaa

a tgGaac tgaGacc

A(35)G

t gaCacc

A(35)C G(30)C

ccgctgg

G(30)T

ccgTtgg

C(24)A

ggadggg

CG(24,23)A

ggadgg

a

G(lO)C

gt

G(lO)T

gt cTtcg

cctcg

C(ll)G

cgtGgtc

C(1 I)A

cgtAgt

T( 12)G

ccgGcg

G(13)T

1.9 _t 0.4

1.6

0.2_tO.l 0.2 * 0.1

0.08

0.8 + 0.3

0.06 0.7

1.4 f 0.3 0.8 & 0.3

0.9

0.3 & 0. 1

0.15

I .(I

2.1 io.5

I .9

2.7 k 0.4

7.1

0.8 & 0.2

0.5 0.5

0.6 k 0.2

c t

accTtcg a c c Atcg

0.5 i_ 0.1

0.3

0.2 * 0. I

0.08

I

0.2

C(14)A

gacAgtc

0.5 _t 0. I

0.6

C(14)T

gacTgtc g acGgtc

0.3 _t 0. I I .of 0.4

0.08

G(13)A

C(14)G ’ The promoter deletions

mutants

are designated

are indicated

are named

in upper-case

h Determined

by listing the wt nt and its position

by the nt deleted, its position letters surrounded

by RNA colony hybridization:

(in parentheses)

by their wt context identical

amounts

(in parentheses),

3-min treatment

ter was first hybridized

to a 3’P-labeled

bl&-specific

after, a high stringency

wash was performed

in lower-case of the different

’ Identical

amounts

experiments

of the different

was changed.

the r.yp. The Phi<,, site-specific

The

mutants

letters. spore suspensions

(5 x 10’ spores/ml,

determined

by viable count) wcrc

63-67 h at 30 a C, and the filter was treated as described after the 3-min incubation

oligo (Fl, 5’-TTCGGACGTGCGGTGGTGCGATCCA),

which totally stripped

0.7

by the nt to which the sequence

of the filter in 0.2 M acetic acid was included

fsr-specific oligo (5’-TGCGATGGTGTCCAACTCAGTCATG). signal and the internal control signal (tsr) were calculated. Values k SD of five independent

followed

and the A symbol. The bent arrow indicate

applied on a nylon filter placed on a R2YE agar plate. The plate was incubated et al., 1990b), except that an additional

0.4 + 0.

the radioactivity

washed,

previously

(Forsman

in IO”,, SDS. The til-

and autoradiographed.

from the filter. The same filter was then hybridized

Therc-

to an “P-lab&d

The autoradiograms were scanned with a densitometer, and the rattos between the h/oF The wild-type/internal control ratio was set to 1, and all other ratios were normalized to that.

are shown.

spore suspensions

(5 x lO’/ml, determined

by viable count) were inoculated

in YEME

medium.

Duplicate

samples

were withdrawn every day. Bla activity was recorded in the supernatant by employing nitrocefin as a substratc (O’Callaghan et al., 1972). Growth was measured by incorporation of [35S]methionine into protein, and samples were processed as described (Forsman and Jaurin, 1987). The peak value of Bla activity was divided by the corresponding cpm value. The wt ratio was set to 1, and all other ratios were normalized to that. Mean values of two independent cultures are shown. d In order to generate random mutations in the Pi,,<,, region, a 50.nt degenerate synthetic oligo extending from nt - 50 to - I in the promoter region of

91 promoter strength; however, a 2-bp change (d nt -23, -24) drastically reduced it (Fig. 4; Table II). A sequence alignment of the P,,, region with that of the closely related

7 2325678 II,

Bla-encoding gene of S. lavendulae revealed particularly close similarity at the -10 and -35 regions (4 out of 6 bp identity for each case) (Forsman, 1991). Taken together,

:-_

the results indicate that Phlu,. has functional sequence motifs at the - 10 (CGTCGT) and the -35 region (ATGAAA), with a spacing of 19 nt. The enzymatic activities of strains carrying each promoter mutant construct were also measured in the supernatants of liquid cultures. Although a few mutant clones showed different relative values with the two methods of assessing activity, the range of variance intrinsic to both

Fig. 3. Primer extension

analysis.

RNA was isolated

of S.fradiue and S. lividam as described

methods was of the same order under the conditions used, and most importantly, both methods gave the same overall pattern of relative expression by the various mutants. However, it cannot be excluded that expression was influenced in some cases by the changed growth conditions. The identified -10 region of P,,, is unusual in that it comprises the first two in a row of three direct repeats with the sequence CGT. Furthermore, the identified -10 and -35 regions of PhluF show a 2 out of 6 bp identity in the - 10 region and a 4 out of 6 bp identity in the -35 region with the consensus sequence of E. coli promoters. Previously, P h,rrFwas shown not to be recognized by the E. coli RNA polymerase (Jaurin et al., 1988). Similarly, when the following promoter mutations: A(39)T, A(39)C, A(35)G, A(35)C, G( lO)T, G( lO)C, C( 1 l)G, C( 1 l)A, T( 12)G, G(13)T, G(13)A, C(14)A, C(14)T, C(14)G, C(14)T/ A(16)T, and C(24)d were subcloned into pACYC184 and then introduced into E. coli, none showed activity, as measured by spraying nitrocefin onto the colonies. This may suggest that none of these promoter mutants improved the promoter identity enough for recognition by any of the different forms of the RNA polymerase holoenzyme existing in E. coli.

from 48-h cultures

by Hopwood

et al. (1985). The

primer extension analysis was carried out as previously described (Forsman et al., 1989). Lane 1, RNA (6ng) isolated from a culture of S. lividans[pJAS

14_Pl](SEP8)(Forsman

and Jaurin,

1987) annealed

with

labeled Fl primer (region of complementarity indicated in Fig. 1) and then extended. Lane 2, RNA (7 ng) isolated from a culture of S.,fradiae annealcd with labeled Fl primer and then extended. T, respectively)

show the nt sequence

Lanes 3-6 (A, C, G and

of the P,,,,

(Fl) as that used in primer extension.

using the same primer

Lane 7, RNA (2 ng) isolated

a culture

of S. lividuns[pflFZOl ] annealed

extended.

Lane 8, labeled Fl primer alone. Arrow indicates

of the extended medium

product

supplemented

(specifying

zsp). All strains

with proline

primer Fl see Table II, footnote

and Ca”

dircctcd resultant

plaques

were used to preparc

with hirF, using BarnHI

from and

the location

(c) Evaluation of the PblaF c factor dependence The PhlrrFmutation constructs on pACYC184 in E. coli (see section b) also contained a 1.6-kb DNA fragment car-

were grown in YED

(Daza

et al., 1989). For

using 97:,

correct

template

containing

and 39; (l”,

each) incorrect nt phosphoramidites at each position. The degenerate oligo was Phlrrf. Further steps were as in the manufacturer’s instructions (Amersham), ‘oligonucleotidesystem’. A sample of the ligation reaction mix was used to transform competent E. coli TGl cells. Pooled phages from the

to a M 13mp 19 single-stranded in vitro mutagenesis

Fl primer

b.

the blaF gene (Fig. 1) was synthesized annealed

with labeled

RF DNA from infected

+ SphI, and inserted

E. coli TGl.

The mixed population

of wt and mutated

promoters

was excised together

into pJASO1 using S. lividunsas host for transformation.

middle position.

’ Eleven synthetic

19.nt oligos complementary to P,,,,,b were synthesized using equivalent amounts of the three ‘wrong’ nucleoside triphosphates at the Each of the eleven oligos was used for in vitro mutagenesis as described above. DNA was isolated from individual plaques and sequenced

with Sequcnase

as directed

by the manufacturer.

vector pJASOl.

Individual

colonies

The promoter

were examined

mutations

were then subcloned,

for Bla activity using nitrocefin.

using BarnHI + SphI, into S. lividam on the Streptom,rces

92

F A

l-l

2.0

J-III T

G

.g

1.0

_5'-CC GAGTG

> 'J

AC *~~TGAA*CCGGTGGGACGG ;

l

Ill

%

F[

n

-25A

A j-J

-35 region

n-l-l G

-10 region

C

ACAGATGAAAC

rCG

TCGTCGTGGATGT-$

-!5

0

1

-'5

-35 region

+1

-10 region

C ;,

T-------i 5~~CCGAGTGACAGATGAAACCGGTGGGACGGGAGGGAC~GTCGTCGTGGATG~_~

-35 region

Fig. 4. The effect on transcription mutants,

of the different P,,,,

(C) single and double mutants

mutants, as determined by RNA colony hybridization: with no effect on transcription. The wt level is 1.0. The wt sequence

appear directly above or below. Double and triple mutants without

a column.

-10 region

The bent arrow indicates

arc connected

with horizontal

lines. The nt substitutions

(A) single mutants, (B) double and triple is shown horizontally. and exchanged nt with no effect on expression

are shown

the tsp.

rying the P 1 (SEP8) promoter (Forsman and Jaurin, 1987) in front of a truncated (the first 310 nt) am& gene.The intention with this was to obtain an internal Streptomyces/ E. co/i-type promoter control on the same constructs as the P ,,,c,p promoter mutants in the S. coelicolor hrd mutants. Hence, firstly these pACYC184 constructs containing both the various PhLIFmutants as well as the P 1 (SEPS) promoter were fused to pJASO1 and introduced into S. lividuns. Bla-producing colonies were identified by screening with nitrocefin. Positive colonies all contained the truncated ampC transcript as measured by RNA colony hybridization by using a radioactive oligo which hybridizes 27-52 nt downstream from the tsp of P 1 (SEP8) (Forsman and Jaurin, 1987). Secondly, the shuttle plasmids from S. lividans were introduced into S. coelicolor strains by transformation. The transformation frequencies were very low, and no ccc form of the plasmid DNAs from the Blaproducing S. coelicolor strains could be isolated. This observation is in agreement with the notion of Kieser and

Hopwood (1991) that SLPl plasmids integrate into the resident SLPl copy in the chromosome of S. coelicolor. However, in contrast to the situation in S. lividuns, only a few of the Th-resistant and BlaF-producing S. coelicolor clones also expressed the truncated ampC transcript as measured by RNA colony hybridization. As the conditions used for selection of the transformed colonies did not select for either integration or maintenance of the truncated ampC gene into the chromosome of S. coelicolor, it is plausible that a partial deletion had occurred in these transformants that eliminated the truncated ampC gene but not the tsr gene or the blaF gene. A similar event for blclF may have occurred in isolated colonies that were Th resistant but did not produce BlaF (data not shown). The promoter activity of PhlcrFin the S. coelicolor strains was quantitated by RNA colony hybridization. Fig. 5 shows RNA colony hybridization against S. coelicolor A 3(2) and S. coelicolor w>hiGwith and without the wt S.,fkdiue Bla-encoding gene. The ratio between tsr expression.

93

1

hybridization

2

against

the different

S. coelicolor hrd dis-

rupted strains and the isogenic S. coelicolor J 1668 with and without the bluF gene. From the results presented in Fig. 6, it is clear that PhlcrFdid not depend on any of the three hrd o factors tested. However, it should be noted that it could not be excluded that the hrd CJfactors may have overlapping specificities that could potentially mask the dependence of PhlaF on an individual hrd ~7factor. Moreover, it could not be excluded either that PhlrrFis recognized G factor specified by hrdB.

Fig. 5. RNA colony hybridization whiG with and without

(B) rsr-specific lane 1, upper,

of S. coelicolor A3(2) and S. coelicolor

the wt blaF gene:

(A) bluF-specific

probe

(Fl),

probe, (C) blaF reverse complement probe. Colonies: S. coelicolor A3(2) containing the wt bluF gene; lane 1,

lower, S. coelicolor A3(2);

lane 2, upper, S. coelicolor whiG containing

the

wt b/OF gene; lane 2, lower, S. coelicolor whiG. For details see Table II.

the internal control used instead of the truncated ampC gene, and bluF expression were approximately the same for both strains. Thus, the whiG mutation did not seem to reduce BlaF expression. To evaluate if P blc,Fwas recognized by either of the hrdA, hrdC, or hrdD r~factors, only the shuttle plasmid carrying the wt blaF gene was introduced into S. coelicolor 51668, J1957,51958 and 51959 strains. Fig. 6 shows RNA colony

1 2

3

4

by the

(d) Sequence comparison of the PhlaFwith Streptomyces n factor-responsive promoters Four different 0 factors of Streptomyces, $‘, d5, a49 and 066, have been well characterized biochemically (Westpheling et al., 1985; Buttner et al., 1988; Brown et al., 1992) and shown to recognize specific Streptomyces promoters. The dagAp4 promoter and the veg promoter of Bacillus show sequence similarities to the E. coli 0”’ promoters and are recognized by a66, and probably also by d5, whereas dagAp2 and dagAp3 are recognized by $‘, and 049, respectively. The two transcribing activities of the S. coelicolor and S. lividuns galactose operon (Westpheling and Brawner, 1989) are both inactive on dS and a49 templates and based on sequence similarities between the gulp2 and dagAp2, it has been suggested that the galp2 promoter may be recognized by 2s. This implies that at least one of the gal promoters must be recognized by a 0 factor distinct from $‘, d5, and 049. Recently, Westpheling et al. (1990) identified a new 0 subunit, do, involved in the recognition of galpl. Inspection of the optimal alignment reveals no close homology of the PblaF to any of the dug or gal promoter sequences. Thus, P,,, is probably not recognized by r?‘, do, d’, 049, or &+j. Moreover, no convincing alignment OfP hloF was found with any known Streptomyces promoter (Strohl, 1992) except with the promoter region of the Blaencoding gene of S. luvendulue (Forsman, 199 1). However, the tsp for the S. lavendulue Bla-encoding gene has not been determined. Taken together, this suggests that PhIuFis recognized by a hitherto unidentified CJfactor which remains to be biochemically characterized. (e) Conclusions

c

30 nt upstream of (I) The PhluF initiated transcription the translational start codon in both S. lividuns and S..fru-

e”

Fig. 6. RNA colony hybridization

diae.

of S. coelicor hrd strains with and with-

out the wt bluF gene: (A) b/OF-specific probe (Fl), (B) tsr-specific

probe,

(C) bluF reverse-complement probe. Colonies: lanes l-4, upper, correspond with S. co&or 51668, S. coelicolor hrdA, S. coelicolor hrdC, and S. coelicolor hrdD, respectively lower, correspond

(all contain

the blaF gene);

lanes l-4,

with S. coelicolor 51668, S. coelicolor hrdA, S. coelicolor

hrdC, and S. coelicolor hrdD, respectively.

For details see Table II.

(2) Sequence motifs located at the -10 and the -35 region and the length of the intervening region were important for the activity of the PhloV Thus, three regions determining the PhluF strength were identified. (3) No influence on transcription of the P,,,c,F.was observed in S. coelicolor whiG or S. coelicolor strains disrupted for hrdA, hrdC, or hrdD.

94

(4) Circumstantial evidence indicates that PhlaF is recognized by an RNA polymerase holoenzyme containing an as yet unidentified cr factor.

tional

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induction

compound. Forsman,

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M., Haggstrom.

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B.. Lindgren,

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Forsman,

ACKNOWLEDGEMENTS

Dr. M.J.

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for kindly

providing

the

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