Differential utilization of Staphylococcus aureus promoter sequences by Escherichia coli and Bacillus subtilis

93

Gene, 48 (1986) 93-100 Elsevier GEN 01794

Differential utilization of Staphylococcus aureus promoter sequences by Escherichia coli and Bacillus subtilis (Recombinant DNA; shuttle vectors; chloramphenicol acetyltransferase; analysis)

gene expression; plasmids; dot-blot

Michael C. Hudson and George C. Stewart * Department of Microbiology, University of Kansas, Lawrence, KS 66045 (U.S.A.) Tel. (913)864-4229 (Received

July 16th, 1986)

(Revision

received

(Accepted

September

September

1 lth, 1986)

12th, 1986)

SUMMARY

Promoter-cloning plasmids were constructed and have been used to isolate transcriptionally active DNA fragments from Staphylococcus aureus. The plasmids contain a chloramphenicol acetyltransferase (CAT) gene of Gram-positive (G + ) origin which lacks both its promoter and the sequence responsible for CAT inducibility. The ability of S. aureus promoters to direct CAT expression in Escherichia coli and Bacillus subtilis was examined. Two classes of staphylococcal promoter sequences have been obtained. Class I DNA fragments direct CAT expression in S. aureus, B. subtilis, and E. coli, while class II DNA sequences direct CAT expression only in the G + hosts.

INTRODUCTION

Mechanisms for the regulation of gene expression in G + organisms have not been characterized as well as those of their G - counterparts. Certain G + bacteria, namely B. subtilis and Streptomyces coelicolor, can effect changes in gene expression by a modification of RNA polymerase through replacement of

* To whom

correspondence

and

reprint

requests

should

be

addressed. Abbreviations:

bp, base

Cm, chloramphenicol;

pair(s);

G -

tive; kb, 1000 bp; Km, kanamycin; sensitivity, tryptic

sensitive;

0

Cm acetyltransferase; G + , Gram-posi-

R, resistance,

Sm, streptomycin;

soy agar; TSB, tryptic

0378-l 119/86/$03.50

CAT,

, Gram-negative;

resistant;

Tc, tetracycline;

‘,

TSA,

soy broth.

1986 Elsevier

Science Publishers

B.V. (Biomedical

the normal (r subunit of the enzyme (Losick and Pero, 198 1; Gilman and Chamberlin, 1983; Westpheling et al., 1985). Studies with other bacterial species have not progressed sufficiently to determine whether a a factor switching mode of gene regulation is common to G + bacteria or is a property unique to the spore formers. The promoter sequences recognized by the minor forms of B. subtilis RNA polymerase differ from the consensus promoter sequence recognized by the major holoenzyme found in vegetatively growing cells (E-o”~) (Moran et al., 1982; Johnson et al., 1983). If minor forms of RNA polymerase play a role in gene expression in other G + bacteria, one might expect to find transcriptional regulatory signals in these organisms which would not be recognized by the B. subtilis E-o~~. These sequences would also Division)

94

likely not be recognized polymerase holoenzyme this enzyme

recognizes

moter sequence

by the predominant RNA found in E. coli (E-a7’), as the same

consensus

pro-

as B. subtilis E-d3.

The G + bacterium

and Quigley (1981). S. uureus chromosomal

was isolated as described (Dyer and Iandolo, 1983). Preparation of competent E. coli cells and transformation

S. cLureu.sis the etiologic agent

was as previously

Ehrlich,

1979). Competent

of a number of diseases in man and animals including

pared as described

mastitis,

S. aureus

nosocomial

infections,

drome, and toxic shock syndrome Although

the cloned

scalded

(Gemmell,

S. uureus cc-hemolysin

et al., 1983), lipase (Lee and Iandolo, toxin

A (Betley

skin syn-

et al., 1984), and

1982). (Kehoe

1985), enterostaphylococcal

protein A (Duggleby and Jones, 1983; Lofdahl et al., 1983) genes are expressed from their own promoters in E. coli, the S. aureus enterotoxin B and exfoliative toxin B genes were found not to be expressed in this host (Ranelli et al., 1985; Jackson and Iandolo, 1986). The lack of expression in the G - organism was believed to be due to poor sequence conservation between the staphylococcal gene promoters and the consensus E. coli/B. subtilis promoter sequence. This paper describes the isolation and preliminary characterization of S. aureus DNA sequences which display promoter activity. Expression directed by staphylococcal sequences has been compared in B. subtilis, S. aureus, and E. coli hosts and a subset of these DNA fragments was found not to promote transcription when introduced into E. coli.

EXPERIMENTAL

AND DISCUSSION

(a) Bacterial strains The bacterial strains used in this study were E. coli strain HB 101 (Boyer and Roulland-Dussoix, 1969), B. subtilis strain AH1 (strain 168 ZeuA8 which carries a chromosomal insert of Tn917; A. Honeyman, personal communication), and the S. aureus 8325-4 derivative strains RN4220 (Kreiswirth et al., 1983) and KUS74 (Breidt and Stewart, 1986). The former is a restrictionless mutant while the latter is a Tn551 mutant which constitutively expresses phospho-jIgalactosidase. (b) DNA isolation

and transformation

Plasmids were isolated (Norgard, 1981) from E. coli and minilysates were prepared as by Holmes

DNA

formed

(Erickson

protoplast

as described

that regeneration

reported

(Dagert

and

B. subtilis cells were preand Copeland,

transformations (Murphy

were

1972). per-

et al., 1981) except

agar was used (Stahl and Pattee,

1983). Selective concentrations of antibiotics were: Cm, 5 pg/ml; Km, 4 pg/ml for B. subtilis and 20 pg/ml for S. aureus;

Sm, 100 pg/ml;

and Tc, 15 pg/ml.

(c) CAT assays Cell lysates were prepared grown in antibiotic-containing

as follows. Cells were TSB to a density

giving an absorbance (AeoO for E. coli, A 540 for S. uureus, A so0 for B. subtilis) of 0.4. Cells from 2 ml of culture were harvested by centrifugation (3090 x g) and resuspended in 175 ~1 WL buffer (25 mM Tris. HCl, 10 mM EDTA, pH 8.0; Norgard, 1981). This cell suspension was transferred to 1.5 ml microfuge tubes. B. subtilis and E. coli cells were treated with 250 pg lysozyme while S. aureus cells were treated with 50 pg lysostaphin (Sigma Chemical Co.). All cultures were incubated for 30 min at 37°C. The cell lysates were then centrifuged (13000 x g) for 10 min and the resulting supernatant was held on ice. CAT assays were performed on the samples by the spectrophotometric method (Shaw, 1979). CAT specific activity is expressed as nmol Cm acetylated/min/mg dry weight of cells. (d) Determination

of plasmid copy number

The procedure used was as described (ScheerAbramowitz et al., 1981) except that unlabeled, purilied plasmid DNA (0.3 pg) was added to each [ 3H]thymidine (ICN Pharmaceuticals, Inc.)-labeled sample to facilitate visualization of the open circular as well as the closed circular plasmid forms. Copy numbers (plasmid molecules per chromosome equivalent) were calculated based upon experimentally determined plasmid sizes and utilizing a value of 4.0 x lo6 bp for the size of the chromosomes of S. aureus, B. subtilis, and E. coli.

95

(e) Dot blot analysis of RNA

moter and sequences repsonsible for induction of the antibiotic resistance. We have modified this plasmid by (1) incorporation of the TcR determinant from pBR322 to provide a better selective marker for cloning into E. cob; (2) positioning the XbaI to EcoRI linker from pUC13 (Messing, 1983) in front of the CAT gene to increase the number of useful cloning sites, and (3) positioning a transcriptional terminator upstream from the cloning sites to reduce background transcription from endogenous plasmid promoters (Band et al., 1983; Brosius, 1984). Two derivative plasmids were constructed (Fig. 1). Plasmid pMH109 retained the replication

was performed as Dot blot hybridization described (Truitt et al., 1985). Nick-translated pC194 (Horinouchi and Weisblum, 1982) was used as the hybridization probe for the CAT-specific sequences. Hybridization conditions used were as described (Berent et al., 1985). (f) Plasmid constructions

The promoter-cloning plasmid pCPP-3 (Band et al., 1983) utilized a CAT gene deleted of its pro-

X BSmSsE

1

X BSmSsE

Tc

Tc

I\

Fig. 1. Maps ofpMH109 1981), which includes entire pSA0501 functions.

and pMHl20.

plasmid

(Gryczan

The CAT gene, 1,

transcription

also carry the TcR determinant

fragment

ofE. coli DNA polymerase

polylinker PvuII;

from pUC13 terminator

Sm, SmuI;

(Messing, preceding

pMH109

1978). pSA0501

terminator, from pBR322

and pBR322

origin of replication

are from pCPP-3

as an EcoRI-AvuI

pMH120

and temperature-sensitive fragment,

(Jalanko

et al.,

contains

the

replication

(Band

et al., 1983). Both

blunt-ended

using the Klenow

site of pCPP-3 which was similarly filled-in. The XbaI to EcoRI The plasmid

sites are as follows: B, BumHI;

areas originated

from pUBll0

in G + hosts. The plasmid

the SmR determinant

in the two vectors.

sites. Restriction site.

functional

which was isolated

1983) was also included the cloning

rbs, ribosome-binding

the 4.0-kb XbaI to PvuII fragment

provides

I, and ligated into the single EarnHI

Ss, SstI; and X, XbuI. The stippled

the CAT determinant.

contains

and an origin of replication

and Dubnau,

plasmids

transcription

The plasmid

the KmR determinant

from either pUBI

pMHl20 Bg, &/II;

or pSA0501

additionally E, EcoRI;

contains

t,

H, HindHI;

while the blackened

a P,

area is

96

functions of pUBll0 (Jalanko et al., 1981) but contained more of PUB 110 than the PvuII-EcoRI fragment found in pCPP-3 (Band et al., 1983). The inclusion of the additional EcoRI-XbuI fragment provided transcription termination functions and thus reduced background expression of CAT (compare pMH104 which lacked this fragment to pMH109 in Table I). The plasmid pMH120 was constructed by replacing the PUB 110 region with the staphylococcal plasmid pSA0501 (Gryczan and Dubnau, 1978; Gryczan et al., 1980). This plasmid confers Sm resistance and is a temperature-sensitive replicon. Thus this plasmid is useful for procedures where curing of the plasmid or chromosomal integration of

TABLE

plasmids carrying homologous inserts is desirable. The 28-bp trp a transcription terminator (Pharmacia, Inc.) was positioned upstream from the CAT gene, which reduced background CAT expression eightfold in E. coli hosts (compare pMH120 with pMHll0 [minus this terminator] in Table I). (g) Isolation and characterization moter-containing

of S. uureus pro-

fragments

The data in Table I were obtained from representative experiments ultilizing pMH 109. The plasmid was digested with SmaI, treated with bacterial alkaline phosphatase, and ligated to &I-cleaved S. uureus chromosomal DNA. AlaI cleaves the

I

Quantitative

analysis

of plasmid

functions

Host

Plasmid

Resistance

organism

Size

CAT

(bp)

specific

COPY number b

Stability” (%)

activity” pSA0501

s. al0Wu.s

Sm

4200

-

40

pUBll0

B. subtilis

Km

4500

-

63

pMH104

E. coli

Tc

7000

0.5

NDd 33

pMH109

E. coli

Tc

7400

0”

pMHl09

B. subtilis

Km

7400

0.3

6

6

pMH109

S. aureus

Km

7400

0

10

88

pMH109-16

E. coli

Tc, Cm

8500

24.5

29

pMH109-16

B. subtilis

Km, Cm

8500

34.4

4

pMH109-16

S. aureus

Km, Cm

8500

8.6

4

pMH109-18

E. coli

Tc, Cm

8700

2.3

49

pMH109-18

B. subtilis

Km, Cm

8700

77.5

6

pMH109-18

S. aureus

Km, Cm

8700

6.1

10

pMH109-20

E. coli

Tc, Cm

8300

3.9

62

pMH 109-20

B. subtilis

Km, Cm

8300

16.0

5

pMH109-20

S. aureus

Km, Cm

8300

4.4

5

pMH1 IO

E. coli

Tc

7700

1.3

ND

pMH120

E. coli

Tc

7800

0.2

28

pMH120

S. aureus

Sm

7800

a Expressed

as nmol Cm acetylated/min/mg

b Expressed

as number

’ Plasmid-carrying aeration

of plasmid

Km-containing

cells (dry weight). Values represent

molecules

strains were grown overnight

at 37°C to a density

of approx.

TSA. % of KmR colonies

per genome

equivalent.

in TSB without

lo9 colony

forming

units/ml.

is given.

= 0.06 nmol/min/mg

cells (dry weight).

averages

1 of two or more determinations.

The values are averages

antibiotic

d Not determined. e Lower limit of detection

ND

selection. Aliquots

0.2 37

of three or more determinations.

were diluted

The cells were then diluted

1: 10 in TSB and grown with

in TSB and plated

on TSA and

S. uureus genome more extensively than does SmaI. Ligated plasmids were introduced into E. coli strain HBlOl by transformation. Clones with plasmid inserts demonstrating promoter activity were obtained by direct selection of transformants on Cmcontaining media. Plasmids containing putative S. uureus promoters were introduced into B. subtilis by competent cell transformation and into S. uureus by protoplast transformation. The level of CAT expression for three representative plasmids, pMH109-16, pMH 109-18, and pMH109-20 is given in Table I. Each plasmid contains an insert approx. l-2 kb in size and CAT assay values were used as an indication of cloned promoter strength. However, at this stage, differences related to translation or enzyme stability could not be distinguished from differences in transcriptional activity. Data shown in Table I demonstrate the absence of measurable CAT expression in clones carrying pMH109. Only B. subtilis exhibits very slight activity. CAT activities for clones carrying pMH 109 or pMH 120 are well below that of their precursors (pMH 104 and pMH 110, respectively) which lack the transcription terminator sequences ustream from CAT. Although pMH109-16, pMH109-18, and pMH109-20 contain cloned S. aureus promoter sequences, CAT activity is highest in B. subtilis for all three plasmids (which is true for all clones which we have thus far characterized). This phenomenon is particularly apparent for pMH109-18 for which there is 13 times the CAT activity in B. subtdis relative to the value obtained with S. uureus. This difference becomes even greater when plasmid copy numbers are considered. The lower enzyme activities obtained with S. uureus are not an artifact resulting from the usage of lysostaphin during cell lysis. The addition of lysostaphin (in addition to lysozyme) to B. subtilis cultures has no effect on resulting CAT yields (not shown). To ascertain whether differences in CAT activities were due to gene dosage, plasmid copy numbers were determined and are also presented in Table I. The plasmids pSA0501 and PUB 110 are maintained at a high copy number in their G + host cells. However, as can be seen in Table I, the copy numbers of these staphylococcal plasmids decrease dramatically with increased plasmid size. For example, with the PUB 1lo-derivatives, the addition of 4.0-4.3 kb of DNA to this plasmid results in a

IO-16-fold reduction in plasmid numbers in the cells. During the construction of pMH120, the addition of 3.6 kb of DNA to the pSA0501 plasmid decreased its copy number 40-fold in S. uureus. Less dramatic reductions in copy numbers have been observed during the construction of B. subtilis chimeric plasmids (Scheer-Abramowitz et al., 1981). However, it has been reported that an increase in the size of PUB 1lo-derived recombinant plasmids strongly reduced their copy numbers and segregational stability in B. subtilis hosts (Bron and Luxen, 1985). The segregational stability of plasmids pMH109 and pMH109-18 in B. subtjlis and S. uureus was determined (Table I). The plasmids are more stably maintained in S. uureus than in B. subtilis. This increased retention was not due to integration of the plasmid into the staphylococcal chromosome at the site of homology to the insert. We have found that Campbell-type integration of plasmids into homologous sites in the staphylococcal chromosome is a very low frequency event (B. Oskouian and G.C.S., unpublished observations). The plasmids are certainly structurally stable and can be maintained in cultures with antibiotic selection. (h) Isolation of S. sureus promoters which do not function in E. coli

Plasmid constructions were as described (section g) except that plasmid transformants of HBlOl were selected on Tc-containing media. TcR clones were toothpick-inoculated onto Cm-containing media. All TcR, CmS E. coli clones were pooled and plasmid DNA was isolated. This plasmid DNA mixture was then introduced into S. uureus protoplasts with Cm selection. The resulting colonies presumably represented clones carrying recombinant plasmids with staphylococcal promoters not recognized by E. coli. These plasmids, when reintroduced into E. coli, did not direct CAT expression, indicating that rearrangements of the plasmid DNA were not responsible for the CAT expression in the S. uureus host. Six representative plasmids which directed CAT expression in S. uureus but not E. coli hosts were introduced by transformation into B. subtilis. The insert fragment size and CAT-specific activities indicated that each of these inserts represented a unique

98

TABLE

II

CAT-specific Plasmid

activities

directed Insert

by ‘staphylococcal size (bp)

inserts

in the three hosts

CAT specific activity a B. sub&

S. aureus

E. coli Ob

pMH109-01

530

36.5

1.1

pMHl09-03

730

8.4

0.5

0

pMH109-012

750

52.3

0.8

0

pMH109-013

73

13.1

0.9

0.2

pMH109-014

610

18.8

1.2

0

pMH109-015

131

30.6

2.9

0

a nmol/min/mg

cells dry weight.

’ Lower limit of assay = 0.06.

fragment from the staphylococcal chromosome. Table II summarizes the results obtained when CAT assays were done with the three host species carrying these plasmids. With the exception of pMH109-013, E. coli clones carrying the recombinant plasmids do not demonstrate detectable CAT activity. However, the plasmid inserts did promote CAT expression in the B. subtilis host. As was the case with the promoters in Table I, B. subtilis demonstrated higher levels of CAT activity than S. aureus, even though the cloned sequences were of staphylococcal origin.

plasmids pMH109-16 and pMH109-013. These two plasmids gave high and low levels of CAT-specific activity, respectively (Tables I and II). However, the amount of cat mRNA consistently did not correlate with the level of CAT-specific activity obtained. The hybridizing species from these two strains was not contaminating plasmid DNA as the hybridizing signal was destroyed when the RNA preparation was treated with RNase (previously boiled to inactivate DNase). The hybridizing species was also not affected by treatment with DNase (data not shown).

(i) RNA dot-blot analysis The failure to detect CAT expression in E. coli could have been a result of a failure to recognize the staphylococcal promoter sequences (a transcriptional failure) or may have been due to a translational block (despite the CAT gene on pMH109 being immediately preceded by a ribosome binding site recognizable in E. co/i). To resolve these possibilities, RNA dot blot analysis was carried out on the E. coli clones carrying the recombinant plasmids. Decreasing amounts of total cellular RNA was spotted onto a nitrocellulose filter, which was then 32P-labeled pC194. The only RNA probed with species with homology to this plasmid probe would be CAT mRNA. CAT gene-specific mRNA was not detected in E. coli clones lacking CAT activity with the exception of low levels seen with pMH109-015bearing cells (Fig. 2). Such sequences were detected in the RNA from E. coli clones harboring the

012 013 014 015 Fig. 2. Dot clones

blot analysis

carrying

amount

of CAT

G + -specific

CAT

of total RNA applied

eliminate

pretreated

DNase

from E. coli

isolated

expression

plasmids.

to the filter ranged

62.5 ng. The last three columns RNA samples

RNA

on the right ofthe figure represent

with RNase (50 pg/ml, heat-treated

activity). The plasmids

carried

on the let? (with the ‘pMH109-’

the designations).

Samples

16Ec and

(see Tables I and II).

to

by the different

clones are indicated controls

The

from 1 pg to

013 serve

deleted from as positive

99

(j) Conclusions

ACKNOWLEDGEMENTS

The results presented demonstrate that S. aureus DNA fragments can be isolated which direct the expression of a promoterless gene. Two classes of promoter-containing DNA fragments were observed. Class I promoters function in S. aureus, B. subtilis, and E. coli and presumably represent promoters whose sequence resembles the consensus E. coli E-07’/B. subtilis E-d3 promoter sequence. Based upon its expression pattern, the cloned ahemolysin gene carries a class I type promoter (Fairweather et al., 1983; Kehoe et al., 1983). Class II promoters are recognized by S. aureus and B. subtilis but not by E. coli. These fragments may (1) carry promoters which require a positive regulatory factor common to the G + bacteria but not found in E. coli; (2) signify a promoter sequence tolerated by the polymerase in the G + organisms but not by the E. coli holoenzyme; or (3) represent promoters recognized by a minor form of 0 factor possessed by both S. aureus and B. subtilis. Each staphylococcal promoter-containing insert that has been characterized is able to direct CAT expression in B. subtilis. In fact, CAT-specific activities are always highest for B. subtilis even though the sequence cloned upstream from CAT is from the S. aureus genome. Determinations of CAT-specific activities were performed on early log-phase B. subtiliscells carrying the recombinant plasmids. The RNA polymerase holoenzymes E-d3 and E-02’ are present in B. subtilis cells at this stage of growth, but promoters recognized by these two forms of Bacillus RNA polymerase are also recognized by E. coli (Moran et al., 1982; Gilman and Chamberlin, 1983). Currently, it is unknown which form(s) of B. subtilis RNA polymerase recognizes the cloned S. aureus sequences. Our results are somewhat surprising in that B. subtilis has been believed to be more stringent than E. coli in the recognition of transcriptional and translational signals from other organisms (Kreft et al., 1978; Moran et al., 1982). The data presented in this paper indicate that significant differences seem to exist between gene expression in S. aureus and E. coli. Studies are currently in progress to determine the nucleotide sequence of these cloned S. aureus promoters as well as the directed start points of transcription.

We thank Dennis Henner for providing us with the plasmid pCPP-3. This work was supported by Public Health Service grant AI21574 from the National Institutes of Health and by University of Kansas General Research allocation 3637-X00038.

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