FEMS Microbiology Letters 110 (1993) 231-238
0 1993 Federation of European Microbiological Societies 037%1097/93/%06.00
Published by Elsevier
231
FEMSLE 05487
ZuxAB gene fusions with the arsenic and cadmium
resistance operons of Staphylococcus aureus
plasmid ~I258
P. Corbisier a, G. Ji b, G. Nuyts a, M. Mergeay a and S. Silver b
’ Flemish Institute for Technological Research (VITO), Laboratory of Genetics and Biotechnology, Mol, Belgium, and
b University of Illitwis at Chicago, Department of Microbiology and Immunology College of Medicine, Chicago, Illinois, USA
(Received 18 February 1993; revision received 20 March 1993; accepted 5 April 1993)
Abstract: pC101, a novel shuttle vector between Escherichia coli and Staphylococcus aureus carrying the lux genes encoding
luciferase from vibrio harueyi, selectable ampicillin and chloramphenicol markers and origins of replication for Gram-negative and
Gram-positive bacteria has been constructed. The inducibility of the arsenic and cadmium operon from S. aureus plasmid ~I258 to
different ions has been tested in E. coli and in S. aureus with two fusions in pC101: an arsB-1uxAB and a cadA-1uxAB
transcriptional gene fusion. Patterns of induction are influenced by the host strain and are slightly different from previous reports
using the b1a.Z gene as reporter gene.
Key words: Staphylococcus aureus; Shuttle plasmid; Luciferase genes; Arsenic; Cadmium; Resistance operon
Introduction
Plasmid ~1258 from Staphylococcus aureus
contains the 3.5-kb cad operon conferring resistance to cadmium and zinc [ll and the 2.7-kb ars
operon conferring resistance to arsenate, arsenite
and antimony [2]. The arsenic efflux resistance
operon consists of three genes in the following
order: arsR, a regulatory gene; arsB, whose gene
product is a membrane protein; and arsC, encoding an arsenate reductase converting intracellular
Correspondence to: P. Corbisier, Flemish Institute for Technological Research (VITO), Laboratory of Genetics and
Biotechnology, Boeretang 200, B-2400 Mol, Belgium.
arsenate to arsenite [2,3]. The cadA resistance
operon contains two genes, the first cadC gene
encodes a soluble protein and the second cadA
gene encodes a membrane ATPase protein. CadA
protein is a member of a protein family known as
P-type ATPases 11,451. Such a protein is not
encountered in cadmium resistance observed in
Gram-negative bacteria although resistance proceeds also through cation efflux [6]. CadA is not
sufficient to confer full resistance to cadmium
and zinc; cadC must also be present [7]. The
function of CadC in Cd2+ efflux is not clear and
the regulator of the cadA operon has not been
identified [8,9]. Gene expression studies in S.
aweuS or Bacillus subtilis have been commonly
realized by gene fusion with the S. aureus blaZ
232
[2,7,10] or cat genes [11] encoding fl-lactamase
and chloramphenicol acetyltransferase, respectively, lux genes encoding luciferase used as reporter genes have been shown to be extremely
valuable in determining gene expression [12] and
provide a very sensitive, non-destructive alternative assay already successfully applied in B. subtilis [13,14].
In this work, we describe the construction of a
novel shuttle vector between Escherichia coli and
S. aureus containing the luxAB genes from Vibrio
harveyi as reporter genes. An arsB-luxAB and a
c a d A - l u x A B transcriptional fusion were constructed and used to study the regulation o f the
ars and cad operons.
Chemicals
Sodium arsenate, sodium arsenite and antimony potassium tartrate were used as oxyanions
(from Sigma Chemical Co, St. Louis, MO). Bismuth sodium tartrate was from RSA Corp. (Ardsley, NY). Cadmium, cobalt, mercury and lead
chloride were from Aldrich Chemical Co.
(Bornem, Belgium).
Plasmid D N A preparations
Mini-preparation of plasmid DNA from E.
coli was done by alkaline lysis method [15] and
rapid isolation of plasmid DNA was done by the
boiling method [16]. Mini-preparation and rapid
isolation of plasmid DNA from S. aureus were
described before [7].
D N A manipulations
Materials and Methods
Bacterial strains
The bacterial strains and plasmids used in this
study are listed in Table 1. Cells were grown in
Luria broth [15] with ampicillin (50/zg ml-1) or
chloramphenicol (5/~g ml-1), when required. Cell
growth was monitored by measuring the culture
turbidity in a OD photometer.
Restriction endonuclease digestion of DNA
was carried out following the manufacturer's instructions. Conversion of 5' or 3' protruding termini to blunt end was carried out with either
DNA polymerase I (Klenow fragment) or T4 DNA
polymerase respectively. Calf intestinal alkaline
phosphatase was used to remove 5'-phosphate of
the digested vector DNA to minimize self ligation
Table 1
Bacterial strains and plasmids
Strains, plasmids
E. coli
HB101
DH10B
S. aureus RN4220
pQF70
pSK265
pGJ103
pGJ501
pGNll4
pC101
pC100
pC200 . . . .
pC300
!,
Genotype or description
Reference or source
supE44 hsdS20(r~ m ~ )recA13ara-14 proA2
lacYI galK2 rpsL2Oxyl-5 mtl-1
F - mcrA A(mrr-hsd RMS-mcr BC)lacZA M 15
A lacX 74 endA1recAl deoR A( ara, leu) araD
galUgalKnupGrpsL
Efficient acceptor of E. coli DNA
luxAB shuttle vector
pC194 derivative with MCS from pUC19
Intact 2.7-kb pi258 ars in pUC19
transcription arsB-luxAB fusion in pQF70
3.5 kb cad.4 cloned into pSK265
E. coli-Pseudomonas-Staphylococcus shuttle
vector containing luxAB genes
Same as pC101 but with luxAB in the opposite
direction of luxAB genes
transcriptional arsB-luxAB fusion in pC101
transcriptional cadA -luxAB fusion in pC101
15
24
25
18
17
2
2
1
This work
This work
This work
This work
233
during subsequent steps. Other techniques were
followed as described [15].
Identification of the cadC-luxAB clones
A 21 long oligonucleotide probe (5'-TCq'TAGGTGTTACGATAGCAA-3') corresponding to
366-386 nt of the cadA operon sequence of Nucifora et al. [1] was labelled at the 5' end with the
T4 polynucleotide kinase with [32p]-7-ATP.
Amp R clones were grown overnight on a Biodyne
Nylon membrane (Pall Biosupport, East Hills,
Bacterial transformation
Competent E. coli HB101 and DH10B cells
were transformed by the CaC12 method [15] and
competent S, aureus RN4220 cells were electroporated [17].
A
~c°RI*~HindlII
R
~c°RI/~alI
_ 1-1indllI
~ v ~ I " - - I ; AORFD
ogC°~
I]
~ORF?~
~ ( ORFA
10.C326~
~Odc~iaureu s
lux A B / /
[PvulIl
" '~ *"
*"
~
~
C
~c°RI'*
~ ~
~
~ Z ~
~.
I I I I I I I I I I I I I I I I
MCS
cad
j,,
~c//bla
.-'~
[Hindlll]
cadC " ~ /
AcadA k'~
51nt \ ~
/
~oriPseudo
~)
NdeI ~ !
[HindlIl]
ori E. Coli
AORFD
o
tl
pC300
10.18 Kb
!l
/I
~t,,\
S. aure,,
\ ~ cat
lux AB / l
/I
~//
Fig. 1. Genetic maps of constructed plasmids. (A) Vector pC101 containing luxAB genes from Vibrioharveyi and the native pMB1
(o) and pRO1600 (e) origins of replication from pQF70 [18] and the additional cloned origin of replication of plasmid pSK265 [17],
resistance genes for chloramphenicol(cat) and ampiciilin(bla). EcoRI ** indicates the presence of a T1 transcription terminator
which prevents read-through from upstream promoters into the luxAB gene. * Non-unique restriction sites in the multiple-cloning
site (MCS). (B) pC200 contains the transcriptional arsB-lux,4B fusion. (C) pC300 contains the transcriptional cadA-luxAB fusion.
234
NY) and hybridized with the probe according to
the manufacturer's instructions.
Results a n d D i s c u s s i o n
Construction of a shuttle vector between E. coli
and S. aureus containing the lux.4B reporter genes
This vector was constructed by cloning the
replication origin of staphylococcal vector pSK265
into the Gram-negative promoter expression vector pQF70 [18] containing the luxA and luxB
genes of Vibrio harveyi, lacking a transcriptional
promoter, but each witff its own ribosomal binding site for protein synthesis initiation and lacking
the genes luxCDE for reduction of fatty acids
into aldehyde and therefore dependent upon exogenously added aldehyde. Plasmid pQF70 was
cut with PvuII, dephosphorylated and ligated with
pSK265 cut with HindIII (to remove its multiple
cloning site) and blunt-ended. The ligation product was transformed into competent E. coli
HB101 cells. Transformants were selected on
ampicillin containing LB plates followed by
checking the plasmid size on a 0.8% agarose
electrophoresis gel. The orientation of the 2.7-kb
Luciferase activity assay
Overnight cultures of cells containing the
arsB-lur,A B or the cadA-luxAB fusion plasmids
were washed twice with LB broth, suspended in
LB and grown at 37°C to mid-log phase. Cells
were induced by addition of increasing amounts
of oxyanions or other cations for 60 to 120 min at
37°C. Uninduced cells were grown for the same
period of time in the absence of heavy metal ions.
Cells were diluted if necessary with ice-cold LB
and 25 /zM n-decyl aldehyde (n-decanal, Sigma)
was added to the sample. After vortexing for 3 s,
the samples were immediately counted using the
3H channel in a Packard Tri-Carb liquid scintillation spectrometer (Packard Instrument Co.,
Downers Grove, IL) for 1 rain with a detection
region selected between 0 and 2000 keV. The
luciferase specific activity was defined as counts
per min (cpm) per OD66o unit.
300
25000"
A
E coli
B
S. aureus
250
200OO
200
~'=
,,ooo
i50
~0
Ioooo
100
I
5000
50
0
0,1
1
i0
100
1000
0,1
1
10
I00
1000
I n d u c e r i o n 0JM)
Fig. 2. Expression of the a r s B - l u x , 4 B fusion in (A) E. coli and in (B) S. aureus. E. coli HB101 and S. aureus RN4220 c e l l s
containing plasmid pC200 were grown and induced by the addition of indicated amounts of A s O ~ - , AsO~-, SbO~-, Bi 3+, and
Mn2+ at 37°C for 70 min. The specificluciferase activitywas measured as described in Materials and Methods. I , arsenate; D,
arsenite; A, antimonite; a, manganese; ©, bismuth.
235
pSK265 insert was checked by double digestion
with PvulI and HindlII. Plasmids containing inserts in both orientations were obtained and
termed pC100 and pC101, respectively (Fig. 1A).
Both plasmids were electroporated into S. aureus
strain RN4220. The stability of pC101 in E. coli
and S. aureus was determined by growing the
cells non-selectively for ten generations. 99% of
E. coli and 100% of S. aureus cells retained the
plasmid.
Construction o f a transcription arsB-luxAB fusion
using pClO1
The intact S. aureus 2.7-kb SalI ars determi-
nant previously cloned into pUC19 forming
pGJ103 [2] was digested with SalI and HindIII to
generate two fragments of 1.262 kb and 1.438 kb.
The smaller fragment containing the promoter,
the intact arsR and a partial deletion of arsB
(first 676 nt corresponding to 225 amino acids)
was isolated by electro-elution from a 0.8%
agarose gel and ligated with pC101, cut with SalI
and HindIII and dephosphorylated. The ligation
product was transformed in competent E. coli
HB101 and ampicillin-resistant clones were selected. The cloned fragment was checked by size
on a 0.8% agarose gel. Three clones were obtained all containing the same arsB-luxAB fusion. This construct was named pC200 (Fig. 1B).
Plasmid pC200 was electroporated into S. aureus
RN4220 and chloramphenicol-resistant clones
containing the pC200 were selected.
Induction o f the arsB-luxAB fusion in E. coli and
S. aureus
E. coli HB101 (pC200) and S. aureus RN4220
(pC200) were induced as described in Materials
and Methods in the presence of increasing concentrations of arsenite, arsenate, antimonite,
manganese and bismuth. The patterns of induction (Fig. 2A) in E. coli HB101 were similar to
those observed by Ji and Silver [2] using a similar
construction pGJ501, a 774-nt D N A fragment
containing the intact arsR and the first 188 nt of
arsB cloned into pQF70. Arsenite was the
stronger inducer followed by arsenate and bismuth. The maximum light emission was obtained
at 10 /xM arsenite. Arsenite, arsenate, bismuth
and antimonite salts were all inducers of the S.
aureus pi258 ars operon as shown with an arsBlacZ fusion in S. aureus [2] and inducers of the E.
coli R773 ars operon as shown by Wu and Rosen
[19] with an arsD-bla gene fusion in E. coli
HBI01. However, we currently do not know why
antimonite did not function as inducer in either
arsB-lux construction on pGJ501 and pC200
plasmid.
When the pC200 plasmid was placed into S.
aureus RN4220 (Fig. 2B), the system was induced
only by arsenite. This arsenite-specific response is
different from pattern of induction for reduced
arsenic uptake by the intact plasmid [20] or using
a different 'reporter' gene with arsB-blaZ gene
fusions in E. coli HB101 [2]. The arsenite-specific
bioluminescent response may be related to the
70 x lower response of the ars-lux fusion in S.
aureus. A lower level of bioluminescence in
Gram-positive genera compared to Gram-negative genera w a s already reported before and explained as a consequence of an inadequate capacity to p r o d u c e / g e n e r a t e reduced flavin mononucleotide in Gram-positive bacteria [13]. It is interesting to note that the S. xylus pSX267 ars operon,
highly homologous to the pi258 ars operon [21] is
also induced by arsenite as shown with an arsB-lip
gene fusion in S. carnosus [22].
The vector plasmid pC101 without insert did
not produce detectable light regardless of addition of heavy metals (data not shown). No measurable light could either be observed with plasmid pC200 when n-decyl aldehyde was not added
to the assay mixture.
Construction o f a transcription c a d A - l ~
fusion
using pClO1
The S. aureus cadA 3.5-kb fragment previously
cloned into plasmid pSK265 forming pGN114 [1]
was digested with Sau96I to generate a 1085-bp
fragment. This fragment containing the promoter,
the intact cadC and the first 51 nt of cadA
corresponding to 17 amino acids was isolated
from a 1% agarose gel, blunt-ended and ligated
with vector pC101 cut with HindIII, blunt-ended
and dephosphorylated. Competent E. coli DH10B
were transformed with the ligation product and
ampicillin-resistant clones were screened by hy-
236
bridization with the 21-nt oligonucleotide radioactive probe (data not shown). The cloned
fragment was also checked by size on a 0.8%
agarose gel. Three clones were obtained containing the desired cadA-lux.4B fusion. Clone 3 was
further analyzed and the construct was termed
pC300 (Fig. 1C). pC300 was electroporated in S.
aureus RN4220 cells and chloramphenicol-resistant clones all contained pC300.
Induction o f the c a d A - l u r A B fusion in E. coli and
S. aureus
The induction of the c a d A - l u x A B fusion by
heavy metals was first tested in E. coli DH10B as
described in Materials and Methods. The system
was only lightly induced by cadmium, bismuth
and lead (Fig. 3A). Other E. coli strains (HB101,
C600, S 1 7 / 1 ) w e r e electroporated with the pC300
and the inducibility of the c a d A - l u x A B fusion
tested with c a d m i u m as inducer (data not shown).
Different Cd2+-induced levels of bioluminescence
were observed depending on the E. coli host
strain: e.g. in E. coli HB101 ceils the bioluminescence levels were about 66 x higher than with
DH10B cells, but the Cd2+-induced/non-induced
bioluminescence ratio remained poor (data not
shown).
In S. aureus RN4220 (pC300), the c a d A - l u x A B
fusion showed a low background in the presence
of any of the inducers (Fig. 3B). C d 2+ w a s found
to be the most efficient inducer, although higher
levels of C d 2+ w e r e inhibitory. (Note that the
strain RN4220(pC300) is cadmium-sensitive, since
it lacks most of the cad.4 operon.) At high Bi 3+
and P b 2÷ concentrations c a d A - l u x A B fusion was
also induced, but at a lower level. This has to be
related to earlier reports on the genetics of plasmid pi258 indicating a gene for marginal resistance to bismuth and lead salts mapping between
bla and cadA [23]. C o 2+, Z n 2+, and Mn 2+ did not
induce the c a d A - l u x A B fusion significantly even
when the cells were exposed to high concentrations of Co 2+, Zn 2+, and Mn 2÷ in the experiments. Yoon et al. [8] constructed a translational
c a d A - b l a Z fusion with the same first 51 nt from
5' end of cadA and also report a /3-1actamase
activity when Cd 2÷, Bi 3÷ and Pb 2÷ were used as
inducers. However, some differences between the
500 1
1200
A
E. coli
I
1000
B
S. aureus
400
!
8®
3O0
_~'r,
600
200
400
100
0,1
1
10
100
1000 0
0,I
1
I0
100
1000
Inducer ion (/aM)
Fig. 3. Expression of the cadA-htrAB fusion in (A) E. coli and in (B) S. aureus. E. coli HB101 and S. aureus RN4220 cells
containing plasmid pC300 were grown and induced by the addition of indicated amounts by the addition of indicated levels of ions
at 37°C for 60 rain and 120 min, respectively.The specific luciferase activitywas measured as described in Materials and Methods.
<3, cadmium; A, bismuth; zx, lead; D, zinc; I1, manganese; e, mercury.
237
p K P Y 1 0 0 a n d t h e pC300 i n d u c t i o n p a t t e r n s w e r e
o b s e r v e d . T h e C d 2+ c o n c e n t r a t i o n giving t h e
m a x i m u m /3-1actamase activity was 10 x lower
t h a n t h e C d 2+ c o n c e n t r a t i o n giving t h e m a x i m u m
light emission. T h e C d 2 + - i n d u c e d light e m i s s i o n
was m u c h h i g h e r t h a n t h e v a l u e o b t a i n e d for
Bi 3+ a n d Pb 2+, w h e r e a s t h e m a x i m u m fl-lactam a s e activity levels w e r e a b o u t t h e s a m e for
C d 2÷, Bi 3+ a n d Pb 2÷. B o t h d i f f e r e n c e s can b e
e x p l a i n e d if t h e m a x i m a l /3-1actamase activity is
s o m e h o w l i m i t e d in t h e / 3 - 1 a c t a m a s e assay so t h a t
/3-1actamase activities b e c a m e u n d e r e s t i m a t e d .
T h e m a x i m u m light e m i s s i o n was o b s e r v e d at 20
/xM C d 2÷, a n d to o b t a i n a s i g n a l / n o i s e r a t i o o f 2
(as c h o s e n limit o f d e t e c t i o n ) 0.5 /xM C d 2÷ (56
p p b ) is r e q u i r e d . E x p o s u r e o f RN4220(pC300)
cells with C d 2÷, Bi 3+, o r Pb 2÷ d u r i n g 120 m i n
i n s t e a d o f 60 m i n a p p r o x i m a t e l y d o u b l e d t h e
m a x i m u m light e m i s s i o n ( d a t a n o t shown) b u t a
180-min C d 2+ e x p o s u r e d i d n o t f u r t h e r i n c r e a s e
t h e light e m i s s i o n ( d a t a n o t shown). N o m e a s u r a b l e light c o u l d e i t h e r b e o b s e r v e d with pC300 if
no n - d e c a n a l was a d d e d to t h e l u c i f e r a s e assay
mixture.
T h e shuttle v e c t o r pC101 c o n t a i n i n g t h e luxAB
as r e p o r t e r g e n e s is a useful tool to d e t e c t p r o m o t e r activities in a w i d e r a n g e o f G r a m - n e g a t i v e
a n d G r a m - p o s i t i v e b a c t e r i a , pC101 c o n t a i n s ind e e d origins o f r e p l i c a t i o n allowing self r e p l i c a tion in E s c h e r i c h i a coli ( p M B 1 ) s t a p h y l o c o c c i
( r e p C ) a n d P s e u d o m o n a s ( p R O 1 6 0 0 ) . T h e differe n t inducibility p a t t e r n s o f pC200 a n d pC300 in
E. coli a n d S. aureus a r e n o t c o m p l e t e l y u n d e r s t o o d a n d a r e p r o b a b l y l i n k e d to t h e d i f f e r e n t
c e l l u l a r b a c k g r o u n d . T h e c a d A - l u x A B fusion can
b e f u r t h e r u s e d to test t h e r e g u l a t i o n o f t h e c a d A
o p e r o n . T h e specificity a n d sensitivity p r o p e r t i e s
o f t h e c a d A - l u x A B a n d t h e a r s B - l u x A B fusions
can b e f u r t h e r e x p l o i t e d to c o n s t r u c t b a c t e r i a l
b i o s e n s o r s for analytical o r e n v i r o n m e n t a l p u r poses.
Acknowledgements
W e t h a n k S.A. K h a n a n d T. M i s r a for suggestions d u r i n g t h e c o u r s e o f this w o r k a n d J.-M.
N u y t e n for t h e o l i g o n u c l e o t i d e synthesis. T h e ex-
periments reported here were supported by grants
f r o m t h e U S D e p a r t m e n t o f E n e r g y ( E n e r g y Biosciences P r o g r a m ) a n d f r o m t h e V l a a m s A c tieprogramma Biotechnologie (VLAB-ETC-003).
References
1 Nucifora, G., Chu, L., Misra, T.K. and Silver, S. (1989)
Cadmium resistance of Staphylococcus aureus plasmid
pi258 results from a Ca2+ efflux ATPase determined by
the cadA gene. Proc. Natl. Acad. Sci. USA 86, 3544-3548.
2 Ji, G. and Silver, S. (1992) Regulation and expression of
the arsenic resistance operon from Staphylococcus aureus
plasmid pi258. J. Bacteriol. 174, 3684-3694.
3 Ji, G. and Silver, S. (1992) Reduction of arsenate to
arsenite by the ArsC protein of the arsenic resistance
operon of Staphylococcus aureus plasmid pi258. Proc.
Natl. Acad. Sci. USA 89, 9474-9478.
4 Silver, S., Nucifora, G., Chu, L. and Misra T.K. (1989)
Bacterial resistance ATPases: primary pumps for exporting toxic cations and anions. Trends Biochem. Sci. 14,
76-80.
5 Tsai, K.J., Yoon, K.P. and Lynn, A.R. (1992) ATP-dependent cadmium transport by the staphylococcal cad.4 determinant in everted membrane vesicles of Bacillus subtilis. J.
Bacterioi. 174, 116-121.
6 Mergeay, M. (1991) Towards an understanding of the
genetics of bacterial metal resistance. Trends Biotechnol.
9, 17-24.
7 Yoon, K.P. and Silver, S. (1991) A second gene in the
Staphylococcus aureus cad.4 cadmium resistance determinant of plasmid pi258. J. Bacteriol. 173, 7636-7642.
8 Yoon, K.P., Misra, T.K. and Silver, S. (1991) Regulation of
the cadA cadmium resistance determinant of Staphylococcus aureus plasmid pi258. J. Bacteriol. 173, 7643-7649.
9 Silver, S. and Walderhaug (1992) Gene regulation of plasmid- and chromosome-determined inorganic ion transport
in bacteria. Microbiol. Rev. 56, 195-228.
10 Chu, L., Mukhapadhayay, D., Yu, H., Kim, K.-S. and
Misra, T.K. (1992) Regulation of the Staphylococcus aureus plasmid pi258 mercury resistance operon. J. Bacteriol. 174, 7044-7047.
11 Goldfarb, D.S., Doi, R.H. and Rodriguez, R.L. (1981)
Expression of Tn9-derived chloramphenicol resistance in
Bacillus subtilis. Nature 293, 309-311.
12 Stewart, G.S.A.B. and Williams, P. (1992) lux genes and
the applications of bacterial bioluminescence. J. Gen. Microbiol. 138, 1289-1300.
13 Karp, M. (1989) Expression of bacterial luciferase genes
from Vibrio harveyi in Bacillus subtilis and in Escherichia
coli. Biochim. Biophys. Acta 1007, 84-90.
14 Jacobs, M., Hill, P.H. and Stewart, G.S.A.B. (1991) Highly
bioluminescent Bacillus subtilis obtained through high-level
expression of a luxAB fusion gene. Mol. Gen. Genet. 230,
251-256.
238
15 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1989) Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY.
16 Holmes, D.S. and Quigley, M . (1981) A rapid boiling
method for the preparation of bacterial plasmids. Anal.
Biochem. 114, 193-197.
17 Jones, C.L. and Khan, S.A. (1986) Nucleotide sequence of
the enterotoxin B gene from Staphylococcus aureus. J.
Bacteriol. 166, 29-33.
18 Farinha, M.A. and Kropinski, A.W. (1990) Construction of
broad-host-range plasmid vectors for easy visible selection
and analysis of promoters. J. Bacteriol. 172, 3496-3499.
19 Wu, J. and Rosen, B.P. (1993) Metalloregulated expression of the ars operon. J. Biol. Chem. 268, 52L58.
20 Silver, S. and Keach, D. (1982) Energy-dependent arsenate
efflux: the mechanism of plasmid-mediated resistance.
Proc. Natl. Aead. Sci. USA 79, 6114-6118.
21 G6tz, F., Zabielski, J., Philipson, L. and Lindberg, M.
(1983) DNA homology between arsenate resistance plasmid pSX267 from Staphylococcus xylosus and the penicillinase plasmid pi258 from Staphylococcus aureus.
22 Rosenstein, R., Peschel, A.; Wieland, B. and G6tz, F.
(1992) Expression and regulation of the antimonite, arsenite, and arsenate resistance operon of Staphylococcus
xy/osus plasmid pSX267. J. Bacteriol. 174, 367-368,
23 Novick, R.P., Murphy, E., Gryczan, T.J., Baron, E. and
Edelman, I. (1979) Penicillinase plasmid of Staphylococcus
aureus: restriction-deletion maps. Plasmid 2, 109-129.
24 Lorow, D. and Jesse, J. (1990) Gibco Focus 12, 19.
25 Kreiswirth, B.N., Lofdahl, M.J., O'Reilly, M., Schlievert,
P.M., Bergdoll, M.S. and Novick, R.P. (1993) The toxic
shock syndrome exotoxin structural gene is not detectably
transmitted by a prophage. Nature (London) 305, 709-712.