The two-component system ArlS–ArlR is a regulator of virulence gene expression in Staphylococcus aureus

The two-component system ArlS–ArlR is a regulator ofvirulence gene expression in Staphylococcus aureus

Benedicte Fournier,* Andre Klier and

Georges Rapoport

Unite de Biochimie Microbienne, URA 2172 du Centre

National de la Recherche Scientifique, Institut Pasteur, 25,

rue du Docteur Roux, 75724 Paris Cedex 15, France.

Summary

Staphylococcus aureus is a major human pathogen

that produces many virulence factors in a temporally

regulated manner controlled by at least two global

virulence regulatory loci (agr and sarA ). We identified

previously a two-component system, ArlS–ArlR, that

modifies the activity of extracellular serine protease

and may be involved in virulence regulation. Here, we

show that mutations in either arlR or arlS increase

the production of secreted proteins [a-toxin (Hla), b-

haemolysin, lipase, coagulase, serine protease (Ssp)]

and especially protein A (Spa). Furthermore, the

pattern of proteins secreted by both mutants was

strikingly different from that of the wild-type strain.

Transcriptional fusions showed that expression of

hla, ssp and spa was higher in both mutants than in

the wild-type strain, indicating that the arl operon

decreases the production of virulence factors by

downregulating the transcription of their genes. The

arl mutation did not change spa expression in an agrA

mutant or in a sarA mutant, suggesting that both the

sarA and the agr loci are required for the action of arl

on spa. Northern blot analyses indicated that the arl

mutation increased the synthesis of both RNA II and

RNA III, but decreased sarA transcription. Finally, arl

was not autoregulated, but its expression was

stimulated by agr and sarA. These results suggest

that the Arl system interacts with both agr and sarA

regulatory loci to modulate the virulence regulation

network.

Introduction

Staphylococcus aureus is a major human pathogen that

causes a wide spectrum of infections, from superficial

lesions to systemic and life-threatening infections, such as

osteomyelitis, endocarditis, pneumonia and septicaemia.

S. aureus produces a range of virulence factors that

contribute to its pathogenicity (Novick, 2000). These

factors include secreted proteins, such as serine protease

(Ssp), nuclease, haemolysins [a-toxin (Hla), b-haemolysin],

enterotoxins, lipase and coagulase, and proteins exposed

on the cell surface, such as protein A (Spa) and fibrinogen-,

fibronectin- and collagen-binding proteins.

These factors are produced co-ordinately in a growth

phase-dependent manner in vitro and are controlled by the

regulator genes agr (Morfeldt et al., 1988; Peng et al.,

1988), sarA (Cheung et al., 1992; Cheung and Projan,

1994), sae (Giraudo et al., 1994), sarH1 (Tegmark et al.,

2000), rot (McNamara et al., 2000) and srrA–srrB

(Yarwood et al., 2001).

The agr locus, which consists of five genes (agrB, agrD,

agrC, agrA and hld ), encodes two divergent transcripts

(RNA II and RNA III), the synthesis of which is initiated by

two different promoters, P2 and P3 respectively. RNA III,

which overlaps the hld open reading frame (ORF), is the

effector molecule of the agr locus (Novick et al., 1993;

Morfeldt et al., 1995). RNA III synthesis is highly

dependent on the activation of the agrBDCA genes,

which are co-transcribed on RNA II. AgrA and AgrC

constitute the response regulator and histidine kinase

components, respectively, of a two-component system

(Novick et al., 1995). RNA III synthesis is regulated by

quorum sensing. Bacteria produce and secrete inducer

molecules that trigger RNA III synthesis when they reach a

threshold concentration. Known autoinducers of RNA III

include the agr-encoded autoinducing peptides (AIP;

encoded by agrD and possibly processed by agrB )

(Balaban and Novick, 1995; Ji et al., 1995) and the

potential RNA III-activating protein (RAP) (Balaban and

Novick, 1995) (Fig. 1).

The second global regulatory locus, sar, located within a

1.2 kb fragment, contains the major 372 bp sarA ORF that

encodes the 15 kDa protein SarA (Cheung and Projan,

1994). This ORF is transcribed by three overlapping

transcripts (sarA, sarC and sarB ) (Fig. 1). These

transcripts have a common 30 end, but are initiated from

three different promoters, sarA (580 bp) at P1, sarC

(840 bp) at P3 and sarB (1150 bp) at P2 (Bayer et al.,

1996; Manna et al., 1998) (Fig. 1).

Tegmark et al. (2000) showed that another gene, sarH1,

is involved in the regulation of virulence factors in

S. aureus. sarH1 encodes a 29 kDa protein, consisting of

two homologous halves that display a high degree ofAccepted 30 April, 2001. *For correspondence. E-mail [email protected]; Tel. (133) 1 45 68 88 09; Fax (133) 1 45 68 89 38.

Molecular Microbiology (2001) 41(1), 247–261

Q 2001 Blackwell Science Ltd

similarity to SarA. The interaction between agr, sarA and

sarH1 in the co-ordinated regulation of secreted virulence

factors and cell wall adhesins of S. aureus is complex. The

transcription of genes encoding secreted proteins (a-toxin,

serine protease) is activated by RNA III, whereas that

of genes encoding cell wall-associated proteins, such as

protein A, is repressed by RNA III (Novick, 2000). SarA

activates a-toxin gene transcription, but represses

transcription of the genes for serine protease and protein

A (Cheung and Ying, 1994; Chan and Foster, 1998)

(Fig. 1). SarA acts partly through the agr regulatory

pathway by binding to agr promoters, stimulating the

transcription of agr (Heinrichs et al., 1996; Morfeldt et al.,

1996; Chien and Cheung, 1998; Rechtin et al., 1999).

However, SarA also affects the expression of certain

virulence genes directly, independently of its effect on agr.

Thus, SarA is a general transcriptional factor, one of the

targets of which is the agr locus (Blevins et al., 1999; Wolz

et al., 2000). Finally, SarH1 is responsible for the agr-

and sarA-dependent repression of protein A synthesis

(Tegmark et al., 2000) (Fig. 1).

The SrrA–SrrB two-component system regulates the

production of both exotoxin (toxic shock syndrome toxin 1)

and surface-associated (protein A) virulence factors in

response to environmental oxygen levels. This regulation

is mediated in part by the agr locus. SrrA–SrrB may act in

anaerobic repression of staphylococcal virulence factors

(Yarwood et al., 2001).

Yet another locus, sae, which also encodes a two-

component system, stimulates the production of a-toxin,

b-haemolysin and coagulase by a pathway that does not

involve agr or sarA (Giraudo et al., 1994; 1996). Thus, the

production of individual virulence factors seems to depend

on the activities of at least five different regulators that

interact to stimulate or repress target gene transcription.

We recently identified a new two-component system,

ArlS–ArlR (Fournier and Hooper, 2000) involved in

several cell activities. Production of a multidrug resistance

efflux pump, NorA, was increased in an arlS transposon

insertion mutant (Fournier et al., 2000). The arlS mutant

exhibited dramatic autolysis as a result of increased

peptidoglycan hydrolase activity. The arlS mutant formed

a biofilm on polystyrene surfaces, probably because of

altered activity of secreted peptidoglycan hydrolases.

Serine protease activity was low in the arlS mutant,

suggesting that this two-component system is involved in

the regulation of virulence factor production in S. aureus

(Fournier and Hooper, 2000).

In this study, we determined the effect of the ArlS–ArlR

system on the production of virulence factors. We

demonstrate that the arl locus is involved in the regulation

of several virulence factors, mainly protein A, and some

Fig. 1. Model of the interactions between regulators and virulence factor genes. Dotted lines represent the transcripts of agr and sar. Thick blacklines represent genes. Arrows and perpendicular bars indicate positive and negative regulation respectively. AIP, autoinducing peptide. Details ofthe model can be found in the text. Dashed lines indicate the probes used for Northern blot hybridization.

248 B. Fournier, A. Klier and G. Rapoport

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 247–261

secreted proteins (a-toxin, b-haemolysin, coagulase,

lipase). The arl locus exerts its effects on virulence factors

mostly via the agr and/or sarA regulatory pathway.

Results

Construction of an arlR mutant

We studied previously the effects of the arlR–arlS locus on

adhesion, autolysis and extracellular proteolytic activity in

a mutant containing a Tn917 LTV1 insertion in the arlS

gene (Fournier and Hooper, 2000; Fig. 2). To study the role

of arlR–arlS in the regulation of virulence gene

expression, we constructed an arlR mutant in which part

of arlR was deleted and replaced by the cat gene (see

Experimental procedures ). The deletion resulted in the

elimination of 126 amino acids (57%) from the predicted

ArlR protein (Fig. 2A). This mutation was then transferred

to the wild-type strain, RN6390, and to other S. aureus

strains (Table 1).

To check that arlR was inactivated, total RNA from wild-

type (RN6390), and arlR mutant (BF21) strains was

analysed by Northern blotting, using two specific probes

(one for arlR and one for arlS ) (Fig. 3). Both probes

hybridized with a 2.7 kb RNA fragment, the expected size

of a combined arlR–arlS transcript. This suggested that

arlR and arlS are in a bicistronic operon. Neither arlR or

arlS transcripts were detected in the arlR mutant,

confirming that arlR was deleted and that replacement of

arlR by the cat gene, which is transcribed divergently, had

a polar effect on the transcription of arlS. Therefore, the

arlR deletion mutant was named the arlRS mutant. A

second 1.5 kb band that was detected in the wild-type

strain, but not in the arlRS mutant, might result from the

use of an alternative promoter or terminator site or from

processing of the 2.7 kb transcript.

Thus, two different mutants (arlS – and arlRS –) were

used to characterize the effects of the arlR–arlS locus on

virulence factor gene expression.

Phenotypic characterization of arlS and arlRS mutants

The effect of inactivation of arlR or arlS on different virulence

factor proteins (secreted and cell wall associated) is

shown in Table 2.

The level of concentration of total extracellular proteins

was three or four times higher in the arlS and arlRS

mutants (BF24, BF26) than in the parent strain (BF23)

(Table 2). To avoid autolysis, we used strains carrying the

atl mutation. Atl is the main autolysin in S. aureus (Foster,

1995). S. aureus strains carrying both arlS and atl

mutations have a very low rate of autolysis, as does the

single atl mutant (Fournier and Hooper, 2000). In strains

containing a transcriptional fusion between the atl

promoter region and lacZ (BF23, BF24 and BF26), b-

galactosidase activity was similar in the culture super-

natants of the parent strain (BF23) and the two mutants

(BF24 and BF26; data not shown), indicating that the

increased release of extracellular proteins was not the

result of bacterial lysis. Furthermore, the inactivation of

arlR and arlS resulted in different patterns of extracellular

proteins (Fig. 4A). In both arlS and arlRS mutants, the

levels of most proteins were higher than those in the wild

type, but the levels of some proteins were lower

(particularly those smaller than 31 kDa). The wild-type

pattern was restored in the mutants complemented with

arlRS (BF25 and BF27).

The activity of several secreted proteins was assessed

Fig. 2. Organization of the arlR–arlS locus.A. Map of the arlR–arlS locus showing the transcription start site (bent arrow) and the putative terminator (hairpin). ORFs are indicated by thickarrows. Numbering is as for the published sequence (Fournier and Hooper, 2000). The black box indicates the replacement of arlR by cat. The site ofTn917 LTV1 insertion is also indicated. Dashed lines indicate the probes used for Northern blot hybridization.B. DNA sequence of the arl promoter region containing the transcription start site as determined by primer extension analysis; the putative 235 and210 sequences are boxed.

Virulence regulation in Staphylococcus aureus 249


Table 1. Bacterial strains and plasmids used in this study.

Strain or plasmid Genotype or characteristicsa Source or reference

StrainsS. aureus

ISP225 Propagating strain of phage f55 (Ps55) Bannatine and Pattee (1996)ISP794 8325 pig-131 carrying prophages f11, f12 and f13 Stahl and Pattee (1983)8325-4 8325, UV cured of prophages f11, f12 and f13 Novick (1967)RN4220 8325-4, nitrosoguanidine-induced restriction mutant used Kreiswirth et al. (1983)

as primary recipient for plasmids propagated in E. coliDU1090 8325-4, pig-131 hla::em O’Reilly et al. (1986b)ISP2094 8325-4, pig-131 hlb::gen Stahl and Pattee (1983c)DU5723 8325-4, spa::EtBr Pattel et al. (1987b)RN6390 8325-4 agr 1 Peng et al. (1988)ALC488 RN6390 sarA::Tn551 Cheung et al. (1997d)RN6112 RN6390 agrA::Tn551 Peng et al. (1988); Kornblum et al. (1990d)KT200 RN4220 sarH1::pKT200 Tegmark et al. (2000e)BF19 RN6390 arlS::Tn917 LTV1 This studyBF20 RN6390 arlS::Tn917 LTV1 complemented with arlRS This studyBF21 RN6390 arlR::cat This studyBF22 RN6390 arlR::cat complemented with arlRS This studyBF23 RN6390 atl::lacZ This studyBF24 RN6390 atl::lacZ arlS::Tn917 LTV1 This studyBF25 RN6390 atl::lacZ arlS::Tn917 LTV1 complemented with arlRS This studyBF26 RN6390 atl::lacZ arlR::cat This studyBF27 RN6390 atl::lacZ arlR::cat complemented with arlRS This studyBF28 ISP794 atl This studyBF29 ISP794 atl::lacZ arlS::Tn917 LTV1 This studyBF31 RN6390 sarA::Tn551 arlS::Tn917 LTV1 This studyBF32 RN6390 sarA::Tn551 arlS::Tn917 LTV1 complemented with arlRS This studyBF33 RN6390 sarA::Tn551 arlR::cat This studyBF34 RN6390 agrA::Tn551 arlS::Tn917 LTV1 This studyBF35 RN6390 agrA::Tn551 arlS::Tn917 LTV1 complemented with arlRS This studyBF36 RN6390 agrA::Tn551 arlR::cat This studyBF37 RN6390 agrA::Tn551 sar::Tn917 LTV1 This studyBF38 RN6390 sarH1::pKT200 This studyBF39 RN6390 sarH1::pKT200 arlS::Tn917 LTV1 This studyBF40 RN6390 sarH1::pKT200 arlR::cat This study

E. coliTG1 F0traD36 lacI q D(lacZ )M15 proAB supE Gibson (1984)

D(hsdM-mcrB )5 (rk–mk

–McrB–) thi D(lac-proAB )DH5a F-f80dLacZDM15 D(lacZYA-argF )U169 deoR recA1 endA1 Gibco BRL

phoA hsdR17 (rk– , mk

–) supE44l – thi-1 gyrA96 relA1Plasmids

pSK950 10.5 kb shuttle plasmid carrying the att site of phage L54a, Niemeyer et al. (1996)replicon of pSC101, Spr (E. coli ) and Ts replicon of pE194, Tcr (S. aureus )

pSKarl 12.9 kb plasmid containing the arlR–arlS locus cloned into pSK950 Fournier and Hooper (2000)pI258 28 kb S. aureus plasmid carrying the b-lactamase gene (blaZ ); Emr; Apr Wang et al. (1987)pYL112D19 7 kb shuttle plasmid carrying the integrase Lee et al. (1991)

gene of phage L54a; Apr (E. coli ); Cmr (S. aureus )pBF50 12 kb shuttle promoterless transcriptional lacZ fusion vector carrying This study

the attP site of phage L54a; replicon of pMB1, Apr (E. coli );temperature-sensitive replicon of pE194, Tcr (S. aureus )

pBFSpa 300 bp fragment containing spa promoter This studycloned upstream of the lacZ gene of pBF50

pBFSsp 280 bp fragment containing ssp promoter This studycloned upstream of the lacZ gene of pBF50

pBFHla 270 bp fragment containing hla promoter This studycloned upstream of the lacZ gene of pBF50

pBFArl 300 bp fragment containing arlR-arlS promoter This studycloned upstream of the lacZ gene of pBF50

a . Em, erythromycin; Gen, gentamicin; EtBr, ethidium bromide; Ap, ampicillin; Tc, tetracyline; Cm, chloramphenicol; Sp, spectinomycin.b . Kindly provided by Timothy J. Foster (Trinity College, Dublin, Ireland).c . Kindly provided by John J. Iandolo (University of Oklahoma, Oklahoma City, OK, USA).d . Kindly provided by Ambrose L. Cheung (Hanover, NH, USA).e . Kindly provided by Staffan Arvidson (Karolinska Institute, Stockholm, Sweden).



in the wild-type strain and in the two mutants in two

different genetic backgrounds: ISP794, a derivative of

strain 8325, which carries three different prophages f11,

f12 and f13; and RN6390, a derivative of strain 8325-4,

which does not carry these prophages (Table 2). Interest-

ingly, the arlS mutation did not have the same effect in

these two backgrounds. In derivatives of strain 8325,

the levels of several secreted proteins (DNase, lipase and

protease) were significantly lower in the arlS mutant

(BF29) than in the parent strain (BF28), as demonstrated

previously for protease (Fournier and Hooper, 2000). The

level of extracellular b-lactamase, which is not a virulence

factor, was also much lower in the arlS mutant (one-fifth of

that in the wild type). In contrast, most of the tested

secreted virulence factors (a-toxin, b-haemolysin, lipase,

coagulase and serine protease) in derivatives of strain

8325-4 were synthesized in reproducibly greater amounts

in the arlS and arlRS mutants (BF24 and BF26

respectively) than in the parent strain (BF23), whereas

b-lactamase production was unaffected. Thus, in the

8325-4 strain background, only the production of virulence

factors was increased by the inactivation of arl genes,

whereas in the 8325 strain background, the production of

other secreted proteins was also impaired as a result of the

presence of either prophages or undefined mutations in

Fig. 3. Northern blot analysis of arlR (A) and arlS (B) transcripts inS. aureus strains RN6390 (wt) and BF21 (arlR –). hu (C) was used asan internal control for the amount of total RNA loaded (seeExperimental procedures ). Specific probes are indicated on the left,and sizes of the transcripts on the right.

Tab

le2.

Phenoty

pic

chara

cte

rizatio

nof

S.

aure

us

str

ain

scarr

yin

gth

eatl

muta

tion

a.

Pro

tein

Ad

(AU

mg

21

pro

tein

)

Str

ain

sG

enoty

pe

ab

-Lacta

mase

b

(mg

ml2

1)

Extr

acellu

lar

pro

tein

(mg

ml2

1)

a-T

oxin

c

(HU

)b

-Haem

oly

sin

c

(HU

)D

Nase

d

(AU

ml2

1)

Lip

ase

d

(AU

ml2

1)

Pro

tease

e

(AU

)C

oagula

se

dC

ell

wall-

associa

ted

Extr

acellu

lar

BF

23

atl

–4

36^

530^

311^

10.6

^0.2

14^

41

20

0.9

15

BF

24

atl

–arlS

–4

98^

13

115^

530^

31.0

^0.4

39^

13

21

100

13

390

BF

26

atl

–arlR

S–

ND

149^

2142^

29

101^

11.4

^0.5

40^

82

1100

7210

BF

28

fatl

–4

68^

8N

D6.0

^1.4

4.8

^1.2

68^

12

1,

11.3

10

BF

29

fatl

–arlS

–0.7

5173^

17

ND

5.5

^0.7

0.9

^0.3

34^

50

,1

8310

a.T

he

phenoty

pes

were

chara

cte

rized

as

described

inth

ete

xt.

Quantit

ativ

ere

sults

are

giv

en

as

am

ean

ofa

tle

asttw

oin

dependentdete

rmin

atio

ns.N

D,notd

ete

rmin

ed.A

sw

ehave

pre

vio

usly

stu

die

dth

eeff

ect

of

the

arl

muta

tion

in8325

derivativ

es

(Fourn

ier

and

Hooper,

2000),

we

com

pare

dtw

odiff

ere

nt

backgro

unds.

Str

ain

sB

F28

and

BF

29

are

derivativ

es

of

8325,

whic

hcarr

ies

the

thre

epro

phages,

where

as

str

ain

sB

F23,

BF

24

and

BF

26

are

derivativ

es

of

8325-4

,w

hic

his

cure

dof

these

thre

epro

phages

(see

text

and

Table

1).

b.b

-Lacta

mase

activ

ityw

as

dete

rmin

ed

by

MIC

sof

am

pic

illin

.c

.H

U,

haem

oly

ticunits

.d

.A

U,

arb

itrary

units

(see

Experim

enta

lpro

cedure

s).

e.

1to

21

indic

ate

sth

ere

lativ

estr

ength

of

sig

nals

on

skim

med

milk

pla

tes

(pro

tease).

0,

no

dete

cta

ble

activ

ity.



8325. We therefore studied the action of the Arl system in

the 8325-4 background.

We also found that the level of production of the cell wall-

associated protein A was much higher in both mutants

than in the parent strain or in the mutant complemented

with arlRS, in which it was very low (Fig. 5, Table 2). Both

cell wall-associated (Fig. 5A) and extracellular (Fig. 5B)

protein A levels were higher in the mutants.

Expression of virulence factor genes in arlS and arlRS

mutants

As inactivation of the arl genes modified the production of

virulence factors, we investigated the effect of arl

mutations on the expression of virulence factor genes.

To determine whether the arl mutations affected transcrip-

tion, we constructed chromosomal transcriptional–repor-

ter gene fusions with the lacZ gene and the virulence

determinant genes spa, hla and ssp. We introduced these

fusions into the wild-type and various S. aureus mutant

strains and assayed for b-galactosidase activity.

The level of expression of spa was 530 times higher in

both arlS and arlRS mutants (BF19 and BF21 respect-

ively) than in the wild-type strain (RN6390) at an OD600 of

1.0 (Fig. 6A). The level of expression of hla and ssp was

about 2.5 times higher in the mutants (BF19 and BF21)

than in the wild-type strain at an OD600 of 4.0 (Fig. 6B and

C). These results confirm the phenotypes documented

in Table 2 and indicate that the arl operon decreases

the production of virulence factors by downregulating the

transcription of their genes.

Interaction of ArlS–ArlR with the regulatory loci agr, sarA

and sarH1

The best-characterized regulatory loci of the virulence

factors are agr, sarA and sarH1. As the Arl system

modifies the transcription of virulence factor genes, we

determined the effect of the inactivation of arl genes on the

Fig. 4. SDS–PAGE gel of total extracellular proteins of S. aureusderivatives grown to OD650 of 3.5.A. Wild-type background; BF23 (wt), BF24 (arlS –), BF25(arlS – complemented with arlRS ), BF26 (arlRS –), BF27(arlRS – complemented with arlRS ).B. agrA mutant background; RN6112 (agrA –), BF34 (agrA – arlS –),BF35 (agrA – arlS – complemented with arlRS ), BF36 (agrA – arlRS –).C. sarA mutant background; ALC488 (sarA –), BF31 (sarA – arlS –),BF32 (sarA – arlS – complemented with arlRS ) and BF33(sarA – arlRS –).

Fig. 5. Western blot analysis of protein A in culture supernatants (A)and in cell wall-associated extracts (B) of S. aureus strains BF23 (wt),BF24 (arlS –), BF25 (arlS – complemented with arlRS ) and BF26(arlRS –).



expression of virulence factor genes in agrA, sarA and

sarH1 mutant backgrounds. It was not necessary to

introduce the atl mutation before measuring the levels of

extracellular proteins in agrA or sarA mutants, because

these two mutations abolished autolysis in arl mutants

(data not shown). Thus, the lysis observed in the arlS

mutant (Fournier and Hooper, 2000) requires the agr or

sarA loci. b-Galactosidase activity in the culture super-

natants of strains ALC488 and BF31, which carried a

chromosomal transcriptional ssp:lacZ fusion, and strains

RN6112 and BF34, which carried a chromosomal

transcriptional spa:lacZ fusion, were not significantly

different (data not shown), indicating that the extracellular

proteins were not released by autolysis.

The pattern of extracellular proteins was similar in the

arl–agrA mutants and the agrA mutant, and in the arl–

sarA mutants and the sarA mutant (Fig. 4B and C). This

suggests that arl represses target gene expression

through agr and/or sarA. Furthermore, serine protease

activity was higher in the double agrA–arl mutant than in

the agrA mutant. In contrast, the serine protease activity of

the sarA–arl mutant was similar to that of the sarA mutant

(Table 3). This suggests that the effect of the arl mutation

on serine protease production depends on sarA but not

agr. Finally, production of cell wall-associated protein A

was not or only slightly affected by the introduction of arl

mutations in the sarA mutant or in the agrA mutant. In

contrast, the addition of arl mutations in the sarH1 mutant

increased the production of protein A (Table 3). Transcrip-

tional fusions also showed that the level of expression of

spa in the arlS–agrA double mutant (BF34) was three

times higher than that in the agrA mutant (RN6112) at an

OD600 of 0.1. The level of spa expression in the double

arlS–sarA mutant (BF31) was similar to that in the sarA

mutant (ALC488) (Fig. 7A). The level of spa expression in

the double arlRS–sarH1 mutant (BF40) was 90 times

higher than that in the sarH1 mutant (BF38) at an OD600 of

0.1 (Fig. 7B). Thus, the effect of the arl mutations on the

transcription of spa observed in a wild-type background

(Fig. 6A) was completely abolished by the sarA mutation,

was dramatically decreased by the agrA mutation and was

unaffected by the sarH1 mutation. This suggests that sarA

and, to a lesser extent, agrA are required for the action of

arl on spa.

ArlR–arlS modifies expression of the agr and sarA loci but

not that of the sarH1 loci

As the arlR–arlS locus seems to affect the expression of

virulence factor genes by interacting with agr and/or sarA,

we analysed the expression of both regulators in the wild-

type strain (RN6390), the arlS mutant (BF19) and the

mutant complemented with arlRS (BF20). Northern blot

analyses using total RNA from late-log phase cells were

carried out with probes specific for the different regulators

(Fig. 8). RNA II synthesis was higher in the arlS mutant

than in the wild-type strain and its mutant complemented

with arlRS (Fig. 8A). Similarly, the production of RNA III

was slightly higher in the arlS mutant than in the wild-type

and the mutant complemented with arlRS (Fig. 8B).

However, analysis of sar transcripts revealed that levels

of sarB (1150 bp) and sarC (840 bp) transcripts were

unaffected, whereas sarA (580 bp) transcript levels were

lower in the arlS mutant than in the wild-type strain and its

Fig. 6. Role of arl in the regulation of spa (A), hla (B) and ssp (C) expression in three S. aureus strains: RN6390 (wt), BF19 (arlS –) and BF21(arlRS –). b-Galactosidase activity measured by chemiluminescence for spa and hla is expressed in relative light units (RLU) mg21 protein.b-Galactosidase activity measured by the colorimetric method for ssp is expressed as specific activity (SA, see Experimental procedures ) mg21

protein. OD600, rather than time, was used as the x-axis because arl mutants grew more slowly than the wild-type strain.



mutant complemented with arlRS (Fig. 8C). Finally, levels

of sarH1 transcripts were unaffected by the arlS mutation

(Fig. 8D). Thus, the arl locus modifies the synthesis of

RNA II and RNA III from the agr locus and of the sarA

transcript from the sar locus, suggesting that arl acts on

virulence gene expression through agr and sar.

SarA and agr stimulate expression of the arlR–arlS locus

As the arl locus acts on the transcription of sarA and agr,

we also studied the effect of sarA and agrA mutations on

expression of the arlR–arlS locus. We first determined the

transcriptional start site of the arl operon, which enabled us

to construct a lacZ transcriptional fusion with a fragment

containing the arl promoter region. Primer extension

analysis with two different primers (data not shown)

revealed that the transcription start site of the arlR–arlS

locus was 141 nucleotides (nt) upstream from the

predicted translation start site of arlR (Fig. 2B). This

predicted start site corresponds to a promoter similar to

the SigA-dependent consensus sequence (Deora and

Misra, 1996).

Transcription of the arl locus increased slightly during

the exponential phase up to the post-exponential phase,

suggesting that activation of the promoter of this operon

was growth phase dependent (Fig. 9). The arlR and arlS

mutations did not alter the expression of the arl operon,

indicating that this two-component system was not

autoregulated. However, inactivation of sarA and agrA

decreased expression of the arl locus (by a factor of two

to six), indicating that both regulators affect transcription

of the arl operon. Thus, agr and sarA activate the

transcription of arl.

Discussion

We have shown that the two-component system, ArlS–

ArlR, is involved in the regulation of transcription of some

virulence genes. The most dramatic effect is the

repression of protein A gene expression. This system

also decreases the synthesis of several other virulence

factors to a lesser extent (a-toxin, b-haemolysin,

lipase, serine protease and coagulase). To our knowledge,

this is the only regulator of S. aureus that downregulates

the expression of all the virulence factors. Interestingly,

some of the effects of arl are mediated by sarA and/or

agr.

The regulation of protein A (SpA) synthesis is complex

and involves at least three different loci (agr, sarA

and sarH1 ). SarA and RNA III are repressors (Cheung

et al., 1997), whereas SarH1 activates spa transcription

(Tegmark et al., 2000) (Fig. 1). SarA seems to repress spa

directly by binding to a specific motif in the spa promoter

(Cheung et al., 1997; Chien et al., 1999). However, aTab

le3.

Phenoty

pic

chara

cte

rizatio

nof

S.

aure

us

str

ain

scarr

yin

gagrA

,sarA

and

sarH

1m

uta

tions

a.

Pro

tein

Ac

(AU

mg

21

pro

tein

)

Str

ain

sG

enoty

pe

Extr

acellu

lar

pro

tein

(mg

ml2

1)

a-T

oxin

b

(HU

)b

-Haem

oly

sin

b

(HU

)D

Nase

c

(AU

ml2

1)

Lip

ase

c

(AU

ml2

1)

Pro

tease

dC

oagula

se

c

(AU

)C

ell

wall-

associa

ted

Extr

acellu

lar

RN

6112

agrA

–5^

1,

0.5

0.8

^0.1

1.0

^0.3

,1

0150

220

1800

BF

34

agrA

–arlS

–20^

1,

0.5

0.5

^0.1

0.0

6^

0.0

2,

12

1150

340

2300

BF

36

agrA

–arlR

S–

37^

1,

0.5

,0.5

0.0

6^

0.0

2,

12

1150

640

970

ALC

488

sarA

–24^

96.5

^0.7

1.2

^0.3

13.0

^2.9

11^

14

13

160

0B

F31

sarA

–arlS

–47^

49.0

^0.1

1.8

^0.2

9.0

^0.9

10^

14

13

370

0B

F33

sarA

–arlR

S–

104^

316^

33.2

^1.1

2.9

^0.6

12^

14

14

240

0B

F38

sarH

1–

ND

ND

ND

ND

ND

ND

ND

0.4

3B

F39

sarH

1–

arlS

–N

DN

DN

DN

DN

DN

DN

D1.7

52

BF

40

sarH

1–

arlR

S–

ND

ND

ND

ND

ND

ND

ND

1.4

63

a.

The

phenoty

pes

were

chara

cte

rized

as

described

inth

ete

xt.

Quantit

ativ

ere

sults

are

giv

en

as

am

ean

of

at

least

two

independent

dete

rmin

atio

ns.

b.

HU

,haem

oly

ticunits

.c

.A

U,

arb

itrary

units

(see

Experim

enta

lpro

cedure

s).

d.

21

to4

1in

dic

ate

sth

ere

lativ

estr

ength

of

sig

nals

on

skim

med

milk

pla

tes

(pro

tease).

0,

no

dete

cta

ble

activ

ity.



recent study (Tegmark et al., 2000) suggested that sarA

and agr repress spa transcription by repressing sarH1

transcription. SarH1 seems to activate spa transcription

directly by binding to its promoter region. In both arl

mutants, spa transcription (Fig. 6A) and the production of

extracellular and cell wall-associated protein A (Fig. 5)

were dramatically increased. The level of expression of

spa was 120 times higher in both arlS and arlRS mutants

than in the wild-type strain and 90 times higher in the

arlRS–sarH1 mutant than in the sarH1 mutant at an OD600

of 0.1, suggesting that the increase in spa expression as a

result of the arl mutations was similar in the sarH1 mutant

and in the wild-type strain. Moreover, production of protein

A in the double arl–sarH1 mutants is three to 23 times

higher than that in the sarH1 mutant (Table 3), and

production of protein A in the arl mutants is eight to 25

Fig. 7. Kinetics of spa:lacZ fusion transcription in S. aureus strains.A. RN6390 (wt), BF19 (arlS –), BF21 (arlRS –), RN6112 (agrA –),BF34 (agrA – arlS –), ALC488 (sarA –), BF31 (sarA – arlS –) andBF37 (agrA – sarA –).B. RN6390 (wt), BF21 (arlRS –), BF38 (sarH1 –) and BF40 (sarH1 –

arlRS –).b-Galactosidase activity measured by colorimetry is expressed asspecific activity mg21 protein. OD600, rather than time, was used asthe x-axis because arl mutants grew more slowly than the wild-typestrain.

Fig. 8. Northern blot analysis of RNA II (A), RNA III (B), sar (C) andsarH1 (D) transcripts in S. aureus strains RN6390 (wt), BF19(arlS –) and BF20 (arlS – complemented with arlRS ). hu (E) wasused as an internal control of the amount for total RNA loaded (seeExperimental procedures ). Specific probes are indicated on the left.



times higher than that in the wild-type strain (BF23)

(Table 2). Differences measured by quantification of

Western blots are underestimated because spots of

mutants were overexposed as a result of the great amount

of protein A. Finally, the arlS mutation does not modify the

level of sarH1 transcripts (Fig. 8D). Taken together, these

data indicate that sarH1 does not significantly alter the

effect of the arl mutations on spa expression and that

sarH1 is thus not involved in the action of arl on spa. The

level of expression of spa in the arlS–agrA double mutant

was three times higher than that in the agrA mutant,

whereas the arl mutation did not alter spa expression in the

sarA mutant background. Therefore, sarA and, to a lesser

extent, agrA are required for the action of arl on spa

transcription. The Arl system increases the level of sarA

transcript (Fig. 8C). SarA has a slight suppressive effect on

spa transcription, independently of sarH1 (Tegmark et al.,

2000). Thus, we can speculate that arl acts on spa

expression through the activation of sarA.

The regulation of serine protease (Ssp), which

represents another class of virulence factors (Novick,

2000), differs from that of protein A. RNA III activates ssp

transcription, whereas SarA strongly represses it (Cheung

et al., 1992; Chan and Foster, 1998; Lindsay and Foster,

1999) (Fig. 1). The doubling in Ssp levels observed in the

atl mutant background (Table 2) was also observed in the

agrA mutant background, whereas no obvious increase

was observed in the sarA mutant background (Table 3).

This suggests that arl requires sarA, but not agr, to alter

the expression of ssp. As SarA downregulates ssp, arl

probably affects ssp transcription through the activation of

sarA (Fig. 1).

Thus, SarA is an important part of the regulatory

pathway that involves arl in the expression of virulence

factors. The control of the sarA locus is complex. Activator

and repressor proteins bind to the sar promoters to

modulate the expression of sarA. SarA is likely to be an

activator for its own expression. In contrast, activation of

SigB probably leads to a downregulation in sarA expression.

The 14 kDa SarR also represses the expression of sarA

(Manna et al., 2001). The activator (SarA) or down-

modulators (SigB and SarR) of sarA expression could be

the target(s) of the Arl system.

The arl locus also modifies agr expression. Studies on

the regulation of agr transcription revealed that it is

autoregulated. Indeed, AgrA and AgrC belong to a two-

component system. Mutations in agrA or agrC decrease

agr transcription (Novick et al., 1993; 1995; Ji et al., 1995),

suggesting that AgrA acts directly on agr promoters.

However, the phosphorylated regulator AgrA has not been

shown to bind to the agr promoter region. Only SarA has

been shown to bind to this region (Heinrichs et al., 1996;

Morfeldt et al., 1996). SarA activates the synthesis of both

RNA II and RNA III (Bayer et al., 1996; Chien et al., 1998)

(Fig. 1). We found that arl decreased transcription of the

RNA II operon (Fig. 8A) and, to a lesser extent, that of RNA

III (Fig. 8B). arl also increased sarA transcription (Fig. 8C).

Thus, arl does not modify agr transcription through sarA. If

this were the case, arl would increase agr transcription.

Thus, it remains unclear how arl affects agr transcription.

As the Arl system has a major effect on exoprotein

production, this system may modify the level of the

secreted inducer molecules (AIP) required to activate the

agr operon.

This study demonstrates the existence of a new

virulence regulator in S. aureus. The arl locus modifies

the expression of virulence factor genes. The effects of the

Arl system on virulence factors depend mainly on the

regulatory loci sarA or agr. The synthesis of several other

unidentified exoproteins appears to be either stimulated or

suppressed by arl (Fig. 4A). This is not surprising, because

two-component systems are known to act on several

target genes (Stock et al., 1989). The modification of the

exoprotein pattern (Fig. 4B and C) and autolysis (data not

shown) observed in arl mutants is not observed in the

arlS–sarA and arlS–agrA mutants. This suggests that

sarA and agr are the key loci for the action of the arl

regulon. Thus, identification of the Arl system provides

new directions for future research in the virulence

regulation of S. aureus.

Fig. 9. Kinetics of arl:lacZ fusion transcription in S. aureus strainsRN6390 (wt), BF19 (arlS –), BF21 (arlRS –), RN6112 (agrA –), BF34(agrA – arlS –), ALC488 (sarA –) and BF31 (sarA – arlS –).b-Galactosidase activity measured by colorimetry is expressed asspecific activity mg21 protein. OD600, rather than time, was used asthe x-axis because arl mutants grew more slowly than the wild-typestrain.



Experimental procedures

Bacterial strains and growth conditions

The bacterial strains used in this study are listed in Table 1.Experiments were carried out with an S. aureus agrA mutant(Emr) (RN6112) (Table 1) rather than an agr mutant (Tetr)(RN6911) because the agrA mutant was erythromycinresistant and thus compatible with the resistance markergenes of the other mutations and plasmids used in this study.RNA II and RNA III are not detectable in the agrA::Tn551strain RN6112 (Morfeldt et al., 1988; Novick et al., 1989);thus, the agrA mutant is similar to the agr mutant.

Escherichia coli strains were used for cloning experiments.They were grown in Luria–Bertani (LB) medium at378C. Staphylococci were grown in trypticase soy broth(TSB) and plated on trypticase soy agar (TSA) at 378C, unlessotherwise stated.

DNA manipulations

DNA was manipulated and CaCl2-competent E. coli cellstransformed according to standard procedures (Sambrooket al., 1989). Plasmid DNA was isolated with the Qiagenmidiprep kit. S. aureus was transformed with plasmid DNA byelectroporation (Fournier and Hooper, 1998). ChromosomalDNA from S. aureus was prepared as described by Stahl andPattee (1983). Transformation with high-molecular-weightchromosomal DNA was carried out as described previouslyusing phage f55 (Stahl and Pattee, 1983). The conditions forpolymerase chain reaction (PCR) were: 5 min at 948C,followed by 25 cycles of 948C for 30 s, 45–518C for 30 s and728C for 30 s to 2 min and, finally, 7 min at 728C. The promoterregions were amplified with the Expand High Fidelity PCRsystem (Boehringer Mannheim). For all other PCRs, Taqpolymerase (Pharmacia) was used.

Plasmids and plasmid construction

The plasmids used in this work are listed in Table 1. Toconstruct an integrative promoterless transcriptional lacZfusion vector, the lacZ gene from pHT304-18Z (Agaisse andLereclus, 1994) was cut with Kpn I and Bam HI and introducedinto pUC18 (New England Biolabs) to give pUHT. pUHT wascut with Kpn I, treated with the Klenow fragment and cut withEco RI to give a 5.7 kb fragment. To remove the Bam HI sitefrom pSK950 (Niemeyer et al., 1996), the plasmid was cut withBam HI, the ends were filled in with the Klenow fragment andthe plasmid religated. The attP site from this plasmid was cutwith Eco RI and Sal I to give a 350 bp fragment. To obtain thethermosensitive replicon of pE194 and the tetracyclineresistance gene, pLTV1 (Camilli et al., 1990) was cut withXba I, the ends filled in with the Klenow fragment and theresulting fragment cut with Sal I to give a 6 kb fragment. Thesethree fragments were ligated together to give pBF50, whichcarried the sequences of pUC18 (replicon and ampicillinresistance in E. coli ), the attP site of phage L54a, thethermosensitive replicon of pE194, the S. aureus tetracyclineresistance gene and three unique sites upstream from thepromoterless lacZ gene (HindIII, Xba I and Bam HI) (Table 1).pBF50 can specifically integrate into the chromosomal attB

site located in the geh gene, which encodes staphylococcallipase. Integration is facilitated by the presence of pYL112D19(Table 1), which carries the L54a int gene, encodingintegrase.

To construct transcriptional fusions of various promoterswith the lacZ gene of pBF50, PCR-generated DNA fragmentswere inserted between the HindIII and Xba I sites of pBF50.PCR was performed with the following primers (restrictionenzyme sites are underlined): 50-AAATTAAAGCTTAGCACATTCA-30 and 50-TACCCTCTAGATGTATTTGTAAAGT-30

(spa promoter); 50-CCAAACAATTAAGCTTCAAAAGTTA-30

and 50-AAACCTCTAGAAAATTTATTTACAA-30 (ssp promo-ter); 50-TTAATCAATAAGCTTAGCTATGTCT-30 and 50-CTATTTTCTAGAACGATTTGAGGAA-30 (hla promoter) and 50-TTCATTCTGCAGTAGTGAAAAGTCA-30 and 50-TACACCTCTAGATACGACTTTTTCTAATAA-30 (arl promoter). For thearl promoter, the PCR product was first inserted between thePst I and Xba I sites of pUC18 and then subcloned by insertionbetween the HindIII and Xba I sites of pBF50. The plasmidscarrying the spa promoter (pBFSpa), ssp promoter (pBFSsp),hla promoter (pBFHla) and arl promoter (pBFArl) were firstintroduced, at 308C into a restriction-deficient strain, S. aureusRN4220 (Table 1), which carried pYL112D19 and wasselected on tetracycline plates (3mg ml21). Transcriptionalfusion plasmids were then integrated into the lipase gene by ashift to 428C. The site-specific integration of plasmids into theS. aureus chromosome was confirmed by loss of lipaseactivity, resulting from disruption of the lipase gene (Lee et al.,1991). Chromosomal DNA of RN4220 strains carrying theintegrated plasmids was then used to transform otherS. aureus strains, using phage f55 and selection on3mg ml21 tetracycline.

Construction of an arlR deletion mutant

As the arlS mutant grows more slowly than the wild-type strain(Fournier and Hooper, 2000), we used a strain in which thearlR–arlS locus was reintroduced into the lipase gene tocomplement the arlR deletion. The arlR–arlS locus wasamplified by PCR and inserted into pSK950 to generatepSKarl (Fournier and Hooper, 2000). The Pst I fragment,containing pE194, was deleted to remove the erythromycinresistance gene, resulting in pCLarl. pCLarl was introducedinto RN4220 at 378C and selected on 3mg ml21 tetracycline togive strain BF38. Integration into the lipase gene was verifiedby loss of lipase activity.

A deletion/replacement mutant of arlR was constructed asfollows: a 2.1 kb DNA fragment containing the arlR–arlSpromoter region and adjacent upstream chromosomal DNAwas amplified from ISP794 chromosomal DNA using primers50-GCTAAACTGCAGACCTAAAGAGAA-30 containing a Pst Isite (underlined) and 50-TACACCTCTAGATACGACTTTTTCTAATAA-30 containing a Xba I site (underlined) frompreliminary sequence data from the University of OklahomaGenome sequencing project. The cat gene of pC194 wasamplified by PCR using the following primers: 50-CCTTAGGATCCAGATAAGAAAGAAA-30 containing the Bam HIsite (underlined) and 50-CGGCATTATCTAGAATTATAAAAGCCA-30 containing the Xba I site (underlined). A 1.7 kbfragment, containing the 30 terminus of arlR and adjacentdownstream chromosomal DNA, including arlS, was obtained



from pSKarl by restriction at the HincII site present in the arlRgene and the Eco RI site of the polylinker. These threefragments were cloned successively into pUC18. Finally,pE194 (Shivakumar et al., 1980), which contained athermosensitive replicon and an erythromycin resistancegene, was inserted into the Pst I site. The resulting plasmidwas introduced into the derivative of strain RN4220 carryingthe arlR–arlS locus in the lipase gene (strain BF38) at 308C byselection on chloramphenicol (5mg ml21) and was integratedby two shifts to 428C. To promote a second recombinationevent, a single colony was used to inoculate TSBsupplemented with 5mg ml21 chloramphenicol and culturedat 308C. The culture was diluted and plated out on TSAmedium to yield isolated colonies. The colonies were thenscreened for Ems and Cmr. We checked that the arlR genehad been deleted by PCR amplification and Northern blotanalysis. The chromosomal DNA of this strain, carrying thearlR deletion, was then used to transform other S. aureusstrains, using phage f55 and selection on 5mg ml21

chloramphenicol.The arlS and arlRS mutants were complemented by

integration of pSKarl, as described previously (Fournier andHooper, 2000).

b-Galactosidase assays

Cells were assayed for b-galactosidase (LacZ) activity usingeither the colorimetric method with ONPG (Miller, 1972) or theAurora Gal XE chemiluminescent reporter assay system(ICN). Bacterial cells were grown to different OD600,harvested, washed and resuspended in lysis mediumsupplemented with 50mg ml21 lysostaphin and 15mg ml21

DNase. For the colorimetric method, 5 mM dithiothreitol (DTT)was also added. The mixture was incubated for 30 min at378C, and the b-galactosidase and protein concentrations ofthe lysis supernatant were then determined. The chemilumi-nescent method involved measurement of b-galactosidaseactivity in a LB 9501 luminometer (Berthold) with a 100mlautomatic injector and a 5 s integration time. b-Galactosidaseactivity measured by colorimetry is expressed as specificactivity [nmol of ONP (o-nitrophenol) min21].

Secreted protein analysis

pI258 (Table 1) was introduced into strains BF23, BF24, BF28and BF29 by electroporation and selected on 0.1mg ml21

ampicillin. b-Lactamase activity was estimated by determiningthe minimal inhibitory concentrations (MICs) of TSA sup-plemented with serial 1:2 dilutions of ampicillin (Fournier andHooper, 1998).

To determine the amount of extracellular protein in theculture supernatant, cells were grown to an OD650 of 3.5.Supernatants were filtered and stored at 2208C. Proteinconcentration was determined by the Bradford method (Bio-Rad). Ten millilitres of culture supernatant was concentratedby precipitation with 5% trichloroacetic acid, separated by10% (w/v) SDS–PAGE and stained with Coomassie brilliantblue.

a-Toxin and b-haemolysin were assayed as describedpreviously (Vandenesch et al., 1991). Briefly, a serial 1:2dilution of the supernatants was carried out, and each dilution

was added to 0.5% whole rabbit or sheep blood, respectively,used as substrates. Haemolytic activity (HU) was determinedby measuring residual turbidity at 540 nm. The 50% lysispoints were calculated by interpolation. Activities (haemolyticunits) are the reciprocal of the dilution that gave 50% lysis.Strain DU1090, which does not produce a-toxin, and strainISP2094, which does not produce b-haemolysin, were usedas controls.

Nuclease (DNase) activity was assayed as describedpreviously (Smeltzer et al., 1993). Briefly, salmon sperm DNA(1 mg ml21) was mixed with culture supernatants. The mixturewas incubated for 30 min at 378C, and the DNA was thenprecipitated by adding trichloroacetic acid to a finalconcentration of 25%. The nuclease activity of the super-natant was determined by measuring the optical density at260 nm. The control consisted of TSB instead of culturesupernatant. Arbitrary units (AU) of DNase activity are definedas OD260(sample)–OD260(TSB), adjusted for the volume ofsupernatant used.

Lipase activity was determined using tributyrin as asubstrate (Smeltzer et al., 1992). The reaction was monitoredby measuring the decrease in optical density at 450 nm as aresult of the hydrolysis of emulsified tributyrin. Arbitrary unitsof lipase activity were calculated as the linear slope of a plot ofOD450 versus time � 100, adjusted for the volume ofsupernatant.

Protease activity was detected as clear zones surroundingcolonies on nutrient agar plates supplemented with 2%skimmed milk powder.

Coagulase activity was determined by mixing 300ml of 1:2serial dilutions of whole-cell cultures with 300ml of rabbitplasma (Difco). Arbitrary units of coagulase activity areexpressed as the reciprocal of the lowest dilution at whichcoagulation disappeared.

Protein A analysis

Cultures were grown to an OD650 of 1.0 and centrifuged. Thecell pellets were used to determine the concentration of cellwall-associated protein A. The culture supernatants werefiltered and used to determine the amount of secreted proteinA. Cell wall-associated proteins were extracted with lysosta-phin in a hypertonic medium (30% raffinose), as describedpreviously (Cheung and Fischetti, 1988). For secreted proteinA, filtered supernatants were concentrated 100-fold bycentrifugation with a Millipore Centriprep concentrator. Cellwall-associated proteins (1–25mg) and secreted proteins(0.1–1.5mg) were resolved on a 10% SDS–polyacrylamidegel, electroblotted onto nitrocellulose Hybond-C Pure andprobed with rabbit anti-staphylococcal protein A antibody(Sigma) at a 1:15 000 dilution. Bound antibody was detectedwith donkey anti-rabbit immunoglobulin G conjugated toperoxidase (Pharmacia) and the ECL Western blottingdetection system (Amersham) (1:20 000). The proteinA-deficient mutant, DU5723, was used as a control.Quantification of signals from Western blots was performedby densitometric analysis of the autoradiograms using thepublic domain National Institutes of Health IMAGE program(version 1.62). Arbitrary units (AU) correspond to theintegrated density measured by the program.



RNA manipulations

RNA was extracted from late-exponential phase (OD600 of1.0) or stationary phase (OD600 of 3.0) cultures. Culture(25 ml) was centrifuged, and the cells were disrupted in aFastPrep disintegrator with 500 mg of glass beads, 400ml of2% Macaloid, 40ml of 10% SDS and 500ml of phenol–choroform–isoamyl alcohol (Derre et al., 1999). The RNA wasprecipitated with ethanol, collected by centrifugation andresuspended in water. The concentration of RNA wasdetermined by measuring absorbance at 260 nm.

Northern blot analysis

Samples containing 3–25mg of total RNA from late-exponential phase cells were analysed by Northern blotting.Samples were denatured, separated on a 1.5% agarose–formaldehyde gel and transferred to Hybond-XL nylonmembrane. Internal fragments of the genes correspondingto hu (50-CAGATTTAATCAATGCAGTTGCAGA-30 and50-TAATGCTTTACCAGCTTTGAATGCT-30), RNA III (50-CAGAGATGTGATGGAAAATAGTTGA-30 and 50-ATTAAGGGAATGTTTTACAGTTATT-30), agrA (50-CAAAG AGAAAACATGGTTACCATTA-30 and 50-CGATGCATAGCAGTGTTCTTTATTT-30), sarA (50-ATGATTGCTTTGAGTTGTTATCAAT-30 and50-ACTCAATAATGATTCGATTTTTTTA-30), sarH1 (50-ATAGTGTTTGATAATGTCATTTATTCA-30 and 50-TGTAAATGATCTTTATCTGCTAAT A-30), arlR (nt 354–807; Fournier andHooper, 2000) and arlS (nt 1263–2326; Fournier and Hooper,2000) were amplified by PCR, radiolabelled with [a-32P]-dCTPusing a random-primed labelling kit (Boehringer Mannheim)and used as probes. These probes are indicated in Figs 1 and2. The filters were hybridized and washed in stringentconditions, as described previously (Sambrook et al., 1989).

The hu transcript was used as an internal control for theamount of RNA (Chien et al., 1999). Its sequence (see primersabove) was obtained on the basis of similarity between HUprotein from S. aureus and the histone-like protein, Hbsu,from Bacillus subtilis (a homologue of the E. coli HU proteins).The sequence was obtained from preliminary sequence datafrom the University of Oklahoma Genome sequencing project.The B. subtilis HBsu protein is equally synthesized duringgrowth in B. subtilis vegetative cells (Micka et al., 1991).

Primer extension

Primer extension analysis was carried out as describedpreviously (Derre et al., 1999), using 70mg of total RNAtemplate from strain RN6390. The primers used were nt 313–342, and nt 378–407 of the sequence published by Fournierand Hooper (2000) (Fig. 2).

Sequence data

Preliminary sequence data were obtained from the Universityof Oklahoma Genome sequencing project website at http://www.genome.ou.edu/staph.html.

Acknowledgements

We would like to thank Staffan Arvidson for providing

S. aureus KT200, Ambrose L. Cheung for strains ALC488and RN6112, Timothy J. Foster for strains DU1090 andDU5723, David C. Hooper for pI258, and John J. Iandolo forstrain ISP2094. This work was supported by research fundsfrom the Institut Pasteur, Centre National de la RechercheScientifique, Universite Paris 7, and Fondation pour laRecherche Medicale. B.F. received a fellowship from theFondation pour la Recherche Medicale.

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