Quorum-sensing and siderophore biosynthesis in Pseudomonas aeruginosa: lasRllasI mutants exhibit reduced pyoverdine biosynthesis

ELSEVIER

FEMS Microbiology Letters 166 (1998) 341-345

Quorum-sensing

and siderophore biosynthesis in Pseudomonas aeruginosa : lasR/lasI mutants exhibit reduced pyoverdine biosynthesis Alain Stintzi a,b, Kelly Evans a, Jean-Marie

Meyer b, Keith Poole ‘.*

.+Department of Microbiology and Immunology, Queen’s University, Kingston. Ont. K7L 3N6 Canada ” Laboratoire de Microbiologic et de GPnPtique. Unite; de Recherche AssociPe au Centre National de la Recherche Scient$que UniversitP Louis-Pasteur. 28 rue Goethe, 67000 Strasbourg. France

No. 1481,

Received 15 June 1998; revised 23 July 1998; accepted 26 July 1998

Abstract

Cell density-dependent gene expression in Pseudomonas aeruginosa is controlled, in part, by the quorum-sensing regulator LasR. IasR null mutants exhibited a reproducible 2-fold decrease in production of the catecholate-hydroxamate siderophore pyoverdine during grown under iron-limiting conditions. Similarly, lasl mutants defective in the biosynthesis of the autoinducer PAI- also exhibited a 2-fold decrease in pyoverdine production which could be largely restored upon addition of exogenous PAI-I. IasR mutants were not altered with respect to expression of the pvdD gene involved in the synthesis of the peptide portion of pyoverdine, indicating that some other pyoverdine biosynthetic gene(s) were affected by the LasRI status of the cell. This represents the first report of quorum-sensing regulation of siderophore production in bacteria and highlights the fact that cell density, while not an essential signal for pyoverdine expression, does enhance production of this siderophore. 0 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Ke.word.s:

Quorum-sensing;

Siderophore;

LasR; Pyoverdine;

Pseudomonas aeruginosu

1. Introduction Pseudomonas ueruginosa, an opportunistic human pathogen, produces two known siderophores, pyochelin [l] and pyoverdine [2]. A mixed hydroxamate-catecholate siderophore, pyoverdine is com-

____ * Corresponding author. Tel.: +l (613) 545-6677;

Fax:

+1 (613) 545-6796;

E-mail: [email protected] 0378-1097 /98/$19.00 0 1998 Federation PII: SO378-1097(98)00352-X

of European

Microbiological

prised of a peptide chain coupled to a hydroxyquinoline moiety, the chromophore responsible for the well-known fluorescence of this siderophore [3]. The genes for pyoverdine biosynthesis have been mapped to three distinct regions of the PA0 chromosome, at min 23, 47 [4] and 66-70 [5] on the recalibrated PA0 map. A 103 kb fragment of chromosomal DNA originating from the 47 min region has been cloned. Referred to as the pvd region [6], this DNA carries several genes shown to be involved in pyoverdine biosynthesis. DNA originating from Societies. Published by Elsevier Science B.V. All rights reserved

the 66-70 min region of the PA0 chromosome has also been cloned. Responsible for the synthesis of a chromophore-like molecule dubbed pseudoverdine, gene(s) in this region are required for pyoverdine biosynthesis [S], presumably the chromophore moiety. Pyoverdine production is stimulated by conditions of iron limitation [7], mediated by a homolog of the Fur repressor protein shown to regulate iron-sidero-

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Fig. I. Pyoverdine production by P ~~ru~ino.vcr strams ML5087 (0) and its IusR mutant K1263 (A) as a function of growth (mcrease in culture density (AC,,,,,)for ML5087 (0) and K1263 (A)) measured in iron-limited minimal succinate medium. Pyoverdine was assessed by measuring the absorbance of spent culture supernatants at 405 nm (A) or by quantitating hydroxamic acids (B). Values shown are normalized per unit ceil density and are representative of three separate experiments.

by P. ~rugmo.ur strains PA01 Fig. 3. Pyoverdine production (solid bar) and its /u.T/ mutant PA@JPI grown without (hatched bar) and with (open bar) 5 pM PAI-I. Values are the mean of three determinations f SD.

phore systems in Eschrrichiu coli [8]. Still, Fur does not appear to regulate pyoverdine biosynthetic genes directly, but rather controls expression of a alternate sigma factor gene, pi&S, whose product is required for the expression of a number of pyoverdine biosynthetic genes [9,10]. Recently, a second regulatory gene, ptxR, has been described which also plays a positive role in pyoverdine production [1 11. In this report, we describe yet another regulatory gene, IusR, whose product modulates pyoverdine production in P. ueruginosu. A so-called quorumsensing regulator, LasR [ 121 together with an acylated homoserine lactone effector molecule called PAI- 1 [ 131 activates expression of a variety of known virulence genes whose expression is cell-density-dependent [14]. PAIis synthesized by the product of the lusl gene [15] and accumulates throughout growth, triggering LasR-mediated expression of target genes at higher cell densities. The observation here that 1usR mutants exhibit reduced pyoverdine production and that PAI- 1 stimulates production of this siderophore clearly indicates that cell density plays a role in siderophore production as well.

A. Stintzi et al. I FEMS Microbiology Letters 166 (1998) 341-345

2. Materials and methods

used in this study included P. ML5087 (ilv-220 thr-9001 leu9001 met-9001 pur-67 aphA) 1161, wildtype PA01 (laboratory designation K867), PAO-JPl (PA01 Alasl: : tet) [17] and K1263 (ML5087 AlasR: : tet). The IasR deletion mutant of P. aeruginosa strain ML5087 was constructed via allelic exchange using plasmid pMG319 according to a previously described protocol [12]. The IasR phenotype was confirmed by assaying for the absence of known LasRdependent exoproducts (e.g. elastase, pyocyanin) as described [ 12,181. The iron-limited succinate minimal medium has been described [7] and was supplemented with amino acids (1 mM) and nucleotides (200 pM) as required. Antibiotics were included in growth media as needed at chloramphenicol, 200 ug/ ml, and tetracycline, 100 &ml. Pyoverdine production by P. aeruginosa growing in iron-limited succinate minimal medium was assayed by measuring the absorbance of cell-free culture supernatants at 405 nm [19] or by quantitating hydroxamate levels in cell-free supernatants according to the method of Gillam et al. [20]. In some experiments, pyoverdine was assessed in overnight cultures only while in others aliquots were taken at various time points following inoculation of the growth medium at Asso of 0.05. In all cases, however, culture density (Asso) was assessed at the time pyoverdine was being measured and siderophore production reported per unit cell density. Where indicated, the chemically synthesized P. aeruginosa autoinducer PAI[13] was added to cultures at the time of inoculation at 5 PM. To assay P-galactosidase production by P. aeruginosa harboring the pMP190 derivative pMP190: :pvdD (PvdD-lacz) [21], bacteria were cultivated overnight in iron-limited succinate minimal medium. P-Galactosidase was then measured using the assay of Miller [22]. Bacterial

aeruginosa

strains strains

limited succinate minimal medium elicited detectable pyoverdine after 4-6 h culture and levels of siderophore (normalized per unit cell density) increased over the next 6-7 h (Fig. 1). The 1asR mutant of ML5087 (K1263) also elicited detectable pyoverdine at 6 h, although levels produced were lower (again normalized per unit cell density) and they increased more slowly with time, such that at any given point they were two-fold lower than that of the LasR+ parent strain. Growth of both strains was comparable and reached saturation after ca. 12 h (Fig. 1). These results were confirmed using two methods to assess pyoverdine production. To determine whether the defect in pyoverdine production in K1263 was due to an influence of LasR on expression of genes involved in the synthesis of the peptide portion of pyoverdine, expression of the pvdD gene was examined (using a pvdD-1acZ fusion) in the two strains. No difference was observed between ML5087 and K1263 (data not shown) indicating that pvdD was not influenced by LasR. 3.2. Decreased production of pyoverdine in a lasl mutant

Since LasR-mediated gene expression is dependent upon the PAIproduct of the Las1 synthase, we also examined the influence of a lasl mutation (in PAO-JPl) on pyoverdine expression. As above, a defect in lasl correlated with a ca. 2-fold decline in pyoverdine levels relative to the wildtype (Fig. 2). Consistent with the positive influence PAI- has on pyoverdine production, we could increase pyoverdine production by the lasl mutant strain by adding exogenous PAI- (Fig. 2). 3.3. Comments The observed

decline in pyoverdine production in indicates that cell density, while not an essential signal for siderophore production, may play a key role in modulating pyoverdine biosynthesis. This is borne out, too, by the enhanced siderophore production seen in strains exposed to PAI-1. It is likely that at higher cell density, the population as a whole might better avail itself of iron made available by complexation with sidero1asR and

3. Results and discussion 3.1. Decreased pyoverdine production in a 1asR mutant P. aeruginosa

ML5087

(LasR+)

growing

in iron-

343

lasl mutants

phore (i.e. the greater the cell numbers, the greater the chance that siderophoreeiron complexes will be ‘found’ by a cell). As such, it would make sense to invest more resources in siderophore production when cell densities are high rather than when they are low. It is interesting to note that a connection between quorum-sensing and siderophore production has recently been described in Burkholdwiu c~c~prrc~iu. although in that instance inactivation of a hsR homolog, cepR. actually increased siderophore production [23]. Although we have not identified the pyoverdine biosynthetic gene(s) controlled by LasR. the observation that pdD expression was not altered in a hR mutant indicates that genes involved in expression of the peptide portion of this siderophore may not be controlled by this quorum-sensing regulator. Expression of pdD is dependent upon an alternate sigma factor, PvdS [9]. and. as such, LasR cannot be acting via PvdS either. The recently described 17~~’locus involved in chromophore biosynthesis [5] (accession number AF002222) is a possible candidate fat LasR control, although attempts at assessing expression of these genes using a pvc,+‘crc,Z fusion have proved futile as a result of the extremely low activity of the ply’ promoter. As /)I‘(’ expression is dependent upon the ptsR gene product [24]. hsR may mediate its effects through this regulator.

NATO in the form of collaborative research award to support travel between the authors’ laboratories is also gratefully acknowledged. A.S. is the recipient of an EAITC (Academic Relations Division of Foreign Affairs and International Trade Canada) fellowship. K.E. is the recipient of a Medical Research Council studentship. K.P. is a Natural Sciences and Engineering Research Council University Research FeiIOW.

References

III

c’ox. C‘.D. citrate

[?I Cos.

C.D.

\crdin

for

D..

mutations

FEMS Cornclis.

A..

Poole,

K..

D..

‘5

biosynthesis

Microbiology

M..

Miyazaki.

and physical

and

199. J.-M..

Krishnapillal. of

ot

P.srfrtk,/,l(,,l~ra

Meyer.

gene(s)

Dean.

V.

(1996)

P.teuc/omono.\ tww

I IX I I 190.

142. H.

mapping

and

m

195

22X.

Mapping

Nakazdwa.

of genes mvolved PAD.

P,vru~lorn~)n~\ wu~~irwwr

T.

(1995)

Genetic

in pyoverdin

produc-

J. Bacterial.

177.

423

431. Meyet-,

J.-M.

ment

of

and

4. Addendum

and Abdallah.

MA.

(1978)

The

Ruorescent

P.cc/rt/[~,llorrrr.v,fh~r~~.ww~ : biosynthesis.

physiochemical

properties.

J. Gen.

pig-

purification

Microbial.

107.

319

328

1x1Prmce.

R.W..

regulation ular

Cox.

(CD.

cloning

and

H.E..

synthesis

i IO1Lconi. lated

Acknowledgments

mw: iol

[I II

175,

Merriman.

in

Ciervo.

factor. A..

etfect

and

Hnmood.

A.N..

Fur

C‘olmer.

qirww

gene. p/v-R. which

[I21 Gambello. xtcriLat xrlptional 3000

3009

I.L.

a gene required

gene

177.

( 1995) for

Clon-

pyover-

PvdS

is probably

2744

2750.

P. (1996)

in

Iron-rcgu-

Pwudomonr~.~ uo’u~i-

on promoter

.I.A.,

Ochsncr.

and characterization

Microbial.

M.J. ion

259X.

and Visa.

PvdS

molec-

activity.

J. Bacter-

2313.

Isolation

Mol.

N.

Coordinate

P,vc,uc/onrowlr.s~rryqiww

and Lament,

the /w/.4

( 1996) duction.

2589

(1993)

A production:

J. Bacterial.

(3%.

of

of

the

of /owls.

transcriptmn

17X. 2299

M.L.

P.teutlornonu.t rrerugirmu:

sigma

L..

of

T.R.

nnd characterization

dint

Vail.

and exotoxin

sequencing

/IO. gene. J. Bactcriol.

[UlCunlilfe. ing

and

of hiderophore

an alternate

The authors thank L. Passador for synthetic PAI1 and B. Iglewski for plasmid pMG319 and strain PAO-JPI. A.S. and J.-M.M acknowledge the financial support of the Association Francaise de Lutte contre la Mucoviscidose. Work in K.P.‘s laboratory was supported by an operating grant from the Medical Research Council of Canada. The Support of

36,

Kourambas.

pyoberdine

in

Lett.

Hohnadel.

Ii0

fluoreh-

209

(1986)

587.

48.

from 104.

production

Microbial. P..

J.-M.

fcrr~c

I

of pyo-

Immun.

Rev.

and Meyer.

pyoverdine

Stintri.

tion

D.

alfecting

[61T~uda.

activity

metabohtes

Microbial.

end 142. 58

Siderophore

Sccondaq

FEMS

wrrr,qiuo.\rl.

Novel

P. (1985)

(1993)

Haa.

,~u~wtr~PAO.

lil

Subsequent to the acceptance of this paper. expression of the I”Y’ operon was shown, using RNase protection assays, to decline 2- to 4-fold in the /r.cR mutant relative to the parent strain (M. Vasil. University of Colorado, personal communication).

H.

pwudomonads.

C..

ferripyochelin

P.r~,[r~l(~~,lono\ocru~i~w.co. Infect.

141Hohnadel.

[il

with

wr~r~qirw.\~,. J. Bacterial.

and Adams.

13x. 131Budrikicww, cent

uptake

(1980) Iron

bq P,rutk,/,lo,lll.s

positively 21.

97

mod Iglewskl,

of the activator

CI.A. of a

and

Vail.

M.1..

P.w~/~monn.~ ww

regulates

exotoxin

A pro-

I IO.

B.H.

(1991)

Clonmg

and char-

P\1,uc/~,n?r,nrr.\rwrrc,yino.w /r/s R gene. a traw of

elastasr

expression.

J.

Bacterial.

173.

A. Stintzi

et al. IFEMS

Microbiology

Letters 166 (1998)

[13] Pearson,

[14]

[I 51

[16]

[17]

[18] [19]

J.P., Gray. K.M., Passador, L., Tucker, K.D., Eberhard. A., Iglewski, B.H. and Greenberg, E.P. (1994) Structure of the autoinducer required for expression of Pseudomonas neruginosu virulence genes. Proc. Nat]. Acad. Sci. USA 91, 197-201. Pesci, E.C. and Iglewski, B.H. (1997) The chain of command in Pseudomonas quorum sensing. Trends Microbial. 5, 132135. Passador, L., Cook, J.M., Gambello, M.J., Rust, L. and Iglewski, B.H. (1993) Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260, 112771130. Okii, M., Iyobe, S. and Mitsuhashi, S. (1983) Mapping of the gene specifying aminoglycoside 3’.phosphotransferase II on the Pseudomonas ueruginosa chromosome. J. Bacterial. 155, 643-649. Pearson, J.P., Everett, C.P. and Iglewski, B.H. (1997) Roles of Pseudomonus aeruginoscl las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J. Bacterial. 179, 5756-5767. Cox, C.D. (1986) Role of pyocyanin in the acquisition of iron from transferrin. Infect. Immun. 52, 263-270. Meyer, J.-M., Halle, F., Hohnadel, D., Lemanceau, P. and

[20]

[21]

[22]

[23]

[24]

341-345

345

Ratefiarivelo, H. (1987) Siderophores of Pseudomonas biological properties. In: Iron Transport in Microbes, Plants and Animals (Winkelmann, G., van der Helm, D. and Neilands, J.B., Eds.), pp. 189-205. VCH, Weinheim. Gillam, A.H., Lewis, A.G. and Andessen, R.J. (1981) Quantitative determination of hydroxamic acids. Anal. Chem. 53. 841-844. Rombel, LT.. McMorran, B.J. and Lamont, IL. (1995) Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads. Mol. Gen. Genet. 246, 519-528. Miller, J.H. (1992) A short course in bacterial genetics. A laboratory manual and handbook for Escherichiu coli and related bacteria, pp. 72-74. Cold Spring Harbor Laboratory Press. Plainview, NY, Lewenza, S. and Sokol, P.A. (1998) Quorum sensing in Burkholderia cepaciu: identification of the LuxRI homologs CepRI, abstr. 16A. In: Abstracts of the UAlUC 8th Biennial Conference on Infectious Diseases, p. 31. Meldrum, A.. Stintzi, A. and Poole, K. (1998) Regulation of pyoverdine biosynthesis in Psrudomonas arruginosa, abstr. B243. In: Abstracts of the 98th General Meeting of the American Society for Microbiology, p. 96.