genome mosaicism is conserved but not unique in pseudomonas aeruginosa isolates from the airways of...

8/13/2019 Genome Mosaicism is Conserved but Not Unique in Pseudomonas Aeruginosa Isolates From the Airways of Young

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Environmental Microbiology (2003) 5(12), 13411349 doi:10.1046/j.1462-2920.2003.00518.x

2003 Society for Applied Microbiology and Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2920Society for Applied Microbiology and Blackwell Publishing Ltd, 200351213411349Original ArticleMicroarray analysis of P. aeruginosa genomesR. K. Ernst

et al.

Received 23 June, 2003; accepted 1 August, 2003. *Forcorrespondence. E-mail [email protected]; Tel. (+1) 206616 5107; Fax (+1) 206 616 4295

Genome mosaicism is conserved but not unique inPseudomonas aeruginosaisolates from the airways ofyoung children with cystic fibrosis

Robert K. Ernst,1,7David A. DArgenio,1

Jeffrey K. Ichikawa,2M. Gita Bangera,2Sara Selgrade,6

Jane L. Burns,3Peter Hiatt,4Karen McCoy,5

Mitchell Brittnacher,1Arnold Kas,6David H. Spencer,6

Maynard V. Olson,6Bonnie W. Ramsey,3Stephen Lory2

and Samuel I. Miller1*,6,7

1Department of Microbiology, University of Washington,

Health Sciences Building, K-140, Box 357710, Seattle,

WA 98195, USA.2Department of Microbiology and Molecular Genetics,

Harvard Medical School, Boston, MA, 02115, USA.3Department of Pediatrics, University of Washington,

Seattle, WA 98195, USA.4Department of Pediatrics, Baylor College of Medicine,

Houston, TX, 77030, USA.5Division of Pulmonary Medicine, Columbus Childrens

Hospital, Columbus, OH, 43205, USA.6Department of Genome Sciences, University of

Washington, Seattle, WA 98195, USA.7Department of Medicine, University of Washington,

Seattle, WA 98195, USA.

Summary

Pseudomonas aeruginosa strains from the chronic

lung infections of cystic fibrosis (CF) patients are

phenotypically and genotypically diverse. Using

strain PAO1 whole genome DNA microarrays, we

assessed the genomic variation in P. aeruginosa

strains isolated from young children with CF

(6 months to 8 years of age) as well as from the envi-

ronment. Eighty-nine to 97% of the PAO1 open read-

ing frames were detected in 20 strains by microarray

analysis, while subsets of 38 gene islands were

absent or divergent. No specific pattern of genome

mosaicism defined strains associated with CF. Many

mosaic regions were distinguished by their low G ++++C

content; their inclusion of phage related or pyocin

genes; or by their linkage to a vgr gene or a tRNA

gene. Microarray and phenotypic analysis of sequen-

tial isolates from individual patients revealed two

deletions of greater than 100 kbp formed during evo-

lution in the lung. The gene loss in these sequential

isolates raises the possibility that acquisition of pyo-

melanin production and loss of pyoverdin uptake

each may be of adaptive significance. Further charac-

terization of P. aeruginosadiversity within the airways

of individual CF patients may reveal common adapta-

tions, perhaps mediated by gene loss, that suggest

new opportunities for therapy.

Introduction

It is a significant challenge to characterize populations of

the bacterium Pseudomonas aeruginosa, an opportunist

by virtue of its physiological and genetic adaptability. The

respiratory tracts of most patients with cystic fibrosis (CF)

become colonized with P. aeruginosa shortly after birth

(Burns et al., 2001) and these bacteria persist resulting in

progressive lung damage (Bodey et al., 1983).

Pseudomonas aeruginosa reaches a high titre in such

chronic infections, with up to 109bacterial cells per ml of

sputum (Singh et al., 2000), and two or more phenotypescommonly are displayed by isolates in individual samples

from CF patients (Burns et al., 2001). Some of these

phenotypes, such as mucoidy, represent adaptations that

are selected within the airway (Boucher et al., 1997;

Spencer et al., 2003) and that are associated with a poor

prognosis (Henry et al., 1992; Emerson et al., 2002).

Much of the genotypic diversity in P. aeruginosaisolates

from the CF patient population, however, is not reflected

by readily visible phenotypes. Previous studies have

shown that each CF patient generally harbours a unique

P. aeruginosaclonal lineage and that islands of chromo-

somal genes are variably present in these strains

(Schmidt et al., 1996; Rmling et al., 1997; Kiewitz et al.,

2000; Arora et al., 2001; Burns et al., 2001; Liang et al.,

2001; Larbig et al., 2002; Spencer et al., 2003). Neverthe-

less, there is accumulating evidence for a highly con-

served chromosomal backbone (Kiewitz and Tmmler,

2000; Spencer et al., 2003; Wolfgang et al., 2003), and

over 80% of the genome of strain PAO1 (Stover et al.,

2000) was found to be shared (with only 0.5% divergence

at the nucleotide level) by three other P. aeruginosa iso-


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1342 R. K. Ernst et al.

2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 13411349

lates, two from CF patients and one environmental strain

(Spencer et al., 2003). We therefore used a PAO1 DNA

microarray to analyse P. aeruginosa isolates from young

children with CF to determine if there were patterns of

genome variation that were specific to the early infections

of CF patients or that changed over the course of these

infections.

Results and discussion

Pseudomonas aeruginosaisolates from young children

with CF

Twenty strains were analysed using the PAO1 DNA

microarray (Table 1). Thirteen strains were minimally pas-

saged isolates from CF patients less than eight years of

age (including sequential isolates from individual patients)

and were collected as part of a study on infection and

inflammation in young children with CF at three regional

CF centres: Seattle, Washington; Columbus, Ohio; and

Houston, Texas (Burns et al., 2001). Whole-genomerestriction analysis indicated that the two strains from

patient 8 were not clonally related whereas the sets of

strains analysed from patient 4 and patient 9 each were

part of a clonal lineage (data not shown). Furthermore,

each patient carried a unique P. aeruginosa lineage.

These results are consistent with a previous restriction

fragment length polymorphism (RFLP) analysis of the

DNA region surrounding the toxA gene (Burns et al.,

2001). For comparison with the 13 CF-associated iso-

lates, seven additional P. aeruginosastrains were analy-

sed: two from a chronic infection in an adult patient with

bronchiectasis, four environmental isolates, and the labo-

ratory reference strain PAK (a human clinical isolate).

DNA microarray analysis of P. aeruginosagenomic

variation

Open reading frames were designated as divergent or

absent by microarray analysis when the corresponding

DNA yielded a spot intensity lower by a factor of two or

more relative to that produced by PAO1 DNA. Hybridiza-

tion was reproducible: multiple experiments using strain

CF250 and different versions of the microarray (with vary-

ing spot density) gave equivalent results. Eighty-nine to

97% of the PAO1 ORFs were detected by microarray

analysis in the 20 P. aeruginosastrains. These results are

consistent with previous evidence for a conserved core

genome (Kiewitz and Tmmler, 2000; Spencer et al.,

2003) including a microarray analysis of diverse clinical P.

aeruginosastrains as well as strain PAK (Wolfgang et al.,

2003).

Table 2 lists the chromosomal regions in which DNA

corresponding to two or more contiguous PAO1 ORFs

was not detected (the data for each gene is given in

Supplementary material, Table S1). Artifacts associated

with single ORFs that produced false negatives in other

microarray analyses (Peters et al., 2003) are therefore

unlikely to be a confounding factor. Single ORFs thatappeared to be detected within otherwise non-detected

regions most likely reflect either variable nucleotide diver-

gence or cross-hybridization. The possibility remains,

however, that in some strains individual genes have trans-

posed to a different location in the chromosome. Only

those regions identified in more than one P. aeruginosa

strain are listed in Table 2; not included are 25 regions,

mostly containing two contiguous ORFs, that were unde-

tected in only one of the strains.

Conserved mosaic genome structure in P. aeruginosa

The genomic regions that varied in their detection by

microarray analysis comprised 38 islands dispersed in the

chromosome (Table 2). Some of the gene islands encode

products known or predicted to play key roles in P. aerug-

inosa biology, including cell surface factors such as O-

antigen polysaccharide as well as a not yet characterized

putative exopolysaccharide encoded by genes PA1383 to

PA1393 (Table 2; Stover et al., 2000). Strikingly, only one

island (encoding a putative DNA restriction and modifica-

Table 1.P. aeruginosastrains analysed.

Patienta Strain Sourceb Reference

CF patient 3 CF250 24 months, BAL J. BurnsCF patient 4 CF171B 9 months, OP J. BurnsCF patient 4 CF488M 88 months, OP J. BurnsCF patient 8 CF1188 18 months, OP (mucoid) J. BurnsCF patient 8 CF1488 27 months, OP (mucoid) J. BurnsCF patient 9 CF416 6 months, OP J. Burns

CF patient 9 CF725 12 months, BAL J. BurnsCF patient 9 CF1213 21 months, OP J. BurnsCF patient 9 CF383c 60 months, sputum

(mucoid)J. Burns

CF patient 9 CF5296 96 months, sputum J. BurnsCF patient 202 CF471 24 months, BAL J. BurnsCF patient 205 CF1153 33 months, OP J. BurnsCF patient 206 CF181 12 months, BAL J. BurnsBronchiectasis Bronch4 adult, sputum (mucoid) ChironBronchiectasis Bronch5 adult, sputum (mucoid) ChironLaboratory strain PAO1 human wound S. LoryLaboratory strain PAK human clinical isolate S. LoryEnvironmental 14886 soil ATCCEnvironmental 25324 air ATCCEnvironmental 43495 air ATCCEnvironmental 700888 biofilm ATCC

a.The isolates from each individual patient are clonally related to theother isolates from that patient except for the two from CF patient 8.The two bronchiectasis isolates are also clonally related.b.Clinical strains were isolated (at the indicated patient ages) from

sputum, the upper airway by a swab of the oropharynx (OP), and thelower airway by bronchoalveolar lavage (BAL). The indicated strainshad a mucoid phenotype.

c. Strain CF383 was designated strain 160 in a whole-genomesequence analysis at 0.57coverage (Spencer et al., 2003).


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Microarray analysis of P. aeruginosa genomes 1343


tion system) was not detected in any of the strains. This

apparent PAO1-specific region, along with six of the other

islands, carried DNA with a relatively low G +C content

(Table 2). These seven islands represent a subset of the

10 chromosomal regions whose low G +C content sug-

gested that they were sites of recent horizontal transfer in

PAO1 (Stover et al., 2000; Wolfgang et al., 2003).

Another previously identified site of horizontal transfer

in PAO1 consists of approximately 7 kbp that includes the

phospholipase gene pldA (PA3487) and a homologue of

a member of the vgrgene family, genes associated with

rearrangement hotspots in the E. colichromosome (Wil-

derman et al., 2001). Over 70% of the P. aeruginosa

strains examined, irrespective of their source, had the

island replaced with approximately 60 bp of conserved

DNA (Wilderman et al., 2001). Whereas the variable pres-

ence of this particular composite genetic element was not

detected by microarray analysis, five other islands were

adjacent to one of the other nine vgrhomologues in the

PAO1 chromosome (Table 2; Croft et al., 2000; Stover

et al., 2000). Therefore, vgrgenes may serve as markers

for islands of P. aeruginosagenomic diversity.

Other islands have also been shown to be absent at a

specific location in the P. aeruginosachromosome and not

replaced by other genes. Of the two PAO1 gene clusters

in tandem that encode pyocin R2 and pyocin F2, both

Table 2.P. aeruginosagenomic diversity relative to strain PAO1.

PAO1 ORFswithin islandsa

Strains:CF/non-CFb

Putative function (and phage ortransposable element related proteins)c Supporting evidenced

PA0098-PA0101 3/3 vgr(PA0095)PA0187-PA0188 1/2PA0202-PA0207 3/5 Polyamine ABC transporterPA0257-PA0261 3/2 IS (PA0257, 258) High % SNPs, vgr(PA0262), tRNAArg(PA0263.1)PA0497-PA0499 4/4 Fimbrial proteins High % SNPs

PA0614-PA0628 2/1 Phage-tail pyocin R2 Variably presentPA0638-PA0646 5/5 Phage-tail pyocin F2 Variably presentPA0688-PA0689 2/2 PhoA homologuesPA0718-PA0728 7/4 Phage Pf1 High % SNPs, tRNAGly(PA0729.1)PA0821-PA0826 2/5 Phage fE125 proteins gp29, gp30, gp31 tmRNA (PA0826.12)PA0978-PA0987 4/5 Pyocin S5; IS (PA0978, 979, 983, 986, 987) Low % G +C, DPA0977-987, tRNALys(PA0976.1)PA1087-PA1096 6/5 Flagellar proteins High % SNPs, DPA1088-1091PA1223-PA1224 1/2PA1239-PA1241 4/2PA1366-PA1372 7/6 IS (PA1368) Low % G +CPA1379-PA1380 3/0PA1383-PA1393 2/2 Exopolysaccharide synthesis, sulphation Low % G +CPA1886-PA1888 5/1 DNA polymerase IIPA1932-PA1939 4/3 Aromatic catabolism; IS (PA1937, 1938)PA2100-PA2106 6/4 Low % G +CPA2183-PA2188 6/4PA2208-PA2227 7/6 Low % G +C, DPA2218-2222

PA2295-PA2296 1/2 ABC transporterPA2333-PA2334 1/1PA2372-PA2373 4/2 vgr(PA2373)PA2387-PA2403 7/3 Pyoverdin biosynthesis High % SNPs cassettePA2565-PA2566 6/0PA2730-PA2736 8/6 Restriction/modification Low % G +C, tRNAPro(PA2736.1)PA3065-PA3067 1/3PA3146-PA3161 6/5 O-antigen biosynthesis Low % G +C cassettePA3366-PA3367 1/1 tRNAArg(PA3368.1)PA3496-PA3514 5/3 DPA3497-3514PA3867-PA3868 7/5 DNA recombinasePA4101-PA4107 2/2PA4192-PA4195 1/1 Amino acid ABC transporterPA4550-PA4556 1/1 Type IV pili High % SNPsPA5086-PA5088 3/2 TPR-repeat proteins vgr(PA5090)PA5264-PA5265 2/2 vgr(PA5266)

a.Chromosomal islands in which DNA corresponding to two or more contiguous PAO1 ORFs was divergent or absent in more than one P.aeruginosastrain. The extent of some of the islands varied between strains.b.Number of strains in which each chromosomal region varied. Clonal strains were treated as one, resulting in eight genomes from CF isolatesand six genomes from non-CF isolates.

c.Selected proteins are described; more complete annotation can be found at http://www.pseudomonas.com.Homologues of transposase genesof IS elements are identified by PAO1 gene number.

d.Some chromosomal regions overlapped with PAO1 DNA either with atypical G +C content or adjacent to a vgrgene, a tRNA gene, or thetmRNA gene (each identified by PAO1 gene number); or with DNA in other P. aeruginosastrains either with a high number of SNPs or absent(D) relative to PAO1 (see text for details).
http://www.pseudomonas.com/http://www.pseudomonas.com/


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predicted to be evolved phage tails, either can be absent

in P. aeruginosastrains (Nakayama et al., 2000). Similarly,

a 17 kbp deletion containing PAO1 genes PA3496 to

PA3514 was documented in three P. aeruginosa isolates

(Spencer et al., 2003). Both of these sites of diversity were

detected using the PAO1 microarray (Table 2). Although

the genes in the latter region are of unknown function, four

of them have homologues in the soil bacterium Acineto-

bacter sp. ADP1 in a region of the chromosome that is

prone to spontaneous deletion (Segura et al., 1999).

Two islands identified by microarray analysis each have

been shown to be members of a set of isofunctional cas-

settes. The O-antigen biosynthetic gene cluster in PAO1

(Table 2) is precisely replaced in the chromosome of other

P. aeruginosa strains by one of a set of gene clusters

(Raymond et al., 2002). This set is predicted to account

for all but one of the 20 recognized serotypes with PAO1

being serotype O5 (Raymond et al., 2002). Similarly, P.

aeruginosaproduces three major types of the siderophore

pyoverdin and one chromosomal island (Table 2) includes

genes for the biosynthesis of the pyoverdin-type specificpeptide moiety as well as the highly variable fpvAgene

(PA2398) encoding the outer membrane receptor for each

pyoverdin type (de Chial et al., 2003; Lamont and Martin,

2003; Ravel and Cornelis, 2003; Spencer et al., 2003).

Although PAO1 produces Type I pyoverdin, production of

Type II pyoverdin appears to be more common in P. aerug-

inosa(De Vos et al., 2001).

Other genomic regions exhibited an even more complex

pattern of variation. In P. aeruginosastrain PAK, a 16 kbp

island within a flagellar gene cluster contains genes

required for flagellin glycosylation (Arora et al., 2001). This

island replaces PAO1 genes PA1088 to PA1091 whereasthe DNA sequence of genes PA1092 to PA1096 diverges

by over 40% compared to their PAK homologues (Arora

et al., 2001). These two contiguous regions varied in their

detection by microarray analysis (Table 2). This variation

is consistent with replacement by the glycosylation island,

particularly given the observation that while PAO1

expresses non-glycosylated b-type flagellin, expression of

glycosylated a-type flagellin is more commonly found in P.

aeruginosa(Arora et al., 2001).

Two other chromosomal regions also appeared to be

hotspots for gene island replacement. Pseudomonas

aeruginosa strains commonly carry PAGI-1, a 49 kbp

island that is absent in PAO1 (Liang et al., 2001). PAGI-1

contains genes potentially involved in oxidative stress

resistance, and in half the analysed cases, replaced PAO1

genes PA2218 to PA2222 (Liang et al., 2001). Suggesting

that this region was a general site of genome mosaicism,

it was undetected in all but one of the analysed strains

using the PAO1 microarray (Table 2). Furthermore, in

other P. aeruginosaisolates, it was the site of insertion of

3 kbp of DNA unrelated to PAGI-1 (Liang et al., 2001) or

deletion of 100 kbp encompassing PA2130 to PA2221

(Spencer et al., 2003). Another region of complex varia-

tion is the 9 kbp containing PA0977 to PA0987 (Table 2).

In PAO1, this island is inserted in the 3 end of a tRNALys

gene, while in a P. aeruginosaisolate from a CF patient,

it is replaced by an insertion of 106 kbp of DNA also found

as a plasmid in other strains (Kiewitz et al., 2000). Some

of the islands inserted at this chromosomal site were

found to carry exoU (Wolfgang et al., 2003). The exoU

gene encodes a lipase cytotoxin that is a substrate of the

Type III secretion system, and whose presence correlates

with the absence of the Type III effector protein ExoS

(Sato et al., 2003; Wolfgang et al., 2003).

Horizontally transferred bacterial DNA is commonly

integrated into tRNA genes or the tmRNA gene on the

chromosome (Williams, 2002; 2003). This process

appears to have contributed significantly to P. aeruginosa

genomic diversity with several of the genomic islands

flanked by these RNA genes (Table 2; Kiewitz et al., 2000;

Larbig et al., 2002; Wolfgang et al., 2003). Furthermore,

these islands carry additional signatures of horizontaltransfer including the presence of putative transposase

genes of IS elements or phage related genes (Table 2;

Wolfgang et al., 2003). The island in PAO1 encompassed

by PA0821 to PA0826, for instance, includes homologues

of three genes of fE125, a Burkholderia thailandensis

bacteriophage (Table 2; Woods et al., 2002).

The pattern of genome mosaicism in P. aeruginosa

isolates from CF patients is not unique

Genotypic and phenotypic characterization of P. aerugi-

nosastrains at various degrees of resolution supports theview that CF patients are infected with environmental

strains (Rmling et al., 1994a; 1997; Schmidt et al., 1996;

Alonso et al., 1999; Pirnay et al., 2002a; Spencer et al.,

2003). The set of chromosomal islands that was absent

or divergent compared to PAO1 by microarray analysis

was also not CF-specific (Table 2), and when strain-relat-

edness was assessed using detection of an island by the

PAO1 microarray as a trait, environmental strains often

clustered with clinical strains (Fig. 1; Wolfgang et al.,

2003). The more frequent lack of detection of some

islands (such as PA2565 to PA2566) in strains associated

with CF (Table 2) will need to be validated by the analysis

of additional strains.

A complete genome sequence remains the definitive

characterization, however. Until a more comprehensive P.

aeruginosamicroarray is constructed, analysis is limited

to comparison with the DNA complement of PAO1.

Pseudomonas aeruginosastrains also may vary in their

gene order. Genome rearrangements, such as the chro-

mosomal inversions documented in strains from CF

patients (Rmling et al., 1997; Kresse et al., 2003) and


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known to occur even during growth in the laboratory (Sto-

ver et al., 2000), are likely to go undetected by microarray

analysis. Furthermore, for chromosomal regions with a

complex pattern of variation, detection by hybridization

may depend on the location of the probe. A whole genomecomparison (Spencer et al., 2003), for instance, revealed

that DNA in several of the gene islands had a high number

of single nucleotide polymorphisms (Table 2), and mosa-

icism at the level of a single gene (oprD) has also been

documented (Pirnay et al., 2002b). Such ambiguity not-

withstanding, Southern blot analysis using short PCR-

generated oligonucleotides was consistent with the lack

of detection by microarray analysis of DNA in two of the

islands: PA3496 to PA3514 and PA1087 to PA1096 (data

not shown).

Large deletions in the P. aeruginosachromosome formedduring evolution in the CF lung

Pseudomonas aeruginosa evolution in the lungs of CF

patients is associated with loss of genetic information. In

some cases, gene inactivation is associated with a gain

of function that confers a selective advantage such as the

mutator phenotype due to loss of MutS (Oliver et al., 2000;

Kresse et al., 2003) or mucoidy as a result of the loss of

the antisigma factor MucA (Boucher et al., 1997; Spencer

et al., 2003). In other cases, such as the auxotrophic P.

aeruginosavariants isolated from the CF lung (Barth and

Pitt, 1995), the role of selective pressure is unclear. Loss

of metabolic capacity may reflect absence of selective

pressure for its maintenance. Alternatively, such loss may

reflect general metabolic streamlining to concentrate

resources on a trait under selection.

It has been noted that P. aeruginosa isolates sampled

later in the chronic lung infections associated with CF

commonly include mutants unable to produce pyoverdin

(De Vos et al., 2001). Another characteristic of these later

isolates is loss of pyocin production and acquisition of

phage resistance (Rmling et al., 1994b). The forces that

drive this diversification of the P. aeruginosapopulation in

CF patients remain to be determined. However, individual

genotypes may represent adaptations by localized sub-

populations of bacteria in the lung, given that P. aerugi-

nosa genotypic diversity does not appear to be rapidly

eliminated by selective sweeps (Spencer et al., 2003).

Furthermore, diversification may be facilitated in those

cases (such as pyoverdin production) where extracellularcomplementation can reduce the cost of loss-of-function

mutations (De Vos et al., 2001).

Similar ambiguity applies to the adaptive significance of

two large chromosomal deletions, revealed by microarray

analysis, that formed during growth of P. aeruginosain the

airway of a CF patient. Strain CF250 was one of five

sequential isolates sampled in the first two years of life of

patient 3, and the only isolate to produce a pigment with

the characteristic brown colour of pyomelanin (Yabuuchi

and Ohyama, 1972). Consistent with this phenotype, the

119 kbp (PA1909 to PA2010) that appeared to be deleted

in strain CF250 included hmgA (PA2009) encodinghomogentisate dioxygenase. Based on studies of

Pseudomonasand other bacteria as well as humans with

alkaptonuria (who have an inborn error in hmgA), pyomel-

anin production is due to extracellular accumulation and

polymerization of homogentisate (Kotob et al., 1995;

Scazzocchio, 1997; Hegedus, 2000; Nosanchuk and Cas-

adevall, 2003). In strain PAO1, transposon insertion muta-

tions in hmgA also resulted in pyomelanin production

(data not shown).

Pyomelanin production has been noted in P. aeruginosa

strains from diverse clinical sources, most commonly in

isolates from urinary tract infections, although it is

unknown if this phenotype is advantageous to the bacte-

rium (Yabuuchi and Ohyama, 1972). Pyomelanin pro-

duced due to loss of HmgA activity may confer benefits

including protection against oxidative stress (Nosanchuk

and Casadevall, 2003). A potential side-effect of pyomel-

anin on human hosts is tissue inflammation (Hegedus,

2000).

The apparent deletion of 189 kbp (PA2273 to PA2409)

in strain CF5296, the latest isolate analysed from CF

Fig. 1. Clustering of P. aeruginosastrains (not clonally related) using

detection of each of 38 gene islands (Table 2) by the PAO1 DNAmicroarray as a trait.


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patient 9 (Table 1), may represent another case where

less is more (Olson, 1999). This deletion eliminates genes

involved in biosynthesis and uptake of pyoverdin, the

major siderophore in P. aeruginosa (Stover et al., 2000;

de Chial et al., 2003; Lamont and Martin, 2003; Spencer

et al., 2003). A potential benefit of such a loss is protection

against killing by pyocins, at least one of which is known

to use the pyoverdin receptor FpvA for uptake (Baysse

et al., 1999; de Chial et al., 2003). Indeed, such resis-

tance represents another potential reason for in vivo

selection of the pyomelanin-producing strain CF250 in

patient 3. Among the pyocin resistant P. aeruginosa

strains that have been selected in vitro, some had an

apparent chromosomal deletion that resulted in produc-

tion of a brown pigment, inhibition of phage replication,

and methionine auxotrophy (Holloway et al., 1973), the

most common auxotrophy in strains associated with CF

(Barth and Pitt, 1995). Pseudomonas aeruginosastrains

vary in their production of and resistance to multiple

pyocins (Michel-Briand and Baysse, 2002) and more than

one clonal lineage of P. aeruginosahas been recoveredfrom both CF patient 3 and patient 9 (Burns et al., 2001).

Consistent with a general role for pyocins in P. aerugi-

nosa population structure, all five pyocins identified in

PAO1 (Michel-Briand and Baysse, 2002; Parret and De

Mot, 2002) are encoded by DNA associated with sites of

genomic diversity. The pyocin R2 and F2 gene clusters

are each variably present, the pyocin S5 gene (PA0984)

is part of the island encompassing PA0978 to PA0987,

and the pyocin S4 gene (PA3866) is adjacent to another

island (Table 2). The pyocin S2 gene (PA1150), encoding

the one remaining pyocin, is part of a genetic element

upstream of toxAwhose presence varies between strains(Pritchard and Vasil, 1990). Although this variation was

not detected by microarray analysis, it has been used as

a genetic marker for strain typing (Pritchard and Vasil,

1990; Burns et al., 2001). Indeed, even though production

of pyocins and pyoverdin can be lost over time in P. aerug-

inosastrains from CF patients (Rmling et al., 1994b; De

Vos et al., 2001), the diversity of both of these extracellular

factors has been used as the basis for typing of these

strains (Fyfe et al., 1984; Meyer et al., 1997; Pirnay et al.,

2002a).

Pseudomonas aeruginosadiversity and its implications forCF lung disease

Analysis of 20 P. aeruginosastrains using the PAO1 DNA

microarray revealed a conserved pattern of genome

mosaicism. Subsets of 38 gene islands were absent or

divergent but no specific pattern was associated with

strains isolated from the airways of CF patients. Neverthe-

less, characterization of the complete set of islands in

isolates from individual young children with CF may lead

to early identification of patients at greater risk of lung

disease that is either more severe or more resistant to

therapy. One factor complicating such predictions is that

adaptation of P. aeruginosa to life in the lung is likely to

be achieved by multiple evolutionary paths. Common

themes, however, are likely to emerge from whole genome

sequence analysis of sequential P. aeruginosa isolates.

The more predictable the course of these chronic lung

infections becomes at the molecular level, the greater the

chance that new therapies will be found to make them

more treatable.

Experimental procedures

DNA microarray analysis

The DNA microarray, affixed onto a glass microscope slide,

consisted of 200 bp to 500 bp PCR products arrayed irre-

spective of their chromosomal location. These gene-specific

products represent 5500 of the 5570 predicted open reading

frames (ORFs) in the genome of strain PAO1, and have been

used previously to analyse gene expression in P. aeruginosagrown as a biofilm (Whiteley et al., 2001).

All P. aeruginosa strains were grown to mid-logarithmic

phase at 37C with aeration in LuriaBertani (LB) medium

and chromosomal DNA was isolated using a DNeasy Tissue

Kit (Qiagen). For use as a probe, chromosomal DNA was

fluorescently labelled with Cy3-dCTP and Cy5-dCTP for

PAO1 DNA and DNA from other strains, respectively. For

labelling, 3 mg of DNA and 1 mg of a semi-random primer

(NSNSNSNSNS) in a final volume of 9.5 ml were heated to

95100C for 5 min and then chilled on ice. To this mixture

was added 4 ml of 10 Klenow buffer (New England Biolabs),

1 ml of dNTP mix (5 mM dATP, 5 mM dGTP, 5 mM dTTP, and

1 mM dCTP), 2 ml (2 nmol) of Cy3-labelled or Cy5-labelled

dCTP (Amersham-Pharmacia), and 20 units Klenow frag-ment of Escherichia colipolymerase (New England Biolabs).

The reaction mixture was incubated at 37C for 1.5 h.

Labelled cDNA was purified using the Qiaquick PCR puri-

fication kit (Qiagen). Labelling efficiency was calculated by

using the A260and either A550for Cy3 incorporation or A650for

Cy5 incorporation. Probes were dried to completion using a

Speed-Vac. The appropriate probe combinations were resus-

pended in 45 ml of hybridization solution (50% formamide, 5

SSC, 5 Denhardts, 0.1% SDS, and 100 mg ml-1 salmon

sperm DNA). Probes were boiled for 3 min, cooled on ice,

and applied to the microarray slide. Slides were covered with

a 24-mm 60 mm (No. 1) coverslip (Corning) and incubated

at 42C in a custom-built humidified chamber for 16 h. After

hybridization, slides were washed once in preheated2 SSC/0.2% SDS buffer at 55C for 10 min, twice in pre-

heated 0.2 SSC/0.2% SDS buffer at 55C for 10 min, twice

in 0.1SSC buffer at room temperature for 2 min, and briefly

with deionised water.

Slides were scanned using a Molecular Dynamics confocal

dual-laser scanner at 532 nm and 633 nm. Data was analy-

sed using custom array analysis software developed by R.

Bumgarner (Spot-On Software package, University of Wash-

ington). Each slide carried the genomic DNA microarray in

duplicate, yielding two data sets that were averaged. Image


7/9



processing, data normalization and error analysis were

performed as previously described (Ichikawa et al., 2000;

Whiteley et al., 2001).

Additional analysis of genomic variation

Pseudomonas aeruginosa genomic DNA was analysed by

multiple-complete-digest restriction fragment mapping (Wong

et al., 1997) using the restriction enzymes NarI and SacII(New England Biolabs). Based on the detection of genomic

islands by microarray analysis, a dendrogram was generated

using XCLUSTER for hierarchical clustering (Eisen et al.,

1998).

Acknowledgements

Support was provided by grants from the Cystic Fibrosis

Foundation including Cystic Fibrosis postdoctoral research

fellowships (R.K.E. and J.K.I.). The NIH also provided sup-

port: RO1AI47938 (S.I.M.).

Supplementary material

The following material is available from

http://www.blackwellpublishing.com/products/journals/

suppmat/emi/emi518/emi518sm.htm

Table S1.Microarray detection of P. aeruginosa genes.

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