Proc. Natl. Acad. Sci. USAVol. 93, pp. 10434-10439, September 1996Microbiology

Large-scale isolation of candidate virulence genes ofPseudomonas aeruginosa by in vivo selection

(in vivo expression/IVET/neutropenic mouse/purEK/Fur)

JINGYI WANG*, ARCADY MUSHEGIANt, STEPHEN LORYt, AND SHOUGUANG JIN*§*Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205; tNational Center for Biotechnology Information,National Library of Medicine, National Institutes of Health, Bethesda, MD 20894; and *Department of Microbiology, University of Washington, Seattle, WA 98195Communicated by John Collier, Harvard Medical School, Boston, MA, May 30, 1996 (received for review April 4, 1996)

ABSTRACT Pseudononas aeruginosa, an opportunistic hu-man pathogen, is a major causative agent of mortality andmorbidity in immunocompromised patients and those with cysticfibrosis genetic disease. To identify new virulence genes of P.aeruginosa, a selection system was developed based on the in vivoexpression technology (IVET) that was first reported in Salmo-neUla system. An adenine-requiring auxotrophic mutant strain ofP. aeruginosa was isolated and found avirulent on neutropenicmice. A DNA fragment that can complement the mutant strain,containingpurEKoperon that is required forde novo biosynthesisof purine, was sequenced and used in the IVET vector construc-tion. By applying the IVET selection system to a neutropenicmouse infection model, genetic loci that are specifically inducedin vivo were identified. Twenty-two such loci were partiallysequenced and analyzed. One of them was a well-studied viru-lence factor, pyochelin receptor (FptA), that is involved in ironacquisition. Fifteen showed significant homology to reportedsequences in GenBank, while the remaining six did not. Onelocus, designated np2O, encodes an open reading frame thatshares amino acid sequence homology to transcriptional regu-lators, especially to the ferric uptake regulator (Fur) proteins ofother bacteria. An insertional np2O null mutant strain of P.aeruginosa did not show a growth defect on laboratory media;however, its virulence on neutropenic mice was significantlyreduced compared with that of a wild-type parent strain, dem-onstrating the importance of the np2O locus in the bacterialvirulence. The successful isolation of genetic loci that affectbacterial virulence demonstrates the utility of the IVET systemin identification of new virulence genes of P. aeruginosa.

Pseudomonas aeruginosa causes a wide range of infections, rang-ing from minor skin infections to serious and sometimes life-threatening complications in immunocompromised patients, orpatients with severe burns and wounds. This organism is also themajor cause of respiratory disease of patients with cystic fibrosis(1, 2). In some infections, such as those of cystic fibrosis patients,P. aeruginosa extensively colonizes the respiratory trackwhere thesymptoms of the disease are primarily due to the toxic effect ofa number of bacterial virulence factors and a subsequent inflam-matory response. Invasive disease, including bacteremia, is how-ever not uncommon in immunocompromised patients, especiallythose with hemolytic malignancies and thermal injury (3).

Infections of humans by opportunistic pathogens, including P.aeruginosa, require for these microorganisms to colonize andpersist in a new environment that drastically differs from theirnormal natural reservoir. The remarkable ability of the P. aerugi-nosa to adapt and thrive in a wide variety of environmentssignificantly contributes to the ability of this bacterium to causea variety ofhuman infections. It is likely that as a part of long-termcolonization or invasion, P. aeruginosa would induce expression ofgenes that encode determinants necessary for survival in the hostenvironment, while repressing genes that encode products that

are unnecessary or even deleterious. Bacteria have evolvedsystems to sense their surrounding environment and respond byselectively expressing appropriate genes. Such signal transductionsystems have been described for several pathogenic bacteria. Thebvg4/bvgS-mediated activation of vag genes and repression of vrggenes in Bordetella pertusis (4, 5), the phoP/phoQ-mediatedregulation of invasion determinants in Salmonella typhimurium(6, 7), and the toxR/toxS/toxT-mediated coordinate regulation ofcholera toxin and adhesin genes in Vibrio cholerae (8) have beenshown to play important roles in infection. It is reasonable toassume that P. aeruginosa genes that are important in theinfection process are specifically induced during colonization.Indeed, many of the known virulence genes of P. aeruginosa areregulated in respond to different environmental signals that arecommonly found in the hosts. For instance, expression of exo-toxin A, the most toxic factor of P. aeruginosa, and pyochelin/pyoverdin receptors, involved in the iron acquisition, are stimu-lated by iron limitation (9-11) which is found in the in vivoenvironment of animal and human tissues.A genetic selection device, called in vivo selection technology

(IVET), has been described for S. typhimurium (12), whichpositively selects for bacterial genes that are specifically inducedduring host infection. The IVET genetic selection device hasseveral important features that are superior to most other selec-tion systems. Selection can be conducted under in vivo conditions(i.e., in experimental animal models), most of which are impos-sible to mimic in vitro. Moreover, random gene fusions to areporter gene were generated on chromosomal DNA which notonly eliminates the high copy number effect compared with usinga plasmid but also avoids generating a chromosomal gene dis-ruption in contrast to transposon insertional fusions which disrupttarget genes.

Characterization of the additional P. aeruginosa genes that arespecifically induced upon infection is of considerable interest, asthis group of genes are most likely to encode virulence determi-nants. In addition, this group of genes may provide excellentmodel systems to study the mechanisms of coordinated generegulation as well as to probe for the in vivo environment.

In this paper, we report development of the IVET system for P.aeruginosa and its application in the neutropenic mouse infectionmodel. We have isolated, sequenced, and analyzed 22 host-inducible genetic loci of P. aeruginosa and confirmed by mutagen-esis that at least one of them indeed affects bacterial virulence.

MATERIALS AND METHODSBacterial Strains and Plasmids. Strains and plasmids used in

this study are listed on Table 1. Cosmid clone bank of the PAKchromosomal DNA in pVK102 was described previously (14).L-agar and L-broth were used for the growth of Escherichia coliand P. aeruginosa at 37°C. Minimal medium A (17) was also used

Abbreviation: IVET, in vivo expression technology.Data deposition: The sequences reported in this paper have beendeposited in the GenBank data base [accession nos. U58364 (purEK)and U58365 (np2O)].§To whom reprint requests should be addressed. e-mail: jins@biomed.uams.edu.

10434

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 93 (1996) 10435

Table 1. Strains and plasmids used in the study

Name Description Ref. or sourceDH5a E. coli, endAl hsdR17 supE44 thi-J recAl gyrA96 relAl A(1acZYA-argF) U169 A-080 dlacZ AM15; recipient for BRL

recombinant plasmidsRZ1032 ung, dut; E. coli host for plasmid to be mutagenized by site directed mutagenesis 13PAK Wild-type clinical isolate of P. aeruginosa David BradleyPAK-SR Spontaneous Smr isolate of the PAK 14PAK-AR1 PAK with chromosomal Tn5G insertion on purEK gene; Gmr This studyPAK-AR2 PAK deleted of the purEK gene; Spr, Smr This studyPAK-np2O PAK with chromosomal disruption of the np2O locus; Spr, Smr This studypBR322 E. coli cloning vector; Apr, Tcr United States BiochemicalpTZ18R E. coli cloning vector; Apr. United States BiochemicalpUC19fl A fl fragment clone in pUC19; Apr, Spr, Smr 15pRK2013Tn5G Tn5G carrying plasmid; Kmr, Gmr 16pSP329 Incompatible group P broad host range vector; Kmr Sandy PorterpVKBK2 A cosmid clone that contain the purEK gene in a 8 KB insert; Kmr This studypVKBK12 A cosmid clone that contain np2O in a 16 kb insert; Kmr This studypSJ9322 A 3-kb EcoRI fragment containing purEK cloned into pSP329; Kmr This studypSJ9333 A 3-kb EcoRI fragment containing purEK gene cloned into pTZ18R; Apr This studypSJ9338 pSJ9333 with two engineered BamHI sites in the purEK gene; Apr This studypSJ9344 A linker DNA containing a BglII site and three way translational stops inserted into the SmaI site of pTZ18R; Apr This studypSJ9347 purEK coding region cloned into the BamHI site of the pSJ9344; Apr This studypSJ9353 The purEK gene in pSJ9338 replaced by "'Q" fragment; Apr, Spr, Smr This studypSJ9443 Selection vector used in this study; Apr, Tcr This studypTZMS12R A 5-kb EcoRI fragment containing np2O locus cloned into the EcoRI site of pTZ18R; Apr This studypTZMS12R-BglII A BglII site was generated in the np2O gene of pTZMS12R; Apr This studypTZMS12R-Q A fl fragment was introduced into the BglII site of the pTZMS12R-BglII; Apr, Spr, Smr This study

for the growth of P. aeruginosa. Adenine was added at 50 ,ug/mlfor a full supplement and 5 ,ug/ml for a limited supplement tosupport the growth of the purEK mutant strains on minimalmedium.DNA Manipulation. Site-directed mutagenesis was conducted

as described (13). DNA sequencing was performed by PCRmediated Taq DyeDeoxy Terminator Cycle sequence on anApplied Biosystems model 373ADNA sequencer. ChromosomalDNA of P. aeruginosa was isolated by phenol extraction method(18) and Southern hybridization was performed as described (19).For sequencing DNA fragments upstream of thepurEK gene,

an oligonucleotide, 5'-CAC GCC AAC CAG TGC GCT CATCG-3', complementary to the 5' end of thepurEK coding region,was used as a primer. DNA restriction sites and open readingframe analysis were conducted by using the DNA STRIDER program.Similarity searches were performed by the BLAST family of pro-grams (20, 21) and Genetics Computer Group programs. Signif-icance of the weak similarities was assessed by motif analysis withMOST program (22). Accession numbers for thepurEK and partialnp2O sequences in GenBank are U58364 and U58365, respectively.

Construction of the IVET Selection Vector pSJ9443. PlasmidpSJ9344 is a derivative of pTZ18R where a linker DNA, 5'-CTAGCT AGC TAG ATC TAG CTA GCT AG-3', containing BglIIrestriction site and a three-way translational stop codons, wasinserted into the SmaI site. Plasmid pSJ9333, a purEK clone inpTZ18R, was subjected to site-directed mutagenesis to generateBamHI sites at both 5' and 3' ends of the gene (see Fig. 1). Theresulting plasmid was named pSJ9338 and the oligonucleotidesused to generate the 5' and 3' BamHI sites were 5'-GTG GCTTGT TGG ATC CTG AGC CCG GGA CGG C-3' and 5'-CAACCCAAG GAT CCG CGGACG GCAATC-3', respectively. ApromoterlesspurEK coding region was isolated from pSJ9338 asa 1.5-kb BamHI fragment and ligated into the BamHI site of thepSJ9344, generating pSJ9347. Finally, a 1.5-kb EcoRI-HindIIIfragment of the pSJ9347 was filled with Klenow enzyme andintroduced into the PvuII site of the pBR322, generating the finalselection vector pSJ9443 as shown in Fig. 2.

Isolation ofAdenine-Requiring Auxotrophs ofP. aeruginosa. ATn5G transposon insertional mutant bank of strain PAK-SR (14,16) were first grown in liquid minimal medium A containing 150[kg of carbenicillin per ml for 5 h and then in minimal medium Acontaining 50 ,tg of adenine per ml for 12 h. Since carbenicillinselectively kills bacterial cells that are dividing, the first growth

condition enriches auxotrophic mutants in general, whereas thesecond growth condition enriches adenine-requiring auxotrophsonly. Adenine-requiring mutants were screened by replica platingcolonies grown on minimal medium containing adenine ontominimal medium without adenine.To generate a purEK deletion strain (PAK-AR2), the 1.5-kb

BamHI fragment of the pSJ9338, containing the purEK codingregion, was replaced with a 2-kb fl fragment from pUC19fQ(BamHI digest) encoding resistance to both spectinomycin andstreptomycin (15). The resulting construct, pSJ9353, was doublecrossed over into the chromosome of PAK by electroporation(23). Colonies that are sensitive to carbenicillin and at the sametime dependent on adenine for growth were picked. Those werefurther confirmed by Southern hybridization analysis.

Generation of a np2O Insertional Mutant Strain of P. aerugi-nosa (PAK-np2O). On a np2O-containing plasmid, pTZMS12R, aBglll site was first introduced at a site 19 base pairs upstream ofthe np2O::purEK fusion junction on the chromosome of theoriginal isolate NP-20 by site directed mutagenesis using anoligonucleotide 5'-CCA GTT CTT ATT TCC CGA GAT CTGCAA CAC GGG ATC-3'. The fl fragment, isolated frompUC19fl as a 2-kb BamHI fragment, was introduced into theBglII site of the pTZMS12R-BglII, generating the np2O genedisruption on a plasmid (pTZMS12R-fQ). The np2O gene muta-tion was then introduced into the chromosome of wild-type PAKstrain by electroporation mediated double cross over (23).

Neutropenic Mice. BALB/c mice weighing 18-20 g (Harlan-Sprague-Dawley) were injected with immunosuppresant cyclo-phosphoamide (Cytoxan from Bristol-Meyers Squibb) at 200mg/kg body weight. After 3 days, the mice were challenged withbacterial cells by intraperitoneal (i.p.) injection. Over night cul-tures of bacterial cells were washed and resuspended in 0.9%NaCl and 5 mM MgCl2. Lethality was based on the animal deaththat occur within 72 h after bacterial challenge. For the in vivoselection, 106 chromosomal cointegrate bank cells were i.p.injected into five neutropenic mice. After 24 h, mice weresacrificed. Animal livers were dissected, homogenized, and inoc-ulated into 500 ml of L broth containing spectinomycin, strep-tomycin, carbenicillin, and tetracycline. The culture was shaken at37°C for 8 h and bacterial cells were recovered by a high speedcentrifugation (1500 rpm, 10 min) after removal of animal liverassociated materials by a lower speed spin (5000 rpm for 10 min).

Microbiology: Wang et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

Cell densities were measured by OD60o and repeated the aboveinfection process for additional rounds of in vivo selection.

RESULTSGeneral Strategy for Construction of the IVET Vector for P.

aeruginosa. The system developed for P. aeruginosa is analogous tothe IVET technology (12) and is based on random integration ofa promoterless purEK operon, encoding an enzyme required forpurine biosynthesis, into the bacterial chromosome of a P. aerugi-nosa strain carrying a chromosomal deletion of thepurEK region.Since the purEK deletion strain is unable to grow in the absenceof purine, growth of the bacteria in environments lacking purineis possible only if the promoterlesspurEK is inserted downstreamof an active promoter. To distinguish genes that are specificallyinducible in vivo from those constitutively expressed, bacteriarecovered from a particular growth condition are screened forgrowth on minimal medium supplemented with a limited amountof purine. Large colonies represent purine-independent growth;therefore, purEK genes are under the control of constitutivelyexpressed promoters in those bacteria. Small colony variantsrepresent bacteria that require purine for growth; therefore,purEK genes are transcribed by promoters that are activate in theselection condition and inactive on minimal medium.The purEK Operon of P. aeruginosa. Adenine requiring

mutants of P. aeruginosa were first isolated. A bank of -3 x104 Tn5G insertional mutants of strain PAK were subjected tocarbenicillin enrichment and screened for adenine-requiringauxotrophes as described in the Materials and Methods. Afterfour cycles of the enrichment, about 2% of the bacterialpopulation were adenine-requiring auxotrophes as determinedby replica plating 200 colonies onto minimal medium A withor without adenine. One mutant strain, designated PAK-AR1,was chosen for further studies.To isolate a complementing DNA clone of the PAK-AR1,

chromosomal cosmid clones of the PAKwere introduced into thePAK-AR1 and selected for growth (colony formation) on min-imal medium A. A cosmid clone, designated pVKBK2, was foundto be able to complement the auxotrophic phenotype of the strainPAK-AR1. This cosmid clone was mapped and DNA fragmentswere further subcloned into a broad host range plasmid pSP329to define the functional gene by complementation. A minimum3-kb EcoRI fragment was found to be able to complement thePAK-AR1 (Fig. 1). The 3-kb EcoRI fragment was further deletedfrom both ends by Bal31 exonuclease enzyme to locate theboundaries of the functional gene. As shown in Fig. 1, thefunctional gene was located between the ends of deletions B andF, as determined by complementation tests.The 1.5-kb DNA fragment located between the deletion B and

F was sequenced in both directions. Two open reading frames,encoding 163 and 382-aa-long peptides that are separated by 35nt, were identified. The two open reading frames are highlyhomologous to PurE and PurK of other microorganisms, respec-tively (24-26). The upstream open reading frame shares 65%identity and 76% similarity to PurE ofE. coli, whereas the secondopen reading frame shows 36% identity and 57% similarity toPurK ofE. coli. The PurE and PurK ofE. coli, encoded by a singlepurEK operon, form a 5'-phosphoribosyl-5-amino-4-imidazolecarboxylase involved in de novo purine nucleotide synthesis (24).Our complementation tests of the PAK-AR1, as described above,indicated that the PurE and PurK of P. aeruginosa are alsoencoded on a single transcript, like that of E. coli.To test if the purEK gene is applicable to the IVET selection

system, the purEK mutant strain PAK-AR1 was tested for viru-lence on neutropenic mice. As shown in Table 2, no animal deathwas observed even with the inoculum of 107 cells, the highest dosetested, whereas infection with as low as 103 wild-type PAKresulted in the death of 50% animals tested, indicating that the invivo environment of the neutropenic mice is deficient of purineand the purEK mutant strain is unable to replicate, giving rise toavirulent phenotype. Therefore, thepurEKoperon can be used in

s P S R S S

I KB-

P R

pSJ9322 (+)

AA (+)

----I AB (+)

AC (-)

AD (-)

AE (+)

AF (+)AG (-)

AH (-)

Functional fragment

5 -BaI[GGATTCI (RBs)

5' -GGTTTCTGAGCCCGGGACGGCGCATTTGAcAGAGGCACGACG-

-(ATG purEK coding region TAA)-

3 ' -BaAIICGGATCC]

-TCGAGGCGTGAGCGATTGCCGTCCGCCCATCC-3'

FIG. 1. Localization of the gene that complements the adeninerequiring mutant of PAK-ARL. A complementing cosmid clonepVKBK2 was subcloned and tested for complementation. A functional3-kb EcoRI fragment was further narrowed by deletion analysis. Twoend sequences of the purEK operon are also shown. The putativeribosome binding site (RBS) is underlined. Two BamHI sites areintroduced at the 5' and 3' ends of the operon (boldface letter) for theisolation of a promoterlesspurEK coding region. +, Complement PAK-AR1; -, cannot complement PAK-AR1; P, PstI; R, EcoRI; S, Sall.

the IVET system construction, where it is suitable for applicationin at least the neutropenic mouse infection model.

Construction of the IVET Selection System for P. aeruginosa.For the construction of the selection vector, a promoter-minuscoding region of thepurEK operon was engineered. Two BamHIsites, one in front of thepurE ribosome binding site and the otherbehind the translational stop codon (TAA) of the purK, weregenerated by site-directed mutagenesis (Fig. 1). The purEKcoding region was isolated as a 1.5-kb BamHI fragment from theresulting plasmid and used in the subsequent steps of the vectorconstruction (see Materials and Methods). The final selectionvector, pSJ9443, was constructed by inserting the EcoRI-HindlIlfragment from the pSJ9347 into the PvuII site ofpBR322 (Fig. 2).Main features of the vector include the followings. (i) A uniqueBglII site where a random Sau3A fragments of the P. aeruginosachromosomal DNA can be inserted. (ii) The plasmid carries aColEl origin of replication which can replicate in E. coli but notin P. aeruginosa. Therefore, chromosomal cointegrates can easilybe isolated by selecting for carbenicillin and tetracycline resis-tance markers encoded on the plasmid after introducing theplasmid constructs into P. aeruginosa strains. (iii) A promoterlesspurEK gene whose ribosome binding site is proceeded by athree-way translational stop codons to ensure transcriptionalfusions, instead of translational fusions, between inserted DNAfragments and the purEK genes.The final selection vector and its use in generating chromosomal

co-integrate library is schematically shown in Fig. 2. ChromosomalDNA of the wild-type P. aeruginosa strain PAK was partiallydigested with Sau3A and fragments ranging from 1 to 8 kb were

Table 2. Virulence tests of P. aeruginosa strains on neutropenic mice

Test 1 Test 2 Test 3,

Bacterial PAK- PAK- IVETinoculum PAK AR1 PAK np2O bank103 cells 2/6 NT 3/6 0/6 NT104 cells 4/6 NT 5/6 2/6 NT105 cells 6/6 0/6 6/6 3/6 0/6106 cells NT 0/6 NT 6/6 1/6107 cells NT 0/6 NT 6/6 4/6Six neutropenic mice were used for each dose of each bacterial strain

tested. Number of dead animals is listed. NT, not tested.

s

10436 Microbiology: Wang et al.

Proc. Natl. Acad. Sci. USA 93 (1996) 10437

HipidiHIEcoRi

pSi9443 ori

RBS purEK

[AGATILUTIAGcl A(;c l A(;2 1

Suo3A tragmentsr tmPAK chromrsnoome

A B

X (Homologous recombination)(EcoRID

ApurEK chrormosome

A B

EcoR[ HodIllpurEEK orI

A f LeTeIi

IEcoRIhIpoirEK chronmosome

A B A B

FIG. 2. The IVET selection vector and its integration into thechromosome. Fragments of PAK chromosomal DNA were randomlyintroduced into the BglII site of selection vector pSJ9443 and theresulting plasmids were single crossed into the PAK-AR2 strain. TheBgIII site is followed by three translational stop codons in three frames.

gel purified and ligated into the BglII site of the selection vectorpSJ9443. The ligation mixture was transformed into E. coli strainDH5a, and at least 5 x 104 individual transformants were pooled.By randomly checking 24 individual transformants, 85% of thepopulation were found to have DNA inserts. Plasmid DNA was

isolated from the pool and electroporated into thepurEK deletionstrain (PAK-AR2, see Materials and Methods) to generate a

chromosomal cointegrate bank. Since the ColEl replication originis not functional in P. aeruginosa, cells with single cross over were

simply selected by carbenicillin and tetracycline. Transformantsconsisting of minimum 2 x 104 colonies were pooled and saved as

a chromosomal cointegrate library.Isolation of in Vivo Inducible Genes. Neutropenic mouse infec-

tion model was used to select bacterial genes that are specificallyinduced in vivo. Since the highest number of infecting bacteriawererecovered from livers of the mice, compared with that from lungs,spleens or hearts (data not shown), we decided to isolate bacteriafrom the livers of the infected animals. First, the virulence of theoriginal chromosomal cointegrate bank was tested to determinethe initial inoculum. As shown in Table 2, 107 cells from the bankcaused death of majority mice tested whereas only one died amongsix mice infected with the inoculum of 106 cells. Therefore, initialinoculum of 106 cells were chosen, which is high enough numberof cells to include the whole chromosomal cointegrate bank (- 104cells) and in the same time keep most of the infected animals alive.

Next, to determine the length of selection time required, therelative replication rate of the wild-type PAK versus the purEKdeletion strain PAK-AR2 was tested. Five neutropenic mice wereinjected with 104 cells of the bacterial mixture, consisting of 1:100ratio of the PAK to PAK-AR2. After 24 h, bacterial cells in theanimal livers were plated on minimal medium A, where PAK can

only grow, and on L-agar plates containing 200 ,ug/ml each of

spectinomycin and streptomycin, where only PAK-AR2 can grow.The relative ratio of the two bacterial cells were calculated fromthe number of colonies grown on the above two kinds of plates.The ratio of PAK versus PAK-AR2 changed from 1:100 to 1:1 by24 h of infection period. Assuming that the two bacterial strains arecleared at the same rate by the host defense system, the change inthe relative ratio of the two bacteria is the result of difference inbacterial replication rates. Our results indicated that cells express-ing the purEK (i.e., PAK) are enriched 100-fold over non-purEKexpressing cells (PAK-AR2) within 24 h, under the in vivo envi-ronment.

Based on the above data, five neutropenic mice were eachinjected with 106 cells of the chromosomal cointegrate bank forthe initial selection. Bacterial cells recovered from animal livers,24 h after infection, were cultured for 8 h in L-broth from which106 bacterial cells were injected into another five neutropenicmice for subsequent round of selection (see Materials and Meth-ods for detail). In total, five rounds of the above selections wereconducted where 104 bacterial cells were injected into the mice inthe final two rounds. Theoretically, cells expressing the purEKgenes were enriched for 1010 times over non-purEK expressingones (102 enrichment in each cycle) which ensure cells in the finalpopulation to have active promoters driving the chromosomallyintegratedpurEK gene under in vivo condition (Fig. 2). From thefinal culture, 2 x 104 cells were plated on minimal medium Acontaining appropriate antibiotics and a limited amount of ade-nine (5 ,ug/ml) to support the growth of desired cells-i.e., thosewith the purEK gene under the control of promoters that areturned off in vitro. Two different sized colonies appeared, largeand small, with the ratio of 500:1 (the bank cells prior to theselection had a ratio of 1:50). Large colonies represent cells withconstitutive promoters driving thepurEKgene whereas small onesrepresent cells having promoters that are specifically induced invivo. A total of 45 small colonies were picked for further studies.

Characterization of the in Vivo Inducible Genetic Loci. Chro-mosomal DNA of the 45 isolates were digested either with EcoRIor HindIII and Southern hybridization was conducted using the1.5-kb purEK operon from pSJ9347 as a probe. A total of 22different hybridization patterns were observed, with several iso-lates identical, suggesting that the 45 isolates were derived from 22different strains (data not shown). To further characterize theisolated loci, upstream DNA fragments that control the expressionof the chromosomally integrated purEK gene were isolated asfollows. Each chromosomal DNA was digested with EcoRI, selfligated, and transformed into E. coli strain DH5a. Because thereis only one EcoRI site in the selection vector, recircularization ofthe vector after EcoRI digestion results in a plasmid that carriesDNA upstream of the purEK gene, origin of replication frompBR322, and the ampicillin resistance gene (see Fig. 2). Plasmidsfrom the resulting transformants were used to sequence DNAfragments upstream of the purEK gene using a primer comple-mentary to the 5' end of the purEK gene (see Materials andMethods). Sequences of o500 bp were obtained from each of the22 isolates, and the products of their conceptual translation in allsix reading frames were used to search the nonredundant sequencedata base (National Center for Biotechnology Information, Na-tional Institutes of Health) by the BLASTX program. Particularattention was paid at the open reading frames that are in the samedirection as thepurEKoperon. Based on the analysis of the partialDNA sequence, one was found identical to the fptA locus of P.aeruginosa, 15 had significant sequence similarities to knownsequences in the GenBank, and 6 did not have signiflcant homol-ogy to any known sequences (Table 3). The fpt4 gene is awell-studied virulence locus, coding for a pyochelin receptor thatis involved in the pyochelin mediated iron acquisition (9).

Characterization of the np2O Locus. The amino acid sequencehomology search indicated that the putative peptide encoded bynp2O locus resembles a number of transcriptional regulators be-longing to the ferric uptake regulatory (Fur) family of bacterialproteins, especially to the N-terminal half of the proteins (Fig. 3).

Microbiology: Wang et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

Table 3. P. aeruginosa loci identified to be inducible during infection of neutropenic mice

Locus Similar proteins in the data bases*

np22np2Onp6

oIp9n1p'0np'3npI9

npi6npI8np]4

np2n1p3np4tzpl2np2J

tip7

tipl, npS, np8, npll, npl5, npl7

Functions and commentsSpecific functional predictions possible on the basis of high similarity

FptA, P. aeruginosa (U03161) Identical to the iron acquisition receptor protein FptAFur, Campylobacter jejuni (Z35165) Transcriptional regulatorPknl, Myxococcus xanthus (P33973) Serine/threonine protein kinase, similar to kinases of Myxococcus

and Streptomyces and to eukaryotic protein kinase CCheY, Salmonella typhimuirium (P06657) Chemotaxis regulatorTfdT, Alcaligenes eutrophus (P42427) Transcriptional activatorDbpA, E. coli (P21693) DEAD-type RNA helicaseYpbl, E. coli plasmid (P03852) Plasmid repressor of primerRedF, E. coli (P06615) (2 ORFs) ResolvaseIlvA, E. coli (P04968) Threonine dehydrataseHisB, E. coli (P06987) Imidazoleglycerol-phosphate dehydrataseOprE, P. aeruginosa (PIR S34969)-plus strand Antisense RNA for a porin

Only generic predictions are possibleYjhQ, E. coli (P39368) Acetyltransferase motifYim4p, S. cerevisisae (P40471) NAD-dependent, eukaryotic-type dehydrogenaseCobI, P. denitrificans (P21639) S-Adenosyl-L-methionine-dependent methyltransferasePIGNADPDL4_1, Sus scrofa (D49386) NAD-dependent, eukaryotic-type dehydrogenaseYhaE, E. coli (P23523) Dinucleotide-dependent dehydrogenase motif

High similarity but no functional predictionECU29581_9, E. coli (U29581) ?

No similarity in the minus strand9

*Only the putative proteins encoded in the same strand as thepurEK operon were considered (minus strand). The highest scoring data base entriesare shown. The accession numbers in SwissProt (starting with P) or in GenBank are given in the parenthesses.

Since iron is an important signal in the regulation of many knownvirulence gene expression, we have further characterized the np2Olocus.A cosmid clone containing the intact np2O locus, pVKBK12,

was identified by colony hybridization of the PAK chromosomalDNA clone bank in E. coli by using the partial upstream sequenceof the np2O as a probe. Restriction sites of the cosmid weremapped, and the np2O locus was subcloned. On a np2O containingsubclone, pTZMS12R, np2O was disrupted by inserting a Qfragment at a site 19 bp upstream of the originalpurEK fusion sitein strain NP-20 and the resulting plasmid was double crossed overinto the chromosome of the PAK strain, as described in theMaterials and Methods. The final np2O insertional null mutantstrain, PAK-np2O, was confirmed to carry a disrupted np2O geneon the chromosome by Southern hybridization using 5-kb EcoRIfragment containing the np2O locus as a probe (Fig. 4).The mutant strain has a similar growth rate as that of wild-type

PAKon minimal mediumA and has no observable phenotype thatcan be distinguished from wild-type PAK strain on minimal or richmedium, indicating that np2O is not an essential house-keepinggene. The mutant strain was then tested for virulence on neutro-penic mice. As shown in Table 2, '100-fold more PAK-np20bacterium was needed to cause similar lethality on mice, comparedwith wild-type PAK, demonstrating that the function of the np2Olocus is important for the bacterial virulence on neutropenic mice.

DISCUSSIONWe have developed a selection system for in vivo induciblepromoters, based on random insertion of the promoterlesspurEKoperon into the chromosome of a P. aeruginosa strain lacking thecorresponding gene. This system is analogous to the IVETselection system described for S. typhimurium (12). The purEK

operon is involved in de novo biosynthesis of purine and thepurEKmutant strain is severely attenuated for growth in the neutropenicmouse model. It has been shown in other bacterial pathogens thatauxotrophic mutants for purine or pyridine are avirulent inanimal models, indicating that these substrates are not readilyavailable in the animal host tissues (31, 32).There are two major differences between the IVET systems of

P. aeruginosa and Salmonella. One is the use of a three-waytranslational stop codons in front of the promoterless purEKgenes in our selection vector. This ensures the generation oftranscriptional fusions between chromosomal genes and thepurEK, instead of translational fusions that might produce non-functional fusion proteins. The other difference is omitting a lacZreporter gene fusion in our vector construction. Inclusion of thelacZ gene in the selection vector significantly decreased thefrequency of chromosomal cointegration, unabling us to generatea reasonable number of independent chromosomal cointegratesfor the bank construction. Accordingly, we have used size dif-ferences of the colonies, on minimal medium containing limitedadenine, as a marker (in place of the blue-white colors) to screenfor promoters that are inactive in vitro.

Isolation of the pyochelin receptor (fptA), known to be induc-ible under an iron-deprived environment, provides further evi-dence that animal host tissues are deficient of free iron due to thepresence of high affinity iron binding proteins, like transferrin andlactoferrin. The importance of iron in the animal infection systemis further demonstrated by the fact that another in vivo induciblegene, np2O, encodes a protein related to a family of transcriptionalregulators, the so called ferric uptake regulator (Fur) family.Disruption of the np2O gene resulted in a significant reduction inthe bacterial virulence, demonstrating that the IVET systemdeveloped in this work is suitable for identification of bacterial

* _ ****** * * * ** ** _ *

Np20: [Q.GVRLT_ELRRRv L VWQSH.x PEOTLE. VLL ETDGRRAAPPTVY LDFLQE H7 a

CJ: QG G L Y T K Q R E V L KT L Y H S D T H Y T P E S L Y K E I K Q A E P D LNLLEE VET SI S

BP: IXNMIGLKAx TIFPRLxX LD IFRK S L RHEIS EE VYRTLIX. IGLAITVYRHLITQF EQ GXLT RSQ

Pa: K A G L x IT L P R V I L Q M L D S A E Q R H K 8 A E V Y KHL X E A R D V Q L A T V Y R V L T Q F E A A G L V R H N

Zc: x K A G L _LPR L x RLxDMO+E..IOLATVYRVL N Q F D D A T R H NZo: KKAGLKr VTLPRL EVLQEPDNNHVSA~ L Y KRLIXD MQGZ E. I GLAT VYR LNQFD IV

FIG. 3. Amino acid sequence alignment of the Np2O homologues. Cj, Fur of Campylobacterjejuni (27); Bp, Fur of Bordetella pertusis (28); Ec,Fur of E. coli (29); Pa, Fur of P. aeruginosa (30). Identical residues are boxed; conservative substitutions, according to schemes PAGST, ILVM,QN, ED, HKR, YFW, and C, are indicated by asterisks (boldface type).

10438 Microbiology: Wang et al.

Proc. Natl. Acad. Sci. USA 93 (1996) 10439

H Ql HI

I

11 kb 1.0

H R H (np2O

1 2 3 4

2.7 0.8

1) R H

-12 kb

-7

-6

- 5 FIG. 4. Insertional mutation of

4the np2O locus. (Upper) DNA mapof the np2O locus. (Lower) Chro-

-3 mosomal DNA of PAK (lanes 1and 3) and PAK-np2O (lanes 2 and4) were digested with either EcoRI

-2 (lanes I and 2) or HindIII (lanes 3and 4) and hybridized with a 5-kb

-1.5 EcoRI fragment containing thenp20 as a DNA probe.

virulence factors. It remains to be investigated whether thereduced virulence of the PAK-np2O is due to the inactivation ofthe np2O product itself or a polar effect of the insertion in the np2Ogene on the expression of the downstream virulence factors.The importance of both fptA and np2O genes in infection was

further suggested in another study where both of them were

shown to be inducible by respiratory mucus of cystic fibrosispatients (J.W., S.L., R. Ramphal, and SJ., unpublished data).Since the ftA locus is known to be inducible by a low iron signal,it is likely that one of the common environmental signals of theneutropenic mice and the cystic fibrosis mucus is iron limitation.However, it is not clear what turns on the expression of the np2Olocus, as our preliminary data ruled out iron being the signalmolecule for this locus (J.W., S.L., R. Ramphal, and S.J., unpub-lished data).Among the other loci that show significant similarity to se-

quences in the data base, several shared high similarity to theknown proteins along the entire lengths of the sequenced inserts;accordingly, the functions of these proteins could be predictedwith high degree of confidence. In the other cases, genericfunctional predictions could be made on the basis of well-definedsequence motifs (Table 3). In the former category, proteins couldbe divided into two broad groups, one composed of the proteinsconcerned with gene regulation or signal transduction, and an-

other consisting of enzymes involved in amino acid biosynthesis.Several loci are especially interesting by virtue of their sequence

similarity to the known genes which provide clues to their functionduring P. aeruginosa infection. For example, the np6 locus, en-

coding a peptide that is similar to serine/threonine protein kinases(PKN) of Myxococcus and Eukaryotes. A secreted protein ofYersinia enterocolitica, YpkA, also shares significant sequencehomology to Eukarytotic Ser/Thr protein kinases and it wasshown to be an indispensible virulence determinnant (33). ThePKN was implicated in the normal morphological development ofM. xanthus (34) and also in the signal transduction of Eukaryotesinvolving different cellular processes (35). SinceP. aeruginosa doesnot undergo a developmental cycle, it is possible that the PKNplays a role in signal transduction within the bacterium as part ofa general environmental signal sensing system. Alternatively, thePKN may be released and may affect host cell signal transductionprocess within epithelial or phagocytic cells. During infection ofhost cells, Y enterocolitica releases a tyrosine phosphatase (YopH)which modifies the activities of several eukaryotic proteins bydephosphorylation (36).

Another locus, np9, which encodes a polypeptide that issimilar to the CheY homologues, including PilG of P. aerugi-nosa, is involved in the pilus biogenesis and twitching motility.Also, the npl4 locus specifies an anti-sense RNA for controllingthe expression of a porin gene. Both of these proteins arerelated to the expression of surface and outer membraneproteins, which may play a role in virulence by some unknownmechanism. Using the IVET system, in vivo induced anti-sensetranscripts have also been found in S. typhimurium and Vibriocholerae, specifying genes involved in 0-antigen synthesis andmotility/chemotaxis, respectively (12, 37).The genetic loci that show no homology to the known genes in

the data base could either be totally new genes or simply represent5' promoter regions of the in vivo inducible genes. These loci couldpotentially encode novel factors that may play important roles inthe bacterial infection process. Importantly, our screen identifiedsix in vivo inducible loci (npl, npS, np8, npll, nplS, and npl 7) thathave no homologues in the data banks; thus it is very likely thatthese are not standard housekeeping or metabolic genes. Theymight represent new virulence factors, which went unnoticed byprevious investigators. To verify their role in virulence, strains ofP. aeruginosa, carrying mutations in each of these loci, arecurrently being constructed, and the effect of these mutations onbacterial virulence will be examined.

This work was supported by Faculty Development Funds of theUniversity of Arkansas for Medical Sciences (to S.J.) and a Pilot andFeasibility grant from the Center for Gene Therapy of the Universityof Washington (to S.L.).1. Bodey, G. P., Bolivar, R., Fainstein, V. & Jadeja, L. (1983) Rev. Infect. Dis.

5, 279-313.2. Gilligan, P. H. (1991) Clin. Microbiol. Rev. 4, 35-51.3. Holder, I. A. (1993) Pseudomonas Aeruginosa: The Opportunist Pathogen-

esis and Disease (CRC, Boca Raton, FL).4. Beattie, D. T., Shahin, R. & Mekalanos, J. J. (1992) Infect. Immun. 60,571-577.5. Finn, T. M., Shahin, R. & Mekalanos, J. J. (1991) Infect. Immun. 59,3273-3279.6. Behlau, I. & Miller, S. I. (1993) J. Bacteriol. 175, 4475-4484.7. Miller, S. I. (1991) Mol. MicrobioL 5, 2073-2078.8. DiRita, V. J. (1995) Two-Component Signal Transduction (Am. Soc. Mi-

crobiol., Washington, DC).9. Ankenbauer, R. G. & Quan, H. N. (1994) J. Bacteriol. 176, 307-319.

10. Lory, S. (1986) J. Bacteriol. 168, 1451-1456.11. Poole, KI, Neshat, S., Krebes, K & Heinrichs, D. E. (1993)1. Bacteriol. 175,

4597-4604.12. Mahan, M. J., Slauch, J. M. & Mekalanos, J. J. (1993) Science 259,686-688.13. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987) Methods Enzymol.

154, 367-382.14. Ishimoto, K. S. & Lory, S. (1989) Pro. Natl. Acad. Sci. USA 86, 1954-1957.15. Totten, P. A. & Lory, S. (1990) J. Bacteriol. 172, 7188-7199.16. Nunn, D. N. & Lory, S. (1992) Proc. Natl. Acad. Sci. USA 89, 47-51.17. Davis, B. D. & Mingioli, E. S. (1950) J. Bacteriol. 60, 17-28.18. Strom, M. S. & Lory, S. (1986) J. Bacteriol. 165, 367-372.19. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A

Laboratoty Manual (Cold Spring Harbor Lab. Press, Plainview, NY), 2nd Ed.20. Altschul, S. F., Boguski, M. S., Gish, W. & Wootton, J. C. (1994) Nat.

Genet. 6, 119-129.21. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990)

J. Biol. Chem. 215, 403-410.22. Tatusov, R. L., Altschul, S. F. & Koonin, E. V. (1994) Proc. Natl. Acad. Sci.

USA 91, 12091-12095.23. Jin, S., Ishimoto, K. & Lory, S. (1994) J. Bacteriol. 176, 1316-1322.24. Watanabe, W., Sampei, G., Aiba, A. & Mizobuchi, K. (1989) J. Bacteriol.

171, 198-204.25. Ebbole, D. J. & Zalkin, H. (1987) J. Biol. Chem. 262, 8274-8287.26. Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness,

E. F., et al. (1995) Science 269, 496-512.27. Wooldridge, K. G., Williams, P. H. & Ketley, J. M. (1994)J. Bacteriol. 176,

5852-5856.28. Beall, B. W. & Sanden, G. N. (1995) Curr. Microbiol. 30, 223-226.29. Schaffer, S., Hantke, K. & Braun, V. (1985) Mol. Gen. Genet. 200, 110-113.30. Prince, R. W., Cox, C. D. & Vasil, M. L. (1993) JBacteriol 175,2589-2598.31. Sigwart, D. F., Stocker, B. A. & Clements, J. D. (1989) Infect. Immun. 57,

1858-1861.32. Stocker, B. A. (1988) Vaccine 6, 141-145.33. Galyov, E. E., Hakansson, S., Forsberg, A. & Wolf-Watz, H. (1993) Nature

(London) 361, 730-732.34. Munoz-Dorado, J., Inouye, S. & Inouye, M. (1991) Cell 67, 995-1006.35. Dijke, P., Franz'en, P., Yamashita, H., Ichijo, H., Heldin, C. H. & Miya-

zono, K (1994) Prog. Growth Factor Res. 5, 55-72.36. Bliska, J. B., Guan, K. L., Dixon, J. E. & Falkow, S. (1991) Proc. Natl. Acad.

Sci. USA 88,1187-1191.37. Camilli, A. & Mekalanos, J. J. (1995) Mol. Microbiol. 18, 671-683.

Microbiology: Wang et al.