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Comprehensive transposon mutant library of

Comprehensive transposon mutant library of Pseudomonas aeruginosa

Michael A.Jacobs*?,Ashley Alwood?,Iyarit Thaipisuttikul?,David Spencer*,Eric Haugen*,Stephen Ernst*,Oliver Will§, Rajinder Kaul*,Christopher Raymond*,Ruth Levy*,Liu Chun-Rong*,Donald Guenthner*,Donald Bovee*,

Maynard V.Olson*?,and Colin Manoil?

*Department of Medicine,University of Washington Genome Center,Box352145,Seattle,WA98195-2145;and Departments of?Genome Sciences and

§Statistics,University of Washington,Seattle,WA98195

Contributed by Maynard V.Olson,September29,2003

We have developed technologies for creating saturating libraries of sequence-de?ned transposon insertion mutants in which each strain is maintained.Phenotypic analysis of such libraries should provide a virtually complete identi?cation of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa,an oppor-tunistic pathogen with a6.3-Mbp genome.The library that was generated consists of30,100sequence-de?ned mutants,corre-sponding to an average of?ve insertions per gene.About12%of the predicted genes of this organism lacked insertions;many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E.coli,we estimate that the actual number of essential genes is300–400.Screening the collection for strains defective in two de?ned multigenic processes(twitching motility and prototrophic growth)identi?ed mutants correspond-ing to nearly all genes expected from earlier studies.Thus,phe-notypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities.The trans-posons used to generate the mutant collection have added features that should facilitate downstream studies of gene expres-sion,protein localization,epistasis,and chromosome engineering. PAO1?MPAO1?mutagenesis?IS phoA?hah?IS lacZ?hah

W hole-genome sequences provide the foundation for the creation of relatively complete collections of strains car-rying defined mutations in individual genes.Such libraries should facilitate the comprehensive identification of genes re-quired for a wide range of biological processes.A nearly complete library of single-gene deletions of Saccharomyces cerevisiae has been assembled by an international consortium using a PCR-based mutagenesis approach(1).Other projects, also following a strategy of gene-by-gene disruption,are under-way for Escherichia coli(E.coli genome project,www. https://www.wendangku.net/doc/e214131718.html,?functional?tnmutagenesis.htm),and have re-cently been completed for Bacillus subtilis(2).

An alternative strategy for generating mutant libraries consists of‘‘random’’whole-genome transposon-insertion mutagenesis followed by sequence-based identification of insertion sites.The approach is cost-effective and applicable to a wide variety of microbes(3,4).Studies with yeast,in which a collection of mutants corresponding to about one-third of the genes were represented,have illustrated that the generation of large,ar-rayed collections of insertion mutants is feasible(5).Other studies with bacteria have analyzed large numbers of transposon insertion mutants to identify genes essential for growth,although the mutants were analyzed within populations rather than being archived in a format allowing additional phenotypes to be examined(6–8).In this report,we describe the generation and initial phenotypic analysis of a near-saturation library of trans-poson insertion mutants of the opportunistic pathogen Pseudo-monas aeruginosa by using technologies that should be applicable to many other bacterial species.P.aeruginosa is a bacterial pathogen that causes a variety of opportunistic infections,in-cluding pulmonary infections in cystic fibrosis patients.The mutant collection that was generated provides?5-fold coverage of predicted genes,corresponding to multiple insertion alleles in most nonessential genes.

Materials and Methods

Transposon Mutagenesis and Colony Selection.Transposon inser-tions in the PAO1chromosome were generated by mating P. aeruginosa PAO1obtained from B.Iglewski(Department of Microbiology,University of Rochester Medical Center,Roch-ester,NY)(referred to as MPAO1)with E.coli strain SM10pir?pCM639(IS phoA?hah insertions)or SM10pir?pIT2(IS lacZ?hah insertions).Mutagenized cells were selected by plating on LB agar containing tetracycline(60?g?ml),chloramphenicol(10?g?ml)for counterselection against the donor strain,and either 5-bromo-4-chloro-3-indolyl phosphate(XP)(40?g?ml)for de-tection of active phoA fusions or5-bromo-4-chloro-3-indolyl-?-D-galactoside(X-gal)(40?g?ml)for detection of active lacZ fusions.After incubation for2–3days at room temperature, tetracycline-resistant colonies were picked by using a Qpix (Genetix,Hampshire,U.K.)colony-picking robot programmed to select white or blue colonies(both colors were picked and mapped).Colonies were arrayed into384-well plates,each well containing90?l of freezing medium(10g/liter tryptone?5g/liter yeast extract?10g/liter NaCl?6.3g/liter K2HPO4?1.8g/liter KH2PO4?0.5g/liter sodium citrate?0.9g/liter(NH4)2SO4?4.4% glycerol)supplemented with20g?ml tetracycline.Plates were incubated for18h at37°C,then frozen and stored at?80°C.

Transposon Insertion Location.Transposon insertion locations were determined by a two-stage semidegenerate PCR and sequencing protocol(Supporting Methods and Table4,which are published as supporting information on the PNAS web site).In the first round of PCR,a specific primer for the transposon sequence is paired with a semidegenerate primer with a defined tail.A0.5-?l aliquot of thawed glycerol stock was added directly to the PCR reagents as template for the first round.In the second round of PCR,a nested transposon primer is paired with a primer targeted to the tail portion of the semidegenerate primer, and0.5?l of PCR product from the first round was used as template.PCR products from the second round were cleaned up by using exonuclease I and shrimp alkaline phosphatase(United States Biochemical),and used as sequencing templates.Se-quencing was performed by using Big Dye Terminator version 3.0(Amersham Pharmacia)and reactions were analyzed with ABI3700autosequencers(Applied Biosystems). Automated sequence analysis was accomplished by using a PERL script that compiled assessment of phred quality scores(9),

?To whom correspondence should be addressed.E-mail:mikejac@https://www.wendangku.net/doc/e214131718.html,.

?2003by The National Academy of Sciences of the USA

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crossmatch(using the Smith–Waterman algorithm)to the PAO1 sequence,and data retrieval from the PAO1annotation table.

Quality Control.Confirmation of the positions of a randomly selected subset of transposon insertions was determined by PCR. Custom primers to the PAO1genome were designed oriented toward the5?end of the transposon.Gel electrophoresis of PCR products was used to determine whether the PCR produced a major product of the expected size.This method was able to confirm the presence of the mapped strain in?95%of wells, including some wells that were identified as containing multiple strains.These‘‘mixed’’wells were more prevalent in replica plates than in the original source plates,and in all cases the mapped strain was retrievable from mixed wells.

Statistical Analysis.To assess the statistical properties of the observed set of insertion sites,we used a neutral-candidate model in which every gene is assumed nonessential and equally likely to be hit,and a neutral-base pair model,in which every base pair is assumed equally likely to define an insertion site.The neutral-candidate model was rejected because variation in gene size had a large effect on the number of times a gene was hit.In both models,the number of times an ORF is hit follows a multinomial distribution with parameters n,p1,...,p k,where n is the number of transposon insertions,p j is the probability of landing in the j th ORF,and k is the number of ORFs.p j was

estimated as the length of the ORF divided by the total length of the genome.The bias corrected estimate of the number of essential genes is377with a standard deviation of77.3.See Supporting Methods for a detailed description of the statistical methods used.

Mutant Phenotype Characterization.Twitching motility and pro-totrophic growth phenotypes were scored by replica printing the entire collection onto LB agar containing tetracycline and chromogenic indicator(identical to the original selection medi-um),Mops minimal agar,Mops minimal agar supplemented with amino acids,vitamins,purines and pyrimidines,and Pseudomo-nas isolation agar(PIA)(see Fig.7).Strains were replicated by using a384-pin plastic replicator(Genetix)or a metal replicator. The replicas were incubated for2days(37°C)and photographed by using high-resolution color digital imaging.Twitching motility was assessed by examining surface colony morphology from the images of the LB and supplemented minimal agar replicas; auxotrophs were scored by a comparison of growth on minimal and supplemented minimal agar.For both phenotypes,two independent blind scorings were carried out by different indi-viduals.All potential mutants identified were included in the analysis.

Results

Mutant Production.A genome-wide random-insertion library was generated for the MPAO1isolate of P.aeruginosa strain PAO1. Two different transposon Tn5IS50L derivatives,IS phoA?hah (10)and IS lacZ?hah,were used to generate mutant strains(Fig.

1).Insertion of either transposon confers tetracycline resistance and leads to a blue colony phenotype on indicator medium when they are positioned in-frame in appropriately expressed genes. Tetracycline-resistant strains were arrayed in384-well plates and assessed for transposon-insertion position and phenotype.In-sertion locations were mapped by using PCR amplification and sequencing of the5?transposon boundary(10).A total of42,240 mutant strains(110plates of384individual mutants)were mapped,corresponding to45,409attempted sequencing reads, and36,154matches to the PAO1genome,for an average success rate of80%(Table1).Elimination of exact-duplicate-insertion locations left30,100unique insertions,split evenly between the two transposons(15,063IS phoA?hah insertions,15,037IS lacZ?hah insertions).Of the unique insertion locations,27,263were within predicted ORFs,corresponding to the89%of the genome comprised of coding sequence(11).The distribution of hits among ORFs did not conform well to a Poisson distribution,but near-saturation was nonetheless achieved(Fig.2).As expected, more hits occurred in larger ORFs(Fig.3),and there was a larger-than-expected zero class.The average number of hits per ORF was5.05,and was5.75among ORFs hit at least once.

Candidate-Essential Genes.Transposon insertions were not recov-ered in678ORFs.Genes may have been missed either by chance, because of sequence-specific insertion rates,or because muta-tions are lethal.The number of genes missed is a small fraction of the total(12%);thus,it is likely that many of these genes are essential for growth on a rich medium.Genes without insertions, designated candidate-essential genes appear to be distributed randomly throughout the PAO1genome(Fig.4).Transposon insertion density is lowest in the area between coordinates1.5 Mbp and3Mbp;the cause for this is

unknown.

Fig.1.Transposons used for insertion mutagenesis.Transposons IS phoA?hah(4.83kbp)and IS lacZ?hah(6.16kbp)are derived from the IS50L element of transposon Tn5and generate alkaline phosphatase(?phoA)or?-galacto-sidase(?lacZ)translational gene fusions if appropriately inserted in a target gene.An outward-facing neomycin phosphotransferase promoter is expected to reduce polar effects on downstream gene expression for appropriately oriented insertions.Cre-mediated recombination excises sequences situated between the loxP sites in each transposon,leaving a63-codon insertion that encodes an in?uenza–hemagglutinin epitope and a hexahistidine metal-af?nity puri?cation tag(together referred to as‘‘hah,’’see ref.10).?phoA, alkaline phosphatase gene;?lacZ,?-galactosidase gene;tet,tetracycline re-sistance determinant;loxP,Cre recognition sequence;P,neomycin phospho-transferase promoter.

Table1.Summary of the results of the transposon library

Data set N Mutants arrayed42,240 Mapped insertion locations36,154 Identical insertions4,423 Unique insertion locations30,100 Insertions inside ORFs27,263 Insertions between ORFs2,837 ORFs hit internally4,892 ORFs never hit internally678 Average hits per ORF 5.05 In-frame insertion locations4,823 Blue colony in-frame insertions2,546 ORFs with in-frame insertions2,582 Mutants scored for colony phenotype42,240 Twitchless mutants709 ORFs with twitchless mutants360 Auxotroph mutants813 ORFs with auxotroph mutants546

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Several models were used to analyze the transposon insertion results.A neutral-candidate ORF model (i.e.,each gene may be hit with equal probability)(12),was rejected after initial trials showed ORF size has an effect on the number of insertions.When a neutral-base pair model (each base pair position in the genome is hit with equal probability and the likelihood of each gene being hit is proportional to its size)is used,the expected number of missed ORFs is 307with a standard deviation of 15.33(see Materials and Methods ).When the data from Fig.2are fitted to this model,it is predicted that a maximum of 5,206ORFs may be hit,leaving 364essential genes.From these analyses,we conclude that it is likely the actual number of essential genes in P.aeruginosa is between 300and 400.

To investigate whether the transposon-insertion positions approximated a random distribution,observed gaps between insertions were compared with simulated gaps from a random positioning of an equal number of insertions in the genome (Fig.5).From this analysis,it is clear that the deviation from a random distribution is significant,and is caused by numerous larger-than-expected gaps between transposon insertions.This result matches the observation that large gaps between insertions contain candidate-essential gene ‘‘clusters.’’

In addition to the 678ORFs never hit,721ORFs were hit only once.We expect this class of ORFs to contain some essential

genes whose functions were not fully disrupted by the insertion (a ‘‘wounded ’’phenotype).Analysis of these single-hit ORFs showed that the hits were distributed approximately evenly throughout the ORFs,with a small bias toward the extreme 3?of the gene.Of the 721ORFs that were hit only once,204are also adjacent to candidate-essential genes.These ORFs were more highly biased toward insertions in their extreme 3?end (Fig.6).Hence,it is likely that some of the single-hit ORFs are essential and,more rarely,that ORFs were hit more than once (see below).

The deduced list of candidate-essential genes was compared with the overall PAO1gene complement for functional repre-sentation.PAO1genes have been grouped into 25functional classes,with an additional class for unknown hypothetical genes (11).When the proportion of candidate-essential genes falling into each class was compared with the whole genome,several categories were highly over-or underrepresented (Table 2).ORFs found most commonly in the list of candidate-essential genes included translation machinery and cell-division control genes (overrepresented by 3.5–4times),whereas underrepre-sented categories included chemotaxis and two-component reg-ulatory systems.

Overall Sequence Conservation of Pseudomonas Candidate-Essential Genes.It is generally observed that significant overlap exists

among sets of essential genes in genomes of Gram-negative bacteria.To examine the overlap between sets of essential genes in genomes of PAO1and E.coli ,ORF translations from these two genomes were compared by using mass BLASTP analyses.Candidate-essential and candidate-nonessential genes from this work were compared with the list of known essential and nonessential E.coli genes in the PEC database

(www.shigen.nig.

Fig.2.Saturation transposon mutagenesis.A total of 110384-well plates of transposon-containing strains of P.aeruginosa were analyzed for transposon insertion location.Insertions were mapped to 27,263locations within ORFs with another 2,837between ORFs.Of the 5,570ORFs in the P.aeruginosa genome,4,892were hit at least once by a transposon insertion.The number of unique insertion locations increased linearly with new strains,whereas the number of ORFs hit approached a

plateau.

Fig.3.Distribution of transposon hits among ORFs.The number of ORFs for which a transposon insertion wasn ’t recovered was 678,and 721were hit only once.The number of times an ORF was hit increases with ORF size.Error bars are one standard deviation in each

direction.

Fig.4.Distribution of transposon insertions and candidate-essential genes.The circular 6.2-Mbp P.aeruginosa genome was hit in 30,100locations with individual transposon insertions.The black circular line represents the ge-nome sequence with the origin of replication at coordinate zero.Bars outside the line represent genes transcribed clockwise,whereas those inside are transcribed counterclockwise.Red bars represent ORFs that contain transpo-son insertions,and green bars represent ORFs not hit (candidate-essential genes).Black marks on the outside of the circle represent transposon inser-tions.The sunburst pattern represents the number of insertions per 10,000bp,with the scale extending from the center.

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M I C R O B I O L O G Y

ac.jp ?ecoli ?pec ?index.jsp)(Table 3).A total of 215ORFs from PAO1have a strict ortholog in the list of known essential genes from E.coli .A majority of these orthologues (133ORFs)are on our list of candidate-essential genes.

The median percent position (5?to 3?)of insertions in PAO1ORFs only hit once was 49.1%,but was 86.7%for those that had a match to an E.coli known-essential gene.This observation suggests that we did recover mutations in some essential genes,but that the positions of these insertions were strongly biased toward the extreme 3?end of the genes.The fraction of apparently wounded genes (i.e.,genes have one or more hits,but

Table https://www.wendangku.net/doc/e214131718.html,parison of the proportion of genes (sorted by function)represented in the list of candidate essentials,versus their proportions in the P.aeruginosa genome

Primary function

No.of genes

No.not hit

Relative representation among candidate essentials

Translation,posttranslational modi ?cation,degradation 14975 4.14Cell division

2611 3.48Cell wall ?lipopolysaccharide ?capsule

8630 2.87Biosynthesis of cofactors,prosthetic groups,and carriers 13244 2.74Transcription,RNA processing,and degradation 4513 2.37Fatty acid and phospholipid metabolism 5715 2.16Nucleotide biosynthesis and metabolism 6014 1.92Energy metabolism

17035 1.69Protein secretion ?export apparatus

8416 1.56DNA replication,recombination,modi ?cation,and repair 8115 1.52Chaperones and heat shock proteins 528 1.26Adaptation and protection

669 1.12Related to phage,transposon,or plasmid 628 1.06Hypothetical,unclassi ?ed,unknown 2,3812610.90Central intermediary metabolism 6570.88Membrane proteins

4340.76Amino acid biosynthesis and metabolism 151130.71Secreted factors (toxins,enzymes,alginate)6050.68Carbon compound metabolism 134110.67Transcriptional regulators 403270.55Putative enzymes

457250.45Antibiotic resistance and susceptibility 1910.43Transport of small molecules 559260.38Motility and attachment

6720.25Two-component regulatory systems 11630.21Chemotaxis 4500.00

Total

5,570

678

Fig.5.Quantile –quantile plot of transposon-insertion gap size distribution.The 30,100-transposon insertion locations in the 6.2-Mbp P.aeruginosa ge-nome were compared with an equal number of random insertions in a simulated 6.2-Mbp genome.The x axis represents the observed size distribu-tion of gaps (the distance between adjacent insertions)and the y axis repre-sents the simulated distribution of gaps.Each point plots the same quantile for both distributions.Line A represents a 1:1relationship,where the points would lie if the observed and the randomly generated data sets were identical.The size distribution of the observed gaps is signi ?cantly larger than that of the random data set.For example,an equal proportion of gaps fell below 3,800bp in the observed data set as fell below 1,500bp in the random data set (represented by line

B).

Fig.6.Hit distribution relative to position within ORF.The proportion of transposon insertions according to their relative position within ORFs is rep-resented in a histogram.Hits are nearly evenly distributed (e.g.,5%of hits occur in the ?rst 5%of ORFs)when all insertions are considered.For ORFs that were hit only once (dark gray),particularly those adjacent to ORFs never hit,the proportion of hits is highly skewed toward the 3?end of the gene.

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none in the first 90%of their ORFs,a total of 109ORFs)from PAO1that have homology to known essential genes was similar to the fraction in our set of candidate-essential genes (21%or 23ORFs).We consider these ORFs additional candidate-essential genes.These results match the mathematical modeling predic-tion that approximately half of our candidate-essential genes are truly nonessential.

Mutant Phenotypes.To examine the use of the strain collection for

mutant identification,we screened for loss of surface (‘‘twitch-ing ’’)motility and inability to grow on minimal medium (aux-otrophy)(Fig.7).These ‘‘reference ’’phenotypes were chosen because their genetic bases are well characterized;hence,we could measure our recovery rate of mutants relative to previous mutational analyses.Twitching motility,which is a pilus-based process dependent on 34identified genes,leads to the produc-tion of colonies with a distinctive lacy edge (13).In the screen for twitching defects,we identified mutations in or immediately upstream to all of the previously identified genes except pilZ ,(Table 5,which is published as supporting information on the PNAS web site).However,an insertion near the 3?end of a functionally unrelated gene immediately upstream of pilZ did cause a nontwitching phenotype,perhaps because of a polar effect on pilZ expression.These results confirm excellent cor-relation between phenotype and genotype.However,numerous insertions in the known twitching motility genes were not detected (Table 5).In the 33genes known to affect surface motility for which we had at least one transposon mutation,12.5–100%of the insertions caused a twitch ?phenotype.The majority of ‘‘missed ’’mutants were in wells containing a mixture of (genetically stable)twitch ?and twitch ?cells.The occurrence of mixed populations in wells was observed to increase through rounds of replica plating,although quality control experiments were able to identify the sequenced strain in most cases.Even low-level contamination with twitch ?cells is expected to obscure the sharp colony edge that defines the twitch ?phenotype.For one gene in which mutants were detected inefficiently (pilS ),further analysis revealed that those undetected alleles we exam-ined had ‘‘leaky ’’phenotypes.Insertions in four genes (chpB –chpE )implicated in surface motility (14)were not associated with significant motility defects.Overall,in the mutant collec-tion,twitchless-phenotype-producing hits occurred in 366ORFs,80of which were confirmed by a second hit.An additional 16produced a phenotype in their only hit.Of the 80‘‘confirmed-twitch ?’’ORFs,26were among the previously known ORFs.An additional 31of these ORFs had been functionally annotated as ‘‘mobility and attachment ’’genes,and another 23had no func-tional annotation.

Intergenic transposon insertions at 39unique locations pro-duced a twitch ?phenotype.Of those,seven were adjacent to genes that produced a twitch ?phenotype when hit internally,and another five were in two large intergenic spaces.The observation that these two large intergenic spaces produced twitch ?phenotypes each time they were hit suggests that genetic information essential for twitching exists there,albeit in se-quences that provide no immediate clues to function.

Insertions resulting in auxotrophic phenotypes were distrib-uted among 546ORFs.Of those,110ORFs that had two or more unique hits that resulted in the same phenotype were considered ‘‘confirmed auxotrophs.’’There were also 21ORFs in which the only hit produced an auxotrophic phenotype.‘‘Auxotroph ’’

Table 3.Homology of PAO1and E.coli candidate-essential genes

Gene class query

Gene class subject

Strict orthologues

PAO1ORFs (5,570)

E.coli known essential genes 215PAO1candidate-nonessential genes (4,892) E.coli known essential genes 82PAO1candidate-essential genes (678) E.coli known essential genes 133E.coli known essential genes (232)

PAO1candidate-essential genes

135

Comparison of predicted translations of PAO1ORFs to E.coli.Strict orthologues were de ?ned as the top BLAST hit that had a match length of at least 75%of the length of the query

sequence.

Fig.7.Replica plating and phenotyping.One 384-well plate of phoA transposon-containing strains was replica plated onto three conditions:rich medium plus indicator (Top ),minimal medium (Middle ),and minimal medium plus supplemented nutrients (Bottom ).(Top )Colony A (arrow)represents a twitch ?phenotype;thus,the mutant is de ?cient in the gene pilY1,a type-4?mbrial biogenesis protein.Colony B (circled)is an auxotrophic mutant (show-ing growth on LB and supplemented,but not minimal,media)and carries an argG (argininosuccinate synthetase)insertion.

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ORFs were10-and6-fold overrepresented in amino acid biosynthesis and metabolism and nucleotide biosynthesis and metabolism genes,respectively.Auxotrophic phenotypes re-sulted from58intergenic hits,all but one of which were adjacent to ORFs that had produced an auxotrophic phenotype at least once.

For the arginine,histidine,isoleucine-valine,leucine,and tryptophan biosynthetic pathways,we compared our results with previous data(Table6,which is published as supporting infor-mation on the PNAS web site).Of the36genes known to be required for these five pathways,mutations in all except one (hisE,the shortest of the36ORFs)were represented in our collection.Mutants with insertions in30of the genes were detected as auxotrophs.In the other five cases,the existence of redundant genes appears to explain our failure to find insertions with auxotrophic phenotypes(Table6).For three histidine-biosynthetic genes,the recovery of auxotrophic insertions in only one of two paralogues implies that these genes(hisC1,hisF1,and hisH1)are primarily responsible for biosynthesis.

Discussion

The genome sequence and associated annotation information for the PAO1strain of P.aeruginosa have been used to facilitate high-throughput generation of a comprehensive mutant library. By using random transposon-insertion mutagenesis,nearly90% of the ORFs in the PAO1genome have been disrupted at least once.This mutant collection is both a significant resource for future research and a source of immediate functional insight into the genome of P.aeruginosa.

Several large mutant collections have provided significant data with regard to mutant phenotypes and essential genes.Our transposon-insertion library is distinct in providing virtually complete coverage of the genome with a set of strains archived in a format that facilitates mutant retrieval and phenotypic analysis.Archived collections of deletion mutants(S.cerevisiae) and inactivating-insertion mutants(B.subtilis)have been created by using gene-by-gene knockout strategies that required the participation of large consortia of laboratories(2).More cost-effective high-throughput technologies,developed here for the analysis of P.aeruginosa,should be directly applicable to a wide variety of bacterial species.

Development of therapeutic agents may be directed by a comprehensive understanding of all of the gene products indi-vidually required for survival of P.aeruginosa.By generating a near-saturation-mutant collection,we have arrived empirically at a list of678candidate-essential genes.Many of the candidate-essential genes are expected(i.e.,they code for nonredundant machinery central to cell survival),whereas others are new, including the263previously unclassified ORFs that are on the candidate-essential gene list.Our statistical and bioinformatic analyses predict that approximately half of the genes on our list are truly essential.

To test the suitability of the P.aeruginosa strain collection for direct phenotypic screening,we identified mutants defective in two previously studied traits(twitching motility and prototrophic growth).In both cases,we identified nearly all genes expected from the earlier studies.Several previously undescribed genes apparently required for twitching motility were also identified. These initial tests show that phenotypic screens of the entire collection are feasible and may produce essentially complete lists of candidate genes implicated in biological processes of interest. Furthermore,the existence of multiple alleles for most genes in our mutant collection meant that low-level contamination did not undermine the phenotypic analysis.

The potential utility of the library extends beyond screens for new mutants.Because the strains in the collection may be readily retrieved,the effects of mutations in any gene can be studied by a‘‘reverse-genetic’’strategy.When a gene attracts interest on the basis of bioinformatic or functional genomic analysis,appropri-ate phenotype tests can be immediately pursued.In addition,the properties of the transposons used to generate the mutant set should facilitate downstream manipulations.Deletion of trans-poson sequences by site-specific(cre–loxP)recombination elim-inates the tetracycline-resistance determinant and facilitates constructing double mutants by using other insertions in the collection.Such double mutants may be useful for epistasis analysis or in engineering genomes with deletions between two insertion sites(15).In-frame insertions of the transposons generate reporter-gene fusions that may be used to study ex-pression.Such fusions may also be readily converted into deriv-atives carrying internal epitope?affinity purification tags for analysis of unfused polypeptides(10).

Infections with P.aeruginosa are the leading cause of death in cystic fibrosis patients,and also lead to several other clinically important infections.The development of new therapies for these infections will be challenging because of the complex biology of P.aeruginosa.The comprehensive mutant library we have constructed will allow an accelerated genetic dissection of traits such as metabolic flexibility and inherent drug resistance that make P.aeruginosa such a tenacious pathogen.

We thank Peter Chapman,David D’Argenio,Larry Gallagher,Arnold Kas,and Doug Passey for technical assistance and advice.We also acknowledge Kenneth Stover and members of his group for sharing unpublished information about a transposon-mutagenesis project on P. aeruginosa that they initiated at Pathogenesis,Inc.(subsequently ac-quired by Chiron).This work is supported by the Cystic Fibrosis Foundation Grants JACOBS03F0and MILLER00V0.

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