Adaptation of the Yeast URA3 Selection System to Gram-Negative Bacteria and Generation of a {Delta}betCDE Pseudomonas putida Strain

A general procedure for efficient generation of gene knockoutsin gram-negative bacteria by the adaptation of the Saccharomycescerevisiae URA3 selection system is described. A Pseudomonasputida strain lacking the URA3 homolog pyrF (encoding orotidine-5'-phosphatedecarboxylase) was constructed, allowing the use of a plasmid-bornecopy of the gene as the target of selection. The delivery vectorpTEC contains the pyrF gene and promoter, a conditional originof replication (oriR6K), an origin of transfer (mobRK2), andan antibiotic selection marker flanked by multiple sites forcloning appropriate DNA segments. The versatility of pyrF asa selection system, allowing both positive and negative selectionof the marker, and the robustness of the selection, where pyrFis associated with uracil prototrophy and fluoroorotic acidsensitivity, make this setup a powerful tool for efficient homologousgene replacement in gram-negative bacteria. The system has beeninstrumental for complete deletion of the P. putida choline-O-sulfateutilization operon betCDE, a mutant which could not be producedby any of the other genetic strategies available.

INTRODUCTION

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Introduction

Homologous gene replacement is a basic tool for the study ofbiological systems, and as such the efficiency of methods forgene knockout generation contributes to the ease with whicha system can be characterized by permitting genetic manipulation.The diversity of bacterial species under study has harboredthe development of a plethora of cloning, expression, and mutagenesistools, but those for the generation of gene knockouts are stillin need of improvement (39). PCR-based one-step techniques forgene replacement and disruption (6, 46, 47) greatly facilitategene knockout construction but thus far have only been shownto work in a limited number of species (i.e., Escherichia coliand Salmonella spp.). For other bacteria, including a largenumber of genera of considerable environmental and biotechnologicalinterest, exchange of DNA segments to and from the chromosomedepends on the use of counterselection markers, but these arefew and of limited efficiency.

The dearth of suitable tools to effect such exchanges limitsthe possibilities of genetic analysis and chromosomal engineeringof most non-E. coli strains. The most widely used counterselectionmarker in gram-negative bacteria is the sacB gene of Bacillussubtilis, whose product, levan sucrase, confers sucrose sensitivityon a large number of gram-negative species (11, 40). Also, inStreptomyces spp., the glkA gene product, glucose kinase, conferssensitivity to 2-deoxyglucose (44). Use of the glkA system inother bacteria has not been reported. In Bacillus subtilis,uracil phosphoribosyltransferase has been used successfullyas a counterselection marker in one-step generation of chromosomalpoint mutations, in-frame deletions, and gene knockouts (9).Uracil phosphoribosyltransferase converts 5-fluorouracil intoa compound, 5-fluoro-UMP, that is metabolized into 5-fluoro-dUMP,which strongly inhibits thymidylate synthetase (28).

Except for sacB-based vectors, none of the counterselectionmarkers have been employed in Pseudomonas putida to any significantextent. Yet, although useful in many cases, sacB is by no meansan optimal gene for this end, as its function depends on thetransport of sucrose. This often has to be forced by addinghigh extracellular concentrations (5% or more) of the sugar,causing undue stress and favoring the rise of mutations thatbypass the stringency of the selection. Having a reliable systemfor allelic exchange and chromosomal engineering of P. putidais a prerequisite to realize the full potential of this speciesin biocatalysis and bioremediation. The same applies to manyother gram-negative bacteria which are not yet amenable to geneticanalysis (34).

In our pursuit of improved genetic tools for P. putida, we cameacross the uracil biosynthesis gene URA3, which has been usedwidely in positive and negative selection in Saccharomyces cerevisiae.URA3 encodes an enzyme, orotidine-5'-phosphate decarboxylase(ODCase), that can catalyze the transformation of 5-fluorooroticacid (FOA, a uracil analogue) into a highly toxic compound (3).Thus, counterselection works on the basis that the presenceof URA3 confers sensitivity to FOA, while URA3-negative cellsare FOA resistant. Alternatively, URA3 as a positive selectionmarker works based on the ability of an exogenous or plasmid-borneURA3 gene complementing uracil auxotrophy of a URA3- negativestrain.

Assuming that URA3/FOA counterselection could work so long asan ODCase activity exists in an organism, successful adaptationof the URA counterselection system has allowed homologous genereplacement in the archaea Halobacterium salinarum (33), Thermococcuskodakaraensis KOD1 (38), and Halorerax volcanii (2). Also, URA3-basedcounterselection has been used in Mycobacterium smegmatis forobtaining unmarked gene integration into the chromosome (20)and in shuttle vectors for the hyperthermophilic archaeon Pyrococcusabyssi (21). The pyr genes are URA homologs, such that adaptationof the URA3 system to bacteria is based on identification ofthe ODCase-encoding pyr gene and isolation of mutants that areFOA resistant and uracil auxotrophs, followed by the use ofallelic exchange to isolate the gene replacement.

Using the complete genome sequence of P. putida KT2440 (27),we identified a single pyrF/URA3 ortholog in the chromosome.This opened the possibility of recreating in P. putida the manyadvantages and utilities of the URA3 system of S. cerevisiae.In this work, we present a complete genetic system for generationof knockouts in gram-negative bacteria that uses the pyrF geneof Pseudomonas putida as a positive and negative marker. Weprove that pyrF-based selection is highly efficient by creatinga P. putida strain entirely deleted of the osmotic stress-relatedoperon betCDE, which could not have been generated by any othermethod.

MATERIALS AND METHODS

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Materials and Methods

Biological materials, culture conditions, and general procedures.
The bacteria and plasmids used in this work are listed in Table1. Pseudomonas putida KT2442 (15) and Escherichia coli strainswere grown at 30°C and 37°C, respectively, in LB or2YT broth (37). P. putida strains were also grown in M9 minimalmedium (37) with citrate (2 g/liter) as the carbon source. Antibioticswere used at the following concentrations: rifampin, 50 µg/ml;ampicillin, 150 µg/ml; kanamycin, 50 µg/ml; streptomycin,50 µg/ml (E. coli) or 100 µg/ml (P. putida); gentamicin,10 µg/ml; and chloramphenicol, 30 µg/ml.

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TABLE 1. Strains and plasmids

For the P. putida ${Delta}$ pyrF TEC1 strain, uracil (Sigma Aldrich, Madrid,Spain) was used at 20 µg/ml. 5-Fluoroorotic acid (FOA;Zymo Research) was used at 250 µg/ml. Plasmids were transferredfrom E. coli to P. putida by tripartite conjugation on membranefilters (0.45 µm, Millipore), including helper strainE. coli HB101(pRK600). After 6 to 8 h of incubation at 30°Con LB agar, the conjugation mixture was plated on minimal selectivemedium as indicated in each case. All oligonucleotides weresynthesized by Sigma-Genosys. DNA constructs were sequencedwith the dideoxy method (ABI-Prism automated DNA sequencer,Perkin Elmer). Endurance to osmotic stress was assayed by platingserial dilutions of cultures onto solid M9 minimal medium supplementedwith citrate and sodium chloride to 0.6 or 0.7 M.

Plasmid and strain construction.
The P. putida ${Delta}$ pyrF TEC1 strain was created as follows. A 1.8-kbregion of the P. putida genome spanning the pyrF gene (identifiedwith sequence information from the Pseudomonas putida genome,http://www.tigr.org) was amplified by PCR with primers 37pyr5(5'-CCGTAAAGCTTGGATGCACTTAGCCTGCAAAAGGAGC-3') and 37pyr3 (5'-TGCAGAATTCCTGAAGCGCGAATGCCCCAAAGGCGTG-3'),which added EcoRI and HindIII sites at the ends of the targetedDNA segment. The resulting product was then cloned into pUC19Not(Table 1), yielding plasmid pUP. This plasmid was cut with Eco47IIIand SmaI, releasing an internal 404-bp segment of the 702-bp-longpyrF gene, and treated with T4 DNA polymerase (Roche AppliedScience) to produce blunt ends at the sequences left attachedto the plasmid. This was ligated with the SmaI fragment frompXYLE10 (42) containing the xylE gene (for catechol 2,3-dioxygenase),yielding pUPX. Plasmid pKPX, used for chromosomal delivery ofthe resulting assembly ${Delta}$ pyrF::xylE, was generated by excisingthe 2,418-bp NotI fragment of pUPX (containing the deleted pyrFgenomic region inserted with xylE) into the NotI site of thesacB⁺ suicide vector pKNG101 (17).

To create a pyrF⁺ broad-host-range plasmid, the P. putida pyrFgene and its promoter (890 bp) was amplified with primers NotIpyrFprom(5'-GATATACAGCGGC CGCATCGGGCCGTGGGCAACGGT-3') and Asc pyrF (5'-GAATGGCGCGCCCCTTACCCACGGATCTC-3'),introducing NotI and AscI sites to the 5' and 3' ends of theamplified segment, respectively. The resulting 882-bp productwas ligated to pGEM-T Easy (Promega), resulting in pGAPN. Digestionof pGAPN with EcoRI (vector sites flanking the pyrF insert)released a 920-bp fragment containing pyrF, which was ligatedwith EcoRI-digested plasmid pKT231 (10) and electroporated intoE. coli X82, a uracil auxotroph (from S. Brenner, through theE. coli Stock Center, CGSC 5889 ID 6952), and plated on M9 minimalmedium (37) supplemented with glucose (2 g/liter), tryptophan(0.1 mM), histidine (1 mg/liter), thymidine (0.5 mg/liter),and kanamycin. The plasmid borne by transformants growing withouturacil was named pKP and consisted of pKT231 with a functionalpyrF insertion.

pTEC, the pyrF⁺ vector for allelic exchange, was generated asfollows. The same primers (NotIpyrFprom and Asc pyrF) employedfor construction of pKP were used to amplify the 870-bp regionof the P. putida pyrF gene and its promoter. A 1.0-kb kanamycinresistance gene was amplified separately from pUT mini-Tn5 Km1(7) with primers 5'MCS Km (5'-ATAAGAATGCGGCCGCTCTAGAAGTACTAGGCCTATCGATGAGCTCCGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAA-3')and 3'MCS Km (5'-TGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAG GTACCAGATCTGATATCACGCGTGAATTCAGAC-3')which flanked the kanamycin resistance determinant with multiplerestriction sites. Finally, a 2.0-kbp fragment from plasmidpGP704 (25) containing the origin of transfer (oriT) of RK2(12, 45) and the vegetative origin of replication (oriV) ofR6K (which functions only in concert with the replication protein encoded by gene pir) (16) was amplified with primers 5'ORIEcoNotAvr (5'-TACGGAATTCGCGGCCGCCCTAGGCCATGTCAGCCGTTAAGTGTTCCTGT-3')and 3'ORI EagApaAsc (5'-CATGTCGGCGCGCCGGGCCCCGGCCGCCGTGGCTCCGGCGTCTT-3'),which add EcoRI and AscI sites to the corresponding DNA. Thethree PCR products (one bearing pyrF, other kanamycin resistance,and the last the oriV/oriT origins) were digested with the cognateenzymes required to produce cohesive ends, ligated, and transformedinto E. coli CC118 ${lambda}$ pir (15). Transformants growing on LB-kanamycinagar plates were found to bear the plasmid shown in Fig. 3,which was subsequently named pTEC (EMBL accession number BN000674).

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FIG. 3. Map of pTEC. (A) The plasmid contains the P. putida pyrF gene and its promoter, a kanamycin resistance cassette flanked by multiple cloning sites, the broad-host-range origin of replication oriR6K, and the origin of transfer mobRK2. Only unique restriction sites are shown. The diagram was drawn with PlasMapper (http://wishart.biology.ualberta.ca/PlasMapper/index.html) (8). (B) Schematic diagram of the cassette cloned into the pTEC NotI site for generating a betCDE deletion mutant. In the cassette, the betCDE genes have been replaced by the gentamicin resistance cassette and lacZ genes while maintaining the flanking sequences, ORF PP0074 and betR.

${Delta}$ pyrF P. putida strain TEC1 was constructed by mobilizing pKPXfrom E. coli CC118 ${lambda}$ pir into P. putida KT2442 by tripartite matingwith E. coli HB101(pRK600) as the helper and selecting exconjugantsin minimal medium plates with citrate and streptomycin. Putativecointegrates were grown in LB without selection, and resolutionof the cointegrate resulting in chromosomal acquisition of thedeleted pyrF gene was selected by plating on minimal mediumplates with citrate, uracil, and FOA (counterselection of sacBwith sucrose never worked in this case).

The plasmid used for deletion of the P. putida putative betCDEoperon, pTGLB, was constructed as follows. A PCR product witha gentamicin resistance cassette and a ß-galactosidasegene flanked by betCDE upstream (ORF PP0074) and downstream(betR) sequences, forming a transcriptional fusion of the betCDpromoter with the ß-galactosidase gene (Galvãoet al., unpublished data) was cloned into plasmid pGEM-T Easy(Promega) to generate pGGLB. pGGLB was cut with NotI, and thedeletion cassette was cloned into the NotI site of pTEC to generatepTGLB.

RESULTS AND DISCUSSION

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Results and Discussion

P. putida pyrF gene and FOA sensitivity.
A BLAST search on the P. putida genome with the P. aeruginosapyrF gene sequence yielded an open reading frame (ORF) with90% identity (locus PP1815 in the P. putida genome). The P.aeruginosa pyrF gene has been shown to encode orotidine-5'-monophosphatedecarboxylase (ODCase) by enzymatic assays of the gene product(43). In P. putida, pyrF is not part of an operon (Fig. 1A),being located between a conserved hypothetical protein and apredicted alcohol dehydrogenase.

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FIG. 1. Generation of ${Delta}$ pyrF P. putida TEC1. (A) Schematic diagram of the P. putida pyrF gene (ORF PP1815) chromosomal context. The locus name of each ORF with assigned or predicted function is shown; hypothetical ORFs are unmarked. (B) Schematic diagram of the P. putida pyrF gene and of the ${Delta}$ pyrF in P. putida TEC1. The xylE gene was cloned in the Eco47III and SmaI sites of pyrF. Primers are shown as arrows, indicating primer combinations used to amplify the pyrF and xylE sequences. (C) PCR analysis of wild-type and ${Delta}$ pyrF strains. Lanes 1 to 3: primers annealing to the pyrF promoter and to the 3' end of the gene were used to amplify the pyrF gene of P. putida KT2442, of the ${Delta}$ pyrF strain P. putida TEC1, and of TEC1 with the pyrF-containing pKP plasmid. Lanes 4 to 6: the chromosomal copy of ${Delta}$ pyrF in P. putida TEC1 carrying pKP was amplified with primers annealing at the 5' end of pyrF and xylE.

In a first step to develop a counterselection system based onpyrF, we tested the sensitivity of P. putida KT2442 to FOA.Plating of up to 10⁹ CFU on solid minimal medium containinguracil and FOA revealed sensitivity to the compound, the spontaneousmutation frequency to FOA resistance being 0.8 x 10^–6.In T. kodakaraensis and H. salinarum, the frequency of FOA resistancearising is 1 x 10^–5 to 5 x 10^–5 and 6.9 x 10^–6,respectively (33, 38). Growth of FOA-resistant colonies on solidminimal medium showed that, contrary to what happens in thesetwo microorganisms, mutation to FOA resistance was not associatedwith uracil auxotrophy. Instead, of 50 FOA-resistant P. putidacolonies tested, all grew in the absence of uracil, such thatresistance is likely to be associated either with FOA uptakeor with the specific reaction catalyzed by the ODCase on FOAbut not on uracil. The particularity in FOA resistance of P.putida is further noted by the fact that the plating of 10⁹CFU on solid medium with FOA but lacking uracil did not yieldany resistant colonies. In any case, these figures were wellwithin the range of what is useful for selection and counterselectionof chromosome segments.

As a prerequisite to set up a counterselection system basedon pyrF, we pursued the generation of a ${Delta}$ pyrF P. putida strainso that pyrF in a plasmid could serve as a marker. To this end,a 2.0-kb region containing the pyrF gene was PCR cloned as describedin the Materials and Methods section, and the subsequent cloningof an xylE cassette was carried out to allow pyrF deletion (Fig.1B). The deletion construct (Fig. 1B) was cloned in pKNG101(17), yielding pKPX, and introduced into P. putida KT2442. Chromosomalintegration of the plasmid was selected by streptomycin resistance.Following growth without selection, a second recombination eventthat maintained the ${Delta}$ pyrF copy was pursued. Efforts to use thesacB gene of pKPX as a counterselection marker in the presenceof 5% or 8% sucrose to force resolution of the cointegratesrepeatedly generated sucrose-resistant spontaneous mutants.On the contrary, plating the streptomycin-resistant exconjugantson minimal medium containing uracil and FOA resulted in therise of FOA-resistant colonies, of which two of eight testedwere uracil auxotrophs. These ura FOA-resistant colonies werealso sensitive to streptomycin and turned yellow when sprayedwith catechol (10 g/liter), the substrate of the xylE gene product,catechol-2,3-dioxygenase (not shown). These properties wereindicative of the successful double recombination of the ${Delta}$ pyrF::xylEsegment into the chromosome of P. putida.

To confirm this, one of the ura clones that was FOA resistant,streptomycin-sensitive, and xylE⁺ (hereafter referred to asP. putida TEC1) was subjected to PCR analysis of the relevantchromosomal sequences. Figure 1C shows that the uracil auxotrophyand FOA resistance of P. putida TEC1 are associated with a bandthat corresponds in size to the deletion cassette with xylE(lanes 2 and 5). Note that when P. putida TEC1 carried a plasmidwith the wild-type pyrF gene, the pyrF::xylE deletion cassettewas not amplified with the pyrF primers, possibly due to preferentialannealing of the primers to the wild-type gene. Indeed, whenevera wild-type copy of pyrF was present, amplification of the pyrF::xylEdeletion cassette was not observed (see also Fig. 5, lane 6).P. putida TEC1 was thus chosen as the pyrF mutant of reference.That pyrF turned out to be far more powerful than sacB to selectresolution of the cointegrate of pKPX with the chromosome ofP. putida provided a good indication of the potential of theURA3 homologue for vector development.

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FIG. 5. PCR analysis of P. putida TEC1 ${Delta}$ betCDE and FOA-resistant cointegrate colonies. Lanes 1 to 5, P. putida TEC1 ${Delta}$ betCDE colonies. Lanes 6 to 10, FOA-resistant colonies carrying a chromosomal insertion of pTGLB. Primers: lanes 1 and 6, anneal at pyrF gene promoter and 3' end (as shown in Fig. 1B); lanes 2 and 7, anneal at pyrF gene 5' end and xylE gene 5' end (as shown in Fig. 1B); lanes 3 and 8, betC internal primers; lanes 4 and 9, kanamycin internal primers; lanes 5 and 10, lacZ internal primers.

To confirm that pyrF encodes the P. putida orotidine-5'-monophosphatedecarboxylase, growth of the wild-type and mutant strains wascompared. Clearly, while the ${Delta}$ pyrF P. putida strain TEC1 failedto grow in standard minimal medium, uracil addition sufficedto make growth of P. putida TEC1 and its wild-type counterpartP. putida KT2442 indistinguishable in liquid medium (Fig. 2A).This suggests that so long as an adequate amount of uracil ispresent, deletion of pyrF does not alter the growth rate. Theuracil auxotrophy of P. putida TEC1 can be complemented by thepyrF⁺ plasmid pKP, carrying pyrF under its own promoter (Fig.2B). That pyrF deletion confers FOA resistance, as should thedeletion of ODCase, was shown by a growth assay on solid medium(Fig. 2C). While P. putida TEC1 is FOA resistant, P. putidaKT2442 is sensitive. Uracil prototrophy and FOA sensitivityin P. putida TEC1 is regained upon complementation with pKP.Thus, the P. putida pyrF gene is a bona fide orotidine-5'-monophosphatedecarboxylase, and this activity is associated with FOA sensitivity.Reversion of the ${Delta}$ pyrF strains back to the wild-type pyrF genotypeis straightforward, as P. putida TEC1 and its derivatives canbe electroporated with the pyrF⁺ pUP plasmid (which does notreplicate in P. putida) (Table 1), and following a round ofantibiotic selection and growth without selection, plated onminimal medium without uracil. Spraying colonies with catecholallows visualization of xylE marker loss (not shown).

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FIG. 2. Uracil auxotrophy and FOA resistance conferred by pyrF deletion is complemented by plasmid-borne pyrF. (A) Strains P. putida KT2442 (squares) and P. putida TEC1 (circles) were grown at 30°C in liquid M9 minimal medium supplemented with citrate in the presence (solid symbols) or absence (open symbols) of uracil at 20 µg/ml. Growth was monitored at 600 nm after inoculation to 0.05 at 0 h. (B) Strains P. putida TEC1 carrying pKT231 (circles) or pKP (pKT231 carrying pyrF under its own promoter) (squares) were grown as in A. (C) Upper panel: Deletion of pyrF confers uracil auxotrophy and FOA resistance. Serial dilutions of P. putida KT2442 or TEC1 were plated on solid M9 minimal medium in the presence or absence of uracil (20 µg/ml) or with uracil and 5-fluoroorotic acid (250 µg/ml). Lower panel: Serial dilutions of P. putida TEC1/pKP were plated as above but with kanamycin, the resistance marker of pKP.

Structure and qualities of pTEC, a suicide plasmid for homologous gene replacement.
On the basis of the data above, we anticipated that the ${Delta}$ pyrFbackground should permit the use of pyrF as a counterselectionmarker in a suicide vector. Thus, the essential elements ofsuch a plasmid were put together by PCR cloning to make pTEC(Fig. 3A). These elements were the pyrF gene under its own promoter,a broad-host-range kanamycin resistance cassette flanked bymultiple cloning sites, facilitating cloning of the desiredgenomic regions through which recombination is to take place,a segment of plasmid pGP704 (25) carrying a conditional originof replication (oriV R6K), and an origin of transfer (oriT)for RK2-mediated mobilization (see Materials and Methods fordetails). This design allows, in a vector of small size, theinsert of any DNA segment of interest and its propagation inspecialized E. coli hosts (such as E. coli CC118 ${lambda}$ pir) engineeredto express in trans the replication protein of plasmid R6K.Finally, pTEC derivatives can be passed to P. putida ${Delta}$ pyrF bybipartite or tripartite conjugation mediated by the tra genesof plasmid RK2 (which can be provided by a helper strain, suchas HB101 carrying pRK600; see Table 1). Formation of a cointegratebetween pTEC derivatives and the chromosome of P. putida TEC1can be selected by plating in minimal medium without uracil(and, if desired, with kanamycin), while its resolution canbe forced by replating in medium containing FOA.

Generation and characterization of a P. putida ${Delta}$ betCDE strain.
The performance and survival of microorganisms, including P.putida, in soil and the rhizosphere of plants relies to a largeextent on their ability to endure desiccation and osmotic stress(18, 24). Choline-O-sulfate has been shown to be an efficientosmoprotectant in several plants (14, 36) and microbial species(4, 13, 14, 23, 26, 32). It is thought that choline-O-sulfateis found in soil, as plants (30), fungi (1, 22), and bacteriahave transport systems for this ester (4, 26, 29). In bacteria,choline-O-sulfate can be transformed into choline (31, 35),which is subsequently transformed into glycine betaine (41),a potent osmoprotectant (5).

We have found that the genome of P. putida bears a putativecholine-O-sulfate utilization operon, named betCDE, for choline-O-sulfateuptake (ORFs PP0075 and PP0076) and conversion to choline (ORFPP0077; Galvão et al., unpublished data). To examinethe role of choline-O-sulfate in osmoprotection of P. putidain a rhizosphere microcosm system, we set out to generate alacZ⁺-traceable ${Delta}$ betCDE strain, the expectation being that sucha deletion would affect P. putida's response to desiccationand osmotic stress. Production of such a strain with a standardsacB-based vector system such as pKNG101 (17) failed repeatedly.Besides the problems inherent in the use of sacB mentioned above,mutations involved in osmotic stress may be particularly difficultto produce with a counterselection system which involves theaddition of $≥$ 5% solute, which causes considerable osmotic pressure.This scenario provided the most appropriate case to test theperformance of the pyrF-based allelic exchange system presentedabove.

To this end, a DNA segment carrying a gentamicin resistancecassette and the ß-galactosidase (lacZ) gene flankedby the genomic sequences upstream and downstream of betCDE (ORFPP0074 and betR) was assembled (Fig. 3B). The ß-galactosidasegene is cloned as a transcriptional fusion to the betCD promoter,as indicated by a small arrow ahead of the lacZ gene (Galvãoet al., unpublished data). This segment was cloned into theNotI site of pTEC to yield plasmid pTGLB. This construct wastransferred to P. putida TEC1 by tripartite conjugation. Recombinationof pTGLB into the chromosome was selected on minimal mediumwith kanamycin and gentamicin but lacking uracil, thus usingpositive selection for uracil prototrophy upon plasmid integration.Following growth in liquid medium supplemented with uracil butwithout antibiotic selection, cells in which a second recombinationevent had taken place (resulting in the betCDE deletion) wereselected by plating on minimal medium with gentamicin, uracil,and FOA (Fig. 4).

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FIG. 4. Schematic map for pyrF-based counterselection for generation of homologous gene recombination with pTEC. Chromosomal integration events by homologous recombination between PP0074 or betR chromosomal and plasmid (pTGLB) sequences are selected by plating on medium with kanamycin (Km) or gentamicin (Gm) or both but lacking uracil. Cells with the plasmid integrated in the chromosome are grown without selection to allow a second recombination event that maintains either the wild-type or the deleted betCDE operon. The ${Delta}$ betCDE mutant is isolated by selecting cells that grow in the presence of FOA, gentamicin, and uracil but are kanamycin sensitive and uracil auxotrophs.

A total of 810 colonies of two morphologies grew when 10⁸ CFUwere plated in these conditions. The major morphotype (760,or 88%) represented ${Delta}$ betCDE deletion mutants, as judged by theuracil auxotrophy and kanamycin sensitivity of 25 colonies analyzed.This represents a respectable double-recombination frequencyof nearly 1.3 x 10^–5. PCR analysis showed that the FOA-resistant,kanamycin-sensitive colonies no longer had pTGLB in the chromosome,as judged by loss of the wild-type pyrF copy, the betC gene,and the kanamycin cassette while maintaining the ß-galactosidasegene in the deletion cassette (Fig. 5, lanes 1 to 5). The secondmorphotype consisted of faster-growing colonies, which werefound to be FOA resistant uracil prototrophs as well as resistantto kanamycin. That these colonies carry mutation to FOA resistancecontaining integrated pTGLB was confirmed by PCR analysis, whichshowed the presence of both wild-type and ${Delta}$ pyrF copies as wellas of the betC gene and the kanamycin cassette (Fig. 5, lanes6 to 10). The frequency at which these FOA-resistant coloniesarose is 2 x 10^–6 (50 FOA-resistant colonies per 10⁸ CFUplated), near that of P. putida KT2442 spontaneous mutationto FOA resistance (see above). That efficient homologous genereplacement is possible through use of pyrF-based selectionis made clear by the fact that the frequency of double-recombinationevents generating knockout strains (1.3 x 10^–5) is atleast 6.5 times higher than that of spontaneous FOA resistance(2 x 10^–6).

To first characterize the P. putida TEC1 ${Delta}$ betCDE strain, itssensitivity to osmotic stress was tested by looking at growthin solid medium containing high salt. Figure 6 shows that the ${Delta}$ betCDE mutant was more sensitive to high salt than P. putidaTEC1, as in the presence of 0.6 M NaCl the mutant grows muchmore slowly than P. putida TEC1 and is not viable in 0.7 M NaCl.Thus, deletion of betCDE renders P. putida more sensitive toosmotic stress. Sensitivity to osmotic stress in the absenceof protectant compounds is interesting despite the fact thatthe putative role of the betCDE gene products is related toosmoprotectant uptake and transformation. Because the betCDEmutant lacks a putative transmembrane protein, BetE (Galvãoet al., unpublished data), it is possible that the internalmembrane is altered, becoming more susceptible to osmotic stress.The betCDE mutant is the basis of further studies under laboratoryconditions as well as in the field.

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FIG. 6. Deletion of betCDE renders P. putida TEC1 susceptible to osmotic stress. Serial dilutions of P. putida TEC1 and TEC1 ${Delta}$ betCDE were plated on M9 minimal medium supplemented with uracil in the presence or absence of 0.6 or 0.7 M NaCl and grown at 30°C for 24 h (0 M NaCl) or 72 h (0.6 and 0.7 M NaCl).

Use of pTEC in other gram-negative bacteria.
Employing pTEC for homologous gene replacement in other gram-negativebacteria requires generating a pyrF background. This requirementis compensated for by the possibility of obtaining such mutantsby plating on FOA, as was done with T. kodakaraensis KOD1 (38)and H. salinarum (33). While full deletion of pyrF has the advantageof avoiding pTEC integration in the pyrF locus, in practicethe use of pyrF-negative, FOA-resistant strains generated byspontaneous mutations implies that $≤$ 50% of cointegrates are inadequateand thus require an additional screening step, easily achievedby PCR. Alternatively, pyrF deletion mutants may be generatedwith pKPX, the pKNG-based suicide plasmid carrying the P. putidagene (see above; Table 1), if there is a high sequence similaritybetween the pyrF genes of the organism being used and P. putida.

A BLAST search with the P. putida pyrF sequence against theEMBL database shows it to be highly homologous to sequencesof several gram-negative bacteria (Pseudomonas syringae, 83%;P. aeruginosa, 82%; Chromobacterium violaceum, 71%; Neisseriameningitidis, 65%; E. coli, 64%; Vibrio vulnificus, 63%; Salmonellaenterica serovar Typhimurium, 64%; Shigella flexneri, 63%; Photorhabdusluminescens, 61%; Yersinia pestis, 61%; and Shewanella oneidensis,60%). Whether these degrees of homology are sufficient to allowhomologous recombination will have to be checked for each organismindividually with the simple test provided by the xylE⁺ markerof the ${Delta}$ pyrF deletion.

Conclusion.
We have developed a vector system that employs the pyrF geneas a selection marker to allow efficient homologous gene replacement.We tested the plasmid in Pseudomonas putida and found that pyrFis a reliable marker in positive and negative selection. Atleast three strategies can be applied for generating gene knockoutswith pTEC. First is the one presented here, where a deletioncassette containing an antibiotic resistance marker is cloneddirectly into pTEC, yielding a knockout strain in which pTECcan be employed again. Second, PCR products of genomic regionsadjacent to the gene of interest may be cloned in the multiplecloning sites flanking the kanamycin resistance cassette inpTEC. Third, a deletion cassette that contains only the genomicregions adjacent to the gene of interest is cloned into pTEC,such that the resulting knockout is antibiotic marker free andadditional deletions can be generated with the same vector system.In all cases, the essential features of the system are pyrF-basedselection, where the initial recombination event is selectedby uracil prototrophy and the double-recombination event isselected by resistance to FOA and by uracil auxotrophy.

ACKNOWLEDGMENTS

David Canovas is acknowledged for providing the pGGLB plasmidand for useful discussions. The help of the E. coli Stock Centerwith strain X82 is greatly appreciated.

This work was supported by the Biocarte and Lindane Contractsof the European Union and by Comunidad Autónoma de Madridgrant 07 M/0075/2002.

FOOTNOTES

* Corresponding author. Mailing address: Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain. Phone: 34 915854536. Fax: 34 91 5854506. E-mail: vdlorenzo{at}cnb.uam.es.

REFERENCES

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