BACKGROUND
the elucidation of the p. aeruginosa strain pao <dig> genome sequence in  <dig>  <cit>  opened the doors for genome- and proteome-scale research with this important opportunistic pathogen.
these include the availability of commercial  p. aeruginosa genechips® for transcriptome analyses  <cit> , as well as the p. aeruginosa pao <dig> gene collection for high-throughput expression and purification of recombinant proteins  <cit> .
a comprehensive transposon mutant library was generated containing over  <dig>  sequence-defined mutants, corresponding to an average of five insertions per gene  <cit> .
however, despite the development of many genetic tools for p. aeruginosa over the past decade  <cit> , isolation of defined deletion mutants is still a relatively tedious process, which relies on construction of deletion alleles, most often tagged with an antibiotic resistance gene, on a suicide plasmid, followed by recombination of the plasmid-borne deletions into the chromosome, usually after conjugal transfer of the suicide plasmid .
merodiploids arising from chromosomal integration of the suicide plasmid are resolved by utilization of a counterselectable marker, most often bacillus subtilis sacb   <cit>  or, less frequently, rpsl  <cit> .
in cases where the antibiotic selection markers are flanked by site specific recombination sites, e.g., the flp recombinase target   <cit>  or cre recombinase   <cit>  sites, they can subsequently be deleted from the chromosome, resulting in unmarked deletion mutants.
unfortunately, the previously described method for one-step inactivation of chromosomal genes in escherichia coli using pcr products  <cit>  does not work in p. aeruginosa, although we have used a modification  <cit>  of this approach where the target gene is first cloned into a plasmid, followed by λ red-mediated recombination of a pcr-generated mutated copy of the gene  <cit> .
here, we describe a more streamlined method for generation of chromosomal deletion mutants.
in this method, a "mutant" fragment containing the 5' and 3' flanking regions of the target gene plus the antibiotic resistance gene is assembled by a modified  <cit>  pcr overlap extension protocol  <cit>  and then cloned into a suicide vector using the gateway recombinational cloning system, similarly to a previously described method  <cit> .
the plasmid-borne deletion allele is then transferred to p. aeruginosa utilizing a rapid transformation procedure and recombined into the chromosome.
the transformants are often merodiploids which are then resolved by sucrose counterselection against the undesired wild-type sequences.
the antibiotic resistance marker is subsequently deleted from the chromosome by flp recombinase.
RESULTS
construction of a gateway-compatible suicide vector
we previously described the pex family of relatively small, fully sequenced suicide plasmids which are now widely used for gene replacement experiments in p. aeruginosa and related bacteria  <cit> .
wolfgang et al.
<cit>  recently described a gateway-compatible pex18gm derivative.
here, we modified pex18ap for use as a gateway destination vector for cloning of mutations marked with a gentamycin resistance  encoding gene.
we prefer using the gmr marker because it is more easily transferable between p. aeruginosa chromosomes by transformation with chromosomal dna fragments when compared to other selection markers  <cit> .
the resulting vector pex18apgw  contains the functional sequences for recombination-based cloning  and the ccdb counterselection marker, while maintaining the sacb counterselection marker for downstream resolution of merodiploids.
generation of a mutant fragment by pcr overlap extension
the amplification of each mutant fragment was achieved in a two step pcr that required  <dig> pcr primers,  <dig> of which were unique for each fragment to be mutated and  <dig> of which were common primers .
the gene-specific portions of the unique primers were designed to yield tm's of 55–57°c and pcr fragments ranging from ~ <dig> to  <dig> bp.
these parameters enabled utilization of the same pcr conditions for different target genes, while at the same time provided for amplification of sufficient chromosomal dna to allow for efficient homologous recombination of plasmid-borne sequence with the chromosome.
the basic strategy for generation of the mutant fragment by pcr overlap extension is illustrated in fig.
<dig>  in pcr <dig>  the four gene-specific primers are used to amplify the 5' and 3' regions of the target gene.
simultaneously, two of the common primers, gm-f and gm-r, are used to amplify the gmr cassette flanked by frt sites from a pps <dig> template.
since the gmr fragment is used for all constructs, it can be prepared in larger amounts ahead of time and stored until needed.
fig.
3a illustrates typical results of pcr <dig> utilizing the example of p. aeruginosa gene pa <dig>  encoding a putative transcriptional regulator of the gntr family.
when we adhered to the prescribed pcr conditions, the amplified fragments were very clean.
it was especially prudent to follow the conditions for pcr amplification of the gmr frt fragment since the frt sites possess significant secondary structures.
the pcr fragments were gel-purified and equal amounts  of each were combined for pcr <dig>  pcr <dig> was allowed to proceed for  <dig> cycles in the absence of exogenous primers, which for pa <dig> yields the intermittent  <dig>  bp 'attb1-pa1520'-frt-gm-frt-'pa1520-attb2' fragment illustrated in fig 3a.
the common gateway primers gw-attb <dig> and gw-attb <dig> were then added and the pcr continued for another  <dig> cycles to obtain the final, recombination-proficient dna fragment, which constituted the predominant pcr product at this stage .
for pa <dig>  the resulting  <dig>  bp attb1-pa1520'-frt-gm-frt-'pa1520-attb <dig> fragment was gel-purified and used for the bp clonase reaction for construction of an entry clone.
bp and lr clonase reactions
the bp and lr clonase reactions  were performed according to invitrogen's  gateway cloning manual.
for the bp clonase reaction pdonr <dig> was used, but only half of the recommended bp clonase enzyme mix.
gmr and kanamycin resistant  dh5α or hps <dig> transformants were selected and the presence of the correct plasmids was confirmed by xbai digestion .
the insert of a correct pdonr-gene::gm entry clone was then recombined into the destination vector pex18apgw using the lr clonase protocol, again using only half of the recommended amount of lr clonase mix.
recombinants were transformed into dh5α and apr colonies were selected.
we noticed that, at least under the conditions we used, many transformants exhibited a apr kmr phenotype, indicating that they either contained both pdonr-gene::gm and pex18apgw-gene::gm or, more likely, a cointegrate of both plasmids.
the presence of the correct pex18apgw-gene::gm in transformants that were only apr was confirmed by xbai digestion.
gene replacement
to expedite gene replacement, an improved electroporation method  <cit>  was used for transfer of the suicide plasmid pex18apgw-gene::gm into p. aeruginosa instead of the traditionally employed conjugation method.
although after transfer into p. aeruginosa double cross-over events can occur frequently , they can also be rare .
in the latter instance, merodiploids formed via integration of the suicide plasmid by a single cross-over event .
the merodiploid state was then resolved via sucrose selection in the presence of gentamycin, resulting in deletion of the wild type gene.
for generation of unmarked deletion mutants, the gmr marker was subsequently removed by deletion of a  <dig> bp fragment using flp recombinase.
the presence of the desired deletion in either the marked or unmarked pa <dig> mutant was verified by colony pcr utilizing the gene-specific up and down primers .
deletion of  <dig> genes encoding transcriptional regulators of the gntr family
we are interested in characterizing the activator involved in regulation of expression of the fabab operon, which encodes two essential enzymes of de novo fatty acid biosynthesis  <cit> .
expression of the fabab operon is repressed by exogenously added fatty acids, most effectively by oleic acid, and preliminary results from our laboratory indicate that this may be due to relief of activation by a transcriptional regulator which binds to a  <dig> nucleotide site located in the fabab promoter region .
this  <dig> bp region is only found and conserved in pseudomonas spp.
its deletion dramatically reduces transcription of the fabab operon and the residual transcriptional activity is no longer fatty acid responsive .
in e. coli, transcriptional activation of the unlinked faba and fabb genes is achieved by fadr, a regulator belonging to a group of transcriptional regulators with the gntr family signature  <cit> .
the pao <dig> chromosome encodes more than  <dig> regulatory proteins with this signature.
of these,  <dig> have not been assigned function.
using the methodology described above for one of these genes, pa <dig>  we deleted  <dig> of the  <dig> genes  in less than  <dig> weeks in strain pao <dig> with a chromosomally integrated faba'-lacz transcriptional fusion and analyzed β-galactosidase expression in the resulting mutant strains .
in exponential phase cells, the overall pattern of faba expression was indistinguishable from the parental strain in all  <dig> mutants, indicating that none of these genes encodes the fabab activator.
we would have expected an expression pattern similar to that observed in the pao1::fabaΔ30'-lacz control strain which contains a deletion of the  <dig> nucleotide putative activator-binding site.
discussion
in our experience, the method described here for generation of unmarked deletion mutants is the fastest available to date.
from start to finish, an individual unmarked p. aeruginosa mutant can be generated and characterized in ~ <dig> days, which is up to one week faster than using our previously described method  <cit> .
however, the main advantage of the method does not lie in individual mutant generation but in the speed by which collective deletion mutant pools can be generated.
for example, we isolated unmarked deletions in  <dig> of the  <dig> genes encoding transcriptional regulators of the gntr family in less than  <dig> weeks.
two genes could not be deleted, presumably because they encode essential proteins.
besides speed, what are some of the other unique features of this method?
first, the deletion of gene sequences is not restricted by the natural availability or synthetic generation of restriction sites that are subsequently used to delete intervening sequences.
with proper primer location, the pcr-based method allows clean deletion of more-or-less the entire coding sequence without fear of generating truncated genes which potentially produce products with undesired side effects.
second, flp excision of the antibiotic resistance gene cassette leaves behind a short  <dig> bp frt scar which does not exhibit any known polar effects on downstream genes.
the method is therefore uniquely suited to study genes organized in polycistronic units.
flp excision is a very straightforward procedure with efficiencies approaching 100%  <cit> .
third, the method is economical, especially in a more high-throughput format.
pcr overlap extension requires only four gene specific primers of reasonable length which keeps oligonucleotide costs to a minimum.
in comparison to traditional methods, it also requires fewer hands-on steps and gene-specific reagents .
CONCLUSIONS
although not yet as fast as the pcr fragment-directed gene replacement method described for e. coli, the pcr- and recombinational cloning based method described opens the door for high-throughput generation of p. aeruginosa deletion mutants on a genome-wide scale.
with appropriate modifications, i.e., choice of appropriate selection markers and gateway-compatible gene replacement vectors, the method should be applicable to the many other bacteria in which the pex vector system can be applied  <cit> .

 the elucidation of the p. aeruginosa strain pao genome sequence in opened the doors for genome- and proteome-scale research with this important opportunistic pathogen. these include the availability of commercial p. aeruginosa genechips® for transcriptome analyses, as well as the p. aeruginosa pao gene collection for high-throughput expression and purification of recombinant proteins. a comprehensive transposon mutant library was generated containing over sequence-defined mutants, corresponding to an average of five insertions per gene. however, despite the development of many genetic tools for p. aeruginosa over the past decade, isolation of defined deletion mutants is still a relatively tedious process, which relies on construction of deletion alleles, most often tagged with an antibiotic resistance gene, on a suicide plasmid, followed by recombination of the plasmid-borne deletions into the chromosome, usually after conjugal transfer of the suicide plasmid. merodiploids arising from chromosomal integration of the suicide plasmid are resolved by utilization of a counterselectable marker, most often bacillus subtilis sacb or, less frequently, rpsl. in cases where the antibiotic selection markers are flanked by site specific recombination sites, e.g., the flp recombinase target or cre recombinase sites, they can subsequently be deleted from the chromosome, resulting in unmarked deletion mutants. unfortunately, the previously described method for one-step inactivation of chromosomal genes in escherichia coli using pcr products does not work in p. aeruginosa, although we have used a modification of this approach where the target gene is first cloned into a plasmid, followed by λ red-mediated recombination of a pcr-generated mutated copy of the gene. here, we describe a more streamlined method for generation of chromosomal deletion mutants. in this method, a "mutant" fragment containing the 5' and 3' flanking regions of the target gene plus the antibiotic resistance gene is assembled by a modified pcr overlap extension protocol and then cloned into a suicide vector using the gateway recombinational cloning system, similarly to a previously described method. the plasmid-borne deletion allele is then transferred to p. aeruginosa utilizing a rapid transformation procedure and recombined into the chromosome. the transformants are often merodiploids which are then resolved by sucrose counterselection against the undesired wild-type sequences. the antibiotic resistance marker is subsequently deleted from the chromosome by flp recombinase.  construction of a gateway-compatible suicide vector we previously described the pex family of relatively small, fully sequenced suicide plasmids which are now widely used for gene replacement experiments in p. aeruginosa and related bacteria. wolfgang et al. recently described a gateway-compatible pex18gm derivative. here, we modified pex18ap for use as a gateway destination vector for cloning of mutations marked with a gentamycin resistance encoding gene. we prefer using the gmr marker because it is more easily transferable between p. aeruginosa chromosomes by transformation with chromosomal dna fragments when compared to other selection markers. the resulting vector pex18apgw contains the functional sequences for recombination-based cloning and the ccdb counterselection marker, while maintaining the sacb counterselection marker for downstream resolution of merodiploids. generation of a mutant fragment by pcr overlap extension the amplification of each mutant fragment was achieved in a two step pcr that required pcr primers, of which were unique for each fragment to be mutated and of which were common primers. the gene-specific portions of the unique primers were designed to yield tm's of 55–57°c and pcr fragments ranging from ~ to bp. these parameters enabled utilization of the same pcr conditions for different target genes, while at the same time provided for amplification of sufficient chromosomal dna to allow for efficient homologous recombination of plasmid-borne sequence with the chromosome. the basic strategy for generation of the mutant fragment by pcr overlap extension is illustrated in fig. in pcr the four gene-specific primers are used to amplify the 5' and 3' regions of the target gene. simultaneously, two of the common primers, gm-f and gm-r, are used to amplify the gmr cassette flanked by frt sites from a pps template. since the gmr fragment is used for all constructs, it can be prepared in larger amounts ahead of time and stored until needed. fig. 3a illustrates typical results of pcr utilizing the example of p. aeruginosa gene pa encoding a putative transcriptional regulator of the gntr family. when we adhered to the prescribed pcr conditions, the amplified fragments were very clean. it was especially prudent to follow the conditions for pcr amplification of the gmr frt fragment since the frt sites possess significant secondary structures. the pcr fragments were gel-purified and equal amounts of each were combined for pcr pcr was allowed to proceed for cycles in the absence of exogenous primers, which for pa yields the intermittent bp'attb1-pa1520'-frt-gm-frt-'pa1520-attb2' fragment illustrated in fig 3a. the common gateway primers gw-attb and gw-attb were then added and the pcr continued for another cycles to obtain the final, recombination-proficient dna fragment, which constituted the predominant pcr product at this stage. for pa the resulting bp attb1-pa1520'-frt-gm-frt-'pa1520-attb fragment was gel-purified and used for the bp clonase reaction for construction of an entry clone. bp and lr clonase reactions the bp and lr clonase reactions were performed according to invitrogen's gateway cloning manual. for the bp clonase reaction pdonr was used, but only half of the recommended bp clonase enzyme mix. gmr and kanamycin resistant dh5α or hps transformants were selected and the presence of the correct plasmids was confirmed by xbai digestion. the insert of a correct pdonr-gene::gm entry clone was then recombined into the destination vector pex18apgw using the lr clonase protocol, again using only half of the recommended amount of lr clonase mix. recombinants were transformed into dh5α and apr colonies were selected. we noticed that, at least under the conditions we used, many transformants exhibited a apr kmr phenotype, indicating that they either contained both pdonr-gene::gm and pex18apgw-gene::gm or, more likely, a cointegrate of both plasmids. the presence of the correct pex18apgw-gene::gm in transformants that were only apr was confirmed by xbai digestion. gene replacement to expedite gene replacement, an improved electroporation method was used for transfer of the suicide plasmid pex18apgw-gene::gm into p. aeruginosa instead of the traditionally employed conjugation method. although after transfer into p. aeruginosa double cross-over events can occur frequently, they can also be rare. in the latter instance, merodiploids formed via integration of the suicide plasmid by a single cross-over event. the merodiploid state was then resolved via sucrose selection in the presence of gentamycin, resulting in deletion of the wild type gene. for generation of unmarked deletion mutants, the gmr marker was subsequently removed by deletion of a bp fragment using flp recombinase. the presence of the desired deletion in either the marked or unmarked pa mutant was verified by colony pcr utilizing the gene-specific up and down primers. deletion of genes encoding transcriptional regulators of the gntr family we are interested in characterizing the activator involved in regulation of expression of the fabab operon, which encodes two essential enzymes of de novo fatty acid biosynthesis. expression of the fabab operon is repressed by exogenously added fatty acids, most effectively by oleic acid, and preliminary results from our laboratory indicate that this may be due to relief of activation by a transcriptional regulator which binds to a nucleotide site located in the fabab promoter region. this bp region is only found and conserved in pseudomonas spp. its deletion dramatically reduces transcription of the fabab operon and the residual transcriptional activity is no longer fatty acid responsive. in e. coli, transcriptional activation of the unlinked faba and fabb genes is achieved by fadr, a regulator belonging to a group of transcriptional regulators with the gntr family signature. the pao chromosome encodes more than regulatory proteins with this signature. of these, have not been assigned function. using the methodology described above for one of these genes, pa we deleted of the genes in less than weeks in strain pao with a chromosomally integrated faba'-lacz transcriptional fusion and analyzed β-galactosidase expression in the resulting mutant strains. in exponential phase cells, the overall pattern of faba expression was indistinguishable from the parental strain in all mutants, indicating that none of these genes encodes the fabab activator. we would have expected an expression pattern similar to that observed in the pao1::fabaΔ30'-lacz control strain which contains a deletion of the nucleotide putative activator-binding site. discussion in our experience, the method described here for generation of unmarked deletion mutants is the fastest available to date. from start to finish, an individual unmarked p. aeruginosa mutant can be generated and characterized in ~ days, which is up to one week faster than using our previously described method. however, the main advantage of the method does not lie in individual mutant generation but in the speed by which collective deletion mutant pools can be generated. for example, we isolated unmarked deletions in of the genes encoding transcriptional regulators of the gntr family in less than weeks. two genes could not be deleted, presumably because they encode essential proteins. besides speed, what are some of the other unique features of this method? first, the deletion of gene sequences is not restricted by the natural availability or synthetic generation of restriction sites that are subsequently used to delete intervening sequences. with proper primer location, the pcr-based method allows clean deletion of more-or-less the entire coding sequence without fear of generating truncated genes which potentially produce products with undesired side effects. second, flp excision of the antibiotic resistance gene cassette leaves behind a short bp frt scar which does not exhibit any known polar effects on downstream genes. the method is therefore uniquely suited to study genes organized in polycistronic units. flp excision is a very straightforward procedure with efficiencies approaching 100%. third, the method is economical, especially in a more high-throughput format. pcr overlap extension requires only four gene specific primers of reasonable length which keeps oligonucleotide costs to a minimum. in comparison to traditional methods, it also requires fewer hands-on steps and gene-specific reagents.  although not yet as fast as the pcr fragment-directed gene replacement method described for e. coli, the pcr- and recombinational cloning based method described opens the door for high-throughput generation of p. aeruginosa deletion mutants on a genome-wide scale. with appropriate modifications, i.e., choice of appropriate selection markers and gateway-compatible gene replacement vectors, the method should be applicable to the many other bacteria in which the pex vector system can be applied. 
the method was applied to deletion of p. aeruginosa genes encoding transcriptional regulators of the gntr family. we describe a method for rapid generation of unmarked p. aeruginosa deletion mutants. with appropriate modifications, the method should be applicable to other bacteria. while maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. unmarked deletion mutants are finally obtained by flp-mediated excision of the antibiotic resistance marker. the plasmid-borne deletions are next transferred to the p. aeruginosa chromosome by homologous recombination.
 traditional gene replacement procedures are still time-consuming. they usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. pcr and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process.  we describe a method for rapid generation of unmarked p. aeruginosa deletion mutants. three partially overlapping dna fragments are amplified and then spliced together in vitro by overlap extension pcr. the resulting dna fragment is cloned in vitro into the gateway vector pdonr and then recombined into the gateway-compatible gene replacement vector pex18apgw. the plasmid-borne deletions are next transferred to the p. aeruginosa chromosome by homologous recombination. unmarked deletion mutants are finally obtained by flp-mediated excision of the antibiotic resistance marker. the method was applied to deletion of p. aeruginosa genes encoding transcriptional regulators of the gntr family.  while maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. with appropriate modifications, the method should be applicable to other bacteria. 