BACKGROUND
in pseudomonas aeruginosa, two acyl-homoserine lactone quorum sensing  systems, lasr-lasi and rhlr-rhli, control the expression of partially overlapping sets of genes.
many of the regulated genes are implicated in virulence and biofilm formation of this opportunistic pathogen.
lasi and rhli are enzymes that synthesize the acyl-hsl signals 3-oxo-dodecanoyl -hsl and butanoyl -hsl, respectively.
lasr and rhlr are receptors that specifically bind the signal generated by their cognate synthases to regulate transcription of target genes  <cit> .
three independent transcriptome analyses identified several hundred such genes  <cit> .
the numbers of identified genes and their expression patterns varied among studies depending on experimental conditions and statistical criteria used.
in our study  <cit>  we noted that the expression of most quorum-controlled genes increased in the stationary phase of growth and could not be advanced to logarithmic phase by the addition of exogenous signal, confirming earlier observations with individual quorum-controlled genes  <cit> .
this suggested that the activation of most quorum-controlled genes is not triggered by the accumulation of signal, and seems to require additional factors.
in accordance with this hypothesis, several other regulatory systems such as the gacas-rsmaz two-component signal transduction pathway  <cit> , the stationary phase sigma factor rpos  <cit> , and mvfr -quinolone signaling  <cit> , have been shown to influence quorum-controlled gene expression in p. aeruginosa.
they are thought to affect las or rhl mediated qs at two distinct levels of signal integration, either at the level of the regulators lasr and rhlr themselves, or at the level of complex qs target promoters  <cit>  .
the stationary phase sigma factor rpos, for example, appears to affect qs gene expression at both levels  <cit> .
furthermore, a recent study showed that there is an inhibitory substance  in complex medium which delays the expression of many quorum-controlled genes  <cit> , but it is not clear at which level this occurs.
because transcripts of lasr, rhlr, and rpos increase at the onset of stationary phase in concert with activation of most quorum-controlled genes  <cit> , it is possible that the abundance of the encoded proteins is critical for target gene induction.
on the other hand, we predict that those late genes for which these regulators are not limiting early in growth are co-regulated by other factors at the target promoter level.
to better understand the network structure of the qs regulon, we expressed lasr, rhlr, and rpos from a heterologous promoter in logarithmic phase and compared quorum-controlled gene expression in the corresponding strains to that of the wildtype by using dna microarrays.
RESULTS
lasr, rhir and rpos levels increase in the stationary phase of growth
our previous transcriptome analyses  <cit> , in addition to other studies with reporter fusions  <cit> , showed that the transcript levels of lasr, rhlr, and rpos increase at the transition from logarithmic to stationary phase during batch culture growth of p. aeruginosa.
immunoblotting with lasr, rhlr, and rpos-specific antisera showed that this expression pattern correlates well with the respective protein levels .
expression strategy
if lasr, rhlr or rpos levels were limiting the expression of qs-controlled genes during logarithmic growth of wildtype p. aeruginosa, early expression of these factors should advance quorum-controlled gene expression from stationary to logarithmic phase.
to test this hypothesis, we constructed strains for regulatable expression of lasr, rhlr, and rpos.
each allele, under the control of an arabinose-inducible arabad promoter, was inserted in single copy into a neutral site of the chromosome of a lasr, rhlr mutant or an rpos mutant, respectively.
this arrangement allowed the careful titration of expression levels in logarithmic phase such that they matched those of the wildtype in stationary phase.
for lasr and rhlr, it also allowed us to assess the impact of each regulator on gene expression independently and uncoupled from the qs hierarchy, where lasr-lasi is required for rhlr-rhli expression  <cit> .
we have thoroughly validated this approach by monitoring target gene expression and regulator protein levels in the engineered strains and in the parent strain throughout growth .
many qs genes can be advanced by early expression of lasr, rhir, or rpos, but not by signal addition
early expression of lasr and rhlr each resulted in the premature induction of  <dig> and  <dig> genes, respectively, during growth of p. aeruginosa in culture .
sixty-seven of the genes induced by lasr and  <dig> of the genes induced by rhlr are among the set of  <dig> quorum-induced genes we identified in a previous study  <cit>  .
early expression of rpos resulted in the premature induction of  <dig> genes.
sixty-one of these genes were identified as quorum-controlled, and  <dig> are among the genes previously identified as rpos-induced  <cit> .
thus, early expression of any of the three proteins revealed genes that are not among the previously characterized qs and rpos regulons.
although this finding warrants further investigation, for the purpose of this study we focused on the set of  <dig> previously identified and well characterized quorum-regulated genes  <cit> .
a heat map shows that the early expression of lasr, rhlr, and rpos induced many genes in logarithmic phase to levels close to those of the wildtype in stationary phase .
nineteen genes were also activated by the addition of 3oc12-hsl and c4-hsl signals alone, but their induction levels were comparatively low and all of them could be induced further by early expression of rhlr .
early expression of rpos often only partially activated those genes that were more highly activated by lasr or rhlr.
because rpos also has a small effect on lasr and rhlr expression  <cit> , it appears that rpos is capable of advancing many genes only indirectly through lasr and rhlr.
overall, there was a good correlation between the genes induced by early expression of lasr or rhlr, and their signal specificity determined by addition of 3oc12-hsl or both 3oc12-hsl and c4-hsl to a signal generation mutant  <cit> .
for example, a cluster of rhl-specific genes was advanced by c4-hsl-rhlr but not by 3oc12-hsl-lasr , whereas several las-specific genes were advanced by 3oc12-hsl-lasr but not c4-hsl-rhlr .
however, there were exceptions.
pa <dig> and pa <dig>  for example, can be advanced by 3oc12-hsl-lasr but not c4-hsl-rhlr  although in a signal synthesis mutant they responded better to both signals than to 3oc12-hsl alone  <cit> .
such genes may require the binding of both regulators to their promoters for activation, or they may require additional factors that are themselves under the control of 3oc12-hsl-lasr.
among the genes significantly induced by early expression of lasr, rhlr, or rpos were 47% of all genes activated early by addition of signals to cells in conditioned medium  <cit> .
the large majority of this subset was induced by early expression of rhlr.
rhlr was one of the genes triggered by media conditioning  <cit>  and it also induced in minimal medium  <cit> .
thus, the advancement of qs in conditioned medium appears to be partially mediated through super-regulation of rhlr.
many other qs genes cannot be advanced significantly by early expression of lasr or rhir
most quorum-controlled genes showed little to no activation by lasr, rhlr or rpos expression in logarithmic phase.
one-hundred and twenty of these genes are late genes.
their quorum-dependent  induction in the wildtype strain is delayed until stationary phase .
these late genes could not be advanced although several are predicted to be directly activated by lasr or rhlr as they possess conserved sequence elements, so-called las-rhl boxes, in their promoter regions  <cit> .
thus it appears that 3oc12-hsl-lasr, c4-hsl-rhlr, and rpos levels are not limiting for the activation of many quorum-controlled genes.
to demonstrate that late quorum-controlled genes can still be activated in stationary phase using our engineered expression strains, and to confirm our microarray results, we measured the logarithmic and stationary phase transcript profiles of several quorum-controlled genes by real-time pcr.
the genes lasa, pa <dig>  pa <dig>  and rhla, which showed little to no quorum activation in logarithmic phase, could be activated in stationary phase cultures of strains expressing lasr or rhlr from the pbad promoter .
both lasa and rhla possess a las-rhl box  <cit> .
pa <dig> is an example of one of the few early genes that are induced fully in the wildtype strain in logarithmic phase.
ectopic expression of lasr resulted in a similar level of induction in stationary phase.
as indicated above, there was generally a good correlation between regulator specificity and acyl-hsl signal specificity .
an exception is pa <dig>  which was 3oc12-hsl specific  <cit>  but responded to both lasr and rhlr expression in logarithmic phase .
this suggests that rhlr-c4-hsl is capable of activating pa <dig>  but this effect is masked in the signal generation mutant because 3oc12-hsl-lasr is required for rhlr expression and 3oc12-hsl-lasr already saturates the pa <dig> promoter.
hence, our expression strategy provides a different assessment of qs promoter specificity because it functions independently of the qs cascade.
direct versus indirect regulation
we hypothesized that most genes advanced by qs are directly activated by qs.
if this hypothesis is true, then we should be able to identify conserved sequence elements, so-called las-rhl boxes, upstream of these genes.
in our previous study, we identified las-rhl box like sequences in  <dig> out of the  <dig> predicted quorum-controlled promoters  <cit> .
many of these promoters are associated with qs advanced genes.
of the  <dig> predicted promoters advanced by lasr,  <dig> contain a las-rhl box-like sequence, and of the  <dig> predicted promoters advanced by rhlr,  <dig> contain a las-rhl box-like sequence.
thus, it appears that genes advanced by rhlr are for the most part also directly regulated by this transcription factor, whereas genes advanced by lasr are mostly indirectly regulated .
however, our data do not provide evidence for indirect regulation by lasr via a regulatory cascade.
candidate regulatory genes mvfr, pa <dig>  and vqsr, which are under direct transcriptional control by lasr  <cit> , are not among the genes activated early .
our previous search of las-rhl box like sequences did not take into account differences between las-specific and rhl-specific binding sites.
it is therefore possible that we missed several actual lasr or rhlr binding sites.
our identification of a subset of genes advanced by either lasr or rhlr in this study afforded the opportunity for a de-novo, unbiased search for separate lasr and rhlr consensus sequences.
we selected those genes most highly induced by lasr alone or by rhlr alone and subjected the presumed upstream regulatory sequences to the pattern discovery algorithm consensus  <cit> .
a sequence pattern that resembles known binding sites was identified for rhlr-advanced but not for lasr-advanced promoters .
this result confirms the notion that many genes advanced by rhlr are activated directly.
most genes advanced by lasr may be activated indirectly as mentioned above, or lasr binding sites may be more complex.
our recent binding studies with purified lasr support the latter.
we noticed a general lack of sequence conservation and identified two subpopulations of las-specific binding sequences based on their distinct interactions with lasr  <cit> .
such heterogeneity undoubtedly complicates the identification of a single consensus sequence.
further biochemical characterization of a larger number of binding sites will be required for a more elaborate sequence alignment.
our alignment of rhlr sequences shows the conserved ctag motif recognized previously  <cit> , and implies several other conserved nucleotides .
similarity to the original las-rhl box sequence alignment is high, reflecting a bias towards rhl-responsive genes in the original alignment  <cit> .
in a second step, the matrix generated with consensus was used to search the upstream regions of all previously identified quorum-controlled genes.
fourty-two predicted qs promoters showed a significant match.
not surprisingly, most sequences had also been identified in our previous study  <cit> .
in addition, we identified sequences with similarity to the rhlr consensus upstream of the following genes: pa <dig>  pa <dig>  pa <dig>  pa <dig>  pa <dig> , and pa <dig> .
of these, the latter three were found to be advanced by rhlr.
CONCLUSIONS
when p. aeruginosa is grown in complex medium, lasr, rhlr and rpos protein levels increase in stationary phase , correlating with the activation of most quorum-controlled genes.
our approach to investigating the advancement of quorum sensing gene expression was to adjust the expression levels of regulators in log-phase cells to match the levels in stationary phase.
this is an overexpression strategy in which proteins are maintained within physiological levels known to exist in p. aeruginosa.
many qs genes could be activated in logarithmic phase by the early expression of lasr, rhlr, and rpos, but not by signal addition.
thus, the levels of these three proteins can be critical in modulating the quorum-response in p. aeruginosa.
several of the genes were induced by more than one regulator, confirming overlapping specificities and co-regulation.
genes that directly and exclusively respond to qs are likely among the subset of genes whose maximal expression can be advanced by increased expression of lasr or rhlr during logarithmic phase.
in fact, our results suggested that most genes advanced by rhlr are directly regulated by this transcription factor, allowing us to build a rhlr consensus sequence.
many other qs genes fail to be advanced by 3oc12-hsl-lasr and c4-hsl-rhlr, although it has been shown that some of these genes are directly regulated by these signal and response systems.
this confirms and extends a recent observation for an individual gene, rhla, which is not significantly activated in logarithmic phase even when c4-hsl-rhlr is present  <cit> .
these results suggest that the corresponding promoters are co-regulated by other transcription factors, likely constituting a network motif known as a multi-input dense overlapping regulon  <cit> .
some of the regulatory inputs may affect translation rather than transcription, as is the case for small regulatory rnas that modulate quorum sensing gene expression  <cit> .
the overall topology allows for specific responses to a multitude of signals, and may provide the basis for the exceptional environmental adaptability of p. aeruginosa.
it also provides a simple explanation for the seemingly discordant sets of quorum-controlled genes identified by two separate groups under different culture conditions  <cit> .
taken together, our results indicate that co-regulation of target genes is an important, and perhaps predominant, feature of the p. aeruginosa qs network in addition to the well established super-regulation of the central components, lasr-lasi and rhlr-rhli.
thus, a thorough understanding of the qs network will necessitate a comprehensive analysis of target promoter architecture, which should include the global identification of transcription factor binding sites.
technologies such as chip-chip, chromatin immunoprecipitation and microarray analysis  <cit> , make this approach feasible.

 in pseudomonas aeruginosa, two acyl-homoserine lactone quorum sensing systems, lasr-lasi and rhlr-rhli, control the expression of partially overlapping sets of genes. many of the regulated genes are implicated in virulence and biofilm formation of this opportunistic pathogen. lasi and rhli are enzymes that synthesize the acyl-hsl signals 3-oxo-dodecanoyl -hsl and butanoyl -hsl, respectively. lasr and rhlr are receptors that specifically bind the signal generated by their cognate synthases to regulate transcription of target genes. three independent transcriptome analyses identified several hundred such genes. the numbers of identified genes and their expression patterns varied among studies depending on experimental conditions and statistical criteria used. in our study we noted that the expression of most quorum-controlled genes increased in the stationary phase of growth and could not be advanced to logarithmic phase by the addition of exogenous signal, confirming earlier observations with individual quorum-controlled genes. this suggested that the activation of most quorum-controlled genes is not triggered by the accumulation of signal, and seems to require additional factors. in accordance with this hypothesis, several other regulatory systems such as the gacas-rsmaz two-component signal transduction pathway, the stationary phase sigma factor rpos, and mvfr -quinolone signaling, have been shown to influence quorum-controlled gene expression in p. aeruginosa. they are thought to affect las or rhl mediated qs at two distinct levels of signal integration, either at the level of the regulators lasr and rhlr themselves, or at the level of complex qs target promoters. the stationary phase sigma factor rpos, for example, appears to affect qs gene expression at both levels. furthermore, a recent study showed that there is an inhibitory substance in complex medium which delays the expression of many quorum-controlled genes, but it is not clear at which level this occurs. because transcripts of lasr, rhlr, and rpos increase at the onset of stationary phase in concert with activation of most quorum-controlled genes, it is possible that the abundance of the encoded proteins is critical for target gene induction. on the other hand, we predict that those late genes for which these regulators are not limiting early in growth are co-regulated by other factors at the target promoter level. to better understand the network structure of the qs regulon, we expressed lasr, rhlr, and rpos from a heterologous promoter in logarithmic phase and compared quorum-controlled gene expression in the corresponding strains to that of the wildtype by using dna microarrays.  lasr, rhir and rpos levels increase in the stationary phase of growth our previous transcriptome analyses, in addition to other studies with reporter fusions, showed that the transcript levels of lasr, rhlr, and rpos increase at the transition from logarithmic to stationary phase during batch culture growth of p. aeruginosa. immunoblotting with lasr, rhlr, and rpos-specific antisera showed that this expression pattern correlates well with the respective protein levels. expression strategy if lasr, rhlr or rpos levels were limiting the expression of qs-controlled genes during logarithmic growth of wildtype p. aeruginosa, early expression of these factors should advance quorum-controlled gene expression from stationary to logarithmic phase. to test this hypothesis, we constructed strains for regulatable expression of lasr, rhlr, and rpos. each allele, under the control of an arabinose-inducible arabad promoter, was inserted in single copy into a neutral site of the chromosome of a lasr, rhlr mutant or an rpos mutant, respectively. this arrangement allowed the careful titration of expression levels in logarithmic phase such that they matched those of the wildtype in stationary phase. for lasr and rhlr, it also allowed us to assess the impact of each regulator on gene expression independently and uncoupled from the qs hierarchy, where lasr-lasi is required for rhlr-rhli expression. we have thoroughly validated this approach by monitoring target gene expression and regulator protein levels in the engineered strains and in the parent strain throughout growth. many qs genes can be advanced by early expression of lasr, rhir, or rpos, but not by signal addition early expression of lasr and rhlr each resulted in the premature induction of and genes, respectively, during growth of p. aeruginosa in culture. sixty-seven of the genes induced by lasr and of the genes induced by rhlr are among the set of quorum-induced genes we identified in a previous study. early expression of rpos resulted in the premature induction of genes. sixty-one of these genes were identified as quorum-controlled, and are among the genes previously identified as rpos-induced. thus, early expression of any of the three proteins revealed genes that are not among the previously characterized qs and rpos regulons. although this finding warrants further investigation, for the purpose of this study we focused on the set of previously identified and well characterized quorum-regulated genes. a heat map shows that the early expression of lasr, rhlr, and rpos induced many genes in logarithmic phase to levels close to those of the wildtype in stationary phase. nineteen genes were also activated by the addition of 3oc12-hsl and c4-hsl signals alone, but their induction levels were comparatively low and all of them could be induced further by early expression of rhlr. early expression of rpos often only partially activated those genes that were more highly activated by lasr or rhlr. because rpos also has a small effect on lasr and rhlr expression, it appears that rpos is capable of advancing many genes only indirectly through lasr and rhlr. overall, there was a good correlation between the genes induced by early expression of lasr or rhlr, and their signal specificity determined by addition of 3oc12-hsl or both 3oc12-hsl and c4-hsl to a signal generation mutant. for example, a cluster of rhl-specific genes was advanced by c4-hsl-rhlr but not by 3oc12-hsl-lasr, whereas several las-specific genes were advanced by 3oc12-hsl-lasr but not c4-hsl-rhlr. however, there were exceptions. pa and pa for example, can be advanced by 3oc12-hsl-lasr but not c4-hsl-rhlr although in a signal synthesis mutant they responded better to both signals than to 3oc12-hsl alone. such genes may require the binding of both regulators to their promoters for activation, or they may require additional factors that are themselves under the control of 3oc12-hsl-lasr. among the genes significantly induced by early expression of lasr, rhlr, or rpos were 47% of all genes activated early by addition of signals to cells in conditioned medium. the large majority of this subset was induced by early expression of rhlr. rhlr was one of the genes triggered by media conditioning and it also induced in minimal medium. thus, the advancement of qs in conditioned medium appears to be partially mediated through super-regulation of rhlr. many other qs genes cannot be advanced significantly by early expression of lasr or rhir most quorum-controlled genes showed little to no activation by lasr, rhlr or rpos expression in logarithmic phase. one-hundred and twenty of these genes are late genes. their quorum-dependent induction in the wildtype strain is delayed until stationary phase. these late genes could not be advanced although several are predicted to be directly activated by lasr or rhlr as they possess conserved sequence elements, so-called las-rhl boxes, in their promoter regions. thus it appears that 3oc12-hsl-lasr, c4-hsl-rhlr, and rpos levels are not limiting for the activation of many quorum-controlled genes. to demonstrate that late quorum-controlled genes can still be activated in stationary phase using our engineered expression strains, and to confirm our microarray results, we measured the logarithmic and stationary phase transcript profiles of several quorum-controlled genes by real-time pcr. the genes lasa, pa pa and rhla, which showed little to no quorum activation in logarithmic phase, could be activated in stationary phase cultures of strains expressing lasr or rhlr from the pbad promoter. both lasa and rhla possess a las-rhl box. pa is an example of one of the few early genes that are induced fully in the wildtype strain in logarithmic phase. ectopic expression of lasr resulted in a similar level of induction in stationary phase. as indicated above, there was generally a good correlation between regulator specificity and acyl-hsl signal specificity. an exception is pa which was 3oc12-hsl specific but responded to both lasr and rhlr expression in logarithmic phase. this suggests that rhlr-c4-hsl is capable of activating pa but this effect is masked in the signal generation mutant because 3oc12-hsl-lasr is required for rhlr expression and 3oc12-hsl-lasr already saturates the pa promoter. hence, our expression strategy provides a different assessment of qs promoter specificity because it functions independently of the qs cascade. direct versus indirect regulation we hypothesized that most genes advanced by qs are directly activated by qs. if this hypothesis is true, then we should be able to identify conserved sequence elements, so-called las-rhl boxes, upstream of these genes. in our previous study, we identified las-rhl box like sequences in out of the predicted quorum-controlled promoters. many of these promoters are associated with qs advanced genes. of the predicted promoters advanced by lasr, contain a las-rhl box-like sequence, and of the predicted promoters advanced by rhlr, contain a las-rhl box-like sequence. thus, it appears that genes advanced by rhlr are for the most part also directly regulated by this transcription factor, whereas genes advanced by lasr are mostly indirectly regulated. however, our data do not provide evidence for indirect regulation by lasr via a regulatory cascade. candidate regulatory genes mvfr, pa and vqsr, which are under direct transcriptional control by lasr, are not among the genes activated early. our previous search of las-rhl box like sequences did not take into account differences between las-specific and rhl-specific binding sites. it is therefore possible that we missed several actual lasr or rhlr binding sites. our identification of a subset of genes advanced by either lasr or rhlr in this study afforded the opportunity for a de-novo, unbiased search for separate lasr and rhlr consensus sequences. we selected those genes most highly induced by lasr alone or by rhlr alone and subjected the presumed upstream regulatory sequences to the pattern discovery algorithm consensus. a sequence pattern that resembles known binding sites was identified for rhlr-advanced but not for lasr-advanced promoters. this result confirms the notion that many genes advanced by rhlr are activated directly. most genes advanced by lasr may be activated indirectly as mentioned above, or lasr binding sites may be more complex. our recent binding studies with purified lasr support the latter. we noticed a general lack of sequence conservation and identified two subpopulations of las-specific binding sequences based on their distinct interactions with lasr. such heterogeneity undoubtedly complicates the identification of a single consensus sequence. further biochemical characterization of a larger number of binding sites will be required for a more elaborate sequence alignment. our alignment of rhlr sequences shows the conserved ctag motif recognized previously, and implies several other conserved nucleotides. similarity to the original las-rhl box sequence alignment is high, reflecting a bias towards rhl-responsive genes in the original alignment. in a second step, the matrix generated with consensus was used to search the upstream regions of all previously identified quorum-controlled genes. fourty-two predicted qs promoters showed a significant match. not surprisingly, most sequences had also been identified in our previous study. in addition, we identified sequences with similarity to the rhlr consensus upstream of the following genes: pa pa pa pa pa, and pa. of these, the latter three were found to be advanced by rhlr.  when p. aeruginosa is grown in complex medium, lasr, rhlr and rpos protein levels increase in stationary phase, correlating with the activation of most quorum-controlled genes. our approach to investigating the advancement of quorum sensing gene expression was to adjust the expression levels of regulators in log-phase cells to match the levels in stationary phase. this is an overexpression strategy in which proteins are maintained within physiological levels known to exist in p. aeruginosa. many qs genes could be activated in logarithmic phase by the early expression of lasr, rhlr, and rpos, but not by signal addition. thus, the levels of these three proteins can be critical in modulating the quorum-response in p. aeruginosa. several of the genes were induced by more than one regulator, confirming overlapping specificities and co-regulation. genes that directly and exclusively respond to qs are likely among the subset of genes whose maximal expression can be advanced by increased expression of lasr or rhlr during logarithmic phase. in fact, our results suggested that most genes advanced by rhlr are directly regulated by this transcription factor, allowing us to build a rhlr consensus sequence. many other qs genes fail to be advanced by 3oc12-hsl-lasr and c4-hsl-rhlr, although it has been shown that some of these genes are directly regulated by these signal and response systems. this confirms and extends a recent observation for an individual gene, rhla, which is not significantly activated in logarithmic phase even when c4-hsl-rhlr is present. these results suggest that the corresponding promoters are co-regulated by other transcription factors, likely constituting a network motif known as a multi-input dense overlapping regulon. some of the regulatory inputs may affect translation rather than transcription, as is the case for small regulatory rnas that modulate quorum sensing gene expression. the overall topology allows for specific responses to a multitude of signals, and may provide the basis for the exceptional environmental adaptability of p. aeruginosa. it also provides a simple explanation for the seemingly discordant sets of quorum-controlled genes identified by two separate groups under different culture conditions. taken together, our results indicate that co-regulation of target genes is an important, and perhaps predominant, feature of the p. aeruginosa qs network in addition to the well established super-regulation of the central components, lasr-lasi and rhlr-rhli. thus, a thorough understanding of the qs network will necessitate a comprehensive analysis of target promoter architecture, which should include the global identification of transcription factor binding sites. technologies such as chip-chip, chromatin immunoprecipitation and microarray analysis, make this approach feasible. 
the induction of most of the genes is delayed during growth of p. aeruginosa in complex medium, cannot be advanced by addition of exogenous signal, and requires additional regulatory components. we determined the effect on target gene expression by microarray and real-time pcr analysis. our results show that many target genes are directly responsive to receptor protein levels. the direct responsiveness of many quorum sensing target genes to receptor protein levels early in growth confirms the role of super-regulation in quorum sensing gene expression. the observation that the induction of most target genes is not affected by signal or receptor protein levels indicates that either target promoters are co-regulated by other transcription factors, or that expression is controlled posttranscriptionally. the majority of promoters advanced by rhlr appeared to be regulated directly, which allowed us to build a rhlr consensus sequence. many of these late genes can be induced by addition of signals early by using specific media conditions. quorum-sensing regulation of gene expression in pseudomonas aeruginosa is complex. to better understand the contributions of super-regulation and co-regulation to quorum-sensing gene expression, and to better understand the general structure of the quorum sensing network, we ectopically expressed the two receptors and another component that affects quorum sensing, the stationary phase sigma factor rpos, early in growth.
 quorum-sensing regulation of gene expression in pseudomonas aeruginosa is complex. two interconnected acyl-homoserine lactone signal-receptor pairs, 3-oxo-dodecanoyl-hsl-lasr and butanoyl-hsl-rhlr, regulate more than genes. the induction of most of the genes is delayed during growth of p. aeruginosa in complex medium, cannot be advanced by addition of exogenous signal, and requires additional regulatory components. many of these late genes can be induced by addition of signals early by using specific media conditions. while several factors super-regulate the quorum receptors, others may co-regulate target promoters or may affect expression posttranscriptionally.  to better understand the contributions of super-regulation and co-regulation to quorum-sensing gene expression, and to better understand the general structure of the quorum sensing network, we ectopically expressed the two receptors and another component that affects quorum sensing, the stationary phase sigma factor rpos, early in growth. we determined the effect on target gene expression by microarray and real-time pcr analysis. our results show that many target genes are directly responsive to receptor protein levels. most genes, however, are not significantly affected, although at least some of these genes are directly regulated by quorum sensing. the majority of promoters advanced by rhlr appeared to be regulated directly, which allowed us to build a rhlr consensus sequence.  the direct responsiveness of many quorum sensing target genes to receptor protein levels early in growth confirms the role of super-regulation in quorum sensing gene expression. the observation that the induction of most target genes is not affected by signal or receptor protein levels indicates that either target promoters are co-regulated by other transcription factors, or that expression is controlled posttranscriptionally. this architecture permits the integration of multiple signaling pathways resulting in quorum responses that require a "quorum" but are otherwise highly adaptable and receptive to environmental conditions. 