BACKGROUND
the burkholderia cepacia complex  is a group of gram negative bacteria that comprises at least fifteen taxonomically related species  <cit> .
bcc strains occupy multiple niches from soil to humans as they have emerged as opportunistic pathogens in patients with cystic fibrosis , chronic granulomatous disease, and other medical conditions associated with a compromised immune system  <cit> .
burkholderia species have evolved large genomes that allow them to deal with a variety of nutrient sources, predation and competition.
the three chromosomes of b. cenocepacia, one of the most common species found in cf patients  <cit> , encode a broad array of catabolic functions.
yet, the contribution of these metabolic capacities to colonization and survival in the host has not been established.
the phenylacetic acid  catabolic pathway is the central route where catabolism of many aromatic compounds converge and are directed to the krebs cycle  <cit> .
it comprises of four steps, namely the pa-coa ligation-activation performed by paak  <cit> , the hydroxylation step for which the paaabcde enzymatic complex is responsible  <cit> , the enoyl-coa isomerization/hydration, ring opening performed by paag and paaz,  <cit> , and the β-oxidation step carried out by paaf and paah,  <cit> .
previously, we initiated the functional characterization of the pa catabolic pathway of b. cenocepacia k56- <dig>  <cit>  and demonstrated that interruption of putative pa-coa ring hydroxylation activity, but not the lower steps of pa degradation, resulted in an attenuated pathogenic phenotype in the caenorhabditis elegans model of infection.
here, we report that the pa catabolic genes of b. cenocepacia k56- <dig> are induced by pa, are negatively regulated by paar, a tetr-type regulator and are subjected to catabolic repression by glucose and succinate.
RESULTS
translational reporter plasmids containing pa catabolic gene promoters are responsive to pa and related compounds
the pa degradation genes are arranged in three separate clusters in b. cenocepacia, namely paaabcde, paafzjgijk <dig> and paahk <dig>  where the paaf gene is divergently orientated from the paazjgijk <dig> cluster  <cit> .
to evaluate whether the upstream regions of the pa catabolic genes contained pa-inducible promoters, translational egfp reporter plasmids containing dna fragments upstream of the first gene of each of the three pa clusters: paaa, paaz and paah were constructed and introduced into b. cenocepacia k56- <dig>  previous results showed that egfp is expressed and remains stable in b. cenocepacia  <cit> .
cells containing reporter plasmids with the paaa, paah, and paaz promoters  fused to the egfp gene, exhibited increased fluorescence when grown in minimal media containing glycerol with pa in comparison with those grown in minimal media containing glycerol without pa .
egpf expression from ppaaa was  <dig>  fold higher when grown with pa compared to glycerol, while the ones from ppaah and ppaaz were each  <dig>  fold higher.
according to the kegg database  <cit>  we expected phenylalanine, phenylacetamide and phenylethylamine to be degraded through the pa catabolic pathway in b. cenocepacia au <dig>  to determine if these aromatic carbon sources induce the pa degradation pathway in b. cenocepacia k56- <dig>  cells containing the ppaaa reporter were grown in media containing these carbon sources.
egfp expression similar to the one shown with pa was observed with phenylalanine, phenylpyruvate or phenylacetamide .
on the contrary, 2-hydroxy-phenylacetic acid did not induce egfp expression, in accordance with this compound not being a true intermediate of the pathway  <cit> .
in addition, we sought to determine whether the pa genes were activated in response to synthetic cystic fibrosis medium , a chemically defined medium formulated according to the contents of cf sputum  <cit> .
our results show that ppaaa reporter activity increases approximately 5-fold when cells are grown in scfm .
the pa catabolic genes are subject to catabolic repression by tca intermediates and sugars
aromatic compound degradation is subject to catabolic repression in the presence of more readily usable carbon sources in other bacteria  <cit> .
therefore, the possible catabolic repression exerted by succinate and glucose was investigated.
strains containing the reporters ppaaa, ppaaz and ppaah or the plasmid pjh <dig> were grown in minimal medium containing pa with or without the additional carbon source and analyzed at one-hour intervals .
b. cenocepacia k56- <dig> harbouring pjh <dig> was used as a control as the dhfr promoter is constitutive in burkholderia species  <cit> .
figure 3a shows that fluorescence increased linearly with optical density in the media types tested, indicating the rate of egfp expression does not change during growth with each of the conditions in b. cenocepacia.
initially, the levels of egfp expression were not affected with the different carbon sources, although at optical densities above  <dig> , fluorescence varied slightly depending on the different carbon sources used.
catabolic repression by glucose on the pa-inducible egfp expression was observed in cells harbouring ppaaa, at approximately an o.d <dig> of  <dig>  where a shift in the slope towards steady levels of fluorescence, suggesting lack of de novo egfp synthesis, was observed .
the same effect was observed with reporters ppaaz and ppaah .
this is contrasted with cells grown in succinate, which exhibited strong silencing of egfp expression at all cell densities .
we concluded that glucose and succinate exert catabolic repression of the pa degradation pathway.
insertional mutagenesis of bcal <dig> results in increased expression of pa-inducible genes
located  <dig> bp downstream of the paaabcde gene cluster and oriented in the same direction are genes bcal <dig> and bcal <dig> .
bcal <dig> is predicted to encode a  <dig> amino acid protein containing a conserved domain of unknown function  while bcal <dig> was annotated as a tetr family regulatory protein.
results of our blast search indicated the n-terminal region of bcal <dig> protein shows 60% similarity to acrr , which is a tetr-like regulator of a multi-drug efflux pump of e. coli  <cit> .
given that a regulator protein homologous to paax, the gntr-type transcriptional regulator of pa degradation in e. coli  <cit>  is not encoded in b. cenocepacia j <dig> genome, we hypothesized that the bcal <dig> gene encoded the regulator of pa catabolism in b. cenocepacia.
the effect of the loss of bcal <dig> function on the regulation on the pa genes was determined by insertional mutagenesis of the bcal <dig> gene to create the strain jnrh <dig>  reporter plasmids containing the ppaaa, ppaah and ppaaz promoters were then conjugated into strain jnrh <dig> and egfp expression was assessed in the presence and the absence of pa. results show that these strains exhibit increased fluorescence regardless of the presence of pa in the culture .
this pa independent activity suggests that bcal <dig> encodes for a negative regulator, whose regulatory ability is abolished in the jnrh <dig> mutant.
interestingly, egfp expression driven by the ppaaa and ppaah promoters in jnrh <dig> was higher in the presence of pa than in reporter strains grown with glycerol only  suggesting a bcal <dig> independent induction of gene expression in the presence of pa.
in order to determine if paaabcde and bcal0211-bcal <dig> were part of the same transcriptional unit, a transcriptional analysis was performed.
total rna was isolated from b. cenocepacia cells grown with lb containing  <dig> mm pa and subjected to rt-pcr using specific primers.
results show that the paaa, paab, paac, paad and paae genes are contained on a single transcript and are thus co-regulated at the transcriptional level .
primers were unable to generate an amplicon between paae and bcal <dig> although an amplicon was generated between bcal <dig> and bcal <dig>  indicating that they are located on the same transcript.
taken together these results demonstrate that paaabcde and bcal0211-bcal <dig> are two separate transcriptional units.
a conserved inverted repeat is necessary for negative control of ppaaa
examination of upstream dna sequences of the pa gene clusters identified near perfect  <dig> bp inverted repeat  sequences located between the putative - <dig> and - <dig> core promoter signals  that resembled operator sites of a tetr regulatory protein  <cit> .
in order to validate the ir sequences found in pa gene promoters as the operator sites of bcal <dig>  translational fusion plasmids containing mutations in the paaa ir were created.
we hypothesized that the sequence is a motif recognized by a tetr-like transcriptional regulator due to it being a dual overlapping inverted repeat, similar to the qacr operator  <cit> .
using plasmid mutagenesis with primers containing mismatched mutations on the 5' ends  that annealed to plasmid pjh <dig> containing the ppaaa reporter we generated plasmids pjh <dig>  pjh <dig> and pjh <dig> .
the plasmid pjh <dig> contains  <dig> mismatch mutations replacing nearly the entire ir within the paaa promoter.
plasmids pjh <dig> and pjh <dig> contain the mutations in the upstream or downstream half of the ir respectively.
these plasmids were then transferred to b. cenocepacia k56- <dig> by triparental mating.
reporter strains were grown in minimal media supplemented with glycerol or pa. cells harbouring plasmids pjh <dig>  pjh <dig> and pjh <dig> exhibited higher levels of relative fluorescence in comparison with k56-2/pjh <dig> when grown with glycerol, demonstrating that the sequence is indeed required for negative control of paaa promoter activity .
cm, chloramphenicol; km, kanamycin; tp, trimethoprim.
arrows represent changes introduced to indicated sequences.
because paax is involved in the regulation of upstream pathways of pa catabolism in other microorganisms through binding a conserved paax box  <cit>  we searched for the consensus ir sequence in the genome of b. cenocepacia.
a position weight matrix   <cit>  of the conserved ir present in the promoter regions of the paaa, paah and paaz plus the divergent promoter of paaf and bcal <dig> was constructed  and used to search the entire genome sequence of b. cenocepacia j <dig>  the coordinate positions of sequences detected up to a cut off score of  <dig>  are listed .
the top scores for the search were the ones for the paaz, paaf, paaa and paah inverted repeats while bcal <dig> ir scored lower at  <dig> .
other sequences with scores that ranked from  <dig>  to  <dig>  did not locate in putative promoters or between - <dig> and - <dig> regions, likely representing false positives.
we concluded that the  <dig> bp ir sequences are specific to the pa catabolic gene clusters.
discussion
in contrast to what has been observed in e. coli and pseudomonas putida  <cit> , the pa genes of b. cenocepacia k56- <dig> are organized into three gene clusters.
we hypothesize that this arrangement may allow regulation of gene expression at different levels.
the observation that egfp expression driven by ppaaa is roughly 3-fold stronger than either the ppaah or ppaaz promoters  is suggestive of a higher requirement for the product of the paaabcde enzymatic complex than the other intermediates.
this could be simply due to the optimal kinetic coupling between the different steps or that the product of the ring hydroxylation complex is used in a second pathway with a yet unknown biological function.
the presence of a poly tract upstream of the paaa - <dig> element  that resembles an up element  <cit>  may likely account for the increased activity.
our results also show that bcal <dig> is necessary for repression of pa dependent activity of the paaa, paah and paaz gene promoters .
therefore, bcal <dig>  encoding for a tetr-type transcriptional regulator is involved in negative regulation of the pa catabolic genes.
since a conserved inverted repeat dna sequence is necessary for pa negative control of paaa gene expression , we hypothesize that bcal <dig> binds the irs located in the core promoter of the paaa, paaz and paah genes to negatively regulate transcription of the pa catabolic genes.
it should be noted however, that the insertional mutagenesis system used to produce jnrh <dig> introduces polar mutations  <cit> .
although the possibility of polar effects on genes downstream bcal <dig> cannot be ruled out, the downstream gene bcal <dig>  encoding a putative gnat family acetyl transferase located several hundred base pairs downstream of bcal <dig> makes the possibility of polar effects unlikely.
on the other hand, bcal <dig> and bcal <dig> are located on the same transcript  and thus are co-regulated at the transcriptional level.
tetr-type proteins are known to regulate their own transcription by self-repression  <cit> .
currently it is unknown if the conserved ir located in the dna leader sequence of the bcal <dig> gene may be involved in regulation of this gene cluster.
whether bcal <dig>  which encodes for a protein of unknown function  is involved in some fashion in the regulation of the pa genes remains to be determined.
b. cenocepacia k56- <dig> containing egfp translational reporters ppaaa were grown for  <dig> hours in m <dig> minimal media supplemented with glycerol or pa. relative fluorescence was determined as described in methods.
data represent the mean from three independent experiments, ± one standard deviation.
catabolic repression of aromatic compound degradation by tca intermediates and glucose has been described in the β-proteobacterium acidovorax sp.
<cit> , and p. putida  <cit>  respectively.
in accordance with these data we found that the pa catabolic pathway of b. cenocepacia k56- <dig> is subject to catabolic repression by glucose and succinate .
interestingly, ppaaa is induced after  <dig> h of growth in scfm probably as a result of the presence of phenylalanine .
this observation is consistent with the recently reported b. cenocepacia global gene expression response to scfm, which shows the induction of the pa catabolic pathway  <cit> .
whether this finding is relevant for pathogenesis of bcc in the cf lung environment remains an unexplored point of interest.
CONCLUSIONS
we show that the pa gene promoters are responsive to pa, scfm, and other compounds expected to proceed via the pa pathway.
we also show the pa gene promoters are negatively regulated by paar, a tetr-type regulator, and are subjected to catabolic repression by succinate and glucose.

 the burkholderia cepacia complex is a group of gram negative bacteria that comprises at least fifteen taxonomically related species. bcc strains occupy multiple niches from soil to humans as they have emerged as opportunistic pathogens in patients with cystic fibrosis, chronic granulomatous disease, and other medical conditions associated with a compromised immune system. burkholderia species have evolved large genomes that allow them to deal with a variety of nutrient sources, predation and competition. the three chromosomes of b. cenocepacia, one of the most common species found in cf patients, encode a broad array of catabolic functions. yet, the contribution of these metabolic capacities to colonization and survival in the host has not been established. the phenylacetic acid catabolic pathway is the central route where catabolism of many aromatic compounds converge and are directed to the krebs cycle. it comprises of four steps, namely the pa-coa ligation-activation performed by paak, the hydroxylation step for which the paaabcde enzymatic complex is responsible, the enoyl-coa isomerization/hydration, ring opening performed by paag and paaz,, and the β-oxidation step carried out by paaf and paah,. previously, we initiated the functional characterization of the pa catabolic pathway of b. cenocepacia k56- and demonstrated that interruption of putative pa-coa ring hydroxylation activity, but not the lower steps of pa degradation, resulted in an attenuated pathogenic phenotype in the caenorhabditis elegans model of infection. here, we report that the pa catabolic genes of b. cenocepacia k56- are induced by pa, are negatively regulated by paar, a tetr-type regulator and are subjected to catabolic repression by glucose and succinate.  translational reporter plasmids containing pa catabolic gene promoters are responsive to pa and related compounds the pa degradation genes are arranged in three separate clusters in b. cenocepacia, namely paaabcde, paafzjgijk and paahk where the paaf gene is divergently orientated from the paazjgijk cluster. to evaluate whether the upstream regions of the pa catabolic genes contained pa-inducible promoters, translational egfp reporter plasmids containing dna fragments upstream of the first gene of each of the three pa clusters: paaa, paaz and paah were constructed and introduced into b. cenocepacia k56- previous results showed that egfp is expressed and remains stable in b. cenocepacia. cells containing reporter plasmids with the paaa, paah, and paaz promoters fused to the egfp gene, exhibited increased fluorescence when grown in minimal media containing glycerol with pa in comparison with those grown in minimal media containing glycerol without pa. egpf expression from ppaaa was fold higher when grown with pa compared to glycerol, while the ones from ppaah and ppaaz were each fold higher. according to the kegg database we expected phenylalanine, phenylacetamide and phenylethylamine to be degraded through the pa catabolic pathway in b. cenocepacia au to determine if these aromatic carbon sources induce the pa degradation pathway in b. cenocepacia k56- cells containing the ppaaa reporter were grown in media containing these carbon sources. egfp expression similar to the one shown with pa was observed with phenylalanine, phenylpyruvate or phenylacetamide. on the contrary, 2-hydroxy-phenylacetic acid did not induce egfp expression, in accordance with this compound not being a true intermediate of the pathway. in addition, we sought to determine whether the pa genes were activated in response to synthetic cystic fibrosis medium, a chemically defined medium formulated according to the contents of cf sputum. our results show that ppaaa reporter activity increases approximately 5-fold when cells are grown in scfm. the pa catabolic genes are subject to catabolic repression by tca intermediates and sugars aromatic compound degradation is subject to catabolic repression in the presence of more readily usable carbon sources in other bacteria. therefore, the possible catabolic repression exerted by succinate and glucose was investigated. strains containing the reporters ppaaa, ppaaz and ppaah or the plasmid pjh were grown in minimal medium containing pa with or without the additional carbon source and analyzed at one-hour intervals. b. cenocepacia k56- harbouring pjh was used as a control as the dhfr promoter is constitutive in burkholderia species. figure 3a shows that fluorescence increased linearly with optical density in the media types tested, indicating the rate of egfp expression does not change during growth with each of the conditions in b. cenocepacia. initially, the levels of egfp expression were not affected with the different carbon sources, although at optical densities above, fluorescence varied slightly depending on the different carbon sources used. catabolic repression by glucose on the pa-inducible egfp expression was observed in cells harbouring ppaaa, at approximately an o.d of where a shift in the slope towards steady levels of fluorescence, suggesting lack of de novo egfp synthesis, was observed. the same effect was observed with reporters ppaaz and ppaah. this is contrasted with cells grown in succinate, which exhibited strong silencing of egfp expression at all cell densities. we concluded that glucose and succinate exert catabolic repression of the pa degradation pathway. insertional mutagenesis of bcal results in increased expression of pa-inducible genes located bp downstream of the paaabcde gene cluster and oriented in the same direction are genes bcal and bcal. bcal is predicted to encode a amino acid protein containing a conserved domain of unknown function while bcal was annotated as a tetr family regulatory protein. results of our blast search indicated the n-terminal region of bcal protein shows 60% similarity to acrr, which is a tetr-like regulator of a multi-drug efflux pump of e. coli. given that a regulator protein homologous to paax, the gntr-type transcriptional regulator of pa degradation in e. coli is not encoded in b. cenocepacia j genome, we hypothesized that the bcal gene encoded the regulator of pa catabolism in b. cenocepacia. the effect of the loss of bcal function on the regulation on the pa genes was determined by insertional mutagenesis of the bcal gene to create the strain jnrh reporter plasmids containing the ppaaa, ppaah and ppaaz promoters were then conjugated into strain jnrh and egfp expression was assessed in the presence and the absence of pa. results show that these strains exhibit increased fluorescence regardless of the presence of pa in the culture. this pa independent activity suggests that bcal encodes for a negative regulator, whose regulatory ability is abolished in the jnrh mutant. interestingly, egfp expression driven by the ppaaa and ppaah promoters in jnrh was higher in the presence of pa than in reporter strains grown with glycerol only suggesting a bcal independent induction of gene expression in the presence of pa. in order to determine if paaabcde and bcal0211-bcal were part of the same transcriptional unit, a transcriptional analysis was performed. total rna was isolated from b. cenocepacia cells grown with lb containing mm pa and subjected to rt-pcr using specific primers. results show that the paaa, paab, paac, paad and paae genes are contained on a single transcript and are thus co-regulated at the transcriptional level. primers were unable to generate an amplicon between paae and bcal although an amplicon was generated between bcal and bcal indicating that they are located on the same transcript. taken together these results demonstrate that paaabcde and bcal0211-bcal are two separate transcriptional units. a conserved inverted repeat is necessary for negative control of ppaaa examination of upstream dna sequences of the pa gene clusters identified near perfect bp inverted repeat sequences located between the putative - and - core promoter signals that resembled operator sites of a tetr regulatory protein. in order to validate the ir sequences found in pa gene promoters as the operator sites of bcal translational fusion plasmids containing mutations in the paaa ir were created. we hypothesized that the sequence is a motif recognized by a tetr-like transcriptional regulator due to it being a dual overlapping inverted repeat, similar to the qacr operator. using plasmid mutagenesis with primers containing mismatched mutations on the 5' ends that annealed to plasmid pjh containing the ppaaa reporter we generated plasmids pjh pjh and pjh. the plasmid pjh contains mismatch mutations replacing nearly the entire ir within the paaa promoter. plasmids pjh and pjh contain the mutations in the upstream or downstream half of the ir respectively. these plasmids were then transferred to b. cenocepacia k56- by triparental mating. reporter strains were grown in minimal media supplemented with glycerol or pa. cells harbouring plasmids pjh pjh and pjh exhibited higher levels of relative fluorescence in comparison with k56-2/pjh when grown with glycerol, demonstrating that the sequence is indeed required for negative control of paaa promoter activity. cm, chloramphenicol; km, kanamycin; tp, trimethoprim. arrows represent changes introduced to indicated sequences. because paax is involved in the regulation of upstream pathways of pa catabolism in other microorganisms through binding a conserved paax box we searched for the consensus ir sequence in the genome of b. cenocepacia. a position weight matrix of the conserved ir present in the promoter regions of the paaa, paah and paaz plus the divergent promoter of paaf and bcal was constructed and used to search the entire genome sequence of b. cenocepacia j the coordinate positions of sequences detected up to a cut off score of are listed. the top scores for the search were the ones for the paaz, paaf, paaa and paah inverted repeats while bcal ir scored lower at. other sequences with scores that ranked from to did not locate in putative promoters or between - and - regions, likely representing false positives. we concluded that the bp ir sequences are specific to the pa catabolic gene clusters. discussion in contrast to what has been observed in e. coli and pseudomonas putida, the pa genes of b. cenocepacia k56- are organized into three gene clusters. we hypothesize that this arrangement may allow regulation of gene expression at different levels. the observation that egfp expression driven by ppaaa is roughly 3-fold stronger than either the ppaah or ppaaz promoters is suggestive of a higher requirement for the product of the paaabcde enzymatic complex than the other intermediates. this could be simply due to the optimal kinetic coupling between the different steps or that the product of the ring hydroxylation complex is used in a second pathway with a yet unknown biological function. the presence of a poly tract upstream of the paaa - element that resembles an up element may likely account for the increased activity. our results also show that bcal is necessary for repression of pa dependent activity of the paaa, paah and paaz gene promoters. therefore, bcal encoding for a tetr-type transcriptional regulator is involved in negative regulation of the pa catabolic genes. since a conserved inverted repeat dna sequence is necessary for pa negative control of paaa gene expression, we hypothesize that bcal binds the irs located in the core promoter of the paaa, paaz and paah genes to negatively regulate transcription of the pa catabolic genes. it should be noted however, that the insertional mutagenesis system used to produce jnrh introduces polar mutations. although the possibility of polar effects on genes downstream bcal cannot be ruled out, the downstream gene bcal encoding a putative gnat family acetyl transferase located several hundred base pairs downstream of bcal makes the possibility of polar effects unlikely. on the other hand, bcal and bcal are located on the same transcript and thus are co-regulated at the transcriptional level. tetr-type proteins are known to regulate their own transcription by self-repression. currently it is unknown if the conserved ir located in the dna leader sequence of the bcal gene may be involved in regulation of this gene cluster. whether bcal which encodes for a protein of unknown function is involved in some fashion in the regulation of the pa genes remains to be determined. b. cenocepacia k56- containing egfp translational reporters ppaaa were grown for hours in m minimal media supplemented with glycerol or pa. relative fluorescence was determined as described in methods. data represent the mean from three independent experiments, ± one standard deviation. catabolic repression of aromatic compound degradation by tca intermediates and glucose has been described in the β-proteobacterium acidovorax sp., and p. putida respectively. in accordance with these data we found that the pa catabolic pathway of b. cenocepacia k56- is subject to catabolic repression by glucose and succinate. interestingly, ppaaa is induced after h of growth in scfm probably as a result of the presence of phenylalanine. this observation is consistent with the recently reported b. cenocepacia global gene expression response to scfm, which shows the induction of the pa catabolic pathway. whether this finding is relevant for pathogenesis of bcc in the cf lung environment remains an unexplored point of interest.  we show that the pa gene promoters are responsive to pa, scfm, and other compounds expected to proceed via the pa pathway. we also show the pa gene promoters are negatively regulated by paar, a tetr-type regulator, and are subjected to catabolic repression by succinate and glucose. 
in this study, we aimed to characterize regulatory elements and nutritional requirements that control the pa catabolic genes in b. cenocepacia k56- the pa catabolic genes of b. cenocepacia k56- are induced by pa and other related compounds, are negatively regulated by paar, a tetr-type regulator, and are subjected to catabolic repression by glucose and succinate. to show that bcal a putative tetr-type regulator gene encodes a regulator for the pa genes in b. cenocepacia, we developed a bcal insertional mutant reporter strain. previously, we initiated the characterization of the phenylacetic acid degradation pathway in b. cenocepacia, a member of the bcc, and demonstrated the necessity of a functional pa catabolic pathway for full virulence in caenorhabditis elegans. translational fusions of the pa degradation gene promoters with egfp were constructed and introduced in b. cenocepacia k56- egfp expression was observed when the reporter strains were grown in minimal media containing glycerol and pa or other compounds expected to proceed through the pa pathway, and in synthetic cf medium. as the pa catabolic pathway of b. cenocepacia appears to be induced during growth in synthetic cystic fibrosis medium, further research is necessary to determine the relevance of this pathway in cf-like conditions and in other host-pathogen interactions. results show that these strains exhibit fluorescence regardless of the presence of pa in the culture. metabolically versatile soil bacteria burkholderia cepacia complex have emerged as opportunistic pathogens, especially of cystic fibrosis. addition of succinate or glucose to the pa containing medium repressed egfp expression.
 metabolically versatile soil bacteria burkholderia cepacia complex have emerged as opportunistic pathogens, especially of cystic fibrosis. previously, we initiated the characterization of the phenylacetic acid degradation pathway in b. cenocepacia, a member of the bcc, and demonstrated the necessity of a functional pa catabolic pathway for full virulence in caenorhabditis elegans. in this study, we aimed to characterize regulatory elements and nutritional requirements that control the pa catabolic genes in b. cenocepacia k56-  translational fusions of the pa degradation gene promoters with egfp were constructed and introduced in b. cenocepacia k56- egfp expression was observed when the reporter strains were grown in minimal media containing glycerol and pa or other compounds expected to proceed through the pa pathway, and in synthetic cf medium. addition of succinate or glucose to the pa containing medium repressed egfp expression. to show that bcal a putative tetr-type regulator gene encodes a regulator for the pa genes in b. cenocepacia, we developed a bcal insertional mutant reporter strain. results show that these strains exhibit fluorescence regardless of the presence of pa in the culture.  the pa catabolic genes of b. cenocepacia k56- are induced by pa and other related compounds, are negatively regulated by paar, a tetr-type regulator, and are subjected to catabolic repression by glucose and succinate. as the pa catabolic pathway of b. cenocepacia appears to be induced during growth in synthetic cystic fibrosis medium, further research is necessary to determine the relevance of this pathway in cf-like conditions and in other host-pathogen interactions. 