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. 2009 Oct 18:9:222.
doi: 10.1186/1471-2180-9-222.

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Jason N R Hamlin  1 , Ruhi A M Bloodworth, Silvia T Cardona
Affiliations

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Jason N R Hamlin et al. BMC Microbiol. .

Abstract

Background: Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) 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 K56-2.

Results: Translational fusions of the PA degradation gene promoters with eGFP were constructed and introduced in B. cenocepacia K56-2. 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 (SCFM). Addition of succinate or glucose to the PA containing medium repressed eGFP expression. To show that BCAL0210, a putative TetR-type regulator gene encodes a regulator for the PA genes in B. cenocepacia, we developed a BCAL0210 insertional mutant reporter strain. Results show that these strains exhibit fluorescence regardless of the presence of PA in the culture.

Conclusion: The PA catabolic genes of B. cenocepacia K56-2 are induced by PA and other related compounds, are negatively regulated by PaaR (named herein), 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 (SCFM), further research is necessary to determine the relevance of this pathway in CF-like conditions and in other host-pathogen interactions.

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Figures

Figure 1
Figure 1
Phenylacetic Acid Responsive PA reporters. B. cenocepacia K56-2 (WT) or JNRH1 (BCAL0210) containing eGFP translational reporters PpaaZ, PpaaA and PpaaH were grown for 18 hours in M9 minimal media supplemented with glycerol (white bars) or PA and glycerol (grey bars). Relative fluorescence was determined as described in methods. Data represent the mean from three independent experiments, with error bars signifying standard deviations.
Figure 2
Figure 2
Activity of PpaaA as a result of growth in M9 minimal media with different carbon sources. B. cenocepacia K56-2 (WT) containing eGFP translational reporters PpaaA were grown for 18 hours in synthetic cystic fibrosis medium (SCFM) or M9 minimal media supplemented with various carbon sources. Gly, glycerol; PA, phenylacetic acid; 2-OHPA, 2-hydroxy-phenylacetic acid; Phe, L- phenylalanine; PhPy, phenylpyruvate; PhAc, phenylacetamide. Relative fluorescence was determined as described in methods. Data represent the mean from three independent experiments, with error bars signifying standard deviations.
Figure 3
Figure 3
Phenylacetic acid genes are subject to Carbon Catabolite Repression. B. cenocepacia K56-2 containing eGFP translational fusions with the dhfr promoter (A), PpaaA (B), PpaaZ (C), and PpaaH (D) were grown for 13 hours in M9 minimal media supplemented with the indicated carbon sources. Error bars represent the standard deviation of three independent cultures.
Figure 4
Figure 4
Genetic and transcriptional organization of the paaABCDE and BCAL0211-BCAL0210 gene clusters. A) Fragment of chromosome 1 of B. cenocepacia J2315 containing the paaABCDE and BCAL0211-0210 gene clusters. The vertical arrow indicates the location of the inserted pJH9. Horizontal arrows represent transcriptional units (see B). B) RT-PCR analysis of the intergenic regions of the paaABCDE and BCAL0211-0210 gene clusters. 500 bp RT-PCR amplified DNA bands correspond to intergenic regions.
Figure 5
Figure 5
Conserved inverted repeat detected in the paaA, paaZ and paaH promoters. DNA Sequences of PpaaA, (A), PpaaH, (B), and PpaaZ, (C), cloned in pJH2. Predicted start codon is highlighted in bold. Putative ribosome binding site is boxed; predicted -10 and -35 regions are highlighted in grey. The detected conserved inverted repeats are underlined with arrows. A putative UP element is indicated with asterisks.
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