cenocepacia K56-2. Previous results showed that eGFP is expressed and remains stable in B. cenocepacia [10]. Cells containing reporter plasmids with the paaA, paaH, and paaZ promoters (P paaA , P paaH , and P paaZ respectively) 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 (Figure 1). eGPF expression from P paaA was 5.7 fold higher when grown with PA compared to glycerol, while the ones from P paaH and P paaZ

were each 2.9 fold higher. Figure 1 Phenylacetic Acid Responsive PA reporters. B. cenocepacia K56-2 (WT) or JNRH1 (BCAL0210) containing selleck kinase inhibitor eGFP translational reporters P paaZ , P paaA and P paaH 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. According to the KEGG database [11–13] we expected phenylalanine, phenylacetamide and phenylethylamine to be degraded through the PA catabolic pathway in B. cenocepacia AU1054. To determine if these aromatic carbon sources induce RO4929097 chemical structure the PA degradation pathway in B. cenocepacia K56-2, cells containing the P paaA 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 (Figure 2). On the contrary, 2-hydroxy-phenylacetic acid did not induce eGFP expression, 3-mercaptopyruvate sulfurtransferase in accordance with this compound not being a true intermediate of the pathway [6]. Figure 2 Activity of P paaA as a result of growth in M9 minimal media with different carbon sources. B. cenocepacia K56-2 (WT) containing eGFP translational reporters P paaA 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. In addition, we sought to determine whether the PA genes were activated in response to Synthetic Cystic Fibrosis Medium (SCFM), a chemically defined medium formulated according to the contents of CF sputum [14]. Our results show that P paaA reporter activity increases approximately 5-fold when cells are grown in SCFM (Figure 2).