Results show that these strains exhibit increased fluorescence regardless of the presence of PA in the culture (Figure 1). This PA independent activity suggests that BCAL0210 encodes for a negative regulator, whose regulatory ability is abolished in the JNRH1 mutant. Interestingly, eGFP expression driven by the P paaA and P paaH promoters in JNRH1 was higher in the presence of PA than in reporter strains grown with glycerol only (Figure 1)

selleck chemicals suggesting a BCAL0210 independent induction of gene expression in the presence of PA. 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. In order to determine if paaABCDE and BCAL0211-BCAL0210 were part of the same transcriptional unit, a transcriptional analysis was performed. Total RNA was isolated from B. cenocepacia cells grown with LB containing 1 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 either and are thus co-regulated at the transcriptional level (Figure 4B). Primers were unable to generate an amplicon between paaE and BCAL0211 although an amplicon was generated between BCAL0211 and BCAL0210, indicating AC220 that they are located on the same transcript. Taken together these results demonstrate that paaABCDE and BCAL0211-BCAL0210 are two separate transcriptional units. A conserved Inverted buy Nirogacestat Repeat is necessary for negative control of P paaA Examination of upstream DNA sequences of the PA gene clusters identified near perfect 15 bp inverted repeat (IR) sequences

located between the putative -10 and -35 core promoter signals (Figure 5) that resembled operator sites of a TetR regulatory protein [21]. In order to validate the IR sequences found in PA gene promoters as the operator sites of BCAL0210, 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 [21]. Figure 5 Conserved inverted repeat detected in the paaA, paaZ and paaH promoters. DNA Sequences of P paaA , (A), P paaH , (B), and P paaZ , (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.