putida CA-3, effectively creating a rate limiting step in substrate use. Indeed, previous work by our group demonstrated that over expression of the styrene active transport protein, StyE, in P. putida CA-3 resulted in an 8 fold increase in transcriptional activation of the upper pathway [24]. The PaaL expression vector was therefore conjugally transferred into wild type cells to give WT-PaaL+, and growth

on phenylacetic acid and PACoA ligase activity assessed. Surprisingly, the observed effect of PaaL over expression in the WT-PaaL+ strain was slower growth on phenylacetic acid compared with the P. putida CA-3 parent and D7-PaaL+ strains, Figure 4. In addition, PACoA ligase activity was found to be approximately 22% lower in the WT-PaaL+ strain compared with wild type AZD4547 P. putida CA-3 (data not shown). It remains unclear whether the reduced activity observed reflects a direct inhibitory impact on the ligase enzyme, or a general toxicity effect within the cells arising from PaaL over-expression and increased phenylacetic acid uptake. Thus, while PaaL expression is click here essential for phenylacetic acid utilisation by P. putida CA-3, it does not appear to represent a rate limiting step in the process. Figure 4 Effects

of PaaL over expression on growth. Growth on phenylacetic acid of P. putida CA-3 wild type (WT) and the wild type and D7 mutant strains harbouring the pBBR1MCS-5 PaaL over expression vector, (WT-PaaL+) and (D7-PaaL+), respectively. Baf-A1 Cloning and bioinformatic analysis of the paaL promoter from P. putida CA-3 The paaL promoter region

was cloned SB-715992 manufacturer from P. putida CA-3, sequenced and analysed for archetypal σ54 promoter features, Figure 5(a) and 5(b)[19, 25]. Analysis of the 458 bp promoter sequence using the search algorithms GenomeMatScan and TRES, failed to identify palindromic or inverted repeat regions, typical of XylR/NtrC family enhancer binding proteins, (EBPs) [19, 26]. EBPs are reportedly essential for transcriptional activation of σ54 promoters and facilitate the integration of promoter activation with host signal responses to environmental cues and physiological states, [27, 28]. Comparative analysis of the paaL promoter with 9 other predicted σ54 promoter sequences from P. putida KT2440, was carried out using the Multiple Em for Motif Elucidation algorithm, MEME [29]. The program quantitatively evaluates background noise in similarly regulated promoters to identify the most conserved motifs among them as potential sites for regulator interactions. One highly conserved motif was identified as common to all sequences, which was identified via the TOMTOM motif comparison tool [30] as a σ54 binding site. The site contained the previously reported GG-N10-GC,-24/-12 consensus sequence found in all σ54 promoters [25, 31].