To date, the only functional characterisation of phenylacetic acid uptake to have been conducted in Pseudomonas was performed with P. putida U [10]. In this GANT61 chemical structure strain the PaaL permease and PaaM membrane proteins were both reported as essential for phenylacetic acid utilisation and were co-ordinately regulated with transcriptional activation of the other 2 catabolic operons. However, the transcriptional profiling presented in Bucladesine purchase Figure 3, provided preliminary evidence that paaL may be differentially regulated in P. putida CA-3, in a σ54 dependent manner. The potential for divergent regulatory mechanisms to influence

transport in different microbial species is perhaps not surprising however, given that the phenylacetic acid transport system is inconsistently reported in the literature. The paaM gene is frequently absent from PACoA catabolons reported in Pseudomonas species [12, 20, 22] while both paaL and paaM are absent from the PACoA catabolon of E. coli W [11]. The authors were unable to identify

any paaM homologue in P. putida CA-3 during this study. Figure 3 PaCoA Catabolon gene transcription analyses. Reverse transcription polymerase chain reaction analysis of P. putida CA-3 parent (WT) and rpoN disrupted mutant (D7) strains, following growth of cultures on styrene (sty), citrate (cit) and phenylacetic acid (PAA), respectively. 16S rRNA amplification acted as a positive control. The paaL, paaF and paaG, gene targets (indicated on the left hand side) learn more were selected as representative genes of the operons for phenylacetic uptake, β-oxidation and ring hydroxylation, respectively. Over-expression of PaaL in wild type P. putida CA-3 and rpoN disrupted Adenosine triphosphate D7 mutant strains To confirm whether the observed paaL gene transcription deficiency was the major contributory factor in the phenylacetic acid negative phenotype of mutant D7, over expression experiments were conducted. The full length 1, 647 kb paaL gene was amplified from P. putida CA-3 and sequenced, (GenBank accession no: HM638062).

The gene was subsequently cloned into the pBBR1MCS-5 expression vector and conjugally transferred into the D7 mutant to give D7-PaaL+. Constitutive expression of PaaL from the pBBR1MCS-5 vector was confirmed by RT-PCR analysis following growth of the host cells on citrate, (result not shown). Growth of D7-PaaL+ on phenylacetic acid was subsequently assessed, with a complete restoration in substrate utilisation by the mutant being observed, Figure 4. Thus, PaaL plays a key role in phenylacetic acid utilisation in P. putida CA-3 and rpoN dependent regulation appears unique to the transport operon within the PACoA catabolon of this strain. Interestingly, previous work by Jurado et al [23] reported that σ54 levels in P. putida remain relatively constant throughout growth, ~80 ± 26 molecules per cell, which barely exceeds the number of genome predicted σ54 dependent promoters in P. putida KT2440.