The integrity of RNA was analyzed by agarose gel electrophoresis. To check for DNA contamination,
samples were analyzed with PCR using primers for benA. First-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume using the Protoscript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). For quantitative real-time PCR (Q-PCR) experiments, primer pairs, as shown in Table 2, were designed based on the published reference genome sequence of P. stutzeri A1501 using the Primer 4 server. Amplicons (100 to 200 bp) and reaction specificity were confirmed by agarose gel electrophoresis and product dissociation MGCD0103 curves. Q-PCR reactions contained 1 μl of cDNA, 10 μl of 2× QuantiTect SYBR Green PCR Master this website Mix (Qiagen, Hilden, Germany), 0.5 μl of each primer (20 μM stock), and 8 μl of RNase-free water. Amplifications were conducted on an ABI PRISM 7000 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) under the following conditions: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 31 s at 55°C, and 31 s at 72°C, followed by a melting-curve program (55°C to 99°C, with a 5-s hold at each temperature). Q-PCR data were analyzed using the ABI PRISM 7000 Sequence Detection System Software
(Applied Biosystems). All cDNA samples were run in triplicate. The expression of l6S rRNA was used as an internal control and the signal was used to normalize variations due to different reverse transcription efficiencies. The comparative CT (threshold cycle) method was used to determine the average fold induction of
mRNA by comparing the CT of the target gene to that of the reference gene, as described previously . The average fold LY3023414 purchase change and standard deviation from three independent RNA samples are reported for each point tested. High-performance liquid chromatography (HPLC) analysis To monitor metabolism, the pcaD mutant and wild-type strains were grown in minimal medium supplemented with benzoate or a mixture of benzoate and 4-hydroxybenzoate. One-milliliter culture samples were centrifuged to pellet cells. Any cells remaining in the supernatant were removed by passage through a low-protein-binding, 0.22 μm pore size, syringe filter (MSI, Westborough, MA, USA). HPLC analysis was performed using an Agilent Technologies (Santa Clara, CA, USA) 1200 series chromatography system. A 20-μl sample of the filtrate was analyzed on a C18 reverse-phase very HPLC column (Agilent Technologies). Elution at a rate of 0.8 ml/min was carried out with 30% acetonitrile and 0.1% phosphoric acid, and the eluant was detected at 254 nm. Under these conditions, the retention times for benzoate, catechol, cis, cis-muconate, and 4-hydroxybenzoate standards were 6.071, 2.388, 3.358, and 2.770 min, respectively. Peak areas corresponding to standard and experimental samples were integrated using the manufacturer’s software package (Agilent Technologies). Acknowledgements We would like to thank Dr. Russell Nicholson and Dr.