Small-Molecule Acetylation Controls the Degradation of Benzoate and Photosynthesis in Rhodopseudomonas palustris

This work shows that the BadL protein of Rhodopseudomonas palustris has N-acetyltransferase activity and that this activity is required for the catabolism of benzoate under photosynthetic conditions in this bacterium. R. palustris occupies lignin-rich habitats, making its benzoate-degrading capability critical for the recycling of this important, energy-rich biopolymer. This work identifies the product of the BadL enzyme as acetamidobenzoates, which were needed to derepress genes encoding benzoate-degrading enzymes and proteins of the photosynthetic apparatus responsible for the generation of the proton motive force under anoxia in the presence of light. In short, acetamidobenzoates potentially coordinate the use of benzoate as a source of reducing power and carbon with the generation of a light-driven proton motive force that fuels ATP synthesis, motility, transport, and many other processes in the metabolically versatile bacterium R. palustris.

R. palustris badL, badM, and aadR (RPA4234, TX73_RS21595) were amplified and purified as described above. The badM and aadR genes were cloned into HindIII and XbaI sites of pBBR1MCS-2, resulting in plasmids pRpBadM3 and pRpAadR3. The badL gene was cloned into KpnI and HindIII sites of pBBR1MCS2, resulting in plasmid pRpBadL3. Ligated plasmids were transformed into E. coli DH5a and plated on LB agar supplemented with kanamycin. Clones of interest were screened via colony PCR using M13 forward and reverse primers.
In-frame deletions of R. palustris badL and badM genes were constructed using described methods (7). Briefly, 1-kb regions upstream and downstream of targeted genes were amplified and fused using overlap extension PCR (8). The PCR products were digested with either EcoRI and HindIII (badL), EcoRI and BamHI (badM), or XbaI and HindIII (badL badM) and cloned into plasmid pK18mobsacB (7). Ligated plasmids were transformed into E. coli DH5a and plated on LB agar supplemented with kanamycin. Clones of interest were screened via colony PCR using M13 forward and reverse primers. Deletion construct plasmids were electroporated into exponentially growing R. palustris (washed twice with 10% glycerol, v/v) and plated on PMsuccinate agar plates + kanamycin. Cells were grown under photosynthetic conditions in an Annoxomat Jar (Spiral Biotech) until single colonies appeared. Cells were streaked to isolation on PM-succinate agar plates + kanamycin and grown under light and anoxia. Multiple colonies were then streaked to isolation for counterselection on PM-succinate + sucrose (10%, v/v, added after autoclaving) and grown under light and anoxic conditions. Single colonies were screened for acquisition of the deletion and deletions were confirmed via DNA sequencing. For construction of DbadL DbadM, both genes were deleted simultaneously in a badL + badM + R. palustris strain. For complementation of genes, plasmids constructed as described above were electroporated into R. palustris and selected for transformation on PM-succinate + kanamycin agar plates.
Proteins were cleaved at 25°C for 3 h with a 1:50 mg:mg ratio of rTEV to target protein. After cleavage, proteins were dialyzed in buffer D [HEPES (50 mM, pH 7.5 at 4°C), NaCl (0.5 M), EDTA (1 mM), and glycerol (20% v/v)], and twice in buffer A. After dialysis, cleaved proteins were applied to a pre-equilibrated 1 mL HisPur Ni-NTA resin. Proteins that did not bind to the column were collected and dialyzed against three buffers with decreasing concentrations of NaCl (400 mM, 0.25 M, 150 mM), with a final buffer composition of [HEPES (50 mM, pH 7.5 at 4°C), NaCl (0.15 M), and glycerol (20% v/v)]. Protein concentrations were determined with a NanoDrop and were flash frozen in liquid N2 and stored at -80°C until used.

HPLC and MS/MS analyses
Reactions containing BadL (10 µM), acetyl-CoA (50 µM), aminobenzoates (100 µM), TCEP (0.5 mM), and sodium phosphate buffer (pH 8 at 25° C) were incubated for 2 h at 37°C. BadL was removed by passing reactions over an Amicon Ultra 0.5 mL 10kDa molecular cut off centrifugal filter (Millipore). The product of BadL aminobenzoate acetylation was resolved by RP-HPLC using a Shimadzu Prominence UFLC with a Kinetex 5µm C18 column (150 mm x 4.6 mm; Phenomenex). The column was equilibrated at a flow rate of 0.5 ml min -1 with 5 column volumes of elution buffer [50 % (v/v, in water) acetonitrile] and 5 column volumes of wash buffer [10 mM sodium phosphate in water (pH 8 at 25° C)]. Samples (100 µl) were injected and the column was developed with wash buffer for 5 min and then a gradient to elution buffer over 10 min. Compounds were detected and compared to standards at 254 nm using a computer-controlled Shimadzu Nexera X2 SPD-30A diode array detector. Data were analyzed using Prism v6 (GraphPad). Fractions were collected in 0.5 mL increments and fractions containing peaks corresponding to acetamidobenzoate were analyzed by MS and MS/MS (Protein and Mass Spectrometry Facility, UGA, Athens, GA, USA). Electrospray ionization (ESI)-MS was performed in acetonitrile and resolved on an Esquire 3000 Plus (Bruker) Ion Trap Mass Spectrometer at 0.3 ml h -1 . Pure standards (3-ABA and 3-ABA Ac , 1 mM) were suspended in DMSO and run as described above.

RNAsnap protocol for isolation of RNA for RT-qPCR
Cell pellets were resuspended in 150 µL of boil solution [EDTA (18 mM), sodium dodecyl sulfate (SDS, 0.025% v/v), RNA-grade formamide (95% v/v), 2-mercaptoethanol (1 % v/v)] in water and transferred to 1.7-mL Eppendorf tubes. Mixtures were incubated at 100°C for 7 min and immediately centrifuged at 16,000g for 5 min. A sample (100 µL) of supernatant was transferred to a fresh 2.0-mL Eppendorf tube containing 400 µL of RNase-free water and 50 µL of 3M sodium acetate, pH 5.2. Following RNA dilution, 1.65 mL of ice-cold ethanol (100%) was added to the reaction mixtures and tubes were incubated at -80°C for at least 1 h. After DNA/RNA precipitation, reaction mixtures were poured into 1.7-mL Eppendorf tubes and centrifuged at 16,000 x g for 1 h. Supernatants were decanted and pellets were washed with 300 µL of ice-cold ethanol (70% v/v). Supernatants were decanted, tubes were immediately centrifuged, excess supernatant was decanted and tubes were allowed to dry upside down on Kimwipes for 20 min. DNA/RNA pellets were resuspended in 100 µL of RNase-free water and centrifuged at 16,000 x g to remove non-soluble debris. Subsequent DNase I treatment was carried out with 90 µL of the DNA/RNA suspension and the Ambion Turbo DNA-free kit (Thermo Fischer Scientific) according to manufacturer's instructions for rigorous DNase treatment. After DNA cleavage, a final sodium acetate-ethanol precipitation was performed as described above. Dried RNA pellets were resuspended in 100 µL of RNase-free water and frozen at -80°C in 20-µL aliquots. A small sample for each RNA prep was sent to the Georgia Genomics Facility (Athens, GA, USA) for quality control analysis using the RNA 600 Nano kit of the Agilent 2100 bioanalyzer. Any RNA samples that had a RNA Integrity Number (RIN) of above 8.0 were used for subsequent qRT-PCR experiments.

cDNA synthesis and quantitative reverse-transcription PCR
Primers for qRT-PCR were designed using primer 3 software and were evaluated for specificity and melting curve. Total RNA (150 ng) from each sample was used with the iScript cDNA synthesis kit (BioRad) following the manufacturer's protocol. The final RNA concentration of the cDNA reaction mixture was 7.5 ng/µL and was used as a template for qRT-PCR. Master mixes were prepared in RNase-free water with FastSYBR green master mix (1X, Applied Biosystems) and gene-specific primers (1 µL of each primer from a 10 µM stock). qRT-PCR reactions (20 µL) were assembled with 15 ng of cDNA (2 µL of the 7.5 ng/µL cDNA) and 18 µL of the abovementioned master mix into a MicroAmpÔ Fast Optical 96-well reaction plate (Thermo Fischer Scientific). The real-time PCR was performed using a 7500 Fast real-time PCR system (Applied Biosystems). Cycle threshold (CT) data were normalized to the fixJ (RPA4248) gene as described elsewhere (9). The normalized DCT values were transformed using 2(e-DCT)/10 -6 (10) and were reported as arbitrary expression units (EU). Mean EU values calculated from technical triplicates of biological triplicates were used to calculate the standard error of the mean in Prism6 software. Statistically significant differences between expression across strains were determined using a Welch's t-test with GraphPad Prism6 software. Figure legends report p values for each sample.

R. palustris pigment analysis
R. palustris strains were grown in triplicate for three days in YP medium aerobically. Cells were sub-cultured (1:30) into 10 mL of PM + benzoate (3 mM) and NaHCO3 (10 mM). The headspace of Balch culture tubes was flushed with O2-free N2 gas, and cultures were grown photosynthetically as described above. At lag (OD660 ~0.20), log (OD660 ~0.6), and stationary phase (OD660 >1) of each replicate, 200 µL was removed from the Balch tube with a sterile needle and syringe. Culture samples (100 µL) were pipetted into a 96-well plate and scanned (A600-1000) using a Spectramax UV-vis spectrophotometer. Replicates were plotted using GraphPad Prism6 software to determine mean and the standard error of the mean (SEM) for each strain.