Department of Microbiology, The University of Iowa, 3-450 BSB, Iowa City, IA 52242, USA
Correspondence
Caroline S. Harwood
csh5{at}u.washington.edu
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ABSTRACT |
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Present address: Department of Microbiology, University of Washington, Box 357242, Seattle, WA 98195-7242, USA.
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INTRODUCTION |
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Rhodopseudomonas palustris is a purple nonsulfur phototroph that has served as a model organism for studies of anaerobic benzoate degradation. It grows on pimelate and other dicarboxylic acids, under both anaerobic and aerobic conditions. Inspection of the recently completed genome sequence of R. palustris strain CGA009 revealed that it has a single cluster of genes predicted to encode all the enzymes that would be required for the -oxidation of fatty acids or dicarboxylic acids (Larimer et al., 2004
; GenBank accession no. BX571963). These genes, annotated as pimFABCDE in the R. palustris genome, are only 3040 % identical at the amino acid level to the genes reported by Parke et al. (2001)
to be required for growth of Acinetobacter spp. on pimelate. Also, some of the predicted enzymic activities differ. Here we report evidence that the R. palustris pim genes are organized as an operon, and are required for maximal anaerobic growth on benzoate and dicarboxylic acids. We also purified the acyl-CoA ligase encoded by pimA, and determined that it was active with a wide range of dicarboxylic acid and fatty acid substrates.
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METHODS |
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Bacterial strains and culture conditions.
R. palustris strains were grown at 30 °C in defined basal medium (PM) aerobically with shaking, or anaerobically in sealed tubes in light, as previously described (Kim & Harwood, 1991). Fatty acid and dicarboxylic acid carbon sources were added to liquid PM medium at a final concentration of either 1·5 or 3 mM, as indicated in the table and figure legends. Pimelate and benzoate were added to a final concentration of 3 mM, and succinate to 10 mM. Anaerobic fatty acid or dicarboxylic acid cultures were supplemented with sodium bicarbonate (10 mM), which was added to PM medium as a sterile solution after autoclaving. Escherichia coli strains DH5
and S17-1 were grown at 37 °C in LuriaBertani broth. Where indicated, R. palustris was grown with 100 µg gentamicin ml1 or 100 µg kanamycin (Km) ml1. E. coli was grown with 100 µg ampicillin ml1, 20 µg Km ml1 or 20 µg gentamicin ml1. Growth rates were determined by measuring turbidity OD660 in a Genesys20 spectrophotometer.
DNA manipulations.
Standard protocols were used for cloning and transformations. All restriction endonucleases and DNA modification enzymes were purchased from New England Biolabs. Shrimp alkaline phosphatase was purchased from Roche Diagnostics. PCR was performed with Pfu DNA polymerase (Stratagene). Chromosomal DNA was purified using a Puregene DNA isolation kit (Gentra systems). DNA fragments were excised and purified from agarose gels using the Qiaquick gel extraction kit (Qiagen), and plasmid DNA was purified with the Qiaprep spin miniprep kit. DNA was sequenced at the University of Iowa DNA core facility by standard automated-sequencing technology.
RNA isolation.
RNA was isolated from R. palustris strain CGA009 using a modification of the RNeasy kit (Qiagen). Cells (30 ml cultures) grown aerobically with 10 mM succinate and 1·5 mM pimelate were harvested at 5000 g at an OD660 of 0·3. The cell pellets were resuspended in 1 ml TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8·0), and incubated with Ready-Lyse Lysozyme (Epicentre) for 15 min at room temperature. Buffer RLT (4 ml; provided with the Qiagen kit) was added to the suspension, and 1 ml aliquots were placed in 2 ml screw-capped tubes containing approximately 1 ml 0·1 mm zirconia/silica beads (BioSpec Products). The suspension was then treated in a Mini-BeadBeater-8 (BioSpec Products) for 1 min periods, with cooling on ice after each period, for a total of 5 min. The broken cells and beads were sedimented by centrifugation, and the supernatants were pooled. The beads were washed by mixing with buffer [100 µl TE (pH 8·0) plus 350 µl RLT] in the 2 ml tubes. After centrifuging a second time, the supernatants were again combined. Ethanol (2·8 ml) was added to the combined cell lysate, and the entire sample was applied to a RNeasy Midi-column. A DNase I digestion was performed on the Midi-column with the Qiagen RNase-Free DNase set. The RNA was eluted from the column, and a second DNase I treatment was performed with RQ1 RNase-Free DNase. The sample was then applied to RNeasy mini-columns to purify the RNA. Manufacturer's instructions were followed for an on-column DNase treatment with the Qiagen DNase set. Three DNase treatments were required to obtain RNA suitable for use in RT-PCR experiments.
RT-PCR.
RT-PCR was performed by using a two-tube protocol. To synthesize cDNA, 0·25 µg template RNA was heated to 65 °C for 5 min to relieve possible secondary structure. The RNA was then transcribed into cDNA using the Omniscript RT kit (Qiagen) and an appropriate primer specific for the pim intergenic regions. The Omniscript was heat-inactivated, and PCR was performed using 2 µl of the reverse-transcription reaction as the template, and primers for the intergenic regions of the pim cluster (Table 1). Negative control reactions were performed, in which the Omniscript reverse transcriptase was omitted, to ensure that the products from the PCR originated from the cDNA, and not from contaminating genomic DNA.
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Expression cloning of pimA.
Primers StartTrc (5'-GCGAAGTGGGATCCATGTCCCATCCCGG-3') and TrcStop (5'-GGTTGGAATTCCGAATTACTTGGTCTGTG-3') containing restriction sites (underlined) were used to amplify the pimA gene. The 1·7 kb product was cloned into pTrcHis (Invitrogen) after digestion with BamHI and EcoRI, to generate pHisLigase. The plasmid insert was sequenced to ensure that mutations were not introduced, and that the full-length gene was present in the His-tag fusion vector.
Purification of PimA.
E. coli DH5(pHisLigase) was grown at 30 °C with aeration to an OD660 of 0·3. The 750 ml culture was induced with IPTG (1 mM) for 4 h. Cells were harvested, washed, and resuspended in 20 ml binding buffer [20 mM triethanolamine (TEA/HCl) buffer (pH 8·0), 0·5 M NaCl, 5 % (v/v) glycerol]. Cells were lysed by sonication. PMSF (0·05 M) was added to the cell paste, and the mixture was centrifuged at 10 000 g for 20 min at 4 °C. The resulting supernatant was then centrifuged at 40 000 g for 90 min at 4 °C, after the addition of DNase I (1 µg ml1), RNase I (1 µg ml1) and PMSF (0·05 M). The supernatant from this high-speed centrifugation was termed the crude cell extract. All subsequent steps were carried out at 4 °C. The crude cell extract (20 ml; 62 mg) was loaded onto a 5 ml HiTrap chelating column (Pharmacia Biotech) that had been charged with 0·1 M NiSO4, and equilibrated with binding buffer. After extensive washing with binding buffer, the column was developed over 120 min using a Bio-Rad Biologic System with a linear gradient of 00·5 M imidazole in binding buffer with a flow rate of 1 ml min1. Fractions (1 ml) were collected, and active fractions determined with the isotopic assay were pooled.
Enzyme assays.
Acyl-CoA ligase (PimA) activity was measured with isotopic and spectrophotometric assays as previously described (Geissler et al., 1988). The two assays gave comparable results with pimelate, but the isotopic assay was more sensitive, and had less variability, than the spectrophotometric assay. For the isotopic assay, the enzymic conversion of [14C]pimelate to [14C]pimelyl-CoA was measured. Pimelate that was not converted to pimelyl-CoA was extracted from the reaction mixture with ethyl acetate at acidic pH, while pimelyl-CoA remained hydrophilic. The amount of unreacted pimelate in the reaction mixture was detected in controls where enzyme was omitted from the reaction, and it was used in calculations of enzymic activity. The reaction mixture contained 2·5 mM MgCl2, 0·5 mM ATP, 0·25 mM reduced CoA (CoASH) and [14C]pimelic acid (200 µM in standard assay for 555 Bq per reaction) in 20 mM TEA/HCl, pH 8·0. The reaction was initiated by the addition of 20 µg enzyme for a final volume of 0·5 ml. For determination of kinetic constants, the above reaction mixture was used with 0·5 mM CoASH. The amount of labelled substrate remained constant at 20 µM for these assays, while the addition of unlabelled substrate was used to achieve the desired final substrate concentration. This assay was suitable for use at all stages of enzyme purification. [1,7-14C]Pimelate (2·223·33 GBq mmol1) was obtained from American Radiolabelled Chemicals.
The spectrophotometric assay measures the formation of AMP by acyl-CoA ligase by coupling the reaction via a series of auxiliary enzymes to NADH oxidation, as previously described (Geissler et al., 1988). The reaction mixture contained 20 mM TEA/HCl (pH 8·0), 2·5 mM MgCl2, 0·5 mM ATP, 0·25 mM CoASH, 10 mM KCl, 10 mM phosphoenol pyruvate, 0·175 mM NADH, substrate (0·25 mM in standard assays), 2 U of the auxiliary enzymes pyruvate kinase and lactic acid dehydrogenase, and 4 U of myokinase (adenylate kinase), in a total volume of 1 ml. The assays were performed using a Beckman DU800 spectrophotometer with a quartz cuvette of 1 cm path length. For determination of kinetic constants, the above mixture was used with 0·5 mM CoASH. The spectrophotometric assay can only be used with pure enzymes due to the very high background activity of NADH oxidation in crude cell extracts.
Kinetic constant determination.
Enzyme activities were measured at a range of substrate concentrations. At least nine concentrations were tested in duplicate on two separate days, and used to generate a HanesWoolf plot. Maximal enzymic velocity (Vmax) and the Michaelis constant (Km) were determined from the plot. These values were then used to calculate Kcat (enzyme turnover rate), which was determined from Vmax/[E], where [E] is the total enzyme concentration, and Kcat/Km (catalytic efficiency).
Southern blotting.
Approximately 3 µg chromosomal DNA was digested with NotI or EcoRI, and separated on a 0·8 % agarose gel. A probe was generated by PCR that was complementary to 1 kb DNA directly upstream of pimF. The probe was labelled with the Ready-To-Go DNA labelling beads (Amersham Biosciences), according to the manufacturer's procedures. Southern hybridizations were performed by standard procedures.
Other procedures.
SDS-PAGE was carried out with 12·5 % acrylamide gels by standard procedures (Ausubel et al., 1990). Separated proteins were visualized by Coomassie blue R-250. Molecular-mass standards were from Gibco-BRL. Protein concentrations of cell extracts and enzyme preparations were determined with the Bio-Rad protein assay reagent.
-Galactosidase activity was measured as previously described (Egland & Harwood, 1999
).
Sedimentation equilibrium centrifugation.
The native molecular mass of the PimA protein was determined using a Beckman XL-1 analytical ultracentrifuge in absorbance mode. All experiments were performed at 12 °C in buffer containing 20 mM TEA/HCl (pH 8·0), 0·5 M NaCl and 10 % (v/v) glycerol. A six-channel centrepiece was used for acquiring absorbance data at 280 nm for a protein concentration range of 28 µM. Data were collected at rotor speeds of 10 000, 12 000 and 14 000 r.p.m. Equilibrium was achieved when three consecutive scans taken 2 h apart were unchanged. Data editing was performed with the WinREEDIT program (version 0.999.0028), and fitting of the data was performed with the WinNONLIN program (version 1.06.0048); both programs were developed at the the National Analytical Ultracentrifugation Facility, University of Connecticut Biotechnology Center; http://www.ucc.uconn.edu/wwwbiotc/AUFMAIN.HTML).
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RESULTS |
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The pim genes are organized as an operon, and are required for optimal growth on benzoate and dicarboxylic acids
To examine the transcriptional organization of the genes in the pim cluster, we carried out RT-PCR using primers designed to amplify the intergenic regions (Table 1). Products were obtained for all of the intergenic regions in the cluster, but not between the upstream regulatory genes rpa3718 and pimF (Fig. 2
). No product was obtained in controls to which no reverse transcriptase was added. These results indicate that the pimFABCDE genes are co-transcribed, and constitute an operon.
To examine the contribution of the pim operon to growth with dicarboxylic acids and fatty acids, we constructed a mutant (CGA151) in which the pimFABCDE genes were deleted from the chromosome. PCR and Southern hybridization experiments (Fig. 3) each verified that the expected deletion had occurred in CGA151. The pimFABCDE deletion mutant grew more slowly than the wild-type parent when pimelate (C7), azelate (C9) or tetradecanedioate (C14) was supplied as the sole carbon source under anaerobic photoheterotrophic growth conditions (Table 2
). The pim operon deletion strain was also impaired in growth on benzoate, a compound that R. palustris degrades only under anaerobic conditions. The pim operon deletion strain was impaired in aerobic growth on pimelate and azelate. We were unable to obtain consistent growth of wild-type R. palustris with other dicarboxylic and fatty acids under aerobic conditions (Table 2
). The pim operon deletion mutant grew anaerobically at wild-type rates on the fatty acid caprylate (C8). This could mean that the pim operon is not involved in the degradation of this fatty acid. Another interpretation is that R. palustris encodes at least two sets of enzymes that can catalyse caprylate degradation, one of which is encoded by the pim operon.
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DISCUSSION |
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The R. palustris genome has genes predicted to mediate conversion of glutaryl-CoA to acetyl-CoA and CO2 (Larimer et al., 2004) (Fig. 1b
). It has a predicted glutaryl-CoA dehydrogenase/glutaconyl-CoA decarboxylase gene (rpa1094), and also encodes predicted 3-hydroxybutyryl-CoA dehydratase (Rpa4339), 3-hydroxybutyryl-CoA dehydrogenase (Rpa4748) and acetoacetyl-CoA thiolase (Rpa0531) enzymes for the conversion of crotonyl-CoA, the product of glutaconyl-CoA decarboxylation, to two acetyl-CoA (Fig. 1b
).
The Pim enzymes are obviously not the only R. palustris enzymes that can catalyse dicarboxylic acid degradation because the pim operon deletion strain is still able to grow, albeit more slowly than the wild-type, on these compounds. The R. palustris genome encodes 45 CoA ligases, 32 flavin-containing dehydrogenases, 26 enoyl-CoA hydratases, many short-chain alcohol dehydrogenases and more than 10 acyl-CoA acetyltransferases. It is likely that combinations of these other -oxidation enzymes can also contribute to the degradation of dicarboxylic acids. In E. coli, the genes encoding enzymes of aerobic fatty acid
-oxidation are scattered around the chromosome (Clark & Cronan, 1996
).
The R. palustris pimE ORF is disrupted by a 1 frameshift at nucleotide 4192036. This mutation is expected to render the gene nonfunctional. Thus an alternate R. palustris protein must catalyse the dehydrogenation reaction that is part of the -oxidation sequence of reactions. It is possible that PimF has 3-hydroxyacyl-CoA dehydrogenase as well as 3-hydroxyacyl-CoA dehydratase activity. PimF possesses the characteristic amino acid motifs to perform both of these functions. A single gene product, FadB, catalyses these two reactions during fatty acid
-oxidation in E. coli (Yang et al., 1988
). Another possibility is that one of the other 54 short-chain alcohol dehydrogenases encoded elsewhere on the R. palustris chromosome has 3-hydroxyacyl-CoA dehydrogenase activity.
The PimA protein appears to have one of the broadest substrate ranges of any acyl-CoA ligase described, as it is active with both dicarboxylic and fatty acids of medium and long carbon chain length. Acyl-CoA ligases involved in the -oxidation of fatty acids, including FadD from E. coli (Kameda & Nunn, 1981
), have been described that are active with a broad range of fatty acids; however, these enzymes were not reported to have been tested for activity with dicarboxylic acids. Pimelyl-CoA is the first committed intermediate in the synthesis of the vitamin biotin, and several pimelate-CoA ligases have been purified and characterized in the course of efforts to develop a biotechnological process for the production of biotin (Binieda et al., 1999
; Ploux et al., 1992
). PimA shares approximately 30 % amino acid identity with these proteins, but the region of identity is mainly confined to the shared AMP-binding domain common to CoA ligases. The described pimelate-CoA ligases have a narrow substrate range, and are specific to pimelate.
A search of the current databases revealed that the only organism with a set of genes that is very similar to the pim genes is the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum (Kaneko et al., 2002). This set of genes lies at nucleotide positions 85677188573337 in the B. japonicum genome. Applying pim designations to these genes, B. japonicum possesses the following: bll7817 (pimD), bll7818 (pimC), bll7819 (pimB), bll7820 (pimA) and bll7821 (pimF). These genes have between 73 and 89 % deduced amino acid identity with the homologous R. palustris genes. Based on this high degree of relatedness, we predict a role for these genes in aerobic dicarboxylic acid degradation by B. japonicum. B. japonicum does not have an anaerobic pathway for benzoate degradation. The pimE gene is absent from the B. japonicum genome pim gene cluster.
An IclR-like regulatory gene, and genes for an ABC transport system related to the branched chain amino acid (Ilv) uptake system of E. coli, are divergently transcribed from the pim cluster in R. palustris (Fig. 2). R. palustris encodes 20 Ilv-type transporters, and we have speculated that these may be specific for various sorts of hydrophobic compounds, as well as for dicarboxylic acids (Larimer et al., 2004
). We predict that the protein products of genes rpa3719rpa3725 are responsible for transporting substrates for the Pim enzymes, and that the multiple periplasmic binding proteins encoded by this gene cluster enable the transport of a range of dicarboxylic and fatty acids. In addition, we speculate the IclR regulator could control both transport and degradation of these compounds. The B. japonicum genome has an identical gene cluster at position 8575477, directly upstream of its putative pim genes, consisting of an IclR-like regulator, and a transport system with multiple periplasmic binding proteins. The R. palustris and B. japonicum regulatory and transport genes share between 76 and 88 % amino acid identity, while two of the three binding proteins in B. japonicum share approximately 7880 % identity with those in R. palustris.
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ACKNOWLEDGEMENTS |
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Received 27 October 2004;
revised 9 December 2004;
accepted 10 December 2004.
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