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Anaerobic Growth of Rhodopseudomonas Palustris on 4 JOURNAL OF BACrERIOLOGY, Sept. 1992, p. 5803-5813 Vol. 174, No. 18 0021-9193/92/185803-11$02.00/0 Copyright ©) 1992, American Society for Microbiology Anaerobic Growth of Rhodopseudomonas palustris on 4-Hydroxybenzoate Is Dependent on AadR, a Member of the Cyclic AMP Receptor Protein Family of Transcriptional Regulators MARILYN DISPENSA,1 CONSTANCE T. THOMAS,2 MIN-KYUNG KIM,1 JOSEPH A. PERROTTA,1 JANE GIBSON,2 AND CAROLINE S. HARWOOD13* Department ofMicrobiologyl* and Center for Biocatalysis and Bioprocessing,3 University of Iowa, Iowa City, Iowa 52242, and Section ofBiochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 148532 Received 17 April 1992/Accepted 13 July 1992 The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris converts structurally diverse aromatic carboxylic acids, including lignin monomers, to benzoate and 4-hydroxybenzoate under anaerobic conditions. These compounds are then further degraded via aromatic ring-fission pathways. A gene termed aadR, for anaerobic aromatic degradation regulator, was identified by complementation of mutants unable to grow anaerobically on 4-hydroxybenzoate. The deduced amino acid sequence of the aadR product is similar to a family of transcriptional regulators which includes Escherichia coli Fnr and Crp, Pseudomonas aeruginosa Anr, and rhizobial FixK and FixK-like proteins. A mutant with a deletion in aadR failed to grow on 4-hydroxybenzoate under anaerobic conditions and grew very slowly on benzoate. It also did not express aromatic acid-coenzyme A ligase II, an enzyme that catalyzes the first step of 4-hydroxybenzoate degradation, and it was defective in 4-hydroxybenzoate-induced expression of benzoate-coenzyme A ligase. The aadR deletion mutant was unaffected in other aspects of anaerobic growth. It grew normally on nonaromatic carbon sources and also under nitrogen-fixing conditions. In addition, aerobic growth on 4-hydroxybenzoate was indistinguishable from that of the wild type. These results indicate that AadR functions as a transcriptional activator of anaerobic aromatic acid degradation. In recent years, the major biochemical strategies used by CoA ligase that activates 4-hydroxybenzoate has been puri- bacteria to accomplish the anaerobic mineralization of ben- fied from cells grown on this compound (11). This enzyme, zene rings have started to emerge (10, 14). Work with several termed aromatic acid-coenzyme A ligase II (aroIl-CoA li- metabolic types, including fermentative bacteria, bacteria gase), is antigenically distinct from benzoate-CoA ligase. that grow by anaerobic respiration, and phototrophic bacte- The identification of genes required for anaerobic 4-hy- ria, suggests that degradation proceeds in two phases (14). droxybenzoate or benzoate degradation should help define First, aromatic ring substituents are removed or modified to individual enzymatic steps in the pathways and would also yield benzoate, 4-hydroxybenzoate, or their coenzyme A be helpful in determining how the pathways are regulated. In (CoA) derivatives. In the second phase, these compounds this report, we describe the generation and characterization enter central catabolic pathways of ring fission. The first ofR. palustrs mutants defective in growth on these aromatic detailed description of anaerobic benzene ring fission arose acids. We identified a fragment of R. palustris DNA that from studies of benzoate metabolism by the phototrophic complements these mutants and characterized a gene, aadR, bacterium Rhodopseudomonas palustris (9), and the main which is required for wild-type growth on benzoate and features proposed have since been validated by studies with 4-hydroxybenzoate. The deduced amino acid sequence of denitrifying bacteria (1, 7, 31), as well as with R. palustris. the aadR product suggests that it belongs to the Crp family Anaerobic benzoate degradation, as presently understood of transcriptional regulators. in R palustris, is shown in Fig. 1. After an initial activation of benzoate to benzoyl-CoA, the general stability of the MATERIALS AND METHODS benzene ring is overcome by reductive steps leading to the dearomatization of the ring (15). Further modifications by a Bacterial strains and plasmids. The bacterial strains and p-oxidation-like sequence of reactions leads, finally, to ring plasmids used are described in Table 1. All recombinant fission (9, 25). Although most of the enzymes of the benzoate plasmids were maintained in Escherichia coli DH5a. pathway have been studied in only a very limited way, the Media and growth conditions. Except where noted, E. coli initial step in benzoate degradation, its thioesterification to and R palustris were grown at 37 and 30°C, respectively. R. benzoyl-CoA, has been examined in some detail (20), and a palustris was grown in a defined mineral medium (PM) (30). benzoate-CoA ligase that catalyzes this reaction has been Carbon sources (3 mM final concentrations, except succi- purified and characterized (13). nate, malate, and acetate at 10 mM) were added at the time 4-Hydroxybenzoate degradation by R palustris also ap- of inoculation from sterile solutions that had been adjusted pears to be initiated by CoA thioester formation (36), and a to pH 7.0. When cells were grown under dinitrogen-fixing conditions, Na2SO4 (0.1%) was substituted for (NH4)2SO4 in PM. R palustris cells were also grown in a complete medium * Corresponding author. (CA), consisting of PM supplemented with 0.2% yeast ex- 5803 5804 DISPENSA ET AL. J. BACTERIOL. O OH 04* SCoA Growth in liquid media was monitored by observing the CoA increase in A660, using a Spectronic 21 spectrophotometer (Bausch & Lomb, Rochester, N.Y.). Antibiotics were used at the following concentrations (in Benzoic acid Benzoyl-CoA micrograms per milliliter): for R. palustris, kanamycin, 100; tetracycline, 100; for E. coli, kanamycin, 100; tetracycline, 25; and ampicillin, 100. DNA methods. Chromosomal DNA was purified as de- 0 SCoA 0 SCoA scribed previously (17) from R. palustris cells that had been o#C OSCoA grown aerobically in 50 ml of CA broth for 3 to 4 days, 4- harvested by centrifugation, and resuspended in 2.8 ml of TES buffer (50 mM Tris, 100 mM Na2EDTA, 20% sucrose, A-3-Cydohexene- 1, 4-Cyclohexadiene- 2, 5-Cydohexadiene- pH 8.0). carboxyl -CoA 1-carboxyl-CoA 1-carboxyl-CoA Plasmid DNA for cloning and sequencing was purified by the method of Lee and Rasheed (32). E. coli was transformed 2114 with plasmid DNA by CaCl2 shock treatment (19). T4 DNA ligase and restriction enzymes were purchased from New 04C ,SCoA England Biolabs, Inc. (Beverly, Mass.) and used as directed by the manufacturer. DNA was dephosphorylated as de- A-1-Cyclohexene- scribed by Ausubel et al. (4) with calf intestinal phosphatase carboxyl-CoA from Boehringer Mannheim Biochemicals (Indianapolis, H20 4 Ind.). Standard methods were used for DNA ligations, agarose gel electrophoresis, and colony hybridizations (34). 04 SCoA O,,C Southern hybridizations were done as described by Ausubel 0OH et al. (4). DNA fragments were purified from agarose gel 2-Hydroxycyclohexane- slices by using the GeneClean kit from Bio 101 (La Jolla, carboxyl-CoA Calif.). Radioactive probes were produced by random primer labeling of purified DNA fragments with the Mul- 2H tiprime random priming kit and [32P]dCTP from Amersham 0 *.SCoA Corp. (Arlington Heights, Ill.). Clone bank construction and storage. CGA009 genomic DNA was partially digested with EcoRI and then size 2-Ketocyclohexane- fractionated in a linear 10 to 40% sucrose gradient (prepared carboxyl-CoA in 1 M NaCl-20 mM Tris [pH 8.0]) by centrifugation for 20 h at 23,000 rpm in a Beckman SW40 rotor at 22°C. Fractions containing DNA fragments in the size range of 20 to 35 kb were pooled for use in clone bank construction. Size- C o SCoA fractionated R palustns DNA was ligated with EcoRI- cbo Pimelyl di-CoA digested, dephosphorylated pLAFR1. The ligated DNA was then packaged into X bacteriophage with a DNA-packaging kit obtained from Boehringer Mannheim Biochemicals. The phage particles were transduced into E. coli S17-1, and colonies were grown on L agar containing tetracycline. The Acetyl-CoA constructed clone bank (consisting of 870 tetracycline-resis- FIG. 1. Proposed pathway of anaerobic benzoate degradation by tant colonies) was stored in microtiter plates. Microtiter R. wells filled with Luria broth were inoculated with single palustris. colonies, and after an overnight incubation and the addition of 20% glycerol (final concentration) to each well, plates were stored at -20°C. tract, 0.5% Casamino Acids, and 10 mM succinate. Luria Matings. R palustns cells to be used as recipients were broth (37) was the complete medium used for E. coli. Media grown aerobically in CA medium for 1 to 2 days. Cultures (3 were solidified with 15 g of agar (Difco Laboratories, De- ml) were harvested and resuspended in 100 to 200 ,ul of CA. troit, Mich.) per liter. An overnight Luria broth culture of E. coli S17-1 donor Cells were usually grown anaerobically in screw-cap tubes harboring an appropriate conjugative plasmid was diluted to or bottles that were completely filled with liquid medium a slightly turbid suspension in PM broth. Matings were (20). Anaerobic growth under nitrogen-fixing conditions was performed by mixing drops of donor and recipient cultures tested in partially filled stoppered tubes containing either a on CA plates, incubating at 37°C overnight, and streaking on nitrogen or an argon atmosphere (21). Solid media were the appropriate selective agar
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