A reductive benzoate pathway is the central conduit for the anaerobic biodegradation of aromatic pollutants and lignin monomers. Benzene ring reduction requires a large input of energy and this metabolic capability has, so far, been reported only in bacteria. To determine the molecular basis for this environmentally important process, we cloned and analyzed genes required for the anaerobic degradation of benzoate and related compounds from the phototrophic bacterium, Rhodopseudomonas palustris. A cluster of 24 genes was identified that includes twelve genes likely to be involved in anaerobic benzoate degradation and additional genes that convert the related compounds 4-hydroxybenzoate and cyclohexanecarboxylate to benzoyl-CoA. Genes encoding benzoylCoA reductase, a novel enzyme able to overcome the resonance stability of the aromatic ring, were identified by directed mutagenesis. The gene encoding the ring-cleavage enzyme, 2-ketocyclohexanecarboxyl-CoA hydrolase, was identified by assaying the enzymatic activity of the protein expressed in Escherichia coli. Physiological data and DNA sequence analyses indicate that the benzoate pathway consists of unusual enzymes for ring reduction and cleavage interposed among enzymes homologous to those catalyzing fatty acid degradation. The cloned genes should be useful as probes to identify benzoate degradation genes from other metabolically distinct groups of anaerobic bacteria, such as denitrifying bacteria and sulfate-reducing bacteria.

Pseudomonas putida flagella were examined. Also, changes in motile behavior in response to chemoattractants were analyzed quantitatively by computer. Reversals in the rotation direction of bundles of polar flagella resulted in changes in swimming direction. Cells swimming in buffer changed direction once every 2 s on average, whereas cells exposed to the attractant benzoate changed direction an average of once every 10 s. The findings show that P. putida responds to temporal gradients of chemoattractant by suppressing changes in the direction of rotation of flagella.Aromatic compounds are a major component of plants and also constitute a substantial proportion of the toxic wastes that are released into the environment (3, 5). Many kinds of bacteria can grow on various aromatic compounds, and several groups of motile soil bacteria, including Psei(domonas, Rhizobium, and Agrobacterium spp., can also sense and respond behaviorally to the presence of aromatics in the environment (1, 8, 17, 18 P. piutida PRS2000 (wild type) (16) was cultivated in defined mineral medium as described previously (8).Cells for light-microscopy observations and videomicroscopy were harvested by centrifugation in the early to midlogarithmic phase of growth and suspended in chemotaxis buffer (50 mM potassium phosphate [pH 7.0], 10 ,uM EDTA) to a density of 4 x 106 to 6 x 106 cells per ml. Approximately 80 to 90% of the cells were motile for at least 1 h.Cells for electron microscopy were harvested in the early to mid-logarithmic phase of growth and suspended in distilled water to a concentration of 108/ml. Cell suspensions were dried onto Formvar-coated grids and shadowed with platinum-palladium (80 and 20%, respectively) at an angle of 200 before examination with a Philips 300 transmission electron microscope operating at 80 kV.High-intensity illumination dark-field microscopy observations were made by using a 450-W xenon short-arc lamp and an oil immersion dark-field condenser, as described previously (13

Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-hydroxybenzoate was purified from 4-hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound.

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.