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Vol. 54, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1988, p. 712-717 0099-2240/88/030712-06$02.00/0 Copyright © 1988, American Society for Microbiology
Anaerobic and Aerobic Metabolism of Diverse Aromatic Compounds by the Photosynthetic Bacterium
Rhodopseudomonas palustris CAROLINE S. HARWOOD'* AND JANE GIBSON2
Department of Microbiology, New York State College ofAgriculture and Life Sciences,' and Section ofBiochemistry, Molecular and Cell Biology, Division of Biological Sciences,2 Cornell University, Ithaca, New York 14853-7201
Received 2 September 1987/Accepted 19 December 1987
The purple nonsulfur photosynthetic bacterium Rhodopseudomonas palustris used diverse aromatic com- pounds for growth under anaerobic and aerobic conditions. Many phenolic, dihydroxylated, and methoxylated aromatic acids, as well as aromatic aldehydes and hydroaromatic acids, supported growth of strain CGA001 in both the presence and absence of oxygen. Some compounds were metabolized under only aerobic or under only anaerobic conditions. Two other strains, CGCO23 and CGDO52, had similar anaerobic substrate utilization patterns, but CGDO52 was able to use a slightly larger number of compounds for growth. These results show that R. palustris is far more versatile in terms of aromatic degradation than had been previously demonstrated. A mutant (CGA033) blocked in aerobic aromatic metabolism remained wild type with respect to anaerobic degradative abilities, indicating that separate metabolic pathways mediate aerobic and anaerobic breakdown of diverse aromatics. Another mutant (CGA047) was unable to grow anaerobically on either benzoate or 4-hydroxybenzoate, and these compounds accumulated in growth media when cells were grown on more complex aromatic compounds. This indicates that R. palustris has two major anaerobic routes for aromatic ring fission, one that passes through benzoate and one that passes through 4-hydroxybenzoate.
Aerobic pathways of aromatic metabolism have been studied extensively in bacteria and show an almost universal requirement for molecular oxygen (7, 11). The biochemical strategies involved in anaerobic aromatic degradation, by contrast, are fundamentally different and are still incom- pletely understood. The earliest and most thorough studies on this topic were carried out with the photosynthetic bacterium Rhodopseudomonas palustris. Nearly 20 years have elapsed since Dutton and Evans (9) proposed a novel ring reduction mechanism leading to anaerobic cleavage of the aromatic ring of benzoate. These studies have been substantiated and elaborated on (14, 15, 17, 30), and work with additional anaerobes, including fermentative anaerobes and nitrate reducers, indicates that the reductive ring cleav- age pathway for benzoate degradation is widespread among microorganisms (2, 18, 24, 31). Recent concern about the environmental fate of industri-
ally produced organic compounds has prompted a resur- gence of interest in the anaerobic degradation of aromatic compounds. Numerous studies have shown that substituted benzoates, including chlorinated, nitro-, and aminoaroma- tics and also aromatic hydrocarbons and phenolic com- pounds, can be broken down under anaerobic conditions by bacteria (3, 5, 6, 13, 19, 25, 32, 33). However, in most cases the metabolic fates of these structurally diverse aromatic compounds have not been delineated. One might ask, for example, whether diverse aromatic compounds are metabo- lized via totally independent catabolic pathways or whether groups of related compounds are degraded to form common intermediates which then undergo ring fission reactions. To begin to address this question, we surveyed several R.
palustris strains for the ability to grow anaerobically on a range of aromatic compounds and we tested a mutant that is blocked in anaerobic benzoate and 4-hydroxybenzoate utili-
* Corresponding author.
zation to see whether it could degrade aromatic compounds which are more complex structurally. The aerobic growth capabilities of R. palustris were also examined. The results reported in this paper show that R. palustris is far more nutritionally versatile with respect to aromatic utilization than had been previously reported. Furthermore, various complex aromatic compounds are metabolized to form either benzoate or 4-hydroxybenzoate before anaerobic cleavage of the aromatic ring. This indicates that the pathways for benzoate and 4-hydroxybenzoate metabolism play a general role as central anaerobic ring fission pathways in R. palus- tris.
MATERIALS AND METHODS
Bacterial strains. Strain CGCO23 is the type strain of R. palustris (ATCC 11168). Strain CGA001 was originally iso- lated by S. Taniguchi and has been maintained in the culture collection of R. K. Clayton at Cornell University, Ithaca, N.Y. Strains CGA033 and CGA047 are derivatives of CGA001 that were obtained after NTG (N-methyl-N'-nitro- N-nitrosoguanidine) mutagenesis. CGA033 is unable to grow aerobically with 4-hydroxybenzoate as the sole carbon source but is wild type with respect to anaerobic growth. Strain CGA047 is unable to utilize either benzoate or 4- hydroxybenzoate under anaerobic conditions but is unim- paired in aerobic growth capabilities. Strain CGDO52 was recently isolated by us from liquid enrichment cultures (4) that had been supplemented with benzoate, inoculated with sewage sludge, and incubated anaerobically in light. Media and growth conditions. Unless otherwise noted, all
strains were grown at 30°C in defined basal medium (22) supplemented with 0.15 mM 4-aminobenzoic acid and 0.1 mM sodium thiosulfate (PM medium). Carbon sources were added to autoclaved medium at the time of inoculation from sterile solutions that had been adjusted to pH 6.8 to 7.0. Anaerobic medium was prepared by bubbling nitrogen gas
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METABOLISM OF AROMATIC COMPOUNDS BY R. PALUSTRIS
through PM liquid medium for 1 h, transferring the medium to an anaerobic glove box (Coy Laboratory Products, Ann Arbor, Mich.), and then dispensing 10-ml aliquots of the medium into culture tubes (Bellco Glass, Inc., Vineland, N.J.) in the glove box. The tubes, containing a nitrogen atmosphere, were sealed with butyl rubber stoppers and autoclaved. Sodium bicarbonate (final concentration, 10 mM) was added to anaerobic growth medium as a sterile solution after autoclaving. Anaerobic cultures were illumi- nated with 40-W incandescent lamps at a distance of 20 to 40 cm. Cells were grown aerobically in liquid medium provided with constant aeration by a gyratory environmental shaker (New Brunswick Scientific Co., Inc., Edison, N.J.). Auxanography was used to screen strains for the ability to
utilize aromatic and hydroaromatic compounds as growth substrates (23). Cells grown aerobically at 37°C in liquid PM medium supplemented with 10 mM succinate were harvested by centrifugation, washed once, and suspended in basal medium to a final density of approximately 5 x 108 cells per ml. Suspended cells were diluted 20-fold into PM medium containing 0.5% (wt/vol) melted Gelrite (Kelco, San Diego, Calif.), and the medium was then immediately poured into petri dishes. Each carbon compound to be tested as a growth substrate was applied to the surface of the solidified medium near the periphery of the petri dish. One compound was tested per plate, and each compound was applied as a spatula-pointful of solid chemical. Auxanographic plates were incubated aerobically in the dark or anaerobically in light in polycarbonate jars to which GasPak hydrogen plus carbon dioxide generator envelopes (BBL Microbiology Systems, Cockeysville, Md.) had been added. Growth in liquid cultures was monitored by measuring the
increase in A660 in a Spectronic 21 (Bausch & Lomb, Inc., Rochester, N.Y.).
Substrate conversion. The absorption spectra of samples of culture fluid that had been centrifuged and diluted 10-fold into 50 mM P04 buffer (pH 7.0) were determined by scanning between 200 and 400 nm with a Beckman DU-7 spectropho- tometer (Beckman Instruments, Palo Alto, Calif.). The me- tabolism of various aromatic substrates during anaerobic growth was also followed by reversed-phase high-perfor- mance liquid chromatography. A Beckman binary gradient model 344 system with a 254-nm fixed-wavelength UV detector was used. The supernatant liquid of centrifuged samples (20 ixl) was injected into a Ultrasphere C-18 column (Beckman Instruments). The mobile phase consisted of acetonitrile and 0.01 N perchloric acid in a linear gradient of 0.6 to 60% acetonitrile. The flow rate was 1.0 ml/min, and the run time was 20 min.
Growth patterns of strain CGA001 in auxanographic plates. To explore the full potential of R. palustris CGA001 for utilizing diverse aromatic compounds, we exposed cells to concentration gradients of potential substrates by the tech- nique of auxanography. Diffusion of a given carbon com- pound from its site of addition effectively exposes cells seeded in plates of agar medium to a range of substrate concentrations, and growth is reflected as the formation of an arc of turbidity (Fig. 1). This approach allowed rapid screening of potential growth substrates, and it also revealed the ability of cells to grow with compounds that are toxic at high concentrations. Cinnamaldehyde, for example, stimu- lated the growth of a narrow arc of cells some distance from its origin of application to the petri dish (Fig. la). Growth
FIG. 1. Growth patterns of R. palustris on auxanographic plates. (a) Growth with cinnamaldehyde, a toxic substrate. (b) Arc of turbid growth observed with the substrate, 4-hydroxycinnamate. (c) Pat- tern of growth with the relativ