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O,O-Dimethyl phosphorodithioate (DMDTP) is an initial breakdown product of organophosphorus pesticides in fields. DMDTP is also released to natural environments by pesticide manufacturers. DMDTP-degrading microorganisms were not known. We isolated two bacteria from activated sludge. One of them, strain TK-l identified as Thiobacillus thioparus, utilized DMDTP as a sole energy source and produced dimethyl phosphate (DMP) and sulfate. The other, strain AK-2 identified as Pseudomonas sp., utilized DMP as a sole energy and carbon source and degraded DMP to inorganic orthophosphate (PJ DMDTP was degraded to Pi by the coaction of the two bacteria.O,O-Dimethyl phosphorodithioate (DM-DTP) is an initial breakdown product of such organophosphorus pesticides as malathion, dimethoate, cidial, formothion, and others in fields. Many investigators have dealt with the degradation of organophosphorus pesticides and have found all pesticides susceptible to hydrolysis. Organophosphorus pesticides are regarded as nonpersistent. However, ionic dialkyl phosphates and sulfur analogs thereof which are the initial products of pesticide breakdown have been reported to be unsusceptible to hydrolysis.!) Some of the breakdown products are toxi<;; to fish.2) Organophosphorus pesticides cannot be regarded as nonpersistent if the toxic breakdown products remain unaltered. The only reported instances of utilization of ionic dialkyl phosphates or sulfur analogs thereof by microorganisms have occurred when the compounds were the sole phosphorus sources. 3 ,4) These instances do not prove that the compounds will be degraded in natural environments, especially in the presence of available orthophosphate.These ionic compounds are also released to natural environments by pesticide manufacturers because these compounds are precursors in the production of pesticides and are abundant in wastewater.In a previous paper,S) we showed that DMDTP was degraded to orthophosphate by activated sludge. The present study was designed to isolate DMDTP-degrading bacteria from the activated sludge. We isolated two bacteria, Thiobacillus thioparus and Pseudomonas sp. By a symbiont of these two bacteria, DMDTP was utilized as a sole energy and carbon source and was degraded to inorganic orthophosphate. MATERIALS AND METHODSChemicals. The organophosphorus compounds used and abbreviations are listed in Table 1. DMDTP ammonium salt was obtained from Aldrich Chemical Co., Milwaukee, U.S.A. DMTP sodium salt was kindly supplied by Sumitomo Chemical Co., Osaka, Japan. The mixture of DMP and MP was obtained from ICN K&K Laboratories, N.Y., U.S.A. All other chemicals were obtained from commercial sources.DMDTP used in degradation tests was purified by preparative layer chromatography as follows. One milliliter of 50% DMDTP was streaked across the width of a chromatoplate of a 2-mm layer of silica gel 60 F 254 (Merck, Art. 5717). After development in a solvent system

Protocatechuate 3,4-dioxygenase was isolated from a gram-positive bacterium, Nocardia erythropolis, the enzyme participates in the phthalate ester metabolism in the bacterium. Cultural conditions for production of the enzyme, the purification procedure, and some properties of the enzyme were studied. A bouillon (beef) mediumwas the most effective amongthose tested for cell growth and enzyme formation. The effect was due to the ring closure type of creatine compounds. Protocatechuate 3,4-dioxygenase was purified from the cell-free extract ca. 1,400-fold and it gave a single band on polyacrylamide gel electrophoresis. The molecular weight was estimated to be ca. 150,000. The optimal pH and temperature were ptt 8.0 and 40°C, respectively. The enzyme was stable in a pH range from 7.6 to 8.6 and below 42°C. The enzyme was inhibited by several metals such as Pb2+, Cd2+and Hg2+. The enzymewas active on a wide range of o-dihydroxyphenyl compounds, in contrast to the high specificity of similar enzymes from gram-negative bacteria (Pseudomonas). The enzyme had a broad absorption band in the visible region with a peak around 450nm, suggesting the presence of non-heme ion(s) bound to the enzyme as a co factor. The spectrum changed markedly upon addition of the substrate, possibly showing the formation of an enzyme-substrate complex. There have been many reports on the oxygenases for aromatic compounds. Protocatechuate 3,4-dioxygenase (EC 1.13.1.3) was first studied by Stanier and Ingraham in 1954,1} and since then a number of investigators have described various methods for purifying the enzyme from many kinds of microorganisms. Among microorganisms, much information is available on the dioxygenase for protocatechuic acid of the genus Pseudomonas, for example, as reported by