REWESWE BJECHLQEEMTSGN {I}? grew? RY fiCIFEMQ'HM (20%.? ANS PSEUDGMQNAS AEMEQEWM 11mm few {Em Dumas :2? pk. D. MKCHiGfiN S?A?E EVNEETERSET? Allen 1"... French 29-68 lW/WWWIWWIW 3129 7872 ‘L This is to certify that the thesis entitled REDUCTIVE DECHLORINATION OF p.p'-DDT BY ESCHERICHIA COLI AND PSEUDOMONAS AERUGINOSA presented by Allen L. French has been accepted towards fulfillment of the requirements for Ph . D 1 degree in My Majo pro Date November 15, 1968 0-169 fl 4 If” {7” I' mmm LIB RA R Y Miclw ‘~ *3 $6 Unlvcrsizy #. 5575* 1m R "1‘86 ABSTRACT REDUCTIVE DECHLORINATION OF p.p'-DDT BY ESCHERICHIA COLI AND PSEUDOMONAS AERUGINOSA By i Allen LE French The metabolism and uptake of Ola-labeled p,p'-DDT (1,1. l-trichloro-Z.2-bis(p-chlorophenyl)ethane) by intact cells of the bacteria Escherichia coli and Pseudomonas aeggginosa were investigated. The phenyl rings of the p.p'-DDT molecules were uniformly labeled with 614 atoms. The cultures were in- cubated for 1. 2, or 3 days in Anderson's minimal synthetic broth medium (Schoenhard, 1961) in the presence or absence of atmospheric oxygen. Autoclaved cultures served as controls. In addition. washed membrane fractions were obtained from g. 22;; by lysozyme treatment followed by osomotic shock. The capacity of cellular components to metabolize P.p'-DDT was investigated. The effect of exogenous Krebs co- factors and intermediates on the metabolism of p.p'-DDT by Particulate components of the bacterial cell was evaluated. The p.p'-DDT and its metabolites were identified by thin-layer and gas-liquid chromatography. Metabolites con- tfiining c1“ were determined by autoradiography of thin-layer . chromatosrams and compared to authentic samples of p.p'-DDT Allen L. French and its metabolites. Quantification was accomplished by gas-liquid chromatography and liquid scintillation counting. Clu-labeled compounds were collected from the column effluent and their radioactivity determined by liquid scintillation counting. Each assay was performed 3 times, and each incu- bant was replicated 2 times. Aerobic and anaerobic cultures of g. ggli and 2. 3933- ginosa degraded p.p'-DDT to p,p'-DDD ( 1.1-dichloro-2.2-bis- (p-chlorophenyl)ethane). The capacity to degrade p.p'-DDT increased with the exclusion of atmospheric oxygen from the incubation medium. Over 90% of the p,p'-DDT was degraded to p,p'-DDD by anaerobic cultures of E. 92;; incubated 3 days. Less than 10% conversion occurred in autoclaved cell cultures incubated anaerobically. The pattern of p.p'-DDT metabolism Iby g. aergginosa was similar to that found in the g. 92;; in- cubations. However, anaerobic cultures of 2. aeggginosa were able to metabolize p,p'-DDT to p,p'-DDD more rapidly. Over 85% of the p,p'-DDT was reductively dechlorinated to p.p'-DDD by anaerobic cultures incubated 2 days. Uptake of p,p'DDT was not increased by its metabolism. After 3 days of incubation, 71% of the radioactivity was asso- ciated with the cells of g. 32;; cultured anaerobically. and 80.1% was associated with the cells of aerobic cultures. Autoclaved cells were able to take up 47.2% of the radio- activity. After 4 hr of anaerobic incubation. neither the particu- late membranes (20,000 g precipatate) nor the non-sedimented Allen L. French components ("shockate" supernatant fraction) of g. 22;; cells. produced substantial amounts of p,p'-DDD. When the above fractions were combined. conversion of p.p'-DDT to p,p'-DDD occurred (29.8%). However, addition of "shockate' supernatant to boiled membrane fractions did not stimulate p.p'-DDD production. When 3 ml of washed membrane fractions (25 mg/ml orig- inal wet weight of cells) were combined with a mixture of Krebs cycle cofactors and intermediates consisting of 2.0 umole each of NAD, NADP. FAD, malate, pyruvate and 0.1 umole each of ADP and inorganic phOSphate and incubated anaerobically for 4 hr, 2.2% conversion of p,p'-DDT to p.p'-DDD occurred. When NAD. NADP, or malate and pyruvate were omitted from the incubations. the conversion was increased by a factor of 10. Addition of FAD (2.0 umole) to washed membrane fractions resulted in the conversion of 22.5% of the p.p'-DDT to p,p'- DDD. However, addition of exogenous FAD to aerobically in- cubated membrane fractions did not stimulate p,p'DDD pro- duction. Based on the results of the membrane studies. the follow— ing possibilities are suggested. Reductive dechlorination of P.p'-DDT occurs in the membranous portion of the bacterial cell and is not cytoplasmic in origin. It is stimulated by c°m-Ponent(s) in the cytoplasm. Reductive dechlorination of . PoP'-DDT does not utilize electrons produced by the oxida- tion of Krebs cycle intermediates and passed through the °Ftochrome system. Reductive dechlorination of p.p'-DDT c 1 u ’ q t I ’ ‘ 1 Q I l v I . t I ‘ ( I ' Q ' t I I e t " I ‘ t l ( ' I a I I I ’ i - v I v “ i I “ v n I l n I l ‘ I I .. ‘ ‘ , «.l Allen L. French is dependent upon the enzymatic reduction of FAD and occurs only under anaerobic conditions. Reductive dechlorination of p,p'-DDT requires electrons produced by the oxidation of an energy source. Reductive dechlorination of p,p'-DDT may require the formation of free radicals. The oxidation of endogenous substrates can produce the half-reduced form of FAD (EADH-. a semiquinone) and may be the active moiety in- volved in the enzymatic reduction of p,p'-DDT. REDUCTIVE DECHLORINATION OF P,P'-DDT BY ESCHERICHIA COLI AND PSEUDOMONAS AERUGINOSA By Allen L.’ ”French A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1968 this : Leelil 1,, {L/ V; / y: -: ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Dr. R. A. Hoopingarner for his assistance and counsel with this study. Appreciation is also expressed to Dr. N. C. Leeling for advice during the course of this study. A special thank you to my wife. Patricia. 11 LIST LIST INTRO LITEH HATER RESUL 3an DIscu LITE}; ”Pm; TABLE OF CONTENTS LIST OF TABLES eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee LIST OF FIGURES eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee INTRODUCTION eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee LITERATURE REVIEW eeeeeeeeeeeeeeeeeeeee-eeeeeeeeeeeee- Microorganisms ISOlated From Animals eeeee-eeeeee Microorganisms ISOlated From 8011 eeeeeeeeeeeeeee Laboratory ISOlateS eeeeeeeeeeeeeeeseeeeeeeeeee-e Degradative Mechanisms eeeeeeeeeeeeeeeeeeeeeeeeee Nonenzymatic Degradation ........................ Animal Degradation eeeeeeeeeeeeeeeeeeeeeeeeeeeeee Biochemical Inhibitions eeeeeeeeeeeeeeeeeeeeeeeee Metabolic Capacity of Isolated Membranes ........ MATERIALS AND METHODS eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Intact Cell Studies eeseeoeeeeeee-eeeeeeeeeeeeeee ExtraCtion & ”Clean-up" eeeeIeeeeeeeeeeeeeeeeeeee Analytical MethOdS eeeeeeeeeeeeeeeeeeeeeeeeeeeeeo C311 Free Studies eeeeeeeeeeeeeeseeeeeeeeeeese-ee RESULTS OF INTACT CELL STUDIES eeee-eeeeeeeeeeeeeeeeee RESULTS OF MEMBRANE STUDIES eeeeeeeeeeeeeeeeeoeeeeeeee DISCUSSION eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeeeeee SUMMABX eeeeeeeeeeeosoeeeeeeeeeeeeeeeeeeeeeeee-eeeeeee LITEMTURE CITE .OOICUOIIIOOOI.OOOIOOOOOI.0.0.0.0.... APPENDIX 0.0.0.000...I.0O...OOIOOOOIOIIOIOODIIOOOOIOIO iii Page iv ou)m\JOWn#nb \» H 4 34 39 LIST OF TABLES Table Page 1 DDT metabolism by intact cells of E. coli incubated aerobically IIOIOIOIIIIOIIIOIIOOOOOOO. 19 2 DDT metabolism by intact cells of E. coli incubated anaerobically IIIOIOIOOOOIIIOIIOOIOIII 19 3 Distribution of radioactivity in cultures Of§00011OI.O....0000...OOOOIIICIOIQOCOOOOOOC. 20 h DDT metabolism by intact cells of 2. aeru- ginosa incubated aerobically ................... 21 5 DDT metabolism by intact cells of E. aeru- ginosa incubated anaerobically ................. 21 6 Effect of membrane and cytoplasm of E. coli on conversion of DDT to DDD .................... 22 7 Effect of exogenous Krebs cycle intermediates and cofactors on DDT metabolism by membrane preparations of E. coli ........................ 24 8 Effect of NAD, FAD. ADP, and inorganic phosphate on DDT metabolism by membrane preparations of E. coli ........................ 25 iv Figure LIST OF FIGURES Preparation of membranes from protoplasts ..... Representative thin-layer chromatogram ........ Autoradiograms of thin-layer chromato- grams of carbon-lh-labeled DDT and carbon-lu-labeled metabolites produced byg. c011 .0O...OO...'0'...IIIOOOOIIOOIOOCODII 3A Anaerobic cultures incubated lmdzdays IOOOIOOOOIIOOOIO...IOOOOOIOIOI SB Anaerobic cultures incubated 2 days, 3 days. and autoclaved cells incubated 30 Anaerobic and aerobic cultures incu- bated 3 days and autoclaved cells incubated 3 days coco-cocoooooovooooooooooo 3D Aerobic cultures incubated 1 and days COIOIOQOOOO.IIOICOIIIOIOCICIIOOIDIIO 3E Aerobic culture incubated 2 and days ClCCC..0...OOIIIOIIOOOIOOIOIUIII.IOI Autoradiograms of thin-layer chromato- grams of carbon-14-labe1ed DDT and carbon- lh-labeled metabolites produced by 2. aeggmosa .0...ICOUIOOIIDIOOOIOOOIOICOOO... 4A rAnaerobic and aerobic cultures incu— bated 2 days and autoclaved cells 1n°ubated2days..O........U..'.I..l....... 4B Anaerobic and aerobic cultures incu- bated 2 days and autoclaved cells 1n°ubated 2 days I.OOOIOOOIIOOIOIOOOIOIOCOO Page 1? 39 40 #1 #2 43 45 #6 47 #8 n u o o o o o a o I o I I o c - Autoradiogram of a thin-layer chromato- gram of carbon-lh-labeled DDT and metab- olites produced by combining cytoplasmic fractions and membrane preparations of .E... 0011 ODOOOOOIIOIOOOIOOOOOOOOOOOOIIIIIIOOIOIO Autoradiograms of thin-layer chromato- grams of carbon-lu-labeled DDT and me- tabolites produced by the addition of Krebs cycle intermediates or cofactors to membrane preparations of E. coli ........... 6A Addition of intermediates and co- factors and the omission of ADP, inorganic phosphate or NAD ................ 6B Addition of intermediates and co- factors, membrane only and omission or intermediates coo-coco...ooooooooooocooo 60 Omission of FAD, NADP and intermediates ... 6D Addition of FAD, ADP and inorganic phosphate OCOIOCOOIOIOIIOOOOIOOO00.0.0.0... 6E Addition of FAD or NAD or FAD, ADP, P04&atmospher1c 02 OOIIOOOOIOOOOQIOIIOIOO Autoradiogram of a thin-layer chromato- gram of carbon-lh-labeled DDT and metab- olites produced by the addition of FAD, ADP and inorganic phosphate to membrane preparations suspended in cytoplasmic fractions IOIOOOOOIOOOIOIOOIOOOIOOOIOOOIIOOIOII vi Page 49 50 51 52 53 55 56 --------------------------------------- ---------------- .......................... IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII uuuuuuuuuuuuuuuuuuuuuu ------------------------------------- INTRODUCTION Kallman and Andrews (1963) were the first to demon- strate that an isolated microorganism could degrade p,p'- DDT (1,1,l-trichloro-Z,2-bis(p-chlorophenyl)ethane) to p,p'- DDD (l,l-dichloro-2,2-bis(p-chloropheny1)ethane). Following this report, interest grew in the role of microorganisms in degradation of "persistent" pesticides. Since p,p'-DDT is extremely stable and has been extensively employed through- out the environment, several investigators have studied its metabolism. Investigators have employed organisms that were obtained from soils, animal feces, intestines, and laboratory strains. Their ability to degrade p,p'-DDT was measured after various incubation intervals in a variety of broth cultures, agar- based suspensions and soils in the presence and absence of oxygen. The results of these investigations are presented in the literature review. Although previous investigations established a wide microbiological spectrum of p,p'-DDT degradative capacity, little is known about the biological mechanism involved in bacterial uptake and degradation of p,p'-DDT. The site of metabolism, cytoplasmic or particulate, is still open to question. The present study was undertaken to answer some 1 2 of these questions and provide information which may prove useful in future investigations. stra cult YCI‘S LITERATURE REVIEW Kallman and Andrews (1963) were the first to demon- strate that microbes could metabolize DDT to DDD when they cultured commercial yeast anaerobically. Eighty-eight % con- version occurred after 3 days compared to only 3% conversion in the boiled controls. They reported that DDE (l,l-dichloro- 2,2-bis(p-chlorophenyl)ethylene) was not metabolized by the yeast. Microorganisms Isolated From Animals Several investigators have demonstrated the metabolism of DDT to DDD by microorganisms isolated from animals. Miskus .33 3;. (1965) reported partial conversion of DDT to DDD in bo- vine rumen fluid. Stenersen (1965) determined the ability of Serratia mgrcescgns and Alcaligenes faecalis, isolated from feces of resistant stableflies, Stomogys calcitrans (L), and laboratory cultures of Escherichia coli, Bacillus brevis and .Aerobacter aerogenes to metabolize Clu-labeled DDT. Ninety % of the DDT had been metabolized to DDD after 3 days of anaer- obic incubation in meat extract bouillon while no conversion was reported in aerobic cultures. Proteus vulgaris, isolated from the intestinal tract of a mouse, was cultured in heart infusion medium with DDT by Baker and Morrison (1965). They found 65% conversion of DDT to DDD in 6 days. Between 6 3 [13: tron 1in iso] bias Prod tri: 4 and 20 days a steady decline in recoverable DDD was noted, suggesting that other metabolites were formed. Mendel and Walton (1966) cultured g. ggli and A. aerogenes, isolated from rat feces, in trypticase soy broth for 2 days with DDT. E. 32;; degraded 35.9% of the DDT to DDD, and A. aerogenes degraded 33% to DDD. Microorganisms Isolated From Soil Chacko 23 El- (1966) tested 9 actinomycetes and 8 fungi from soil for their ability to degrade DDT in a nutrient me- dium. None of the fungi displayed any appreciable capacity to degrade DDT while 6 actinomycetes did produce DDD. A max- imum of 25% was degraded by Streptomyces aureofaciens in 6 days. ‘ Matsumura and Bousch (1968), employing an unspecified liquid medium containing Clu-labeled DDT, incubated 18 soil isolated variants of the fungus Trichoderma viride anaero- bically for 3 days. Of the 18 variants tested, 8 cultures produced both DDD and dicofol (l,1—bis(p-chlorophenyl)2,2,2- trichloroethanol) as their major metabolite, 3 produced DDD and 1 produced DDE and DDD. Six variants displayed no abil- ity to degrade DDT under the conditions tested. The authors indicated the presence of unknown water soluble metabolites. Johnson (1967) cultured 27 species of pathogenic and saprophytic bacteria associated with plants, anaerobically, in thioglycolate medium containing DDT for 7 or 14 days. Only the strict aerobe Sarcina lgtga and the anaerobe gigg- tridium sporogenes failed to convert DDT to DDD. None of ‘ 5 the organisms tested displayed any capacity to degrade DDT to DDD when cultured aerobically. Guenzi and Beard (1967) recovered 34% of the Cln-labeled DDT which had been added to soil and maintained anaerobically for 4 weeks. The major metabolite was DDD (62%) while only #% was recovered as other products. Although the authors in- cubated autoclaved soil containing DDT, no values were pre- sented. Eartha 23 AA. (1967) measured the effect of DDT and DDD on carbon dioxide and nitrite production in the soil. The compounds at 150 and 1500 ppm had no appreciable effect on carbon dioxide production but were found to slightly in- crease nitrification as measured by nitrite production. Laboratory Isolates Wedemeyer (1966) tested E. coli. A. aerogenes and Kleb- siella pneumoniae for their ability to anaerobically degrade DDT in trypticase soy broth or thioglycolate medium. Maximum conversion to DDD (80%) was achieved by A. aerogenes cultures after an unspecified incubation period. In subsequent reports Wedemeyer (1967 a and 1967 b), using 2 day A. aerogenes cul- tures, identified 4 additional metabolites, DDMU (l—chloro- 2,2-bis(p—chlorophenyl)ethylene), DDMS (l-chloro-2,2-bis(p- chlorophenyl)ethane), DDNU (unsym-bis(p-chlorophenyl)ethylene), and DDE. When the cells were incubated in mineral media con- taining methionine as a carbon source, only DDD was recovered after 100 hr incubation. Degradative Mechanisms Plemmer 93 3;. (1968), employing deuterated DDT, con- vincingly demonstrated that DDE was not an intermediate in the metabolism of DDT to DDD. After incubating A. aerogenes anaerobically for 2 days in trypticase broth containing deuterated DDT, 2-deuterioethane was found to be present in the recovered DDD. Ninety-two Z conversion was reported with DDD being the only metabolite. Wedemeyer (1966) employed sonically disrupted cells of A. aerogenes and selected inhibitors to ellucidate the biol- ogical mechanism involved in reductive dechlorination of DDT to DDD. Cell suspensions were sonically disrupted in 0.07 M phosphate buffer (pH 7) and added to buffer to which DDT in an acetone solution had been added resulting in a final con- centration of 5 ppm. After incubating overnight, anaero- bically, an average of 70% conversion to DDD occurred. No other metabolites were reported, and no conversion was found in the boiled controls. Cyanide, nitrate, ferricyanide, mal- onate, antimycin A and an atmosphere of carbon monoxide com- pletely inhibited DDD production. The carbon monoxide effect was completely reversed by exogenous cytochrome 0 plus ascor- bate. Based on the nature of the inhibition, the author con- cluded that reduced cytochrome oxidase was probably the agent of reductive dechlorination. In subsequent work, Wedemeyer (196? a) increased the incubation time to 2 days and deter- mined the influence of temperature, pH and exogenous energy sources on the metabolism of DDT by cell free preparations of *- 7 A. aerogenes. The preparation of the cell free system was essentially the same as reported previously. However, he did reduce the acetone concentration to 0.5%, doubled the mass of the preparation and increased the volume of cell free preparation utilized. Five metabolites of DDT were identified; DDD, DDE, DDMU, DDMS, and DDNU with DDD and DDNU being the major metabolites. The recovery of DDT from aerobic incubations averaged 92%. Ninety-five % remained un- changed in the boiled controls. When cultured anaerobically, the relative distribution of metabolites varied with both temperature and pH but not with different carbon sources. Each metabolite was synthesized and incubated with the cell free preparation. Based on the results of these studies, the author proposed the following pathway: DDT -9 DDD —9 DDMU —9 DDMS —9 DDNU. DDE was not degraded further, while DDA (2.2-bis(p-chlorophenyl)acetate) was produced from DDNU, and DBP (#,4'-d1ch1 L “ ) from DDA but not from DDT. The conversion of DDD to DDMU was inhibited by cyanide, fluo- ride, iodoacetate and malonate. DDMS conversion to DDNU was inhibited by malonic acid while DDA to DBP was not inhibited by any of the agents employed. Nonenzymatic Degradation A number of workers have shown nonenzymatic conversion of DDT to DDD. Castro (196#) demonstrated that dilute solu- tions of Fe++ can be oxidized at room temperature by alkyl +++ halides. including DDT, to the corresponding Fe halide C a. 8 complexes. Miskus gt 3;. (1965) showed partial conversion of DDT to DDD in hemoglobin and hematin solutions. Ott and Gunther (1965) established that DDT can be converted to DDD when injected in a stainless steel gas chromatographic column at 228°C. Farrow g§_§;. (1966) showed conversion of residual DDT to DDD during canning of spinach. Ecobichon and Saschen- brecker (1967) observed conversion of DDT to DDE, DDD and other undetermined metabolites in frozen chicken blood. To obtain samples, the blood was repeatedly thawed over a twelve week period. Animal Degradation DDE, DDD and dicofol have been reported as metabolites of DDT in insects, while DDD production has been reported as common in mammals. A DDA derivative has been produced by rats, and DDE production has been reported in man (O'Brien, 1967). When rats were fed DDT, DDD was recovered from the liver (Datta, gt 5;., 196“; Klein, 23 5A., l96#; Peterson and Robinson, 1964; Mendel and Walton, 1966). However, when DDT “was administered by interperitoneal injection, no conversion to DDD occurred (Baker and Morrison, 1964; Mendel and Walton, 1966). If the excised livers of DDT injected rats were al- lowed to putrify then DDD was recovered (Baker and Morrison, 1964; Peterson and Robinson, 1964; Mendel and Walton, 1966). Significantly, bacteria isolated from intestinal tracts and feces of animals have shown the ability to degrade DDT to DDD (Baker, et a1., 1965; Stenersen, 1965; Mendel and Walton, 1966; ,_-. ‘ 9 Brunberg and Beck, 1968). Mendel and Walton concluded that the microflora of the intestinal tract were responsible for the conversion of DDT to DDD in the rat. However, Morella (1965) isolated microsomes from rat liver that degraded DDT to DDD, and DDT-metabolizing activity was increased after intraperitoneal injections of DDT. The inductive effect of DDT and its metabolites on rat liver microsomes have also resulted in increased epoxidation of Aldrin (1,2,3,4,lO,10,- hexachloro-1,4,4a,5,8,8a-hexahydro-1,4-endo-exo-5,8-dimeth— anophthalene), (O'Brien, 1967; Gillett, 1968). DDT has been shown to induce NAD kinase in Tritoma infestans (Agosin, 1967) and to induce the synthesis of messenger RNA and overall pro- tein synthesis (Litvak, 1968). Peterson and Robinson (1964) proposed the following pathway of DDT metabolism in rats: DDT -+ DDD -9 DDMU —9 DDMS -9 DDNU -9 DDA. The pathway was deduced from the metab- olism of orally administered doses of DDT and DDT metabolites. It should be noted, however, that not all metabolites were recovered when DDT was the initial substrate. Biochemical Inhibitions DDT and many non-insecticidal derivatives.have been re- ported to inhibit the cytochrome oxidase activity in muscle homogEnates of the American roach, Periplaneta americana (Morrison and Brown, 1954), in meal worm homogenates, Pyralis farinalis L. (Ludwig, et a1., 1955), by sub-cell particles from the housefly, Musca domestica L. (Sacklin, et a1., 1955) ob1 Het 10 and in muscle homogenates of mealworm and housefly (Barsa and Ludwig. 1959) and lactate dehydrogenase (Sova, 1966). DDT also inhibits the oxidation of Kreb cycle intermediates and oxidative phosphorylation catalized by sub-cell particles obtained from houseflies (Sacklin, 23 3A., 1955) and glyco- lytic pyruvate production in cell free preparations of tho- racic leg muscle obtained from Triatoma infestans (Agosin, 1961). DDT has been reported to inhibit oxidative phosphory- 1ation of rat liver metochondria (O'Brien, 1967) and housefly mitochondria (Gregg, 23 El" 1964). However, in most cases concentrations greater than 0.001 M were required. Metabolic Capacity of Isolated Membranes That the Krebs cycle is the pathway of terminal respir- ation in bacteria was first established by cell-free extracts (Kornberg, 1959). Weibull (1953) was the first to success- fully employ lysozyme to dissolve the cell wall of Bacillus megaterium to produce protoplasts. Yoshida 23 5;. (1960) demonstrated that the sub-cellular membrane system procuced by lysozyme treatment followed by osmotic shock could pro- cruce large membrane fragments of g. 29;; capable of incor- poration of Cln—labeled amino acids into protein. The au- thors also established the necessity of magnesium ions for membrane activity. Utilizing a similar method of preparation, Mizuno 23 El- (1961) demonstrated the capacity of isolated bacterial membranes to oxidize Krebs cycle intermediates, carbohydrates and casamino acids. The authors further noted 11 that the oxidations were stimulated by addition of the "shock- ate" supernatant. This was confirmed by Gray 23 El- (1966) who was able to identify the cytochromes b, a, ag, e and 0 associated with g. 22;; membrane fractions, and Cox gt 3;. (1968), who employed membranes isolated from a ubiquinone- deficient mutant to study the oxidation of malate. Nagata 23 5;. (1966) and Yoshida 32 El- (1966) reported the ability of isolated g. 92;; membranes to incorporate P32 into nucleic acids, Clu-labeled amino acids into protein and to catalize oxidative phosphorylation. ———— MATERIALS AND METHODS Intact Cell Studies For uptake and metabolism studies with whole cells, sterile Anderson's minimal synthetic broth medium was inoc- ulated with Escherichia 32;; or Pseudomonas aergginosa and incubated for 19 hr at 37°C with shaking. At the end of the growth periods, measurements of cell masses were made by ob- serving their optical densities at 650 mp with a Bausch and Lamb Spectronic 20. Their dry weights were read from a cali- bration curve relating optical density at 650 my to dry weight in mg/ml. The cells were harvested by centrifugation at 12,000 g for 5 minutes. The cells were washed by resus- pending in 0.85% saline and recentrifuged. The experimental inocula consisted of 425 mg (dry weight) of washed cells. The cells were resuspended in 500 ml ali- quots of sterile minimal medium to which had been added 0.1 ml of acetone containing 2.22 x 105 dpm of 01'+ ring labeled p,p'-DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane). The cultures were shaken at 37°C for 1,2, or 3 days under aerobic or nitrogen atmospheres. Controls consisted of auto- claved cells to which Clu-labeled DDT was aseptically added. 12 A l3 byt and were util Vite 13 Extraction & "Clean-up" After incubation the cells were separated from the medium by centrifugation at 12,000 g for 5 minutes. The superna- tants were extracted 3 times with 100 ml volumes of hexane and concentrated to 25 m1 aliquots. Interfering materials were removed from the concentrates by column chromatography utilizing 10 g aliquots of Florisil and Celite (5:1) deacti- vated with water ( 15% ). The effluents were concentrated to 0.5 ml and assayed for Cl# content with a Mark I liquid scintillation computer (Nuclear-Chicago Corporation). Super- natants were assayed for Cl“ content before and after extrac- tion. The cells were extracted 3 times with acetone. The extracts were taken to dryness and "cleaned-up" as above. The effluents were concentrated to 0.5 ml and assayed for C1“ content. Analytical Methods DDT and its metabolites were identified by thin-layer and gas-liquid chromatography. Thirty pg of p,p'-DDT, o,p'- DDT (1,1,l-trichloro-Z,o-chlorophenyl-2-p-chlorophenyl- ethane), DDD, and DDE were spotted on silica gel H thin-layer chromatographic plates (Brinkman Instrument Co.) with 15,000 dpm of each experimental concentrate and developed twice through 15 cm. Autoradiograms were produced by exposing Kodak medical X-ray film to the plates for 4 days. After development of the X-ray films, the chromatograms were sprayed lightly with a 0.1% alcoholic Rhodamine B solution and A 14 treated with sodium carbonate to resolve the authentic stan- dards (Johnson and Goodman, 1967). Quantification was accomplished by gas-liquid co-chroma- tography utilizing a 6 ft. X i in. glass column containing 80-100 mesh Gas Chrome-Q (The Anspec Co.) coated with 11% DC QF-l (Applied Science Laboratories, Inc.) and OV-l7 (Applied Science Laboratories, Inc.) in a ratio of 1.3:1.0. Base line separations of the standards were achieved by a column temp- erature of 190°C, detector temperature of 200°C and nitrogen flow rate of 20 cc/min. A Packard Model 850 gas fraction collector employing cartridges filled with Pyrex glass wool was used to collect Cl“vlabeled components from the effluent stream of the column. Following injection of the sample, collections were made at 5 minute intervals for a total of 75 minutes. The glass wool was removed from the cartridges and the entraped radioactivity determined by liquid scin- tillation counting. The retention times of the Clu-labeled components were compared to the retention times of the 4 authentic standards mentioned above. Cell Free Studies For cell free studies, active g. 32;; membranes were prepared by the method of Nagata 23 2A. (1966). Washed in- tact cells (0.25 mg/ml dry weight) were incubated at 30°C for 30 minutes with gentle shaking in a medium consisting of 3 parts 0.9 M sucrose in pH 8.0, 0.05 M Tris (2-amino- 2-(hydroxymethyl)-l,3-propanediol), 1 part 0.0071 M EDTA 15 (ethylenediaminetetraacetic acid), and 1 part of lysozyme (Sigma Corporation) solution at 0.6 mg/ml. The incubation mixture was centrifuged at 15,000 g for 5 minutes and the "protoplasts" harvested. Membranes were obtained from pro- toplasts as illustrated in Figure 1. Incubations were carried out in 10 ml Warburg flasks containing 0.1 g glass beads (15 p) and 350,000 dpm of DDT- Clu was added in acetone solution to the surface of the beads and the acetone evaporated prior to the addition of 3 m1 a1- iquots of membrane suspension. The desired Krebs cycle in- termediates and cofactors were added to the side arms of the flasks and the contents emptied into the incubation mixtures after 5 minute periods of temperature and atmospheric equil- ibration. Controls consisted of boiled membrane suspensions. Nitrogen atmospheres were maintained throughout the incuba- tion period. Extraction of C14 metabolites, their identifi- cation and quantification were carried out as described pre- viously. The Clu-labeled p,p'-DDT was obtained from Nuclear-Chi- cago Corporation. The p,p'-DDT (unlabeled) was obtained from City Chemical Corp., the o,p'-DDT isomer from Geigy Chemical Corp., and the DDD from City Chemical Corp. The p,p'-DDE was prepared by alkaline dehydrochlorination of p,p'-DDT and Alumina chromatography (Sternburg and Kerns, 1952). The Krebs cycle intermediates, malonate and pyruvate, as well as the cofactors NAD (nicotinamide adenine dinucleotide), NADP (nicotinamide adenine dinucleotide phosphate), FAD ‘ 16 (flavin adenine dinucleotide), and ADP (adenosine diphos- phate), were obtained from the Sigma Corporation. Values presented in the results are means of two repli- cate experiments . 1? PROTOPLASTS Homogenized with a Teflon homogenizer in ice cold pH 7.6, 0.05 M Tris containing 0.005 M MgClg. ("Shockate") Centrifuged at 20,000 x g for 20 min. at l.0°C. "Shockate" Precipitate "Shockate" Supernatant I saved) Resuspended in 0.05 M Tris pH 7.6, 0.005 M MgClz at 12.5 mg/ml of original dry weight. Centrifuged at 20,000 x g for 20 min. at l.0°C. Precipitate Supernatant (membrane) (discarded) Resuspended in 0.05 M Tris pH 7.6, 0.005 M MgClZ at 25.0 mg/ml original dry weight. Figure 1. Preparation of membranes from protoplasts. (Nagata. _e_t 9A.. 1966) RESULTS OF INTACT CELL STUDIES Cells cultured anaerobically were able to convert sub- stantially more p,p'-DDT to p,p'-DDD than those cultured aero- bically (Tables 1 and 2). In both cases the conversion achieved in the third day of a 3 day incubation period approx- imately equaled the conversion achieved in the previous 2 days. Although the aerobic cultures converted 70% less DDT to DDD, they did convert approximately 10% more DDT to DDD than autoclaved cells incubated anaerobically for 3 days (Table 2). DDE was produced by E. coli cultured aerobically and anaerobically. However, these values did not substan- tially exceed those obtained from autoclaved cells, nor did the values change with incubation time. The levels of re- covered o,p'-DDT did not change with time or incubation con- ditions. All 3 metabolites occurred at low levels in the stock DDT-cl” (Table 1). The distribution of recovered radioactivity between cells and medium is presented in Table 3. Although bacteria both in aerobic and anaerobic cultures were able to concen- trate the radioactivity progressively with time, slightly more radioactivity was concentrated by cells cultured aero- bically. After 3 days, 96% of the radioactivity found in the medium was DDD in anaerobic cultures, while 70% of the radioactivity was DDT in the medium of aerobic cultures. 18 19 Table 1. DDT metabolism by intact cells of E. coli incu- bated aerobically. % 014 found as DDT metabolites Time p.p'- 0.p‘- p.p'- p.p'- Fraction (days) DDE DDT DD DDT Unknown Medium 1 5.1 1.6 12.9 75.4 5.0 Cells 1 7.2 0.0 15.2 74.6 3.0 Medium 2 3.9 2.7 13.5 76.6 3.3 Cells 2 8.1 1.7 17.4 70.4 2.4 Medium 3 2.0 1.1 24.0 70.2 2.7 Cffils 3 2.1 1.5 22.4 70.3 3.9 C -Stock 0 3.5 1.7 0.8 91.8 2.2 Table 2. DDT metabolism by intact cells of E. coli incu- bated anaerobically. % C14 found as DDT metabolites Time p.p'- 0.p‘- p.p'- p.p'- Fraction (days) DDE DDT DDD DDT Unknown Medium 1 2.0 1.8 39.2 53.0 4.0 Cells 1 7.0 1.0 22.3 62.0 7.6 Medium 2 3.4 1.8 54.2 39.1 1.5 Cells 2 5.4 2.0 35.6 54.3 2.? Medium 3 1.0 1.1 96.1 0.9 0.8 Cells 3 2.2 2.0 92.4 3.0 0.4 Control mediuma 3 2.9 1.2 10.3 82.7 3.3 Control cells 3 8.6 2.0 9.0 75.1 5.3 a Autoclaved cultures were employed as controls. 00000000 nnnnnnn 20 Table 3. Distribution of radioactivity in cultures of _. co . % radioactivity Culture Time(days) Medium Cells Aerobic 19.0 1.0 2.3 5707 9-7 80.3 Anaerobic 2.0 18.0 1.0 39.0 9.0 71.0 2.8 47.2 UWNI—‘KDNH Anaerobic control With autoclaved E. coli, 58% of the recovered radio- activity was associated with the medium, while 47.2% was associated with the cells. The pattern of DDT metabolism by g. aeguginosa was sim- ilar to that found in the g. 32;; incubations(Tables 4 and 5). However, anaerobic cultures of P. aeggginosa were able to produce nearly as much DDD in 2 days as the g. 32;; cul- tures were able to produce in 3 days. In addition, 2-day-ae- robic g. aeggginosa cultures were able to produce slightly more DDD from DDT than l-day-anaerobic E. 22;; cultures. The percent recovery of Clu-labeled metabolites extracted from the medium varied. The highest recovery was attained in l-day-aerobic cultures (97%) and the lowest from anaerobic cul- tures incubated 3 days (71%). This non-extractable radio- activity attained a maximum of 4% of the total radioactivity. An average of 84% of the extracted radioactivity was recovered from injected samples by glass wool trapping. 21 Table 4. DDT metabolism by intact cells of P. aeggginosa incubated aerobically. — z 014 found as DDT metabolites Time p.p'- o.p'- p.p'- p.p'- Fraction (days) DDE DDT DDD DDT Unknown Medium 2 2.8 0.8 27.0 57.4 2.0 Cells 2 2.2 1.1 0.1 55.1 1.5 Table 5. DDT metabolism by intact cells of g. aeggginosa incubated anaerobically. 5 014 found as DDT metabolites Time p.p'- 0.p'- p.p'- p.p'- Fraction (days) DDE DDT DDD DDT Unknown Medium 2 2.1 0.7 87.4 6.4 3.4 Cells 2 3.3 1.1 86.5 6.9 2.2 Control medium 2 6.2 2.5 4.0 85.3 2.1 Control cells 2 1.5 1.0 5.7 88.3 3.5 RESULTS OF MEMBRANE STUDIES Experiments were designed to acertain the site of DDT metabolism in E. coli. Both the cytoplasmic fraction ("shock- ate“ supernatant) alone and the cytoplasmic fraction plus boiled membrane fraction displayed little ability to degrade DDT to DDD (Table 6). On the other hand, cytoplasmic frac- tions plus unboiled membranes produced substantially more I DDD (29.8 vs. 2.4 and 3.8%). Thus the membrane of bacteria u is the site of reductive dechlorination of DDT and the cyto- plasm contains an essential factor(s). Table 6. Effect of membrane and cytoplasm of E. coli on con- version of DDT to DDD. % c1” found as DDT metabolites p p'- o p'- p.p'- p.p'- Componentsa DDE DDT DDD DDT Unknown Membrane only 0.4 1.8 4.6 90.5 2.7 Membrane & Cytoplasmb 0.3 1.3 29.8 61.9 6-3 cYtoplasm only 0.1 2.0 2.4 92.5 3. Boiled membrane & cytoplasm 4 0.3 1.5 3.8 90.0 4.4 DDT-C -1 & Burreirbon 0.4 1.9 0.6 93.9 3-2 a 1 acts) consisted of Tris The membrane fractions (3 ml a1 buffer containing membranes at 25.0 mg ml (original dry weight of cells). b The membrane ppt. were resuspended in cytoplasmic frac- tion at 25.0 mg/ml (original dry weight of cells). 22 .L 23 In the presence of NAD, NADP, ADP, FAD, inorganic phose phate, malate, and pyruvate, the level of DDD recovered was not substantially different from that of the membranes alone (Table 7). When NAD, NADP, or the Krebs cycle intermediates (malate and pyruvate) were not included in the incubation mixtures, increases in DDD production occurred. Omission of ADP plus inorganic phosphate or FAD from the incubation mix- ture gave no increase in DDD production. Significantly, substantial DDD production was achieved only in those incu- bants containing FAD, ADP, and inorganic phosphate. Since addition of exogenous ADP plus inorganic phosphate or FAD did enhance DDD production by isolated membranes, experiments were conducted to determine the effects of these components singly or in combination (Table 8). Membranes plus exogenous FAD, ADP, and inorganic phosphate or membranes plus FAD only produced over 4 times the DDD then membranes incubated with exogenous FAD plus inorganic phosphate (Table 8). Thus the addition of exogenous FAD to membrane prep- arations enhances DDD production. Increasing the exogenous FAD from 2 to 81pmole did not result in a substantial in- creases in DDD production. The addition of ADP plus inorganic phosphate, or FAD, or FAD plus ADP and inorganic phosphate to membrane resus- pended in cytoplasmic fractions did not increase DDD produc- tion beyond that attained by membrane and cytoplasmic combin- ations only (Tables 6 and 8). These results suggest that the availibility of endogenous enzymes and/or substrates were ‘ 24 limiting the rate of DDD production. The addition of FAD, ADP, and inorganic phosphate to membrane fractions incubated aerobically did not enhance DDD production. Thus FAD enhancement of DDD production is depen- dent on anaerobic conditions. This suggests that normally op- erating oxidative pathways preclude the reductive dechlorin- ation of DDT. Table 7. Effect of exogenous Krebs cycle intermediates and cofactors on DDT metabolism by membrane prepara- tions of E. coli. % 014 found as DDT metabolites p.p'- 0.p‘- p.p‘- p.p'- Componentsa DDE DDT DDD DDT Unknown Membrane only 0.4 1.8 4.6 90.5 2.7 All Cofactors & Chos.b 0.2 1.3 2.2 93.2 3.1 Cofactors & Chos.(0.lX) 0.1 1.6 7.9 86.3 4.1 minus FAD 1.1 1.8 4.9 86.4 5.8 minus ADP & P04 0.0 1.2 5.9 89.1 3.8 minus Malate & Pyruvate 0.4 1.4 21.4 73.7 3.1 minus NAD 0.5 1.6 26.8 65.6 5.5 minus NADP 0.8 1.6 21.1 63.3 7.2 minus Chos. (control)° 0.1 0.4 'l;3 9 .7 3.5 a Each incubant contained 3 ml of membrane fraction. b Two pmole each of NAD, NADP, FAD, malate, pyruvate, and 0.14pmole each of ADP and inorganic phOSphate. Components consisted of 3ml boiled membrane fraction plus exogenous cofactors. Malate and pyruvate were not added. 25 Table 8. Effect of NAD, FAD, ADP, and inorganic phosphate on DDT metabolism by membrane preparations of E. coli. is 014 found as DDT metabolites p.p‘- 0.p‘- p.p'- p.p'- Componentsa DDE DDT DDD DDT Unknown NAD 0.3 1.3 7-7 87.4 3.3 FAD 0.2 1.2 22.5 72.6 3.5 ADP .2. P04. 0.5 2.1 5.2 88.3 3.9 FAD, ADP & P04 0.5 1.7 20.5 74.2 3.1 4X FAD, ADP & P04 0.7 0.8 23.1 71.1 4.3 Cytoplasm & FAD,ADP,P04 0.9 1.6 28.9 62.6 6.0 Cytoplasm & FAD 0.1 1.3 26.2 68.8 3.6 Cytoplasm, ADP & P04 0.5 1.3 27.9 65.1 5.2 Aerobic Atmosphere, FAD, ADP, & P04 0.5 1.6 2.9 90.0 5.0 a Each incubant contained 3 m1 of membrane fraction. uuuuuuuu nnnnnnnn DISCUSSION The results of the whole cell studies carried out in this investigation are in general agreement with the obser- vations made by other investigators, that is, the conversion of DDT to DDD is inversely related to the supply of atmo- spheric oxygen available to the bacteria. However, the present investigation also demonstrated aerobic conversion of DDT to DDD by bacterial cultures. Two investigators have reported that no conversion of DDT to DDD occurs in aerobic bacterial cultures (Stenersen, 1965; Johnson, 1967) while others have reported the contrary (Chacko, 23 §;.. 1966; Wedemeyer, 1966). Metabolic differences between species may account for this discrepancy. On the other hand, shaking may not have provided sufficient oxygen to maintain an aero- bic state with the bacterial populations employed in this investigation. The levels of DDE exceeded that of the DDT-C1)+ stock solution. However, the DDE content did not increase with incubation time and did not vary significantly from the levels found in autoclaved cells. Similar results have been obtained by other investigators employing other micro- organisms (Kallman and Andrews, 1963; Stenersen, 1965; Wedemeyer, 1966; Plemmer, 32 §;., 1968). Guenzi and 26 2? Beard (1967) reported a slight increase in recoverable DDE after incubating nonsterile soil, 4 weeks, anaerobically, with DDT. Autoclaved cells displayed a limited capacity to con- vert DDT to DDD under anaerobic conditions. Of those in- vestigators that referred to autoclaved control experiments, none reported conversion of DDT to DDD. Contamination of the control cultures cannot be categorically eliminated as the control cultures were not plated after incubation. The observations by Castro (1964) that dilute solutions of Fe++ porphyrins can dechlorinate DDT and Miskus (1965) that de- chlorination can be accomplished by hemoglobin and hemitin solutions at room temperature, demonstrate non-biologically catalized degradation can occur with relatively mild con- ditions. This may account for all the DDE and a fraction of the DDD extracted from aerobic and anaerobic cultures and for the presence of DDE and DDD in autoclaved cultures. The non-hexane-extractable radioactivity in anaerobic E. 22;; cultures represented 4% of the radioactivity after 3 days. Stenersen (1965), Guenzi and Beard (1967), and Matsumura and Bousch (1968) also indicated the presence of nonextractable radioactivity associated with water phases. This residual activity may represent water soluble metabo- lites. The autoradiograms of anaerobic cultures of E. EQAA. incubated 3 days, and P. aeggginosa, incubated 2 days, pos- sessed 1 slightly exposed spot of extremely low Rf, probably 28 indicating a high degree of polarity, which did not corre- spond to any of the standards. This lends support to re- ports of Wedemeyer (1967 a) and Bousch and Matsumura (1968) that products other than DDD occurred as minor metabolites of bacterial degradation of DDT. Anaerobic, aerobic and autoclaved cells concentrated DDT and its metabolites (Table 3). Eighty %, 71%, and 47% of the total C14 was extracted from the cells of aerobic, anaerobic and autoclaved cultures respectively, after 3 days of incubation. The volume occupied by 0.25 mg (dry weight) of E. 22;; is approximately 1.0 pd (Roberts, 23 gl.. 1963). At the population levels employed in this research, the bac- teria occupied a volume of approximately 3.5 pl/ml of culture. Relating this information to the distribution of extractable radioactivity presented in Table 3, a more dramatic repre- sentation of the radioactive distribution can be seen. That is, 80% of the extractable radioactivity was associated with less than 0.4% of the incubation volumes in aerobic cultures, and 71% of the extractable radioactivity was associated with 0.4% of the incubation volumes in anaerobic cultures. Washing the bacterial pellets by resuspension in 0.85% sodium chloride solution released an insignificant amount of radioactivity. Since a rather large initial inocula of lag-phase bacteria were employed in these experiments (425 mg/experiment), the increases in population were not sufficient to alter the optical density of the medium. Thus living cells A ((17). .II 1“, 29 do concentrate DDT and its metabolites, but this is not a requisite for uptake as autoclaved cells displayed this capacity as well (Table 3). The ability to metabolize DDT does not enhance its uptake under the conditions employed in this investigation. In addition, most of the DDD pro- duced by the bacteria remained associated with the cells. However, the role of the medium cannot be discounted. If media of high lipid content were employed, the partitioning of DDT and its metabolites may show different characteristics. After cellular lysis, neither the particulate membrane fraction nor the soluble fraction (cytoplasm) could produce significant amounts of DDD (Table 6). If, however, these 2 fractions were combined, conversion of DDT to DDD occurred. Upon boiling the membrane fraction for 5 minutes and com- bining the boiled membranes with the "shockate" supernatant, one could no longer obtain significant conversion. These observations, plus the fact that the addition of FAD to the membrane fraction enhanced DDD production, suggests that the capacity to metabolize DDT to DDD resides in the membranous portion of the bacterial cell and is not cytoplasmic in ori- gin. Since the cell walls were depolymerized and made solu- ble by the action of lysozyme (Salton, 1960), it most probably plays no direct role in this aspect of DDT degradation. The membrane fraction plus NAD, NADP, pyruvate, malate, FAD, ADP, and inorganic phosphate converted little DDT to DDD. However, if NAD, NADP or the 2 Krebs cycle intermediates I 4 l I v I v I x z c l t i I I ' l 30 were deleted from the incubation components, significant increases in DDD production occurred (Table 7). Thus the presence of potential Krebs cycle activity inhibits DDD pro- duction with the conditions utilized in this study. Since membrane preparations of E. 92;; are capable of metabolizing Krebs intermediates (Mizuno, 33 El-- 1961; Gray, 32 gl.. 1966; Cox, 32 5A., 1968), and furthermore, contain the cytochromes b, a, a2 and c (Gray, 23 él-v 1968), one would not expect the results obtained in this investi- gation. If indeed, reduced cytochrome a3 (cytochrome oxidase) is the enzyme responsible for the conversion of DDT to DDD (Wedemeyer, 1966) then the deletion of major components of the Krebs cycle should not enhance DDD production. On the contrary, their metabolism should contribute electrons to the cytochrome system and maintain them in a reduced state. The involvement of FAD in enzymatic electron transfer processes is well documented (White, 33 El-v 1965; Slater, 1966; Wellner, 1967). Addition of exogenous FAD to the mem- brane fraction significantly enhanced DDD production (Table 8). However, addition of FAD to aerobically incubated mem- brane fractions did not stimulate DDD production. Thus anaerobic conditions are a requisite to FAD enhancement of DDD production. The results of this investigation suggest that under anaerobic conditions FAD may be a cofactor re- quired for the enzymatic conversion of DDT to DDD. Secondly, the results suggest that FAD may function as a cofactor in an electron transfer process not directly involved in the 31 immediate reduction of DDT to DDD, but in an electron trans- fer process or processes necessary for the ultimate reduc- tion of DDT. Further investigation would be required to es- tablish the role of FAD in DDT reduction. Four-fold increments of exogenous FAD added to membrane fractions failed to significantly increase DDD production be- yond that obtained by the addition of 2 umole aliquots (Table 8). This suggests that another factor or factors are limiting the rate of DDT reduction. Cytoplasmic stimulation of DDT reduction by membrane preparations was not increased by addi- tion of exogenous FAD. The stimulating factor or factors that were present in the cytomplsmic fraction ("shockate" supernatant) are unknown. The isolation and characterization of this factor or factors required for DDT reduction would contribute valuable information concerning the metabolic processes involved in DDT reduction. h: SUMMARY Aerobic and anaerobic cultures of E. 3%; and E. gogg- ginosa degraded DDT to DDD. This conversion was inversely related to the supply of atmospheric ongen available to the bacteria. Autoclaved cells produced substantially less DDD. The levels of DDE and o,p'-DDT produced after 3 days did not exceed the control levels. Anaerobic, aerobic and autoclaved cells concentrated DDT and its metabolites. The magnitude of 014 uptake was not related to the ability of the cells to metabolize DDT. Thus the ability to concentrate DDT is a passive process. The membrane fraction or the cytoplasmic fraction (20,000 g' "shockate" supernatant) degraded little DDT to DDD. The combined fractions were able to dechlorinate more DDT. Addition of cytoplasmic fractions to boiled mem- brane fractions did not enhance the reductive dechlorination of DDT. When NAD, NADP,FAD, ADP, malate, pyruvate, and inor- ganic phosphate was added to membrane fractions, the levels of recovered DDD did not exceed the'levels of DDD produced by membrane fractions only. When NAD, NADP, or malate and pyruvate were omitted from the incubation components, in- creases in DDD production occurred. Addition of exogenous 32 [37,: "i" I 33 FAD to membrane fractions resulted in increased DDD pro- duction under anaerobic conditions. The results of the membrane studies indicate the following: 1. Reductive dechlorination of DDT occurs in the mem- branous portion of the bacterial cell and is not cytoplasmic in origin. 2. Reductive dechlorination of DDT is stimulated by com- ponents in the cytoplasm. 3. Reductive dechlorination of DDT does not utilize electrons produced by the oxidation of Krebs cycle inter- mediates and passed through the cytochrome system. 4. Reductive dechlorination of DDT is dependent upon the enzymatic reduction of FAD and occurs only under anaer- obic conditions. 5. Reductive dechlorination of DDT requires electrons produced by the oxidation of an energy source. 6. Beductive dechlorination of DDT may require the formation of free radicals. The oxidation of endogenous substrates could produce the half-reduced form. (if ‘EAD (FADE. , a semiquinone) and may be the active moiety involved in the enzymatic reduction of DDT. LITERATURE CITED LITERATURE CITED Agosin, M. , Scaramelli, N., and Neghme, A. 1961. Interme- diary carbohydrate metabolism of Triatoma infestans. Comp. Biochem. Physiol. 2:143-159. , Ilividky, J., and Litvak, S. 1967. The induction of NADkinase by DDT in Triatoma infestans. Can. BiOGhem. 458619-626. Baker, P.S. , and Morrison, F.0. 1964. DDD in mouse tissue. J. Breakdown of DDT to Can. J. Zool. 42:324-325. , and Morrison, F.0. 1965. Conversion of DDT to DDD by Proteus vulgaris, a bacterium isolated from the in- testinal flora of a mouse. Nature 205:621-622. Barsa, M.C. , and Ludwig, D. 1959. Effects of DDT on the respiratory enzymes of the mealworm, Tenebrio molitor L. and of the housefly, Musca domestica L. Ann. Entomol. Soc. 52:179-182. Bertha, R. . Lanzilotta, R.P., and Framer, D. 1967. Stability and effect of some pesticides in soil. J. Appl. Micro- biol. 15:67-75. Brunberg , R.C. , and Beck, V. 1968. Interaction of DDT and the gastrointestinal microflora of the rat. J. Ag. and Food Chem. 16:451-453. Castro, C.A. 1964. The rapid oxidation of Iron (II) por- phyrins by alkyl halides. A possible mode of intox- ication of organisms by alkyl halides. J. Amer. Chem. Soc. 86: 2310-2311. ChaOkO' COI. ’ LOGkWOOd, JoLe ' 311d. zabik’ Me 1966. Chlor- inated hydrocarbon pesticides: degradation by microbes. Science 154:893-895. Collins, J.A.. , and Langlois, B.E. 1968. Effect of DDT, Dieldrin and Heptachlor on the growth of selected bac- teria. Appl. Microbiol. 16:799-800. Cox, G.B. , Snoswell, A.M., and Gibson, F. 1968. The use of a ubiquinone-deficient mutant in the study of malate oxidation in E. coli. Biochim. Biophy. Acta 153:1-12. 34 I a u o _ . O 1 I : u . I _ \ .._ m ' I r:. c e a o a u n . 35 Datta, P.R., Laug, E.P., and Klein, A.K. 1964. Conversion of P,P'-DDT to P,P'-DDD in the liver of the rat. Science 145:1052-1053. Ecobichon, D.L.. and Saschenbrecker, PM. 1967. Dechlor— ination of DDT in frozen blood. Science 156:663-664. Farrow, E.P., Eldins, E.R., and Cook, R.N. 1966. Conver- sion of DDT to TDE in canned spinach. J. Ag. Food Chem. 14:430-434. Gillett, J.W. 1968. No effect level of DDT in induction of microsomal epoxidation. J. Ag. Food Chem. 16:295-297. . Regulation of metabolism in facultative bacteria. Gray, C.T., Wimpenny, J.M.T., Hughes, D.E.. and Mossman, M. 19 Biochim. Biophys. Acta 117:22-32. Gregg, C.T., Johnson, J.R., Heisler, C.R., and Remmert, L.F. 1 . Inhibition of oxidative phosphorylation and re- lated reactions in insect mitochondria. Biochim. Biophys. Acta 82:340-343. Guenzi, 14.0., and Beard, W.E. 1967. Anaerobic biodegrada- tion of DDT to DDD in soil. Science 156:1116-1117. Hayes Jr. , W.J. 1965. Review of the metabolism of chlorin- ated hydrocarbon insecticides especially in mammals. Ann. Review Pharmacology 5:27-52. Johnson, B.T., and Goodman, R.N. 1967. Conversion of DDT to DDD by pathogenic and saprOphytic bacteria associ- ated with plants. Science 157:560-561. Kallman, B.J., and Andrews, A.K. 1963. Reductive dechlor- ination of DDT to DDD by yeast. Science 141:1050-1051. Klein, A.K., Laug, E.P., Datta, P.R., Watts, J.0., and Chen, J.'I'. 1964. Reductive dechlorination of DDT to DDD and isomeric transformation. J. Assoc. Official Ag. Chem. 47 : 1129-1145. Kornberg, H.L. 1959. Aspects of terminal respiration in microorganisms. Ann. Rev. Microbiol. 113:49-55. Litvak, S., Litvak, L., and Pobete, P. 1968. Evidence for the DDT-induced synthesis of messenger ribonucleic acid in Triatoma infestans. Comp. Biochem. Physiol. 26:45- 56. Ludwig. D., Barsa, M.C., and Cali, T. 1955. The effect of DDT on the activity of cytochrome oxidase. Ann. Ento- 36 Martin, J.P. 1967. Influence of pesticides on soil microbes and soil properties. ASA Special Publication 8. Soil Science of America Inc. 95-108. Matsumura, F., and Bousch, G.M. 1968. Degradation of in— secticides by a soil fungus, Trichoderma viride. J. Econ. Entomol. 61:610-612. Mendel, J.L., and Walton, M.S. 1966. Conversion of P,P'- DDT to P,P'-DDD by intestinal flora of the rat. Science 151:1527-1528. Miskus, P., Blair, D.P., and Casida, J.E. 1965. Conversion of DDT to DDD by bovine rumen fluid, lake water, and reduced porphyrins. J. Ag. Food Chem. 13:481-483. Mizuno, S., Yoshida, E., Takahashi, H., and Maruo, B. 1961. Experimental proof of a compartment of "energy-rich-P” in a subcellular system from Pseudomonas flourscent. Biochim. Biophys. Acta 49:361-3 1. Morello, A. 1965. Induction of DDT-metabolizing enzymes in microsomes of rat liver after adminstration of DDT. Can. J. Biochem. 43:1289-1293. Morrison, P.E., and Brown, A.W.A. 1954. The effects of in- secticides on cytochrome oxidase. J. Econ. Entomol. 47:723-730. Nagata, E., Mizuno, 3., and Maruo, B. 1966. Preparation and properties of active membrane systems from various species of bacteria. J. Biochem. 59:404-410. O'Brien, R.D. 1967. Insecticides Action and Metabolism. Academic Press, New Tori. Ott, D.E.. and Gunther, F.A. 1965. DDD as a decomposition product of DDT. Residue Rev. 10:70-84. Perry, A.S., and Sacktor, B. 1955. Detoxification of DDT in relation to cytochrome oxidase activity in resis- tant and susceptible houseflies. Ann. Entomol. Soc. 48:329-333. , 1960. Biochemical aspects of insect resistance to chlorinated hydrocarbons. Miscellaneous Pub. Entomol. Soc. Amer. 2:119-137. Peterson, J.E., and Robison, Wm. H. 1964. Metabolic products of P,P'-DDT in the rat. Toxicology Appl. Pharmacol. 6: 321-327. 37 Plemmer, J.E., Kleamey, P.C., and VanEndt, D.W. 1968. Mech- anism of conversion of DDT to DDD by Aerobacter arogenes. J. Ag. Food Chem. 16:594-597. Roberts, R.B., Abelson, P.R., Bolton, E.T., Cowie, D.B., and Britten, R.J. 1963. Studies of Biosynthesis in Escher- ichia coli. Carnegie Institution, Washington. Sacklin, J.A., Terriers, L.C.. and Remmert, L.F. 1955. Effect of DDT on enzymatic oxidation and phosphorylation. Science 122:377-378. Salton, M.B.J. 1960. The properties of lysozymes. Ann. Rev. Microbiol. 115:82-99. Schoenhard, D.E. 1961. Basic Conce ts and Experiments fl Burgess Pu lishing Co. , Minneapolis. Microbiology. Slater, E.C. 1966. Flavin and Flavoproteins. Elsevier Pub- lishing Co.. New IorE. Lactate dehydroginase activity: effect in Science 154:1661- Sova, 0.3. 1966. vitro of some pesticidal chemicals. 1662. Stenersen, V.H.J. 1965. susceptible stableflies and in bacteria. 60—661. Sternburg, J. , and Kearns, W. 1952. Chromatographic sepa- ration of DDT and some of its known and possible metab- olites. J. Econ. mtol. 45:505-509. Wedemeyer, G. 1966. Dechlorination of DDT by Aerobacter aerogenes. Science 152:647. , 1967 a. Dechlorination of l,l,l-trichloro-2,2-bis (p-chlorophenyl)ethane by Aerobacter aero enes. 1. Met- abolic products. J. Appl. Micro'Eiol. 15:569-574. , 1967 b. Biodegradation of dichlorodiphenyl tri- chloroethane: Intermediates in dichlorodiphenylacetic acid metabolism by Aerobacter aerogenes. J. Appl. Microbiol. 15:149 I49 5. Weibull, C. 1953. Isolation of protoplasts from Bacillus e aterium by controlled treatment with lysozyme. J. m Bac teriol. 66:688-695. Wellnzg, D. 1967. Flavoproteins. 9- e White, A., Handler, P., and Smith, E.L. 1964. Princi 1es g_f_ Biochemistry. 3rd ed., McGraw-Hill BooE Co.. New York. DDT metabolism in resistant and Nature 207: Ann. Rev. Biochem. 36: 38 Ioshida, E., Mitsui, H., Takahashi, H.. and Maruo, B. 1960. Amino acid incorporation by a bacterial cell free sys- tem. J. Biochem. 48:251-261. APPENDIX Figure 2. Representative thin-layer chromatogram. Mobil phase; hexane. Chromogenic agent: 0.1% Rhodamine B. Each point of origin was spotted with 30.0 pg each of p,p'- DDE, o,p'-DDT, p,p'-DDT, and p,p'-DDD. From left to right authentic carbon-1h-labe1ed DDT (1); 3ml of membrane frac- tion + 2.0 pmoles each of NAD, NADP, FAD, and 0.1 pmoles each of ADP & inorganic P04 (2); duplicate experiment using the components of number 2 (3); 3 m1 of membrane fraction + 0.2 nmoles each of NAD, NADP, FAD, malate and pyruvate and 0.01pmoles each of ADP & inorganic P04 (4); duplicate ex- periment using the components of number 4 (5); 3 ml of mem- brane fraction (6); duplicate experiment using the compo- nents of number 6 (7); authentic carbon-14-labeled DDT (8). The spots at Rf 0.60, 0.51, 0.4#, and 0.26 correspond to DDE, o,p'-DDT, p,p'-DDT, and DDD, respectively. The auto- radiogram obtained from this chromatogram is presented in Figure 6B. 40 Figure 3. Autoradiograms of thin-layer chromatograms of carbon-lh-labeled DDT and carbon-lb-labeled metabolites produced by _E_I. coli. 41 Figure 3A. Anaerobic cultures incubated 1 and 2 days. From left to right carbon-lh-labeled DDT (1); medium of an anaerobic culture incubated 1 day (2); bacteria of an anaer- obic culture incubated 1 day (3); medium of an anaerobic cul- ture incubated 1 day (4); bacteria of an anaerobic culture incubated 1 day (5); bacteria of an anaerobic culture incu- bated 2 days (6); medium of an anaerobic culture incubated 2 days (7); authentic carbon-lh-labeled DDT (8). The upper and lower spots correspond to p,p'-DDT and p.p'-DDD, respectively. 42 Figure BB. Anaerobic cultures incubated 2 days, 3 days, and autoclaved cells incubated 3 days. From left to right authentic carbon-14-1abeled DDT (1); medium of an anaerobic culture incubated 2 days (2); bac- teria of an anaerobic culture incubated 2 days (3); medium of an anaerobic culture incubated 3 days (“)3 bacteria of an anaerobic culture incubated 3 days (5); medium of an anaerobic autoclaved culture incubated 3 days (6); bacteria of an anaerobic autoclaved culture incubated 3 days (7); authentic carbon-lh-labeled DDT (8). The upper and lower spots correspond to p,p'-DDT and p,p'-DDD, respectively. 1+3 Figure 3C. Anaerobic and aerobic cultures incubated 3 days and autoclaved cells incubated 3 days. From left to right authentic carbon-l#-1abeled DDT (1); medium of an anaerobic culture incubated 3 days (2); bac- teria of an anaerobic culture incubated 3 days (3); medium of an anaerobic autoclaved culture incubated 3 days (4): bacteria of an anaerobic autoclaved culture incubated 3 days (5); medium of an aerobic culture incubated 3 days (6); bac- teria of an aerobic culture incubated 3 days (7); authentic carbon-lfl-labeled DDT (8). The upper and lower spots cor- respond to p,p'-DDT and p,p'-DDD, respectively. . v I ‘0 O A Figure 3D. Aerobic cultures incubated l and 2 days. From left to right authentic carbon-lu-labeled DDT (l); bacteria of an aerobic culture incubated 2 days (2); medium of an aerobic culture incubated 2 days (3); medium of an aerobic culture incubated 1 day (a). bacteria of an aerobic culture incubated 1 day (5); medium of an aerobic culture incubated 1 day (6); bacteria of an aerobic culture incu- bated 1 day (7); authentic carbon-14-labeled DDT (8). The upper and lower spots correspond to p,p'-DDT and p,p'-DDD, respectively. ’45 Figure 3E. Aerobic culture incubated 2 and 3 days. From left to right authentic carbon-1h-labeled DDT (1); medium of an aerobic culture incubated 2 days (2); bac- teria of an aerobic culture incubated 2 days (3); medium of an aerobic culture incubated 3 days (4); bacteria of an aerobic culture incubated 3 days (5); authentic carbon-14- labeled DDT (6). The upper and lower spots correspond to p,p'-DDT and p,p'-DDD, respectively. . . i . .— . o . . .3 0903'! v 46 Figure 4. Autoradiograms of thin-layer chromatograms (bf carbon-14-labe1ed DDT and carbon-14-1abe1ed metabolites produced by g. aergginosa. sand alites 47 r3 Figure 4A. Anaerobic and aerobic cultures incubated 2 days and autoclaved cells incubated 2 days.‘ From left to right authentic carbon-l4-labeled DDT (1); medium of an anaerobic culture incubated 2 days (2); bac- teria of an anaerobic culture incubated 2 days (3); medium of an aerobic culture incubated 2 days (4); bacteria of an aerobic culture incubated 2 days (5); medium of an anaer- obic autoclaved culture incubated 2 days (6); bacteria of an anaerobic autoclaved culture incubated 2 days (7); au- thentic carbon-l4-labe1ed DDT (8). The upper and lower spots correspond to p,p'-DDT and p,p'-DDD, respectively. h-z.’ 48 -iéi A a . ' ‘ Figure 4B. Anaerobic and aerobic cultures incubated 2 days and autoclaved cells incubated 2 days. From left to right authentic carbon-l4-labeled DDT (1); medium of an anaerobic culture incubated 2 days (2); bac- teria of an anaerobic culture incubated 2 days (3); medium of an aerobic culture incubated 2 days (4); bacteria of an aerobic culture incubated 2 days (5); medium of an anaero- bic autoclaved culture incubated 2 days (6); bacteria of an anaerobic autoclaved culture incubated 2 days (7); authentic carbon-l4-labe1ed DDT (8). The upper and lower spots cor- respond to p,p'-DDT and p,p'-DDD, respectively. 49 Figure 5. Autoradiogram of a thin-layer chromatogram of carbon-l4 labeled DDT and metabolites produced by combining cytoplasmic fractions and membrane preparations of E. coli. From left to right authentic carbon-14-labe1ed DDT(1); 3 ml of boiled membrane + cytoplasmic fraction (2); dup- licate experiment using the components of 2 (3); 3 ml of membrane + cytoplasmic fraction (4); duplicate experiment using the components of number 4 (5); 3 ml of cytOplasmic fraction only (6); duplicate of 6 (7); authentic carbon-l4- 1abeled DDT (8). The upper and lower spots corresponded to p,p'-DDT and p,p'-DDD, respectively. u h a 50 Figure 6. Autoradiograms of thin-layer chromatograms of carbon-l4-labeled DDT and metabolites produced by the add- ition of Krebs cycle intermediates or cofactors to membrane preparations of E. coli. gram M by the ad'- to membra; 51 00.00000 ‘ Figure 6A. Addition of intermediates and cofactors and the omission of ADP, inorganic phosphate or NAD. From left to right authentic carbon-l4-labeled DDT (1); 3 m1 of membrane + 2.0‘pmole each of NAD, NADP, FAD, malate. pyruvate, & 0.1 pmole each of ADP & P04 (2); duplicate ex- periment using the components of 2 (3); 3 ml of membrane + cofactors & intermediates minus ADP & Pot (4); duplicate experiment using the components of 4 (5); 3 ml of membrane + cofactors & intermediates minus NAD (6); duplicate ex er- iment using the components of 6 (7): authentic carbon-l labeled DDT (8). The upper and lower spots correspond to p,p'-DDT and p,p'-DDD, respectively. [III 444.] .1 ; 31.»- I ha. alga“. 52 Figure 6B. Addition of intermediates and cofactors, membrane only and omission of intermediates. From left to right authentic carbon-l4glabe1ed DDT (1); 3 m1 of membrane fraction + 2.0 mole each of cofactors minus intermediates (2); duplicate experiment using the compon- ents of 2 (3); 3 ml of membrane + 0.2‘pmoles each of inter- mediates and cofactors & 0.01'pmole each of ADP & P04 (4); duplicate experiment using the components of 4 (5); 3 ml of membrane only (6); duplicate experiment using the com- ponents of 6 (7); authentic carbon-14-labeled DDT (8). The upper and lower spots corresponded to p,p'-DDT and p,p'-DDD, reSpectively. 'E".‘""“’“‘ ‘3 53 Figure 6C. Omission of FAD, NADP and intermediates. From left to right 3 ml of membrane + 2.0 umole of cofactors & intermediates minus FAD (l); duplicate ex eriment using the components of 1 (2); authentic carbon-1 -labeled DDT (3); 3 ml membrane + intermediates, cofactors, ADP & P04 minus NADP (4); duplicate experiment using the components of 4 (5); 3 ml of membrane + cofactors, ADP & P04 minus intermediates (6); duplicate experiment using the compon- ents of 6 (7); authentic carbon-l4-labeled DDT (8). The upper and lower spots correspond to p,p'-DDT and p,p'-DDD, respectively. ‘ )lllIl 1.. .. r.-. 3.3;); «NJ: I Qua—IL A .00..... ~ ‘; fi .‘ Figure 6D. Addition of FAD, ADP and inorganic phosphate. From left to right authentic carbon-l4-1abe1ed DDT (1); 3 ml of membrane + 2.0 pmole FAD (2); duplicate exper- iment using the components of 2 (3); 3 m1 of membrane + 0.1.pmole each of ADP & P04 (4); duplicate experiment using the components of 4 (5); 3 ml of membrane + FAD, ADP & P04 (6); duplicate experiment using the components of 6 (7); authentic carbon-14-labeled DDT (8). The upper and lower spots correspond to p,p'-DDT and p,p'—DDD, respectively. 55 A Figure 6E. Addition of FAD or NAD or FAD, ADP. P04 & atmos- pheric 02. From left to right authentic carbon-14-labeled DDT 01); 3 ml of membrane + 8.0 pmmle FAD & 0.1.pmole each of ADP & P0 (2); duplicate experiment using the components of 2 (3); 3 m1 of membrane + FAD, ADP & P04 with atmospheric 02 (4); duplicate experiment using the components of 4 (5); 3 ml of membrane + 2.0 pmole of NAD (6); duplicate exper- iment using the components of 6 (7); authentic carbon-l4 labeled DDT (8). The upper and lower spots correspond to p,p'-DDT and p,p'-DDD, respectively. Fla 56 .0000... A O. l c. 0 Figure 7. Autoradiogram of a thin-layer chromatogram of carbon-l4-labe1ed DDT and metabolites produced by the add- ition of FAD, ADP and inorganic phosphate to membrane pre— parations suspended in cytoplasmic fractions. From left to right authentic carbon-14-labeled DDT (1); 3 ml of membrane in cytoplasm fraction + 2.0 umole FAD, 0.1 pmole each of ADP & P0 (2); duplicate experiment using the com onents of 2 (3); 3 ml of membrane in cyto- plasm + FAD ( I; duplicate experiment using the components of 4 (5); 3 m1 of membrane in cytoplasm fraction + ADP & P0 (6); duplicate experiment using the components of 6 (7); authentic carbon-l4-1abeled DDT (8); The upper and lower spots correspond to p,p'-DDT and p,p'-DDD, respectively. - Anna... "I(fli‘lmiflflll‘lfl‘flfl‘lfilTil—)4“