, ...... A STUDY OF THE TERMINAL RESPIRATION 0F THIOBACILLUS TBIOPARUS BI Robert.Chauncey Cooper AN ABSTRACT A study has been made on the terminal respiration of Thio— bacillus thioparus. Whole cell suspensions showed some oxygen up- take in the presence of tricarboxylic acid cycle intermediates. This uptake appeared to be directly proportional to the endogenous rate. Unsupplimented cell free extracts demonstrated no activity against these organic intermediates Evidence has been presented for the presence of various en— zymes of the tricarboxylic acid cycle. No direct evidence could be obtained for the presence of cis-aconitase or fumarase. Tests for the condensing enzyme, citrogenase, were inconclusive. Citrate was shown to be active in the metabolism of 1} thioparus but the actual mechanism involved in its formation was not clear. The en- zyme, citratase, appeared to be absent. From the results obtained it appears that 3, thioparus has a terminal respiration similar to that found in many heterotrophic 0 ‘51 inc of a “3'30 ect b1 “i310 bjec bemietfl Bio grown 08. items 80 5.111 ‘3 derg, «in e acne , Unisersi .S. c 1952 Grade dies , chi gen ELS. in etiology merienc °. aduate 1‘ sea 1963, acter og' 053.5 , caliiow e conege 1 52,53 t “ichig tate ited .k y a dical 9919 t W chigan State tudies Of ciegce v.~wrrfi TO JMJ. ACKNOWLEDGEMENT The writer wishes to express his sincere appreciation to Dr. W.L. Mallmann without whose assistance this thesis would not be possible. "i‘BE‘l‘S ‘YM’U‘S 0? 00‘s introducti \ tera Refiei 4 General \‘etlio 24 Eesuits Section 0101“» Characteristics 2% Sectio s Respiration indies 33 Section 1 t ‘Y H: ixc' 01c tions 41 iscussio Conclusion 66 Suave» 60 Cited 62 mm 3 Regardless of the term u“ed, all the definitions imply a high degree of synthetic ability by these organisms since the bulk if not all the organic material of the cell originates from inorganic sources. How do the organisms that fall into the above classification carry out their metabolic processes? Except for their special ability in assimilating inorganic carbon, it may be incorrect to assume that their basic metabolic patterns are different than those found in heterotrophic organisms. 0n the assumption of uniformity in nature it would be advantageous to approach the metabolism of autotrophic bacteria by considering it in terms of metabolic pat- terns already known to exist in other microorganisms. It may be -best to seek activities that are common to chemolithotrophs and heterotrophs. Some small similarities do exist such as the exist- ence of certain lithotrophic activities which are common to hete- rotrophs, as in Escherichia £211 for example, in which hydrogen + carbon dioxide___=. formate or snccinate + carbon dioxide__;—.alpha- ketoglutarate (Utter and Wood,195l). Some heterotrophic bacteria can utilize carbon dioxide as the main hydrogen acceptor for anae- All these facts indicate that definite similarities in the meta- bolism of these organisms do exist. However, at present there are fev actual facts concerning the metabolic patterns of autotrophic microorganisms. Knowledge of these patterns would clarify the relationship of these organisms to other bacteria and how they fit into the evolutionary scheme of bacterial development. Information of this kind may also be use- ful as another step in shoving metabolic continuity among various forms of life. The purpose of this work is to give some evidence that a chemolithotrophic organism, Thiobacillus thioparus, has a terminal oxidation system similar to many heterotrophic microorganisms. LITERATURE REV”, The genus Thiobacillus includes nine species (Bergy 1957) of chemslithotrophic bacteria which oxidize sulfur compounds to sul— fate. Some members of this group are facultative autotrophs but the majority are obligate autotrophs. The four common species of this genus are Thiobacillus this arus, I; novellus, 3} denitrifi- cans, and I, thiooxidans. Winogradsky ( 1887, 1888) described certain sulfur bacteria, Thiothrix 52,, and this work gave impetus to the study of the sulfur bacteria. Nathanson.(I903) described a group of bacteria found in sea water which oxidized hydrogen sulfide or sodium thie- sulfate and reduced carbonic acid to form intracellular materials. These organisms grew on a completely inorganic medium and did not accumulate free sulfur intracellularly which differentiates them from those described by Winogradsky. Beijerinck (1904) confirmed Nathansons work using a completely inorganic medium with thiosul- fate as the energy source and named the organisms Thiobacillus. In: this instance be isolated two organisms and described them as 3? wmv‘,“ a ’VV' \ consisting of a gram negflti‘re protoplasm containing one or more large vacuoles and that the “III are actively motile. These organisms appear “ be wide-spread in soil and fresh and and sea water. Bunker (1936) reports 1. thigarus to be widely distributed in coastal waters, Iarine muds, fresh water and soil. These organisms are not found in all soils as reported by Starkey (1935) who found sulfur oxidizing bacteria in only two out of twenty-nine samples tested. The position of these organisms in bacterial taxonomy has been questionable for some years. Breed and Conn (1938) stated that the general assumption that such bacteria are the most primitive form of life may well be erroneous and moved that these autotrophs be. taken out of the Nitrobacteriacae and considered either as an inde- pendent family or as a tribe in the family Bacteriacae. Bergy's Manual listed these organisms in the Nitrobacteriacae group until 1957 at which time they were given new status in the order Pseudo- monadales, family Thiobacteriacao. This last classification seems most reasonable because their gram reaction, size, shape and" fla- gellation places them in close relation to the pseudomonads. The thiobacilli are of practical significance in a number of ways. They play a role in the sulfur cycle in which they oxidize reduced sulfur to sulfate which in turn can be reduced by such or- ganisms as Desulfovibrio pp. Gleen and Quastel (1953) feel that the thiobacilli as well as some heterotrophs supply intermediates to soil which may play a beneficial role in the manganese up-take of certain plants. Reports have been made that l. thiooxidans “Tr—f causes the corrosion of sulfur—containing pipe line seals. Frederick and Starkey(1948)state that this is a minor problem, but have shown that thiobacilli will attack some pipe sealing mixtures. Certain members of the group are thought to cause the corrosion of concrete (Parker and Prisk51953) and 2, denitrificans has been re- ported by Robotonova_g§_g;4 (1950) to break down crude asphalt. The acidic properties of mine drainage is usually ascribed to mem— bers of the thiobacilli (Colmer and Hinkle91947). Temple and Delchamps (1953) have postulated a sequence of synergistic events which cause acid mine water. The hypothesized synergism exists between !, ferrooxidans, an organism which can oxidize iron or thic- sulfate, and.2, thiooxidans as follows: Non-biological: FeS2 + séo + 3% 02 a FeSO4 + H2804 Biological—(T: ferrooxidans): 2Fe304+é02+u so - Fe2(SO 2 4 ‘ *Héo 4)3 As the Fe2(504)3 is formed it reacts with finely divided py- rite present as follows: Non-biological: Fe2(SO4)3 + FeS2 = aseso4 + as Biologica1(!} thiooxidans): s + 1&02 + H20 . 23* + so4 mF».r-m—_ , oxidation of elemental sulfur. Beijerinck (1904) proposed the following as the over-all reaction carried out by 1, thioparus when thiosulfate is oxidized: O (1) 110.23203 + log = Ha2804 + s This is a rather simple equation and has been proposed in more detail by Starkey (1935) as: O (2) 5Na28203 + H20 + 402 _ smaso4 + H2804 + 45 This equation seems to hold true for all the strictly aerobic spe- cies of thiobacillus. Thiobacillus denitrificans under anaerobic conditions utilizes nitrate rather than oxygen in the following manner (Foster, 1951): (a) oxno + as + 2H20 - K 280 + 4KBSO4 + 3N 3 4 2 All of these oxidations result in the formation of sulfate with the production of acid. In the case of equations (1) and (2) sulfur is also an end product. From their experiments with I, thiooxidans Vogler and Umbreit (1941 a,b) concluded that the organism must be in direct physical contact vith the sulfur particle before sulfur oxidation can take q-_.~._..fl. (1956) demonstrated that elemental sulfur was oxidized more rapid— ly in shaken than stationary medium. This indicates that direct contact may not be required as proposed by Umbreithg_ El, Knaysi (1943) found vacuoles in cells of I, thiooxidans to contain both volutin and sulfur and proposed that these materials might be mis- taken for fat globules. It seems curious that these organisms which use sulfur in their metabolism should in turn produce free sulfur as an end pro- duct. Starkey'(1935a) demonstrated that 40 percent of the oxida- tion products of I, thioparus was elemental sulfur and 60 percent sulfate. He concluded that this elemental sulfur arises by a specifi— ic biological reaction which is part of the mechanism of thiosulfate oxidation. Work has been done since which indicates that the sul- fur is produced by a non-biological mechanism. Tamiya .33.31‘ (1941) state that sulfur is produced by a non-biological decompo- sition of pentathionate to tetrathionate and sulfur. He sums up the oxidation of thiosulfate in the following reactions: (4) $20; + 0.2502 + 0.5n20 . 0.5540; + ofl (5) 2340; = $30; + $50; (6) 550; . $40; + 5° (7) $30; + n20 . s20; + so; + 23* (8) $306 + 202 + 2n- - + 20 a 3804 + 4H Only reactions (4) and (8) are thought to be biological. The renmiinder are carried on by spontaneous chemical reactions. 'Vishniac (1952) and Vishniac and Santer (1957) agree with Tamiya g£__l. that sulfur produced by I, thioparus is a result of purely non-biological mechansims, but disagree in that they feel thiosul- fate oxidation proceeds through the intermediate formation of te- tra, tri and dithionates. Using 15 thioparus these workers have postulated the following pathway for sulfur oxidation: Extracellular Intracellular (1) (2) (6) In this scheme sulfide enters the cell (9) and is oxidized to sulfur (l). The sulfur undergoes an enzymatic condensation vith sulfite (2) to form thiosulfate which is oxidized (3) to form te— trathionate. Thetrathionate is oxidized (4) to trithionate and ‘41 form trithionate and pentathionate. This reaction is catalized by thiosulfate. The pentathionate, in the presence of thiosulfate will produce sulfur and tetrathionate (8). This scheme will ex- plain all the phenomena noted in growing cultures. Reaction (1) has been observed in other organisms; reactions (8) and (4) have been observed in 15 thioparus. Reactions (7) and (8) are known in— organic phenomena. Reactions (2),(5),(6) and (9) are conjecture on the part of the authors. The method by which elemental sulfur gets into the cell is o- pen to much speculation. Starkey (1937,1937a) has reported the ability of 2, thioparus and T, thiooxidans to reduce elemental sul- fur to hydrogen sulfide. This reaction may point to the Presence of active sulfhydryl groups in the sulfur bacteria such as found in glutathione. This may be the means of transport in reaction (9) above and may be schematically represented as follows: Glutathione . SH + HS. Glutathione + s° . Glutathione . s—s. Glutathione + 23* + s‘ Starkey stated that this was an unlikely mechanism because, accord- ing to his calculations, it was thermo—dynamically impossible. T. 'thiooxidans could oxidize sulfur in the absence of CO2 and sub— sequently these same cells will fix 002 in the absence of sulfur. The amount of €02 fixed in the absence of sulfur is limited to about 40uL 002/100 gms. of bacterial nitrogen. This work showed that the oxidation of sulfur and the fixation of CO were indepen- 2 dent though related entities. This worker also felt that some of the oxygen of the final product, sulfate, arises from carbon diox— ide. Vogler and Umbreifi (1943) continued this work and indicated that the oxidation of sulfur is coupled with the transfer of inor- ganic phosphate from the medium to the cells and that CO2 fixation returns inorganic phosphate to the medium.This was apparent in their results which showed that the oxidation of sulfur in the absence of 802 removed inorganic phosphate from the medium and similarly the same cells, freed of sulfur, and in a 002 atmosphere, added inor— ganic phosphate to the medium. It was postulated that the energy of sulfur oxidation was trapped and transported in the form of phos- phate esters. Newburgh (1954) repeated this work in regards to the uptake of inorganic phosphate but felt that the simultaneous pre— sence of 002, oxygen and sulfur is required since he could not show it to be independent of any of these substances. he also showed that CO2 fixation can be separated from sulfur oxidation but that no inorganic phosphate liberation could be demonstrated. Baalsrud and Baalsrud (1952) report in their work with various spe- {WNW' i'ya: : _ however, had been observed during the oxidation of thiosulfate. lhnbreit (1954) repeated the work he had done in 1943 since many questions had been raised about his conclusions. This later work upheld his original hypothesis. LePage and Umbreit (1943) isolated a number of organic ma- terials from I: thiooxidans. Among the isolates was a compOund very similar to adenosine triphosphate (ATP). Later (1943a) these workers defined this compound as ATP and stated that it contained ribose-3-phosphate rather than the usual ribose—S-phosphate of bac— teria. However,Barker and Kronberg (1954) made a very careful analysis of the ATP found in 23 thiooxidans and discovered it to be adenosine-5—triphosphate. They could find no evidence for any other type of ATP. Vishniac and Ochoa (1952) suggest that the oxidation of sulfur may be coupled to electron carriers which in turn may re- duce 002 within the cell. Oxidation of these electron carriers could yield high energy phosphate compounds and this esterification could account for the disappearance of phosphate from the medium. A diagramatic representation of such a scheme follows: oxidation _ x03. X0112 002 602 reduced The "I" in the above scheme represents the electron carrier. The 13 .\ — -m'"-— 'reoxidation of which may yield the energy for 002 fixation. This electron carrier could possibly be an iron containing system since Vogler gt_gl. (1942) have shown that azide and similar compounds inhibit sulfur oxidation. More recently Marqulies gt_gl, (1958) have showu that T, thioparus consumes less oxygen when oxidizing thiosulfate in the presence of buffers other than phosphate. Thio- sulfate and tetrathionate disappear and chromatography shows the accumulation of sulfur containing compounds not found when phos- phate is present. This seems to indicate that phosphate is in- volved in the oxidation of some intermediate which is produced dur— ing the oxidation of thiosulfate to sulfate. The organism's system of sulfur oxidation and energy exchange appears to be extremely inefficient. As stated previously, I} 32127 22:23 produces 40 percent sulfur and 60 percent sulfate. The pro- duction of the former appears to be a great energy loss. In con— tinuing this vork Starkey (1935) calculated that the machine ef- ficiency of this organism was quite low since the cell utilizes only 4.7 percent of the energy liberated in the oxidation of sul- fur. The photosynthetic process, calculated in the same manner, is 80 percent efficient, a considerable difference. Starkey also Organisms to heterotrophs by the isolation of known metabolic in- termediates. Starkey (1925) demonstrated that glucose disappears from the sulfur medium during active growth but is not removed in the absence of sulfur. He also found that ammonia is the only source of nitrogen. Urea, peptone and amino acids could act as neither a nitrogen nor carbon source. Vogler (1942) showed that I, thiooxidans has an endogenous oxygen uptake in the absence of sulfur. He stated that this indicated the utilization of organic materials which must have been previously synthesized by the chemo- synthetic process. Such growth factors as nicotinic acid, panto- thenic acid, biotin, riboflavin, thiamin and pyridoxine were iso- lated from cell extracts by O'Kane (1942). None of these compounds alone or in combination stimulated growth when added to the growth medium. Because of the endogenous respiration in 1} thiooxidans, LePage (1943)proposed that there must be a storage substance which sustains life. He determined this to be a polysaccharide containing‘ glucose,mannose and some other reduced material.In the same experi— ments in which LePage and Umbreit (1943) isolated ATP they also isolated, from T, thiooxidans: fructose 1—6 diphosphate, phospho- glyceric acid, fructose-G—phosphate, glucose-S—phosphate, glucose- l—phosphate and presumptive evidence for the presence of coenzyme I. From this work they concluded that the internal metabolism of acids were tested. They either inhibited respiration or had little or no effect. Rittenberg gt §}¥(1950) succeeded in obtaining a mu— tant of 2, thiooxidans by ultraviolet irradiation which required yeast extract for growth. This again points to a heterotroph-like metabolism. Skarznski £t_gl.(1956) isolated a new cytochrome for 1. thioparus which they called cytochrome S and which appeared to be different from any known cytochrome. Aubert £t_gl.(1958,1958a) and Milhaud gt_gl,(1958) demonstrated a cytochrome C in E. denitri— ficans. This is of interest since this organism is a facultative anaerobe and the cytochrome transports electrons to nitrate or oxy— gen depending upon the method of growth. All of this work keeps adding to the general fund of knowledge but it has not been well or— ganized into any definitive scheme. Until recently the mechanism of carbon dioxide fixation in chemoautotrophic bacteria had remained obscure. BOWever, the work of Calvin on photosynthesis has opened the door to this problem. Calvin and Massini(1952) working with algae and using 01402 looked for the first compound they could find in which the labeled carbon dioxide was incorporated. This compound was phosphoglyceric acid. Bassham and co-workers (1954) demonstrated that ribulose 1-5 di- phosphate was the acceptor compound for CO in green plants. These 2 ' “‘3‘ — ’ ‘_ .20....04um. Hz... 2. ou>40>z. mm430m402 “.0 cumin... NI... «9.20.02. mauzhzucf. z. cumin: “3...... .In. ummOoEan VIIV .zaaémoSmEv .3..- $323. a 5.33.: 2.3 umjguxmzéf It umo.¢...N .:....umo khmuzooum N um<4oo4xu .z...umo¢:§¢u a x x , t u. .9 ~ .23.. 3235. a < V .zaémgaec u av: u / .8 £9.33. 23 u. umzm04 cuorna «.4... .zmhu. inauuhizmofia u. 2.3. 5'...“ 2:3: :2: out9". ' ‘0‘: r".‘P-M-~s suasv.-r~v- ,.-_ 4‘ Weave-0.. Reactions with: Isocitrate (aerobic) test 0.31 0.69 control l -- 0.65 Isocitrate (anaerobic) test i —— 0.00 control % -——- 0.66 ”L.wu-.me-.“.~_fl--M."mN-_Mm-MM 47 the reaction of isocitrate, TPN and cell free extract. This is indicated by the production of a spot which has the same Bf as known alpha—ketoglutarate. No glyoxylate was found under aerobic or anaerobic conditions which would indicate that isocitratase was not present. The spots giving an Bf value in the area of 66‘ are due to excess phenylhydrazone. PART C The purpose of these experiments was to demonstrate succinic dehydrogenase activity in T, thioparus. Two methods were used both based on the reduction of methylene blue in the presence of cells and succinate. These methods were described by Burris (1945). The first employs the Thunberg technique. The Thunberg tube con- tained.2 ml of 0.04M succinate, 0.1 ml of 1-1000 methylene blue, 2 ml of 0.1B phosphate buffer, pH 7.4, and one ml of cells. The cells were washed from 48 hour agar slants. The tube was evacuated for 10 minutes and incubated at 306 until the dye was reduced. The second method was carried out in agar. This medium contained 0.5 m1 of 0.004I.succinate, 0.5 ml of 1-1000 methylene blue, 2 ml of 2 percent agar in 0.1M.phosphate buffer at pH 7.4. The agar was melted and cooled to about 450 and added to a four inch test tube containing the other ingredients plus one ml of cells and allowed to solidify. The tube was incubated at 306 until the leuco form appeared. 48 These tests were quite qualitative since whole cells had to be used. Whole cell suspensions contain so much sulfur that photo- metric measurements are impossible. In the Thunberg tube the cells took 45 minutes to completely reduce the methylene blue while the control without succinate was reduced in 135 minutes. The same results were obtained using the agar method except that the en- dogenous never reached the complete leuco form. These results indicate the presence of succinic dehydrogenase in these cells since the dye is reduced considerably faster in the presence of succinate. The endogenous reduction is most probably due to the presence of colloidal sulfur which is being oxidized and in turn reducing the methylene blue. These same tests were carried out using acetone powders and cell free extracts. The results were all negative. In most or- ganisms the enzyme succinic dehydrogenase is associated with the cell wall. This would also seem to be the case with 2, thioparus because of the negative results with acetone powders and cell free extracts. Acetone drying appears to have a drastic effect on cell permeability which would indicate an effect on the cell wall. It would probably follow then that enzymes associated with the cell wall would be affected as well. Malic dehydrogenase determinations were made using the method 49 of Mehler 312;. (1948). This method is based on the absorption of ultraviolet light at 340m/a by reduced lPN. One ml of cell free extract (1 mg protein per ml) was added to a quartz curvett con- taining 0.1]! tris buffer, pl! 7.4, lOpMOlGS L—malate and 1.5,“,moles of RN oxidized. The total volume was brought to 3 ml with buffer. The reaction was carried out at room temperature and the change in optical density with time was recorded. The results of these experiments are illustrated in FIGURE 6. The amount of IPN reduction was quite small; a total of 0.1081» moles in five minutes. The overall reaction was relatively rapid reaching the peak of activity in two minutes. The reaction rate at zero time was approximately a change in optical density of 0.4 per minute or 0.19/4. moles IPN reduced per minute. However the fact that activity was present and not the rate or amount of NH reduc- tion is the important thing here. No activity was obtained with this system in the presence of TPN indicating that the malic dehy- drogenase of these cells is IPN specific. A number of attempts were made to demonstrate fumarase ac- tivity in this organism. The method of Backer (1950) was employed in which the production of fumarate from L-malate is followed by measuring the absorption of ultraviolet light between 240 to 800m». Fumarate absorbs ultraviolet light at this wave length in propor- tion to its molar concentration. One ml of cell free extract was added to a curvett containing lOp-m01es of L-malate and 0.ll|' tris 50 FIGURE 6. THE RENCTION 0F ll’N BY CELL [BEE EXTRACTS 0F TRIOBACILLUS TBIOPARUS IN THE PRESENCE OF L— nun-E. 1 .20 r J ). L". CD 2 M O .J € 2 .— fl. . 10 P l 1 1 + 2 l3‘ 4: 5 TIME IN MINUTES 51’ ‘buffer, pH 7.4, with a final volume brought to 3 ml with the buf- fer. In all these experiments no activity was observed. Since phosphate is a known activator of fumarase, the tris buffer used above was replaced with 0.1! phosphate buffer, pH 7.4, but the re— sults were still negative. The wave length used with this method presents the same problem as was encountered.with attempts to demonstrate aconitase activity. The cell free extracts of 2, thioparus absorb highly at these wave lengths and this absorption might.mask fumarase activity if the activity were very small. A series of experiments was carried out in order to find evidence for the production of citric acid from acetate, pyruvate and oxalacetate. One ml of cell free extract was added to warburg vessels containing 0.IH tris buffer, pH 7.4, and 10 moles of sub- strate. The total volume was brought to 3 ml with buffer.. The reactions were carried out with shaking at 300 for 60minutes. The reaction was stopped by the addition of 0.5 ml of 10 percent phos- photungstic acid in 0.5! HCL. The precipitate formed.was fil- tered off using Whatman #50 paper and the reantant filtrate tested for citric acid. The method employed for the determination40f citric acid was that of Ettinger 2!: :1, (1952) in which any citrate 52 present is converted to the penta bromoacetone by the addition of- KHnO4 and KBr. The excess KMnO4 was removed with 3202 and the bromoketone extracted with Neheptane. The heptane layer was then added to a pyridineéKOB mixture and heated to 806 for four minutes and.immediately cooled to 00. This last step results in the de- velopment of a red color in the pyridine layer, the intensity of which is proportional to the concentration of citrate present. The entire procedure with the exception of heating the pyridine mixture was carried out at 50. These experiments indicated that citrate was formed by cell free extracts of I, thioparus in the presence of pyruvate and oxalacetate but never in the presence of acetate alone. Attempts to show the presence of the condensing enzyme, citrogenase, by demonstrating an increase in citrate production when pyruvate or acetate plus oxalacetate were added to the cell free extract were uncertain in that quantitative results varied from one run to an- other. Citrate was produced in all cases; however, in one instance a mixture of pyruvate and oxalacetate or acetate and oxalacetate would produce more citrate than pyruvate or oxalacetate alone. In the majority of cases the citrate produced in the presence of the afore mentioned compounds would only slightly exceed, or not exceed at all, that of oxalacetate alone. The over-all trend of these reactions indicate that these cells produce as much citrate from oxalacetate as they do from the combination of acetate and 53 oxalacetate or pyruvate and oxalacetate. It should be reiterated, however, that these results were erratic and that the only defi- nite information obtained was that these cells can produce citrate in all cases except in the presence of acetate. This method is supposedly specific for citrate; however dur- ing the course of these experiments the pyruvate and oxalacetate controls consistently developed a red color some times more intense than others. These non—specific reactions may be the cause of the discrepancies in the results obtained. 54 DISCUSSION AND CONCLUSION Using standard Warburg techniques it was shown that the or- ganism used in this study rapidly oxidizes thiosulfate. The en- dogenous respiration rate, which is often quite high in whole cell suspensions due to residual sulfur, may confuse the picture of oxygen uptake and give misleading results. This same situation has been discussed rather thoroughly by Baalsrud and Baalsrud (1954) working with Thiobacillus denitrificans. When cell free extracts are employed,the oxidative activity is reduced to a con- siderable degree, about one-tenth the activity of whole cells as measured by thiosulfate oxidation. Respiration studies carried out in the presence of tricar- boxylic acid cycle intermediates showed some interesting results when whole cells were used. The rate of oxygen uptake was by no means equal to that in the presence of thiosulfate but under cer- tain conditions a considerable amount of oxygen was removed by 3} thioparus in the presence of these intermediates. Again as in thiosulfate oxidation studies, there was a high endogenous rate due to the carry-over of sulfur. It appeared that the oxygen up- take was directly related to the amount of oxygen removed by on- dogenous respiration. The higher the endogenous rate the greater the oxygen uptake. However, the ratio of oxygen uptake in the 55 presence of substrate to the endogenous oxygen uptake appeared to he a relatively constant ratio. Thiosulfate oxidation, on the other hand, indicated more independence from the endogenous rate in that the ratio of substrate oxidation to endogenous activity became smaller as the endogenous rate became higher. This relationship of endogenous respiration to substrate oxidation may be speculated upon in the following manner. This organism being an autotroph normally does not metabolize organic materials. However, Vogler £t_gl. (1942) have presented some evi- dence that the addition of C4 dicarboxylic acids enhances the res- piration rate of 2, thiooxidans. It may be possible that a truly resting cell can only transfer the compound across the cell wall very slowly but if an active oxidation such as a high endogenous rate is being carried out the "permease system" is activated and the compounds gain access to the cell interior more readily. An analogous situation is that reported by Gale (1948) in which glu- tamic acid could not enter into cells of staphlococci unless glu- cose was also present. Cell free extracts of 3} thioparus did not possess any acti— vity against these organic intenmediates. This result does not necessarily mean that the cells are incapable of utilizing these compounds. A number of reasons for this inactivity may be as fol- lows: the activity as measured with thiosulfate oxidation is re- duced about ten-fold, there is a large dilution and loss of order, a loss of required co-factors and probably denaturation of some of pmte in. The only conclusion to be drawn at this point is that whole cells show an increase in oxygen uptake in the presence of tricar- boxylic acid cycle intermediates and that this uptake appears to be dependent on the endogenous rate. The respiration studies gave no evidence for the presence or absence of a tricarboxylic acid cycle in this bacterium. Therefore, a series of experiments was carried out to obtain evidence for or against the presence of certain reactions common to the afore men- tioned cycle. Isocitric dehydrogenase determinations were quite successful. It was by far the most active enzyme determined. Aconitase could not be demonstrated directly but it was shown that IPN is reduced, at different rates, in the presence of citrate and cis—aconitate as well as isocitrate. These reactions were all TPN specific. More evidence for the presence of isocitric dehydrogenase was found by the demonstration that alpha-ketoglutarate is the product of this dehydrogenase activity and also that alpha-ketoglutarate is an intermediate in these reactions. From the experiments performed it would appear that the en- zyme, isocitratase, does not play a role here since glyoxylate could not be demonstrated when isocitrate and cell free extracts were reacted together under anaerobic conditions. Obviously these negative results are not conclusive since there may be certain 575 v- .7, a. V- -7- co-factors or conditions necessary in this particular organism about which we are unaware. Succinic dehydrogenase activity was measured qualitatively by methylene blue reduction and it would be reasonable to assume that succinate arises from alphaqketoglutarate although this lat- ter reaction was not'measured. In the typical tricarboxylic acid cycle succinic dehydrogenase activity results in the formation of fumarate. However, fumarase activity could not be demonstrated in cell free extracts of this organism yet malic dehydrogenase acti- vity could be shown. This latter reaction was not very strong since only 0.hp.mole of DEN was reduced. However the reaction was positive and was specific for’nPN. From this experiment one would assume fumarate to be an intermediate since both succinate and ma1-. ate are oxidized by the cell extracts and fumarate is normally the bridge compound between these two. Attempts to show the presence of the condensing enzyme were disappointing. Citrate was produced by cell free extracts in the presence of pyruvate and oxalacetate but not in the presence of acetate. Attempts to show the citrogenase enzyme by demonstrating an increase in citrate production in the presence of pyruvate and oxalacetate or acetate and oxalacetate were not conclusive since the results were erratic. In the majority of cases it seems that as much citrate is produced from oxalacetate and cell free extract as when oxalacetate is in combination with pyruvate or acetate. These results indicate that citrate is apparently an intermediate 58 in these cells and that oxalacetate alone or pyruvate alone may have to be in some active state before it will react to form cit- rate. Whether the citrate is fonned by a typical condensation reaction of an active acetate and oxalacetate or by the condensa- tion of two pyruvate molecules as reported in Aspergillus gigs: (Cleland gt_gl, 1964) or by some other mechanism remains to be seen. From these results it seems reasonable to conclude that these organisms have a terminal respiration which is very similar to that found in heterotrophic microorganisms. The data obtained appear to agree, in most particulars, with the well established tricarboxylic acid cycle. The weakest points in this conclusion are to be found in the lack of direct evidence for the enzymes aconitase and fumarase and in the inconclusive results in deter- mining the condensing enzyme, citrogenase. More work will be re- quired to clarify these points. This organism shows some respiratory activity on these com- pounds. The activity appears to be related to the endogenous res- piration rate and may indicate a relationship between cell per - meability and state of activity or presence of an essential sub- strate. 59 , rim" — .as‘i. _1 I; ‘s-‘ I. SUMMARY Thiobacillus thioparus grew quite slowly and all attempts to stimulate the growth were negative. The rate of growth was depen- dent on the size of the original inoculum. Whole cells of this organism rapidly oxidized thiosulfate as measured by the standard Warburg technique. Cell free extracts possessed a diminution of oxidative activity of about one-tenth that found in whole cells. Whole cell suspensions showed some oxygen uptake in the pre- sence of tricarboxylic acid cycle intermediates. This uptake ap- peared to be directly proportional to the endogenous rate. Cell free extracts contained no activity against these organic inter- mediates. Evidence was presented for the occurrence of various enzymes of the tricarboxylic acid cycle. No direct evidence could be ob- tained for the presence of cis-aconitase or fumarase. Tests for the condensing enzyme, citrogenase, were inconclusive. Citrate was shown to be active in the metabolism of 2, thioparus but the actual mechanism involved in its formation was not clear. The enzyme, citratase, appeared to be absent. From these results it appears thatuz, thioparus has a ter- minah respiration similar to that found in many heterotrophic 60 —' H‘J‘r' Inicroorganisms. This respiration is the same as or closely relat- ed.to the conventional tricarboxylic acid cycle of Krebs. 61 LITERATURE CITED Ajl, S.J. 1950 Acetic acid oxidation by Escherichia coli and Acre- bacter aerogenes. l. Bacteriol., 59, 499. bacteria. VI comparative patterns of acetate oxidation by citrate grown and acetate grown Aerobacter aerogenes. ._J_. Gen. 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