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The influence of iron on the metabolism of actinomyces with special reference to the rate of proteolysis… Hicks, Winnifred Odetta 1941

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THE INFLUENCE OP IRON ON THE METABOLISM OF ACTDTOICCES WITH SPECIAL REFERENCE TO THE HATE OP PROTEOLYSIS I I MILK Winnifred Odetta Hicks A Thesis submitted i n P a r t i a l Fulfilment of The Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE i n the Department of. AGROKOM The University of B r i t i s h Columbia A p r i l , 1941. AGE¥OWLl.I)GIffiM! The writer wishes to acknowledge the assistance received, from the National Research Council during the year 1939-40o Laboratory f a c i l i t i e s were provided by The University of B r i t i s h Columbia throughout the period of investigation* The writer ^also wishes to express her deep appreciation to Dr. D. G-. Laird for invaluable advice and c r i t i c i s m during the course of the study* TABLE OF CONTENTS Page IHTRODUCTIOF 1 REVIEW OF LITERATURE... 2 EXPERIMENTAL .................... 5 Part A P r o t e o l y t i c Studies 5 Media...............*.. 5 Preliminary Experiments. V Procedure... . 8 Stimulatory Effect of Iron. 9 S t a b i l i t y of Actinomyces on a S o i l Medium. 14 Effect of S o i l Ash 18 Effect of Trace Elements.......... 20 Discussion and Summary - Part A. 24 Part B - Studies on Oxygen Requirements 28 Methylene Blue Studies. 28 Influence of Iron under Anaerobic Conditions........ 35 Discussion and Summary - Part B..................... 38 Part C ^ Enzyme Studies. 41 Influence of Iron on the Proteolytic Enzyme Complex® 41 Discussion and Summary - Part C...................v" 45 GMERAL SUMMARY * 47 BIBLIOGRAPHY 50 THE INFLUENCE OF IKON ON THE 1ETABOLISH OF ACTINOHICES - WITH SPECIAL REFEEENCE TO THE RATE OF PROTEOLYSIS IN MILK I. p.TRpPUOTIOg : A s t r i k i n g p e c u l i a r i t y of the Actinomyces, as commonly observed, i s t h e i r v a r i a b i l i t y in morphology and cu l t u r a l characteristics. Whether or not t h i s i s an inherent character of the members of the group, or mere-* l y the result of the a r t i f i c i a l conditions under which the cultures are grown i n the laboratory, has never been made clear. I f the l a t t e r i s the case, the lack of some essential factor might well he the cause of the variation encountered. It i s reasonable to suppose that i f the different species are naturally unstable, they would remain so under a r t i f i c i a l conditions i n the laboratory* I f , on.the other hand, they are normally stable, i t should be possible to determine the factor which would maintain them at a high and consistent l e v e l of efficiency* In an endeavor to s t a b i l i z e the actinomycetes, the various factors which might conceivably exercise an influence on t h e i r proteolytic a c t i v i t y were reviewed. Recalling that the s o i l contains a wide variety of metallic elements, many of which stimulate the physiological a c t i v i t y of certain microorganisms, and recalling that iron i s essential for the formation of the respiratory pigments i n both plants and animals, i t was decided to study the influence of trace elements with particular reference to iron. Since the decomposition of protein i s one of t h e i r most important functions i n nature, proteolytic a c t i v i t y was selected as the basis for determining their efficiency and response to trace elements* I I . REVIEW OF LITERATURE. The genus Actinomyces has i n the past been given many appellations, such, for instance, as Streptothrix, Nocardia, Ray Fungi, Oospora, Discomyees, and Cladothrix. Oohn (10), who i n 1875 f i r s t isolated an Actinomyces, referred to i t as Streptothrix Foesteri. The name "Actinomyces was f i r s t employed by Harz (23), who isolated and described Actinomyces bovis in 1877. Trevisan (47) applied the term "Nocardia" to a saprophytic form, but according to Waksman (53), there i s no authority for separating the p a r a s i t i c and saprophytic forms. Domec (14) and Drechier (15) made morphological studies on the oasis of which the l a t t e r c l a s s i f i e d them with the Hyphomycetes. They showed that the terms "Oospora", "Streptothrix" or "Discomycetes" cannot oe applied since the actinomycetes are u n i c e l l u l a r . Waksman (50) agreed with Isiadson (35), who suggested that they form a special group of fungi to be c l a s s i f i e d separately. Olaypole (9) con-cluded that this group should be looked upon as representing the ancestral type of both the higher fungi and the true bacteria, while i n 1939 Newcomer (36) demonstrated nuclear morphology by the Feulgen technique which showed a s i m i l a r i t y to the fungi rather than to bacteria. The saprophytic forms of Actinomyces were more or less ignored prior to 1900. Beijerinck (4) i n 1900 studied actinomycetes i n nature and investigated th e i r a b i l i t y to form quinone. Muntor (33) and Fousek (19), Hiltner and Stormer (28), Conn ( l l ) and Krainsky (32) investigated t h e i r numbers and di s t r i b u t i o n throughout the s o i l p r o f i l e , Glaypole (9) and Sanfelice (42) were perhaps the f i r s t to record the great v a r i a b i l i t y i n morphology and cultural characteristics of these microorganisms, while Krainsky (32), Waksman and Curtis (57), and Conn (12), (13), came to the conclusion that pigment production was variable and hence an i n -sufficient basis for characterization of the species. Skinner (44), (45), i n 193S and 1939 discussed the tyrosinase reaction of the actinomycetes i n relation to pigment production, and i n the same year Kedrovskii (31) concluded, i n agreement with the earlier workers, that pigmentation should not constitute a basis for classification,, Katznelson (30) des-cribed a thermophilic strain which was subject to autolysis, while Hassegavm, et a l (24) suggested mutation i n t h i s group of microorganisms. Waksman (56) i n 1940 revised his system of c l a s s i f i c a t i o n , which i s now based on morphology rather than on a wide variety of cu l t u r a l character-i s t i c s . The physiological requirements and responses of actinomycetes have been studied q u a l i t a t i v e l y by many workers with a view to c l a s s i f i c a t i o n , while quantitative measurements have been rarely reported. Waksman (52) in studying the proteolytic action of these organisms i n milk with a view to di f f e r e n t i a t i o n between the species, measured amino nitrogen production using the micro-apparatus of Van Slyke and the F o l i n aeration method, and came to the conclusion that the variations encountered, although often very s t r i k i n g , were of a quantitative rather than a qualitative nature. The temperature requirements of the actinomycetes have oeen more or less f u l l y investigated. According to Waksman (55), the optimum for most species i s 25° to 28° C, with a maximum of 40° C and a minimum "between 18° and 20° C. Krainsky (32) reported that most species grew oest at 30° C, while a few showed optimum growth at 35° C and A. citreus has an optimum at 26° 0. Domec (14) and Foulerton and Jones (18) studied the thermal death points of the spores and mycelial growth* Giloert (20) i n 1904 isolated a thermophilic species with an optimum tem-perature of 55° 0. The oxygen requirements of the species do not appear to he well understood. The earlier workers believed the pathogenic types to be anaerobic and the saprophytic types, aerobic. Beijerinck (4) c l a s s i f i e d them as facultative anaerobes, while Musgrave, Clegg and Polk (34) stated that the actinomycetes are neither s t r i c t aerobes nor s t r i c t anaerobes. Waksman (50), (53) concluded that they are not s t r i c t anaerobes, but that some may be able to thrive under semi-anaerobic conditions. A search of the l i t e r a t u r e has f a i l e d to reveal any direct information regarding the oxidation-reduction potentials of the saprophy-t i c forms, but Hagan (22) described the formation of hydrogen peroxide by A. neerophorus on exposure to a i r . Among the papers of. a widely diverse nature which have appeared i n recent years i s that of V/aksman and Woodruff (61), who reported the production of bacteriostatic and bactericidal substances by s o i l actinomycetes, and of Patrick, et a l (.40) who described a species of Actinomyces which attacked xylan strongly, but which did not attack xylose or any of the common carbohydrates or alcohols. ITo publication has appeared, as far as the writer i s aware, re l a t i v e to the u t i l i z a t i o n of iron i n the metabolism of the actinomycetes. f • : : " ? I " ' ' ' 'I n • • • if • " • • - 5. -However, the importance of the heavy metals i n the n u t r i t i o n of fungi and certain bacterial species has Deen known for a considerable period of time. Gottheil (21) as early as 1901 used traces of metallic iron for the c u l t i v a t i o n of s o i l bacteria. lyem, et a l (29) stated that the addi-tion of iron oxide to the s o i l caused an increase i n the numbers of bac-t e r i a , fungi, and actinomycetes present. Foster (17) i n 1940 ably reviewed the l i t e r a t u r e on the necessity of the heavy metals i n the n u t r i t i o n of the fungi, while Steinberg (46) showed that certain cations are essential for bacteria associated with nitrogen f i x a t i o n , and Roberg (41) indicated their importance for the optimum growth of the green algae. The considerable volume of l i t e r a t u r e dealing with the necessity of the heavy metals for the pathogenic bacteria i s considered outside the scope of t h i s study. The increasing interest i n the heavy metal n u t r i t i o n of a l l types of microorganisms, and the fact that iron i s of prime importance i n many l i f e processes of plants and animals, prompted an investigation of the influence of t h i s element on the physiology of representative cultures of Actinomyces. I I I . EXPERIIMTIL Following considerable preliminary investigation proteolytic a o i l i t y appeared to be the most suitable measure of a c t i v i t y . Actinomycetes representative of species found i n the upland g l a c i a l s o i l of coast regions of B r i t i s h Columbia were used for this purpose. PART A - PROTEOLYTIC STUDIES Media Skim milk was chosen as the oasic medium for measuring the rate of proteolysis, since, despite i t s heterogeneity of constitution, i t i s a convenient source of native protein and supports good growth of a l l strains of Actinomyces (59), (60)» Although freshly skimmed milk from the University farm was f i r s t employed as the medium, i t s use was abandoned early i n the study when the nitrogen content was found to vary somewhat from time to time. Skim milk powder from the Fraser Valley Milk Producers" Association plant in- Vancouver was substituted, and used at a concentration of 10 grams per 100 ml. of d i s t i l l e d water. The nitrogen content of milk prepared i n t h i s v/ay has remained r e l a t i v e l y constants While there i s considerable variation i n the analyses of milk from individual cows, the average composition as presented by the Associates of Rogers (2) i s ; Water 87.27 per cent Casein 2.95 " " Albumin .52 " " Fat 3.66 ,f " Lactose 4.91 " " Ash .69 " n According to Babcock (3), the ash has the following constitutions KgO 25.02 per cent CaO 20.01 " 11 l a 2 0 10.01 " " IfeO 2.42 " " FegQg .13 ". '* . P 20 5 24.29 " " CI 14.28 " " S0 3 3.84 " " Of the trace elements, copper ranges between 0.2 to 0.8 ppm. of whole milk, zinc between 3.6 and 5.6 ppm., s i l i c a about 2 ppm., and Iffig, 3 to 4 ppm. In addition s traces of aluminum, manganese and iodine are said to occur. The non-protein nitrogenous constituents include urea, amino nitrogen, creatin, creatinin, and u r i c acid. Traces of thiocyanic acid, choline and methyl guanidine have also been reported. While i t i s admitted that milk i s not a perfect medium, due to the presence of iron, trace elements, and the so-called growth factors, yet i t appeared to be quite suitable for the problem under investigation. Stock cultures, isolated in. the autumn of 1937, have been carried continuously on sodium asparaginate glycerol agar of the following constitutions Later i n the study a duplicate set of stock cultures was transferred to and maintained i n s t e r i l e s o i l tubed i n convenient quantities to which s t e r i l e water was added as required to maintain the a c t i v i t y of the cultures. Preliminary Experiments In preliminary experiments, which have oeen reported i n unpublished data (27), a comparison of the a c t i v i t y of various soluble iron, salts resulted i n the selection of f e r r i c n i t r a t e as the source of iron for use i n subsequent studies. In the case of a l l salts tested, f e r r i c iron gave stronger stimulation than the corresponding ferrous s a l t , and f e r r i c nitrate was more active than the sulphate, chloride, or ammonium c i t r a t e . The optimum concentration of the f e r r i c ion was shown to l i e between 40 and 50 gammas per ml. of milk. Since the concentration curves f a l l off gradually, either there i s no definite t o x i c i t y l i m i t , or the iron i s Sodium asparaginate Dipotassium phosphate Dextrose Glycerol Agar Water, d i s t i l l e d pH 1.0 gm. 1.0 " 1*0 " 10.0 " 15.0 " 1000 cc. approximately neutral rendered insoluble through combination with some component of the milk. Since Waksman (53) has pointed out the influence of the type of inoculum used i n quantitative studies of the actinomycetes, various methods of inoculation were compared, and i t was found that the most consistent results were obtained when 0.5 ml* of a water suspension prepared from seven-day slope cultures were used* In later work, the influence of the incubation temperature on the rate of hydrolysis of milk protein was studied, and although Waksman (52) reported that the greatest ammonia production, measured with brom cresol purple as recommended by Clark and Lubs |7), (8) took place at 37° G, the cultures under study showed the greatest color change at 28° C* In l i g h t of the above findings, the standard procedure, as described below, was evolved* Procedure Skimmed milk prepared from milk powder was put up i n 500 ml. quantities in l i t e r conical flasks and s t e r i l i z e d at 12 pounds pressure for 25 minutes. Stock solutions of iron i n d i s t i l l e d water were pre-pared containing 1 mg. of the metallic ion per ml. of solution. The iron enrichments were added to the milk prior to autoclaving, precautions being taken to secure uniform dispersal i n the medium before subjecting to heat. Each culture as required was seeded on several slopes and incubated at 28° C for seven days. A t y p i c a l culture was then selected, 3 ml. of s t e r i l e water were added and the growth carefully worked off with a loop to form a uniform suspension. 0.5 ml. of the water suspension was used as inoculum for each flask. The foregoing milk cultures were incubated at 28° C for seven days. At the end of that time t o t a l nitrogen was determined on 5 ml. aliquots of the control, after the method of Orla-Jensen (39)• The decomposition of protein was measured as t r i c h l o r a c e t i c acid soluble nitrogen, or non-protein nitrogen, after the method of Eagles and Sadler (16), a modi-f i c a t i o n of the procedure of Wasteneys and Borsook (62)• The procedure for the non-protein nitrogen determination i s as follows? Add 8 drops of.formalin to the 300-ml. culture; treat with 40 ml. of 20 per cent t r i c h l o r a c e t i c acid and allow to stand for one hour. F i l t e r off the precipitate and take two 70-ml. aliquots of the f i l t r a t e . Heat the 70-ml. quantities i n a water bath for three hours, cool, f i l t e r , and make up to volume. Using two 10-ml. aliquots from each f l a s k , determine nitrogen content by the Kjeldahl method. A l l data are presented as non-protein nitrogen expressed as per cent of t o t a l nitrogen. Stimulatory Effect of Iron An experiment was set up i n such a manner as to enaDle the procuring of information respecting (l) the variation i n proteolytic a c t i v i t y within a species which may be expected from time to time; (2) differences between the species; and (3) the influence of iron on the rate of pro-te o l y s i s . That the twelve cultures of Actinomyces used i n the study are di s t i n c t species cannot be stated with absolute assurance, but each does at least represent a pure l i n e . For purposes of discussion they may be considered as species. They were isolated from the s o i l eight months prior to the commencement of the experiment. - 10 • -The data as presented i n Table I, while inconclusive i n some respects, set forth certain s p e c i f i c information. A study of each species i n "milk alone" over an eighteen months' period reveals that, while some species, notably A-5, A-7, A-8 and A - l l are r e l a t i v e l y con-sistent i n the production of non-protein nitrogen, others, p a r t i c u l a r l y A-l and A-2, vary over a wide range. A-7, for instance, varies only from 7.5 to 8.4 per cent while A-E shows the maximum variation of 7.6 to 37.7 per cent. That t h i s indicates the extent of variation which may normally be expected i n a laboratory study of actinomycetes i s very doubtful for reasons which w i l l be apparent l a t e r . It i s interesting to observe that a l l the species with the exception of A-3 and A-7 show a reduction i n proteolytic a c t i v i t y over the period of the experiment. This decrease i n a c t i v i t y i s most noticeable i n the case of cultures A - l , A-2, A-6 and A-10, which exhibited strongest proteo-l y t i c a c t i v i t y at the time of the f i r s t determination, while A-5, A-8, A-9, A - l l and A-l2 show a small but significant decrease. Thus, the hydrolytic a b i l i t y of the species i n Experiment 5 i s on the average at a d i s t i n c t l y lower level than i n Experiment 1. This decrease i s more pronounced between Experiments 1 and 3 than between Experiments 3 and 5. I f the average percentage increase i n non-protein nitrogen i s calculated for a l l cultures on the basis of the control, i t w i l l be seen that a decrease from 146 per cent to 49 per cent has taken place during the period of time elapsing between Experiments 1 to 5. Although certain i r r e g u l a r i t i e s are seen to occur i n the data, i t would thus appear that as the cultures are carried on an a r t i f i c i a l medium, their proteolytic a c t i v i t y tends to decrease u n t i l i t reaches a more or less constant le v e l where the a c t i v i t y of each species i s at a minimum. In the l i g h t of t h i s data i t Table I Response of Species of Actinomyces to Iron over a Two-Year Period * Species .Ho. :Experiment 1 8 months' cultures Milk Milk + Fe Experiment 2 18 months* cultures Milk Milk + Fe Experiment 3 20 months' cultures Milk . Milk + Fe 1 • ! .T Experiment 4 23 months* cultures Milk Milk * Fe Experiment 5 26 months'cultures Milk Milk + Fe Average i n Milk A v e r -i n Milk + . Fe i i v e r -age % Stimu lation A - l l 24.5 48.0 19.6 16.4 8.0 10.9 10.0 18.4 6.9 7,8 . 13.8 20.3 47.1 A-2 • 37.7 36.6 7.6 10.4 12.5 15.0 XX * 2 19.5 9.3 9.1 15.6 18.1 16.0 A-3 7.9 8.0 8.7 8.7 12.0 12.0 10.2 8.7 8.6 9.6 9.5 9.4 — A-4 10.7 14. 8 8.4 11.6 8.0 13.3 11.4 .8.1 14.1 9.3, 13.4 44.1 A-5 12.0 22.6 * 11.8 15.1 9.8 10.0 11.4 12.9 9.1 12.3 10.8 14.5 34.2 A~6 20.1 25.5 19.1 12.6 11.4 11.8 12.4 14.1 10.7 10.6 14.7 14.9 1.3 A-7 7.6 9.5 8.4 7.6 7.5 7.9 7.5 8.7 7.7 . 6.7 7.7 8.1 5.1 A-8 13.2 11.8 11.8 11.8 17.8 13.2 18.5 10.6 16.6 11.8 16.1 36.4 A-9 12.5 23.5 .. 10.1 14.8 7.6 8.8 8.7 7.5 10.3 9.6 9.8 12.8 30.6 A-10 19.2 17.3 16.3 10.6 7.8 9.7 9.2 12.7 8.8 11.1 14.2 12.3 -A - l l 8.7 15.3 8.3 10.6 6.3 6.9 6.6 6.8 6.9 9.2 7.3 9.7 32.9 A-12 12.0 16.0 8.3 24.8 6.4 12.4 12.2 13.4 9.2 18.9 9.6 17.1 78.1 Average 15.5 21.5 11.3 12.9 9.1 11.4 10.3 12.8 8.8 11.3 11.0 13.8 27.1 Q 6.3 6.3 5.8 5.8 6.0 6.0 6.1 6.1 5.9 5.9 Average % In-crease •: 146.0 241.1 94.8 122.4 51.6 90.0 68.8 109.8 49.1 91.5 over G • -SJ * A l l figures represent non-protein nitrogen as per cent of t o t a l nitrogen. ** Control, uninoculated. appears reasonable to suppose that had the experiment been undertaken when the microorganisms were freshly isolated from the s o i l , greater proteolytic a c t i v i t y would have been noted, resulting i n a wider range of v a r i a b i l i t y within the species than has been observed above. It i s to be noted too that i n Experiment 1 the cultures i n "milk alone" differed markedly i n their a b i l i t y to hydrolyse milk protein. For instance, A-7 showed 7.6 per cent non-protein nitrogen, whereas the corresponding figure for A-2 was 37.7 per cent. At the conclusion of the experiment there was r e l a t i v e l y l i t t l e difference between the species, as i s apparent from the extremes A-l and A - l l with 6.9 per cent each and A-6 with 10.7 per cent. When the average for non-protein nitrogen pro-duction i s considered the spread i s 15.6 to 7.3 per cent, as compared with 37.7 to 7.6 per cent i n Experiment 1. The species which showed the highest i n i t i a l a c t i v i t y , Ar-1, A-2, A-6 and A.-10 are s l i g h t l y higher than the remaining species, while A-7 and A - l l are the least active. A-3 i s peculiar i n that on the average i t has increased s l i g h t l y i n protein hydrolysing a b i l i t y . One might conclude from the data presented that, while A - l , A-2, A-6 and A-10 are extremely active, some of the species, notably A-3 and A-7 are weakly proteolytic in nature as they appear i n t h i s experiment. Such a conclusion, however, i s scarcely warranted at t h i s time, since, as has already been stated, the stock cultures had been carried on a laboratory medium for some eight months pr i o r to the commencement of the study. I t i s suggested that, during that time A-3, A-7 and l-ll may have largely lost t h e i r protein hydrolysing power* The data as presented perhaps indicates that sodium asparaginate glycerol agar lacks some constituent essential for the maintenance of high" proteolytic a c t i v i t y . An examination of the data with respect to the influence of iron i n the i n i t i a l experiment indicates an even greater v a r i a b i l i t y "between the cultures than was apparent with milk i t s e l f , the presence of iron stimulating such species as A - l , A-5 and A-9 to a marked degree, and showing no effect on the rate of protein breakdown i n the case of A-2, A-3 and A-10. The tendency of the species to decrease i n proteolytic a c t i v i t y as they are carried on the a r t i f i c i a l medium, while not as con-sistent as with milk alone, i s even more marked. While the average percentage increase of the cultures when calculated on the basis of the control drops from 146 to 49 per cent i n milk, i t drops from 241 per cent to 91.5 i n the presence of iro n , a decrease 1.5 times as great. Hence the iron has apparently accentuated the variation i n proteolytic a c t i v i t y which was recorded within a given species i n milk alone. The species do not give a consistent response to iron as determined over the eighteen months* period, but, considering the average percentage stimulation for the different cultures, i t w i l l be seen that A-4 and A-l2 show the strongest stimulatory effect, as compared with A - l , A-5, A-10 and A - l l , which responded to iron i n Experiment 1. The response to iron by A-8 as determined by experiments not reported herein i s perhaps more marked than this data would indicate, and hence i t i s regarded, with A-4 and A-12, as the species showing the greatest stimulatory effect upon the addition of iron to the basic milk medium. - 14 -The extreme v a r i a b i l i t y of species of Actinomyces has also been reported by Waksman (53), who found str i k i n g quantitative variation when studying amino nitrogen production by various species. In view of the data presented i n Table I i t would seem possible that the microorganisms once removed from their natural habitat-the soil-and carried on a r t i f i c i a l media for an extended period of time, change to unstable physiological forms, which change may result i n quantitative variation. I t was therefore decided to carry the cultures i n s t e r i l e s o i l i n an attempt to restore any lost measure of a c t i v i t y and to maintain them at a constant physiological l e v e l . S t a b i l i t y of Actinomycetes on a S o i l Medium Quantities of fine sandy loam were placed i n large test tubes and s t e r i l i z e d for three hours at 15 pounds pressure on three successive days. After testing the s o i l for s t e r i l i t y , tubes were inoculated with a water suspension of the twelve cultures and incubated at 28° C, s t e r i l e water being added as required to maintain the moisture content of the s o i l . The a c t i v i t y of representative species was determined at intervals over a f i f t e e n months' period. The inoculum i n each case was prepared by adding a small portion of the s o i l culture to a 3-ml. melted agar medium tube, sloping, and allowing to harden. After a week's incubation, cultures prepared i n t h i s way exhibited some surface growth which was transferred to a fresh agar slope to be used for inoculation of the milk cultures after a further seven-day incubation period. Table I I presents a summary of the data ootained with representative cultures after they had been carried i n s o i l for periods of three months, twelve months, and f i f t e e n months. Table I I Effect of Maintaining Stock Cultures on S t e r i l e Soil-as measured "by Response to Iron* Species Fo. . 3 months on S o i l Milk Milk + Fe Per Gent Increase 12 '• Milk months on S o i l Per Cent Milk * ;Fe Increase 15 Milk months on S o i l Per Gent Milk -t- Fe Increase A - l 12.2 11,7 13.0 12.9 A-E 13* 3 8.1 ISe 5 54.3 A-3 17.6 17.9 A-4 12.2 18.3 50.0 22.4 33.6 50.0 19.0 31.0 63. A-5 24.5 30.8 25.7 11.6 14.4 24.1 A-6 14.2 14.4 18.2 19,8 8.7 17.0 20.2 18.8 A-7 '. .• • 9.3 9.1 7.8 7.9 8.1 11.0 36.7 A-8. 15.6 31.5 101» 22.9 42.0 86. 20.3 39.5 94.5 A-9 A-10 18.2 24.6 35.1 17.6 22.2 26.1 A - l l 8.6 8.6 10.8 11.6 7.4 9.1 11.8 29.6 A-12 8.4 16. 3 94. 11.6 25.3 118.0 8.8 16.6 88.0 C ** 6.0 . 6.0 5.9 5.9 6.0 6.0 ** Control, Uninoculated. * A l l figures represent non-protein nitrogen as per cent of t o t a l nitrogen. From Table I I i t i s apparent that the differences i n a b i l i t y to degrade milk protein which were observed with regard to the various c u l -tures i n the i n i t i a l experiment reported i n Table I, and which tended to disappear as the cultures were carried on the laboratory medium, have begun to reappear. While some of the strains, notably A-l and A-2, have not reached the level of proteolytic a c t i v i t y f i r s t reported, cultures A-3, A-4, A-5 and A-8 far exceed i t , and cultures A-6 and A-IO approximate the o r i g i n a l figures closely. A-7, A - l l and A-12 are remarkaole i n that l i t t l e change i n the rate of degradation of milk protein can be recorded. In the presence of iron, cultures A-4, A-8 and A-12 show a strong and con-sistent stimulation, contrasting with the v a r i a b i l i t y of their response as recorded in the previous table. The other cultures studied show l i t t l e or no stimulatory effect. With the exception of A-5, the figures denoting nonprotein nitrogen as percentage of t o t a l nitrogen agree closely for the sets completed at twelve and at f i f t e e n months, both i n the absence and i n the presence of iron. That this i s an important con-sideration i s apparent when the di v e r s i t y of the data i n the preceding table i s recalled. The extreme v a r i a b i l i t y of the actinomycetes, which has been reported Dy so. many investigations, has been largely overcome, and i t i s suggested that by maintaining isolations of these microorganisms on s t e r i l e s o i l , the great d i f f i c u l t i e s encountered i n any study of th i s group may be at least to some extent eliminated. The tendency on the part of the cultures to show decreased a c t i v i t y after numerous transfers on an a r t i f i c i a l medium, and to regain their l o s t a c t i v i t y after prolonged contact with s o i l has also been observed with regard to gelatin liquefaction. Giant colonies of the actinomycetes were seeded on plates of 15 per cent gelatin and incubated for a period of ten days, after which time the width of the liquefied c i r c l e on the plate was measured. This technique was f i r s t carried out i n July, 1938, using the cultures after they had "been carried, i n the stock medium for six months, and repeated i n February, 1941, using the same cultures which had been carried on the laboratory medium during the intervening time, and also the corresponding cultures freshly isolated from the s o i l medium after having been i n contact with i t for a period of f i f t e e n months. The data are presented i n Table .•III. Table I I I Comparison of the Rate of G-elatin Liquefaction of Species of Actinomyces carried on an A r t i f i c i a l Medium and i n S t e r i l e S o i l Species A r t i f i c i a l Medium A r t i f i c d a l Medium S o i l Medium No. 8 months' cultures 26 months' Cultures 26 months' cultures Width of li q u e f i e d Width of Liquefied Width of Liquefied zone i n Cm. _ zone i n Cm. zone i n Cm. : A-l ' • ' liS-A-2 .6 .7 1.7 A-3 ' * " • .'.4.; .7 ' A-4 • 2.4 1.9 2.4 , A-5 .5 .5 1.5 A-6 .9 1.0 1.0 A-7 2.0 1.0 1.0 A-8 1.2 3.1 A-9 .5 .5 .5;. A-10 1.0 .6 1.0 A - l l 1.3 .4. .8 A-12 .7 .7 1.0 An examination of the data reveals that the behavior of the cultures closely p a r a l l e l s that recorded i n Tables I and I I . After contact with the a r t i f i c i a l medium for some time, the cultures are seen to decrease i n a c t i v i t y or to show no change, but the cultures freshly isolated from the s o i l medium are s i g n i f i c a n t l y more active, with the exception of cultures A-6, A-7, A-9 and A-12. Of these, A^7, A-9 and A-12 showed l i t t l e appreciable change i n the preceding data, while A-6 decreased s l i g h t l y and then regained i t s former proteolytic a c t i v i t y after maintenance on s o i l . The relative rates of starch hydrolysis, while not correlating com-ple t e l y with proteolytic a c t i v i t y , indicate that the same general tendency may apply to metabolic functions other than those concerned with protein degradation. I t would thus seem probable that a higher le v e l of bi o l o g i c a l a c t i v i t y results when the actinomycetes are maintained under natural con-ditio n s , and that the v a r i a b i l i t y which has been reported with regard to quantitative studies of these microorganisms may be largely eliminated through the use of s o i l as the stock medium. Effect of Soil, Ash ' "/ Since the b i o l o g i c a l a c t i v i t y of cultures of actinomycetes was con-siderably increased through contact with the s o i l medium, and since iron, a material of almost universal di s t r i b u t i o n i n s o i l s , exhibited a stimu-latory effect on certain of the cultures, i t was decided to investigate the possible influence of other metallic constituents on the proteolytic a b i l i t y of representative cultures. Since the p o s s i b i l i t y that the increased a c t i v i t y of cultures i n contact with the s o i l medium was due to substances of an organic nature was considered, i t was decided to remove s o i l organic matter by i g n i t i o n , and to test the stimulatory effect of the resultant ash. Two-gram samples of s o i l of the same sample as that on which the cultures had been maintained were ignited i n porcelain crucibles u n t i l t h e i r weight remained constant after continued heating. The ash was transferred to 500 ml, of milk i n l i t e r flasks and autoclaved with the milk to f a c i l i t a t e solution of at least part of the oxides formed on ig n i t i o n . The flasks were then inoculated and incubated i n the" usual manner. Cultures A-4, A-8 and A-12 from the laboratory medium were tested i n t h i s way. The data are presented i n Table IT. Table IV . Response of Species A-4, A-8 and A-12 to the Presence of S o i l Ash Species No. Wt. of Ash i n grams. Non-protein Nitrogen as Per Cent Total Nitrogen Per Cent Increase Milk... . . Milk +. Ash A-4 11.0 21.7 97.2 A-8 1.5 10.3 ••"• 14.7 42.7 A-12 1.5 9.7 11.3 16.5 0* 6.3 6.3 ' * Control, uninoculated. A comparison of the response of the species to s o i l ash, and to f e r r i c n i t r a t e as reported i n Table I, reveals that they d i f f e r consid-erably. The response of A-4, with an average stimulation of 44.1 per cent from f e r r i c nitrate,increases to 97.2 per cent when i n contact with s o i l ash. This i s higher than i t s response to iron after contact with the s o i l medium, when the percentage stimulation was 50 to 63 per cent. The response of A-8 to s o i l ash i s only s l i g h t l y greater with an increase from 36.4 per cent to 42.7 per cent for the more varied enrichment, but this figure i s less than half the percentage stimulation recorded for iron when the stock cultures were carried on a s o i l medium. A-12, on the - 20 -other hand, shows only 16.5 per cent stimulation from the s o i l ash as compared to an average stimulation of 78.1 per cent as reported i n Table I, and of 100 per cent when kept i n contact with s o i l . I t should be noted here that A-12 was among those species exhibiting no increase in proteolytic a c t i v i t y after extended contact with the s o i l medium, while both A-4 and A-8 showed substantial increases. Effect'of Trace Elements Since i t seemed possible that some other constituent of the ash was exerting a stimulatory effect, a qualitative analysis of t h i s s o i l was carried,out. Twenty grams of the s o i l were digested with hydrochloric acid according to the method of Van Bemmelen and Hissink(49), the organic matter was removed from the f i l t r a t e by evaporation with n i t r i c acid, and the extract tested by the qualitative procedures of IToyes (37) supplemented by floyes and Bray's (38) procedures for the rare elements 0 Since a hy-drochloric acid digestion had been used i t was not possible to test for the metals of Group I. The amounts of the various elements were roughly estimated, and are reported as "absent", "trace" or "heavy" i n Table V. It i s f u l l y realized that the procedures used are not as sensitive as some of the micro-analytical methods which have been evolved, but they may be carried out with ra p i d i t y and by using a large sample and concentrating the f i l t r a t e , any trace elements which would be available and present i n the s o i l solution should be recovered. Very heavy pre-ci p i t a t e s of manganese, iron, and aluminum were obtained, and the possi-b i l i t y that trace elements were occluded and carried down with these materials should not be overlooked. Table .? Summary of Metallic Elements found i n Upland Glac i a l S o i l , using Qualitative Analytical Procedures Element Occurrence Element Occurrence Lead absent Manganese heavy B i smuth absent Iron heavy Copper trace Nickel trace Cadmium trace ^'Cobalt trace Ar senic absent Vanadium absent Antimony trace Zirconium absent Tin trace Titanium heavy Selenium absent Strontium absent Tellurium absent Barium absent Aluminum heavy Calcium heavy Chromium absent Magne slum heavy .• Zinc trace - 22 -The effect of these metals on the proteolytic a c t i v i t y of representative species of actinomycetes was determined, using the con-centration and source of the cation as outlined i n Table 71. Table VI Source of Metallic Constituents and Concentrations used to Determine thei r Accumulative Effect on Actinomyces i n Milk Metal Salt Used Concentration of Cation per ml. of milk iron Fe{SF03)5.9H2Q 9.90 gammas manganese MnS04.2H20 6.22 " copper CuSp4.5H20 3.31 " cobalt Co(F0 3) 2.6H 20 3.31 " aluminum A1G13.6H2G. 3.31 " zinc 2nCl 2 , 3;31 " • t i n , SnCl2.2H2G • 3.31 » nickel M(F0 3) 2.6H 20 3.31 " cadmium Gd (F0 3] 2.4H 20 3.31 " Y/hen the accumulative effect of these metallic ions as reported i n Table VII i s compared with that of 47.6 gammas of iron, i t ' w i l l be seen that certain species respond more strongly to the varied enrichment. A-4 shows a response of 209.5 per cent i n the l a t t e r case, while the aver-age stimulation was 44.1 per cent i n Table I and 50 to 63 per cent after the cultures had been returned to the s o i l . The a c t i v i t y of A-8 i s - 23 -Table VII Response of Species of Actinomyces to traces of Certain Metals Culture Non-Protein Nitrogen as Per cent Total Nitrogen Per Cent No. Milk Milk + Metals Increase A-4 9 & 5 29.4 209.5 A-5 8.2 9.4 11.6 A-6 9.6 11.8 , A-7 7.2 11.5 59.7 A-8 10.3 23.6 129.1 A-10 9.0 9.8 8.0 A - l l 6.9 7.7 11.6 A-12 9.9 9.8 -c* 6.1 6.1 * Control, uninoculatecL stepped up from an average of 36.4 per cent over an eighteen months5 period to 129.1 per cent, an increase over the a c t i v i t y of the s o i l c u l -tures, which responded from 86 to 101 per cent. A-7, which did not show any appreciable stimulation from iron alone, shows a significant increase of 59.7 per cent from the varied enrichment. On the other hand A-12, which did not respond to any extent to the addition of s o i l ash to the basic medium, showed no increase from the various metals added above. The a c t i v i t y of the other species tested (A-5, A-6, A-10 and A - l l ) was i n -fluenced only s l i g h t l y by either the varied enrichment or by iron alone. In the case of species A-4 and A-8, the concentration of iron added was too low to account for the large increases i n non-protein nitrogen recorded i n Table VII. The immediately preceding experiments have indicated the influence of other metals i n the physiology of actinomycetes. Apparently some other metal or metals are important i n the metabolism of A-7, v/hich ex-hi b i t s l i t t l e or no stimulation from iron alone. Cultures A-4 and A-8, which have shown a consistent response to iro n , exhibit an additional effect from the presence of other cations. A-12, on the other hand,is not stimulated by the metals added at these concentrations, although show-ing large increases i n non-protein nitrogen i n the presence of 47.6 gammas of f e r r i c n i t r a t e . The requirements of the other species studied apparently do not include the materials outlined i n Table YJ, although the slight increases reported may suggest a response at a higher concentration* While no conclusions can be drawn as regards the necessity of particular metals for a given species, such requirements are suggested. At the same time, the increase i n proteolytic a c t i v i t y reported i n Table I I appears to be due, not to the organic f r a c t i o n , but to the wide variety of metallic constituents normally present i n the s o i l . 'Discussion and Summary...- Part .A The proteolytic a c t i v i t y of cultures of actinomycetes and their response.to the addition of traces of iro n i n soluble form were measured by determining the non-protein nitrogen produced i n milk cultures. In preliminary experiments, the cultures were shown to respond i n the greatest degree to f e r r i c nitrate added at the rate of 47.6 gammas of cation per ml. of milk, although no toxic effect was ooserved at higher concentrations. Cultures incubated at 28° C. for seven days exhibited the maximum response to iron, the percentage stimulation increasing up to this time, then gradually decreasing. The quantitative variation en-countered i n the non-protein nitrogen production of the cultures made i t apparent that a l l experimental procedures and methods must he closely checked before comparable data could he obtained. The source of inoculum and method of inoculation affected the rate of proteolysis and the con-sistency of the cultures, and the results were found to be most s a t i s f a c -tory when 0.5 ml. of a suspension of mycelial growth i n water was used. Although the standard procedure was followed with extreme care, and the duplicate determinations of non-protein nitrogen checked satisfactor-i l y , variations too significant to be overlooked i n a quantitative study were recorded when the proteolytic a c t i v i t y of the species was determined at different times. A series of experiments over an eighteen months* period indicated that, i n general, the protein degrading power of the species tended to decrease as stock cultures were carried on the labora-tory medium. Thus the average non-protein nitrogen production of the twelve cultures i n milk dropped from 146 per cent to 49 per cent during the eighteen months' period. Considerable differences i n proteolytic a c t i v i t y between the species were observed i n i t i a l l y ; A-3, A-7 arid A - l l exhibited only s l i g h t a c t i v i t y , whereas A - l , A-2, A-6 and A-10 were very active i n th i s regard; but as they were carried on the a r t i f i c i a l substrate, their comparative acti¥ity appeared to become equalized and minimized. Thus the range recorded for the different species decreased from 37.7 - 7i6 per cent to 10.7 - 6.9 per cent during the course of the study. The addition of iron to the milk medium accentuated the differences between the species as a result of stimulation on the part of certain cultures,, Considering the effect of the iron over the eighteen months* period, the same tendency for decreased a c t i v i t y , v/hile not as regular as i n milk i t s e l f , was observed, the average percentage stimulation on the basis of the control dropping from 241.1 per cent to 91.5 per cent. Whereas the response of the individual cultures was extremely variable, the average percentage stimulation indicated that A - l , A-4, A-8 and A-12 responded most strongly to iron® Thus i t became apparent that, speaking generally, the a c t i v i t y of the species was progressively decreased i n accord with the length of time the stock cultures were i n contact with the a r t i f i c i a l medium u n t i l they reached a more or less constant l e v e l . A l l cultures had been carried i n stock on the a r t i f i c i a l medium for eight months prior to the commencement of -the work. In t h i s time i t i s possible that cultures A-5, A-7, A - l l and A-12 may have reached a constant le v e l of a c t i v i t y and hence varied only s l i g h t l y during the course of the study. Since i t i s of the utmost importance to maintain the s t a b i l i t y of the species, they were, returned i n pure culture to their natural habitat, the s o i l . The proteolytic a c t i v i t y of isolations made at the end of a year was then determined and i n nearly a l l cases was shown to have increased considerably; and moreover, with but one exception, the non-protein n i -trogen determinations checked s a t i s f a c t o r i l y when cultures were compared at different times. Evidently contact with this widely diverse material had, i n the course of twelve months, raised the l e v e l of b i o l o g i c a l a c t i v -i t y and maintained i t so that fresh isolations showed high and consistent results. That such a conclusion i s important i s obvious when the numerous reports of variation and inconsistency occurring i n the l i t e r a -ture concerning t h i s group are recalled. A-4, A-8 and A-12 responded strongly to the addition of iron, i n contrast to their inconsistent reaction when carried on the a r t i f i c i a l medium. The influence of s o i l ash on the proteolytic a b i l i t y of the three cultures showing the strongest response to f e r r i c n i t r a t e suggested that other metals might have an influence i n maintaining certain cultures i n a high state of a c t i v i t y , since A-4 responded more strongly to the presence of s o i l ash than to iron alone. By qualitative analysis t h i s s o i l was found to- contain a' wide variety of metals which, when added i n trace amounts to milk, indicated that at least certain cultures responded to metallic substances other than iron. Cultures A-4, A-6, and A-8 were stimulated to a greater degree by the more' varied enrichment, but A-12, despite the fact that i t responded to iron alone, was not stimulated appreciably by s o i l ash or by the various trace elements added. This species did not exhibit any increase i n proteolytic a c t i v i t y after con-tact with the s o i l medium and apparently reacts only to iron at a higher concentration than was dissolved from the s o i l ash or was added with the varied enrichment. I t has thus been shown that the cultures of actinomycetes under study vary markedly i n their a b i l i t y to degrade milk protein; that iron stimulates the proteolytic a c t i v i t y of certain of the cultures; that, i n general, continuous c u l t i v a t i o n on an a r t i f i c i a l medium impairs their hydrolytic a b i l i t y ; and that the maintenance of the stock cultures i n s t e r i l e s o i l restores to a large degree their lost a c t i v i t y . Certain other metals appear to show a stimulatory effect, but since the study i s mainly concerned with the influence of iron on proteolysis, a study of other metallic substances has not been undertaken. PART B - .STUDIES. -ON:OXYGEN" REQUIREMENTS Preliminary experiments involving the use of shake agar cultures demonstrated differences i n the oxygen requirements of the actinomycetes under study. In view of the fact that Brown and Baldwin (5) and Allyn and Baldwin (l) reported the influence of oxidizing and reducing sub-stances on the growth levels of various b a c t e r i a l species, and demonstrated the opposite effects of ferrous and f e r r i c iron i n the oxidation-reduction potential of a medium, i t was believed that the differences rioted i n the oxygen requirements of the cultures might afford a possible explanation for the stimulatory effect of f e r r i c nitrate as n o t e d i n P a r t A« Methylene Blue Studies The influence of methylene blue and f e r r i c n i t r a t e on the growth levels of the various species i n sodium asparaginate glycerol agar sim-p l i f i e d by omitting dextrose, were studied. Water solutions of methylene blue and f e r r i c nitrate were s t e r i l i z e d separately and added aseptically, to tubed 10 ml. quantities of the medium immediately prior to inoculation with a water suspension of the required culture. The series were i n -cubated at 28° C for a period of ten days. An examination of the data as presented i n Table VIII reveals specific differences between the cultures. A l l the "control" cultures with the exception of A-2, A-7 and A - l l produced only surface growth, while the named cultures showed sub-surface growth, and hence a greater tendency towards anaerobiosis. The presence of methylene blue i n a l l Table T i l l The Influence of Methylene Blue and Ferric t i t r a t e on the Growth Levels'of Species of 'Actinomyces i n Shake Agar Cultures Culture Mo* Control Methylene Blue .0001$ Methylene Blue oQQZfo Fe 10 y per ml. A-l Surface sub-surface* sub-surface 5 mm. sub-surface* A-2 sub-surface* sub-surface'- a few colonies through tube sub-surface* -colonies through tube surface and sub-surface a few colonies through tube :' A-3 heavy: surface surface and sub surface sub-surface* heavy surface. - a few colonies through tube A-5 heavy surface surface and sub-surface sub-surface* - a few colonies through tube heavy surface ' A-6 ' heavy surface surface and sub^ surface - a few colonies through tube sub'-surface 5 mm. many colonies through tube heavy surface - a few small colonies through tube A-7 surface and sub-surface, many colonies through tube sub-surface* sub-surface* many small c o l -onies through tube surface and sub-surface A-6 heavy surface numerous small colonies through tube f a i r l y heavy through tube surface - small c o l -onies through tube A-9 heavy surface surface - a few c o l -onies through tube large colonies through tube . heavy surface A-10 heavy surface surface and sub-surface numerous colonies• . . • through tube heavy surface - a few large colonies through tube A - l l A-12 surface and sub-surface many colonies through tube heavy surface sub-surface - a few colonies through tube surface and sub-surface sub-surface* - many co 1 onie s t hro.ugh tub e sub-surface* - many small colonies through tube surface and sub-surface - a few colonies through tube surface - small c o l -onies through tuoe * Denotes a heavy growth ring l-2mm.. below the surface of the medium. cases allov/ed growth to occur deeper i n the medium, the higher concentra-tion being more effective i n this regard. The addition of iron at a concentration of 10 garnnas per ml. had no effect on the growth levels of A-5 and A-9, but tended to bring growth to the surface i n the case of A-2, A-7 arid A - l l . In a l l other cases, the effect of iron i s similar to that of methylene bluej i n other words, i t too tended to lower the level of growth i n the shake agar cultures. It would appear that methylene blue, by functioning as a hydrogen acceptor, f a c i l i t a t e s the energy exchange i n this simple synthetic medium, thereby permitting a l l cultures to show discernible growth at a lower l e v e l than i n the controls. In the presence of iron, the growth lev e l i s lowered i n the case of the more aerobic cultures, while with the so-called anaerobic cultures, A-2, A-7 and A - l l , the growth i s brought to the surface. Ho explanation of this observation can be offered at the present time. Attempts to define, by the use of a suitable range of oxidation-reduction indicator dyes, as recommended by Clark (6) and discussed by Hewitt (26),. the actual potential produced by the various species, proved unsuccessful, due to abundant pigment formation by the organisms when milk constituted the medium and to high t o x i c i t y of the dyes when a synthe-t i c medium was used. At the same time1, a non-toxic dye concentration was too low to allow observation of the color changes. Methylene blue, however, proved to be highly satisfactory for this purpose?due to the fact that the color change i s sharply defined i n milk, and the potential of the medium several days after inoculation l i e s more or less i n the v i c i n i t y of the change i n color of this indicator. I t has therefore been - 31 -employed to determine the reducing power of the various species and to study the effect of iron on the potential attained during the growth of the microorganisms. Preliminary experiments demonstrated that a l l the strains exerted a f a i r l y strong reducing action, as measured by methylene blue at a concentration of 1:100,000. The a c t i v i t y of the cultures was then determined at higher concentrations: Methylene blue was added to milk to give concentrations of 1:50,000, 1:10,000 and 1:1,000, and s t e r i l i z e d i n 10-ml. quantities. Table IK records the data at the end of a five-week incubation period. Considering their tolerance to higher concentrations of methylene blue, the species under study are observed to divide themselves into three groups: (a) those withstanding a con-centration of 1:1,000 - A-2, A-7, A-10 and A - l l ; (b) those withstand-ing a concentration of 1:10,000 - A - l , A-3, A-5, A-9; and (c) those withstanding a concentration of 1:50,000 -A-4, A--6, A-8 > A-12. A study of the influences of iron on methylene blue reduction by the various species was undertaken. To this end.methylene blue was added to milk at a concentration of 1:100,000 and the milk was divided into three parts. One-third was tubed i n 10-ml. quantities to serve as a control; to a second portion was added a water solution of f e r r i c n i t r a t e to give a concentration of 53 gammas per ml. of milk; the remain-ing one-third was tuoed,and to each tube was added a l i t t l e reduced iron, as recommended by Scott (43) and Hastings and McCoy (25) for the main-tenance of a reduced potential i n a medium. This material after s t e r i l i z -ation with the milk maintained the indicator i n a reduced condition as the leuko compound except for a narrow surface band which re-oxidized to methylene blue on cooling. Table Z records the data as obtained with this series after a f i v e day incubation period. Table IX The Tolerance of Species of Actinomyces to Varying Concentrations of Methylene Blue i n Milk Species l o . 1:50,000 1:10.000 1:1,000 A-l A-2 A-3 A-4 A-5 A-6 A-7 A-8 A^9 A-10 A - l l A-12 Heavy surface ring. Complete reduction below pigment zone. White surface ring. Heduction complete at bottom. Black surface growth. Se-duction complete below pigment. Whole tube digested. Serum greenish. Surface ring and growth down sides. Reduction complete i n lower half of tube. Reduction complete at bottom. Blue surface ri n g . Reduction complete at bottom. Whole tube digested. Serum yellowish. Black surface growth. Se-duction complete below pigment. Brown surface growth. Re-duction complete below pigment. Blue-green surface r i n g . Re-duction complete i n lower half of tube. Yellow surface growth.,. Whole tube digested. Surface growth and colonies down sides of tube. Complete reduc-ti o n at bottom of tube, White surface ring. Reduction complete at bottom. Yellow colonies down sides. 75$ reduction at bottom. Growth down sides. Slight reduction at bottom. Slight surface ring. Slight reduction at bottom.. Small blue colonies down sides. Reditction complete at bottom; p a r t i a l through rest of tube. Slight growth down sides. Reduction complete at bottom; p a r t i a l through lower half of tube. Small colonies down -g- of tube. Reduction complete at bottom. Yellow colonf ies down sides of tube. Reduc-tion complete at bottom. Small blue colonies down sides. Mo re-duction. Small blue colonies near bottom of tube. Fo reduction. Small colon-ies near bot-tom of tube. 50% reduction at bottom, i Table X The Influence of Fe r r i c Nitrate and Reduced Iron i n the Reduction of Methylene Blue i n Milk by Species of Actinomyces Culture No» Methylene Blue ; Methylene Blue + F.e(JT0R).„ Methylene Blue + Reduced Iron.. . A - l complete reduction complete reduction at bottom - 75$ reduction above no re-oxidation A-2 > .'• no reduction no reduction no re-oxidation A-3 complete reduction 50$ reduction through-out tube sl i g h t re-oxidation A-4 no reduction ' no reduction s l i g h t re-oxidation A-5 sli g h t reduction at bottom no reduction p a r t i a l re-oxidation A-6 no reduction no reduction complete re-oxidation A-? ; sli g h t reduction at bottom sl i g h t reduction at bottom no re-oxidation A-8 no reduction sli g h t reduction at bottom complete re-oxidation A-9 no reduction no reduction no re-oxidation A-10 ... 90$' of tube reduced 80$ of tube reduced no re-oxidation A - l l complete reduction at bottom complete reduction at bottom blue surface band 50$ reduced A-12 no reduction no reduction complete re-oxidation - 34 -It w i l l De observed that, considering the a b i l i t y to reduce or to discharge the color from methylene blue, or to re-oxidize methylene white, the species f a l l into three groups.* (l) Those exerting a strong reduc-ing action i n the medium,- Species A - l , A-7, A-10 and A - l l ; these effected at least p a r t i a l reduction of methylene blue and showed no a b i l i t y to oxidize the reduced iron tubes. (2) Those exerting no reducing action i n the medium - Species A-2, A-5 and A-9; these did not reduce methylene blue i n the given time and showed no a b i l i t y to oxidize the reduced iron tubes. A-3 i s also placed i n th i s group since i t effected both reduction and a slight re-oxidation. (5) Those exerting an oxidizing action -Species A-4, A-6, A*-8, A-12j with these cultures there i s no reduction of methylene blue but a more or less complete re-oxidation of methylene white. This c l a s s i f i c a t i o n of the species agrees with the i r tolerance to methylene blue as ascertained i n Table IX, with the exception of A-l and A-2. On the basis of tolerance to methylene blue A-2 appeared more anaerooic than A - l ; but i n the above data the order i s reversed. Since A-2 showed sub-surface growth i n shake agar cultures (Table T i l l ) , i t was decided that i t should be placed with A-7, A-10 and A - l l as species showing the strongest tendency towards anaerobiosis. The presence of f e r r i c n i t r a t e i n many cases tended to prevent as complete reduction of methylene blue as i n the "control" cultures, i n d i -cating that the oxidation-reduction potential of the medium had been raised. The three cultures showing the greatest response to f e r r i c n i t r a t e i n the degradation of milk protein are among the most aerobic. This per-haps suggests that the presence of iron raises the potential of the medium so that the microorganisms can grow down to a greater depth i n the medium, the resu l t being greater proteolytic a c t i v i t y . Influence, of,Iron xmder Anaerobic Conditions As a result of these studies i t was decided to determine the effect of iron on the a c t i v i t y of A-8, chosen as an aerobic species, and A - l l , chosen as an anaerobic species., when placed under anaerobic conditions. Heavy mineral o i l , added to the surface of the milk flasks p r i o r to ster-i l i z a t i o n to form a layer about a half an inch thick, was used as a means of preventing the absorption of atmospheric oxygen by the milk after s t e r i l i z a t i o n . While i t i s possible that complete exclusion of a i r cannot be obtained i n this manner, at least a considerably reduced oxygen tension results. 1 Table XI A Comparison of the Response of Species A-8 and A - l l to the Addition of Ferric If i t rate to Milk under Aerobic and Anaerobic Conditions species No. A-8 A-8 A-8 A-8 A - l l A - l l A - l l A - l l Iron Added •r. 47.68 47.68 47.68 47.68 C - uninocu-lated L _ O i l 150 ml, 150 ml. 150 ml. 150 ml. Non-protein Nitrogen as per cent of Total Nitrogen 9.6 21.7 607 8.3 7.8 8.0 7.8 9x8 6.1 Per cent Increase 26.0 23.8 2.5 25.6 The data as presented i n Table XI show cl e a r l y that the a c t i v i t y of A-8 i s materially lessened under anaerobic conditions, when there was l i t t l e stimulation from iron. Whereas the percentage stimulation i n aerobic culture was 126 per cent, i t dropped to 23.8 per cent upon the exclusion of a i r . The anaerobic species, A - l l , on the other hand, showed l i t t l e or no stimulation from iron under aerobic conditions, but was stimulated by 25.6 per cent i n the absence of atmospheric oxygen. This increase i s believed significant,since A - l l was noteworthy i n that i t did not respond to iron at this period of the study. Apparently the presence of iron i n anaerobic culture f a c i l i t a t e s the exchange of oxygen in the case of A - l l but has l i t t l e effect on the aerobic A-8 when atmospheric oxygen lias been excluded. I f such i s the case, a moderate supply of a i r should increase the a c t i v i t y of the iron i n the case of A-8 and should decrease i t s effectiveness when A - l l i s used. Experiments were set up to prove or disprove t h i s p o s s i b i l i t y . Flasks were prepared as above; i n addition to adding the o i l p r i o r to autoclaving, 150-ml. quantities of s t e r i l e o i l were added to two of a series of flasks at definite intervals during the cooling of the milk, i n an endeavor to l i m i t the amount of a i r absorbed by the milk during cooling. These were then inoculated by placing 0.5 ml. of a water sus-pension of the required seven-day culture below the surface of the o i l , , and incuoated at 28° C for one week. The data reported i n Table XII indicate that, when species A-8 i s used as the source of inoculum, the response to iron becomes greater as atmospheric oxygen i s absorbed by the culture medium. Whereas no response was noted when more or less complete exclusion of a i r was effected, an 18.7 per cent increase was recorded when the milk had been allowed to stand for twenty hours p r i o r Table XII The Influence of Aerobic to Anaerobic Conditions on the A c t i v i t y of A-8 Treatment with O i l Non-protein Nit per cent of .. .Nitrog rogen as Total en • Per Cent Milk Alone Milk + Iron Increase Ko o i l 9.2 16.1 72.7 Added before s t e r i l i z a t i o n 6*3 6.2 «. Added 4 hours after s t e r i l i z a t i o n 7.2 7.9 9.7 Added 8 hours after s t e r i l i z a t i o n 8.0 8„3 Added 20 hours after s t e r i l i z a t i o n 8.0 9 © 5 18.7 Control - uninoculated 6.0 6.0 to the addition of the o i l . While this figure i s much lower than the 72.7 per cent increase obtained i n the cultures exposed to the atmosphere during the incubation period, the increase would suggest that oxygen i s essential for the highest proteolytic efficiency of A-8, and that iron gives i t s maximum stimulatory effect i n the presence of a i r . From Table XIII,the response of A - l l under identical conditions shows the opposite trend. This species exhibits i t s maximum response to iron under most nearly anaerobic conditions, while the presence of a i r depresses i t s stimulatory effect. Thus, when oxygen was excluded, an increase of 35.3 per cent was recorded, whereas no stimulation or only negligible response resulted when even small amounts of a i r were absorbed by the medium. While this increase does not approach that recorded for A-8 under optimum conditions, i t i s believed s i g n i f i c a n t , since A - l l showed l i t t l e response to ir o n i n other experiments performed at t h i s Table XIII The Influence of Aerobic to Anaerobic Conditions on the A c t i v i t y of A - l l Treatment with O i l lion-protein Nitrogen as per cent of Total Nitrogen Per Cent Increase Milk Alone Milk + Iron No o i l 6.7 6.8 1.5 Added before s t e r i l i z a t i o n •6.5 8.8 55.5 Added 4 hours after s t e r i l i z a t i o n 5.5 Added 8 hours after s t e r i l i z a t i o n 6.7 6»7 -Added 20 hours after s t e r i l i z a t i o n 6.5 6.7 3.0 Control - uninoculated 6.0 6.0 time. Thus species A-8 and A - l l are d i r e c t l y opposite i n their reaction to these conditions. On the basis of the data presented i n t h i s section, i t would appear that response to iron i s largely based on oxygen requirement. The aerouio species are stimulated by the addition of iron to the medium, whereas those species i n which a stronger reducing a c t i v i t y was demonstrated, exhibit l i t t l e or no response to iron under aerobic conditions, but when ai r i s excluded from the culture, significant stimulation occurs* The difference between the oxygen requirements of the species would appear to be one of degree only, since a l l the cultures w i l l grow i n the presence of a i r , but iron does not appear to have i t s maximum stimulatory effect on the more anaerobic species except i n the absence of oxygen. Discussion and SummaryPart B A study of the stimulatory effect of iron on proteolytic a c t i v i t y as - 39 -determined i n Part A indicated certain differences between the various species. When the stock cultures had been stabilized through contact with s o i l , A-4, A-8 and A-12 responded markedly to the presence of iron, while the other species were inconsistent or showed l i t t l e stimulatory effect. This appeared to suggest some fundamental difference between the species. Preliminary experiments i n shake agar cultures coupled with th e i r general cultural characteristics suggested certain differences i n oxygen requirements, and these differences were believed to afford an explanation, for the varying response to iron. In shake agar cultures three species, A-2, A-7 and A - l l , exhibited sub-surface growth and hence the greatest tendency toward anaerobiosis. The presence of methylene blue allowed growth i n the case of a l l species to grow deeper i n the medium, whereas the presence of f e r r i c n i t r a t e allowed the more anaerobic species to grow at the surface, and forced the species which i n the controls exhib-ited surface growth, to develop below the surface of the agar. This data strengthened the conviction that iron played some fundamental part i n the respiratory complex, and that the oxygen requirements of the species determined the conditions under which the enrichment would be most effective. Accordingly, a study of the oxidation-reduction requirements of the species was undertaken, using methylene blue, the indicator which proved most satisfactory for the purpose. On the basis of tolerance to methylene blue and their reduction and re-oxidation of the dye, the spe-cies were divided into three groups: those exerting a strong reducing action - species A-2, A-7, A-10 and A - l l ; those exerting no reducing action - species A-4, A-6, A-8 and A-12; and those with an intermediate reducing power - species A - l , A-3, A-5 and A-9. The presence of f e r r i c nitrate at the concentration used i n milk throughout the study retarded i n many cases the rate of reduction of the methylene blue, suggesting that i t had some effect i n r a i s i n g the potential of the medium. This would account for the lower growth level observed i n shake agar cultures with species A - l , A-3, A-4, A-5, A-6, A-8, A-9, A-10 and A-12, but affords no reason for the surface growth with A-2, A-7 and -A - l l . In an effort to find some explanation for t h i s observation, species A-8 and A - l l were chosen for study under anaerobic conditions as secured • through providing a layer of mineral o i l on the surface of the culture,, A-8, an aerobic species showing strong response to iron i n the presence of a i r , was not stimulated by iron under anaerobic conditions, but showed increased stimulation with the amount of a i r absorbed by the freshly ster-i l i z e d milk. A - l l , on the other hand, responded to iron under conditions of anaerobic culture, while showing no significant response when oxygen was present. This species, i t i s true, had shown response to f e r r i c n i -trate i n the data presented i n Table I, but i t s reaction was inconsistent, and in the control cultures run at the time of these experiments not more than 3*0 per cent stimulation was recorded, whereas under anaerobic con-ditions an increase of 25.6 - 35.3 per cent was noted and i s believed s i g n i f i c a n t . I t i s d i f f i c u l t to state at the present time as to whether or not Warburg's theory that, respiration or the u t i l i z a t i o n of molecular oxygen by l i v i n g c e l l s i s a process resulting from catalysis by iron, i s a p p l i -cable i n the case of the data recorded. However, the importance of iron . . . . . . 41 - • for the respiratory enzymes as reviewed by the University of Wisconsin Biochemists (48), and the opposite reactions of the aerobic and the more or less anaerobic species, would suggest a fundamental difference i n the respiratory enzyme complexes of A-8 and A - l l . In view of the data outlined above there appear to be certain rather s t r i k i n g differences and s i m i l a r i t i e s among the twelve species under study, but detailed explanations cannot be set forth at the present time regard-ing the actual function of the iron enrichment with respect to the oxida-tion-reduction requirements of the individual species. PART C - MZY1E STUDIES While i t has been demonstrated i n the preceding section that iron i s effective in. stimulating proteolysis through i t s influence on the oxidation-reduction potential of the medium, i t was s t i l l thought possible that i t might play a part i n influencing the production of the proteolytic enzyme complex. Waksman (51) takes as part of his system of c l a s s i f i c a t i o n the action of the species i n milk, dividing them into f i v e major classes, based on the i r a b i l i t y to bring about the c l o t t i n g of milk and to effect protein breakdown. I t i s noteworthy that no species isolated i n t h i s laboratory over a three-year period has had the a b i l i t y to form a clot i n milk. In certain instances, however, species have been observed to c l o t milk i n the presence of a greater amount of iron than i s optimum for the stimulation of proteolysis. He stated that, while the optimum temperature for growth l i e s be-tween 25 and 28° 0 for the saprophytic groups, the maximum rate of pro-teolysis takes place at 37° G when hydrolysis of protein i s measured by - 42 -ammonia production using brom cresol purple as the indicators The species used i n the present study, however, i n every case showed maximum ammonia production at 28° 0, and three cultures, A-5, A-9, and A-12, refused to grow at 37° C. In the same paper Waksman described methods for iso l a t i n g a rennet-l i k e enzyme from a well hydrolyzed milk culture by preci p i t a t i o n with alcohol, and for separation of a crude enzyme complex capable of proteo-l y t i c action by treating vegetative growth oy the "acetone-dauerhefe" method. He reported a f a i r l y complete separation of the two enzyme sys-tems by these procedures.but no confirmation of t h i s has been obtained with the species studied herein. The material prepared by either method clotted s t e r i l e milk occasionally^but no consistent behavior could be discovered- on the other hand, both preparations brought about peptoniza-tion of the milk protein, suggesting that no complete separation of the two enzymes had been effected. Since the preparation of an alcohol precipitate provided an active proteolytic complex, t h i s method was used to determine the effect of iron on i t s a c t i v i t y . Two-months* old cultures of A-8 and A - l l i n milk and i n milk containing 47.6 gammas of iron, i n which the casein was almost completely hydrolyzed, was f i l t e r e d , the f i l t r a t e precipitated with 95 per cent alcohol, and the precipitate so obtained f i l t e r e d o f f , washed with alcohol and ether, and dried over sulphuric acid. Weighed portions of the dried precipitate were added to 10-ml. quantities of s t e r i l e milk and s t e r i l e milk containing 47.6 gammas of iron as f e r r i c n i t r a t e . 1-ml. of toluene was added to prevent bacterial growth and the tubes were incubated at 28° C for a period of four days, after which time non-protein nitrogen was determined. Since only small amounts of the f i l t r a t e s were available for the t o t a l nitrogen determinations, the micro-Kjeldahl method was used after i t had been found to give almost identical results with the macro-method. The data are presented i n Table XIV. Table XI? .Response of. Various Proteolytic Enzyme Preparations to the Presence of Iron Source of Enzyme gms.'of Enzyme added Son-protein Kitrogen as per cent of Total Nitrogen Milk M l k + Fe A-8 .05 13.1 15.7 a X 24.9 28.4 A-8 Fe* • .05 50.1 43.9 - » X 55.7 50.5 A - l l . * X 18.0 15.4 .2 23o o 24.9 A - l l Fe* .1 25.5 24.4 .2 38.2 35.7 * Cultures A-8 and A - l l grown i n milk containing 47.6 gammas of iron were the enzyme sources. A study of Table XIT reveals that iron does not appear to stimulate the proteolytic enzyme complex per se, since no significant stimulation was found with, any of the preparations studied. The material from the parent iron cultures, on the other hand, i s more active than a corres-ponding amount of enzyme from a culture to which no iron had been added, suggesting that the iron stimulates production of the proteolytic enzyme complex. While i t must he taken into consideration that the alcohol precipitate from the cultures containing iron was smaller, and hence the enzyme complex was more concentrated, t h i s large increase i n a c t i v i t y - 44 -appears to be s i g n i f i c a n t , at least i n the case of species A-8. Thus i t may be concluded that f e r r i c n i t r a t e brings about greater production of the proteolytic enzyme complex, and that t h i s stimulatory effect i s responsible,in part at least^for tfee large increases i n non-protein n i -trogen recorded for certain species e a r l i e r i n the study. The p o s s i b i l i t y that iron was stimulating not only the enzyme com-plex, but also the amount of growth, was considered. Since the physical nature of milk does not allow the separation of vegetative growth from the medium, the species were cultured on sodium asparaginate glycerol f l u i d medium both i n the presence and i n the absence of f e r r i c n i t r a t e . The nature of the growth of actinomycetes i n f l u i d media made i t convenient to f i l t e r off the colony mat and determine i t s weight i n the manner used for study of the fungi. Accordingly, 100 ml. quantities of sodium asparaginate glycerol f l u i d medium of the' same constitution as the s o l i d medium used for carrying the stock cultures (with, of course, the omission of the agar) were placed i n 500 ml. fla s k s . A water solution of f e r r i c n i t r a t e was added to certain of the flasks to give a concentration of 5.68 gammas of iron per ml. of medium, and after s t e r i l i z a t i o n , they were inoculated with representative species. After a ten-day incubation period at 28° C, the medium was f i l t e r e d through a weighed paper and the weight of vegetative c e l l s determined. The data i s presented i n Table XT. The presence of i r o n apparently does not influence the amount of vegetative growth produced by the cultures to any appreciable extent, although A-8 does show some increase. However, the increase reported i n Table XT with regard to A-8 has not been confirmed i n duplicate experi-ments, and hence i t i s probably correct to conclude that iron exerts no - 45 -Table XT Influence of Iron on the Amount of Vegetative Growth Produced by Species of Actinomycetes Culture . No. Weight of Tegetative Growth i n mgms. . Sasic medium Basic medium + Fe Per Cent Increase A-4 26.2 25.2 -A-5 24.5 • 9.8 . A-6 34.5 31.0 A-7 25,8 26.2 1.6 A-8 21.7 26.7 23.0 : V; A - l l ' 23.4 26.3 14.0 A-12 25.7 22.7 -stimulatory effect upon the amount of growth produced by the various species. Discussion and Summary - Part. C / Since increased proteolytic a c t i v i t y had been reported i n the case of certain species i n the presence of iron, i t was decided to determine i t s stimulatory effect on the proteolytic enzyme complex. Species A-8 arid A - l l were chosen for t h i s phase of the investigation. A-8, when carried on s o i l , responded markedly to f e r r i c n i t r a t e and was quite active p r o t e o l y t i c a l l y . A - l l , on the other hand, was not stimulated appreciably by iron, and did not hydrolize milk protein to any great extent. Milk cultures of the species with and without iron were used as the source of the enzyme material, which was prepared by precipitation with alcohol. The data, while inconclusive i n some respects, demonstrated that f e r r i c n i t r a t e , at the concentration to which species 1-8 i t s e l f responded, had l i t t l e or no effect on the a c t i v i t y of the enzyme preparation. At the same time, the actual production of the enzyme"complex was stimulated •markedly, i n view of the fact that preparations from parent cultures with and without iron differed widely i n a b i l i t y to bring about non-protein nitrogen formation. While i t i s recognized that the enzyme material from the parent iron culture was more concentrated than that without i r o n , at the same time, the large increases reported with A-8 i n the presence of iron would lead one to conclude that the amount of enzyme produced i n the presence of iron was considerably greater than that formed i n i t s absence. A similar study of A - l l supports the observations made as regards A-8, but since t h i s species was not as active p r o t e o l y t i c a l l y and did not show large responses to iron, the data i s perhaps not as clear-cut. A comparison of the actual weight of,vegetative growth from the various species i n the presence and i n the absence of iron prompted the conclusion that t h i s metal had no stimulatory effect on the amount of colony mat produced. It would appear that, while iron has no effect on the amount of ve-getative growth produced, and does not stimulate the proteolytic enzyme complex per se, i t does perform a function i n increasing the amount of enzyme complex formed i n a given milk culture. Thus, iron apparently stimulates certain species under aerobic conditions both by increasing the amount of proteolytic enzyme complex produced, and, as was pointed out i n Part 3, through i t s function with regard to the oxidation-reduction requirements of the species. - 47 -IT. GENERAL STffiffflRY The influence of iron on the metabolism of Actinomyces was i n v e s t i -gated, using non-protein nitrogen production i n milk as the basis of study. Twelve cultures representative of species normally found i n the upland g l a c i a l s o i l of coast regions of B r i t i s h Columbia were selected for the study. Significant variations recorded between experiments performed at different times were shown to follow definite trends. Speaking generally, the proteolytic a c t i v i t y of the species decreased i n accordance with the length of time the stock cultures had been maintained on an a r t i f i c i a l medium. The large differences recorded between species i n the i n i t i a l experiments tended to become equalized and minimized, u n t i l , after an eighteen months* period, l i t t l e difference i n non-protein nitrogen pro-duction was observed. The addition of f e r r i c nitrate to the milk medium resulted i n a larger spread of a c t i v i t y between the species at the com-mencement of the work, due to the stimulatory effect of iron i n certain instances. But this effect was inconsistent, and a c t i v i t y decreased u n t i l a minimum l e v e l was reached. On the average, a slight stimulatory effect from iron was s t i l l apparent. The data suggested the u n s u i t a b i l i t y of the laboratory medium for maintaining the a c t i v i t y and s t a b i l i t y of the cultures over a period of time. In an effort to overcome the d i f f i c u l t y , the species were returned to their native habitat - the s o i l - i n pure culture. Isolations at the end of a year displayed, i n most instances, a greatly increased hydrol-y t i c a c t i v i t y , and three species, A-4, A-8 and A-12, responded markedly to iron. Apparently contact with this complex natural medium had largely overcome the i n s t a b i l i t y of the species, raising them to a higher l e v e l of biological a c t i v i t y when the extreme v a r i a b i l i t y of the cultures was reduced to the minimum. By providing s t e r i l e s o i l as the medium for the maintenance of the stock cultures, and by using fresh isolations for quantitative studies, v a r i a b i l i t y , which has heretofor been considered an inherent p e c u l i a r i t y of the actinomycetes, may be largely overcome. Since s o i l ash was shown to have a stimulatory effect on certain of the species, their response to a wide variety of trace elements was studied. The accumulative effect of the metals added indicated that, while A-12 responded only to iron, A-7 was stimulated by some other cation, and A-4 and A-8 showed an increased response when other metals were added with iron. The efficiency of s o i l i n maintaining the species at a high l e v e l of biological a c t i v i t y thus appeared to be due to the variety of metallic elements i t contained. The p o s s i b i l i t y that iron might bring about i t s stimulatory effect through i t s influence on the varying oxidation-red-uction requirements of the species was considered. Accordingly, i t was shown that, while an aerobic species responded to iron i n the presence of oxygen, a less aerobic species was stimulated only under anaerobic conditions. Certain fundamental differences i n the respiratory mechanism of the contrasting species were thus suggested. No stimulatory effect of iron on crude proteolytic enzyme pre-parations could be demonstrated, but the presence of the metal appeared to stimulate proteolytic enzyme production, at least i n the case of those cultures exhibiting strong hydrolytic a c t i v i t y . 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