Received for publication June 10, can be considered to be naturally occurring. dwarf colony types. It has been difficult to determine

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1 Western OXIDATIVE ASSIMILATION OF AMINO ACIDS BY SALMONELLAE IN RELATION TO GROWTH RATES J. L. STOKES' AND H. G. BAYNE Regional Research Laboratory,2 Albany, California, and Department of Bacteriology an(d Public Health, Washington State University, Pullman, Washington Received for publication June 10, 1960 Many species of bacteria form variants which grow slowly on agar media and produce small or dwarf colonies that are only a few tenths of a millimeter or less in diameter. These small colony variants may be isolated directly from nature as in the case of Salmonella typhosa (Jacobsen, 1910; Morris, Barnes, and Sellers, 1943), Corynebacterium diphtheriae, and other bacteria (Morton, 1940). They can also be produced in the laboratory by growing normal, large colony type, bacterial strains of Shigella sp., Salmonella sp., Staphylococcus aureus, Escherichia coli, and other genera and species in media containing toxic inorganic salts or toxic organic compounds such as antibiotics, and also by allowing the cultures to age (Clowes and Rowley, 1955; Stokes and Bayne, 1958a). The dwarf colony variant may differ from the large colony, parent strain in inability to ferment some carbohydrates or to synthesize essential growth factors, or in serological and other properties. Wlhere loss of ability to synthesize known growth factors is involved, large colony growth can be obtained by the addition of adequate amounts of the required vitamins or amino acids to the medium (Weinberg, 1950; Stokes and Bayne, 1958a). In other instances the cause of slow growth has not been determined and decreased permeability to nutrients has been postulated, although the possibility of need for unknown stimulatory growth factors has not been entirely excluded (Clowes and Rowley, 1955; Stokes and Bayne, 1957, 1958a). Some species such as S. pullorum, S. abortusovis, and S. typhisuis, normally grow slowly and form small colonies on solid media.. Such species 1 Present address: Washington State University, Pullman, Washington. 2A laboratory of the Western Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. can be considered to be naturally occurring dwarf colony types. It has been difficult to determine the cause of slow growth of these strains or to increase their rate of growth (Stokes and Bayne, 1957; 1958b). Recently, however, comparative experiments on the oxidation of amino acids by slowly growing and rapidly growing strains of salmonellae have disclosed some interesting differences between the two types which may provide an explanation for the marked difference in growth rates. These experiments are described here. MATERIALS AND METHODS The salmonellae used included: S. oranienburg, S. typhimurium, S. worthington, S. newport, S. derby, and S. anatum as representative of the normal, rapidly growing salmonellae and several strains of S. pullorum as representative of the slowly growing salmonellae. For the manometric experiments, the cultures were grown on trypticase soy agar plates at 35 C for 18 hr, removed with distilled water, centrifuged, and suspended in sufficient 0.1 M phosphate buffer, ph 7.1, to give a reading of 450 on the Klett-Summerson photometer (red filter). Conventional manometric methods were used to measure the rate and extent of oxidation of amino acids. The main compartment of each Warburg vessel received 2 ml of cell suspension and the side cup received 0.1 ml or 0.2 ml of a 0.02 M solution of the amino acid, i.e. 2 or 4,umoles. In the center well was placed 0.2 ml of 10 per cent KOH and a strip of filter paper to absorb CO2. The gas phase was air and the bath temperature 30 C. In some experiments carbon dioxide formation also was measured, and this was done by the direct method. The L isomers of the amino acids were employed except for the DL isomers of leucine, isoleucine, valine, methionine, and phenylalanine. Aspartic acid and glutamic acid were neutralized 118

2 1961] ASSIMILATION OF AMINO ACIDS BY SALMONELLAE 119 prior to use. Tyrosine was brought into solution with a small amount of NaOH and cystine with a small amount of HCI. Rates of oxidation of the amino acids are recorded as A1 of 02 consumed per vessel per hr. The small, endogenous 02 consumption has been subtracted. Since all of the cell suspensions were of the same optical density, approximately the same amount of cell material was used in each experiment and therefore the data for the different salmonellae strains are comparable. Dry weight determinations made on several of the cell suspensions gave values of 2.3 to 2.7 mg of cell material per ml of suspension. Additional details of technique will be described later. RESULTS AND DISCUSSION Oxidation of amino acids. Trypticase soy agar is composed largely of peptides and amino acids. Consequently, aerobic growth of the salmonellae on this and similar media probably involves the oxidation of the substrate amino acids as a major metabolic process. It was for this reason that the oxidation of amino acids by the slow and fast strains was investigated. The course of a typical series of oxidations by S. worthington is shown in figure 1. Alanine and serine are oxidized rapidly and the process is complete in about 40 to 80 min. The oxidation of glucose, included for comparative purposes, is somewhat more rapid than that of the amino acids but this is not always the case with the salmonellae strains. Glutamic acid is oxidized more slowly than the other amino minutes Figure 1. Oxidation of 4 umoles of alanine, serine, and glutamic acid and 2 jamoles of glucose by Salmonella worthington (endogenous 02 uptake not subtracted). TABLE 1 Rates of oxidation of amino acids by a rapid and a slow growing strain of Salmonella Amino Acids 02 Consumed per Vessel S. oranienburg S. pullorum j.d/hr idl/hr Alanine Serine Threonine Proline Aspartic acid Glutamic acid Lysine Histidine Cystine Arginine Glycine Leucine Isoleucine Valine Methionine Phenylalanine Tyrosine Tryptophan Glucose acids. The endogenous oxygen consumption is relatively small. The rates of oxidation of 18 amino acids by three rapidly growing strains of salmonellae, S. oranienburg, S. typhimurium, and S. worthington, were determined and also those of three slowly growing strains of S. pullorum. The results were very similar for all of the strains of each type. Data for one representative of each type are given in table 1. The first group of six amino acids, alanine, serine, threonine, proline, aspartic acid, and glutamic acid, is oxidized rapidly by the fast growing strains and most of them are oxidized as rapidly as glucose. In contrast, S. pullorum oxidizes only alanine and serine rapidly. The second group of five amino acids, lysine, histidine, cystine, arginine, and glycine, is oxidized slowly by the rapidly growing strains and either slowly or not at all by S. pullorum. The last group of seven amino acids, leucine, isoleucine, valine, methionine, phenylalanine, tyrosine, and tryptophan, is not oxidized by any of the

3 120 STOKES AND BAYNE [VOL. 81 TABLE 2 Extent of oxidation of alanine and serine by rapidly growing Salmonella Organism Alanine Oxidation Serine S. oranienburg S. typhimurium S. worthington S. newport S. derby S. anatum salmonellae. Similar results were obtained with several additional representatives of the two types of salmonellae. In general, therefore, the rapidly growing strains oxidize more amino acids and many of these more rapidly than the slowly growing strains. This pattern is consistent with the different growth rates of the two types on proteinaceous media and suggests that the more rapid growth of the fast strains may be due to their greater ability to metabolize amino acids. More extensive data on the oxidation of amino acids by salmonellae and also data on the decarboxylation and transamination of amino acids are presented in a separate paper (Bayne and Stokes, 1961). Oxidative assimilation of amino acids. A probably more significant difference between the slow and fast strains appeared when the extent rather than the rate of oxidation of the amino acids was determined. The extent of oxidation is expressed in per cent and is based on the actual amount of 02 consumed compared to the theoretical amount of 02 necessary for complete oxidation of all of the added amino acid. The data in table 2 show the extent of oxidation of alanine and serine by six species of rapidly growing salmonellae. Every strain consumed only 50 to 60 per cent of the amount of 02 needed for complete oxidation. This was not unexpected. It has been established that nonproliferating cell suspensions of microorganisms oxidize carbohydrates, amino acids, and other organic compounds in a manner which does not consume the theoretically maximal amount of 02 or produce the theoretically maximal amount of C02 and H20. The oxidation is incomplete in the sense that part of the substrate, which may vary from 20 to 85 per cent, is converted into cellular material, the latter having approximately the empirical composition of carbohydrate (CH20)n. This process of oxidative assimilation has been observed with all microorganisms so far examined and also with all substrates tested with the sole exception of formate which is completely oxidized (Clifton, 1950). Van Niel and Anderson (1941) have shown, however, that there is no assimilation when homofermentative lactic acid bacteria ferment sugars. Therefore the incomplete oxidations of the amino acids by the rapidly growing salmonellae fitted the normal pattern of oxidations of organic compounds by nonproliferating suspensions of bacteria and it could be assumed that the 40 to 50 per cent of the alanine and serine unaccounted for in the oxidations had been assimilated. There was an unexpected and remarkable difference, however, when the extent of oxidation of alanine and serine by strains of S. pullorum was determined. This is indicated by the data in table 3. Instead of normal, incomplete oxidations, both amino acids were oxidized to the extent of essentially 100 per cent. This appears to be a general property of S. pullorum strains since it occurred with each of the six cultures. Such complete oxidations are normally obtained with resting cell suspensions of microorganisms only when the oxidations are carried out in the presence of inhibitors of oxidative assimilation such as sodium azide, 2, 4-dinitrophenol, methylene blue, and other inhibitors of synthesis (Clifton, 1950; Stokes, 1952). No inhibitors were added to our suspensions. The reaction mixtures contained only washed cells, phosphate buffer, and amino acids. It must be concluded, therefore, that S. pullorum is unable to transform alanine and serine into cellular material due to some unknown metabolic derangement although the organism can readily oxidize those amino acids. Confirmation of the absence of assimilation in the oxidation of alanine by S. pullorum was obtained from experiments in which both 02 consumption and C02 production were simultaneously measured. The C02 values, like those for 02 were approximately the theoretically maximal amounts to be expected from oxidation of alanine. The respiratory quotient was l.0. Therefore, the results are in agreement with the requirements of the equation for the complete oxidation of alanine, CH3CHNH2COOH * 3C02 +

4 1961] ASSIMILATION OF AMINO ACIDS BY SALMONELLAE 121 TABLE 3 Extent of oxidation of alanine and serine by Salmonella pullorum Strain Alanine Oxidation Serine S. pullorum S. pullorum S. pullorum BC15L S. pullorum 75K S. pullorum S. pullorum H20 + NH3. In contrast to S. pullorum, the rapidly growing strains of S. oranienburg and S. typhimurium consumed O2 and produced C02 in the oxidation of alanine to the extent of only 55 per cent of theory for complete oxidation. Their respiratory quotients, however, were also 1.0.' Hydrogen peroxide has been shown to accumulate in the absence of catalase, during oxidation of alanine and other amino acids by an L-amino acid oxidase obtained from animal tissues (Blanchard et al., 1944). There was a possibility, therefore, that the high 02 consumption by S. pullorum in the oxidation of alanine and serine might be due to the formation and accumulation of H202 rather than to lack of assimilation of the amino acids. Oxidations of alanine, however, carried out in the presence of active solutions of crystalline beef catalase yielded the same high oxygen values as the oxidations without catalase. In a typical experiment, 275 IdI of 02 were consumed by S. pullorum in the oxidation of 4,moles of alanine without added catalase and 266,ul of 02 in the presence of catalase. Since the amount of 02 needed for complete oxidation of 4,umoles of alanine is 269,ul, oxidation in both instances was complete. Therefore, H202 accumulation can not be a complicating factor in our experiments. Also, as will be described later, radioactivity experiments with C14-labeled material confirmed the phenomenon of oxidative assimilation by the rapidly growing salmonellae and the lack of assimilation by the slowly growing S. pullorum strains. Oxidative assimilation or the absence of assimilation by the fast and slow strains is independent of (i) cell concentration in the range of to 2-fold the normally used cell concentration, (ii) the ph in the range of ph 5.0 to 8.0, and (iii) the medium used to grow the cells. Yeast extract agar, trypticase soy broth, and trypticase soy agar were used. Also, both slow and fast strains oxidize the D isomers of alanine and serine and the assimilation patterns are the same as with the L isomers. The inability of S. pullorum to assimilate alanine and serine could markedly reduce its rate of growth since these are the only two amino acids which the organism can metabolize rapidly. The oxidation of alanine and serine would not lead, at least directly, to the synthesis of cell material and would be in that sense, a wasteful process. Since S. pullorum does grow on trypticase soy and nutrient agar, although slowly, there must be assimilation of some of the slowly oxidized amino acids. The slow metabolism of the latter could thus limit the rate of growth of S. pullorum. As shown by the representative data in table 4, the slowly oxidized amino acids are indeed assimilated by S. pullorum. Threonine, proline, cystine, aspartic acid, and glutamic acid, all of which are oxidized slowly by S. pullorum, are assimilated to the extent of 20 to 30 per cent. The only exception is glycine which, like alanine and serine, is not assimilated. The rate of oxidation and assimilation of these five amino acids could permit, therefore, slow but appreciable growth. Accumulation of acetate. Of a total of 10 strains of S. pullorum examined for possible oxidative assimilation of alanine and serine six strains showed no assimilation. The remaining four strains, however, appeared to assimilate large amounts of the two amino acids, 50 to 70 per TABLE 4 Assimilation of amino acids which are oxidized slowly by Salmonella pullorum 02 Con- Substrate sumed per Oxidation Vessel Id/hr % Threonine Proline Cystine Aspartic acid Glutamic acid Glycine

5 122 STOKES AND BAYNE [VOL. 81 cent, and resembled, in this respect, the rapidly growing salmonellae. This relatively large number of exceptions among strains of S. pullorum appeared to limit the applicability of oxidative assimilation of amino acids as a regulator of growth rate. It was noted, however, that the four divergent strains, in contrast to the other six strains and also to the rapidly growing salmonellae, were unable to oxidize acetate. The latter compound is a possible intermediate in the oxidation of both alanine and serine and may arise by oxidative decarboxylation of previously formed pyruvate (Stumpf and Green, 1944). Thus oxidation of alanine by salmonellae could proceed as shown in diagram 1. The catabolism CH3CHNH2COOH CH3COCOOH + NH3 CH3COCOOH CH3COOH + CO2 CH3COOH s 2CO2 + 2H20 CH3CHNH2COOH CO2 + 2H20 + NH3 Diagram 1 of serine could also lead to pyruvate by way of enzymatic dehydration and subsequent spontaneous deamination (Chargaff and Sprinson, 1943). Since the divergent strains of S. pullorum could not oxidize acetate, it seemed possible that acetate accumulated in the cell suspensions during the oxidation of alanine and serine and that this process rather than assimilation was responsible for the incomplete oxidations. To determine whether there is indeed an accumulation of acetate in the case of the divergent strains, oxidations of alanine were carried out on a scale large enough to permit the isolation and identification of acetate. In a typical experiment 100 ml of cell suspension of a divergent strain of S. pullorum, prepared in the usual manner, were mixed with 500 jamoles of alanine in a cotton-stoppered, 1-liter Erlenmeyer flask. The mixture was incubated, with shaking, at 28 C. To measure 02 consumption and to determine completion of the oxidation, a control manometric experiment was carried out, simultaneously, with 2 ml of the same cell suspension and 10,moles of alanine. The cell suspensionsubstrate relation, therefore, was the same, quantitatively, in the Warburg vessel as in the large flask. The latter was removed from the incubator for analysis shortly after the manometric experiment indicated completion of the oxidation. The l... TABLE 5 Accumulation of acetate in the oxidation of alanine by Salmonella pullorum Strain Alanine oxidized,,umoles for complete oxidation, 33,600 19, consumed,,ua... 15,950 8,964 Extent of oxidation, % Acetate expected,,umoles Acetate found, /.moles Recovery, % Distillate TABLE 6 Comparison of acetic and propionic acid distillation and titration Total Acid Distilled Unknown Acetic Propionic ml N N % cells were removed by centrifugation. The supernatant liquid was adjusted to ph 8.0 with 1 N NaOH and concentrated to about 5 ml on a steam bath. The concentrate was adjusted to ph 2 with 5 N H2S04 and steam-distilled until 200 ml of distillate were collected. Two 20-ml samples of the distillate were titrated with 0.01 N NaOH to determine total volatile acid. The remainder of the distillate was used to analyze for acetic acid by Duclaux distillation and comparison of the resulting titration curves with those for known acetic acid (Stokes, 1949), by the lanthanum nitrate color test, and finally, by isolation and characterization of acetate as the silver salt. All four divergent strains behaved similarly. Data for two of them are presented in table 5. In agreement with previous results, the amount of oxygen consumed was only about one-half that required for complete oxidation of all of the alanine and this suggests that half of the alanine was assimilated. However, volatile acid accumulated in the cell suspensions during alanine oxi-

6 1961] ASSIMILATION OF AMINO ACIDS BY SALMONELLAE 123 dation. The acid was identified by Duclaux distillation. Of the volatile acid fraction, 100 ml were distilled and four consecutive 20-ml portions were collected and individually titrated with 0.01 N NaOH. For comparison, solutions of acetic acid and of propionic acid were distilled and titrated under exactly the same conditions as the unknown volatile acid. Representative results obtained are shown in table 6. Thus the unknown volatile acid fraction contained acetic acid and only acetic acid. The presence of the latter was confirmed by positive lanthanum nitrate tests. Also, the volatile acid was isolated in crystalline form as the silver salt by precipitation with dilute AgNO3. The crystals had the same characteristic plate structure and refractive index as that of authentic silver acetate. The total amount of acetic acid which accumulated during alanine oxidation was calculated from the titration values obtained on the aliquots of the volatile acid fraction. There was good agreement between the amount of acetic acid isolated from the cell suspensions and the calculated amount present based on the oxygen consumption data. The recoveries of acetic acid were in the range of 85 to 100 per cent. There can be no doubt, therefore, that the four divergent strains of S. pullorum, like the other six strains, are unable to assimilate alanine during oxidation. What appears to be assimilation is actually an incomplete oxidation of alanine in which acetate accumulates because the organisms are unable to oxidize it. These results indicate, also, that acetate formation is probably an intermediate stage in the oxidation of alanine by salmonellae. There was no accumulation of acetate in the oxidation of alanine by any of our strains of rapidly growing salmonellae nor could this be expected since all of the strains readily oxidize acetate. In addition to the 10 strains of S. pullorum already described, six other strains were obtained and examined. These were like the majority of the previous strains in that they oxidized alanine completely without assimilation or accumulation of acetate. Inability to assimilate alanine and probably also serine appears to be a general property of S. pullorum since it occurred, without exception, in a total of 16 strains randomly chosen. Oxidation and assimilation of tricarboxylic acid TABLE 7 Extent of oxidation of tricarboxylic acid cycle and other compounds by rapidly and slowly growing strains of Salmonella Substrate Acetate... Pyruvate... Lactate... Succinate... Fumarate... Malate... Oxaloacetate Glucose... Rapid strains S. orani- S. typhienburg murium Oxidation Slow strains S. pullo- S. pullorum, 3083 rum, cycle intermediates. The previous results suggest that acetate is an intermediate in the oxidation of alanine. Strains of S. pullorum, therefore, which do not assimilate alanine and which can oxidize acetate should also be unable to assimilate acetate. This aspect was investigated in a series which the rate and extent of of experiments in oxidation of acetate, pyruvate, most of the members of the tricarboxylic acid cycle, and also lactate and glucose were determined. The acids were used in the form of their sodium salts except for lithium lactate. Usually 4,umoles of each compound were added. Some of the data obtained are given in table 7. The rapidly growing strains of salmonellae oxidized most of the tested compounds at a fairly rapid rate. The rates of 02 consumption per vessel are not given in the table but, for example, with S. oranienburg, they ranged from 54 for oxaloacetate to 216 for lactate. The oxidations were over after about 40 to 60 per cent of the theoretically maximal amount of oxygen was consumed. This indicates that roughly 50 per cent of each compound was oxidatively assimilated. The slowly growing strains of S. pullorum also oxidized most of the compounds readily. The /l of 02 Consumed per vessel per hr by strain 3083 ranged from 102 for malate to 138 for oxaloacetate. However, in contrast to the rapidly growing salmonellae, S. pullorum oxidized acetate completely without assimilation, as predicted.

7 124 STOKES AND BAYNE [VOL. 81 TABLE 8 Oxidation and assimilation of radioactive acetate by Salmonella Strain 02 Manometry Radioactivity Con - suned per Oxida- Assim Assim- Vessel tion ilation Added In celiailated MI/hr % % counts/min % S. typhimuriumi ,000 16, S. pullorum ,000 4,500 7 Pyruvate and lactate, which are probably metabolized via acetate, were also completely oxidized. Assimilation occurred, however, during the oxidation of succinate, fumarate, malate, oxaloacetate, and glucose, and roughly to the same extent as with the rapidly growing salmonellae. With S. pullorum, therefore, assimilation does not occur in the oxidation of C2 and C3 compounds but appears in the oxidation of C4 and higher compounds (table 4). Citrate, ketoglutarate, and glyoxylate were oxidized either very slowly or not at all by the various salmonella strains. A total of six compounds have been shown to be oxidized by S. pullorum without assimilation: alanine, serine, glycine, lactate, pyruvate, and acetate. The oxidation of the first five compounds may proceed through acetate as an intermediate. Lack of assimilation of these compounds may be due, therefore, to some peculiarity in the metabolism of acetate which prevents transformation of acetate to cellular material and thus leads to complete oxidation. It appears as if the cells contain, naturally, an inhibitor of assimilation which uncouples oxidation and synthesis. Oxidation of glycine may proceed, via formate (Paretsky and Werkman, 1950) and this may also account for the absence of assimilation with glycine. Radioactive tracer experiments on assimilation. To obtain more direct evidence of oxidative assimilation or lack of it by the salmonellae, oxidations were carried out with labeled acetate, C'4H3COONa, with S. typhimurium and S. pullorum strain Assimilation by S. typhimurium should result in marked radioactivity of the cells whereas the absence of assimilation with S. pullorum should leave these cells essentially unlabeled after oxidation. 2-C'4-labeled anhydrous sodium acetate (0.6 mg) was dissolved in water along with sufficient unlabeled acetate to make a 0.02 M solution. The oxidations were carried out in the usual manner with 4,moles of acetate. When the oxidation was over, the contents of the vessel were removed quantitatively with the aid of a small amount of water. The cells were collected by centrifugation and washed thoroughly with four changes of water to remove any externally adhering radioactive material. The washed pad of cells was suspended in 2.2 ml of water. Amounts of this suspension which varied from 0.05 to 0.2 ml were uniformly distributed on stainless steel planchets and dried. The radioactivity of the cells was measured with a thin end-window Geiger tube and an autoscaler. Radioactivity counts were made also on the original solution of acetate to determine the total number of counts used in each oxidation. The manometric and radioactivity data are presented in table 8. According to the manometric data, S. tryphimurium oxidized 71 per cent of the acetate and assimilated 29 per cent. The corresponding radioactivity results show that the cells, after oxidation of the labeled acetate, are very radioactive and that 25 per cent of the original radioactivity of the acetate has been assimilated. This figure is in good agreement with the 29 per cent assimilation derived from the manometric, oxygen consumption data. In contrast, S. pullorum oxidized virtually all of the acetate and only a small amount of radioactivity was present in the cells after the oxidation. The results with radioactive acetate support, therefore, the previous conclusions on oxidative assimilation by the salmonellae, based on manometric data. ACKNOWLEDGMENTS We are indebted to Professor C. B. van Niel for many helpful discussions and suggestions during the course of these investigations and to Dr. Francis T. Jones of the Western Regional Research Laboratory for assistance in the isolation and characterization of crystalline silver acetate. SUMMARY In an attempt to find an explanation for the great difference in growth rates of normal, rapidly growing salmonellae and slowly growing strains of Salmonella pullorum, the rate and extent of oxida-

8 1961] ASSIMILATION OF AMINO ACIDS BY SALMONELLAE 120' tion of amino acids by resting cell suspensions of both types were determined and compared. Significant differences were found. The rapidly growing strains, Salmonella oranienburg, Salmonella typhimurium and others, oxidize more amino acids and many of them more rapidly than the slowly growing strains of S. pullorum. Moreover, alanine and serine-the only two amino acids oxidized rapidly by S. pullorum-are oxidized completely, without assimilation. Only those amino acids which are oxidized slowly, such as proline and cystine, are assimilated by S. pullorum. In contrast, the rapidly growing strains oxidatively assimilate alanine, serine, and all of the other amino acids oxidized either rapidly or slowly. It is suggested, therefore, that the rate of oxidation and extent of assimilation of amino acids may control the rate of growth of salmonellae Ṡome strains of S. pullorum which cannot oxidize acetate accumulate this coinpound during the oxidation of alanine. In addition to alanine and serine, S. pullorum cannot oxidatively assimilate glycine, lactate, pyruvate, and acetate. Since acetate miiay be an intermediate stage in the oxidation of these compounds, lack of assimilation may be due to some peculiarity in the metabolism of acetate which prevents synthesis of cellular material. In general, assimilations by S. pullorum do not occur in the oxidation of C2 and C3 compounds but appear in the oxidation of C4 and higher compounds. The nonoccurrence of assimilation with S. pullorum in the oxidation of so many compounds appears to be unique among microorganisms. REFERENCES BAYNE, H. G., AND J. L. STOKES 1961 Amino acid metabolism of salmonellae. J. Bacteriol., 81, BLANCHARD, M., D. E. GREEN, V. NOCITO, AND S. RATNER Amino acid oxidase of animal tissue. J. Biol. Chem., 155, CHARGAFF, E., AND D. B. SPRINSON 1943 Studies on the mechanism of deamination of serine and threonine in biological systems. Chem., 151, J. Biol. CLIFTON, C. E Assimilation by bacteria. Chapter 17 in Bacterial physiology. Edited by C. H. Werkman and P. W. Wilson. Academic Press, Inc., New York. CLOWES, R. C., AND D. ROWLEY 1955 Genetic studies on small-colony variants of Escherichia coli K-12. J. Gen. Microbiol., 13, JACOBSEN, K. A Mitteilungen uber einen variablen Typhusstamm (Bacterium typhi mutabile), sowie uber eine eigentumliche hemmende Wirkung des gewohnlichen Agar, verursacht durch Autoklavierung. Centr. Bakteriol. Abt. I., 56, MORRIS, J. F., C. G. BARNES, AND T. F. SELLERS 1943 An outbreak of typhoid fever due to the small colony variety of Eberthella typhosa. Am. J. Public Health, 33, MORTON, H. E Corynebacterium diphtheriae. A correlation of recorded variations within the species. Bacteriol. Rev., 4, PARETSKY, D., AND C. H. WERKMAN 1950 The bacterial metabolism of glycine. Arch. Biochem., 25, STOKES, J. L Fermentation of glucose by suspensions of Escherichia coli. J. Bacteriol., 57, STOKES, J. L Inhibition of microbial oxidation, assimilation, and adaptive enzyme formation by methylene blue. Antonie van Leeuwenhoek. J. Microbiol. Serol., 18, STOKES, J. L., AND H. G. BAYNE 1957 Growth rates of Salmonella colonies. J. Bacteriol., 74, STOKES, J. L., AND H. G. BAYNE 1958a Dwarf colony mutants of salmonellae. J. Bacteriol., 76, STOKES, J. L., AND H. G. BAYNE 1958b Growthfactor-dependent strains of salmonellae. J. Bacteriol., 76, STUMPF, P. K., AND D. E. GREEN Amino acid oxidase of Proteus vulgaris. J. Biol. Chem., 153, VAN NIEL, C. B., AND E. H. ANDERSON 1941 On the occurrence of fermentative assimilation. J. Cellular Comp. Physiol., 17, WEINBERG, E. D Vitamin requirements of dwarf colony variants of bacteria. J. Infectious Diseases, 87,