STUDIES ON CERTAIN SPECIES OF BACTERIA ASSIGNED TO

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1 STUDIES ON CERTAIN SPECIES OF BACTERIA ASSIGNED TO THE GENUS CHROMOBACTERIUM' JAMES P. GILMAN" Department of Biology, Kent State University, Kent, Ohio Received for publication May 20,1952 Certain of the pigment producing bacteria are placed in the genus Chromobacterium. In Bergey's Manual (Breed et al., 1948), Chromobacterium violaceum, C. amethystinum, and C. ianthinum are placed definitely in this genus, whereas C. chocolatum, C. orangium, C. iodinum, and C. viscosum are placed in the appendix of the genus. Due to the limited study and to the lack of detailed description of these latter species, it has been doubted that the classification was correct. Cruess-Callaghan and Gorman (1935) published descriptions of C. violaceum and other violet bacteria, and Grimes (1927) described C. viscosum, and Davis (1939) described C. iodinum. Tobie (1945) suggested that the best classification of pigment producing bacteria would be one based on biochemistry. He suggested that all bacteria producing yellowish-green or brownishgreen fluorescent pigments, which were soluble in water or diffusible in media, or a pigment of a phenaine derivative regardless of color, or both, should be placed in the genus Pseudomonas. With the history of the classification of these species in mind, it was the objective of this study to describe more fully the relationships between the species in question and, by so doing, either enhance a more logical cla#sification of the doubtful species or substantiate their present classification. EXPERIMENTAL METHODS Chromobacterium violaceum (from L. F. Rettger, Yale University), C. violaceum (from human lesions), C. violaceum, strain Lewitus,3 C. chocolatum, C. orangium, C. iodinum, and C. viscosum ' Based on data submitted in a thesis in partial fulfillment of the requirements for the degree of Master of Arts, Kent State University, Kent, Ohio. 2 Present address: Third Army Medical Laboratory, Fort McPherson, Georgia. ' Elsewhere in this article these three strains of C. violaceum will be referred to as strains 553, 6357, and 7461, respectively. were obtained from the American Type Culture Collection, Washington, D. C. C. amethystinum was obtained from Dr. W. J. Dowson, Cambridge, England; and C. ianthinum was obtained from Dr. A. C. Thaysen, Trinidad, British West Indies. The cultures of Sarcina lutea and Pseudomonas aerugsnosa also were obtained from the American Type Culture Collection. In all tests, standard controls were maintained by simultaneous testing of uninoculated media under equal conditions. To determine the morphological and physiological characteristics the following standard determinative tests were used: For cell and colony morphology all species were examined at different ages, from eighteen hour cultures to ninetysix hour cultures, grown at temperatures from 4 C to 37 C, and on nutrient, glucose, and yeast agar. C. viscosum was grown also on lactose, maltose, raffinose, and calcium lactate agar. Mode and conditions of growth and pigmentation were determined by pour plates and streak plates using the same media and conditions as above. Motility was determined by hanging drop preparations from twenty-four hour cultures grown at optimum temperatures for the species in question. To determine fermentation of carbohydrates, -arabinose, glucose, sucrose, lactose, maltose, raffinose, dextrin, D-mannitol, and salicin, in phenol red broth base, were used. Other standard determinative tests included growth in nutrient broth, gelatin stab, hydrogen sulfide production, starch hydrolysis, utiliation of tryptophan, production of catalase and reductase, utiliation of citrate, nitrate reduction, and litmus milk. In addition to the above standard tests for classification, in order to determine whether or not there were any relationships between the pigments in question and to determine if the pigments were related to pigments of known composition, spectrophotometric transmittancy 48

2 STUDIES OF BACTERIA IN CHROMOBACTERIUM curves were obtained of the unknown pigments and those of known composition. All strains of C. violaceum, C. amethystinum, C. iarnthinum, C. chocolatum, C. orangium, and C. iodinum were grown on nutrient agar slants, and their pigments were extracted with ethyl alcohol, with the exception of C. iodinum whose pigment was extracted with water. C. viscosum was grown on calcium lactate agar because of the excellent surface pigment production obtained and the comparatively small amount of diffusion into the media. The pigment was extracted with water. S. lutea and P. aeruginosa also were grown on nutrient agar and the pigments extracted with ethyl alcohol and water, respectively. Then solutions were pipetted aseptically into sterile culture tubes, centrifuged, and filtered through a Seit filter to exclude any cellular or extraneous matter. After centrifugation and filtration, the solutions were pipetted aseptically into sterile absorption cells for final testing. Control samples of the pure solvents were collected in the same manner after being introduced onto uninoculated media. The pigmented solutions were run in corex absorption cells against the pure solvents on the model DU Beckman spectrophotometer at wavelengths of every 10 mg, from 250 mg to 1,000 m,a. These data were plotted then on a logarithmic graph (Mellon, 1950) to show the complete transmittancy curves of the various pigments, the locations of the transmittancy, and absorption peaks being compared to show possible relationships. RESULTS The strains of C. violaeum, C. amethystinum, and C. ianthinum agreed with the description in Bergey's Manual (Breed et al., 1948) with the following exceptions: C. violaceum produced acid from glucose only, C. amethystinum produced acid from glucose, C. ianthinum gave complete peptoniation in litmus milk and produced a pigment that was first greenish-yellow, then reddishyellow, and finally reddish-violet in seventy-two hours; all species uilied citrate, were catalase positive, and wealdy reductase positive with the exception of C. ianthinum, which was catalase negative and strongly reductase positive. C. iodinum agreed with the description given by Davis (1939) except that citrate was utilied, and catalase and reductase were produced. C. viscosum agreed with the description given by Grimes (1927) except that nitrates were reduced to nitrites, and catalase and reductase were produced. Following is the complete description of C. chocolatum as the result of the various determinative tests: Morphology-Short rods occurring singly, at times appearing in short chains, nonmotile, endospores and capsules absent, gram positive. Pour plate colonies are circular, entire, smooth, raised, opaque, and chocolate colored. Agar slant cultures are filiform with undulate margins and ciliate edges. Cultural reactions-growth is good from 20 C to 30 C with slow growth at 37 C, and no growth at 4 C. Pigmentation is best at 25 C with the original dark orange pigment becoming deep brown in forty-eight hours. At 37 C, the pigment remains orange. An orange sediment and a slight turbidity are produced in nutrient broth, and gelatin is liquefied slowly. Biochemical reactions-hydrogen sulfide is not produced, starch is hydrolyed, indole is not formed, nitrates are not reduced, catalase is not produced, but reductase is, and there is no apparent reaction in litmus milk but perhaps an extremely slow acid production. Acid is produced, without gas, from glucose, maltose, lactose, sucrose, raffinose, L-arabinose, dextrin, and D-mannitol. Salicin is not fermented. It is thought that C. chocolatum dissociates into C. orangium. At times, small areas of the former remain orange on subculture and may be picked with a needle and subcultured. The resulting colonies have the appearance of C. orangium, and will not revert on subculture. It is suggested that C. orangium be referred to as C. chocolatum var. orangium. DISCUSSION Judging these species from a morphological and physiological basis, it is noted that there are three distinct groups of organisms represented, namely: C. violaceum, C. amethystinum, and C. ianthinum in one group, C. chocolatum and C. orangium in another, and C. iodinum and C. viscosum in the third. In the first group, all the organisms are gram negative, motile, form acid from glucose only, are catalase and reductase positive, reduce nitrates to nitrites, do not hydrolye starch, do not produce hydrogen sulfide or indole; the 49

3 50 JAMES P. GILMAN [vol. 65 exception being C. ianthinum whjeh differs in catalase and reductase production and in the reduction of nitrates. All produce a similar pigment with regard to color except C. ianthinum, and all of the pigments are soluble in alcohols but not in water. More study is needed on the chemical composition of these pigments before C. iarnhinum can be placed definitely in or out of the genus, but, for the present, it should remain there. The second group differs in that the members are gram positive, nonmotile rods. Both species 0.98 O.95r U I~ ( , duce pigments that diffuse into the media, and are soluble in water but not in alcohols. In this last respect they differ greatly from the other species studied. It is possible to substantiate further the above differences in classification by examining the spectrophotometric curves of the various pigments. It may be seen upon inspection of figure 1 that the three strains of C. violaceum and C. amethystinum produce substantially the same pigment. All four pigments examined have trans- I I I I I I 2S WAVELENGTH (mrr,) Figure 1. Spectrophotometric curves showing transmittancy peaks at approximately 450 m,u and absorption peaks at approximately 410 nip, 490 mu, and 580 mnu. IEGEND A - ChromDbacterium violacui, strairns 553, 6357, B - Chromobacterium aethystim C - Chromobacterium ianthinum I I I l I I I I I I I I hydrolye starch, do not reduce nitrates, are catalase negative and strongly reductase positive, produce acid from all carbohydrates tested except salicin, do not affect litmus milk, and produce pigments that are different from those produced by the other related organisms. From this, it seems that these organisms should be placed in a genus other than Chromobacterium. Although C. iodinum and C. viwcosum are different with respect to their actions in litmus milk and in the utiliation of citrate, they are both gram positive rods, have similar reactions in carbohydrate broth, are catalase positive and strongly reductase positive, reduce nitrates, promittancy peaks at approximately 450 mp and absorption peaks at approximately 580 my inferring that the composition of the pigments is the same. The curve of C. ianthinum, figure 1, differs enough so as to make it difficult to place it in any of the groups although the absorption peaks at 410 m; and at 490 m; indicate that the pigment might be carotenoid, thus explaining the early yellowness or, later, the redness of the pigment. Upon examination of the transmittancy curves of C. chocolatum and C. orangium, figure 2, it is seen that there are three absorption peaks at,ib A.

4 STUDIES OF BACTERIA IN CHROMOBACTERIUM 51 U 4 C/) WAVELENGTH (rmra) Figure 2. Spectrophotometric curves showing absorption peaks at approximately 410 mm, 440 mu, 470 mju, 510 mm, and 570 mus. *A 1- ii F H 080 H /S / m~~~~~~~~dgnd C B A Chromobactr1im lodinum B - Chromobacterium viscosum C - Psoudomonas aeruginosa I I I I C l l I I _I WAVELENGT.H (m,a) Figure 3. Spectrophotometric curves showing transmittancy and absorption peaks in the ultraviolet at approximately 300 mp and 350 mp, respectively.

5 52 JAMES P. GILMAN [VOL. 65 approximately 440 ms, 470 ma, and 510 mp. Although the former has one ximbre absorption peak at 570 mp, it may be seen th* the two pigments are similar. There is no obvious relationship between these pigments and the others in question. S. lutea, figure 2, shows three absorption peaks common to C. chocolatum and C. orangium at 440 mi,n 470 mju, and 510 m;, indicating that a relationship is present between these three organisms. S. lutea produces a pigment known to be carotenoid (Sobin and Stahly, 1942); and lycopene, another carotenoid, shows similar absorption peaks at 470 m, and 510 my (Mellon, 1950). Although the transmittancy curves of C. iodinum and C. viscosum, figure 3, are not completely similar, there is a relationship shown in respect to the general location of transmittancy peaks in the ultraviolet region. This shows that each has a transmittancy peak in the ultraviolet at approximately 300 mp& and an absorption peak in the ultraviolet at approximately 350 m;p. This relationship is strengthened further by the fact that P. aeruginosa produces a pigment known to be a phenaine derivative (Tobie, 1945), and that phenaine derivatives have this distinctive peak of absorption in the ultraviolet, figure 3. It is known further that the pigment of C. iodinum is a phenaine derivative (Tobie, 1945). Also, C. visco8um and P. aeruginosa show similar transmittancy peaks at 510 mu. Thus it is seen that these three pigments show a relationship and that C. viscosum may be a phenaine derivative. Also, they are all three soluble in water but not in alcohols and they produce yellowish-green and brownish-green pigment that diffuses into the media. If we accept Tobie's (1945) suggestion, it seems reasonable to classify C. iodinum and C. viscosum in the genus Pseudomons. ACKNOWLEDGMENTS The author is indebted to Dr. Charles B. Sumner for advice and suggestions concerning this study, to Dr. Emanuel Hertler for technical assistance, and to Dr. Walter C. Tobie for his kind advice in the editing of this manuscript. SUMMARY A study has been made of the properties of several species assigned to the genus Chromobacterium, and the following conclusions have been reached. The physiological properties and the transmittancy curves of the pigments of the species can be grouped into three general groups of diverse nature; only one of which satisfies the description of the genus Chromobacierium. C. violaceum, C. amethystinum, and C. ianthinum are similar species that fit the description of the genus Chromobacterium and should be retained in the genus; however, further study is needed concerning the classification of C. ianthinum. C. chocolatum and C. orangium do not show a relationship with the other species studied, but they do present a relationship with Sarcina lutea which produces a carotenoid pigment and with lycopene, another carotenoid. They should be assigned to another genus that better fits their characteristics. C. iodinum and C. vi8cosum share many mutual properties and are not similar to the other species studied. Their pigments show similar solubilities and transmittancy curves and indicate that they are phenaine derivatives; they appear to be related to some pigments produced by Pseudomonas aerugino8a. Thus these species should be removed from the genus Chromobacterium and assigned to the genus Pseudomonas. REFERENCES BREED, R. S., MURRAY, E. G. D., AND HITCHENS, A. P Bergey's manual of determinative bacteriology. 6th ed. The Williams and Wilkins Co., Baltimore, Md. CRUESS-CALLAGHAN, G., AND GORMAN, M. J On the characteristics of Chromobacterium violaceum and some allied species of violet bacteria. Sci. Proc. Roy. Dublin Soc., 21, DAVIS, J. G Chromobacterium iodinum (n. sp.). Zentr. Bakt. Parasitenk., II Abt., 100, GRIMES, P. I An aerobic capsulated bacterium chromogenic on sugar media. Zentr. Bakt. Parasitenk., II Abt., 72, MELLON, M. G Analytical absorption spectroscopy. John Wiley & Sons, Inc., New York, N. Y. SOBIN, B., AND STAHLY, G. L The isolation and absorption spectrum maxima of bacterial carotenoid pigments. J. Bact., 44, TOBIE, W. C A proposed biochemical basis for the genus Pseudomonas. J. Bact., 49,