Characterization of Bacteria by Their Degradation of Amino Acids

APPLIED MICROBIOLOGY, Oct. 1968, P. 1591-1595 Copyright 1968 American Society for Microbiology Vol. 16, No. 10 Printed in U.S.A. Characterization of Bacteria by Their Degradation of Amino Acids M. J. PICKETT AND M. M. PEDERSEN Department of Bacteriology, University of California, Los Angeles, California 90024 Received for publication 15 July 1968 A procedure for detecting the degradation of amino acids by microorganisms is described, and examples of its use in the characterization of bacteria are presented. The procedure involves inoculating a buffered solution of amino acids with a suspension of bacteria, incubating, chromatographing a sample of the suspension, and detecting degradation in terms of absence of ninhydrin-positive spots. Few amino acids, in contrast to carbohydrates in a solution of 0.2% ninhydrin in acetone, and heated and carboxylic acids, have been used as substrates at 100 C to develop the spots. in characterizing bacteria since, with few exceptions, there has been no practical assay. The few containing three amino acids with different RF values Concentration of amino acids. Aqueous pools, each exceptions are those instances in which the (1), were prepared. Dilutions were made to give samples for chromatography containing 1.00, 0.75, product of degradation is readily detected by a 0.50, and 0.25 Ag of each amino acid. The least concentration of each amino acid which gave a readily color test, e.g., indole from tryptophan and cadaverine from lysine, or in which alkalinization visible spot with ninhydrin was then used in subsequent studies. occurs when the substrate is -degraded, e.g., one of the procedures for detecting degradation of ph. We initially assumed that two (or more) ph lysine. optima would be found for degradation of each amino We report here a procedure which permits acid, one representing deamination and the other simultaneous detecting of the degradation of decarboxylation. To determine these optima, pools several amino acids. The procedure consists of 1-5 (Table 1) of amino acids were prepared in volumes of 0.5 ml in tubes (13 by 100 mm). inoculating a buffered solution of amino acids Each pool was in 0.1 M buffer, the buffers being with a suspension of bacteria, incubating this citric acid-phosphate at intervals of 0.4 from ph 3.6 suspension until each amino acid has been completely removed, chromatographing a sample to 8.8. The tubes of buffered amino acids were steri- to 6.0, and phosphate at intervals of 0.4 from ph 6.4 from the suspension on a strip of filter paper, and lized by autoclaving. Each tube was then inoculated developing the chromatogram with ninhydrin. with 0.1 ml of bacterial suspension, incubated for 16 The procedure permits various combinations of hr at 37 C (40 hr for Mycobacterium phlei), decontaminated by heating either at 60 C for 60 min or 100 amino acids in any one pool, the restrictions being, for a given pool, that the constituent amino C for 10 min, and centrifuged. A control (uninoculated) tube was processed along with each experimental tube. A 5-jliter sample of the supematant acids have unique Rp values, that the ph of the pool be optimal for degradation of each of the fluid was then chromatographed to detect disappearance of amino acids from that pool. Results were amino acids, and that there be no interference among those amino acids, e.g., one inhibiting recorded as 4+ (complete disappearance of the utilization of another. ninhydrin-positive spot), 3+ (nearly complete disappearance), 2+ (approximately 50% disappearance), MATERIALS AND METHODS 1+ (slight fading of spot), and (no diminution of - Chromatography. Chromatography was carried out the spot). Organisms used to determine ph optima on rectangles of Whatman 3MM paper (20 by 9 were cm). Escherichia coli, Proteus vulgaris, Serratia The papers were marked with four dots, 2 marcescens, cm apart Klebsiella pneumoniae, Pseudomonas and 3 cm from one end, and with a "front" line 7 cm aeruginosa, Salmonella typhimurium, Staphylococcus from the opposite end. Five microliters of sample was aureus, Bacillus subtilis, Listeria monocytogenes, applied to each spot. The samples were chromatographed with n-butanol-glacial acetic acid-water (120: were also determined for the additional pools of Streptococcus faecalis, and M. phlei. Later, ph optima 30:50, v/v) until this solvent reached the front line, amino acids (Table 1). about 1.5 hr. The papers were then air-dried, dipped Bacterial inocula. Bacteria were grown on Infusion 1591

1592 PICKETIT AND PEDERSEN APPL. MICROBIOL. Agar (Difco) slants for 24 hr at 37 C (M. phlei was incubated for 48 hr), harvested with sterile distilled water, centrifuged, and resuspended in water to give a concentration of approximately 1010 organisms per ml. Some organisms, particularly Pseudomonas and Flavobacterium, were recentrifuged and resuspended in order to remove ninhydrin-positive material which had occluded when the cells were centrifuged only once. Each inoculum for an amino acid test was 0.1 ml from such a suspension. RESULTS Table 2 records RF values and the minimal concentrations needed for detecting 24 amino acids by means of paper chromatography. f-alanine was examined but was not included, since it was scarcely visible on the chromatograms at alkaline ph. The effect of ph and duration of incubation on the degradation of each amino acid was examined by incubating the organisms listed above in the amino acid pools and recording the degree TABLE 1. Aqueous pools of amino acids of degradation (Tables 3 and 4). With all of the amino acids examined and for each organism, the ph was usually not critical; relatively broad plateaus for degradation of the amino acids (if degraded at all) were obtained. From such data an optimal ph for degradation of each amino acid was approximated (Table 5). New pools of amino acids (Table 6) for further studies were prepared according to their optimal ph and compatible RF values, adjustment of ph being affected by aqueous solutions of NaOH or HCI. Using these pools, we appraised the reproducibility of the procedure by repeated tests on several strains of bacteria (Table 7). A ninhydrinpositive substance arising from the inocula interfered with the recordings for some amino acids. However, our results were readily reproducible with most of the amino acids. Two kinds of information may be obtained from these amino acid tests, namely, (i) recogni- Pool Amino acid Pool Amino acid 1 2 3 4 5 6 7 L-Alanine D D D hydrochloride D L-Histidine hydrochloride D D DL-Valine L-Cysteine hydrochloride L-Arginine hydrochloride D DL-,8-Phenylalanine D-Histidine D-Glutamic acid D-Valine D-Leucine DL-a-amino-n-butyric acid 8 9 10 11 12 13 14 15 D-Asparagine D-Methionine L-Serine L-Alanine D-Tryptophan D-Phenylalanine L-Histidine L-Valine L-Arginine D-Threonine

VoL. 16, 1968 TABLE 2. Concentrations and RF valuesa for the amino acids used Concentration Amino acid RF} RFC mg/ml mm Alanine... 0.50 3.6 30 32 a-amino-n-butyric acid.. 0.50 4.8 40 43 Arginine hydrochloride.. 0.80 3.8 15 14 Asparagine... 1.00 6.7 12 15 Aspartic acid... 0.50 3.8 23 21 Cysteine... 0.80 6.7 8 12 Cystine... 0.50 2.1 5 9 Ethionine... 0.80 4.9 58 60 Glutamic acid... 0.50 3.4 28 26... 0. 30 4.0 23 23 Histidine... 0.80 3.8 11 15 Hydroxyproline... 1.50 12.0 22 25 Isoleucine... 0.50 3.8 67 70 Leucine... 0.80 6.0 70 68 Lysine hydrochloride... 0.50 2.7 12 13 Methionine... 0.50 3.3 50 53 Ornithine hydrochloride.. 0.50 3.0 12 12 Phenylalanine... 0.50 3.0 60 59 Proline... 0.50 4.3 34 37 Serine... 0.50 4.8 22 20 Threonine... 0.30 2.5 26 26 Tryptophan... 1.20 5.9 50 47 Tyrosine... 1.20 6.6 45 42 Valine... 0.50 4.2 51 54 a Butanol-acetic acid-water (12:3:5) solvent. b Smith (1). c This paper. TABLE 3. Effect of ph and time of incubation on degradation ofalanine and serine by Salmonella typhimuriuma ph Alanine CHARACTrERIZATION OF BACrERIA Serine 6 hr 16 hr 6 hr 16 hr 3.6 0 0 0 2+ 4.0 0 0 2+ 4+ 4.4 0 2+ 3+ 4+ 4.8 0 4+ 4+ 4+ 5.2 0 4+ 4+ 4+ 5.6 0 4+ 4+ 4+ 6.0 0 4+ 4+ 4+ 6.4 3+ 4+ 4+ 4+ 6.8 3+ 4+ 4+ 4+ 3+ 4+ 4+ 4+ 7.6 3+ 4+ 4+ 4+ 8.0 3+ 4+ 4+ 4+ 8.4 4+ 4+ 4+ 4+ 8.8 4+ 4+ 4+ 4+ a Symbols: 0, no diminution of ninhydrin-positive spot; 1+, slight fading of spot; 2+, approximately 50% disappearance; 3+, nearly complete disappearance; 4+, complete disappearance of spot. TABLE 4. Effect ofph on degradation of proline by three species of bacteria incubated for 16 hra ph S. faccalis B. subtilis P. vulgaris 3.6 3+ 2+ 0 4.0 3+ 2+ 0 4.4 2+ 2+ 0 4.8 0 3+ 0 5.2 0 3+ 0 5.6 0 3+ 2+ 6.0 0 3+ 2+ 6.4 0 3+ 2+ 6.8 0 0 4+ 0 0 4+ 7.6 0 0 4+ 8.0 0 0 4+ 8.4 0 0 4+ 8.8 0 0 4+ a Symbols as in Table 3. 1593 tion of strains within a taxon, in the sense that phage-typing detects strains within a Salmonella serotype, and (ii) supplementary data for defining taxa at the species or generic level. The first test shows (Table 7) that the six strains of S. choleraesuis differ in their degradation of L- histidine, glycine, and L-lysine, and probably also of L-cystine. Although P. stutzeri and P. pseudoalcaligenes have some common features, they can readily be distinguished by their degradation of carbohydrates (unpublished data) and amino acids (Table 8). DIscussIoN Our initial premise that degradation of amino acids by bacteria can be reproducibly detected by means of paper chromatography has been substantiated. Several details of the procedure can be clarified only after extensive trial. As already noted, reproducibility of results is poor with a few amino acids; such instances merit further examination with higher concentrations of substrate. Further experience with these and additional amino acids may also lead to periods of incubation different from the 16 hr recommended here. However, it is apparent (Table 3) that reproducible results may not be obtained with either markedly shortened or irregular periods of incubation. With shorter periods of incubation, variations in the amount of inoculum may also become undesirably critical. With the 16-hr period of incubation and the five strains of Enterobacteriaceae, we found no qualitative change in results when the inoculum was reduced to 20% or increased to 200% of that recommended. There was some evidence of competitive utilization of amino acids existing in pools., for

1594 PICKElT AND PEDERSEN APPL. MICROBIOL. example, was completely utilized by P. aeruginosa and M. phlei in pool 14, but only incompletely or not at all in pool 7. Again, this implies that reproducible results will be obtained only when a standardized procedure is followed. Since all bacteria do not have the same optimal ph for utilization of a given amino acid, e.g.,jfor proline decarboxylase (Table 3), it may be desirable to use that amino acid at more than one TABLE 5. ph. We elected to include proline in both pool D (ph ) and pool I (ph ). Reproducibly negative results were thus obtained with B. subtilis in pool D, and reproducibly positive results in pool I. Contrariwise, poorly reproducible results should be anticipated with B. subtilis and proline at ph near but not at the optimum, e.g., ph 4.4. Although naturally occurring amino acids have been examined here, the chromatographic pro- Optimal ph for detecting, chromatographically, the degradation of amino acids by representative microorganisms Amino acid ph Amino acid ph L-Alanine... D-Leucine... 7.6 DL-a-amino-n-butyric acid.. 8.0..... 8.4 L-Arginine.. 7.0, 4.4..., 5.8 D-Asparagine... 6.2 D-Methionine.... 8.0, 6.0.7.8, 6.2...5.6.. 6.4... D-Phenylalanine. 7.6, 5.0... 7.4.7.4... 6.0., 5.2, 4.0... 6.6 L-Serine.6.6 D-Glutamic acid... 7.0 D-Threonine.6.4... 5.4.6.8...... 7.8, 6.8, 4.6., 7.0, 4.4 D-HiStidine L-HiStidine..., 4.4.... 8.0 D-Valine.7.6, 5.8... 6.4 L-Valine... 6.4... 8.0, 6.0 TABLE 6. Additional pools of amino acids prepared according to ph optima Pool ph Amino acids Pool ph Amino acids A F L-Serine G D-Threonine B L-Histidine hydrochloride L-Valine DL-a-Amino-n-butyric acid H hydrochloride C L-CyStine I D-Asparagine D hydrochloride J 4.0 D-Histidine hydrochloride E

VOL. 16, 1968 CHARACTERIZATION OF BACTERIA 1595 TABLE 7. Reproducibility of results and strain variations in chromatographic tests for degradation of amino acids by Salmonella choleraesuis Reproducibility Amino acid ph Strain 60b 60d 60f 60g 60h 60i Reproducible Incompletely reproducible Poorly reproducible L-Serine D-Asparagine DL-a-Amino-nbutyric acid L-Valine L-Histidine D-Threonine +-±+ ++++ +4--_ + - ++ ++4+ + 4-- - a Positive results obtained on all 4 days of test. b Positive results obtained on the 1st and 4th, negative on the 2nd and 3rd days of test. TABLE 8. Degradation ofamino acids by two strains each of Pseudomonas stutzeri and P. pseudoalcaligenes P. stutzeri pseudo Anino acid ph alcaligen_s K-97 K-100 K-94 K-99 + + _ L-LeUCinle + + _ + + _ + + + + cedure for detecting degradation is equally applicable to purines, pyrimidines, and synthetic amino acids. (Purines and pyrimidines would be ---- detected under ultraviolet light before treatment with ninhydrin.) Such techniques are now proving useful for characterization of Mycobacterium species. ACKNOWLEDGMENTS These studies were initiated by Susan Walshire with support to the University of California from National Science Foundation Undergraduate Research Participation grant GY 938, and by Madeleine Kraut with support to Claremont College from Public Health Service grant 5 Ti GM 945-404 from the National Institute of General Medical Sciences. LITERATURE CITE 1. Smith, I. 1960. Chromatographic and electrophoretic techniques. Interscience Publishers, Inc., New York.