Scrofulaceum Isolates. and Mycobacterium Mycobacterium
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Uric acid utilization by Mycobacterium intracellulare and Mycobacterium Downloaded from scrofulaceum isolates. J O Falkinham 3rd, K L George, B C Parker and H Gruft J. Bacteriol. 1983, 155(1):36. http://jb.asm.org/ Updated information and services can be found at: http://jb.asm.org/content/155/1/36 on February 13, 2012 by TECH SERVICES/SERIALS RECVG These include: CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://jb.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ JOURNAL OF BACTERIOLOGY, July 1983, P. 36-39 Vol. 155, No. 1 0021-9193/83/070036-04$02.00/0 Copyright © 1983, American Society for Microbiology Uric Acid Utilization by Mycobacterium intracellulare and Mycobacterium scrofulaceum Isolates Downloaded from JOSEPH 0. FALKINHAM 111,1* KAREN L. GEORGE,' BRUCE C. PARKER,1 AND HOWARD GRUFT2 Department ofBiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061,1 and Center for Laboratories and Research, New York State Department of Health, Albany, New York 122012 Received 10 December 1982/Accepted 3 April 1983 Forty-nine human and environmental isolates of Mycobacterium intracellulare and Mycobacterium scrofulaceum were tested for their ability to growon uric acid http://jb.asm.org/ and a number of its degradation products. Nearly all (88 to 90%) strains used uric acid or allantoin as a sole nitrogen source; fewer (47 to 69%) used allantoate, urea, or possibly ureidoglycollate. Enzymatic activities of one representative isolate demonstrated the existence of a uric acid degradation pathway resembling that in other aerobic microorganisms. Mycobacteriosis outbreaks in starlings (1), Furthermore, we examine the degradation path- on February 13, 2012 by TECH SERVICES/SERIALS RECVG endemic mycobacteriosis in other birds with way in one isolate. simultaneous isolation of Mycobacterium avium from feces-contaminated soil and mud of avian MATERIALS AND METHODS habitats (16), and recovery of related mycobac- Bacterial strains. Of the 49 isolates of M. intracellu- teria from still other birds and their habitats (20) lare or M. scrofulaceum used in this study, 27 were recovered from infected humans by the Mycobac- provide cumulative evidence that nontubercu- teriology Laboratory of the New York State Depart- lous mycobacteria of the M. avium, Mycobacte- ment of Health, and 22 were isolated from fresh, rium intracellulare, and Mycobacterium scrofu- brackish, or salinei waters of the eastern United States laceum (MAIS) group may utilize uric acid, a (7). The isolates were identified to the species level major avian nitrogen excretion product, or cer- and then assigned to one of the eight biotypes, based tain degradation products of uric acid as sources on their pigmentation and on the results of semiquanti- of carbon or nitrogen or both. tative catalase and urease tests (8, 12). Isolates sharing Previous metabolic studies have focused on two characteristics with either M. intracellulare, i.e., degradation and utilization of uric acid or its nonpigmented, urease negative, and catalase negative nontu- (-- -), or M. scrofulaceum (+ + +) were recorded as degradation products by rapidly growing, members of that species, although of different bio- berculous mycobacteria (14) or on degradation types. One M. scrofulaceum isolate, W200 (+++ +), of allantoin or urea (3, 5, 17-19). In these latter recovered from Lake Borgne, east of New Orleans, studies, the isolates which showed uricolytic La., was used to study the enzymes of uric acid activity were primarily rapidly growing nontu- degradation. berculous mycobacteria. Klemperer et al. (9) Media. Strains were grown in Middlebrook and demonstrated decomposition of radioactively la- Cohn 7H9 medium containing 0.5% (vol/vol) glycerol beled uric acid by various mycobacteria, includ- and 10o (vol/vol) OADC Enrichment (BBL Microbiol- ing several MAIS isolates, and concluded that in ogy Systems, Cockeysville, Md.). The basal medium used contained 0.5 g each of KH2PO4 and Mycobacterium butyricum degradation is oxida- MgSO4 * 7H20 per liter and 0.1 M glycerol. To this tive, involving the intermediate formation of was added one of seven substrates, uric acid, allan- allantoin, allantoic acid, ureidoglycollate, and toin, allantoic acid, ureidoglycollate, urea, L-gluta- urea, as suggested earlier by DiFonzo (6) for mate, or (NH4)2SO4, as the sole nitrogen source to a non-MAIS mycobacteria. Rohrscheidt et al. (13) final concentration of 0.02 M (19). In some experi- cast doubt on these claims by showing that ments, the concentrations of uric acid, allantoic acid, degradation in other rapidly growing mycobacte- ureidoglycollate, and urea were lowered to 0.002 M to ria did not follow the oxidative pathway. How- eliminate precipitate formation. ever, in none of these studies was a detailed Preliminary tests established that liquid or solidified of uric acid and its medium (Bacto agar, 20 g/liter; Difco Laboratories, examination degradation Detroit, Mich.) gave similar results and that no signifi- pathway in MAIS isolates conducted. cant differences in growth rates were observed at the In this report we describe the ability of 49 different nitrogen source concentrations. human and environmental isolates of M. intra- Cell extracts. Cells grown to late log phase in the cellulare and M. scrofulaceum to utilize uric basal medium were collected by centrifugation at 5,000 acid and its degradation products for growth. x g for 20 min at 4°C, washed twice, and suspended in 36 VOL. 155, 1983 URIC ACID DEGRADATION BY MYCOBACTERIA 37 1/10 the original volume of 0.04 M potassium phos- concentrations of extracts were determined by the phate buffer (pH 7.2). Cells were disrupted with a method of Lowry et al. (10). French pressure cell until less than 10% of the cells were intact, as judged by microscopic examination. The suspension was then centrifuged at 10,000 x g for RESULTS Downloaded from 20 min at 4°C, and the supernatant was used for Utilization of uric acid and degradation prod- enzyme assays. Assays were performed only on fresh ucts. extracts. Preliminary experiments indicated that iso- Enzyme assays. Uricase (urate:oxygen oxidoreduc- lates of M. intracellulare and M. scrofulaceum tase, EC 1.7.3.3) activity was measured by the disap- could utilize uric acid or one of its degradation pearance of uric acid, which was assayed spectropho- products as a sole nitrogen source, although not tometrically as described by Mahler (11). as a sole carbon source (data not shown). Of the Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) 49 isolates, 88% utilized uric acid and 90% activity was determined by measuring the utilization of utilized allantoin as sole nitrogen sources (Table allantoin. Allantoin was determined by conversion to 1). Fewer isolates grew on allantoic acid, urei- http://jb.asm.org/ glyoxylate by alkaline-acid hydrolysis with heat. The doglycollate, and urea. resulting glyoxylate was transformed to glyoxylic acid- Because of the long periods of incubation phenylhydrazone upon reaction with phenylhydra- grow zine, which was oxidized in the presence of strong acid required to these microorganisms in the and potassium ferricyanide. Azo coupling and decar- basal medium (see above), the stability of each boxylation of the oxidized product yielded the red 1,5- substrate was carefully monitored. The only diphenylformazan, whose concentration was mea- apparently unstable substrate during the incuba- sured spectrophotometrically (21). tion periods used (even in enzyme analyses) was The combination of allantoicase (allantoate amidino- ureidoglycollate. Because of this instability, the on February 13, 2012 by TECH SERVICES/SERIALS RECVG hydrolase, EC 3.5.3.4) and allantoate deiminase [allan- isolates which appeared to utilize ureidoglycol- toate amidinohydrolase (decarboxylating), EC 3.5.3.9] late may have been growing on released urea. activities was assayed by the enzymatic disappearance Although differences in utilization of these of allantoic acid. Allantoic acid was converted to glyoxylate by acid hydrolysis (2, 21). nitrogen sources were found between M. intra- Ureidoglycollate lyase (ureidoglycollate urea-lyase, cellulare and M. scrofulaceum (Table 1) and EC 4.3.2.3) activity was determined by the utilization differences among their biotypes occurred (data of ureidoglycollate, measured before and after the not shown), these differences were not signifi- reaction period by conversion to glyoxylate with heat cant at the 0.05% level (chi-square test). Also, (21). no significant differences were noted between Urease (urea amidohydrolase, EC 3.5.1.5) activity the human isolates and those recovered from was measured by determining the amount of ammonia eastern waters. M. scrofulaceum isolate W200, formed enzymatically from urea by the phenolhypo- chlorite method of Russell (15). which grew on all substrates (albeit slowly on was Because commercial samples of allantoic acid con- urate), chosen for detailed studies of growth tained ureidoglycollate, glyoxylate, and ammonia and characteristics and enzyme activities. because ureidoglycollate samples contained glyoxy- Generation time with uric acid and degradation late and ammonia, appropriate control mixtures were products. The generation times for isolate W200 prepared, and the initial concentration of glyoxylate, on various substrates, as judged by the increase