Chromosomal Rearrangements Associated with Morphological ...

Vol. 172, No. 3

JOURNAL OF BACTERIOLOGY, Mar. 1990, p. 1276-1283

0021-9193/90/031276-08$02.00/0 Copyright C) 1990, American Society for Microbiology

Chromosomal Rearrangements Associated with Morphological Mutants Provide a Means for Genetic Variation of Candida albicans ELENA P. RUSTCHENKO-BULGAC,l* FRED SHERMAN,"2 AND JAMES B. HICKS3 Departments of Biochemistry' and Biophysics,2 University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, and Scripps Clinic and Research Foundation, La Jolla, California 920373 Received 20 March 1989/Accepted 8 December 1989

At frequencies as high as 1.4%, the pathogenic yeast Candida albicans spontaneously gave rise to morphological mutants exhibiting more than 20 different types of abnormal colonies; approximately two-thirds of the mutants were stable, while the other one-third were unstable and produced mixtures of different colonial forms at very high rates. Abnormal electrophoretic karyotypes were observed for all of the 14 mutants that were examined, indicating that they were associated with different types of single and multiple gross chromosomal rearrangements. Because C. albicans is asexual and does not go through a meiotic cycle, we suggest that the high frequency of chromosomal rearrangements provides a means for genetic variation in this organism.

Laboratories), 2% Bacto-Peptone, and 2% glucose, with 2% Bacto-Agar when required for solid medium (37). LBC synthetic medium (21) was initially developed to monitor yeast-hypha transition, and its modified version was also used for revealing colonial morphology (38). LBC medium consisted of 5 g of (NH4)2SO4, 0.2 g of MgSO4 7H20, 2.5 g of K2HPO4, 5 g of NaCl, 12.5 g of glucose, 0.5 g of alanine, 1.3 g of leucine, 1 g of lysine, 0.1 g of methionine, 0.0714 g of ornithine, 0.5 g of phenylalanine, 0.5 g of proline, 0.5 g of threonine, 1 mg of biotin (Sigma Chemical Co.), and 1 liter of distilled water, with 20 g of Bacto-Agar when required for solid medium. The biotin was filter sterilized and added after the other ingredients were autoclaved at 120°C for 15 to 20 min. Solid LBC medium was used to detect colony morphology; liquid LBC medium was used to grow cultures for detecting cell morphology. DNA samples for separating chromosomes were prepared from cells grown on either YPD or LBC liquid medium. Electrophoretic protocols. Orthogonal-field-alternation gel electrophoresis (OFAGE) (5) and transverse altering field gel electrophoresis (TAFE) (The Beckman GeneLine TAFE System, Beckman Instruments, Inc.) (41) were used to resolve C. albicans chromosomes. Three different running conditions of OFAGE were applied to improve the separation. The first condition consisted of constant 7.5 V/cm, approximately 90 mA at the start of the run, a 180-s pulse time, 10°C of 0.5x TBE (26) cooling buffer, 1.3% agarose, and a 70-h running time. The second condition was the same except for a 250-s pulse time, 1% agarose, and a 50-h running time. The third condition included constant 3 V/cm, approximately 30 mA at the start of the run, a 1-h pulse time, 11°C, 1% agarose, and a 5-day running time. These three conditions were applied to separate gels. The following conditions were used for TAFE: constant 100 V; approximately 70 mA at the start of the run; a 2-, 4-, 7-, and 10-min pulse time each for 1 day; 12°C of 1x TAE (26) cooling buffer; and 1% agarose. The techniques for staining and photographing the gels were the same as those previously described (5). Sample preparation. Chromosome-sized DNA was prepared from cultures grown to 2 x 107 to 4 x 107 cells per ml as described by Carle and Olson (6).

Since 1935, numerous investigators have reported that the pathogenic yeast Candida albicans produces abnormal colonies, which have been referred to as R type (29), rough variants (1, 7, 8, 46), lethal variants, membranous variants or wrinkled colonies (22), and minute-rough variants (15) as well as by special terms such as star, ring, fuzzy, etc., denoting specific colonial types (38). Such unusual colonies can be isolated from nature, can arise spontaneously in the laboratory (1, 2, 7, 8, 13, 15, 22, 29, 38, 40, 46), or can be induced by UV irradiation or chemical agents (14, 19, 33, 38). The genetic basis for their formation was previously unknown (see reference 32). In this paper, we call mutants arising from a normal strain morphological mutants if they have altered colony form, color, size, or texture (28), although this term has also been used to designate morphogenesis mutants (31). We report here the isolation and characterization of spontaneous morphological mutants and demonstrate that such morphological mutants are associated with different types of multiple chromosomal aberrations. Because the morphological mutants arise spontaneously at a high frequency, we suggest that chromosomal aberrations provide a means of genetic variation in this pathogenic amictic microorganism. While this article was in preparation, Suzuki et al. (43) described a morphological mutant with two additional bands in the electrophoretic karyotype. This mutant, in turn, produced revertantlike smooth colonies with differently altered karyotypes at a high frequency of 5 x 10-3. MATERIALS AND METHODS Strains. A collection of spontaneous mutants was isolated from C. albicans 3153A (standard strain) which has also been designated ATCC 32354, 300, and 3153A-SG (13; H. R. Buckley, unpublished result). The haploid strain 867 and diploid strain 7255/4C of Saccharomyces cerevisiae are standard laboratory strains (P. Wakem and F. Sherman, unpublished results) that were used for references of DNA content and for chromosomal markers in pulsed-field electrophoresis. Media. YPD medium contained 1% yeast extract (Difco *

Corresponding author. 1276

GENETIC VARIATION OF C. ALBICANS

VOL. 172, 1990

TABLE 1. Occurrence of morphological mutants from independent cultures after various incubation and storage times Culture

1 2 3 4

5

colof coloxamnies 4.2 3.2 4.7 1.0 4.2 8.1 3.5

104 104 x 104 x x

X

1 wk at

2 wk at

220C

220C

Approx at

mo

40C

4.8x 10-4 3.1x 10-4 6.0x 10-4

10

28 45

83 116

82

Strain Stabi tayiy no.

(%)b

Amt of DNA/cell

SD

(fg)

frequency

"I

20

104

x 104 x 103 x 104

3

TABLE 2. Properties of the 14 morphological mutantsa Strain or culture no.

No. of mutants after:

No. of

1277

4.5

x

C. albicans 5 4 5

1O-3

2.0x 10-3 1.4x 10-2 2.3x 10-3

A portion of the plates was incubated at 220C for several weeks and then stored for 3 months at 40C. a

Hybridization probes. The following probes were used for DNA. DNA hybridization to assign C. albicans chromosomes: BEN4 (Benr), LYS2, SOR2, and SOR9 (23); IH399 (an undefined fragment inserted in the plasmid pIH399; R. Poulter, unpublished result); ADE2 (17); URA3 (12); and HIS3 (36). Labeling with [a-32P]ATP by the random priming method, hybridization, and preparation of autoradiograms were carried out by standard methods (9, 26). DNA content. The DNA content of cells grown in YPD medium was determined by a diphenylamine method modified (T. Zamb, Ph.D. thesis, Illinois Institute of Technology, Chicago, 1978) (C. Giroux, unpublished results) from previous procedures (3, 4, 11). Diphenylamine assays were carried out in triplicate on samples of 8.9 x 107 cells from C. albicans cultures grown to 1.2 x 107 to 9.8 x 107 cells per ml. These assays were also carried out in triplicate on samples of approximately 2.5 x 108 and 1.25 x 108 cells, respectively, of the haploid and diploid strains of S. cerevisiae cultures grown to similar levels. Cells were washed twice in a solution of 0.2 N perchloric acid and 50% ethanol and then subsequently extracted with a solution of ethanol-ether (3:1, vol/vol). The pellets were further extracted with 1.5 N perchloric acid. DNA in the supernatant was assayed colorimetrically with the complete diphenylamine reagent containing 99 parts of 4% diphenylamine in glacial acid and 1 part of 1% paraldehyde. Standard stock solutions were prepared by dissolving known weights of highly polymerized calf thymus DNA (type I, sodium salt; Sigma) and by verifying the concentrations spectrophotometrically. Cell densities were determined by counting from 1,100 to 1,400 cells with a hemocytometer; buds equal to or greater than one-third the size of the mother cell were considered independent cells. Cell photographs. C. albicans strains were grown by shaking in 5 ml of liquid LBC medium at room temperature until the middle-logarithmic phase. Washed cells were fixed to the surface of polylysine-coated slides and covered with 90% glycerol to diminish the halo. Photographs were prepared with a Leitz Laborlux D microscope, using phase contrast, and with an Olympus photomicrographic system (model PM-1OAD). RESULTS Isolation of morphological mutants. A collection of 384 morphological mutants of spontaneous origin was obtained from five separate cultures of C. albicans 3153A (a standard strain) plated on LBC medium (see Materials and Methods). Each of these five cultures was derived from a separate

2 4 3 1 5 3

3153A ml m2 {m3 m4 {rm5 t m6 m7 m8 m9 mlO rmll

{m12 4 m13 1 m14 S. cerevisiae haploid (867) S. cerevisiae diploid (7255/4C)

100 68 100 100

44 52 43 63

100 100 90 100 95 100 100 100 94 97 75

48 46 43 58 47 47 52 47 58 51 85 17 34

± 8 ±3 ± 10 ± 1 ±4 ± 1 ± 1 ± 8 ± 1 ± 5 ± 11 ±1 ±6 ±6 ± 21 ± 3 ± 4

a Mutants ml to m14 were derived from one of the five independent cultures designated above. Mutants m2, m7, m8, mlO, and m13 were initially and subsequently stable mutants; mutant ml was unstable, having two colonial forms; the stable mutant m9 and the unstable mutant m14 were each derived from very unstable mutants that gave rise to colonies having at least six different morphologies. The three clonally related pairs of mutants, indicated by brackets, were derived as different morphological forms from three unstable strains that yielded colonies having more than two forms. b Percentage of the major class of colonies after at least 100 colonies were examined.

normal colony, thus ensuring that mutants arising from separate cultures would be the results of independent, spontaneous events. Approximately 4 x 104 colonies from each culture were examined for abnormal morphologies after the plates were incubated for various times at 22°C (room temperature) and after a portion of these plates was stored at 4°C, as summarized in Table 1. Because of the wide range of growth rates and because the morphologies were manifested after different periods of incubation, the conditions for detecting the maximum number of morphological mutants were not optimized. However, increased numbers were observed after increased times of incubation and storage, resulting in frequencies as high as 1.4% (Table 1). Previously, the frequency of morphological mutants was also noted to increase after storage (46) (D. Howard, unpublished result). Colonial morphologies. Stabilities and characteristic morphologies for a subset of 223 mutants obtained from the five independent cultures incubated for 1 and 2 weeks at room temperature were determined (Table 1). The mutants were repeatedly subcloned for two to six cycles on LBC plates that were subsequently incubated at room temperature for 2 to 4 weeks. Each of 152 mutants was found to yield predominantly a uniform population of a characteristic abnormal colony and was considered stable. In contrast, 71 mutants were unstable, each giving rise to mixtures of various types of colonies, including instances of normal-looking colonies and often at least six different colonial types. BrownThomsen (2) reported earlier that some unstable C. albicans strains gave rise to five colonial forms. Details of the properties of the unstable morphological mutants are forthcoming (manuscript in preparation). Approximately 20 different types of colonial morphologies could be easily distinguished after the 152 stable mutants were examined (data not shown). Some of them resembled morphological mutants and natural variants isolated by

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RUSTCHENKO-BULGAC ET AL.

J. BACTERIOL.

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-BDIIs I. ___ _ FIG. 1. Colonial morphologies of the normal strain 3153A (+) and the mutants ml to ml4. The mutant m5 is not shown because it appeared normal (Table 2).

others (1, 7, 8, 13, 15, 22, 29, 38, 39, 46), although better comparisons would require parallel platings on identical media and incubations with identical conditions. Mutants ml through m14. Fourteen distinguishable mutants designated ml through m14 were arbitrarily chosen from the most unusual-looking ford-Is. Only mS, a revertant, had a normal appearance. DerivE nons, relationships, and stabilities of the strains are presented in Table 2; photographs of typical colonies are shown in Fig. 1. To verify that the mutants truly originated from the parental strain 3153A, DNA samples were fingerprinted, using the middle-repeat sequence Ca3 as a hybridization probe. The hybridization pattern of this probe in EcoRI digests of C. albicans has been shown to be a useful method for identifying stra;, and species (40; unpublished data). The 3153A isolates and all mutants from this study exhibited the identical pattern and are considered clonally related (data not shown). Cell populations. Cell cultures that were grown in liquid LBC medium at room temperature to approximately the middle-logarithmic phase were examined microscopically, thus revealing the features of actively growing cells while preventing microheterogeneity that can occur with plategrown cells and within colonies. Under these conditions, which were specifically designed to maintain C. albicans as pure yeast indefinitely (21), the normal strain 3153A exhibited a uniform cell population of oval cells having mostly one or no bud and lacking aggregates (Fig. 2A). This normal culture also contained a small fraction (