Arch. Microbiol. 104, 225-231 (1975) - 9 by Springer-Verlag 1975 Physiological and DNA Characterization of Candida maltosa, a Hydrocarbon-Utilizing Yeast S. A. MEYER, K. ANDERSON, and R. E. BROWN American Type Culture Collection, Rockville, Maryland M. TH. SMITH and D. YARROW Centraalbureau voor Schimmelcultures, Delft G. MITCHELL and D. G. AHEARN Georgia State University, Atlanta, Georgia Received April 14, 1975 Abstract. Selected yeasts classified as Candida sake van Uden et Buckley were examined for their physiological, morpho- logical and immunological properties and their DNA relat- edness. Candida maltosa Komagata, Nakase et Katsuya is herein recognized as a species separate from C. sake. Candida maltosa was distinguished from C. sake and from C. tropicalis by insignificant DNA reassociation. In addition, C. maltosa was distinguished from C. sake by its higher maximal growth temperature and lower guanine plus cytosine content of its DNA and from C. tropicalis by its failure to utilize soluble starch for growth and its resistance to cycloheximide. The species C. cloacae and C. subtropicalis are placed in synonymy with C. maltosa. Key words: Candida maltosa - C. tropicalis - C. cloacae- C. subtropicalis - C. sake- DNA Relatedness - GC Contents - Yeasts Hydrocarbon Utilization - Serology of Yeasts. A variety of yeasts, particularly species of Candida, are known to assimilate alkanes (Komagata et al., 1964; Klug and Markovetz, 1967; Bos and de Bruyn, 1973). Among those giving the greatest cell crops per unit of substrate are representatives of C. subtropicalis, C. tropiealis and isolates grouped with C.sake by van Uden and. Buckley (Lodder, 1970). These three species have similar fermentation and assimilation patterns. Bos and de Bruyn (1973) examined 29 strains included in C. sake. The type strain grew weakly on alkanes, whereas strains originally described by Koma- gata et al. (1964) as C. maltosa .and C. cloacae grew well. Nakase and Komagata (1971) stated-that these two yeasts should be treated as separate species as they grow at higher temperatures (40-42~ than C.sake. Later, Nakase et al. (1972) distinguished C. subtropicalis from C. tropicalis by its higher GC con- tent, agglutination reactions, proton-magnetic-reso- nance spectrum of the cell-wall mannan and inability to assimilate soluble starch. The assimilation and fermentation reactions described for C. subtropicalis coincide with those given for C. sake by Lodder (1970). To determine the correct classification of the above- mentioned species, we have examined the morpho- logical, physiological, and immunological properties and the DNA relatedness of selected isolates. Materials and Methods Organisms and Morphological and Physiological Character- &tics. The cultures examined are listed in Table 1. The mor- phological and physiological characterization of the yeasts was performed according to the methods in Lodder (1970). The assimilation tests were incubated on a shaker for 21 days. Growth on hydrocarbons was determined by the method of Markovetz and Kallio (1964) and in yeast nitrogen base (Difco) with 1.0 ~ hydrocarbon. Immunological Character&tics. Antisera were produced in rabbits and homogenate' antigen prepared according to the procedure of Stickle~et al. (1972). A micro-immunodiffusion technique of double-diffusion in two dimensions was used as described by Ibrahim and Hammon (1968). Adsorption-in-gel was accomplished by the method of Ibrahim and Hammon (1969). The slides and templates used were identical to those employed for immunodiffusion. The central well was first filled with the" adsorbing antigen, which was allowed to diffuse for 2 hrs at 35- 37~ C. The residual antigen was then removed by aspiration and the central well was refilled with the antiserum to be adsorbed. Simultaneously, the outer wells were filled with the test antigens and the slides were incubated at 35-37~ Observations for precipitin bands were made at 12- to 18-hr intervals. DNA Base Composition and DNA Reassociation. DNA was isolated and purified according to the methods previously described (Meyer and Phaff, 1969). DNA base composition (expressed as the mean molar percent of the guanine and cytosine content, ~GC) was determined by the thermal denaturation method and formula [~GC = (TM-69.3)/