YUZO YAMADA AND MANAMI AKITA. Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Shizuoka University, Shizuoka 422, Japan

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1 J. Gen. App!. Microbiol., 30, (1984) AN ELECTROPHORETIC COMPARISON IN STRAINS OF GL UCONOBACTER OF SPECIES' ENZYMES YUZO YAMADA AND MANAMI AKITA Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Shizuoka University, Shizuoka 422, Japan (Received March 22, 1984) Forty-three strains of Gluconobacter species were examined for the electrophoretic comparison of six enzymes, namely, glucose-6-phosphate dehydrogenase (NADP-dependent, EC ), 6-phosphogluconate dehydrogenase (NADP-dependent, EC ), glucose dehydrogenase (NADP-dependent, EC ), alcohol dehydrogenase (NADdependent, EC ), aldehyde dehydrogenase (NADP-dependent, EC ), and catalase (EC ). From the electrophoretic patterns of the enzymes, these organisms were unequivocally divided into two groups, Groups I and II. There was no relation between the two groups; the similarity value was 0 %. Group I consists of organisms with a high guanine-plus-cytosine content of DNA ranging from 58.1 to 62.8 mol %, a range of 4.7 mol %. Group II is composed of organisms with a distinctly lower guanine-plus-cytosine content of DNA ranging from 54.2 to 57.6 mol %, a range of 3.4 mol %. Such a grouping was supported earlier by a DNA-DNA hybrid experiment. On the basis of the electrophoretic patterns of the enzymes and the DNA-DNA hybridization, we propose a new species, Gluconobacter cerinus, sp. nov., nom. rev., for the organisms classified in Group II. The organisms in the genus Gluconobacter Asai 1935 emend, mut, char. Asai, Iizuka, and Komagata 1964 are characterized by polar flagellation (2, 3), lack of oxidation of lactate and acetate (4, 5), the ubiquinone-10 system in the respiratory quinone (5-7), and the C18:1 acid type in the cellular fatty acid composition (8). In Bergey's Manual, 8th Edition (5), the genus Gluconobacter is composed of the single species, Gluconobacter oxydans, which is divided into four subspecies oxydans, industrius, suboxydans, and melanogenes. In addition to the four subspecies, G. oxydans subsp. sphaericus was reported by AMEYAMA (9). In a previous paper (10), one of the authors (Y.Y.) measured deoxyribonucleic 1 Taxonomic Studies on Acetic Acid Bacteria and Allied Organisms VIII, see ref. 1. Address reprint requests to Dr. Y. Yamada Part IX. For Part

2 116 YAMADA and AKITA VOL. 30 acid (DNA) base composition in twenty representative strains of the genus Gluconobacter and calculated the composition to be from 54.2 to 62.8 mol % guanine plus cytosine, a range of 8.6 mol %. Such a wide range of the guanine-plus-cytosine content of DNA led us to conceive that the genus Gluconobacter might be heterogeneous from the taxonomic point of view and that the organisms would be better classified in more than one species. The DNA-DNA hybrid experiment confirmed that the genus Gluconobacter can be genetically divided into at least two groups (1). This paper deals with the electrophoretic patterns of the enzymes from forty-three strains included in the genus Gluconobacter and proposes Gluconobacter cerinus sp. nov., nom. rev., for the organisms characterized by the lower guanineplus-cytosine content of DNA. MATERIALS AND METHODS Microorganisms and cultivation. Forty-three strains of acetic acid bacteria classified in the genus Gluconobacter were used in the experiment (Table 1). The bacterial names in quotation marks are not on the Approved Lists of Bacterial Names, 1980 (11). The microorganisms were grown aerobically for 24 hr at 30 in a medium (100 ml) containing glucose 0.5 %, glycerol 1.5 %, peptone 0.5 %, yeast extract 0.5 %, and malt extract 0.1 % (ph 6.8) dispensed into 500-m1 conical flasks, as described in a previous paper (8). Preparation of enzymes. The intact cells harvested by centrifugation were suspended in 0.05 M Tris-HCl buffer (ph 7.8) containing 0.57 mm ascorbic acid, 0.64 mm cysteine-hci, and 0.4 M sucrose, and disrupted by sonication for 10 min at 140 W. The enzyme solution was prepared by centrifugation at 12,500 rpm for 60 min, and the precipitate was discarded. Electrophoresis and staining of enzymes. Six enzymes were examined: glucose-6-phosphate dehydrogenase (NADP-dependent, EC ), 6-phosphogluconate dehydrogenase (NADP-dependent, EC ), glucose dehydrogenase (NADP-dependent, EC ), alcohol dehydrogenase (NAD-dependent, EC ), aldehyde dehydrogenase (NADP-dependent, EC ), and catalase (EC ). Malate dehydrogenase (NADP-dependent, EC ) in G. oxydans subsp. oxydans NCIB 9013 and "G. cerinus" IFO 3267 was reported in a previous paper (12) and is omitted here because the representatives of the genus Gluconobacter lack the tricarboxylic acid cycle (13). The malate dehydrogenase activity of the Gluconobacter species was actually not so active as that of the Acetobacter species. The NADP-dependent glucose dehydrogenase was recognized in a wide variety of Gluconobacter species. The electrophoresis and staining procedures of the enzymes were done as described by URAKAMI and KOMAGATA (14). A 7.5% polyacrylamide gel (130>< 135 x 2 mm, the so-called 8.3 gel) slab was used in the experiment. The enzyme solution ( ,~g of protein) was applied to

3 1984 Electrophoretic Enzyme Patterns in Gluconobacter Species 117 Table 1. The examined strains of Gluconobacter species.

4 118 YAMADA and AKITA VOL. 30 the gel slab. The loaded gel slab was subjected to electrophoresis at 15 to 20 ma per gel slab with a marker of bromophenol blue (0.001%) for 5 to 6 hr at 5. The dehydrogenase bands were stained with nitroblue tetrazolium, and the catalase bands were stained with 3,3-diaminobenzidine tetrachloride. The relative electrophoretic mobilities (Rm values) of the enzymes were obtained between the bromophenol blue and the enzyme bands. The similarity values in the electrophoretic patterns of the enzymes were calculated by the following formula based on the procedures of BAPTIST et al. (15) : s(%)=ns/(ns+nd) x 100 (s, similarity value; NS, the number of enzymes with identical relative mobility; Nd, the number of enzymes with different relative mobility). Chemicals. Acrylamide (monomer, EPI-7) was obtained from Nakarai Chemicals, Ltd., Kyoto, Japan. N,N'-Methylenebisacrylamide ( ) and N,N,N',N'-tetramethylethylenediamine ( ) were from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Glucose-6-phosphate, 6-phosphogluconate, NAD, and NADP were the products of Oriental Yeast Co., Ltd., Tokyo, Japan. RESULTS AND DISCUSSION The relative electrophoretic mobilities of the six enzymes are shown in Table 2. In the forty-three strains examined, all six enzymes were present. However, in some strains the activities of the NAD-dependent alcohol dehydrogenase and NADP-dependent aldehyde dehydrogenase were not detected. Although the oxidation of ethanol to acetic acid is physiologically catalyzed by the particlebound alcohol and aldehyde dehydrogenases in acetic acid bacteria, the reaction is not necessarily important in the organisms classified in the genus Gluconobacter (16). In fact, there are a few strains which do not produce acetic acid from ethanol (3,16). The Rm values of glucose-6-phosphate dehydrogenase were distributed from 0.45 to "Gluconobacter cerinus" IFO 3266 and G. oxydans subsp. sphaericus IFO had the lower Rm values of 0.41 and 0.35, respectively. The Rm values of 6-phosphogluconate dehydrogenase were divided into two groups, namely, from 0.45 to 0.49 and from 0.35 to 0.37 (Fig. 1). The intermediate Rm values of 0.42 and 0.43 appeared in some strains. The group of the Rm values from 0.45 to 0.49 contained the organisms with a high guanine-plus-cytosine content of DNA such as G. oxydans subsp. oxydans NCIB 9013, and the group of the Rm values from 0.35 to 0.37 contained the organisms with a low guanine-plus-cytosine content of DNA such as "G. cerinus" IFO The Rm values of glucose dehydrogenase were widely distributed from 0.40 to Alcohol dehydrogenase was mostly found at the Rm values from 0.32 to Addehyde dehydrogenase gave, on the whole, two stained bands. In a previous paper (12), we reported three Rm values for the aldehyde dehydrogenase, 0.23, 0.32, and 0.34 in G. oxydans subsp. oxydans NCIB 9013 and 0.35, 0.42, and 0.45 in "G. cerinus" IFO 3267.

5 1984 Flectrophoretic Enzyme Patterns in Gluconobacter Species 119 Of the three, the Rm values corresponding to 0.23 in G. oxydans subsp. oxydans NCIB 9013 and to 0.35 in "G. cerinus" IFO 3267 were omitted in this work, since such a band still appeared in G. oxydans IFO 3287 which had no detectable aldehyde dehydrogenase activity. The Rm values of catalase made three clusters: from 0.17 to 0.19, from 0.25 to 0.28, and over The Rm values were mostly found at 0.19 and at 0.25 to Based on the Rm values shown in Table 2, the similarity values in the electrophoretic patterns were calculated. As shown in Figs. 2 and 3, the genus Gluconobacter was completely divided into two groups, Group I containing G. oxydans subsp. oxydans NCIB 9013 and Group II containing "G. cerinus" IFO Between the two groups, there was no correlation in the electrophoretic patterns of the six enzymes. The similarity value was found to be 0 % between Groups I and II. In Group I, G. oxydans subsp. oxydans NCIB 9013 and "G. capsulatus" IFO 3462 were very similar to each other in the electrophoretic enzyme patterns. Among the four strains, G. oxydans IFO 3189 and IFO 3287 and the above-mentioned two strains, the similarity value was calculated to be 83 %. The watersoluble brown pigment-producing strains, "G. melanogenus" IFO 3292, IFO 3293, and IFO 3294, "G. rubiginosus" IFO 3244, and G. oxydans subsp. sphaericus IFO were all included in Group I. However, G. oxydans subsp. sphaericus IFO was unique in its electrophoretic patterns. Its similarity value with other strains was calculated to be only 17 %. "Gluconobacter cerinus" IFO 3266, "G. albidus" IFO 3250, and "G. dioxyacetonicus" IFO 3273 were contained in Group I. From the results obtained above, Group I was found to consist of the organisms with DNA base composition ranging from 58.1 to 62.8 mol % guanine plus cytosine, a range of 4.7 mol %. The other twenty-nine strains appeared in Group II. These strains had at least a 50 % similarity value within the group, which was divided into three small subgroups. Group II consisted of the organisms with DNA base composition from 54.2 to 57.6 mol % guanine plus cytosine, a range of 3.4 mol %. The previously reported DNA-DNA hybrid experiment showed that the genus Gluconobacter is also divided into at least two groups (1). When G. oxydans subsp. oxydans NCIB 9013 was labeled with [6-3H]thymidine, homology indexes of more than 45 % were calculated in "G. albidus" IFO 3250, "G. capsulatus" IFO 3462, "G. melanogenus" IFO 3293 and IFO 3294, G. oxydans IFO 3189 and IFO 3287, G. oxydans subsp. sphaericus IFO 12467, "G. roseus" IFO 3990, and "G. rubiginosus" IFO These strains are included in Group I. Other strains which had homology indexes below 40 % are members of Group II. Conversely, homology indexes over 51 % were obtained in "G. albidus" IFO 3251, "G, cerinus" IAM 1832, "G, gluconicus" IFO 3171, "G. nonoxygluconicus" IFO 3275, and "G. suboxydans" IFO 3172 and IFO 3255 using the labeled "G. cerinus" IFO 3267 DNA. Very recently, GossELE et al. (17) have demonstrated that the organisms in-

6 120 YAMADA and AK[TA VOL. 30 Table 2. The electrophoretic mobilities of enzymes in the strains of Gluconobacter species.

7 1984 Electrophoretic Enzyme Patterns in Gluconobacter Species 121 Table 2. Continued. eluded in the genus Gluconobacter are divided into two groups on the basis of phenotypic features. Their Phenon A is comprised of G. oxydans subsp. suboxydans ("G. albidus") IFO 3251 and IFO 3253, G. oxydans subsp. suboxydans ("G. cerinus") IFO 3262, IFO 3263, IFO 3264, IFO 3265, IFO 3267, IFO 3268, IFO 3269, and IFO 3270, G. oxydans subsp. suboxydans ("G. dioxyacetonicus") IFO 3271 and IFO 3274, G. oxydans subsp. suboxydans ("G. gluconicus") IFO 3285 and IFO 3286, G. oxydans subsp. suboxydans ("G. nonoxygluconicus") IFO 3276, G. oxydans subsp. suboxydans ("G. suboxydans") IFO 3289, IFO 3290, IFO 3291, IFO 3255, IFO 3257, and IFO 3258, and G. oxydans subsp, industrius ("G. industrius") IFO 3260, and so on. All the strains mentioned above belong to our Group II. They described the uniqueness of G. oxydans subsp. suboxydans ("G. cerinus") IAM 1832 in the phenotypic features and included the organism in neither their Phenon A nor B. However, our two studies on the DNA-DNA

8 122 YAMADA and AKITA VOL. 30 Fig. 1. Polyacrylamide gel slabs stained for six enzymes from some strains of Gluconobacter species. (a) glucose-6-phosphate dehydrogenase, (b) 6-phosphogluconate dehydrogenase, (c) glucose dehydrogenase, (d) alcohol dehydrogenase, (e) aldehyde dehydrogenase, (f) catalase. hybrids and the electrophoretic patterns of enzymes showed that "G. cerinus" IAM 1832 and IFO 3267 are very closely related to one another; the homology index and the similarity value were calculated to be 91 % and 83 %, respectively. Actually, the two strains have a similar electrophoretic protein pattern, A'1 (17). Although there is some disagreement between their grouping and ours, it is obvious that their Phenon A and our Group II have similar characteristics in the classification of the organisms in the genus Gluconobacter. Included in their Phenon B are G. oxydans subsp. suboxydans ("G. cerinus") IFO 3266, G, oxydans subsp. suboxydans ("G. dioxyacetonicus") IFO 3273, G. oxydans subsp. melanogenes ("G. melanogenus") IFO 3293, G. oxydans subsp, oxydans NCIB 9013, G. oxydans subsp, oxydans (G. oxydans) IFO 3287, G. oxydans subsp. sphaericus IFO 12467, and G. oxydans subsp. melanogenes ("G. rubiginosus") IFO All of these strains are members of our Group I. Gluconobacter oxydans subsp, melanogenes ("G. melanogenus") IFO 3294 is contained in their Phenon A (17). GILLIS and DE LEY (18) apparently did not measure the DNA base composition of the strain.

9 1984 Electrophoretic Enzyme Patterns in Gluconobacter Species 123 Fig. 2. The dendrogram of the strains of Gluconobacter species based on similarity values in the electrophoretic enzyme patterns (Group I). Fig. 3. The dendrogram of the strains of Gluconobacter species based on similarity values in the electrophoretic enzyme patterns (Group II). Their strain IFO 3294 is regarded as a non-producer of a water-soluble brown pigment (17). Our strain IFO 3294 produces such a pigment, and has a high guanine-plus-cytosine content of DNA (62.0 mol %) (10). Probably, their strain

10 124 YAMADA and AKITA VOL. 30 IFO 3294 has been mixed up. It is demonstrated above that the DNA base composition of our Group I ranges from 58.1 to 62.8 mol % guanine plus cytosine and our Group II ranges from 54.2 to 57.6 mol % guanine plus cytosine. GossELE et al. (17) have not discussed their groupings, Phenons A and B, in relation to the DNA base composition. Apparently, they examined the DNA base composition in only three strains of their Phenon A (18). Probably, their Phenon A is composed of the organisms with lower guanine-plus-cytosine content of DNA, and their Phenon B is comprised of the organisms with higher guanine-plus-cytosine content of DNA. Phenons A and B are discriminated from each other only by the phenotypic character of nicotinic acid requirement; the former does not require nicotinic acid for growth but the latter does (17). In addition to the requirement of nicotinic acid, the electrophoretic patterns of the six enzymes distinctly differentiate these two groups, Group I (or Phenon B) and Group II (or Phenon A). The DNA-DNA hybridization studies also supported such groupings (1). "Gluconobacter suboxydans" IFO 3172 in our Group II is derived from the strain ATCC 621 (=NCIB 621), the type of G. oxydans subsp. suboxydans. Previously, we determined the DNA base composition of the strain IFO 3172 to be 56.4 mol % guanine plus cytosine (10). However, GILLis and DE LEY (18) calculated that of G. oxydans subsp. suboxydans NCIB 621 (=ATCC 621) to be 61.2 mol % guanine plus cytosine. The difference is 4.8 mol % between their determination and ours. "Gluconobacter suboxydans" (G. oxydans subsp. suboxydans) IFO 3172 is included in our Group II (or probably their Phenon A). However, G. oxydans subsp. suboxydans NCIB 621 is a member of their Phenon B (or probably our Group I). Considering the origin of the strain IFO 3172, a certain confusion can be expected occasionally. The strain IFO 3130 of "G. suboxydans" is a member of our Group I. As has been described above, it is apparent that the genus Gluconobacter can be divided phenotypically and genetically into two groups, Group I (or Phenon B) and Group II (or Phenon A). Contrary to the opinion of GossELE et a1. (17), the two groups in the genus Gluconobacter should be separated from each other at the species level. We here propose a new species, Gluconobacter cerinus sp. nov., nom. rev., for the organisms with the lower guanine-plus-cytosine content of DNA. Gluconobacter cerinus (ex Asai 1935) Yamada and Akita sp. nov., nom. rev. Gram-negative rods, occurring singly, in pairs or in chains, x µm. Polarly flagellated when motile. Forms glossy, smooth, and entire colonies. Grows on mannitol agar. Produces gluconate, 2-ketogluconate, and 5-ketogluconate from glucose, dihydroxyacetone from glycerol, and acetic acid from ethanol. Produces neither water-soluble brown pigment on glucose-yeast extract-calcium carbonate nor 2,5-diketogluconate from glucose. Grows mostly

11 1984 Electrophoretic Enzyme Patterns in Gluconobacter Species 125 Table 3. Some characteristics differentiating Gluconobacter cerinus from Gluconobacter oxydans. on ribitol, xylitol, and L-arabitol. Requires pantothenic acid but not nicotinic acid for growth. DNA base composition ranges from 54.2 to 57.6 mol % guanine plus cytosine. Possesses Q-10 system. Cellular fatty acid is of C18;1 acid type. Habitats are mainly fruits and flowers. Type strain is IFO 3267 (=ATCC 19441). Etymology: cerinus Latin adjective, wax-colored. ASAI (19) first gave his strain 24 (1AM 1832) isolated from an apple the name

12 126 YAMADA and AKITA VOL. 30 of "Gluconoacetobacter cerinus" in a subgenus of the genus Gluconobacter. Later, AsAI et al. (3) pointed out that the strain IAM 1832 produces little acetic acid from ethanol. According to GOSSELE et al. (17), the strain IAM 1832 is atypical and does not belong to either Phenon A or B. However, the strain IAM 1832 and IFO 3267 are genetically closely related (1). In fact, the two strains have a similar electrophoretic protein pattern, A' l (17) as well as a high similarity value (83%) in the electrophoretic enzyme patterns mentioned above. Gluconobacter cerinus is phenotypically distinguished from G. oxydans not only by not requiring nicotinic acid and by the electrophoretic protein patterns, but also by the electrophoretic patterns of enzymes, especially 6-phosphogluconate dehydrogenase (Table 3). Gluconobacter cerinus forms the cluster which has the lower guanine-plus-cytosine content of DNA from 54.2 to 57.6 mol %, a range of 3.4 mol %. In contrast, G. oxydans occupies the remaining cluster which has the higher guanine-plus-cytos~ne content of DNA ranging from 58.1 to 62.8 mol %, a range of 4.7 mol %. REFERENCES 1) Y. YAMADA, N. ITAKURA, M. YAMASHITA and Y. TAHARA, J. Ferment. Technol., 62, in press (1984). 2) E. LEIFSON, Antonie van Leeuwenhoek: J. Microbiol. Serol., 20, 102 (1954). 3) T. AsAI, H. IIzuKA and K. KoMAGATA, J. Gen. Appl. Microbiol.,10, 95 (1964). 4) R. H. VAUGHN, In Bergey's Manual of Determinative Bacteriology, 7th Ed., edd by R. S. BREED, E. G. D. MURRAY and N. R. SMITH, The Williams & Wilkins Co., Baltimore (1957), p ) J. DE LEY and J. FRATEUR, In Bergey's Manual of Determinative Bacteriology, 8th Ed., ed. by R. E. BUCHANAN and N. E. GIBBONS, The Williams & Wilkins Co., Baltimore (1974), p. 251, ) Y. Yamada, K. Aida and T. Uemura, Agric. Biol. Chem., 32, 786 (1968). 7) Y. YAMADA, K. AIDA and T. UEMURA, J. Gen. Appl. Microbiol., 15, 181 (1969). 8) Y. YAMADA, M. NUNODA, T. ISHIKAWA and Y. TAHARA, J. Gen. Appl. Microbiol., 27, 405 (1981). 9) M. AMEYAMA, Int. J. Syst. Bacteriol., 25, 365 (1975). 10) Y. YAMADA, T. ISHIKAWA, M. YAMASHITA, Y. TAHARA, K. YAMASATO and T. KANEKO, J. Gen. Appl. Microbiol., 27, 465 (1981). 11) V. B. D. SKERMAN, V. MCGOWAN and P. H. A. SNEATH, Int. J. Syst. Bacteriol., 30, 225 (1980). 12) Y. YAMADA, M. AKITA, T. KODA and Y. TAHARA, J. Gen. Appl. Microbiol., 29, 327 (1983). 13) V. H. CHELDELIN, Metabolic Pathways in Microorganisms, John Wiley & Sons, Inc., New York (1960). 14) T. URAKAMI and K. KOMAGATA, J. Gen. Appl. Microbiol., 27, 381 (1981). 15) J. N. BAPTIST, C. R. SHAW and M. MANDEL, J. Bacteriol., 99, 180 (1969). 16) T. AsAI, Acetic Acid Bacteria Classification and Biochemical Activities, University of Tokyo Press (1968). 17) F. GOSSELE, J. SWINGS, K. KERSTERS and J. DE LEY, Int. J. Svst. Bacteriol., 33, 65 (1983). 18) M. GILLIS and J. DE LEY, Int. J. Syst. Bacteriol., 30, 7 (1980). 19) T. ASAI, Nippon Nogeikagaku Kaishi,11, 610, 674 (1935).