Agric. Bioi. Chem., 44 (6), 1443 - 1446, 1980 1443

Short Communication organism began to grow on o-, L­ and L- after long incubation in a Evolution of D-Arabinose, L-Xylose medium containing these as a and L-Ribose Utilization in source. In this report we describe some Mycobacterium smegmatis: experiments on the evolution of pathways for Mutants with a Novel growth on these novel pentoses. " Reductase" Growth was observed after one day of incubation when o-xylose, L-arabinose, 0­ , L-lyxose, o-, L-arabitol, xylitol, Ken IZUMORI, * Yumiko WATANABE ribitol, o-, o-, o-, L­ and Shigeru SUGIMOTO** , L-, sorbitol or mannitol Department of Food Science, Faculty of Agriculture, were used as the sole source ofcarbon. After a Kagawa University, long time lag of eight to ten days, growth of Miki-cho, Kagawa 761-07, Japan cells appeared in the medium containing 0­ Received December 20, 1979 arabinose, L-xylose, o-ribose, L-ribose or 0­ . Other tested, L-glucose, L­ Mycobacterium smegmatis was noted for its galactose, L-mannose, o-, L-fucose, dul­ ability to produce D-ribose whose citol, and , did not serve as Downloaded from https://academic.oup.com/bbb/article/44/6/1443/5970676 by guest on 29 September 2021 existence was first reported by the authors. l ) growth substrates for M. smegmatis. Also the organism seemed to be unusual The mutants that grew on o-arabinose, L­ among microorganisms in that the inducer­ xylose, L-ribose, o-ribose and o-galactose were specificity of pentose was relatively selected after long incubation and maintained broad.2) By comparing the structures of on the slants of Ogawa medium consisting of inducers of D-Iyxose isomerase, it was postu­ 1.0 g of KH2P04 , 1.0 g of sodium glutamate, lated that the f3-o-lyxose should be the inducer 200 g of chicken egg, 109 of glycerol, 0.12 g of of the enzyme.3 ) brilliant green and 110 g of water. The mutant The metabolism of various pentoses in strain selected for growth on o-arabinose (0­ Mycobacterium smegmatis seemed to proceed Ara + mutant), that was transferred to Ogawa by way of the corresponding ketopentoses. medium several times, was inoculated in the Namely, o-xylose, L-arabinose, o-lyxose and mineral salts medium2)with various sugars. As o-ribose isomerase activities were found in the shown in Fig. 1, o-Ara + mutant could grow cells grown on o-xylose, L-arabinose, o-Iyxose on o-arabinose without long time lag. Long and o-ribose, respectively.2) It seemed likely time lag also disappeared when the o-Ara + that L-Iyxose was metabolized by o-ribose mutant grew on not only o-arabinose but also isomerase which is active on and able to be L-xylose and L-ribose. However, the mutant 4 induced by L-lyxose. ) Of eight pentoses, 0- still grew on o-ribose and o-galactose after a o arbinose, L-xylose and L-ribose were not able long lag, and could not grow on other sugars to serve as carbon sources for growth. 0­ which the parent strain could not utilize as a was not found in the cells growth substrate. incubated with o-arabinose or L-fucose.2) The The same growth experiments were carried inability to utilize L-fucose by M. smegmatis out on varibus mutants: L-Xyl +, L-Rib +, 0­ implies that this organism is deficient in L­ Rib + and o-Gal+ mutants. The lengths of fucose (o-arabinose) isomerase. time lag of these mutants for growth on During studies on growth of M. smegmatis various sugars were determined and are shown on various pentoses, we discovered that the in Table I. L-Xyl+ and L-Rib+ mutants grew on o-arabinose, L-xylose and L-ribose without * To whom correspondence should be addressed. ** Present address: Taiyo Foods Co. Ltd., Tsuchida long lag, but still a long time lag was observed 136, Okayama-shi, Okayama 703, Japan. in the medium with o-ribose or o-galactose. In 1444 K. !ZUMORI, Y. WATANABE and S. SUGIMOTO

assayed by the use of o-[U-14C]-arabinose as 5 1.0 reported previously for Klebsiella aerogenes. ) The aCtivity in the cells of o-Ara+ mutant

~ E c grown on o-arabinose or various sugars was o a determined at about 41lmol per g of dry cells ~ 4 ~ 0.5 z per min and that was about five to ten times ~ "a larger than that in the parent strain grown on ~ ~ various sugars. The uptake activity was in­ hibited by the simultaneous addition of va­ :::!::'!:::::!::::::!::::5~~·~=-~~. oto .. .. -"=1'0- __---l rious non-radioactive . These TIM [, DAY S) results suggested that o-arabinose was in­ FIG. I. Growth Curves Obtained for D-Ara + M. corporated by the nonspecific permease smegma/is on Various Sugars. system. Symbols: (0), D-arabinose; (L), I.-xylose; (D), I.-ribose; o-Arabinose isomerase activity in the cells (.), D-ribose; (A), D-galactose. of o-Ara + mutant grown on o-arabinose was assayed at various pH with or without Mn2+, 2 2 the 0-Rib + and o-Gal+ mutants, a long lag Mg + or Co +. Under any reaction conditions disappeared only when the mutants grew on 0­ no activity of o-arabinose isomerase was ribose and o-galactose, respectively, and still detected in the cells. Nor was o-arabinose Downloaded from https://academic.oup.com/bbb/article/44/6/1443/5970676 by guest on 29 September 2021 needed long incubation to grow on 0­ isomerase detected in the cells incubated with arabinose, L-xylose and L-ribose. L-fucose which was a substrate and inducer of It seemed possible that in o-Ara +, L-Xyl + o-arabinose (L-fucose) isomerase in Klebsiella 6 and L- Rib + mutants a similar enzyme system aerogenes. ,7) o-Arabinose kinase and 0­ was working on the metabolic pathways of 0­ arabinose dehydrogenase activities were not arabinose, L-xylose and L-ribose. found in the cells grown on o-arabinose, We tried to find a novel metabolic pathway either. for utilization of o-arabinose in o-Ara+ The crude extract of o-Ara + mutant grown mutant. o-Arabinose uptake activity was on o-arabinose was found to contain 0-

T ABLE I. TIME LAG OF GROWTH OF MUTANT STRAINS ON VARIOUS SUGARS

Time lag of growth on various sugars" Carbon source Parent D-Ara + o-Gal+

o-Arabinose 7-8 1 1 1 8-9 7-9 I.-Arabinose I I 1 1 1 I D-Lyxose I I 1 1 1 1 I.-Lyxose I I 1 1 1 I o-Ribose 7-9 7-8 9-10 8-10 1 9-10 I.-Ribose 7-8 1 1 1 5-6 5-6 D-Xylose I I I 1 I I I.-Xylose 8-9 I 1 1 9-10 7-9 D-Arabitol 1 I 1 1 1 I I.-Arabitol 1 1 1 1 1 1 Ribitol I I I I I I Xylitol I I 1 I 1 I o-Glucose I I 1 I 1 1 D-Galactose 8-10 8-9 10-12 9-10 10-12 2 D-Mannose I I I I I I

" Oays required before any increase in growth could be measured. Evolution of D-Arabinose, L-Xylose and L-Ribose Utilization in M. smegmatis 1445 arabinose reductase activity that required

NADPH2 as a coenzyme. Specific activity was determined as 3.0 x 10-3 units per mg of protein. The optimum pH ofthe reductase was found at 6.0. Substrate specificities for various woo sugars were determined and are shown in Table II. The reductase found in the cells did

not seemed to be specific for o-arabinose, and ~ u500 4 a higher activity was found against D-lyxose o - B o and L-ribose. Almost the same amount of the 4 4 reductase was detected in the cells of o-Ara + mutant grown on other sugars. Thus the reductase seemed to be constitutively pro­ duced in o-Ara + mutant. The reaction pro­ --;~" -",-.,~115-- duct of o-arabinose reductase was determined o!:-'- -- iO DISTANCE FROf-1 ORIGiN (eM' with crude extract and o-[U-14C]-arabinose as the substrate. As shown in Fig. 2, o-arabitol FIG. 2. Radioactivity of Products from O-[U_14C]­ was detected when NADPHz was added to the Arabinose by Enzyme Reaction with Crude Pentose Reductase. reaction mixture. A little reductase activity Downloaded from https://academic.oup.com/bbb/article/44/6/1443/5970676 by guest on 29 September 2021 also was determined in the cells of the parent After enzyme reaction, the radioactive products were chromatographed in the solvent, methylethyl ketone, strain. However, the amount of the activity acetate, saturated borate solution (8: I: I), on Toyo No. was less than five percent of that of o-Ara + 50 paper. Then the paper was cut into O.S-cm strips, and mutant. the radioactivity was determined with a liquid scintil­ Studies on experimental evolution of bac­ lation spectrometer. Also shown are cold o-arabinose teria have been extensively done during recent and D-arabitoJ. S 13 A, complete reaction mixture; B, without NAOPH ; C, years - ) and reviewed by Hegeman and 2 14 15 boiled enzyme used. Rosenberg ) and WU. ) There are two dif­ ferent pathways for o-arabinose metabolism by mutants selected for growth on 0­ L-fucose isomerase acting on L-fucose and D­ 6 7 16 arabinose. In o-Ara + Klebsiella aerogenes, the arabinose. . . ) On the other hand, in D­ mutation allowing o-arabinose utilization is Ara + , D-arabinose induces associated with the constitutive synthesis of an the synthesis of three normally associated with L-fucose catabolism, L-fucose isomerase, L-fuculokinase and L--l­

TABLE II. SUBSTRATE-SPECIFlCITY OF CRUDE phosphate aldolase, and all three dual sub­ PENTOSE REDUCTASE FROM D-ARA + strate specificity.17.IS) In the parent strain and Mycobacterium smegmatis D-Ara + mutant of Mycobacterium smegmatis, L-fucose was not utilized as a growth substrate Activity Activity Pentose e~)" e~)" and no activity of L-fucose isomerase was found in the cells. In D-Ara + Klebsiella 6 o-Lyxose 100 o-Mannose 10 aerogenes PRL-R3 ) and K. aerogenes M-7,5) L-Ribose 61 o-Galactose 8 D-arabinose was incorporated with cells by L­ L-Xylose 29 D-Glucose 6 o-Xylose 27 L-Glucose 6 fucose (o-arabinose) permease which was o-Ribose 25 L-Mannose 4 specific for D-arabinose and L-fucose. In D­ o-Arabinose 14 L-Galactose I Ara + M. smegmatis, however, D-arabinose L-Arabinose 10 L-Fucose 4 seemed to be incorporated by non-specific L-Lyxose 9 L-Rhamnose 4 permease. o o-Lyxose, 100%. With the demonstration of constitutive 1446 K. IZUMORl, Y. WATANABE and S. SUGIMOTO production of D-arabinose reductase in the So we are now in the process of purifying the cells of D-Ara + mutant, the major growth reductase to determine the properties of the characteristics on o-arabinose become in­ enzyme and confirming the origin of the novel terpretable. In M. smegmatis, four pentitols enzyme. 2 were good substrates for growth. ) Ifthe novel Acknawledgment. This work was supported in part pentoses were reduced to certain pentitols by by a grant from the Ministry of Education of Japan. the reductase produced constitutively, the strain should be able to utilize the novel REFERENCES pentoses as growth substrates. As shown in the growth experiments, it was I) K. Izumori, A. W. Rees and A. D. Elbein, J. Bial. Chern., 250, 8085 (1975). assumed that D-arabinose, L-xylose and L­ 2) K. Izumori, K. Yamanaka and A. D. Elbein, J. ribose should be metabolized by a similar Bacterial., 128, 587 (1976). metabolic system in D-Ara +, L-Xyl + and L­ 3) K. Izumori and K. Yamanaka, FEBS Letters, 77,133 Rib + mutants. From the fact that the non­ (1977). specific pentose reductase was found in not 4) K. Izumori, M. Mitchell and A. D. Elbein, J. Bacterial., 126, 553 (1976). only o-Ara+ mutant but also L-Xyl + and L­ 5) K. Izumori and K. Yamanaka, J. Bacterial., 134, 713 Rib +. mutants, constitutively, the metabolic (1978). pathway of these novel pentoses should be 6) E. J. Oliver and R. P. Mortlock, J. Bacterial., 108, postulated as follows. o-Arabinose, L-xylose 287 (1971). Downloaded from https://academic.oup.com/bbb/article/44/6/1443/5970676 by guest on 29 September 2021 and L-ribose were incorporated by the non­ 7) E. J. Oliver and R. P. Mortlock, J. Bacterial., 108, specific aldose permease and reduced by the 293 (1971). 8) G. T. Cockes, J. Aguilar and E. C. C. Lin, J. reductase to o-arabitol, xylitol and ribitol, Bacterial., 118, 83 (1974). respectively. 9) A. J. Hacking, J. Aguilar and E. C. C. Lin, J. L-Ribose was utilized by o-Ara +, L-Xyl + Bacterial., 136, 522 (1978). and L-Rib + mutants as a growth substrate. 10) C. B. Inderlied and R. P. Mortlock, J. Mol. Evol., 10, These strains were the first organisms that III (1977). 11) T. T. Wu, Biochirn. Biophys. Acta, 428, 656 (1976"). were found capable of growth on the un­ 12) T. T. Wu, J. Gen. Micrabial., 94, 246 (1976). natural pentose, L-ribose, as a sole source of 13) T. T. Wu, E. C. C. Lin and S. Tanaka, J. Bacterial., carbon. 96,447 (1968). Our present study demonstrates another 14) G. D. Hegeman and S. 1. Rosenberg, Ann. Rev. strategy how a new catabolic pathway ofnove! Micrabial., 24, 429 (1970). 15) T. T. Wu, CRC Critical Reviews in Microbial., pentoses can be constructed by a derepressing September, 33 (1978). enzyme which may fortuitously act on novel 16) K. P. Camyre and R. P. Mortlock, J. Bacterial., 90, pentoses. However, the origin ofthe derepress­ 1157 (1965). ing enzyme, non-specific pentose reductase 17) E. J. LeBlanc and R. P. Mortlock, J. Bacterial., 106, that has a high activity against unnatural 82 (1971). pentoses, o-lyxose and L-ribose, is not clear. 18) E. J. LeBlanc and R. P. Mortlock, J. Bacterial., 106, 90 (1971).