X5%
F. IV. MINOR,G. A. GREATHOUSE, H. G. SHIRK,-1.AI. SCHWARTZ
tran from this same train.'^!^^ Our subsequent preparations from this strain have contained 2 distinct structural types of dextran, one of which (fraction L or L-R)apparently was identical with previous preparations and the other (fraction C or S-R)26was an entirely new type having a high content of 1,3-like links. Characterization of dextran products from 12 different colonies picked from a plated culture of B-T42 failed to reveal evidence of variation or mutation in the culture. These products had somewhat different proportions of the 2 structural types, l,(i-links from 72-T3';1, and intrinsic viscosities near 0.20. The preparation reported in Table I (heterogeneous group), which was obtained from a large-scale fermentation of the original culture, had a higher content of the anomalous fraction and, therefore, showed lower 1,B- and higher 1,8-like linkage contents and higher T1 ' SCOSity and rotation. The strains B-T42 and B-1142 (class C ) had a common origin but different subsequent histories. Our R-1142 dextran consisted almost exclusively of the anomalous fraction. -1pparently these cultures have been changing, B-1142 more than B-742. '
1.7'2j Published iniormation" was t h e basis for our identifying strain N R K L 8.7421'6 ("number 5" of H u c k e r l and "culture 4" of T a r r and Hibbertd) with t h e strain whose dextran was subjected t o methylationAnother source ( P h . D . thesis of structure analysis by Levi, el ai." I. Levi, hIcGill University, 19.12) now has disclosed t h a t this methylation ntudy wasmade on dextran from either "culture l" or "culture 2.."p Fou,ler, e l al,,b and later T. H . Evans (Ph.D. thesis, McGill University, 1941), carried o u t methylation-structure analysis on dextran from "culture 4" b u t obtained results almost identical with those obtained by Levi, ct ai., on t h e other dextrdn.I1
[CONTRIBUTIOS FROM
AND
11.HARRIS
Vol. Tli
X similar change in another strain is indicated by published data13,53 and by our results on dextran from this strain (B-1375, Table I, class C). Bacterial Classification and Dextran Type.-The fact that methylation analysis gave no evidence of branching in dextrans from 2 strains of Leuconostoc d e r n f r a n i ~ u r nhas ~ ~ ~led ~ ~to ~ the apparent espectation that all such strains would produce essentially a straight-chain type of dextran.54 Our observations indicate that although several of our strains of L . dextrnnicum produced dextrans with low percentages of non-1,ii-links and long gums (B-640, -1143 and -1146), others produced dcstrans with high percentages of non-l,(i-links ant1 short gums (B-1420, -1141 and -1373). Dextral1 B-1193 had 95% l,B-links, but short gum. Acknowledgment.-We should like to express our gratitude to the numerous individuals and organizations named in Table I who gave us cultures utilized in this investigation. IVe are deeply iiidebted to Dr. E. J . Hehre for advice and encouragement and to members of this Laboratory, as follows: Dr. K.T. XIilner for coordination of this project; C . H. vanEtten, T. .A. 1lcGuire and XIar>11-eile for the nitrogen, phosphorus arid ash analyses; and Lenora J. Rhodes and Geraldine Bryant for assisting with production of the cultures and ol the media, respectively. ( 5 3 ) S . A . Barker, E . J. Bourne, G. T. Bruce and A i . Stacey, C k e m i s t r y atid Industry, 1136 (1952). (51) S. A. Barker and E. J, Bourne, Q i k n i i . Kr7's. ( L o i i d o i i j , 7 , .ili (1953).
PEORIA, ILLISOIS
HARRIS RESEARCH LABORATORY A S D S A l . I O S A L RESEARCH COUNCII,]
Biosynthesis of C14-Specifically Labeled Cellulose by Acefobacferxylinum. 11. From ~-Mannitol-l-C'~ with and without Ethanol' BY
FRANCIS IV. I \ I I K O R , GLENNA%.
GREATHOUSE,? HAROLD(>. SITIRK,' LIILTON HARRIS
.ANTHONY
11. SCIILVARTZ
AND
RECEIVEDMAY10, 1954 Cellulose-C" was biosynthesized by .4cetobecter x y ~ i n u n zemployiiig D-tnannitol-1 as the sole labeled nutrient. Lahel distribution in the D-glucose from the bacterial cellulose showed 84-96y0 of the activity was located a t positions 1 and 6, with the residual activity being a t positions 2 t o 5 . These data indicate that some of the scission products from the original D-Mannitol became oriented in the cellulose. The labeled cellulose had lower specific radioactivity than the D-mannitol-IC14 supplied. T h e presence of ethanol in the culture media, although i t increased the yield of cellulose as well as its C" content, did not affect the distribution of the label among the carbons of the glucose making u p the cellulose.
Introduction This paper is the second in a series concerned with the abilitv of Acetobacter xvlinum to Droduce cellulose-CL4 irom various substrates, cossibly providing some information regarding the mechanisms involved in cellulose formation. Presented are results of experiments in which D-mannitolwas the sole labeled additive to a medium suitable for the organism to produce cellulosic pellicles. -1nalyses reported include the distribution of the C14-label among the various culture products as well as the spread of radioactivity i l l ( 1 , This series of papers is based on work supported b y the Atomic Energy Commission under contract AT(30-1)-9IJ with the Harrii Research 1.ahoratories , 2 ) Sdtii,nal Research CI~UIICII \!.a~l1111gtii112.i 11. C
the purified glucose from the hydrolyzed C"celldose. Experimental Culture Conditions.-The cultures were grown in tiiedia containing 0.3y0of KH2P04plus other ingredients, as shown in Table I . Each culture was inoculated from actively growing stock. Culture was incubated a t 30' and harvested 7 days following inoculation; while the others were grown a t room temperature (20 t o 2 3 " ) and harvested 14 days after inoculation. The culture vcssel, including accessorj- apparatus for collecting CO?, was the same as that previously described.3 Analyses.-The methods for purifying the cellulose and for determining the C'*-content of culture fractions were identical with those described in the preceding paper of this series.3 Except for the procedure involving lactic acid ~
~
~~
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13) 17 IV. h l i n r ) r , G . A . C,renthouw H ( > . S h i r k . .A \ I 11. I I ~ r i n s ,I'irrs J < , c x 11.. \ 7 6 . I i i i X I [ ! I 31 ,
Sr1iiznl-t~.iu
by filtration through cheesecloth arid the yellow-brown aqueous solution was heated to 80-90" for one hour to precipitate proteins. After clarification by centrifuging, the solution was evaporated under reduced pressure to 10 1. Much color and the ionic constituents were removed by passing through columns of Dowex-.50 and Duolite A3 ionexchange resins (80-100 mesh). The pale yellow effluent solution was concentrated (< 30') to a thin sirup which was mixed with two volumes of ethanol a t 70". T h e voluminous precipitate of slimy and pectic substances was filtered off and extracted twice with 1.5 1. of boiling 60% ethanol to extract a n y coprecipitated perseitol. The combined alcoholic filtrates were concentrated t o a thick sirup (ca. 400 g.) a t reduced pressure. This was taken up in 800 ml. of boiling methanol and stored a t 10" for crystallization of perseito15 which continued several days. The crude perseitol mas dissolved in a minimum volume of hot 80% methanol, decolorized with charcoal, and allowed to crystallize. The yiel: after one further crystallization was 55 g., m.p. 187-188 . Paper chromatographic examination of the mother liquor from the crude perseitol showed t h a t the solution contained mannoheptulose and copious amounts of fructose, glucosc and sucrose. T o remove these, the solution was freed of methanol by evaporation, diluted with water to 2 1. and fermented with baker's yeast for 24 hours a t 37". The yeast was filtered off and the alcohol and some water removed b y evaporation a t reduced pressure. The residue was again diluted with water and the fermentation repeated. After three such fermentations, the solution contained very little hexose. The residue was concentrated in vacuo t o a thick sirup and diluted with four volumes of hot methanol. Almost pure mannoheptulose separated on cooling and was recrystallized from 85% methanol. The yield of pure mannoheptulose was 160 g., m.p. 160-152', with a n appreciable amount of recoverable sugar remaining in the mother liquors. Identification of Mannoheptulose Phosphate in Avocado Leaves.-The concentration of sedoheptulose-7-phosphate .ll and it was suspected that in many plants is near mannoheptulose monophosphate concentration may 1)c even lower. Therefore, labeled intermediates of C"O2 reduction were prepared in the manner generally used in thii Laboratory.e After five minutes, photosynthesis in C140? was stopped suddenly by plunging the leaf into liquid nitrogen. The frozen leaf was ground and the powder dumped into boiling 80% ethanol for extraction. Two-dimensional (phenol-water, butanol-propionic acid-water) chromatograms were prepared from such extracts. Glucose-6-phosphate and sedoheptulose-7-phosphate are not generally separable in these solvents. Identification of the sugars iii this monophosphate area is accomplished by dephosphoryl( 5 ) L. hlaquenne, Cornpf. r e n d . , 107, 583 (1888); A i i n . chim. Pizrs., (1890). ( 6 ) A. A . Beoson. J . A . Rassham, Irl. Calvin. T. C . Corirlale, . ' 1 .\. Haar and \Xr. Stepka, THISJ O I . R N h T . , 72, 1710 (1950).
[e] 19, .5
