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Glucan biosynthesis in Bacillus stearothermophilus
ARCHIVES
OF
RIOCHEMISTRY
Glucan
hND
BIOPHYSICS
Biosynthesis I. Properties
149,
252-258
in Bacillus
(1972)
stearothermophilos
of the Polysaccharide’
SARA H. GOLDERIBERCP Institulo
de Investiqaciones
Bioquimicas Received
“Pundacidn Campomar” Argentina
Judy 8, 1971; accepted
November
Obligado
2490, Buenos
Aires
(28),
18, 1971
A glucan which contains w-l,4 and presumably a-l,6 bonds has been isolated from stearothernrophilus 1503-4R by trichloroacetic acid a spontaneous variant of Bacillus extraction and precipitation with et,hanol. It resembles glycogen in solubility, characteristics of the iodine complex and internal chain length. It has longer average and external chains and also different electrophoretic mobility on glass fiber paper. The product synthesized in &,-o in the presence of accept~or and ADP-glucose, by cellfree extracts containing glucan synthetase, has elongated external chains. The newly added glrlcosyl residues are a-l,-1 linked. The glucan acc~lm~dates in stationary phase cells. No polysaccharidr nor synt hctic activity were detected in wild-i ype cells. st,ationary phase, harvested and disntpt,ed as already described (1). The 20008 slrpernat,ant was precipitated wit,h one volrune of 107; t,richloroacet,ic acid, and the pellet, was washed twice with 57; irichloroacetic acid. The combined fIllids were precipitated with one volllmc of ethanol, and t,he resldting sllspension was left, overnight at O-4”. After centrifllgat,ion, the precipitatr was taken up in :I small vo111n~c of water, treated with one volume of 2 s KOIf and left, at 37” overnight (a). After low speed c,clll,rifugatioll to discard any insoluble mat.crinl. the polysaccharide was pllritied by several rcprrcipitations wit,h 1)~ vo111mc~ of et,hanol. III most cases, the sollltion in water of the first rthauolic precipitate obl,ained after alkalinr: treal,ment was centrifllgrd at. 3000!/, ihc sediment resllspended in water and again ccntrifllgcd at I he S:IIIIP speed. The procedllre was rc~pcal.rd OIIW more and t,hc final sediment, was discarded CC). These combined sllpernatant s were prrcipit,:rt~cvl with one vollune of c~t,hanol (with the addit ion cjf ace~:rtc or K(:li, a drop of sat~~rntrd ammonilun the precipitat
CLUCAN
FROM
grown to the stationary phase in a culture medium containing [3H]-glucose. The product was isolated as described above, using unlabeled glucan as carrier. Glucan synthet.ase ‘Vethods ant/ chemiculs. (ADP-glucose: 0l-1,4-glucan n-4.glucosyltransferase, .1SC 2.4.1.a) was prepared and tested as alread)- described (I), or obiained from B. stearofhwnophilus spheroplasts (7, 8). The hydrolytic activity of the extracts was determined by measuring the disappearance of polysaccharide in a mixture containing ‘25 rg glycogen or glllcan, 0.1 *t glycylglycine buffer, pII 8, and ~nxyme, in a final volume of 50 ~1. Aft,er incttbating 15 min at 65”, 50 ~1 water and 50 ~1 1O70 trichloroacet,ic acid were added, t,he mixtllre was centrifllged and the remaining glyeogen or glucan was estimated in an aliquot, of the supernatant, with the iodine method (14). Control experiments withollt incltbation were also performed. A preparation of rat mrlscle glycogen synthetase (UDP-glucose: glycogen a-4.glZhlcosyltr:msferase, RC 2.4.1.11) free of endogenous glycogen was a kind gift of Dr. R. Staneloni. The enzyme activity was measured with UDP-[14C]glucose as indicated (a-1,4by Piras et ab. (9). Amyloglucosidase glucan glucohydrolasc, EC 3.2.1.3) from Rhitopus (obtained from Sigma) was used according to Lee and Whelan (lo), at 55”. Polysaccharide chain length was estimated
B.
253
sfearothermophilus
Wavelength
[run)
FIG. 2. Absorpt’ion spectra of t,he iodine complex of 0.017~ solutions of B. sleurothermophilus liver glycogen and potato amyglucan, rabbit lopectin. Itsing t,he periodate met’hod of Fales (11). pAmylolysis was measured according to Kjelberg and Manners (12) and the maltose liberated was determined with Park and Johnson’s method (4) as modified by Sigal et al. (3). Total carbohydrate was measured either with phenol-sulfuric acid, or the anthrone method (13). Glucose was used as standard. Purified glucan was e&mated with phenolsulfuric acid, or using the iodine method of Krisman (14), with a standard solution of glncan previously determined with phenol-sulfuric acid. Protein was assayed according to Lowry ct al. (15).
1. Carbohydrate content during growth of I?. slearothermophilus. B. stcarothcrmophilus cells were grown as described (1) and at the points indicated samples were withdrawn for absorbance reading at 700 nm (01, tot.al carbohydrate estimat,ion (13) per milligram of dry weight (O), and isolation and determination of glucan (as described in “Methods”) per milligram of dry weight (A). FIG.
cycle
The iodine-polysaccharide complex was prepared as indicated by Krisman and the spect.ra recorded with a Unicam SP 800 spectrophotometer. Electrophoresis on glass fiber paper was performed according to Fuller alld Northcote (Ifi), at 9-12 V/cm depending on the strip width (to avoid excessive heat), for 2.5 hr. The strips were scanned in a Packard radiochromatogram scanner, model 385. Glycogen, glncan and amylopectin were det,ected by spraying wit,h t.he iodirteCaC12 reagent (14). A brown color was obtained wit,h rabbit liver glycogen, B. sfearolhermophilus glucan gave a more reddish shade and potato amylopectin a violet color. The colors began to fade after a few minutes. Rabbit liver glycogen and ADP-ghlcose were obtained from Sigma, potato amylopectin was prepared and kindiy donated by Dr. C. 15. Cardini and ADI’-[‘4C]-glucose was purchased from Sew England Nllrlear Corp. L
DISCUSSION
Accumulation of qlucan in cultures. Glucarr is vxtrnctcd from cells in the st:itionarJ
254
GOLDEMBERG
in logarithmic phase extracts, as already reported (1). This is also demonstrated in Fig. 1, which shows total carbohydrate and glucan content per milligram of dry weight of cells throughout the growth cycle. It can be seen that the polysaccharide begins to accumulate at the end of the exponential phase. It represents approximately 5 % of the dry weight in the stationary phase. No polysaccharide was detected in wildtype cells, even with different culture media or lowering the growth temperature to 55”. It has been reported that certain microorganisms accumulate greater quantities of polysaccharides at lower temperatures (17). Solubility of the product. The material is soluble in water, but after freezing and t’hawing a sediment, forms which redissolves by n-arming. The polysaccharide gives an opalescent solution, with a higher turbidity than a glycogen solution of the same concentration. The glucan is also soluble in strong alkali or acid, and precipitat’es with ethanol, similarly to glycogen. Iodine spectrum. Figure 2 sho\vs the absorption spectra of the iodine-polysaccharide complexes obtained with the B. glucan, rabbit liver glysfearothermophilus cogen and potato amylopectin. It can be sc’en t,hat, the glucan absorption spectrum is vrry similar to that of the rabbit liver glycogen, with the maximum somewhat displaced (475 nm) and a little higher. Chain length parameters. Table I shoTITs the chain length determinat)ions and percent conversion to maltose by @-amylase (w TABLE: ~~.
__~
B. stearolhermophilus
Rabbit liver B. wAegaterium E. coli A. aerogenes Ncisseria perfiava
Arthrobacter Agrobacterium
Potato
a. 21
16 9-11 10-13 12, 13 11, 12 7-9
lunzejaciens
amylopectin
I
PROPERTIES OF GLYCOGENS”
~-
Sourceof polysaccharide
1,4-glucan maltohydrolase, EC 3.2.1.2). A comparison with glycogens from ot,her sources is included, as well as the x max (wavelength of peak absorption) of the iodine complex. The values obtained for the B. stearothermophilus glucan average chain length, external chain length and percentage pamylolysis resemble those of amylopectin, while internal chain length and Xmax of the iodine complex, as ~11 as the solubility properties, are more akin to those of glycogen. There are no sharp differences in nature between glycogen and amylopcctin, c.g., there are reports of samples of rabbit liver glycogen with avrragc chain length of 18 (20, 22, 23) and a fraction of the pol.vsaccharide from the Golden Bantam varietv of sweet corn has an avc’rage chain length glucan of 11 (24). R. stearothermoph.ilus seems to belong to the glycogen family of polysaccharides. The other bacterial glycogens described in the litcrat’urc are in general rather compact structures, with very short internal chain lengths. The glucan described in this paper appears to bc a much looser and flexible molecule according to the detwmined chain lrngth parameters. Priming ability. The polysaccharide isolated from the thcrmonhilic bacterium serves as acceptor for B. stearothermophilus glucan synthctase (1) ; some preparations of the glucan give slightly higher incorporations than rabbit liver glycogen of the same conThe bacterial polysaccharidc centration. serves also as sccrpt’or for animal glycogen
13 23
~-AlTlyhSe limit (%I
ECL
59
15 10 6-8
49 43-46 47-56 46-64 55-59 23-37 52 53
819 8-10 8-10 4-6 9 15
ICL 5 5 1, 2 l-3 1-3 1, 2 2, 3 3 7
Iodin;;;&x 475 460 520 420-455 470485 red brown 420440 470 520
Ref. ___-~--
18 3 19,3 20 2, 21 21
a Abbreviations used are as follows: ?% = average chain length; ECL = external chain length; ICI, = internal chain length; Xmax = wavelength of peak absorption of iodine-polysaccharide complex.
GLUCAK
FROM B. stearothermophilus
synthetase, being 77 % as efficient as rabbit liver glycogen when tested at equimolar concentrations (expressed as glucosyl residues). Electrophoretic mobility. Some diffrrences of the B. stearothermophilus glucan with rabbit liver glycogen and potato amyloprctin can be detected by elect’rophoretic mobility in borate buffer using glass fiber paper. Figure 3A shows that thr B. stearothermoph.ilus polysaccharide has an Mglycogenof about 0.80, while amylopectin gives a spot at the origin and a second diffuse one in the glycogen-glucan area. In vitro synthesized product. It has been previously shown that the product formed by R. stearothermophilus glucan synthetase liberates maltose with /3-amylase, indicating the presence of a-l,4 bonds (1). The existence of a-1 ,6 linkages in the purified glucan is suggested by t,hr attack with amyloglucosidase and t,hn characteristics of the absorption spectrum of the complex formed
255
with iodine. The structure of the product synthesized in vitro was analyzed in an experiment of double labeling. For that purpose, rH]glucan was obtained as described under “Methods.” It was used as acceptor for a preparation of glucan synthetase containing no endogenous primer, with ADP-[Wlglucose as substrate. The product was isolated and submitted to /3amylolysis; t#he dist’ribubion of thcl label between the p-limit dextrin and the maltose liberated was studied. The results are shown in Table II. It can be seen that 100 % of the [14C]glucosyl residues added to the acceptor by the glucan synthetase are liberat’ed into the supernatant by p-amylase. On the other hand, the latter enzyme att,acks only 72 % of the [3H]glucose from the acceptor. This is as expected in a polysaccharide of the glycogenamylopectin type, which contains a-l,4 and a-l,6 bonds. In this experiment the glucan synthetase preparation only lengthened branches of the acceptor. Branching enzyme
- +
FIG. 3. Electrophoretic mobility of the product formed in vitro. The product formed incubating several enzyme preparations under the conditions already described (1) was submitted to electrophoresis on Whatman glass fiber paper as indicated in “Methods.” The dried strips were scanned and then sprayed as described in “Methods.” A) Standards. a: B. stearothermophilw glucan, b: rabbit liver glycogen, c: potato amy1opectin.B) Stationary phase extract, 40 pg protein. C) Logarithmic phase extract, 25 fig protein. D) Same as C, with 150 ag protein. E) Same as C, with rabbit liver glycogen (3 pmoles glucosyl residues). F) Same as C, with B. stearothermophilus glucan (0.43 rmole glucosyl residues). G) Same as C, with potato amylopectin (0.3 Imole glucosyl residues). H) Same as C, adding the supernatant of boiled B enzyme extract.
25G
GOLDEMBERG
might br present in the c>xtracts but perhaps it, xw not actiw bccausr t,hc c>xtcrnal chains rlf thcx glucnn had not, been lcngthencd c~nough under thcb conditions used. The in w’fw product, is a gIuc:~n with longrr ext,ernsl chnins.3 Iiigurc 3 she\\-s the> clectrophorctic mobilit,y of the product synthesized in vitro in the abscncc (B, C and D) or prescncc (E, I’, G and H) of added acceptors. Figure 3B rcprwbnts the pattern obtairwd \\-ith ~1 stxtionar\- phaw extract, C and D with t,wo different concentrations of a logarithmic phase pwparat,ion. Wlm~ sufficicnt~ amounts of cindogcnous primer arc present (B and D), radioactivity appears in the product,, which rcmnins at, the origin. Wit11 wry small amounts of t~ndogc~nousprimer (extract used in C) thcrc is no incorporation. The addition of diffwrnt~ accrptors to tllis extract, kads to the formation of products whicll migrake diffrwntly. Rabbit liwr glycogc>n (E) givrs :I radioactivr product, lo&cad in tlw glycogcw :lrw, purificad gluc:m (1;) givw :L spot1in tjllc> n small one glnc;~Il ZOIl( (:lralYeuncn 03) and at the origin, and \vith :~m,vlopwtin (G) the r:ldioactivit,\, appwrs at, thr origin. Thr :lddit’ion to prcynration C of a11 Aiquot of th(b rxtrwt uwl in B, prwiously innctivatrd by brat and wntrifugrd (H), giws thr same pattern :IS B or I). Th(l product obtaiwd lvith rabbit, livw glycogc>n (I?) can bt> diffwcwtk~tc~d from thcl onr formed in the: prcwnw of glucan, eithclr nat,ive (B, D, H) or purifkd (1:). Tt is wid& that, tllr isolation and purification mc%hod uwl has altewd it’ somon-ll:kt,. Thr nntiw product might’ br mow am~loJ”,ctin-lil~(~. ( Ttilimtion 0s the &can. Many bacteria :w l;no\~~nto accun-&tc~ glycogcn as endog(~110118 sow-cc of cwrgy for ccl1 mnintrnancr ~rocc~ssc~s (L&i, z(j). It is gcmrrally :wYpted th:lt c~~l1ul:tr wwrvrs :LW synthrsizcd when growth is limited, for instanw as bacteria cwt,w- tllc, stationary phaw. This limit,ation rnav b(: dw to nitrogcln scarcity, low pH, sulfur or phosphorus dcficic~ncy, in tlic of rxcrss carbon sourw (‘27). prcwriw :: ?;oic :rtidccl in proof : Preliminary resells obtained in o\tr laboratory indicate the cxistencc of branching activity in t.he bactcrinl extracts, when different conditions were llscd.
Omissions from complete system“
Radioactivity incorporated :‘I1 (cpm)
&Amylolysis of the product ~~ ~___ W (cpm) 3II (yo) “C (“0)
None ii80 3530 73 100 Qlucan 350 .~A1~P-glllcosc 6150 ~~.. 72 lkzyme 7500 io 71 _____ __n The complele system contains, in a final volume of 50 ~1, 0.1 3Cglycylglycinc buffer, pll S; 50 mx rnercaptoetllallol; enzyme from logarithmic phase, prepared from spheroplastjs and fract.ionated with ammonilml slrlfate, 49 pg protein; 0.46 pmole (g111cosyl residlles) [“ll]-glucan (7800 cpm), and 1 mar ADP-[‘4(:]-glr~cc~se (15,000 ~1x11). Incubations were carried OII~ :L( 05” for 15 ruin, Ihe system was complct ed where necessary, and I he product was isolated as descrihcd (1). An aliqllot was submitted to crystalline sweet po~,nlo Bamylsse action (12) and after overnight inclbtion at 37” lmder tol11c11c V:LI)OI’S, the &dest,rin was precipitated with 3 vol of met~hanol and w:~slrcvl. J:adioact,ivity was dckrmined in the prccipit atcd p-limit destrin and the comhillrtl sllprrnat,:mis hy means of :t liqliid scini ill:il,ic,n sl~ertromckr.
I?igurcl 1 shops that in II. .sfea~othel~,nophilus thcrcl is :m :~ccumulntion of giuca.11 in t,lir stationary- pli;w 2nd no polys:~ccharid~: in t,hcl logarit,limic ph:w, \vliw th: cnrrgrt~ic nods ar(l maximal. It may,- be assumcbdt’h:kt some mwhanism for its utilization exists in thcl wll. This possibility wits t&cd in a11ia vice caxpwimcwt shown in Fig. 4. 011s \v(w grO\\-II
t(J
tllc‘
st&tk~rl:l~~*
I)li:kx’,
:ultl
tlr!,
weight, gluc:m :md protck content wrc drt,crminrd in :L s:Lmplr (-1). I+sh prewnrmcd mcldium WM t8hw :Ldded in a sixfold CXCPSSand thcl incubnbion W;IS cont~inuc~d. Wlica growth \v:w rwumcd :Iftc>r :L short timr, two furthc~r snmplw wrc taken (13 and C) to assay the same p:wnwtws. It’ c:m b(l sc(xr1 that, whc~ri crlls wrr actiwly gro~virlg again (C), a largc~ fall in gluci~n corit8cxnt,was obscirvrd. This is in accord with t,hcxrwults obtuiwd in Icig. 1, in wllich thaw: is 110 glucan in thrb logarit~hmic phwr. Ho\\-cwr, iI Spite (Jf th’ hr#’ (liSapJ~(‘:u:lnc(: of &cm obsrrwd bctwwn B and C in Icig. 4, the in f&o hydrolytic activity from oxtrilcts
1.0
2.5
1
c!8-
20
Q6-
15
04-
l.0.
0.4 -
0.5-
-
FIG. 4. Utilization of B. stearofhelmophil?s glucan. Conditions as for Fig. 1. At point A, a sixfold excess of fresh medium prewarmed to G5” was added and incubation immediately resumed. (0) log absorbance at, 700 nm (the origin of the upper scale on the ordinate at the left was displaced to get a bet,ter continuity of the growth curve); (III) Nmoles of glucan per milligram dry weight; (WI) prnoles of glucan per milligram of protein.
obtaiwd at thcw points \V:LS very small wlm~ twtcxd \vith rabbit livtr glycogcw or purified glucan. Wild-type logarithmic phase extracts, on the contrary, show-cd a wry high hydrolytic activity (JII both polysncchnridrs (8). ~Iorcovw, n-hen :L prtyar:~tion of thcl wild-typo strain was add(bd to :m ext’ract’ of th(> wriant, the> radioactivity incorporutcd into thcl product II-W much loo er. The, possibility that thcl hydrolytic clnzymo(s) from that variant wlls is more unst.abl(~ than thnt of thcl \\ild-type bactwia or has diffcrcnt rquiwmc~nts, cann(ot be dkwrdcd. Ko phosphorolytic activit,y could be d(kccttld in any of thr strains. Thcl propwt’y of thwmophilic bactc~rin of
rcyroducing optimally at, t8wnperaturcs at which mesophiles :ire killrd, has not yet been fully clarific~d. T\vo general thcorics have been put for\\ard. Orw attributes it to an incrcawd thwmostability of the proteins (2s); thcl second suggc>sts a rapid rqvntlrwis of thcl lrclat,-damaged wllular components (29). Thcl glucan stored by R. slear,othe~nlophiIus in t,hcl stationary phase: might function as :L sourw of carbon subst!r:tt,rs for rcsynthwis of d(sgradcd wllular componwts, brkg used at :I wry fast, r:k in the c:spo~wntial ph:w as slio~\-n in Yig. 4. Whc~ gro\vt’h is limited, the ncwssity for a rapid supply of cnwgy diminishes and tllc glucan begins to accumul:Ltc~. This I)ol?-s:Lccli:lritl(: could Ii:~w
20‘8
GOLDEMBERG
a pllysiological rok similar t,o glycogen in mesophilic bactjcria, tha looser structural being perhaps an addit,ional advantagr for vcyv rapid utilization during active growth. Ko polysaccharidt~ nor synthetic activit#J have bwn dctwtc>d in wild-type cells. This failurc~ might bc dw t’o thcl very high hydrolytic activity obwrwd in these bacteria, and/or different requirements for synthct,ic activity. Thrsc~ possibilities arc under stud! in our laboratory. ACKNOWLEDGMENTS The author wishes to thank Dr. Luis F. Leloir, Dr. I. I>. .4lgranat,i and the other members of the Instituto de Investigaciones Bioquimicas for helpful sllggestions and criticisms.
1. GOLDEMHERG, S. H., .ZND ALGRliNBTI, 1. D., Biochim. Biophys. Acta 177, 166 (1969). 2. GHOSH, II. P., AND PREISS, J., Biochim. Biophys. Ada 104, 274 (1965). 3. SIGIL, N., ~?:\TTANISO, J., .\ND %:GEL, I. FI., Arch. Bioch~em. Biophys. 108, 440 (1964). 4. PAI+ J. T., AND JOHNSON, M. J.. .J. Biol. Chem. 181, 149 (1949). 5. HUGGISTT, 9.8~.C:., .IND NIXON, D. A., Biothem. J. 66, 12P (1957). 6. Duno~s, M., GILLITS, K. A., HAMILTON, J. K., RAPIERS, P. A.. .\ND SMITH, F., Anal. Chem. 28, 350 (1956). 7. BODM.\N, II., AND WELKER, N. E., J. Bacleriol. 97, 9‘24 (1969). 8. GOLDE~IBERG, S. H., to be published. 9. PIRAS, R., ROTHMAN, I,. B., AND Cxm, E., Biochemistry 7, 56 (1968).
10. LSIG, 14:. Y. c., .1ND bvHEL.IS-, w. J., Arch. Bioch,em. Biophys. 116, 162 (1966). 11. F.~LI!x, F. N., Anal. Chem. 31, 1898 (1959). 12. KJ~LI~BRG, O., .\ND M.\vNI,:Rs, D. J., J. Chem. S’oc. 4596 (1962). 13. SPIRO, I<. (:., in “Met,hods in Enzymology” (I<. F. Neufeld and V. Ginsburg, eds.), Vol. 8, p. 4. Academic Press, New York (1966). 14. KILISW.IN, C. It., Anal. fliochem. 4, 17 (1962). 1.5. LOWRY, 0. H., I~~S~HR~VGH, N. J., F:\RK, A. L., .\ND I:.~ND.ILL, 11. .J., J. Biol. Chcm. 193, 265 (1951). 16. FULLILR, K. W.,, .u-D NOI~THCOTI’:, 1~. H., Biochem. J. 64, 657 (1956). 17. F.\KRWLL, J., .IND Itos~, A., iinnu. fZer. Microbial. 21, 101 (1967). 18. B.\RRY, C., (:.IVARD, I:., RIILHAUD, G., AND AUB&RT, J. P., Ann. Inst. Pusleur 84, 605 (1953). 19. KINDT, T. J., .\ND CONHAD, H. F;., Biochemistry 6, 3718 (1967). 20. BARKW, S. A., BOURNE, I<. J., AND STACW, M.. J. Chem. Sot. 2884 (1950). 21. ZEVI~NHUIZEN, L. P. T. hf., Mededelingen Landbouwhogeschool, Wageningen, 10-66, 39, (1966). 22. H.\WORTH, W. N., HIRST, If. L., ~NI) ISHERWOOD, F. A., J. Chem. Sot. 577 (1937). 23. H.~Ls~LL, T. (+., HIRST. R. L., AND JONIOS, J. K. N., J. Chem. Sot. 1399 (1947). 24. D~oN~H, W., Al~~ WHISTLEK, U.. L., J. Biol. Chem. 181, 889 (1949). 25. CHEN, G. S., AND SEGE:L, I. II., Arch. Biochem. Biophys. 127, 164 (1968). 26. D.awtss, E. A., AND Rrr~uo~s, D. W., Annu. Rev. Microbial. 16, 241 (1962). 27. EIDICLS, I,., ‘INI) Pnixss, J., Arch. Biochem. Biophys. 140, 75 (1970). 28. KOFBLI.:R, H., Racferiol. Rev. 21, 227 (1957). 29. ALLEN, M. TS., Backrio!. Rev. 17, 125 (1953).
OF
RIOCHEMISTRY
Glucan
hND
BIOPHYSICS
Biosynthesis I. Properties
149,
252-258
in Bacillus
(1972)
stearothermophilos
of the Polysaccharide’
SARA H. GOLDERIBERCP Institulo
de Investiqaciones
Bioquimicas Received
“Pundacidn Campomar” Argentina
Judy 8, 1971; accepted
November
Obligado
2490, Buenos
Aires
(28),
18, 1971
A glucan which contains w-l,4 and presumably a-l,6 bonds has been isolated from stearothernrophilus 1503-4R by trichloroacetic acid a spontaneous variant of Bacillus extraction and precipitation with et,hanol. It resembles glycogen in solubility, characteristics of the iodine complex and internal chain length. It has longer average and external chains and also different electrophoretic mobility on glass fiber paper. The product synthesized in &,-o in the presence of accept~or and ADP-glucose, by cellfree extracts containing glucan synthetase, has elongated external chains. The newly added glrlcosyl residues are a-l,-1 linked. The glucan acc~lm~dates in stationary phase cells. No polysaccharidr nor synt hctic activity were detected in wild-i ype cells. st,ationary phase, harvested and disntpt,ed as already described (1). The 20008 slrpernat,ant was precipitated wit,h one volrune of 107; t,richloroacet,ic acid, and the pellet, was washed twice with 57; irichloroacetic acid. The combined fIllids were precipitated with one volllmc of ethanol, and t,he resldting sllspension was left, overnight at O-4”. After centrifllgat,ion, the precipitatr was taken up in :I small vo111n~c of water, treated with one volume of 2 s KOIf and left, at 37” overnight (a). After low speed c,clll,rifugatioll to discard any insoluble mat.crinl. the polysaccharide was pllritied by several rcprrcipitations wit,h 1)~ vo111mc~ of et,hanol. III most cases, the sollltion in water of the first rthauolic precipitate obl,ained after alkalinr: treal,ment was centrifllgrd at. 3000!/, ihc sediment resllspended in water and again ccntrifllgcd at I he S:IIIIP speed. The procedllre was rc~pcal.rd OIIW more and t,hc final sediment, was discarded CC). These combined sllpernatant s were prrcipit,:rt~cvl with one vollune of c~t,hanol (with the addit ion cjf ace~:rtc or K(:li, a drop of sat~~rntrd ammonilun the precipitat
CLUCAN
FROM
grown to the stationary phase in a culture medium containing [3H]-glucose. The product was isolated as described above, using unlabeled glucan as carrier. Glucan synthet.ase ‘Vethods ant/ chemiculs. (ADP-glucose: 0l-1,4-glucan n-4.glucosyltransferase, .1SC 2.4.1.a) was prepared and tested as alread)- described (I), or obiained from B. stearofhwnophilus spheroplasts (7, 8). The hydrolytic activity of the extracts was determined by measuring the disappearance of polysaccharide in a mixture containing ‘25 rg glycogen or glllcan, 0.1 *t glycylglycine buffer, pII 8, and ~nxyme, in a final volume of 50 ~1. Aft,er incttbating 15 min at 65”, 50 ~1 water and 50 ~1 1O70 trichloroacet,ic acid were added, t,he mixtllre was centrifllged and the remaining glyeogen or glucan was estimated in an aliquot, of the supernatant, with the iodine method (14). Control experiments withollt incltbation were also performed. A preparation of rat mrlscle glycogen synthetase (UDP-glucose: glycogen a-4.glZhlcosyltr:msferase, RC 2.4.1.11) free of endogenous glycogen was a kind gift of Dr. R. Staneloni. The enzyme activity was measured with UDP-[14C]glucose as indicated (a-1,4by Piras et ab. (9). Amyloglucosidase glucan glucohydrolasc, EC 3.2.1.3) from Rhitopus (obtained from Sigma) was used according to Lee and Whelan (lo), at 55”. Polysaccharide chain length was estimated
B.
253
sfearothermophilus
Wavelength
[run)
FIG. 2. Absorpt’ion spectra of t,he iodine complex of 0.017~ solutions of B. sleurothermophilus liver glycogen and potato amyglucan, rabbit lopectin. Itsing t,he periodate met’hod of Fales (11). pAmylolysis was measured according to Kjelberg and Manners (12) and the maltose liberated was determined with Park and Johnson’s method (4) as modified by Sigal et al. (3). Total carbohydrate was measured either with phenol-sulfuric acid, or the anthrone method (13). Glucose was used as standard. Purified glucan was e&mated with phenolsulfuric acid, or using the iodine method of Krisman (14), with a standard solution of glncan previously determined with phenol-sulfuric acid. Protein was assayed according to Lowry ct al. (15).
1. Carbohydrate content during growth of I?. slearothermophilus. B. stcarothcrmophilus cells were grown as described (1) and at the points indicated samples were withdrawn for absorbance reading at 700 nm (01, tot.al carbohydrate estimat,ion (13) per milligram of dry weight (O), and isolation and determination of glucan (as described in “Methods”) per milligram of dry weight (A). FIG.
cycle
The iodine-polysaccharide complex was prepared as indicated by Krisman and the spect.ra recorded with a Unicam SP 800 spectrophotometer. Electrophoresis on glass fiber paper was performed according to Fuller alld Northcote (Ifi), at 9-12 V/cm depending on the strip width (to avoid excessive heat), for 2.5 hr. The strips were scanned in a Packard radiochromatogram scanner, model 385. Glycogen, glncan and amylopectin were det,ected by spraying wit,h t.he iodirteCaC12 reagent (14). A brown color was obtained wit,h rabbit liver glycogen, B. sfearolhermophilus glucan gave a more reddish shade and potato amylopectin a violet color. The colors began to fade after a few minutes. Rabbit liver glycogen and ADP-ghlcose were obtained from Sigma, potato amylopectin was prepared and kindiy donated by Dr. C. 15. Cardini and ADI’-[‘4C]-glucose was purchased from Sew England Nllrlear Corp. L
DISCUSSION
Accumulation of qlucan in cultures. Glucarr is vxtrnctcd from cells in the st:itionarJ
254
GOLDEMBERG
in logarithmic phase extracts, as already reported (1). This is also demonstrated in Fig. 1, which shows total carbohydrate and glucan content per milligram of dry weight of cells throughout the growth cycle. It can be seen that the polysaccharide begins to accumulate at the end of the exponential phase. It represents approximately 5 % of the dry weight in the stationary phase. No polysaccharide was detected in wildtype cells, even with different culture media or lowering the growth temperature to 55”. It has been reported that certain microorganisms accumulate greater quantities of polysaccharides at lower temperatures (17). Solubility of the product. The material is soluble in water, but after freezing and t’hawing a sediment, forms which redissolves by n-arming. The polysaccharide gives an opalescent solution, with a higher turbidity than a glycogen solution of the same concentration. The glucan is also soluble in strong alkali or acid, and precipitat’es with ethanol, similarly to glycogen. Iodine spectrum. Figure 2 sho\vs the absorption spectra of the iodine-polysaccharide complexes obtained with the B. glucan, rabbit liver glysfearothermophilus cogen and potato amylopectin. It can be sc’en t,hat, the glucan absorption spectrum is vrry similar to that of the rabbit liver glycogen, with the maximum somewhat displaced (475 nm) and a little higher. Chain length parameters. Table I shoTITs the chain length determinat)ions and percent conversion to maltose by @-amylase (w TABLE: ~~.
__~
B. stearolhermophilus
Rabbit liver B. wAegaterium E. coli A. aerogenes Ncisseria perfiava
Arthrobacter Agrobacterium
Potato
a. 21
16 9-11 10-13 12, 13 11, 12 7-9
lunzejaciens
amylopectin
I
PROPERTIES OF GLYCOGENS”
~-
Sourceof polysaccharide
1,4-glucan maltohydrolase, EC 3.2.1.2). A comparison with glycogens from ot,her sources is included, as well as the x max (wavelength of peak absorption) of the iodine complex. The values obtained for the B. stearothermophilus glucan average chain length, external chain length and percentage pamylolysis resemble those of amylopectin, while internal chain length and Xmax of the iodine complex, as ~11 as the solubility properties, are more akin to those of glycogen. There are no sharp differences in nature between glycogen and amylopcctin, c.g., there are reports of samples of rabbit liver glycogen with avrragc chain length of 18 (20, 22, 23) and a fraction of the pol.vsaccharide from the Golden Bantam varietv of sweet corn has an avc’rage chain length glucan of 11 (24). R. stearothermoph.ilus seems to belong to the glycogen family of polysaccharides. The other bacterial glycogens described in the litcrat’urc are in general rather compact structures, with very short internal chain lengths. The glucan described in this paper appears to bc a much looser and flexible molecule according to the detwmined chain lrngth parameters. Priming ability. The polysaccharide isolated from the thcrmonhilic bacterium serves as acceptor for B. stearothermophilus glucan synthctase (1) ; some preparations of the glucan give slightly higher incorporations than rabbit liver glycogen of the same conThe bacterial polysaccharidc centration. serves also as sccrpt’or for animal glycogen
13 23
~-AlTlyhSe limit (%I
ECL
59
15 10 6-8
49 43-46 47-56 46-64 55-59 23-37 52 53
819 8-10 8-10 4-6 9 15
ICL 5 5 1, 2 l-3 1-3 1, 2 2, 3 3 7
Iodin;;;&x 475 460 520 420-455 470485 red brown 420440 470 520
Ref. ___-~--
18 3 19,3 20 2, 21 21
a Abbreviations used are as follows: ?% = average chain length; ECL = external chain length; ICI, = internal chain length; Xmax = wavelength of peak absorption of iodine-polysaccharide complex.
GLUCAK
FROM B. stearothermophilus
synthetase, being 77 % as efficient as rabbit liver glycogen when tested at equimolar concentrations (expressed as glucosyl residues). Electrophoretic mobility. Some diffrrences of the B. stearothermophilus glucan with rabbit liver glycogen and potato amyloprctin can be detected by elect’rophoretic mobility in borate buffer using glass fiber paper. Figure 3A shows that thr B. stearothermoph.ilus polysaccharide has an Mglycogenof about 0.80, while amylopectin gives a spot at the origin and a second diffuse one in the glycogen-glucan area. In vitro synthesized product. It has been previously shown that the product formed by R. stearothermophilus glucan synthetase liberates maltose with /3-amylase, indicating the presence of a-l,4 bonds (1). The existence of a-1 ,6 linkages in the purified glucan is suggested by t,hr attack with amyloglucosidase and t,hn characteristics of the absorption spectrum of the complex formed
255
with iodine. The structure of the product synthesized in vitro was analyzed in an experiment of double labeling. For that purpose, rH]glucan was obtained as described under “Methods.” It was used as acceptor for a preparation of glucan synthetase containing no endogenous primer, with ADP-[Wlglucose as substrate. The product was isolated and submitted to /3amylolysis; t#he dist’ribubion of thcl label between the p-limit dextrin and the maltose liberated was studied. The results are shown in Table II. It can be seen that 100 % of the [14C]glucosyl residues added to the acceptor by the glucan synthetase are liberat’ed into the supernatant by p-amylase. On the other hand, the latter enzyme att,acks only 72 % of the [3H]glucose from the acceptor. This is as expected in a polysaccharide of the glycogenamylopectin type, which contains a-l,4 and a-l,6 bonds. In this experiment the glucan synthetase preparation only lengthened branches of the acceptor. Branching enzyme
- +
FIG. 3. Electrophoretic mobility of the product formed in vitro. The product formed incubating several enzyme preparations under the conditions already described (1) was submitted to electrophoresis on Whatman glass fiber paper as indicated in “Methods.” The dried strips were scanned and then sprayed as described in “Methods.” A) Standards. a: B. stearothermophilw glucan, b: rabbit liver glycogen, c: potato amy1opectin.B) Stationary phase extract, 40 pg protein. C) Logarithmic phase extract, 25 fig protein. D) Same as C, with 150 ag protein. E) Same as C, with rabbit liver glycogen (3 pmoles glucosyl residues). F) Same as C, with B. stearothermophilus glucan (0.43 rmole glucosyl residues). G) Same as C, with potato amylopectin (0.3 Imole glucosyl residues). H) Same as C, adding the supernatant of boiled B enzyme extract.
25G
GOLDEMBERG
might br present in the c>xtracts but perhaps it, xw not actiw bccausr t,hc c>xtcrnal chains rlf thcx glucnn had not, been lcngthencd c~nough under thcb conditions used. The in w’fw product, is a gIuc:~n with longrr ext,ernsl chnins.3 Iiigurc 3 she\\-s the> clectrophorctic mobilit,y of the product synthesized in vitro in the abscncc (B, C and D) or prescncc (E, I’, G and H) of added acceptors. Figure 3B rcprwbnts the pattern obtairwd \\-ith ~1 stxtionar\- phaw extract, C and D with t,wo different concentrations of a logarithmic phase pwparat,ion. Wlm~ sufficicnt~ amounts of cindogcnous primer arc present (B and D), radioactivity appears in the product,, which rcmnins at, the origin. Wit11 wry small amounts of t~ndogc~nousprimer (extract used in C) thcrc is no incorporation. The addition of diffwrnt~ accrptors to tllis extract, kads to the formation of products whicll migrake diffrwntly. Rabbit liwr glycogc>n (E) givrs :I radioactivr product, lo&cad in tlw glycogcw :lrw, purificad gluc:m (1;) givw :L spot1in tjllc> n small one glnc;~Il ZOIl( (:lralYeuncn 03) and at the origin, and \vith :~m,vlopwtin (G) the r:ldioactivit,\, appwrs at, thr origin. Thr :lddit’ion to prcynration C of a11 Aiquot of th(b rxtrwt uwl in B, prwiously innctivatrd by brat and wntrifugrd (H), giws thr same pattern :IS B or I). Th(l product obtaiwd lvith rabbit, livw glycogc>n (I?) can bt> diffwcwtk~tc~d from thcl onr formed in the: prcwnw of glucan, eithclr nat,ive (B, D, H) or purifkd (1:). Tt is wid& that, tllr isolation and purification mc%hod uwl has altewd it’ somon-ll:kt,. Thr nntiw product might’ br mow am~loJ”,ctin-lil~(~. ( Ttilimtion 0s the &can. Many bacteria :w l;no\~~nto accun-&tc~ glycogcn as endog(~110118 sow-cc of cwrgy for ccl1 mnintrnancr ~rocc~ssc~s (L&i, z(j). It is gcmrrally :wYpted th:lt c~~l1ul:tr wwrvrs :LW synthrsizcd when growth is limited, for instanw as bacteria cwt,w- tllc, stationary phaw. This limit,ation rnav b(: dw to nitrogcln scarcity, low pH, sulfur or phosphorus dcficic~ncy, in tlic of rxcrss carbon sourw (‘27). prcwriw :: ?;oic :rtidccl in proof : Preliminary resells obtained in o\tr laboratory indicate the cxistencc of branching activity in t.he bactcrinl extracts, when different conditions were llscd.
Omissions from complete system“
Radioactivity incorporated :‘I1 (cpm)
&Amylolysis of the product ~~ ~___ W (cpm) 3II (yo) “C (“0)
None ii80 3530 73 100 Qlucan 350 .~A1~P-glllcosc 6150 ~~.. 72 lkzyme 7500 io 71 _____ __n The complele system contains, in a final volume of 50 ~1, 0.1 3Cglycylglycinc buffer, pll S; 50 mx rnercaptoetllallol; enzyme from logarithmic phase, prepared from spheroplastjs and fract.ionated with ammonilml slrlfate, 49 pg protein; 0.46 pmole (g111cosyl residlles) [“ll]-glucan (7800 cpm), and 1 mar ADP-[‘4(:]-glr~cc~se (15,000 ~1x11). Incubations were carried OII~ :L( 05” for 15 ruin, Ihe system was complct ed where necessary, and I he product was isolated as descrihcd (1). An aliqllot was submitted to crystalline sweet po~,nlo Bamylsse action (12) and after overnight inclbtion at 37” lmder tol11c11c V:LI)OI’S, the &dest,rin was precipitated with 3 vol of met~hanol and w:~slrcvl. J:adioact,ivity was dckrmined in the prccipit atcd p-limit destrin and the comhillrtl sllprrnat,:mis hy means of :t liqliid scini ill:il,ic,n sl~ertromckr.
I?igurcl 1 shops that in II. .sfea~othel~,nophilus thcrcl is :m :~ccumulntion of giuca.11 in t,lir stationary- pli;w 2nd no polys:~ccharid~: in t,hcl logarit,limic ph:w, \vliw th: cnrrgrt~ic nods ar(l maximal. It may,- be assumcbdt’h:kt some mwhanism for its utilization exists in thcl wll. This possibility wits t&cd in a11ia vice caxpwimcwt shown in Fig. 4. 011s \v(w grO\\-II
t(J
tllc‘
st&tk~rl:l~~*
I)li:kx’,
:ultl
tlr!,
weight, gluc:m :md protck content wrc drt,crminrd in :L s:Lmplr (-1). I+sh prewnrmcd mcldium WM t8hw :Ldded in a sixfold CXCPSSand thcl incubnbion W;IS cont~inuc~d. Wlica growth \v:w rwumcd :Iftc>r :L short timr, two furthc~r snmplw wrc taken (13 and C) to assay the same p:wnwtws. It’ c:m b(l sc(xr1 that, whc~ri crlls wrr actiwly gro~virlg again (C), a largc~ fall in gluci~n corit8cxnt,was obscirvrd. This is in accord with t,hcxrwults obtuiwd in Icig. 1, in wllich thaw: is 110 glucan in thrb logarit~hmic phwr. Ho\\-cwr, iI Spite (Jf th’ hr#’ (liSapJ~(‘:u:lnc(: of &cm obsrrwd bctwwn B and C in Icig. 4, the in f&o hydrolytic activity from oxtrilcts
1.0
2.5
1
c!8-
20
Q6-
15
04-
l.0.
0.4 -
0.5-
-
FIG. 4. Utilization of B. stearofhelmophil?s glucan. Conditions as for Fig. 1. At point A, a sixfold excess of fresh medium prewarmed to G5” was added and incubation immediately resumed. (0) log absorbance at, 700 nm (the origin of the upper scale on the ordinate at the left was displaced to get a bet,ter continuity of the growth curve); (III) Nmoles of glucan per milligram dry weight; (WI) prnoles of glucan per milligram of protein.
obtaiwd at thcw points \V:LS very small wlm~ twtcxd \vith rabbit livtr glycogcw or purified glucan. Wild-type logarithmic phase extracts, on the contrary, show-cd a wry high hydrolytic activity (JII both polysncchnridrs (8). ~Iorcovw, n-hen :L prtyar:~tion of thcl wild-typo strain was add(bd to :m ext’ract’ of th(> wriant, the> radioactivity incorporutcd into thcl product II-W much loo er. The, possibility that thcl hydrolytic clnzymo(s) from that variant wlls is more unst.abl(~ than thnt of thcl \\ild-type bactwia or has diffcrcnt rquiwmc~nts, cann(ot be dkwrdcd. Ko phosphorolytic activit,y could be d(kccttld in any of thr strains. Thcl propwt’y of thwmophilic bactc~rin of
rcyroducing optimally at, t8wnperaturcs at which mesophiles :ire killrd, has not yet been fully clarific~d. T\vo general thcorics have been put for\\ard. Orw attributes it to an incrcawd thwmostability of the proteins (2s); thcl second suggc>sts a rapid rqvntlrwis of thcl lrclat,-damaged wllular components (29). Thcl glucan stored by R. slear,othe~nlophiIus in t,hcl stationary phase: might function as :L sourw of carbon subst!r:tt,rs for rcsynthwis of d(sgradcd wllular componwts, brkg used at :I wry fast, r:k in the c:spo~wntial ph:w as slio~\-n in Yig. 4. Whc~ gro\vt’h is limited, the ncwssity for a rapid supply of cnwgy diminishes and tllc glucan begins to accumul:Ltc~. This I)ol?-s:Lccli:lritl(: could Ii:~w
20‘8
GOLDEMBERG
a pllysiological rok similar t,o glycogen in mesophilic bactjcria, tha looser structural being perhaps an addit,ional advantagr for vcyv rapid utilization during active growth. Ko polysaccharidt~ nor synthetic activit#J have bwn dctwtc>d in wild-type cells. This failurc~ might bc dw t’o thcl very high hydrolytic activity obwrwd in these bacteria, and/or different requirements for synthct,ic activity. Thrsc~ possibilities arc under stud! in our laboratory. ACKNOWLEDGMENTS The author wishes to thank Dr. Luis F. Leloir, Dr. I. I>. .4lgranat,i and the other members of the Instituto de Investigaciones Bioquimicas for helpful sllggestions and criticisms.
1. GOLDEMHERG, S. H., .ZND ALGRliNBTI, 1. D., Biochim. Biophys. Acta 177, 166 (1969). 2. GHOSH, II. P., AND PREISS, J., Biochim. Biophys. Ada 104, 274 (1965). 3. SIGIL, N., ~?:\TTANISO, J., .\ND %:GEL, I. FI., Arch. Bioch~em. Biophys. 108, 440 (1964). 4. PAI+ J. T., AND JOHNSON, M. J.. .J. Biol. Chem. 181, 149 (1949). 5. HUGGISTT, 9.8~.C:., .IND NIXON, D. A., Biothem. J. 66, 12P (1957). 6. Duno~s, M., GILLITS, K. A., HAMILTON, J. K., RAPIERS, P. A.. .\ND SMITH, F., Anal. Chem. 28, 350 (1956). 7. BODM.\N, II., AND WELKER, N. E., J. Bacleriol. 97, 9‘24 (1969). 8. GOLDE~IBERG, S. H., to be published. 9. PIRAS, R., ROTHMAN, I,. B., AND Cxm, E., Biochemistry 7, 56 (1968).
10. LSIG, 14:. Y. c., .1ND bvHEL.IS-, w. J., Arch. Bioch,em. Biophys. 116, 162 (1966). 11. F.~LI!x, F. N., Anal. Chem. 31, 1898 (1959). 12. KJ~LI~BRG, O., .\ND M.\vNI,:Rs, D. J., J. Chem. S’oc. 4596 (1962). 13. SPIRO, I<. (:., in “Met,hods in Enzymology” (I<. F. Neufeld and V. Ginsburg, eds.), Vol. 8, p. 4. Academic Press, New York (1966). 14. KILISW.IN, C. It., Anal. fliochem. 4, 17 (1962). 1.5. LOWRY, 0. H., I~~S~HR~VGH, N. J., F:\RK, A. L., .\ND I:.~ND.ILL, 11. .J., J. Biol. Chcm. 193, 265 (1951). 16. FULLILR, K. W.,, .u-D NOI~THCOTI’:, 1~. H., Biochem. J. 64, 657 (1956). 17. F.\KRWLL, J., .IND Itos~, A., iinnu. fZer. Microbial. 21, 101 (1967). 18. B.\RRY, C., (:.IVARD, I:., RIILHAUD, G., AND AUB&RT, J. P., Ann. Inst. Pusleur 84, 605 (1953). 19. KINDT, T. J., .\ND CONHAD, H. F;., Biochemistry 6, 3718 (1967). 20. BARKW, S. A., BOURNE, I<. J., AND STACW, M.. J. Chem. Sot. 2884 (1950). 21. ZEVI~NHUIZEN, L. P. T. hf., Mededelingen Landbouwhogeschool, Wageningen, 10-66, 39, (1966). 22. H.\WORTH, W. N., HIRST, If. L., ~NI) ISHERWOOD, F. A., J. Chem. Sot. 577 (1937). 23. H.~Ls~LL, T. (+., HIRST. R. L., AND JONIOS, J. K. N., J. Chem. Sot. 1399 (1947). 24. D~oN~H, W., Al~~ WHISTLEK, U.. L., J. Biol. Chem. 181, 889 (1949). 25. CHEN, G. S., AND SEGE:L, I. II., Arch. Biochem. Biophys. 127, 164 (1968). 26. D.awtss, E. A., AND Rrr~uo~s, D. W., Annu. Rev. Microbial. 16, 241 (1962). 27. EIDICLS, I,., ‘INI) Pnixss, J., Arch. Biochem. Biophys. 140, 75 (1970). 28. KOFBLI.:R, H., Racferiol. Rev. 21, 227 (1957). 29. ALLEN, M. TS., Backrio!. Rev. 17, 125 (1953).