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hnh thnh mng sinh hc (biofilm) bi mt chng Bacillus subtilis sn sinh -polyglutamat.
Masaaki Morikawa, Shinji Kagihiro, Mitsuru Hakuri Kazufumi Takano, Steve Branda, Roberto Kolter v Shigenori Kanaya B mn vt liu v khoa hc s sng, trng k thut sau i hc,i hc Osaka, Nht Bn. B mn vi sinh vt hc v di truyn hc phn t ,trng y khoa Harvard, Boston, Hoa K. Cc cht gian bo c sn xut bi Bacillus subtilis B-1, mt chng ngoi mi trng hnh thnh rt mnh floating biofilms(mng sinh hc ni), c tinh sch v c xc nh cha ch yu -polyglutamat (-PGA), mt phn t c khi lng trn 1000 kDa. C s hnh thnh biofilm v s sn xut -PGA bi Bacillus subtilis B-1 c tng ln vi s tng nng Mn2+ hoc glixerol . -PGA c tng hp trong pha sinh trng ca nui cy tnh,v biofilms floating cng c hnh thnh. Tuy nhin trong iu kin nui cy lc -PGA c to ra trong mt pha khng sinh trng. Khi B. subtilis B-1 c sinh trng trong mt h thng nui cy vi hiu kh, s hnh thnh floating biofilms v s sn xut -PGA gim ng k.iu ny cho thy trong nui cy tnh s gim nng oxy lin quan n cc bc u tin trong s tng hp floating biofilms .Phn tch proteomic ca cc protein mng chng minh rng flagellin, oligopeptide permease v tin protease Vpr l cc protein chnh c sn xut bi cc t bo trong mt floating biofilm v mt khun lc.

GII THIU Biofilm hay mng sinh hc l cc qun x vi khun c nhng vo cht gian bo v gn vo mt b mt.S hnh thnh biofilm din ra trong rt nhiu trng hp,v phn ng li vi cc tn hiu a dng ca mi trng (Otoole v cng s).V d biofilm c th hnh thnh trn b mt rn,hoc trn b mt ca mi trng lng.Trong trng hp sau floating biofilm cn c gi l mng phim(pellicles).Cc khun lc m sinh trng trn mi trng bn rn cng c th c xem nh mt dng ca biofilm. Cc quan st v m v hin vi cc biofilm vi khun tit l cc cu trc c trt t cao ,chng bin mt khi cc thnh phn ca cht gian bo b loi b nh l mt nh hng ca t bin (Branda v cng s, 2005). Cc cht gian bo gp phn vo c ch n nh ca biofilm, cho php n chu c ng k cc lc ko ln, v biofilm c ch ra l c cha cc polysaccharide,protein v axit nucleic (Branda v cng s, 2005).Trong s cc polysaccharide hin din trong biofilm th : Pseudomonas aeruginosa cha alginate, Pel v Psl ; Escherichia coli v Salmonella typhimurium cha cellulose;in Klebsiella pneumoniae v Enterobacteraerogenes cha mt tetrasaccharide lp li ca D-glucose, L-fucose v axit D-glucuronic ;Sphingomonas spp cha gellan ; v streptococci cha levan (b-D-fructan) (Boyd & Chakrabarty, 1995; Danese cng cng s., 2000; Friedman & Kolter, 2004; Kiska & Macrina,1994; ONeill cng cng s., 1986; Yamazaki cng cs., 1996).Biofilm ca Vibrio cholerae O1 c ch ra l cha mt polysaccharide cu to bi N-acetyl-D-glucosamine, Dmannose, 6-deoxy-D-galactose v D-galactose (Wai v cs, 1998).

Trong vi nm gn y Bacillus subtilis ,mt trc khun gram dng sinh bo t, tr thnh mt sinh vt m hnh cho cc nghin cu s hnh thnh biofilm .Ngi ta ch ra rng cc chng hoang di ca Bacillus subtilis thng hnh thnh floating biofilm mnh hn so vi cc chng xut pht t chng Bacillus subtilis 168 thun ho phng th nghim. In the highly structured floating biofilms and colonies formed by the wild strain B. subtilis 3610,sporulation displays a high degree of spatio-temporal organization, occurring predominantly at the tips of aerial projections. iu ny cho thy rng s hnh thnh biofilm c th l mt phn trong qu trnh pht trin ca sinh vt ny. B. subtilis 3610,mt thnh phn chnh ca cht gian bo trong cc khun lc v floating biofilm l cc exopolysaccharide (EPS) c sn xut bi cc locus eps (Branda v cng s,2001, 2005).Tuy nhin cc c tnh v m ca cc khun lc v floating biofilm hnh thnh bi cc chng hoang di khc nhau ca B. subtilis c th khc nhau ng k .Trong trng hp khi chng ti so snh B. subtilis 3610 vi B. subtilis B-1,mt chng ngoi mi trng c phn lp t m du (Morikawa v cs, 1992). y chng ti nhn thy rng PGA l mt cht trng hp ngoi bo chnh, v s gim st nng oxy l mt tn hiu cho s hnh thnh floating biofilm trong B. subtilis B-1. Chng ti cng chng minh rng, flagellin, oligopeptide permease v tin protease Vpr l cc protein mng chnh c sn xut bi cc t bo ca floating biofilm. PHNG PHP Mi trng nui cy Luria (L-) broth, cha (l-1) 10 g Bacto tryptone (Difco), 5 g cao nm men (Difco) v 10 g NaCl, c s dng chung cho nui cy cc vi khun khc nhau . L-agar l mt mi trng bn rn cha 1-2% agar trong L-broth. Mi trng E l mt mi trng c s dng kch thch s sn xut -PGA (Cromwick & Gross, 1995), n cha (l-1): 20 g L-glutamic acid, 12 g citric acid, 80 g glycerol,7 g NH4Cl, 0-5 g MgSO4.7H2O, 0-5gK2HPO4, 0-15 g CaCl2.2H2O,40 mg FeCl3.6H2O v 148 mg MnSO4.H2O. PH ca mi trng E c iu chnh n 7.4 bng NaOH 2M. S lng glycerol v MnSO4 c iu chnh nh nu Hin vi in t qut (SEM) Pellicles c t nh nhng trn mt tm knh c ph poly-L-lysine.Cc mu vt c c nh vi glutaraldehyde v OsO4, loi nc trong ethanol, isoamyl acetate v phn on CO2 ,v ph vi bch kim (Glauert, 1975) S quan st c thc hin vi mt knh hin vi in t qut Hitachi S800. Chun b mu v phn tch cht gian bo T bo B. subtilis B-1 (Morikawa et al., 1992) c nui qua m trong L-agar ,cc khun lc c tch,huyn ph trong NaCl 0.9%.iu ny gip chng ti thu c gn nh ton b cc cht trng hp ngoi bo tinh khit qua mt qu trnh 2 bc n gin : loi b t bo bng ly tm (30 000 g 4 oC trong 30 pht), v kt ta trong ethanol bng cch thm vo 3 ln th tch ethanol lnh ni trn mt.Bc kt ta vi ethanol c lp li 2 ln v sn phm thu c c lm kh trong chn khng. Mt phn ca vt liu tinh sch c thu phn trong HCl 5M 100 oC trong 24 gi,v cc sn phm c phn tch trn gel silica 60 hiu sut cao bng phng php sc k l mng (HPTLC) ,vi mt hn hp dung mi ethanol/nc (63 : 37).Cc phn cn li c ha tan trong D2O (4% w / v), v phn tch bi 1H-NMR v 13C-NMR(cng hng t ht nhn) (UNITY-INIVA 600; Brucker). Nhng phn tch cho thy rng cc vt liu thu hi ch yu cha duy nht -PGA. Cc ng phn DL trong thnh phn ca glutamic axit c xc nh bi phn tch HPLC (sc k lng cao p)ngc pha,,sau khi dn xut ho vi tinh khit Marfey (1% trong acetone) 40 oC trong 90 pht. Ct sc k HPLC l mt COSMOSIL 5C18-AR (i.d. 4-6 mm, di 20 cm; Nacalai Tesque), v dung dch thi l methanol/20mM m natri axetat (PH 4)/acetonitrile (2:7:1).Cc phn on c gim st bng cch o hp th bc sng 340nm.S ra thi cc vi tr v cc vng ca nh hp th c so snh vi D-glutamic acid and L-glutamic acid chun.Khi lng phn t c xc nh bng sc k lc gel (TSKgel G4000SWxL )(i.d. 7-8mm, di 30 cm; Tosoh) ,vi 0-2 M m Natri phosphate

(PH 6-9) v 0-2 M Na2SO4.S tch -PGA c theo di bng o hp th bc sng 220 nm.Mc sn xut -PGA trong nui cy c c lng da vo so snh vng nh trong sc k lc gel vi cc mu chun,hoc bng cch cn vt liu kh sau khi tinh sch. Cc th nghim biofilm B mt rn c s hnh thnh biofilm c pht hin bng phng php nhum tm tinh th (CV) vi cc ci bin nh(OToole v cs., 1999). Tm tt,mt hn hp nui cy qua m c pha long sao cho OD600 nm trong khong 0-3,v 1l c b sung thm 99 l mi trng E trong mi ging ca a nha plastic chun 96 ging.a ny c gi yn 37 oC trong 8 gi.Sau , cc mng phim b mt v mi trng nui cy c cn thn loi ra khi ging .Mi ging c ra nh nhng 2 ln vi nc ct, cc t bo cn li v cht gian bo c nhum vi 150 ml ca dung dch tm tinh th 1 % trong 25 pht nhit phng,sau khi ra 2 ln vi nc ct,tm tinh th bm vo biofilm c ho tan trong 150 ml DMSO ,v nh lng bng o hp th ca n bc sng 507 nm. Chun b v phn tch protein mng Cc t bo c thu hoch, v ra sch v huyn ph trong m phosphate 10 mM (pH 7.0),sau khi sinh trng trong nui cy tnh ,lc hoc agar trong 14h 37oC.Trong nui cy tnh ,t bo hnh thnh floating biofilm trn b mt (hnh 1).Huyn ph t bo c x l vi lysozyme (1mg.ml-1) trong 3h 37oC ,sau lng 1 pht trn . Sau khi loi b t bo nguyn vn bi ly tm 8.000 g trong 10 pht.Phn dch ni trn mt c phn on tip bng siu ly tm 9000 g trong 1h. Phn dch ni v kt ta thu c ln lt tng ng l cc thnh phn ca t bo cht v mu protein mng t bo.Cc mu protein mng t bo c phn tch bng in di trn gel polyacrylamide 12% c SDS 1%.Cc bng protein c quan st bng phng php nhum vi Coomassie brilliant blue R-250.Bn gel cha mt bng protein c phn gii bi trypsin ,sau phn tch trnh t amino acid vi phng php LC-MS/MS (sc k lng kt hp khi ph lin tc) (Taplin Biological Mass Spectrometry Facility, Harvard Medical School, Boston, MA, USA) v Sequest (in t nhit). KT QU V THO LUN Cu trc ha hc ca cc cht trng hp ngoi bo sn xut bi B. subtilis B-1 B. subtilis B-1,mt chng c phn lp t m du, pht hin c hnh thnh mng phim (floating biofilm) mnh v biofilm lin kt b mt rn trong nui cy tnh.N cng hnh thnh cc cu trc trn khun lc trong mi trng L-agar m ni bt l cc mng bm trung tm ca chng.Ngc li B. subtilis 168 hnh thnh cc khun lc nhn,v khng hnh thnh mng phim (pellices) r rt hoc cc biofilm lin kt b mt rn trong mi trng L- broth.Cc quan st hin vi in t qut (SEM) ca pellicle sn xut bi Bacillus subtilis B-1 ch ra rng cc t bo c ng gi dy c bn cnh v nhng vo trong mt cht nn (hnh 2).Trnh t gen 16S rRNA ca Bacillus subtilis B-1 (EMBL/GenBank/DDBJ s nh danh AB213262) ging vi trnh t tng ng ca Bacillus subtilis 168 ti 99.6%. Cc cht trng hp ngoi bo sn xut bi cc khun lc ca Bacillus subtilis B-1c tinh sch bng cch kt ta vi ethanol,v cho kt qu m tnh i vi c hai th nghim axit uronic v anthrone,cho thy rng, nguyn liu khng cha mt lng ng k polysaccharide (May & Chakrabarty, 1994).Thm vo ,sn phm tinh sch khng cha mt lng ng k protein hoc axit nucleic,iu ny c chng minh bi s thiu hp th cc bc sng 280 v 254 nm.Khi mu b thy phn ,phn tch vi HPTLC v nhum bng cch phun vi 0-1% dung dch ninhydrin ethanol ,sau sy 100 oC.Mi im mu n c quan st ti mt vi tr tng ng ca axit glutamic.Vt liu sau c phn tch bng 1H- v 13C-NMR.Bn cht ha hc ca cc nh c ch ra trong bng 1.1. Tt c cc kt qu ny ch ra rng vt liu chng ti phn tch c cha ch yu PGA .Lin quan n cc vng nh glutamic acid ,sau khi phn tch bng HPLC, ch ra rng t l ca D- v L- glutamic acid cu thnh cc -PGA dao ng t 9:1 n 4:1 sau 12h nui cy 37 oC trong mi trng Lbroth.Khi lng phn t ca -PGA phn tch c xc nh trn 1000 kDa bng phng php sc k lc gel .Cn ch rng B. subtilis 3610 khng sn xut -PGA. S sn xut -PGA,v s hnh thnh biofilm

Gn y,stanley v lazazzera (2005), bo co rng km theo s chuyn gen quy nh kim sot s tng hp PGA t mt chng hoang di sang chng thun ha B. subtilis 168,l kh nng c th hnh thnh mng sinh hc (biofilm) Tuy nhin s xa b ywsC,mt gen m ha cho s tng hp -PGA,trong chng hoang di B. subtilis RO-FF-1 khng dn n s st gim ng k s hnh thnh mng sinh hc lin kt b mt . Kt qu ny chng minh rng s sn xut -PGA l khng cn thit i vi s hnh thnh biofilm bi B. subtilis.T cc kt qu ny,chng ti hng s quan tm ti vic xc nh mt s lin quan nu c gia s sn xut -PGA v s hnh thnh biofilm B. subtilis B-1.Cc bo co ca cc tc gi khc ch ra rng nhng thay i trong nng ca MnSO4 v glycerol nh hng ti s sn sinh -PGA bi Bacillus licheniformis ATCC 9945A (Cromwick & Gross, 1995; Ko & Gross, 1998).Chng ti tm thy cc kt qu tng t B. subtilis B-1 (hnh 3a,b) .Quan trng hn, iu ny cho php chng ti cho thy rng c mt s tng quan trc tip gia s sn xut -PGA v hnh thnh mng sinh hc trong B.subtilis B-1 (Hnh 3c),s tng quan ny cng c thy mt chng B. subtilis khc, B. subtilis natto BEST 195,mt chng sn xut -PGA cp bnh thng (s liu khng c ch ra, Itaya & Matsui,1999).Chng ti tch dng gen ywsC t B. subtilis B-1 v c gng to ra mt chng xut pht t chng B-1 b mt gen ywsC do t bin,nhng chng ti khng th lm iu ny. kim tra xem liu s hnh thnh pellicle l mt nguyn nhn hay mt kt qu ca s s sn xut PGA,ng hc tng trng v s sn xut -PGA bi B.subtilis B-1 c so snh trong nui cy tnh v nui cy lc.Cc t bo khng hnh thnh pellicle trong nui cy lc l do cc tc ng ca stress do lc ct lin tc.S sn sinh -PGA trong nui cy tnh bt u 6h sau khi cy ging,khi m s hnh thnh pellicle vn cha bt u.Hn na , -PGA cng c sn xut trong nui cy lc,mc d sau mt pha lag kh di,khong 12h sau khi cy ging.Cui cng ,sau 24h mc sn xut -PGA l tng t nhau gia nui cy tnh v nui cy lc.Cc kt qu ny ch ra rng s hnh thnh pellicle khng phi l mt yu cu ca s sn sinh -PGA B-1. S gim st nng oxy ha tan v s hnh thnh biofilm Chng ta bit rng s sc kh mnh c hiu qu cho sn lng -PGA cao (Cromwick v cs., 1996). kim tra nh hng ca nng oxy ha tan i vi vic tng hp -PGA trong khi pellicle ang hnh thnh, mt h thng nui cy vi hiu kh (MACS), thu c bng cch s dng cc ng Teflon microporous Poreflon (Sumitomo Electric Polymer M) c s dng (hnh 5b).MACS cho php chng ti lm thng kh ca mi trng m khng thy c s si bong bng.Khi MACS c s dng,cc t bo sinh trng trong pha lng trong khong 9 ting m khng hnh thnh pellicle.Kt qu ny tri ngc vi s quan st thy t bo bt u hnh thnh pellicle khong 6 ting sau khi cy ging trong h thng nui cy tnh,v hu nh khng c t bo sinh trng trong pha lng sau (hnh 5a).Nhng quan st ny ch ra rng s suy gim oxy trong nui cy tnh lin quan n cc bc u tin trong s hnh thnh floating biofilm bi B.subtilis B-1. Phn tch cc protein mng trong biofilm v cc t bo ph du (planktonic cell) c ci nhn su hn na v s khc bit sinh l gia biofilm v cc t bo ph du,cc protein mng c chun b t cc pellicle,t bo ph du v cc khun lc c phn tch tng i bi in di SDS,sau nhum vi Coomassie brilliant blue (hnh 6).R rng l cc mu protein ca biofilm v khun lc n gin hn so vi protein ca t bo ph du.Cc protein vi kch thc 77 (P77), 57 (P57) v 36 kDa (P36) c sn xut ch yu bi cc t bo ca pellicle v cc khun lc khi so snh vi cc t bo ph du .Phn tch bi Sequest ,da trn mt phn trnh t amino acid ca cc protein ny,ch ra rng P77,P57 v P36 tng ng l mt tin protease ngoi bo nh (Vpr;P29141) ,mt oligopeptide permease (OppA; CAA39787/Spo0K, AAA62687),v mt flagellin hon chnh (Hag; P02968).Con ng pht tn hiu ComX-ComP-ComA l mt con ng phn ng chnh ti thiu trong B. subtilis, v n iu khin s tng hp -PGA(Comella & Grossman, 2005; Stanley &Lazazzerra, 2005).S tch ly pheromon ComX kch thch enzyme histidine kinase mng ComP,kt qu l s t phosphoryl ha.S phosphoryl ha ComP nhng nhm phosphat cho cht iu ha p ng ,dn ti s hot ha ComA ph thuc cht hot ha.CSF (yu t kch thch kh nng,xut pht t sn phm ca gen phrC) l

mt pheromon khc cng kch thch s hot ha ComA(Lazazzera, 2000).CSF c ch RapC,mt yu t iu ha m s phin m ca ComA.CSF c sn xut t tin CSF sau khi c x l bi protease serine ngoi bo nh Vpr, Epr v Apr (subtilisin E) (Lazzazerra,2006).CSF c vn chuyn vo trong t bo bi mt h thng vn chuyn bng lin kt vi ATP (ABC) cha oligopeptide permease (OppA/Spo0K).S quan st OppA/Spo0K (P57) v tin Vpr (P77) ch yu biu hin trong mu pellicle v khun lc cho thy rng mt h thng quorum-sensing c cc chc nng tch cc trong cc t bo ny .iu th v l P36 c tm thy chnh l flagellin,v n c sn xut vi mt lng ng k bi cc t bo trong pellicle v trong khun lc.Mc d Chagneau v Saier chng minh rng lng roi l cn thit cho s hnh thnh biofilm,mt phn tch DNA microarray ca biofilm B. subtilis ch ra rng s biu hin ca gen m ha cho flagella b c ch (Chagneau & Saier, 2004; Stanley v cs., 2003).Mt bo co khc ch ra rng khng c s khc bit ng k trong mc biu hin ca gen m ha cho flagellin trong cc t bo ca biofilm,khi so snh vi cc t bo ph du (Ren v cs,2004).Nhng mu thun ny vn cn tn ti ch c gii quyt.Tuy nhin,s tn ti ca lng roi trong cc t bo ca biofilm cng c xc nhn bi quan st hin vi in t sau khi nhum uranyl acetate(khng ch ra).T cc kt qu ca chng ti c th cho rng lng roi trong cc t bo trong biofilm ca chng B-1 c sn xut cho mt mc ch khc ngoi gip t bo bi .Trong trng hp ca Aeromonas hydrophila AH-3,s sn sinh roi bn c ch ra l gia tng c bit trn cc mi trng bn rn v b mt,v quan trng i vi s dnh bm v s hnh thnh biofilm (Canals v cs., 2006). LI CM N Cc tc gi rt bit n Gio s M. Itaya (Keio University) vui lng cung cpB. subtilis natto BEST 195, v E. Oiki (i hc Osaka) h tr tuyt vi trong quan st hin vi in t qut SEM. ti ny c h tr bi s ti tr t Institute for Fermentation, Osaka, v J-Power, , v Hc vin Y t quc gia . TI LIU THAM KHO Boyd, A. & Chakrabarty, A. M. (1995). Pseudomonas aeruginosa biolms: role of the alginate exopolysaccharide. J Ind Microbiol 15,162168. Branda, S. S., Gonzalez-Pastor, J. E., Ben-Yehuda, S., Losick, R. &Kolter, R. (2001). Fruiting body formation by Bacillus subtilis. Proc Natl Acad Sci U S A 98, 116211626. Branda, S. S., Vik, S. L., Friedman, L. & Kolter, R. (2005). Biolms:the matrix revisited. Trends Microbiol 13, 2026. Canals, R., Altarriba, M., Vilches, S., Horsburgh, G., Shaw, J. G.,Tomas, J. M. & Merino, S. (2006). Analysis of the lateral agellar gene system of Aeromonas hydrophila AH-3. J Bacteriol 188, 852862. Chagneau, C. & Saier, M. H., Jr (2004). Biolm-defective mutants of Bacillus subtilis. J Mol Microbiol Biotechnol 8, 177188. Comella, N. & Grossman, A. D. (2005). Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes contolled by the quorumsensing transcription factor ComA in Bacillus subtilis. Mol Microbiol 57, 11591174. Cromwick, A. M. & Gross, R. A. (1995). Effects of manganese (II) on Bacillus licheniformis ATCC 9945A physiology and gamma-poly(glutamic acid) formation. Int J Biol Macromol 17, 259267.