20

Chng 2

Cu trc v chc nng ca t bo vi sinh vtI. Sinh vt nhn s1. Cc phng php v k thut nghin cu 1.1. Phng php quan st t bo Nh knh hin vi quang hc (knh hin vi thng) vi phng i 1500 - 2000 ln, c bit nh knh hin vi in t thng (TEM) v knh hin vi in t qut (SEM) m khoa hc c th thy c t bo, cu trc siu hin vi ca vi khun vi ng knh khong 1m. - Quan st vi sinh vt trn tiu bn sng: vi sinh vt gia lam v lamella, nhum mc nho thy r mng nhy (capsule), y l phng php hay dng cho nhng vi sinh vt nui cy trn mi trng lng vi knh hin vi thng, quan st c kh nng vn ng ca chng.Bng 2.1: Mt s phng php nhum mu v nguyn tc s dngPhg php nhum mu +Nhum n (xanh methylene, carbolfuchsin, tinh th tm, safranin) +Nhum phn ly - Gram - Ziehl - Nielsen Nguyn tc s dng Dung dch ru hoc nc ca cc kim, dng quan st hnh dng vi sinh vt, cch sp xp t bo Vi cc phn ng khc nhau vi thuc nhum c th phn bit c chng Chia cc vi khun thnh 2 nhm ln: Gram dng gi mu tinh th tm, Gram m mt mu khi ty do s nhum mu ph hng safranin Dng phn bit cc loi Mycobacterium v mt s loi Nocardia. Vi khun acid nhum vi carbolfuchsin v x l vi dung dch ru acid, vn gi mu . Vi khun khng acid s mt mu do s nhum mu ph l xanh methylene. Dng pht hin s c mt ca mng nhy, bi v polysaccharide mng nhy khng bt mu thuc nhum bao quanh t bo vi khun nhum mu t S dng pht hin bo t vi khun. khi dng thuc nhum lc malachite vi tiu bn c un nng, thuc nhum s thm nhp vo ni bo t v lm chng nhum mu lc, khi b sung bng safranin s lm phn bao quang bo t nhum mu hng. Dng pht hin tin mao vi khun, s dng thuc lm phng tin mao ri sau nhum bng carbolfuchsin

+Nhum c bit - Nhum m (negative) -Nhum ni (endospore) -Nhum (flagella) tin bo

mao

21

- Quan st vi sinh vt trn tiu bn c nh v nhum mu: Phng php nhum Gram v Ziehl - Nielsen cho php nhn bit 2 nhm vi khun Gram dng va Gram m, hnh dng ca bo t, cc vt th n nhp nh ht d tr polyphosphate (ht d nhim sc, ht volutin), cc git m, glycogen Nhng tiu bn c nh ny c quan st bi gic ln X 90 hoc X 100 (bi gic dng du, vt knh c vng en). Tham gia vo c ch nhum mu c cu trc ca thnh t bo v bn cht cc hp cht ca sinh cht khc nhau hai loi vi khun. quan st nhng cu trc siu hin vi ngi ta dng knh hin vi in t TEM v SEM, c th thy c nhng cu to rt nh vi ln vi nanometre. Nhng vi khun Gram+ c mi lin h chng loi pht sinh gn gi nhau, y ngi ta chia thnh hai nhm ph: nhm c hm lng (G + X)% cao hn 50% nh cc Actinomycetales, Corynebacterium, Cellulomonas v nhm c (G + X)% thp hn 50% nh Clostridium, Bacillus, Staphylococcus, Nhng vi khun Gram- c mi quan h chng loi pht sinh cch xa nhau v y ngi ta phn ra rt nhiu nhm ph.Bng 2.2: Mt s tnh Gram m Tnh cht Phn ng vi ha cht nhum Gram cht khc bit gia vi khun Gram dng v

Gram dng Gram m gi mu tinh th tm, mt mu tm khi ty do t bo c mu ra, nhum mu ph tm hoc ta safranin hay Fuchsin Lp peptidoglucan dy, nhiu lp mng, ch c mt lp c khng c Acid techoic Lp pha ngoi thnh khng c c Lp lipopolysaccharide rt t hoc khng c nhiu, hm lng cao Hm lng lipid v thp (vi khun acid c cao (to thnh lp lp lipid mng lin kt ngoi thnh) lipoprotein vi peptidoglucan) Cu trc gc tin mao hai vng a gc bn vng a gc To c t ch yu l ngoi c ch yu l ni c t t (exotoxins) (endotoxins) Chng chu vi tc nhn kh nng chng chu kh nng chng chu vt l cao thp Mn cm vi lysozyme rt mn cm, d b tan t mn cm (cn phi vi enzyme ny x l ph lp mng ngoi ca peptidoglucan) Mn cm vi Penicillin v cao thp sulfonamide

22

Mn cm vi Streptomycine, Chloramphenicol, Tetracycline Kt hp vi thuc nhum kim Mn cm vi cc cht ty anionic Chng chu vi mui Natri Chng chu vi kh hn

thp

cao

cao, cht ch cao cao cao

thp, lng lo thp thp thp

1.2. Phng php tch ly cc thnh phn ca t bo Khi cn nghin cu cc thnh phn ring bit ca t bo, ngi ta phi tch ly cc thnh phn ny nh siu m, enzyme lm tan thnh, cc khng sinh tc ng vo thnh, dng p sut thm thu gy co nguyn sinh, dng sc p c hc, dng siu li tm hoc li tm trong ng gradient Nh cc my o quang ph (Spectrophotometer) chng ta bit r rng v nhanh chng s lng t bo trong dch huyn ph. Nh phng php sc k giy hoc sc k ct chng ta c th thu c cc hp cht ring bit. 2. Hnh dng vi khun v vi khun c Hnh dng vi khun khc nhau gia loi ny v loi khc, i vi nhng vi khun a hnh (polymorphysme) th hnh dng c th khc nhau trong cc giai on sng khc nhau ca chu k sinh trng. Nhng hnh dng chnh ca vi khun: - Cu khun (Coccus): Khi phn chia theo mt phng v dnh nhau ta c song cu khun (Diplococcus), hoc lin cu khun (Streptococcus), phn chia hai phng v dnh vi nhau ta c t cu khun (Tetracoccus), phn chia 3 phng v dnh nhau ta c bt cu khun (Sarcina) hoc phn chia theo nhiu phng ta c t cu khun (Staphylococcus). - Trc khun: bao gm trc khun khng sinh bo t (nh E.coli) v trc khun sinh bo t nh Bacillus, Clostridium vi kch thc khong 2-3 x 1m. - Xon khun: Spirillum, Campylobacter, xon th vi cc vng khc nhau: Spirochaeta, Leptospira vi kch thc 1 x 5-500m. - X khun: gm nhng vi khun thuc b Actinomycetales trong c cc ging quan trng nh Streptomyces, Micromonospora, c kch thc 1-2 x 100-500m.

23

Hnh 2.1: Cc nhm hnh thi chnh ca vi khun

- Vi sinh vt hnh sao nh ging Stella v vi sinh vt hnh vung nh ging Haloarcula, mt loi "vi khun" a mn thuc vi sinh vt c.

24

3. S cu to t bo vi khun Nh knh hin vi in t khoa hc bit rt r cc t chc di mc t bo ca vi khun nh lp mng nhy, thnh t bo, mng sinh cht, cht nguyn sinh v cc thnh phn quan trng khc.

Hnh 2.2: M hnh v s cu trc t bo vi khun

1. Thnh t bo, 2. Mng sinh cht, 3. Th nhn, 4. Mexosome, 5. Cht d tr, 6. Sinh cht, 7. Bo t, 8. Tin mao, 9. Khun mao, 10. Khun mao gii tnh, 11. Bao nhy, 12. Tng dch nhy, 13. Ribosome, 14. Th n nhp, 15. Plasmid. 3.1. Mng nhy (capsule) Nhiu vi khun c bao bc bn ngoi bng mt lp mng nhy c bn cht ha hc l polysaccharide ca mt loi gc ng (homopolysaccharide) hoc ca nhiu gc ng khc nhau (heteropolysaccharide), mt s vi khun trong v ny cn cha mt t lipoprotein. Khi lm kh, ngi ta xc nh c 90 - 98% trng lng ca mng nhy l nc. Mng nhy c tc dng hn ch kh nng thc bo, do tng cng c lc i vi vi khun gy bnh, do cu trc ha hc ca mng nhy l polysaccharide v t lipoprotein nn c lin quan n tnh khng nguyn ca vi khun gy bnh. Mt khc, khi mi trng ngho cht dinh dng mng nhy c th cung cp mt phn cc cht sng cho t bo, trong trng hp mng nhy teo i. Mt khun lc gm cc vi khun c mng nhy nhn thy dng nhn bng - dng S (smooth), cn khun lc gm cc vi khun t hay khng hnh thnh mng nhy s c dng nhn

25

nheo - dng R (rough), i vi nhng vi khun trong iu kin mi trng qu d tha carbon, nh rnh cc nh my ng, th mng nhy rt dy v to ra khun lc nhy nht - dng M (mucoid). V d nh my ng thng gp Leuconostoc mesenteroides c nhiu mng nhy dy gp 20 ln chiu ngang ca t bo vi khun. Nhng mng nhy nh vy c gi l nhng mng nhy ln (macrocapsule) cn nhng mng nhy nh hn 0,2m (ngha l khng nhn thy trn knh hin vi thng, m ch nhn thy trn TEM hay SEM) c gi l mng nhy nh (microcapsule). C nhng vi khun ch hnh thnh mng nhy trong cc iu kin nht nh, v d Bac. anthracis (vi khun gy bnh nhit than) ch hnh thnh mng nhy trong mi trng c protein ng vt, Diplococcus pneumoniae (gy bnh vim mng phi) ch hnh thnh mng nhy khi xm nhp vo c th ngi v ng vt. Mun quan st mng nhy ngi ta lm tiu bn m vi mc nho hoc nhum n bng thuc nhum kim. 3.2. Thnh t bo Ngy nay khoa hc bit kh r v thnh t bo cc c th nhn s. Da vo s nghin cu cu trc phn t ca thnh t bo, kiu trao i cht m ta c th xp chng vo hai gii: vi sinh vt c vi loi thnh t bo c bit v kiu trao i cht khc thng v vi khun vi ba nhnh tin ha: nhnh c thnh t bo dy l cc vi khun Gram dng, nhnh c thnh t bo mng l cc vi khun Gram m v nhm tiu gim, khng c hay thnh t bo rt mng. Nh cc phng php tch ly t bo nh lc dch huyn ph vi khun vi cc ht thy tinh c ng knh 0,1 mm hoc p dch huyn ph qua mng c l nh hn ng knh t bo, hoc a t bo vo mi trng u trng gy co nguyn sinh ri sau qua siu ly tm, ngi ta c th thu c lp thnh t bo kh tinh khit. Thnh t bo ca cc vi sinh vt c rt khc bit vi thnh t bo ca vi khun v hon ton khc vi c th nhn chun. i vi vi khun, thnh t bo chim khong 20 - 30% trng lng kh ca t bo, c bit Corynebacterium diphteria thnh t bo chim ti 76 - 78% trng lng kh ca t bo. Thnh t bo vi khun Gram dng dy 150 - 800A0 Cn vi khun Gram m lp thnh murein mng hn: t 50 - 180A0. Hp cht c bn ca thnh t bo vi khun l hai cht d cao phn t (heteropolyme): glucopeptid v acid teichoid. Glucopeptid (hay cn gi l peptidoglucan, mucopeptid, murein) l khung rn chc gi vng hnh dng vi khun, khi thy phn glucopeptid ta s oc 2 hp cht vi s phn t

26

Gram nh nhau l: N-acetyl glucosamin v acid N- acetyl muramic, chng lin kt vi nhau qua lin kt 1,4 glucozid. Enzyme lysozim ct lin kt glucozid gia C1 cn li ca acid Nacetyl muramic v C4 cn li ca N-acetyl glucosamin (lin kt 1,4). Cc gc N- acetyl muramic lin kt vi nhau qua dy ni peptid, to ra mng li chng cht nh t ong, trong thnh phn ca N- acetyl muramic c mt ca cc acid amin vi trng lng phn t Gram: 2 D.L.alanin, 1 D. glutamic v 1 acid diamin. Acid diamin ny Sta. aureus l lizin, E.coli l acid L diaminpimelic (ADP), cc acid diamin ny c th kt hp vi mch peptid ca chui bn, do m hnh thnh mt mng li murein chc chn. i vi nhm vi khun tiu gim thnh t bo (Mycoplasmatales, Mollicustes, Tenericutes) ngi ta khng tm thy hp cht murein, nhng vi khun ny khng c thnh rn chc cho nn chng c th thay i hnh dng, phn ln chng l nhng c th a hnh nh cc dng PPLO, Mycoplasma (vi khun nh nht c th nui cy trn mi trng). Trong thnh t bo vi khun cn c mt loi hp cht c bit l acid teichoic, hp cht thy nhiu vi khun Gram dng (c th chim ti 50% trng lng kh ca thnh), vi khun Gram m hin cha tm thy hp cht ny. Acid teichoic lin kt vi acid muramic qua mch phosphodieste. Acid teichoic c hai loi ribiteichoic v glycerin teichoic. Thnh t bo vi khun c chc nng quan trng l gi hnh dng n nh ca t bo , tham gia vo vic duy tr p sut thm thu, s phn bo, tham gia vo qu trnh nhum Gram...Vi khun Gr+ Vi khun Gr-

Hnh 2.3: M hnh cu trc thnh t bo vi khun Gram(+) v Gram(-)

27

3.3. Mng sinh cht Mng sinh cht ca vi khun v cc vi sinh vt c c th thy c nh co nguyn sinh, lp mng ny di knh hin vi i pha l lp c dy khong 7,5nm, nm ngay di lp thnh hoc cc lp mng ngoi, n bao bc ton b khi cht nguyn sinh. Khoa hc gi n l mng c s v c cu to ging hu ht cc mng trong t bo nh mng nhn, mng ty th, lc lp, mng li ni cht, mng bo tng Phn tch ha sinh cho thy mng sinh cht gm 3 loi phn t: lipid (ch yu dng phospholipid - chim khong 30 - 40%), protein (gm rt nhiu h enzyme, trong c cc h permease, thnh phn protein ny c th chim ti 60 - 70%) v mt t hp cht glucid.Cc phospholipid c th l phosphatidiglycerol v/hoc l phosphatidylethanolamin. Cc phospholipid di knh hin vi in t gm 2 lp nm gia, trong sut trong khi cc lp protein bn ngoi c mu m ti. Cc phn t phospholipid c u a nc (hydrophyle) gm c choline - phosphate v glycerol cn ui k nc (hydrophone) l cc phn t acid bo. Do cu trc cc phn t phospholipid nh trn nn chng phi sp xp cc ui k nc vi nhau v u a nc quay ra pha ngoi v pha trong sinh cht, chnh cch sp xp ny c li nht cho chc nng vn chuyn cc cht (a cc dng proton nh cc enzyme), chc nng h hp Chc nng ch yu ca mng sinh cht l tm bnh phong ngn tr dng cc cht ra cng nh cho i qua cc hp cht t pha ngoi vo. N m bo t bo hp th c cc cht dinh dng, cc nguyn t c li cho qu trnh trao i cht. N la chn cho i qua c cc phn t cht hu c loi nh ng thi ngn cn cc hp cht phn t ln. i vi cc phn t b k nc nh O2, N2, CH4, N2O, H2 hoc cc phn t c cc nhng khng tch in nh H2O, urea, CO2,mng sinh cht th hin nh mt mng vt l cho i qua theo quy lut vt l hc. i vi cc phn t c kch thc ln quan trng i vi t bo v a nc th mng cho i qua theo c ch khuch tn theo nng cht ha tan, c th l khuch tn th ng t nng cao n nng thp hoc khuch tn tch cc ch ng nh cc enzyme vn chuyn permease, nh m c th tp trung vo trong cc hp cht cn thit cho t bo.

28

C mt cch vn chuyn khc l do s thay i v tr cc nhm chc nng, v d khi phn t glucose i vo c phosphoryl ha v gii phng vo bn trong mng t bo l hp cht glucoso - phosphate. Mng sinh cht cha cc enzyme sinh tng hp kim sot cc khu kt thc tng hp lipid ca mng v cc hp cht kin to thnh t bo. Cui cng, mng sinh cht l ni nh v ca nhiu enzyme tham gia tng hp ATP. cc c th nhn chun, cc enzyme ny c mt trong ty th. Cc chui h hp ca mng sinh cht ca vi khun v vi sinh vt c lm chc nng tng t nh mng trong ca ty th. 3.4. Sinh cht v Ribosome Sinh cht ca c th nhn s gm c 80 - 90% l nc, nc c th trng thi t do (chim phn ln) lm nhim v ha tan cc cht v to nn dung dch keo vi cc cht cao phn t. Nc trng thi kt hp (chim phn nh) thng lin kt trong cc vi cu trc nh protein, lipid v hydratcarbon. Phn cn li ca sinh cht l lipoproteid (chim 10 20%). H keo ca sinh cht bao gm 2 pha: pha th nht l dung dch mui khong v cc hp cht ha tan c bn cht l lipoproteid, pha th 2 l pha huyn ph gm cc ht nucleoprotein, lipid v nhiu loi ht c kch thc rt khc nhau. Khi cn non, ang sinh trng, cht nguyn sinh c cu to ng nht v bt mu ging nhau. Khi trng thnh, trong cht nguyn sinh xut hin cc vt th n nhp, khng bo kh lm cho sinh cht c dng huyn ph ln nhn, bt mu khng ng u v c tnh chit quang khc nhau. Sinh cht ca vi khun c pH bnh thng l 7 - 7,2. nghin cu sinh cht, ngi ta dng siu li tm cao tc tch cht nguyn sinh v cc cu trc siu hin vi ring ra. * RNA v Ribosome Mt t bo vi khun cha trung bnh khong 18.000 ribosome, ht 70S (S l ch u ca Sverberg - 10-3 cm/giy trong siu li tm) vi ng knh t 10 - 30nm, trng lng phn t 3.106 daltons. Mi ribosome, khi gim nng Mg2+ ca dung dch s tch ra thnh 2 tiu phn trong siu li tm: tiu phn ln (50S) v tiu phn be (30S). tiu phn ln lin kt vi tiu phn b bng mi lin kt trung gian RNA - protein v protein protein. Cc ribosome cha phn ch yu l RNA (63%) v phn kia l protein (37%). Ngoi RNA v protein, ribosome c th cn cha mt lng nh lipid v enzyme ribonuclease, lepxinaminopeptidase, galactosidase v mt t cht khong giu Mg v ngho Ca.

29

S t hp ca tiu phn ln vo tiu phn nh bi lin kt bn trong ribosome, lin kt RNA - protein, y c 2 site (cht) c bit c vai tr quan trng trong qu trnh dch m (translation) t chui RNA sang protein. Cc site ny gi l site "P" (nh peptidyl) v "A" (nh aminoacyl). Ribosome l c quan tng hp protein ca t bo, nhng ch c mt s nh ribosome (khong 5 - 10% tng s ribosome) dng lin kt vi RNAm i hi phi c s tham gia ca nhiu yu t lm di chui polypeptide. Mt trong cc yu t l EF-2 thng thy cc vi sinh vt c v c th nhn chun, nhng khng thy vi khun, cc yu t ny thay i v tr phin m ca histidine (cho diphtamide) lm mn cm vi c t diphteria, y l mt s khc bit na gia cc vi sinh vt c v vi khun. 3.5. Cc ht d tr vi khun cui pha trng thnh, trong t bo vi khun xut hin nhng ht c ln v thnh phn ha hc khc nhau. Kch thc v s lng cc ht ny ty thuc vo loi vi khun v iu kin nui cy chng. Trong khi sinh trng, vi khun tch ly dn cc cht d tr hu c v v c, cc cht d tr ny t n kch thc nht nh th hnh thnh nn ht d tr (vt th n nhp) c th thy di knh hin vi. Cc cht d tr carbon o vi khun thng thy l glycogen, tinh bt, poly-- hydroxy butyrate Cc ht d tr carbon d dng nhn thy khi nhum bng dung dch c iod, hp cht ny nhum cc cht ang trung hp (polymer) khng phn nhnh ca glucose (tinh bt) thnh mu xanh thm v cc hydratcarbon phn nhnh (glucogen) thnh nu. Cc hp cht poly-hydroxy butyrate c nhum mu bi Soudan en nh mu cc git m. - Cc ht d nhim sc (metachomatic granulation) hay ht volutin c tm thy ln u xon khun Spirillum volutans. Chng bt mu vi thuc nhum kim nh xanh methylene, xanh toluidine thnh mu ta trong khi sinh cht ca vi khun li c mu xanh. Cc ht volutin c dng hnh trn, ng knh c th t ti 0,3m, c th tan trong nc nng 800C, trong dung dch kim hoc dung dch NaCl 5%, trong dung dch HCl 1Nkhng tan trong ru, ester, chlorofor. Ht volutin l nhng ht d tr polyphosphate v c. - Cc vi khun Chromatium, Beggiatoa c th tch ly lu hunh bn trong t bo do oxy ha H2S gii phng ra S. Cc vi khun oxy ha st c th tch ly st trong sinh cht di dng Fe3O4.

30

- Cc ht carboxysome thng thy Cyanobacteria, mt s vi khun mu ta, vi khun nitrat ha, vi khun oxy ha lu hung nh Thiobacillus, l nhng ht ribulose diphosphat carboxylase (hay carboxydis mutase) c bao bc bi s gp np ca mng sinh cht, c ng knh khong 50 - 100nm. - Tinh th dit cn trng thng thy mt s loi vi khun sinh bo t nh Bacillus thuringiensis, Bacillus dechrolimus, tinh th ny xut hin khi hnh thnh bo t, chng c bn cht l protein v ngy nay c dng dit su hi. 3.6. Cc th mang mu v sc t vi khun quang hp c quan thc hin qu trnh quang hp l cc th mang mu (chromatophores) ( to v thc vt l lc lp chlorophaste) v cu trc siu hin vi ca cc th mang mu khc vi lc lp. Mt khc bn cht ca cc sc t quang hp cng khc bit, to v thc vt l dip lc t (chlorophyll) trong khi vi khun quang hp l cc sc t gn vi chlorophyll m ngi ta gi l khun dip lc t (bacteriochlorophyll). Halobacterium halobium (c th sng trong mi trng mn) c cha bacteriorhodopsine mng sinh cht, sc t gn ging vi sc t vng mc mt. Ngoi ra, chng ta c th gp cc sc t sau: - Vitamine K2 (hp cht quinon) Bacillus subtilis v Bacillus cereus. - Sc t carotenoid chng tia t ngoi Corynebacterium. - Pyocyanine, violaceine Chromobacterium violaceum, sc t c hot tnh khng sinh. - Mt s sc t to thnh mu c trng ca khun lc nh zeaxanthine (carotenoid), sc t vng Staphylococcus aureus; xanthophyll v sarcinaxanthine (carotenoid) l loi sc t Sarcina. - Sc t pyocyanine xanh pyoverdine xanh lc hung quang P. aeruginosa. - Dn xut sc t pyrolic Serratia marcescens. 3.7. Khng bo kh y l mt loi ti cha y kh thng gp nhiu loi thuc 3 nhm vi khun quang hp: vi khun lam (Cyanobacteria), vi khun ta v vi khun lc. Khng bo kh gip vi khun quang hp tri l lng trong nc v ni ln mt nc.

31

3.8. Cht nhn ca vi khun DNA ca t bo vi khun chim khong 1 - 2% trng lng kh ca chng, l hp cht cha thng tin di truyn ch yu ca t bo. Cht nhn ca vi khun khng c mng bc, hnh dng rt khc nhau v ch c 1 si gm 2 mch DNA. Chiu di ca n trong t bo E.coli o c l 1mm, tc l gp 500 - 1000 ln chiu di ca vi khun. Vng th nhim sc c nh v ti mt im trn mng sinh cht lc sp phn chia. ln DNA vo khong 5.106 pb (cp base nit) vi trng lng phn t vo khong 3.109 dalton (4,5.108 i vi Mycoplasma, 1.109 i vi Acholeplasma). Khong thy protein histone kiu t bo nhn chun m ch c cc polyamine nh specmidin v specmine lm chc nng cng c n nh DNA. Tuy nhin, nhng nghin cu gn y cho thy Thermoplasma (mt loi vi sinh vt c - Archaea) tm thy histone. Vng DNA xon kp ca vi khun thng c gi l th nhim sc vi khun hay genophore. Nhng nghin cu trn knh hin vi in t cho thy genophore ca vi khun c nhiu vng, mi vng nh c khong 400 cp base nit, nhiu vng to thnh bi xon. E.coli c 50 bi xon v c gn 500 vng trong mt bi, iu gii thch ti sao chui DNA ca vi khun di ti 1mm v gm khong 5.106 pb. - E. coli tm thy t nht l 4 loi protein lin kt vi DNA, l nhng protein kim, bn nhit nn rt gn gi vi histone ca cc sinh vt nhn chun: + Hu c hnh thnh bi 2 tiu phn v (nh phn protein ProteinII) nn rt gn vi histone H2B. + H gn ging vi histone H2A. + H1 v HLP1, nhng protein lin kt DNA ny c chc nng iu ha khng c hiu trong qu trnh sao m. S c mt ca cc protein trn lm cho cu trc th cp DNA ca E. coli vng chc hn. Mc d ch c mt th nhim sc i vi mt t bo vi khun, nhng trong mi trng nui cy lin tc khi c s sinh sn nhanh, c th dn ti s c mt ca 2 thm ch 4 th nhim sc trong mt t bo vi khun ang phn chia. - Ba loi DNA polymerase c chit t E. coli: + DNA polymerase I: lm chc nng khi phc, sa cha b sung, xc tc b sung desoxyribonucleotide vo u ca chui DNA. Phn ng ch xy ra khi DNA c inh v mng sinh cht.

32

+ DNA polymerase II c hot tnh ngoi nhn, n cn thit cho hot ng ca RNAm. + DNA polymerase III l loi c hot tnh nht trong 3 loi polymerase. - Cc DNA-ligase xc tc hnh thnh cc u ni 2 mch DNA - cu phosphodiester gia nhm hydroxyl 3' u mch v nhm phosphate 5' u mch kia. DNA-ligase c th thc hin cc chc nng sau: + Sa cha, ct b mt on ca mch trong DNA xon kp. + ng v to thnh vng cc phn t DNA xon kp mch thng. + Hn cc on DNA trong qu trnh ti t hp di truyn. + Tham gia vo qu trnh nhn DNA cng vi DNA polymerase. Cc enzyme tho mch ca chui xon kp lm cho cc mch DNA trong chui xon kp c tch ra nhn ln. t hai mch lm khun tng hp hai mch mi b sung to thnh hai chui DNA xon kp hon ton ging nhau. y l c ch nhn i DNA bo m tnh di truyn ca cc th h vi khun. Cc thng tin di truyn t DNA c sao m (transcription) sang mRNA v sau c dch m (translation) thnh chui polypeptide hay enzyme. Bng phng php phng x t ghi (autoradiogragh) khoa hc c th chp c qu trnh nhn ln ca nhim sc th E.coli v nhiu c th nhn s khc. DNA ca c th c cu to c trng, s lng adenine bng s lng thymine v s lng guanine bng s lng cytosine. T l (G+X)/(A+T) l mt ch s sinh ha quan trng gip phn loi n ging v loi trong phn loi sinh ha (Chemotaxonomy), v d ch s Chargaff ca Micrococcus luteus l 4 v ca Clostridium perfringens l 0,34. 3.9. Plasmid Ngoi cc gen nm trong genophore ra, t bo vi khun (v mt s loi nm men) c th cha cc yu t di truyn ngoi th nhim sc, chng c th t nhn ln. V vy m nm 1952, Lederberg gi chng l plasmid ch tnh cht c lp ca chng vi cc gen nm trn th nhim sc. Khi nim vt cht di truyn ngoi th nhim sc bao gm DNA ca cc ty th, lc lp (nu c), cc plasmid, cc yu t gii tnh, cc yu t "dit", mt s prophage Khoa hc xc nh plasmid c rt nhiu loi vi khun nh E. coli, cc trc khun ng rut v vi khun Gram m, t cu khun

33

Staphylococcus aureus, x khun Streptomyces coelicolor v Rhizobium melitoli Plasmid l phn t DNA vng kn 2 mch, him thy 2 mch thng, nm ngoi th nhim sc, c kch thc nh (bng khong 1/100 th nhim sc ca vi khun, gn ging mt prophage), c kh nng t nhn ln c lp vi t bo v chng c phn sang cc t bo con khi nhn ln cng vi s nhn ln ca t bo. Cc plasmid c th tng ln hoc gim i khi c yu t bt li nh nhit , thuc mu, khng sinh, cc cht dinh dng Cc plasmid c th trng thi ci vo th nhim sc, c kh nng tip hp hoc khng tip hp, c th c mt hoc nhiu bn sao cng loi ngay trong mt t bo vi khun. Cc plasmid khng phi l yu t nht thit phi c i vi s sng t bo, nhng khi c mt, chng em li cho t bo nhiu c tnh chn lc qu gi nh c thm kh nng phn gii mt s hp cht, chng chu vi nhit bt li, chng chu vi cc khng sinh Thng th cc plasmid nhn ln khi t bo nhn ln hay t bo tip hp, nhng khng c ngha s nhn ln ca plasmid ph thuc vo s nhn ln ca t bo. trong trng hp cng nhn ln, chc phi c c ch kim sot m bo ng thi s nhn ln, s lng bn sao v s phn chia ng u cc bn sao cho hai t bo con, cho n nay c ch ny cn cha sng t. C nhng plasmid c th chuyn t t bo cho sang t bo nhn nh tip hp (tip hp c), ngc li c plasmid khng tip hp c. Cc plasmid qua t cu khun Staphylococcus ch c th chuyn sang t bo nhn nh ti np. Mt s plasmid (v d yu t gii tnh F) c th xm nhp vo th nhim sc v tr c bit c trt t nucleotide b tr vi on nucleotide trn plasmid, nhng site nh vy gi l nhng yu t gia nhp (IS), nhng IS ny gm khong 1000bp v thy E. coli nh IS 1 (768bp) v IS 2 (1327bp). Nh vy, cc yu t gia nhp (yu t in vo - IS) l on nucleotide nh (700 - 1500bp) thng ch m ha s chuyn v (transposition) (to ra cc site - t ct t trc ca DNA). Cc yu t di truyn vn ng (Transposon - Tn) l nhng on nucleotide di hn (vi ngn n vi vn bp) m ha kh nng thay i tnh cht (nh chng chu) thng nh v bn cnh IS. Cc gen vn ng ny c th di chuyn trong ni b mt th nhim sc hoc gia cc th nhim sc. Cc yu t Tn thy E.coli nh Tn5 (5700bp) chng chu Kanamycine, Tn681 (2100bp) sinh c t rut, Tn2571 (23000bp) chng chu Chloramphenicol, Tn3 (4597bp) chng chu Ampicillin. Tn s ca s chuyn v (Transposition) cng nh tn s ca cc t bin t pht ngu nhin (10-5 - 10-7).

34

3.10. Tin mao (Flagelles), tim mao (Cils) v nhung mao (Pili) Tin mao thng thy Vibrio, Spirillum v nhiu loi vi khun Gram m. S lng tin mao c th t 1 - 30 si ty thuc vo loi vi khun. Mt tin mao c chiu di t 6 - 30m v ng knh 10 30nm (12nm Proteus, 20 - 25nm Vibrio v Pseudomonas). Khi tin mao ngn th ngi ta thng gi l tim mao. Tin mao c cu to t mt loi protein gn vi Keratin m ngi ta gi l flagelline, protein c trng lng phn t khong 40.000 (trong c cc acid amin ch yu l arginine, lysine, acid aspartic, acid glutamic v tyrosine), nhng protein ny c tnh khng nguyn (H, khng nguyn ng nhit). Tin mao gip cho vi khun chuyn ng, khi vi khun di ng tin mao xoy vo nc hoc mi trng lng (c th ti 100 vng trong 1 giy), trong khi tim mao chuyn ng nh que gt. Mc d vy, trong vi sinh hc ngi ta thng dng tin mao v tim mao vi cng mt ngha, chng khc vi nhung mao (pili) l nhng si mnh v ngn hn nhiu, thng c xung quanh t bo Gram m v t thy t bo vi khun Gram dng. Ngi ta chia nhung mao (pili hoc fimbria) lm 2 loi: loi nhung mao ph thng (Type I) v loi nhung mao gii tnh (Type II). Loi nhung mao ph thng phn b vi s lng ln trn b mt t bo vi khun (vi trm), ngi ta cho rng loi nhung mao ny c lin quan n tnh cht kt dinh mu ca vi khun, loi nhung mao ny ngn hn nhung mao gii tnh (loi II c th di ti 10m), s lng nhung mao gii tnh rt t, khong 1 - 4, u cng c ch phnh ra, nhung mao loai II c vai tr quan trng trong qu trnh tip hp (conjugation) gia 2 t bo vi khun. Trong s cc si (tim mao v nhung mao) trn b mt t bo vi khun th tin mao c nghin cu k hn v ngi ta chia chng lm 2 nhm phn b: phn b cc (polaire) v phn b xung quanh (peritriche). S chuyn ng ca t bo vi khun c th t ti 10m/s (tc l trong 1 giy vi khun c th i c khong cch di gp 10 ln n) nn i hi mt nng lng rt ln (khong 2% nng lng trao i cht ca t bo). B my xoay tin mao gc nm trong mng sinh

35

cht v thnh t bo gm 2 vnh khuyn, vnh trong gm 16 n phn protein c th dch chuyn rt nhanh nh dng proton (H+) i vo. Cch sp xp tin mao l mt tiu chun trong phn loi hnh thi (Morphology taxonomy) vi khun: - n mao cc (Monotriche polaire hay parapolaire) c trng cho nhiu loi Vibrio hoc chm mao cc (Lophotriche) c trung cho nhiu loi Pseudomonas, Chromatium v Thiospirillum. - Lng cc (Amphitriche hay Cephalotriche) c th chm mao lng cc nh cc loi Spirillum. - Chu mao (Peritriche) hay tin mao sp xp xung quanh thn t bo vi khun c trng cho nhiu loi Prote 3.11. Ni bo t vi khun (Endospores) Mt s vi khun cui giai on sinh trng, khi cht dinh dng mi trng cn kit v cht qua trao i c hi qu nhiu, hoc do c s thay i t ngt cc iu kin sinh trng c kh nng hnh thnh bo t bn trong t bo, c gi l ni bo t (endospores). Mi t bo vi khun ch to mt ni bo t nn loi bo t ny khng phi l bo t sinh sn, khc vi cc loi nh bo t (conidiospores), bo t ti (ascospores) hay bo t m (basidiospores) nhiu loi nm, chng l nhng bo t sinh sn v tnh hay hu tnh. Nhng vi khun c kh nng hnh thnh ni bo t gm nhiu loi thuc cc ging Bacillus, Clostridium, Desulfotomaculum, Sporosarcina, Sporolactobacillus, Thermoactinomyces. V bo t c trng bng s c mt ca acid dipicolinic (di dng dipicolinate calcium), hp cht ny c th chim ti 10 - 15% trng lng kh ca bo t. Vai tr ca hp cht dipicolinate calcium lm cho bo t chng chu c nhit cao, khi th nghim ngi ta thay calcium bng strontium th kh nng chu nhit ca bo t thu c km i. Mt hp cht khc ca v bo t mi tm thy l acid L.N-succinylglutamic khng c trong t bo sinh dng v ch c tng hp khi hnh thnh bo t, ngay giai on u khi hnh thnh vch ngn DNA mi vi mt t nguyn sinh cht, hp cht ny gip cho bo t bn nhit. Khi hnh thnh bo t, t bo c th mt i n 70% nc. Mt s vi khun khi hnh thnh bo t c th tn ti rt lu trong t nhin, v d Bac. anthracis c th sng tim sinh trong nhiu nm. Trong mt s cht c t bo vi khun b cht rt nhanh nhng bo t c th tn ti c kh lu, v d trong phenol c th vn sng trong 15 ngy, trong HgCl2 1% tn ti

36

trong 2 ngy. Thi gian hnh thnh bo t khc nhau ty loi (t vi gi n 20 gi). Khi a bo t vo mi trng thun li pht trin, bo t s c hng lot thay i tch cc v mc mm, qu trnh mc mm gm 3 giai on ch yu: hot ha, nt v v mc ra. mun quan st c bo t ngi ta dng phng php nhum n hoc kp. 4. X khun v vi khun ni bo Nhng vi khun thuc b Actinomycetales (nhm bc thp v bc cao) l nhng vi khun c hnh si Gram dng, si c th phn nhnh, thng gp chng trong t v nc. Nhng x khun rt gn vi nm v hnh dng h si pht trin trong c cht nh b "r" hp th cht dinh dng, h si mt c cht to nn khung ca khun lc v h si kh sinh tn cng bng nhng cung mang bo t. l nhng loi thuc ging Streptomyces, mt ging khai thc c khong 50% s khng sinh hin bit, ng knh si khong 1- 2m (nh hn nhiu so vi ng knh si nm 7 - 9m). X khun cng ging nh vi khun l cha c nhn c bao bc bng mng, thnh t bo x khun khong cha cellulose hay chitine m cha hp cht in hnh ca vi khun l glucopeptide. X khun cng c qu trnh sinh sn phn bo theo kiu amitose (phn bo khng t), loi vi khun ny cng cha c gii tnh, ngha l cha c s phn ha thnh t bo c, ci v c th hnh thnh hp t tng phn nh tip hp, ti np v bin np, x khun cng b phage tn cng v gi l Actinophage. Streptomyces l mt ging x khun bc cao c Waksman v Henrici t tn nm 1943. Chng c v tr tin ha cao trong s cc ging thuc vi khun Gram dng, c tr s % mol Guanine v Xytozin rt cao (69 - 77% (G + X) trong DNA). X khun sinh sn v tnh bng bo t. Trn u si kh sinh ca Streptomyces hnh thnh cung bo t v chui bo t. Si mang bo t c nhng hnh dng khc nhau ty theo loi: thng-ln sng (Retiflexibilis), xon (Spirales) hoc c mc, vng (Retinaculiaperti) on tn cng ca cung mang bo t bng phng php ct khc (segmentation) hay kt on (fragmentation) m hnh thnh nn chui bo t. Bo t x khun (Arthrospores bo t t) hnh bu dc, hnh lng tr hoc hnh cu vi ng knh khong 1,5m. Mng bo t x khun c th nhn (smooth), gai (spiny), tc (hairy), khi u (warty), np nhn (rugose) ty thuc vo loi x khun. Nhng tiu tit ny c th thay i theo quy lut trn mi trng nui cy, thng th x khun c tiu tit

37

c th c mng nhn trn mi trng c ngun nit hu c, trn mi trng c m v c v glucose cc bo t d biu hin cc tiu tit r nht. Mu sc ca khun lc v h si kh sinh cng rt khc nhau ty theo nhm Streptomyces, mu sc ny cng c th bin i khi nui cy trn mi trng khc nhau, chnh v vy m y ban quc t v phn loi x khun (ISP) nu ra cc mi trng chun v phng php chung phn loi nhm vi sinh vt ny. Hin nay khoa hc m t c khong 600 loi x khun bc cao ny. - Rickettsia l mt ging ca Rickettsiaceae, Rickettsiales, y l vi khun k sinh ni bo, c 3 nhm (Tribus): Rickettsiae, Ehrlichiae v Wolbachiae vi 2 ging ch yu l Rickettsia v Coxiella, k sinh ng vt c xng. Nhng vi khun ny cng c 2 loi acid nucleic, thnh t bo cng c hp cht c trng ca thnh t bo vi khun, sinh sn bng cch chia i khi trong t bo ng vt. - Chlamydia l mt ging ca Chlamydiaceae, thuc b Chlamydiales, l nhng vi khun k sinh bt buc, Gram m v c nhiu im ging Rickettsia, trong ng vt c xng sng chng khng to ra ATP ring m s dng ATP ca vt ch, kch thc ca chng rt nh (0,3 - 0,45m) v th trong mt thi gian di trc y ngi ta coi chng l mt loi virus (virus kim tnh Van Royen). - Mycoplasma thuc h Mycoplasmaceae, h c nht ca b Mycoplasmales (i khi l Mycoplasmatales - Freundt, 1955). Chng l nhng vi khun khng c thnh t bo, thn mm d bin hnh, bt ng. Trong b Mycoplasmales c cc dng vi khun Mycoplasma dng L v PPLO. y l nhng vi khun thuc Tenerecutes, lp Mollicutes, l lp vi khun tiu gim thnh, vi khun c kch thc nh nht khoa hc hin bit (0,1 - 0,3m) v vy chng d qua mng lc vi khun, chng l tc nhn gy cc bnh h hp, tit niu - sinh dc. 5. Vi sinh vt c (Archaea) Cc vi sinh vt c m trc y gi l cc vi khun c (Archaeobacteria) l nhm c th nhn s c sm nht (khong 4 t nm trc y), c Woese R. v Woese C.R. tch ra thnh mt nhnh tin ha ring. Nhng c th cn li hin nay thng sng trong cc iu kin khc thng. Nhng nghin cu gn y v nhit v pH ti u ca vi khun a nhit v Archaea cho php chia vi sinh vt c thnh 2 nhm: Crenarchaeota v Euryarchaeota. Nhm Crenarchaeota l nhng vi sinh vt c k kh bt buc, a nhit v a acid (Thermoacidophiles, Thermophiles anaerobies stricts).

38

Nhm Euryarchaeota l nhng vi sinh vt c a mn, sinh methane (methanogenes) v mt vi loi k kh a nhit.Bng 2.3: So snh mt s tnh cht gia vi khun v vi sinh vt c Tnh cht Bacteria Archaea Thnh phn thnh t bo Murein Pseudomurein, protein, polysaccharide Lipid ca mng Glycerol, acid Glycerol, ete bo, aster isopranyl Hnh dng t bo c loi + (hoc khng) hnh vung v dt Ni bo t (endospore) + (hoc khng) Pseudouridine hoc Cht " nhnh" ca RNAt Ribothymidine L.methylpseudouridine Hnh thnh cht kch thch + methyonyl RNAt Cc intron trong gen + C Polymerase - RNA loi + nhn chun C coenzyme c bit + Nhit sinh trng ti a 900C 1100C C quang hp phc tp C th C th sinh methane + Chu trnh Calvin c s C th dng khi c nh CO2

Nhng vi sinh vt c (s liu ch yu da trn methanogenes) l nhng n bo nh khong 1m c thnh t bo gm nhng hp cht c bit (thay v murein y c acid talosaminuronic, ester ca acid bo mch khng thng v glycerol, s phn nhnh ca chui dang ether (-O-) m khng phi dng ester (-CO-O-) (lipide ether-phytanyl), c nhiu lin kt mui v hydro trong protein, c pseudouracil v tr uracil trong RNAt, khng c dihydrouracil, thymine trong vng bn ca RNAt, cht nhn phn tn trong sinh cht vi lng nh (ch bng khong 1/3 so vi cht nhn ca E.coli). DNA ca chng giu hm lng G + X (mi lin kt 3 hydrrogen), ribosome loi c th nhn s, c intron, c coenzyme c bit (M), hu nh khng c s n nh v quinon v cytochrome. Chnh nhng c im gip cho vi sinh vt c sng trong nhng iu kin khc thng: nhit cao (c loi sng c 2500C) di 265 atm, chng chu vi lysozyme C 3 nhm sinh l v sinh thi quan trng l: * Cc c th sinh methane (methanogenes), y c l l nhnh c xa nht, cc lp nc su, y, k kh, mt s loi tm thy trong

39

ng tiu ha ca ng vt nhai li. Phn loi c th sinh methane da ch yu vo kh nng v c ch sinh methane ca chng, gm c 5 b: - Methanobacteriales, c cc loi: Methanobacterium ivanovii, M. thermoautotrophicum, M. smithii, M. fervidus, M. sociabilis. - Methanococcales, c cc loi Methanococcus voltae, M. vannielii, M. thermolithotrophicus. - Methanomicrobiales, c loi Methanomicrococcus sp. - Methanosarcinales, c cc loi Methanosarcina mazeii, M. barkeri, M. thermophila, M. acetivorans, Methanothrix sp. - Methanopyrales c loi Methanopyrus sp. Nhng c th sinh methane c th thc hin nhng c ch khc nhau ty theo loi (hu ht l c th k kh bt buc): CO2 + 4H2 CH4 + 2H2O (G10 = -135,4 KJ/mol) CH3COOH CH4 + CO2 (G10 = -32,5 KJ/mol) HCOOH + 3H2 CH4 + 2H2O CH3OH + H2 CH4 + H2O (G10 = -112,5 KJ/mol) CH3NH2 + H2 CH4 + NH3 (CH3)2S + 2H2 2CH4 + H2S Ngi ta cho rng nhng c th sinh methane cng vi c th dinh dng methane (methanotrophes) lm tun hon nguyn t carbon trong thi k kh quyn khng c hoc rt t oxy. S bin i t CO2 thnh CH4 c bit vi sinh vt c c 4 giai on: + 2H 2H+ 2H+ 2H+ 2e2e2e2eCO2 HCOOH HCHO CH3OH CH4 acid formic H2O formandehyde methanol H2 O Trong khi , qu trnh oxy ha methane c th dinh dng methane (methanotrophes) c thc hin theo c ch nh sau: CO2 CH4 2H+ H CH3OH PQQ HCHO HCOOH CO2

PQQH2 NAD+ NAD+H+ NAD+ NAD+H+2eC th ng ha

40

* Cc c th a mn (halophiles) nh Haloarcula, Halobacterium. Nhng c th ny sng trong mi trng c nng mui cao ( bin, cc m mui), hp thu cht dinh dng ch yu l do chnh lch gradient nng mui to ra, qu trnh quang hp y kh c bieet nh Bacteriorhodopsine, hp cht lin kt trong mng sinh cht ch khng phi l khun dip lc t (bacteriochlorophyll). * Cc c th a nhit, a acid (Thermoacidophiles), l nhng c th sng ngun t - nc nng, vng ni la, chng l nhng c th hiu kh hoc hiu k kh.V d: Sulfolobus acidocaldaricus c nhit sinh trng ti a l 900C v ti u l 750C, pH ti u l 2,5. Thermoplasma acidophilum c nhit sinh trng ti a l 650C v ti u l 600C, pH ti u l 1,5. 6. Nguyn tc phn loi c th nhn s C th nhn s ngy cng c pht hin nhiu hn, v vy nhn bit v s dng chng ta cn phi t tn cho n v phi phn loi chng. Taxonomy l khoa hc nghin cu s a dng ca vi sinh vt v mi lin h vn c tn ti gia chng. Khoa hc nh loi ny bao gm 3 lnh vc khc nhau: Classification (xp loi), Nomenclature (t tn) v Identification (nhn bit).Bng 2.4. Bng nh loi cc nhm ln c th nhn s (Da ch yu vo Gergey's Manual, 1984, tn siu gii c xut) Tn nh loi Nhm c th + Siu gii nhn s (Super C th nhn cha c mng kingdom procaryote) A. Gii vi khun (Kingdom C th c thnh t bo vi hp cht murein Bacteria) I. Ngnh I: Gracilicutes C th nhn s c thnh c trng vi khun Gram m Lp 1: Scotobacteria 1. Vi khun ha dng v c (Chimiolithotrophes) 2. Myxobacteria 3. Vi khun c bao (Trichome). 4. Vi khun dnh kt v vi khun c chi 5. Spirochaete 6. Vi khun Gram m, hiu kh v vi hiu kh 7. Vi khun Gram m, k kh khng bt buc

41

8. Vi khun Gram m, k kh bt buc 9. Rickettsia v Chlamydia Lp 2: Vi khun quang hp 10. Vi khun quang dng khng thi oxy (Onoxyphotobacteria) Lp 3: Vi khun quang hp thi 11. Cyanobacteria oxy (Oxyphotobacteria) II. Ngnh II: Firmicute C th nhn s c thnh vi khun gram dng Lp 1: Fimibacteria 12. Vi khun Gram+, khng sinh bo t 13. Vi khun Gram+, sinh bo t Lp 2: Thallobacteria 14. Vi khun tia v ch (Actinomycetales) (x khun) III. Ngnh III: Tenericutes 15. Mycoplasmes B. Gii sinh vt c (Kingdom Archaea) Mendosicutes Lp 1: Archaea C th nhn s khng c thnh, hoc c thnh vi hp cht c trng Pseudomurein, kiu sinh l rt c bit. tng chim a s trn hnh tinh cch y 3 t nm v trc, hin nay ch cn li vi nhm: Methanogens, Halophiles v Thermoacidophiles

S xp loi (Classification) thit lp cc nhm nh loi (cc taxon) cn c vo nhng tieu chun hnh thi v phn t cu to t bo. S t tn (Nomenclature) l a ra mt tn cho mt nhm, mt c th theo nguyn tc ca Linaeus gm 2 t, mt t latin ch ging v t k tip ch loi. V d tn ca trc khun c kh l Bacillus subtilis. S nhn bit (Identification) ch nh cc loi nghin cu vo mt trong cc loi (cc taxon) m t. Phn loi hc vi khun ngy nay s dung phng php nh loi hnh thi (Taxonomy phenetic), hay s dng phng php nh loi s (Taxonomy numeric), phng php ny s dng mt lng ln cc tnh trng v so snh cc tnh cht rt ra s ging nhau gia hai c th, trong ch s Jaccard l d hiu v thng dng nht trong nghin cu i cng. SAB = h s ging nhau gia A v B nS+ SAB = nS+ = s cc c im ging nhau nS+ + nd nd = s cc c im khc nhau.

42

Vi khun hc cn s dng phng php nh loi di truyn (Taxonomy genetic), 3 tiu chun c s dng y l ch s (G + X)% (h s Chargaff), t s lai DNA - DNA, s lng v trt t cc nucleotide trong 16sRNAt v 5sRNAt. ch s lai DNA - DNA l mt tiu chun bt buc phn tch v tra cc loi vi khun, cho n nay cc loi ca h vi khun ng rut Enterobacteriaceae cho lai vi nhau v thit lp c mi lin h chng loi pht sinh gia chng. Theo Woese (1981) trt t nucleotide trong 16sRNAt l mt ch s quan trng xc nh s gn gi ca hp cht protein v t quy nh mi quan h chng loi pht sinh gia cc nhm vi khun. Chnh nh phn tch 16sRNAt m hnh thnh mt hng nh loi mi - nh loi chng loi pht sinh (polygenetic taxonomy), nh c th tch cc vi sinh vt c ra khi vi khun v chng minh n l nhnh tin hoa c lp khc xa x khun. Cc phng php nh loi sinh ha (chimiotaxonomy) ngy nay c s dng rng ri, nh cc hp cht polymer ca thnh t bo vi khun (bao gm vi khun Gram dng, Gram m v Cyanobacteria) l peptidogulcan (murein), hp cht c trng ny khng thy nhm vi khun tiu gim thnh Mycoplasmes v cc vi sinh vt c. Cc vi sinh vt c c hp cht thnh c trng l pseudomurein, trong acid muramic c thay bng acid talosaminuromic. Cc mc n v phn loi dng trong vi sinh vt hc l: Clone biotype species genus familia order classe division kingdom - Dng t bo (clone): l mt qun th vi khun (hay vi sinh vt) c c t mt t bo b m ban u nh s phn chia v tnh. - Dng (biotype): mt nhm c th c chung c im di truyn tuyn chn v nhng im ch yu, dng c c t tuyn chn cc dng, y l n v hay dng trong cng ngh vi sinh. - Chng (ni - souche): l t thng dng trong k thut thc hnh, l taaoj hp nhng c th c c do s cy chuyn t mt khun lc vi khun. - Loi (species): tp hp nhng c th ging nhau v hnh dng, cch sng, c th sinh sn bnh thng gia chng v thng khng c kh nng ny vi cc c th thuc loi khc. - Ging (genus): tp hp cc vi khun trong bng xp loi di h, trn loi, tp hp cc loi rt gn nhau. - H (familia): tp hp cc ging c mt s c im chung.

43

- B (order): mt nhnh trong bng xp loi vi khun, bao gm mt s h gn nhau nm di lp. - Lp (class): mt nhnh trong bng xp loi vi khun, bao gm vi khun trong mt s b gn nhau. - Ngnh (division): tp hp cc vi khun mt s lp c chung mt s c im c bn. II. Vi sinh vt nhn chun. 1. Vi nm Cc c th nm vi cu to thnh t bo, kiu trao i cht v h enzyme khc bit vi cc c th nhn chun khc (thc vt, ng vt) v khc xa vi c th nhn s nn t lu c xp thnh mt gii ring l gii nm (Kingdom Fungi). C 3 danh t thng dng ch nm l: nm men (Levures, Yeasts), nm mc (Moisissures) v nm ln hay nm c qu th. Trong vi sinh vt hc, vi nm c hiu l cc nm c c th hin vi, bao gm hai nhm: - Nm n bo hay nm men. - Nm si hay nm mc. 1.1. Nm men (Levures, Yeasts) Nm men l nhm nm c th n bo, nhn chun, hin vi. Nm men c th thuc v 3 lp nm l nm ti (Ascomycetes), nm m (Basidiomycetes) v nm bt ton (Deuteromycetes). Trong s 75.000 loi nm hin bit th c hn 500 loi nm men thuc khong 50 ging. Nm men c hnh trng, qu da chut ng ring l hoc tp hp thnh si d gy, kch thc t bo t 7 - 10m. Trn mi trng sng, c nhng loi nm men sinh cc sc t c trng nh , vng Nm men sinh sn v tnh bng m chi hoc phn chi, gia qu trnh ny c th sinh sn hu tnh. Nm men c 3 dng chu trnh sinh hc: - Chu trnh n bi - lng bi nh loi Saccharomyces cerevisiae. - Chu trnh u th lng bi nh loi Saccharomycodes ludgyzii. - Chu trnh u th n bi nh loi Schizosaccharomyces octosporus. Nh knh hin vi in t khoa hc thy c s khc nhau v thi gian hnh thnh thoi v sc trong sinh sn v tnh nm men phn i v nm men m chi.

44

Nm men nhn i th trong chu k phn bo cng c cc giai on G1, S, G2 v M nh sinh vt nhn chun. Trong khi mng nhn cha c pht tn, cc thoi vo sc ca mitose c hnh thnh trong nhn v th nhim sc c a v hai cc, sau mi hnh thnh vch ngn to thnh hai t bo ring. nm men m chi c cc pha G1 v S bnh thng, nhng thoi v sc y c hnh thnh rt sm ngay cui pha S lm cho pha G2 khng bnh thng (ngn li) v trong khi cha hnh thnh hai nhn thnh t bo bt u gp li. C ch tch cc th nhim sc phn bo c t ca nm men l nh cc thoi v sc m cc th nhim sc trt v hai u c din ra trong nhn. Trong khi phn bo v t ca cc vi khun, cc th nhim sc con c hnh thnh vn nh ln mng sinh cht v ch c tch ra nh s pht trin v phn vch ca mng. So snh cu to t bo nm men v t bo vi khun ta thy c s tin ha nhy vt t c th nhn s chuyn thnh c th nhn chun. Cng vi s tin ha v nhn v c ch phn chia nhn (nhn c mng, c cc th nhim sc, phn bo c t) c th nhn chun xut hin nhiu c quan t khng thy cc c th nhn s nh ty th, lc lp ( c th nhn chun quang hp) Nghin cu cc c im ca c quan t ta thy rt nhiu im ging c th nhn s nh DNA mch vng (DNA nhn mch thng), khng c protein histone (th nhim sc trong nhn c histone), ribosome 70 S (trong t bo l 80S) v phn bo trc phn trong khi t bo phn bo gin phn. Mt khc, khoa hc cn tm thy mt t loi ng vt nguyn sinh nh Microsporidia v Giardia l nhng n bo nhn chun k kh cn li cho n nay, khng c ty th, nhng c th ny i din cho mt nhnh tin ha tiu gim khng ty th ca cc c th nhn chun, k kh so vi nhnh tin ha pht trin ca cc c th nhn chun hiu kh. Con ng tin ha hnh thnh ty th trong cc c th nhn chun hiu kh c thc y nhanh hn khi hm lng oxy trong kh quyn tng ln. Ngi ta cho rng mt con ng tin ha hnh thnh ty th trong t bo nhn chun l do s tip xc ca cc vi khun ni cng sinh (endosymbiotic bacteria), nhng t bo nhn chun mang trong mnh vi khun hiu kh ni cng sinh, sau nhng thi k ni cng sinh ko di, c s chuyn gen t vi khun vo nhn v ch cn li mt s gen t tr trong ty th, l t bo nhn chun hiu kh mang ty th nh ngy nay. Ngoi nhng cu to c bit ca nm men so vi vi khun, thnh t bo ca nm cng l mt nt rt c trng. Thnh t bo nm c n 80%

45

l polysaccharide mang tnh khng nguyn, trong cc hp cht mannan, glucan v chitine l rt quan trng, phn cn li l protein c th n 10 20%, trong gn mt na l mannoprotein, chng to thnh 3 lp ca thnh t bo nm men. Cc polymer ca thnh t bo c thay i cht t ph thuc vo tng nhm nm.Bng 2.5: Bng phn nhm n gin cc nm menLp (Classes) B H Saccharomycetaceae H ph Schizosaccharum vcetoideae Nadsonioideae Lipomycetoideae Saccharomycetoideae Ging Schizosaccharomyces Hanseniaspora Lipomyces Debaryomyces Hansenula Kluyveromyces Pichia Sacharomyces Coccidiascus Filobasidium Leucospiridium Sirobasidium TremallaCryptococcoideae

Nm ti (Ascomycetes)

Endomycetales

Spermophthoraceae Filobasidiaceae Leuvures Sirobasidiaceae Tremellaceae Cryptococcaceae

Nm bt ton Nm m (Deuteromy-cetes) (Basidiomycetes)

Ustilagi -nales Tremellales

RhodotoruloideaeTrichospororoideae

Sporobolomycetaceae

Brettanomyces Candida Cryptococcus Torulopsis Rhodotorula Trichosporon Sporobolomyces

Nm men chim mt v tr c bit trong cng nghip thc phm: lm n bt m, nu ru, lm ru vang, lm pho mt, sn xut sinh khi ch protein (trong nm men c th cha n 40% m ca trng lng kh). Ring sn xut bnh m hng nm th gii tiu th 1,7 triu tn nm men bnh m.

Blastomycetales

46

1.2. Nm mc Nm mc l loi nm si in hnh. Cng nh nm men, nm si l nhng c th d dng, mt s sng cng sinh vi thc vt, khi cng sinh vi to n bo hoc tp hp n bo th hnh thnh a y (Lichens). Mt s nm k sinh trn ng vt v thc vt gy nn cc bnh nm rt kh cha. Nhiu nm sng hoi sinh s dng rc thi hu c ng vt v thc vt hoc ph hoi thc n, vt dng hng ngy. Chng thng c nhng enzyme phn gii rt mnh nh h enzyme phn gii cellulose, phn gii pectin, cc enzyme amylase, protease, lipase Con ngi t lu bit s dng mt c li ca nm mc trong vic ch tng, nc chm, sn xut khng sinh, to cc enzymeBng 2.6: Bng phn loi n gin mt s ging nm mcNgnh Amastigomycota Ngnh ph Zygomycotina (Zygomycetes) Ascomycotina Lp Zygomycetes Plectomycetes Pyenomycetes Hemiascomycetes Basidiomycotina (Basidiomycetes) Hemibasidiomycetes B Mucorales Eurotiales Spahaeriales Endomycetales (nm men) Urenidales Ustilaginales Ging Mucor Rhizopus Emericella (A. nidulans) Neurospora (N. grassa) Eremothecium Puccinia Ustilago Candida Geotrichum Aspergillus (A. flavus, A. niger) Penicillium P. votatum, P. camenbertii, P. roquefortii)

Deuteromycotina (Deuteromycetes)

Hyphomycetes (dng nm men = Blastomycetes) Hyphomycetes

Moniliales

Si nm c th c vch ngn nh cc lp nm bc cao (Ascomycetes, Basidiomycetes, Deuteromycetes) hoc hnh ng trong si c nhiu nhn m ngi ta gi l si cng bo (coenocytis). Nhng loi nm si khng vch ngn thuc v cc nm bc thp nh Oomycetes

47

v Zygomycetes. Cc vch ngn khng ngn cch hon ton gia cc t bo ca si m chng thng lin h vi nhau qua l vch. Mt bo t khi ri vo mi trng thun li s ny mm v to thnh khun lc gm h si pht trin su vo c cht ht thc n (si c cht - SM), si c cht to thnh khung ca khun lc v si kh sinh mang cc cung bo t (si kh sinh - AM) Cc nm bc thp c th sinh ra cc ng bo t mt roi (chytridiomycetes) hoc hai roi (Oomycetes) trong chu trnh sinh sn ca mnh. Cc nm bc cao nh loi Aspergillus v Penicillium c th hnh thnh cu tip hp gia hai t bo ca hai si (+ v -), l hin tng sinh sn cn tnh. Cc cung mang bo t pht trin t mt loi si kh sinh, c th phn nhnh hoc khng, trn u cung bo t c th hnh thnh ti mang bo t (Mucor, Rhizopus) vi cc bo t ti sinh sn v tnh hoc trn u cung bo t bng phng php m chi m sinh ra cc bo t nh (conidie hay conidiospore), cc cung sinh bo t c th tp hp li thnh thng hay khoang nh bo t (pycnide). i khi cc bo t c hnh thnh bng cch phn t ca si (Geo trichum) m ngi ta gi l bo t t (athrospore). Cc bo t hu tnh c hnh thnh nh qu trnh hu tnh kt hp cc t bo c v ci (cc giao t) hoc cc si khc gii tnh, hoc do s hp nht hai nhn trong si cng bo (coenocytis) hnh thnh hp t, sau nh gim nhim m hnh thnh cc ti bo t vi cc bo t ti. cc nm m (Basidiomycetes) qu trnh hnh thnh m v cc bo t m l giai on cui cng, qu th nhn thy c bng mt thng, trong khi phn ln chu trnh pht trin dng si mc. cc loi nm m qu trnh hp nhn xy ra mun hn so vi qu trnh hp cht nguyn sinh. Giai on si lng nhn (mt t bo c 2 nhn) tn ti kh lu, ch giai on hnh thnh m mi c t bo c nhn l 2n. * Tng qut v nm:Bng 2.7: Cc lp nm thng gpLp nm Oomycetes Loi si khng c vch ngn (coenocytic) Bo t v tnh bo t chuyn ng Bo t hu tnh Oospora Ni chnh sng V d Allomyces

thy sinh, nhiu loi gy bnh cho c, mc sng khoai ty t, phn gii cht hu c thc vt

Zygomycetes

coenocytic

bo t ti, i khi bo t nh (conidia)

Zygospora (bo t tip hp)

Mucor, Rhizopus

48

Ascomycetes

c vch ngn, mt s n bo c vch ngn, mt s n bo c vch ngn, mt s n bo

nh bo t (m chi)

bo t ti (ascospore)

t, phn gii cht hu c thc vt t, phn gii cht hu c thc vt t, thc vt v trn c th ng vt

Neurospora, Saccharomyces, Morchella Agaricus, Amanita Candida, Trychophyton, Epidermophyton

Basidiomycetes

Uredospora, (m chi)

conidia

bo t m (basidiospore) cha thy

Deuteromycetes

bo t nh, bo t t (arthrospora)

Bng 2.8: Cc kiu bo t nmV d ging nm Saprolegnia Aspergillus, Penicillium Mucor, Rhizopus Coccidioides Candida Kiu bo t Bo t v tnh (ng bo t) Zoospores) nh bo t (Conidiospore) Tnh cht Bo t n, c roi, chuyn ng Bo t n hoc tp hp thnh chui c hnh thnh trn cung bo t (Conidiospore) Bo t c hnh thnh trong ti (bo t v tnh) Bo t c hnh thnh bng cch chia t cc si kh sinh Thnh dy, bo t n, c hnh thnh bng phn i hay chi ging nm men c hnh thnh trong ti (bo t hu tnh), thng 4 - 8 bo t trong mt ti Pht trin tn cng ca m (thng 4 bo t) Bo t ln c hnh thnh trong mt thnh dy (bo t hu tnh) Bo t pht trin trong Oogonium (nguyn bo trng)

Bo t ti v tnh (Sporangiospores) Bo t t (Arthrospores) Bo t dy (Chlamydospore), bo t mm (Blastospore) Bo t ti

Saccharomyces, Neurospora Agaricus Rhizopus Saprolegnia

Bo t m (Basidiospores) Bo t tip hp (Zygospores) Bo t non (Oospore)

Cc loi nm m n c nh cc ging nm m (Agaricus bisporus), nm rm (Volvariella volvacea), mc nh (Auricularia), nm hng (Lentinus), nhn nh (Tremella)ang tr thnh i tng ch yu trong cng ngh nui trng nm n. Phn loi nm mc ch yu da vo cc tnh trng hnh thi: cu to si mang bo t, cu to bo t v mt s tnh trng sinh l sinh ha.

49

2. Vi to Vi to l to hin vi c sc t quang hp. Vi to n gin nht l c th n bo, hoc tp hp n bo, c th c roi nh Clamydomonas, Peridium v Euglena (to mt), hoc khng c roi nh Chlorella (to lc), Diatomia (to ct). Cc vi to thng gp hn l cc c th a bo hoc tp hp n bo, nh cc tp on Volvox, Pediastrum, Scenendesmus (thuc nhm Archethalle) hoc phc tp hn c b phn nh bm v b phn dng ng nh cc si mnh phn nhnh hoc khng (c th c vch ngn to thnh cc t bo tng i c lp hoc khng c vch ngn nh mt ng cng bo (coenocytic). Nhng to ny sinh sn bng cch phn chia nhng t bo l gia hoc bng cch rng t bo u cng (Sphacelaria, Ectocarpus), chng sinh sn hu tnh bng tip hp ng giao (hai giao t bng nhau) hoc d giao (hai giao t khc nhau). Cc sc t quang hp v h tr cc nhm to khc nhau th khc nhau, hin nay ngi ta bit 6 nhm to vi cc sc t c nghin cu tng i k. Bn ging to lc l Clamydomonas (n bo 2 roi), Gonium (tp hp n bo 2 roi), Pandorina (tp hp n bo, pha ngoi cn c 2 roi, pha trong mt roi) v Volvox (tp hp n bo, pha ngoi c 2 roi lm chc nng di ng cho c tp on, pha trong t bo mt roi lm chc nng quang hp, h hp) l mt v d r nt chng minh s tin ha t t chc n bo ln t chc a bo phn ha th s.Bng 2.9: Sc t v mt s tnh cht ca cc nhm to khc nhauNgnh to Chrysophy Euglenoph Chlorophy ta (to vng, yta (to mt) ta (to lc) to silic) Dip lc a v b Carote -noid v mt t Oxycaroten Lutein, Zeaxanthine, Neoxanthine, Violaxanthine Astaxanthine, Neoxanthine Mt s tnh cht T bo 2 roi, sinh sn vo tnh bng chia i hoc sinh sn hu tnh, cht d tr l tinh bt, thnh t bo ch yu l cellulose n bo c 1 roi (mt s c 2 3 roi), sinh sn v tnh bng chia i hoc hu tnh, cht d tr l m v loi tinh bt paramylon, khng c thnh t bo Phn lp l n bo, mt s nh dng si c 1 - 2 roi, sinh sn v tnh hoc hu tnh, cht d tr l du v lecucosin vi silic, thnh t bo thm pectin, silica

a v b

Lutein, Fucoxanthine, Diadinoxanthine, Diatoxanthine

a v c

50

Phaeophyta (to Pyrophyta nu) (To la, to gip)

a v c

Dinoxanthine, Diadinoxanthine, Peridinine Tucoxanthine, Lutein, Diatoxanthine, Xanthophylls

n bo 2 roi bn, sinh sn v tnh bng phn i, cht d tr l tinh bt, thnh t bo l cellulosse

Rhodophyta (to )

a bo, kch thc ln, hai roi khc bit bn, sinh sn v tnh bng ng bo t, sinh sn hu tnh bng giao t chuyn ng, cht d tr l Laminarian, thnh t bo c cellulosse v acid alginic Phycocyanine, Hu ht a bo, kch thc ln, Phycoerythrine, bt ng, sinh sn v tnh bng Neoxanthine, Lutein, bo t, sinh sn hu tnh bng Zeaxanthine, giao t, cht d tr l tinh bt, Violaxanthine thnh sinh cht ch yu l cellulosse

a v c

3. ng vt n bo Ton b ng vt c chia lm hai mc t chc: ng vt n bo (Protozoa) v ng vt a bo (Metazoa) (theo phn gii truyn thng th l hai gii ph). ng vt n bo (i khi ngi ta gp vo nhm ny c nhng ng vt hin vi dng si nhiu nhn) l nhng c th n bo nhn chun, thng dinh dng hu c, mt s nh quang dng. Nhng ng vt n bo u tin c Leeuwenhoek A.V pht hin ra ngay t th k XVII nhng c nghin cu vo th k XVIII bi Joblot L. v nhiu tc gi khc. Theo h thng phn loi hin nay th ng vt dn bo c chia lm 4 nhm: 1. Sarcomastigophora vi cc nhnh Sarcodina hay Rhizopodes (Rhizopodes v Actinopodes), nhnh Mastigophora hay Flagelles, nhnh Opalinata (cng c ti liu hp nht Rhizopodes v Flagelles vo dng Rhizoflagelles). 2. Sporzoa (k sinh trn ng vt, mt hoc nhiu vt ch). 3. Cnidospora (k sinh trn ng vt c xng v khng xng). 4. Ciliophora hay cilie (roi ngn - cils, c hai loi nhn: nhn to v nhn b). Vi hn 30.000 loi c m t, ng vt n bo sng t v nc, nhiu ng vt n bo c vai tr quan trng cc lp bn hot tnh ti cc trm lc nc thi.

v

a v d

51

Bng 2.10: Mt s nhm ng vt n bo v tnh cht ca chngMt s tnh cht Ni sng V d Mt hoc nhiu roi Nc ngt, k sinh Trypanosoma, (flagella), t bo c th trn ng vt Giardia, chia dc. Leishmania Sarcodina Dng amip, gi tc, Nc ngt v mn, Amoeba, khng roi, chia i. k sinh trn ng vt Entamoeba Sporozoa Thng bt ng, mt s K sinh s cp trn Plasmodium (gy c th trn, b, chia ng vt chn t, bnh st rt cn i, k sinh ng vt, su tc nhn truyn bnh Malaria), b. k sinh Toxoplasma Ciliophora Nhiu roi ngn (tim Nc ngt v mn, Paramecium, mao-cilia), chia i k sinh trn ng vt, Balantidium ngang, mi t bo trong d con ca thng c 2 nhn, nhn ng vt nhai li ln v nhn b lm chc nng khc nhau. Cnidophora Hnh thnh chui bo t K sinh trn ng vt Nosema gy bnh nh si phnh ra v ct c xng v khng tm gai (Pebrina) khc. xng. Sau y l so snh mt s tnh cht ca cc nhm vi sinh vt Bng 2.11: So snh mt s tnh cht ca cc nhm vi sinh vt Tnh cht Vi khun Nm To ng vt n Ghi ch bo Loi t nhn s nhn chun nhn chun nhn chun bo Kiu dinh ha d dng ha d dng quang t ha d dng tnh cht dng (mt s quang hu c dng hu c s ng dng) a bo, n bo a bo (tr mt s n n bo n bo nm men) bo v mt s a bo Cch sp ring l, mt n bo, si n bo, tp ring l, tp vch hp si v hp xp t bo s hnh thnh khng ngn v si c bt u hnh tp hp vch ngn thnh m Phng quang hp, hp th, thc tnh cht hp th php thu hp th hp th bo s ng nhn thc n Tnh cht phn bo v bo t hu sc t quang chuyn ng c trng t (trc phn) tnh v v hp v sc t tnh h tr Thnh t Murein Hemicellulose cellulose khng c hoc bo v chitine lipoproteid pH ti u 6,5 - 7,5 3,8 - 5,6 gn trung trung tnh tnh cht Nhm Mastigophora

52

Nhu cu O2 Cht d tr chnh S loi hin bit

k kh n hiu kh cc loi polysaccharide 4000

hiu kh glucogen 80.000 (tt c gii nm)

tnh hiu kh tinh bt 15.000 (ch tnh to n bo)

hiu kh glucogen v nhiu loi polysaccharide 30.000 (ch tnh ng vt n bo)

s ng tnh cht s ng

Cu hi n tp chng 21. V s cu to t bo vi khun, trong ghi r cc cu to bt buc phi c v cu to khng thng xuyn ph thuc vo nhm vi khun. 2. So snh cu to thnh t bo vi khun v vi sinh vt c. Ni r vai tr ca thnh trong hot ng sng v trong phng php nhum Gram. 3. V s cu to mng sinh cht ca vi khun, ni r chc nng vn chuyn cc cht qua mng. 4. Bn cht ca cc vt th n nhp, cu to ca chng v kh nng nhum mu. 5. Cht nhn ca vi khun, nhng pht hin mi trong vn genophore ca c th nhn s. 6. Plasmid c th nhn s, vai tr v chc nng. 7. Mng nhy v tin mao, cu to v chc nng cc loi. 8. Ni bo t, cu to v nhum mu. 9. Nu mt s v d vi khun sinh bo t v khng sinh bo t, ng dng ca chng trong cng ngh vi sinh. 10. V s cu to t bo nm men. 11. Cu to thnh t bo nm. 12. Cc chu trnh sinh hc ca nm men, i din nm mc. 13. Nguyn tc v phng php phn loi vi sinh vt. 14. nh ngha v cho v d cc khi nim sau: bo t v tnh, bo t hu tnh, ni bo t, bo t nh, bo t ti, bo t m, si nm c vch ngn, si cng bo, si hai nhn. 15. Nu mt s nm c li v gy hi. 16. Cc nhm to, cu to t bo v thnh t bo. 17. ng vt n bo, cu to c trng khc vi vi khun. 18. So snh tng qut s khc bit ca vi khun, nm, to n bo v ng vt n bo.

53

Chng 3

Cc khi nim c bn v virusVirus l cc tc nhn rt nh c th gy bnh mi c th sng. Do cu to rt n gin nn mun nhn ln chng bt buc phi k sinh trong t bo v nh b my tng hp ca t bo.

I. c im ca virusKch thc nh. Virus c kch thc rt nh t 10nm n 300nm trong khi kch thc ca vi khun khong 1000nm v kch thc ca hng cu l 7500nm. V vy virus ch c th quan st c trn knh hin vi in t.

Hnh 3.1: Hnh thi ca mt s virus1.i xng a din: [A] polio-, wart-, adeno-, rota-; [B] herpet. 2.i xng xon: [C] khm thuc l; [D] cm;[E] si, quai b, parainfluenza; [F] di; 3.i xng hn hp:[G] poxvirus; [H] phage T chn

Genome virus ch cha mt loi acid nucleic, c th l DNA hoc RNA, c th dng thng hoc khp kn, chui n hoc chui kp. Genome phn on hoc khng phn on. L dng sng khng c hot tnh trao i cht. Virus khng c ribosome hot ng hoc khng c b my tng hp protein. Cho nn mc d mt s virus c enzyme ring cu mnh nhng virus ch c th nhn ln

54

trong t bo sng, iu khin b my tng hp ca t bo phc v cho mnh to thnh cc ht virus mi.

II. Cu trc virusVirus c cu to rt n gin, bao gm li l acid nucleic, tc genome nm pha trong cn pha ngoi c bao bc bi v protein, v protein bo v genome khi s tc ng ca cc yu t mi trng v d nh nuclease trong mu. V protein c gi l capsid. Capsid c cu to bi cc n v hnh thi l capsome. Capsome li c cu to bi cc n v cu trc l protome. Protome c th l monome (ch c mt phn t protein) hoc polyme (nhiu phn t protein). Capsid v acid nucleic c gi l nucleocapsid.

Hnh 3.2. Cu trc c bn ca virionLi l acid nucleic, v l capsome l protein, hp li thnh nucleocapsid. Nucleocapsid c bao bc bi lp v ngoi (lipoprotein) vi cc gai. A.S virus a din n gin nht, mi mt hnh a din l tam gic u. nh do 5 cnh hp thnh. Mi cnh cha 3 capsomer B. S ca virus hnh que vi cu trc i xng xon (virus khm thuc l). Capsomer sp xp xon xung quanh si acid nucleic dng xon c.

55

Mt s virus cn cha v ngoi, bao bc bn ngoi capsid. V ngoi c bn cht l lipoprotein cha khng nguyn ca virus. V ngoi mt phn bt ngun t mng sinh cht ca t bo ch khi virus chui ra ngoi theo li ny chi. mt s virus, v ngoi c ngun gc t mng nhn ca t bo. Ht virus nguyn vn cn c gi l virion. Virus c 3 kiu cu trc Cu trc hnh khi. Capsid c cu trc hnh khi 20 mt tam gic u. Cu trc xon. Nucleocapsid dng ko di. Cc capsome sp xp xung quanh theo chiu xon ca acid nucleic. a s virus c cu trc xon c v ngoi bao bc nucleocapsid xon. Cu trc phc tp. Cu trc hn hp va dng khi va dng xon. V d phage c u dng khi, ui dng xon trng nh con nng nc.

III. Nui cy virusDo virus ch sinh sn bn trong t bo sng nn phi c cc phng php c bit nui cy chng. C 3 h thng chnh dng nui cy virus trong phng th nghim. Nui cy m t bo. Cc t bo c ngun gc t cc m ca ngi hay ng vt c nui trong bnh cha mi trng nhn to, cho pht trin v dng lm ngun nguyn liu cy virus. Phi g. Mt s virus c th nhn ln trong t bo phi g 6- 13 ngy. Ngy nay phng php nui ny c thay th bi t bo nui cy m. Tuy nhin trong sn xut mt s loi vaccine, phng php ny vn c s dng. ng vt thc nghim. Trc y phng php ny c dng ph bin phn lp v nghin cu virus. Cc ng vt c s dng l chut, th, kh, chn...Tim hn dch nghi l c virus vo ng vt v quan st bnh cnh lm sng. Hin nay phng php ny vn c dng phn lp mt s virus. IV. nh hng ca virus ln t bo Virus c th tc ng ln t bo theo 4 cch sau: Gy cht t bo. Kt qu ca vic nhim virus l lm cho t bo b hu hoi, dn n lm cht t bo (CPE- Cytopathic effect).

56

Chuyn dng. T bo b nhim virus nhng khng cht m chuyn t trng thi bnh thng sang trng thi c bit, thnh cc t bo u hoc ung th. Nhim tim tng. Virus tn ti bn trong t bo trng thi hot ng tim n nhng khng nh hng r rt n chc nng ca t bo. Gy ngng kt hng cu. Mt s virus trn b mt v ngoi c cha protein gy ngng kt hng cu (Haemaglutinin) gn trn b mt cc t bo nhim. Khi thm hng cu vo th hng cu s b kt dnh bi cc t bo nhim.

V. Phn loi virusVirus c phn loi da theo c im hnh thi, bn cht ca genome (DNA hay RNA), c hay khng c v ngoi, v tr lp rp... U ban quc t phn loi virus quy nh: H virus c tip v ng l viridae, h ph virinae v chi- virus. Sau y l mt s virus gy bnh.Bng 3.1. Virus gy bnh cha genome DNA Nhm virus Pox Herpes Tn virus Variola Molluscum (u mm) Herpes simplex Varicella zoster Cytomegalo EB ( Epstein- Barr), HH6 Virus adeno Vim gan B Papiloma Virus JC B19 Tn bnh u ma u mm ly Herpes Thu u zona (shingles) Nhim trong tho hip min dch Bnh bch cu n nhn ly nhim Bnh ngoi ban t ngt Vim hng Vim kt mc Vim gan Mn cc Vim cht trng no nhiu tin trin Ban truyn nhim, cn bt sn

Adeno Hepadna Papova Parvo

57

Nhm virus Orthomyxo Paramyxo Corona Rhabdo Picorna Calici Toga Flavi Bunya Reo Arena

Bng 3.2. Virus gy bnh cha genome RNA Tn virus Tn bnh Virus cm cm;Hp bo h hp Si Quai b Virus corona Cm Vim nhim ng h hp Si Quai b Gy nhim ng h hp SARS Virus di Bnh di Entero Vim no, bi lit Rhino Cm lnh Vim gan A Vim gan SRSV (virus c cu trc dng Vim d dy, rut trn nh- small round structure virus) Alpha (virus arbo nhm A) Vim no Rubi St xut huyt Rubeon (si c) Flavi (virus arbo nhm B) Vim no St xut huyt Vim gan C Vim gan Mt s virus arbo Vim no St xut huyt St, vim thn Rota Bnh ng tiu ho Vim mng no m ri mng Vim mng no mch lympho bo Virus Machupo Virus Junin Virus lassa St xut huyt Ung th t bo T HTLV-I, II U lympho Lit AIDS HIV- 1, 2 Virus Marburg St Marburg Virus E bola St xut huyt Ebola

Retro

Filo

VI. nh hng ca tc nhn vt l, ho hc n virus- Nhit cao: a s virus b bt hot 560C trong vng 30 pht, hoc 1000C trong vi giy.

58

- Nht thp: a s virus bn nht lnh nn c th bo qun lu 700C. Mt s virus b bt hot trong qu trnh lm ng lnh hoc tan bng. - Kh hn: Kh nng chu kh hn ca virus khc nhau tu loi. Mt s sng st, mt s b bt hot nhanh iu kin kh hn - Bc x t ngoi: Virus b bt hot bi tia t ngoi - Chlorofoc, ete v cc dung mi khc: Cc virus c v ngoi cha lipid s b bt hot, cn khng cha lipid s bn vng. - Cc cht oxi ha v cht kh. Virus b bt hot bi di tc dng ca formaldehyt, clo, iot v H2O2. - propiolacton v formaldehyd l cc ho cht c dng bt hot virus trong sn xut vaccine, song a s virus khng b bt hot bi phenol. - Cht kh trng virus: Tt nht l dng dung dch hypoclorua (mt cht n mn) v glutaraldehyt (l cht c th gy mn cm v kch thch gy kh chu chy nc mt cho ngi dng).

VII. Cc bnh do virusVirus l tc nhn gy bnh quan trng cho ngi, ng vt, cy trng v vi sinh vt. a s cc bnh thng gp ngi l do virus. Hu ht chng gy bnh th nh, bnh nhn t bnh phc sau mt thi gian nht nh. Nhiu loi tn ti thm lng trong c th. Chng nhn ln nhng khng gy bt k triu chng no. Tuy nhin vic nhim virus thng th nh, nhng i khi c th gy bnh trm trng nhng ngi mn cm bt thng. Mt s do virus gy bnh rt nng v thng c t l t vong cao.

VIII. Con ng ly nhim ca virus vo c thVirus vo c th theo 4 con ng chnh: - Ht th: Qua ng h hp - n ung: Qua ng tiu ho (d dy- rut) - Xm nhp qua da, vt xc nim mc (qua quan h tnh dc), truyn mu, tim chch, phu thut cy ghp hay do cn trng hoc ng vt cn. - Bm sinh: Do m truyn qua nhau thai sang con

59

IX. S xm nhp C ch gy bnh ch yu ca virus l s xm nhp. Bnh sinh ra do virus lan truyn trc tip ti cc m v c quan trong c th. S sinh sn ca virus trong t bo s git cht t bo (tuy nhin c trng hp khng git t bo). Tc ng gy hu hoi t bo c gi l CPE (cytopathic effect) s dn n tn thng v hu hoi chc nng ca cc m v c quan, ri t biu hin ra cc du hiu, triu chng.

X. Cc qu trnh nhn ln ca virusVirus khng c hot tnh trao i cht m s dng b my trao i cht ca t bo tng hp cc thnh phn thit yu ca mnh, sau lp rp to ra cc ht virus con ging nh nguyn bn. V vy ngi ta thng s dng thut ng nhn ln (nhn bn) ca virus thay cho t sinh sn. S nhn ln ca virus c th lm tan t bo gi l chu trnh sinh tan hoc c th gn genome ca mnh vo nhim sc th ca t bo, tn ti lu di di dng tim n m khng l cht t bo gi l chu trnh tim tan. Qu trnh nhn ln ca virus din ra theo 7 giai on 1. Hp ph a. Gn th th c hiu ca mnh ln th th nm trn mng sinh cht ca t bo. V c tnh c hiu cao nn ch c virus nht nh mi gn ln c cc t bo nht nh. b. S hp ph c tng cng khi c mt ca ion Mg2+ hoc Ca2+ 2. Xm nhp a. Thng thng virus xm nhp vo t bo theo c ch m bo. - Virus khng c v ngoi : Mng t bo lm vo bao ly virus to khng bo tm thi. Tip khng bo dung hp cng vi mng li ni cht gii phng nucleocapsid. - Virus c v ngoi: V ngoi ca virus dung hp vi mng sinh cht ri y nucleocapsid vo trong m khng to khng bo. V ngoi virus ho vi mng sinh cht m khng chui vo t bo cht . - Mng t bo lm vo bao ly virus cng v ngoi, to khng bo. Sau mng khng bo (c ngun gc t mng t bo) dung hp vi v ngoi ca virus ri y nucleocapsid vo t bo cht.

60

Hnh 3.3: Cc phng thc xm nhp ca virus vo t bo1-Virus c v ngoi: a) Dung hp vi mng sinh cht, y nucleocapsid vo t bo. V ngoi virus nm trn mng sinh cht (v d virus paramyxo, herpes). b) Xm nhp theo li m bo, to khng bo. Dung hp vi mng li ni cht hoc endosom tiu ha ri gii phng nucleocapsid (v d virus cm, toga v rabdo) 2.Virus khng c v ngoi. Xm nhp theo li m bo, to khng bo, dung hp vi endosom tiu ha, tin hnh ci v v gii phng acid nucleic (v d virus polio, adeno v reo).

3. Ci v Enzyme tiu ho ca t bo t lysosome tin hnh phn gii v protein cu virus gii phng acid nucleic hoc genome vo t bo cht. 4. Phin m - To thnh mRNA ca virus hoc phn t dng sao chp ca genome (RF- replicative form) - Thc hin nh enzyme ca t bo hoc ca virus - Din ra theo c ch iu khin phc tp: Kiu phin m trc v sau khi sao chp acid nucleic hon ton khc nhau.

61

Nhiu genome virus cha promoter v enhancer c tc dng kch thch qu trnh phin m. Bn phin m u tin thng c ct ni (splicing) loi b cc on intron nm xen gia cc exon. Phin m i khi xy ra theo c ch chng lp. Trong cng mt gen c nhiu im khi u v nhiu im kt thc, nh to ra nhiu protein t cng mt on acid nucleic - V c bn mRNA ca virus c nhng c im sau y (nhng cng c th khc): Cha trnh t khi u C gn m u 5 u 3 c gn ui polyA 5. Tng hp cc thnh phn ca virus 5.1. Tng hp protein ca virus mRNA ca virus c phin m trn ribosome ca t bo to ra 2 loi protein - Protein cu trc l protein capsid, protein v ngoi v protein trong li. - Protein khngcu trc l enzyme cn cho sao chp genome. Protein khng cu trc tm thy trong ht virus, tr mt s trng hp c bit v d enzyme phim m ngc c trong virus HIV hoc virus vim gan B cha DNA polymerase, mt s virus RNA cha RNA polymerase 5.2.Tng hp acid nucleic ca virus - Genome ca virus con c sao chp t genome ca virus m. Trong trng hp genome l mch n th khun l mch b sung mi to thnh ca genome m. - Phn ln qu trnh sao chp c thc hin nh polymerase (replicase) do virus m ho. i vi mt s virus DNA th qu trnh tng hp c thc hin nh enzyme ca t bo. 6. Lp rp - Genome v protein mi c to thnh lp rp vi nhau to nn ht virus mi. a s trng hp protein capsid lp rp to thnh cu trc

62

rng gi l tin capsid (procapsid) sau do chuyn ng Brao, acid nucleic chui vo cu trc ny ri hn kn li. - Lp rp c th xy ra trong nhn t bo, trong t bo cht hoc ngay st mng sinh cht (i vi a s virus c v ngoi). Mng sinh cht bao ly nucleocapsid to v ngoi. 7. Gii phng Virus c th lm tan t bo chui t ra ngoi hoc i vi virus c v ngoi th chui ra t t theo li ny chi.

XI. Genome virus C th l DNA hoc RNA m khng bao gi cha c hai. C dng chui n hoc chui kp. Dng thng hoc khp kn (ng vng). Virus cha genome DNA kp thng c kch thc ln, virus cha genome DNA n thng c kch thc nh. Genome RNA kp tt c u phn on (trong mi ht virus c nhiu on). Cn phn ln genome RNA n khng phn on (tr virus cm v HIV). Genome RNA n c trnh t nucleotit ging trnh t ca mRNA th c quy c l genome (+), cn ngc li c gi l genome (-). Genome ca HepDNAaviridae (v d virus vim gan B) khi sao chp phi thng qua RNA trung gian (DNA RNA- DNA). Acid nucleic ca virus c kch thc ln. C trng lng phn t ln. M ho cho nhiu protein. M cho nhiu enzyme cn cho qu trnh nhn ln. Acid nucleic ca virus c kch thc nh. C trng lng phn t nh. Do d kh nng m ho to protein hn ch. Nhiu trng hp virus phi c gen chng lp. nhn ln phi dng mt s enzyme ca t bo.

63

Kh nng gy nhim. i vi nhiu loi virus, acid nucleic ch c kh nng ly nhim khi c a vo trong t bo. V d khi c gii phng khi v capsid, bn thn acid nucleic c th t thc hin qu trnh gy nhim t bo, bt u chu trnh gy nhim hon chnh nhn ln. Genome ca cc virus cha RNA (-) khng c kh nng gy nhim. Mun nhn ln chng phi phin m thnh RNA (+).

XII. Protein ca virusTu thuc vo thi gian to thnh m protein ca virus c chia lm 3 loi Protein c tng hp ngay sau khi nhim c gi l protein sm tc protein khng cu trc. y l enzyme cn cho sao chp acid nucleic . Protein c tng hp mun hn gi l protein mun, thng l protein cu trc to capsid, v ngoi v protein li. Protein phn gii, thng l lyzozym gip virus gii phng ra khi t bo. Ba loi protein c iu ho tng hp mt cch hp l. Protein sm l enzyme xc tc nn ch cn mt lng nh, cn protein cu trc th phi tng hp mt lng ln.

XIII. Cc phng thc nhn ln ca virusQu trnh nhn ln ca virus trong t bo ht sc phc tp. Mi nhm virus c cch nhn ln ring. Virus c chia lm 6 nhm da vo genome v cch thc tng hp mRNA. Sau y l vi nt n gin ho qu trnh nhn ln nu bt s khc nhau gia cc nhm. Cc virus c genome DNA 1. Nhm 1. virus DNA kp V d virus vaccinia, herpes simplex, adeno, papiloma. 1.1. Dch m C 2 loi mRNA c tng hp . mRNA sm: c to thnh trc khi tng hp DNA virus, ch yu m ho cho cc enzyme tham gia vo qu trnh tng hp acid nucleic ca virus,

64

mRNA mun: to thnh sau khi tng hp DNA virus , ch yu m ho cho cc protein cu trc nh v capsid, v ngoi 1.2. Tng hp DNA virus - Cc enzyme Enzyme sao chp DNA ch yu l DNA polymerase ph thuc DNA.Cc virus c kch thc ln hn (v d vaccinia, herpes simplex) th t tng hp DNA polymerase ring cho mnh.Cc virus c kch thc nh (v d adeno, papiloma) th s dng DNA polymerase ca t bo - Khun: DNA ca virus con c tng hp trn khun genome DNA ca virus m. - V tr tng hp: trong nhn t bo (tr virus pox). - DNA mi to thnh Dng lm khun to mRNA mun cn cho tng hp protein mun. Lm khun to mch tngb cn cho tng hp nhiu genome ca virus mi. 1.3. Tng hp protein virus Qu trnh gm 2 giai on tu thuc vo s tng hp mRNA. - Tng hp protein sm: y l cc enzyme (DNA polymerase ph thuc DNA ) cn cho sao chp DNA. - Tng hp protein mun Din ra sau khi tng hp DNA Ch yu l cc protein cu trc to v capsid v v ngoi V tr tng hp : Protein c tng hp trn ribosome trong t bo cht , sau c vn chuyn ti v tr lp rp. 1.4. Lp rp Qu trnh lp rp genome vi protein to thnh ht virus mi xy ra cc v tr khc nhau bn trong t bo. - Trong nhn t bo: V d virus Herpes simplex, adeno, papiloma. Ring virus herpes hnh thnh v ngoi bng cch ny chi qua mng nhn. Mng nhn trc c gn glycoprotein do virus m ho ti

65

cc v tr c hiu. Virus c bao bi mng nhn khi ra khi nhn, sau kt hp vi mng li ni cht sn hoc b my Golgi ra khi t bo. - Trong t bo cht: V d virus vaccinia nhn ln hon ton trong t bo cht ti cc cm ribosome. 1.5. Virus c genome DNA kp c bit Virus vim gan B (HBV) c chu trnh nhn ln c bit v phc tp. Genome gm 2 mch khng bng nhau. Mch (-) di, mch (+) ngn. Cha enzyme DNA polymerase. Sau khi nhim, DNA c gii phng vo nhn. Phin m xy ra trong nhn nh RNA polymerase ca t bo to ra nhiu loi mRNA trong c RNA kch thc ln c coi l tin genome mt dng trung gian to genome. Cc RNA i ra t bo cht tng hp protein ca virus nh protein li v polymerase. Enzyme ny c 3 hot tnh ( DNA polymerase, enzyme phin m ngc v ribonuclease H). Tip RNA tin genome lin kt vi DNA polymerase v protein li to ra ht li (virus cha hon chnh). Enzyme phin m ngc tin hnh chuyn RNA tin genome thnh mch DNA (-), sau hu ht RNA tin genome b phn hu nh ribonuclease H. Ch mt on RNA c gi li dng lm mi cho DNA polymerase tng hp mch DNA (+) t khun DNA (-) to chui DNA kp. Tip l hon thin nucleocapsid. 2. Nhm 2. Virus DNA n. Ch c mt h duy nht l Parvoviridae. Cc virus cha DNA n thng c genome nh. Tin hnh sao chp trong nhn nh DNA polymerase ca t bo to DNA kp trung gian, gi l dng sao chp (RF-replicative form). RF va dng lm khun tng hp DNA n genome va dng phin m to mRNA sau dch m tng hp protein . Mt s virus c khim khuyt nn mun nhn ln cn s h tr ca cc virus khc. Cc virus c genome RNA. Do vt liu di truyn l RNA nn virus c c ch sao chp khng ging vi cc c th sng khc. Genome RNA ca virus c cc dng phin m sau y.

66

- RNA n, (+): Genome ca virus chnh l mRNA. - RNA n, (-): mRNA c phin m t genome ca virus m . - RNA kp, phn on: mRNA ca virus c phin m ring t mi on RNA genome ca m s dng transcriptase lin kt vi mi on. Virus RNA c cc c im sau: Do t bo ch khng c RNA polymerase ph thuc RNA nn enzyme ny bt buc phi c m ho bi genome virus v thng c mt trong ht virus trng thnh. Gen m ho cho RNA polymerase thng l gen ln nht trong genome v c lp hon ton vi nhn t bo ch trong sao chp v phin m. Do vy khc vi virus DNA gy nhim eucaryota, rt nhiu virus tin hnh nhn ln hon ton trong t bo cht. Cc enzyme RNA polymerase ph thuc RNA hot ng khng chnh xc nh polymerase ph thuc DNA v khng c kh nng c sa (proofreading), nn c tn s t bin rt cao, khong 10-3- 10-4 base, qua mi chu k sao chp xut hin mt t bin, gp 3-4 ln so vi virus DNA. iu ny dn n 3 h qu: Tn s t bin virus rt cao nn nu virus c chu k nhn nhanh th s bin i khng nguyn cng din ra nhanh, v th tnh c cng pht trin rt nhanh. iu ny c c l do virus RNA thch nghi rt nhanh vi s thay i ca iu kin mi trng hoc t bo ch. Mt s virus RNA t bin nhanh n ni chng to thnh v tn ti cc qun th cha cc genome khc nhau ngay trong mt vt ch. Vic xc nh chng mc phn t ch c th da vo cc trnh t chim a s hoc trung bnh. Do nhiu t bin c hi cho s nhn ln ca virus, nn tn s t bin t gii hn cao hn kch thc genome RNA, vo khong 10 nucleotit. Sau y l mt s v d v qu trnh nhn ln ca virus RNA. 3. Nhm 3. Virus RNA n, (+) V d virus gy bnh bi lit polio. Lc u khng c qu trnh phin m, v RNA genome c trnh t nucleotid ging vi trnh t ca mRNA nn lm lun chc nng ca mRNA.

67

C kh nng gy nhim v genome m ho c cho tt c cc protein cn thit ca virus. 3.1. Dch m Genome RNA (+), dng lm mRNA tng hp polypeptit tin cht ln sau nh protease phn ct thnh cc phn t nh c chc nng khc nhau nh: Protein cu trc to capsid. RNA polymerase ph thuc RNA cn cho qu trnh sao chp (t bo khng c enzyme ny) Protease phn ct peptit ln. Protein gn vo u 5' ca genome (thay cho m) gi l protein VPg, cn cho khi u sao chp. 3.2. Tng hp RNA ca virus S to thnh genome mi xy ra trn khun RNA dng sao chp (RF). Dng ny c to thnh bng cch tng hp RNA (-) b sung trn khun RNA (+) m. Genome RNA (+) c tng hp trn khun RNA (-) ca dng sao chp RF

Hnh 3.3: Chu trnh n gin ho qu trnh nhn ln ca virus RNA (-), n

68

RNA polymerase ph thuc RNA tham gia tng hp c RF v c genome RNA (+) mi . RNA (+) mi c tng hp c nhng chc nng sau: Lm khun tng hp nhiu RF ri t tng hp nhiu RNA genome. Dng lm genome cho cc virus mi Dng lm mRNA 3.3. Lp rp Protein c to thnh sau khi phn ct polypeptit tin cht s lp rp vi RNA genome mi trong t bo cht to ra virus mi Vi virus polio th s nhn ln hon ton xy ra trong t bo cht 13.3.4. Gii phng Virus ph v t bo t ngt t ra ngoi . 4. Nhm 4. Virus RNA n, (-) V d virus cm hoc cm. Chu trnh nhn ln c minh ho hnh 3.4

Hnh 3.4: Chu trnh n gin ho qu trnh nhn ln ca virus RNA (+), n

c im qu trnh phin m c th tm tt nh sau:

69

Virus RNA (-) lun mang theo RNA polymerase ph thuc RNA v t bo khng c enzyme ny. mRNA c tng hp trong nhn t bo t genome RNA (-) ca m nh enzyme RNA polymerase ph thuc RNA c trong ht virus . Virus cm cha genome nhiu on do mi mRNA to thnh c dng tng hp mt loi protein. 4.1. Tng hp RNA genome. Khc vi phin m to mRNA, mun tng hp genome RNA (-) trc ht phI tng hp chui RNA trung gian lm khun. Qu trnh ny khng cn mi. Tip tng hp RNA (-) genome trn mch khun trung gian. 4.2. Tng hp protein. Protein ca virus bao gm: Enzyme transcriptase (RNA polymerase ph thuc RNA ). Protein v ngoi gm 2 loi u c gn glucoz (glycosyl ho). Mt loi c hot tnh haemaglutinin hoc neuraminidase v mt loi c hot tnh dung hp hoc tan mu. 4.3. Lp rp Nucleocapsid ca virus c lp rp mng nhn t bo v to v ngoi khi ny chi qua mng sinh cht. Virus cm l virus RNA nhiu on, n. Mi on phin m cho mt m RNA ring t tng hp thnh cc phn t protein c chc nng khc nhau. RNA mi ca virus c tng hp trong nhn t bo. Qu trnh ny cn c s tham gia phin m cu t bo ch. Protein virus c tng hp trong t bo cht ri di chuyn ti b mt t bo. Ti y glycoprotein v ngoi cng c gn vo cc v tr nht nh. Genome RNA mi c vn chuyn n b mt t bo v lp rp vi protein mi tng hp ca virus. Ht virus c bao v ngoi khi ny chi qua mng sinh cht 5. Nhm 5. Virus RNA kp Nhm ny bao gm cc virus Reo v Rota. Tt c cc virus RNA kp u c genome nhiu on. Mi on phin m cho mt mRNA tng hp mt protein ring.

70

Ht virus cha RNA polymerase ph thuc RNA . Phin m thc hin theo c ch bo th v khng i xng, ngha l ch to thnh mRNA. Mi mRNA li c sao mt ln to chui RNA kp, cng chnh l genome (s nhn ln c minh ho hnh sau ).

Hnh 3.5: Chu trnh n gin ho qu trnh nhn ln ca virus Retro (RT: Enzyme phin m ngc)

Hnh 3.6: Chu trnh n gin ho qu trnh nhn ln ca virus RNA kp (RdRp: Enzyme RNA polymerase ph thuc RNA)

71

6. Nhm 6. Virus Retro cha genome DNA n, (+) Rt nhiu nhng khng phi tt c virus retro c kh nng to khi u. Khi nhn ln khng git cht t bo m c th chuyn dng thnh t bo ung th. Genome l 2 phn t RNA n, (+), gn vi nhau pha u (dng dime). Cha 3 gen chnh l gap (m cho protein li), gen pol m cho polymerase phin m ngc (RT) v gen env m cho protein v ngoi. Ngoi ra cn c mt s gen iu ho. 6.1.Qu trnh nhn ln Picorma v flavivirus

1i genome RNA (-)

5

Genome RNA (+)

2Enzyme sao chp

6

Tin polyprotein

4

3

7

Protein cu trc v khng cu

8

Virus miHnh 3.7. S nhn ln ca virus RNA chui dng, khng to thnh cc mRNA genome. Sau khi xm nhp v ci v (bc 1), RNA genome c s dng trc tip tng hp cc polyprotein kch thc ln, cc protein cu trc v khng cu trc (bc 2 v 3). Protein khng cu trc (enzyme) xc tc sao chp RNA thng qua s tng hp RNA chui m (i genome) (bc 5 v 6). Qu trnh sao chp to ra nhiu genome RNA mi phc v cho tng hp cc protein ca virus (bc 6 v 7) v cho lp rp to virus mi (bc 8). Mi tn m th hin s tng hp mc cao hn.

72

Gm 2 giai on: - Giai on u Phin m nh enzyme phin m ngc to chui lai RNA/ DNA. Chuyn chui lai RNA/DNA thnh chui DNA kp. Enzyme RT c hot tnh ribonuclease H phn gii mch RNA. Cn mch cDNA dng lm khun tng hp mch DNA b sung. Ci xen phn t DNA kp mi tng hp vo nhim sc th ca t bo to ra provirus. - Giai on hai. RNA ca virus phin m nh enzyme ca t bo. Bn sao RNA c 2 chc nng: va l m RNA tng hpj protein virus, va l genome ca virus mi. 6.2. Tng hp protein mRNA c phin m t DNA provirus tin hnh tng hp protein trn ribosome nm trong t bo cht, bao gm: Enzyme phin m ngc, protein li, protein v ngoi. 6.3. Lp rp Nucleocapsid c lp rp t genome RNA mi to thnh vi protein di tr ra b mt t bo. V ngoi c hnh thnh khi virus ny chi qua mng sinh cht.

73

Atro, calici, toga, corona v arterivirus

1Di genome mRNA

6

i genome RNA (-)

5 2Enzyme sao chp

Genome RNA (+)

7Protein cu trc v tin polyprotein

9

Tin polyprotein khng cu trc

4 8

3

10

Protein khng cu trc

Protein cu trc

11

Virus mi

Hnh 3.8. S nhn ln ca virus RNA dng, km theo tng hp RNA di genome. Sau khi xm nhp v ci v (bc 1), RNA genome c dng trc tip lm mRNA tng hp protein khng cu trc, trong c enzyme RNA polymerase ph thuc RNA (RdRp) ( bc 2 v 3). RdRp xc tc sao chp RNA, tng hp chui RNA m (i genome) c kch thc (tng ng RNA genome) ( bc 4 v 5). T chui m lm khun tin hnh sao chp to ra nhiu chui RNA genome mi (bc 5 v 6). mRNA di genome (c kch thc nh hn genome) tin hnh dch m to protein cu trc ( bc 7 v 8). Genome mi to thnh tin hnh dch m to nhiu protein khng cu trc (bc 9 v 10) v sau lp rp vi protein cu trc to virus mi ( bc 11). Ch thch: Bc 7. Dch m ca mRNA di genome; Bc 8. Phn ct nh protease v x l sau dch m; Bc 10. Phn ct nh protease

74

Phabdo, filo, borna, paramyxo, orthomyxo v mt s bunya

1RNA (+) i genome

5 2Protein capsid v

RNA genome

6

mRNA

3 4

7 8

Protein cu trc v khng cu trc

Virus mi

Hnh 3.9. S nhn ln ca virus RNA chui n, m . Sau khi xm nhp v ci v mt phn( bc 1), RNA tin hnh phin m nh RNA polymerase ph thuc RNA do virus mang theo to mRNA (bc 2) cho tng hp protein cu trc v khng cu trc (RdRp) ( bc 3). Enzyme RdRp tin hnh sao chp RNA thng qua bc trung gian l to RNA chui dng (i genome) (bc 4 v 5). i genome (chui dng) c dng lm khun tng hp nhiu RNA genome (bc 5). Mt phn RNA genome c dng tin hnh phin m mnh, to ra mRNA cn cho tng hp protein (bc 7), mt phn dng lp rp vi protein to virus mi (bc 8).

75

Virus Reo v Birna

1mRNA trong ht di mc virus

9 5

Genome RNA kp trong ht di mc virus

4 8

2

6

mRNA virus

3

7 10

Protein cu trc v khng cu trc

Virus mi

Hnh 3.10. S nhn ln ca virus RNA chui kp. Sau khi xm nhp v ci v tng phn (bc 1), cc on RNA chui kp vn nm trong li tin hnh phin m nh enzyme RdRp gn li tng hp mRNA (bc 2) ri t tng hp protein (bc 3). Cc protein ny lp rp quanh mRNA to ht di mc virus (bc 4). Sau mRNA trong cc ht ny tin hnh sao chp to RNA genome chui m tham gia to thnh RNA genome chui kp (bc 5). Cc ht di mc virus c to thnh li tip tc tham gia vo qu trnh tng hp m RNA (bc 6), tng hp protein (bc 7), sao chp (bc 8 v 9), lp rp vi protein cu trc to thnh virus mi (bc 10) Ch thch: Bc 1. Xm nhp v ci v Bc 4. Lp rp to ht di mc virus. Bc 10. Lp rp to virus mi.

76

Virus Retro

1DNA kp

3

cDNA (i genome)

2

RNA genome

4DNA ci xen (provirut)

5

RNA cha ch bin

6RNA virus sau ch bin

9

7

7 8

Cc bn sao DNA kp ca virus

Protein cu trc, khng cu trc v protein gy ung th

Virut mi Hnh 3.11. S nhn ln ca virus Retro.Sau khi xm nhp v ci v tng phn (bc 1), RNA genome c phin m thnh cDNA (i genome), sau thnh DNA chui kp nh enzyme phin m ngc (bc 2 v 3). Nh enzyme integrase, DNA ca virus ci xen vo nhim sc th t bo ch (bc 4). C hai loi enzyme trn u nm trong li ca virus. Nh RNA polymerase ca t bo, provirus phin m to RNA (bc 5) l tin cht ca mRNA (bc 6) tng hp protein (bc 7) v lp rp to virus mi (bc 8).

Cu hi n tp chng 31.Hy gii thch cc thut ng sau y: capsome, capsid, nucleocapsid, capsid c cu trc xon, khi a din, virus c cu trc phc tp, v ngoi. 2.Virus khc vi plasmit v khc vi t bo im no. 3.Virus c th tc ng ln t bo nh th no ? 4.Nu cc giai on nhn ln ca virus.

77

5.Ti sao virus ch c th nhn ln trong cc t bo nht nh ? 6.Trnh by cc phng thc xm nhp ca virus vo t bo ch. 7.Hy nu cc loi genome ca virus, Th no l RNA(+), RNA().Th no l genome chng lp. 8.Dng sao chp (RF) khc vi genome im no ? 9.Ti sao cc virus RNA(-) nht nh phi mang theo enzyme RNA polymerase. 10.Hy tm tt qu trnh nhn ln ca cc virus AND n, AND kp,RNA(+), RNA(-).

78

Chng 4

Dinh dng, sinh trng v pht trin ca vi sinh vtI. Dinh dngTrong qu trnh sng, t bo vi sinh vt tin hnh trao i cht khng ngng vi mi trng chung quanh. T bo vi sinh vt tuy rt nh, nhng v hp thu cc cht dinh dng v thi ra cc sn phm trao i cht qua ton b b mt, cho nn cng trao i cht ca chng l rt ln. Cc cht dinh dng vo t bo qua mng v c chuyn ho to thnh nhng cht ring bit cn thit cho vic xy dng t bo. Nh qu trnh ng ho cc t bo mi c th sinh trng, pht trin tng sinh khi, ng thi to ra cc sn phm trao i cht. S bin i cc cht dinh dng bao gm nhiu phn ng ho sinh khc nhau nh h enzyme theo con ng trao i cht, hoc to ra nhng cht l thnh phn ca t bo (qu trnh ng ho) hoc to ra nng lng sinh hc cn thit cho hot ng sng (qu trnh d ho). Nhng cht dinh dng l nhng hp cht phn t nh c th i qua mng vo bn trong t bo vi sinh vt v tham gia vo hai loi phn ng sinh ho: - Bin i d ho lm xut hin nhng sn phm c cu trc n gin hn. Nhng bin i d ho ny cung cp cho vi sinh vt nng lng chuyn ho dng ATP hoc nhng hp cht giu nng lng khc. Mt s nhng sn phm d ho c thi i, mt s khc lm vt liu hoc lm tin cht cho cc phn ng ng ho. - Bin i ng ho, m bo s tng hp ca thnh phn mi c cu trc phc tp hn v phn t lng cao hn. Qu trnh ny thng c gi l ng ho hoc sinh tng hp. iu kin ch yu sinh tng hp cc thnh phn t bo vi sinh vt l cung cp mt lng thch hp ca nhng hp cht c phn t lng nh, nh cc acid hu c hoc amino acid, c th lm nguyn liu hay tin cht cho cc phn ng ng ho hay phn ng sinh tng hp. Khi trong mi trng c nhng hp cht - vt liu th vi sinh vt s trc tip s dng. Nhng khng phi bao gi trong mi trng cng c sn nhng hp cht - vt liu cn cho qu trnh sinh tng hp. Mun c t bo vi sinh vt bt buc phi t sn xut ly bng cch t bin i nhng thnh phn ca mi trng nui cy. nhng vi sinh vt d dng sng bng cht hu c,

79

mt s tin cht c hnh thnh trong cc phn ng d ho, n sn sinh ra ATP cng mt s ln cc hp cht khc nhau ca carbon. Cc cht dinh dng ca vi sinh vt ch yu ly mi trng xung quanh. Cc mi trng dinh dng nhn to cn cung cp y nng lng, cc vt liu xy dng t bo v m bo cho hiu sut sinh tng hp cao. Thnh phn mi trng gm c cc ngun n carbon, nit, cht khong, cc nguyn t vi lng v cc cht kch thch sinh trng. Vic la chn cc ngun dinh dng v nng ca chng trong mi trng ph thuc vo c tnh sinh l ca tng chng tng loi vi sinh vt v iu kin nui cy chng.

II. Mi trng dinh dng v iu kin sinh trng1. Mi trng dinh dng 1.1. Ngun thc n carbon Ty nhm vi sinh vt m ngun carbon c cung cp c th l cht v c (CO2, NaHCO3 , CaCO3... ) hoc cht hu c. Gi tr dinh dng v kh nng hp th cc ngun thc n carbon khc nhau ph thuc vo thnh phn ha hc, tnh cht sinh l ca ngun thc n v c im sinh l ca tng loi vi sinh vt. Hu nh khng c hp cht carbon no m khng b hoc nhm vi sinh vt ny hoc nhm vi sinh vt khc phn gii. Khng t vi sinh vt c th ng ha c c cc hp cht carbon rt bn vng nh cao su, cht do, du m, parafin, kh t nhin. Nhiu cht hu c v khng tan c trong nc hoc v c khi lng phn t qu ln cho nn trc khi c hp th, vi sinh vt phi tit ra cc enzyme thy phn (amylase, cellulase, pectinase, protease, lipase...) chuyn ha chng thnh cc hp cht d hp th (ng, amino acid, acid bo...). Ngi ta thng s dng ng lm ngun thc n carbon khi nui cy phn ln cc vi sinh vt d dng. nui cy cc loi vi sinh vt khc nhau ngi ta dng cc nng ng khng ging nhau. Vi vi khun, x khun thng dng 0,2 - 0,5% ng cn i vi nm men, nm si thng dng 3 - 10% ng. Hu ht vi sinh vt ch ng ha c cc loi ng dng ng phn D. Nhng phn ln cc ng phn ca ng n trong t nhin u l thuc loi D. Cc hp cht hu c cha c C v N (peptone, nc tht, nc chit ng, nc chit nm men, nc chit i mch, nc chit gi u...) c th s dng va lm ngun C va lm ngun N i vi vi sinh

80

vt. Vi vi sinh vt d dng, ngun thc n carbon lm c hai chc nng, ngun dinh dng v nng lng. 1.2. Ngun dinh dng nit (N) ngha ch yu ca cc cht dinh dng cha nit i vi vi sinh vt l cung cp N to thnh cc nhm amin (-NH2) v nhm imin (=NH) trong phn t amino acid, cc nucleotide, purine v pirimidine, trong nhiu vitamin... Ngun nit d hp th i vi vi sinh vt l NO3- v NH4+. Mui nitrate l ngun thc n nit thch hp i vi nhiu loi to, nm si v x khun nhng t thch hp i vi nhiu loi nm men v vi khun. Sau khi vi sinh vt s dng ht gc NO3- cc ion kim loi cn li (K+, Na+, Mg2+..) s lm kim ha mi trng. trnh hin tng ny ngi ta thng s dng mui NH4NO3 lm ngun nit cho nhiu loi vi sinh vt. Tuy nhin gc NH4+ thng c hp th nhanh hn gc NO3-. Vi sinh vt cn c kh nng ng ha rt tt nit cha trong cc thc n hu c. Cc thc n ny s va l ngun carbon va l ngun nit cung cp cho vi sinh vt. Vi sinh vt khng c kh nng hp th trc tip cc protein cao phn t. Rt nhiu vi sinh vt c kh nng sn sinh protease thy phn protein thnh cc hp cht phn t thp c kh nng xm nhp vo t bo vi sinh vt. Ngun nit hu c thng c s dng nui cy vi sinh vt l peptone (l ch phm thy phn khng trit ca mt ngun protein no ) chng khc nhau v lng cha cc loi polypeptide v lng cha amino acid t do. Ngoi ra phn t N2 t do cng c mt nhm nh vi sinh vt s dng gi l nhm vi sinh vt c nh nit (nitrogen fixing microorrganisms). Chng c h enzyme nitrogenase c th b gy ni ba ca nit phn t iu kin nhit v p sut bnh thng. 1.3. Ngun dinh dng khong Khi s dng cc mi trng thin nhin nui cy vi sinh vt thng khng cn thit b sung cc nguyn t khong. Trong nguyn liu dng pha ch cc mi trng ny (khoai ty, nc tht, sa, huyt thanh, peptone, gi u...) thng c cha cc nguyn t khong cn thit i vi vi sinh vt. Ngc li, khi lm cc mi trng tng hp (dng nguyn liu l ha cht) bt buc phi b sung cc nguyn t khong cn thit. Nhng nguyn t khong m vi sinh vt i hi phi c cung cp vi liu lng ln c gi l cc nguyn t i lng. Cn nhng

81

nguyn t khong m vi sinh vt ch i hi vi nhng liu lng rt nh c gi l cc nguyn t vi lng. Phosphorus (P) bao gi cng chim t l cao nht trong s cc nguyn t khong ca t bo vi sinh vt (nhiu khi P chim n 50% so vi tng s cht khong). P c mt trong cu to ca nhiu thnh phn quan trng ca t bo (acid nucleic, phosphoprotein, phospholipid, ADP, ATP, UDP, UTP, CDP, CTP, NAD, NADP, flavin...; mt s vitamin nh thiamine, biotin...). m bo ngun dinh dng P ngi ta thng s dng cc loi mui phosphate v c. Vic b sung phosphate vo cc mi trng dinh dng ngoi tc dng cung cp P cn c tc dng to ra tnh m ca mi trng. Lu hunh (S) cng l mt nguyn t khong quan trng trong t bo vi sinh vt. S c mt trong mt s amino acid (cysteine, cystine, methionine), mt s vitamin (biotin, thiamine...) cysteine, cystine v mt tripeptide l glutation khng ch tham gia vo cu trc protein m cn c vai tr quan trng trong cc qu trnh xy ha kh. Cc hp cht hu c c cha S dng xy ha thng c tc dng c i vi t bo vi sinh vt. Trong khi cc mui sulphate v c vi nguyn t S trng thi xy ha li c c th vi sinh vt ng ha rt tt. Mt s vi sinh vt c th dng c thiosulphate lm ngun thc n S. Mt s vi sinh vt khc li i hi cc thc n cha S dng kh (H2S, cystine, cysteine...). Fe l nguyn t rt cn thit gip vi sinh vt c th tng hp mt s enzyme loi porphiryl cha st (nh cytochrome, cytochrome oxydase, peroxydase, catalase...). Mt s vi sinh vt t dng quang nng cn s dng Fe tng hp ra cc sc t quang hp c cu trc porphiryl (chlorophyll, bacteriochlorophyll). Mg l nguyn t c vi sinh vt i hi cung cp vi lng kh cao. Mg mang tnh cht mt cofactor, chng tham gia nhiu phn ng enzyme c lin quan n qa trnh phosphoryl ha. Mg2+ c th hot ha cc enzyme nh hexokinase, ATP - ase, carboxylase, cc enzyme trao i protein, cc enzyme xy ha kh ca chu trnh Krebs... Mg cn c vai tr quan trng trong vic lin kt cc tiu phn ribosome vi nhau. Ca mc du l nguyn t t tham gia vo vic xy dng nn cc cht hu c nhng n c vai tr ng k trong vic xy dng cc cu trc tinh vi ca t bo. Ca ng vai tr cu ni trung gian gia nhiu thnh phn quan trng ca t bo sng (nh gia ADN v protein trong nhn, gia cc nucleotide vi nhau, gia ARN v protein trong ribosome). Ca rt cn thit i vi vic hnh thnh cc cu trc khng gian n nh ca nhiu bo quan nh ribosome, ti th, nhn...

82

Zn cng l mt cofactor tham gia vo nhiu qu trnh enzyme. Zn c tc dng ng k trong vic hot ha cc enzyme nh carbonanhydrase, enolase, pyrophosphatase, phosphatase kim ... Cng c mt s nguyn t ha hc chng ta cha hiu r v vai tr sinh l ca chng, trong s phi k n l K. K l nguyn t chim t l kh cao trong thnh phn khong ca t bo vi sinh vt, nhng cho n nay ngi ta cha tm thy K tham gia vo bt k thnh phn no ca nguyn sinh cht, cng cha tm thy bt k enzyme no c cha K. K thng tn ti dng ion K+ mt ngoi ca cu trc t bo. Mt phn ng k K tn ti trng thi lin kt ha l khng bn vi protein v cc thnh phn khc ca nguyn sinh cht. K lm tng ngm nc ca cc h thng keo do nh hng n cc qu trnh trao i cht, nht l cc qu trnh sinh tng hp. K cn c th tham gia vo qu trnh tng hp mt s vitamin v c nh hng n qu trnh h hp ca t bo vi sinh vt. Na v Cl cng l nhng nguyn t m nhiu vi sinh vt i hi vi mt lng khng nh, nhng cho ti nay ngi ta vn cn bi