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Herbaspirillum Frisingense
Herbaspirillum Frisingense
Herbaspirillum Frisingense
Phn lp
Herbaspirillum frisingensesp.nov.
Thc vt
GSF3
S.pectinata
R c ra sch
GSF36
S.pectinata
Thn c ra sch
GSF20
M.sinensiscv.Giganteus
Thn c ra sch
GSF28
GSF22, 24, 27, 35
M.sacchariflorus
R c ra sch
M.sacchariflorus
Thn c ra sch
M.sacchariflorus
L c ra sch
M.sacchariflorus
R c ra sch
P.purpureumcv.Cana Africa
R c ra sch
37
P.purpureumcv.Gramafante
Thn c ra sch
61B
P.purpureumcv.Guacu
Thn c ra sch
114B
P.purpureumcv.Guacu
R c rasch
72B
P.purpureumcv.Merker
Thn c ra sch
P.purpureumcv.Merker239
P.purpureumcv.Mineiro
R c ra sch
Thn c ra sch
75B, 118B
P.purpureumcv.Mineiro
R c ra sch
112B, 115B
P.purpureumcv.Mott
Thn c ra sch
106B
P.purpureumcv.Mott
R c ra sch
P.purpureumcv.Piracicaba
Thn c ra sch
78B
P.purpureumcv.Piracicaba
R c rasch
36B, 41B
P.purpureumcv.Roxo
Thn c rasch
48H
P.purpureumcv.s/pelo
Thn c ra sch
95B
P.purpureumcv.s/pelo
R c ra sch
P.purpureumcv.Taiwan
P.purpureumcv.Taiwan
Thn c ra sch
R c ra sch
16B
P.purpureumcv.Merker
R c rasch
80B
P.purpureumcv.Cameroon
R c ra sch
81B
P.purpureumcv.Roxo
R c ra sch
91B
P.purpureumcv.Piracicaba
R c ra sch
Herbaspirillum seropedicae
c im kiu hnh.
c im hnh thi c nghin cu bng knh hin vi tng phn pha v knh hin
vi in t truyn sut (TEM). Cc loi roi c xc nh bng cch quan st nhng roi
c nhum m vi uranyl acetate di knh hin vi in t. Hnh thi khun lc,
sc t v kh nng di ng c kim tra trn mi trng c b sung chit xut
nm men (0.05%) NFB, bao gm 6 ml dung dch cht ch th bromthymolblue 0.5%
(Baldani et al., 1986). Hot ng t li thnh nhm c quan st trong mi trng
thch mm NB (0.8% agar) bng cch o ng knh ca mt vng trung tm ca
cht c cy t NFB lng c cy qua m sau 5 ngy ti 35o C. Nhit tng
trng ti u c kim tra bng cch s dng mi trng bn rn JNFb vi pH
c iu chnh n gi tr yu cu sau khi kh trng trong ni hp. Mt cht cy
gm c 20l ca mt t bo c nui cy pht trin trong mi trng DYGS ci bin
(Rodrigues Neto et al., 1986), c tn l 1/2 DYGS (g l-1: dextrose, 1.0;
malate,1.0; peptone, 1.5; chit xut nm men, 2.0; MgSO4.7H2O, 0.5; L-axit glutamic,
1.5; pH 6.0), c thm vo mi bnh v th nghim v s bin i axetylen c
thc hin sau 72h- 30oC. Nhit tng trng ti u c nh gi trong mi
trng lng 1/2 DYGS.
Nhng c tnh sinh l c sng lc bng API20NE v API50CHE (bio Mrieux)
v BIOLOG-GN MicroPlate m hnh ging v s chuyn ho ngun carbon, theo
hng dn ca nh sn xut.
Kh nng hnh thnh mng mng (vi hiu kh, kh nng c nh dinitrogen ph thuc
vo tng trng) ca cc chng c chn vi ngun carbon khc nhau c phn
tch. Do , malate thnh phn trong mi trng bn rn JNFb c thay th
bi N -acetyl-D-glucosamine, L-arabinose, D-fructose, D-glucose, meso-inositol, Lrhamnose, malate, meso-erythritol v L-tartrate, tng ng. Mi mi trng lp li 3
ln c cy vi 10 l mi trng lng cy qua m trong 1/2 DYGS v 30
o
C trong mt khong thi gian 3-4 ngy.
Bng chng v kh nng c nh m c nh gi thng qua kh nng kh
acetylen v khuch i gen nifD.
Kh nng c nh m trong khng kh ca nhng chng Herbaspirillum c
kim tra bng cc th nghim kh acetylen c in (Burris, 1972). V mc tiu ny m
dng Mb11 c cy vi mt th tch l 15 ml trong nm mi trng tng ng
NFB bn c c nit t do. L 23ml c ng kn bi vch cao su v 0.1 dung tch
(th tch khng kh) acetylen c thm sau khi cy 36 h. Lng ethylene c o
mi 30 pht trong thi gian 4 h. Tt c thi k c thc hin 300C trong iu kin
ti, trnh di di cc l ny. Ethylene c o bng cch s dng mt loi h thng sc
k kh Hewlett Packard 5890A c trang b u d ion ha ngn la v ct mao
qun (HayeSep N; Supelco). S tnh ton c da trn vng nh v cc phn mm
CHEMSTATION (Hewlett Packard) c s dng phn tch d liu. i vi
vic xc nh s lng t bo, pha long theo th t ca mi trng bn rn ng nht
c cy trn mi trng 1/2 DYGS v c m sau khi 300C trong 5 ngy.
Ngoi ra, vic la chn cc chng phn lp c kim tra s hin din
ca gen nifD bng PCR vi mi v iu kin phn ng theo Stoltzfus et
al. (1997). Cc sn phm phn ng c phn tch bi in di trn gel agarose ,
nhum vi ethidium bromide v kch thc bng c tnh bng cch so snh vi 100
bp bc thang chun (Gibco-BRL).
S lai b gen ADN-ADN. Khi lng phn t cao ca ADN c chun b theo
phng php ca Mayer &Schleifer (1978), b qua cc bc kt ta CTAB.
Lp li i vi nhng mu c khi lng cao phn t ADN l 1 v10 g v nhng
mu ny c bin tnh bng cch trong kim (200 ml 0.4 M NaOH, 20 pht,
nhit phng) v c nh trn mt mng nylon khng tch in(Quiabrane Nylon;
Diagen) s dng mt n v thm tch im chn khng (mt k thut lai AND)
(Schleicher & Schuell). Sau khi lm kh b lc chng c nung trong 20 pht 80
o
C. lai ADN-ADN, phng php Tjernberg et al. (1989) c th c thay i. Cc
u d ADN tng ng (0.3 g) c gn nhn vi [-32] dCTP bi k thut tng hp
nhng mch t gy (nick translation), theo hng dn ca nh sn xut (GibcoBRL). Cc b lc c trc trong 10 ml dung dch gm 35%(V/v) Formamide, 2x
SSC, gii php 5x dung dch Denhardt, 1% glycine, 1.5 mg ADN trong tuyn c b
ml-1(3x 20 s sng m) 6 gi nhit lai. Nhit c la chn ti 25 oC, m
nhit lai c la chn trn l di im bin tnh nhit (TM(2SSC)) ca mu d
ADN. i vi mt herbaspirilla hm lng G+C nh nht l 61 mol% TM(2 SSC) trong
98.4oC. V th, trong cc th nghim ca chng ti nhit lai chun c la chn l
53oC v cc yu cu nghim ngt v iu kin phn ng c iu chnh ng 30%
(v/v) formamid (-0.70C/1% formamid). i vi cc dung dch lai 0.1mg c gn vi
mu d ADN v 700 g AND ca tuyn c b c pha trn vi 1.5 ml formamid
v lm bin tnh trong 15 pht nhit phng. Sau , cc thnh phn sau y thm
vo c nng cui cng: SSC x2, 1x dung dch Denhardts v 0.3% SDS, nc
c thm vo to thnh th tch cui cng l 5 ml. Dung dch trc khi c
thay th bng 5 ml dung dch lai. Sau khi lai qua m ti 530C mng c ra 2-3 ln
trong 10-15 pht vi 15 ml dung dch cha 2xSSC/0.1% SDS v 3-4 ln cho 10-30
pht vi mt dung dch cha 0.1x SSC/0.1% SDS nhit lai. Cui cng, cc cht
lng c thot khi b lc, n c bao bc trong mt bc nha v tip xc
vi phim X-ray trong s hin din ca lc tch cc trong 3-72h ti1000C.
Nhng tn hiu ca n c pht x c th nh lng c mt t my qut phng
vi phn mm phn tch (RFLP-Scan; Scanalytics). T l phn trm lin kt ca
ADN d tng ng tng ng vi 100% lin kt ca ADN ng dng sau khi
xem xt s lng ca b lc AND gii hn [tnh c mt sau khi lai vi mt hn
hp oligonucleotide c s dng ph bin, trnh t rADN ca vi khun (Lee et
al., 1993)]. Trong trng hp ny phng php lai c thc hin ti 500C, theo th
thut c m t bi Kirchhof & Hartmann (1992). Cc gi tr ny l nn tng -
sa cha v tnh ton gi tr trung bnh ca cc ln lp li.
Hm lng G+C. Cc hm lng G+C ca cc ADN phn lp c xc nh bng
phng php bin tnh nhit (Johnson, 1989). Cc m t s lc v s bin tnh nhit
c ghi nhn trc quang bng chng trnh o quang ph (Gilford 250) v hm
lng G+C c tnh bng phng trnh ca Owen & Lapage (1976).
Phn tch pht sinh loi da trn trnh t 16S rADN.
Trnh t 16S rADN c quyt nh thng mi ha bi DSMZ, Braunschweig,
c. Trnh t 1465-1485 bp ca chng c la chn v cc kiu chng ca
Herbaspirillum seropedicae (LMG6513T) v Herbaspirillum rubrisubalbicans (LMG
2286T) c khuch i bng PCR (Rainey et al., 1996) v sn phm tinh ch c
gii trnh t bng cch s dng ABI PRISM Dye Terminator Cycle Sequencing
Ready Reaction Kit (Applied Biosystems). Cc kt qu trnh t c thm vo mt
lin kt trong khong 6000 trnh t tng ng t vi khun c cng b bng
cch s dng cng c lin kt ca cc gi phn mm ARB (http:
\\www.mikro.biologie.tu-muenchen.de). Phn tch pht sinh loi c thc hin bng
cch p dng phng php ma trn khong cch (distance matrix) (ARB, Felsenstein,
Jukes-Cantor), phng php h tin n mc ti a (maximum- parsimony) (ARB) v
phng php hp l cc i (maximum-likelihood) (fastADNml; Maidak et al., 1996)
nh m t ca Wittke et al. (1997) trn b d liu khc nhau.
u d oligonucleotide.
c thit k nhn bit chi v loi- bng u d oligonucleotide c trng, d liu
trnh t 16S rADN c s dng. Cc trnh t ph hp c sng lc thnh
phn cu to c trng duy nht i vi tng loi v chi bng cch s dng cc cng
c thit k mu d (Probe design) ca gi phn mm ARB . Ngoi ra, cc chng ny
c phn tch trong th nghim lai vi vng trnh t III ca mu d 23S rADN ch
(Baldani et al., 1996) theo phng php m t bi Kirchhof & Hartmann (1992). iu
kin lai to ch nghim ngt cho cc u d 23S rADN-ch c th nghim
trong th nghim lai dot-blot bng cch s dng nhit lai khc nhau. iu kin lai
ti ch cho cc u d oligonucleotide 23S rADN ch mi c ti u ha bng
cch tng dn nng formamide trong dung dch m nh m t ca Manz et
al. (1992), bao gm cc chng loi c lin quan cht ch ca - Proteobacteria trong
cc bi kim tra.
Phn tch cc chng bng fingerprinting PCR.
S a dng v gen cc chng c kim tra trong cc phng php phn tch PCR
figerprint, p dng mt chui trnh t lp i lp li ng vt c v (LINEs, long
interspersed; Smida et al., 1996) thu c mi GRK (5h-GAG TTT GGC AAA GAC
CC-3h) (Kirchhof et al., 1997). Cc mu bng (pattern) kt qu c hin th trn gel
agarose 1.5%, c nhn thy bng vic nhum ethidiumbromide.
KT QU V THO LUN
c im hnh thi.
Trong sut qu trnh phn lp vi khun c nh nit t Miscanthus spp v Spartina
pectinata c trng c, v ging Pennisetum purpureum c trng Brazill, c
57 chng vi khun to mng mng (pellicles) trong mi trng bn rn NFb v JNFb,
6
(b)
c tnh sinh l
Hnh dng ca Herbaspirillum frisingense ph hp c m t (Baldani et al.,
1986), khi phn lp tt c cc loi Herbaspirillum cho thy cc enzyme catalase,
oxidase v urease hot ng. Genatin khng c th thy phn. Trong tt c cc th
nghim phn lp th NO3- c th chuyn thnh NO2-, nhng khng th chuyn thnh
N2. S dng h thng a BIOLOG GN (chn lc 22 chng), cc chng a ra c c
trng l h hp v trao i cht vi ph rng ca ca ng v alcohol: adonitol, Larabinose, D-fructose, L-fucose, galactose, D-glucose, glycerol, mannitol, mannose,
7
carbon, acid amin. Tt c cc loi l Gram m v m tnh cho s trao i cht ca di- v
trisaccharides. ND, Khng xc nh.
Herbaspirillum
sp.
Herbaspirillum
seropedicae
Herbaspirillm
rubrisubalbicans
Herbaspirillum
frisingense
15 500607
15500607
141805
07
ND
ND
ND
ND
ND
ND
34
30
3037
ND
ND
53 80
57 68
6070
ND
ND
L-Rhamnose
meso-Erythritol
61 65
+
ND
+
61
c im
3a
3b
c tnh sinh l
Nhit tng trng ti
u (C)
pH tng trng ti u
C nh N2
Ngun Carbon s dng
N-Acetyl-Dglucosamine
meso-Inositol
Adipate
Hm lng G + C ADN
(mol%)
64 65
62 63
Bng 3. Mng mng hnh thnh trong mi trng bn rn khc nhau bi cc dng Herbaspirillum c
ngun carbon khc nhau v khng c ngun nit tr trng hp quynh.
+ + +, Mng mng dy trn b mt mi trng; + +, Mng mng hnh thnh di bmt mi trng
vi mm;+, Mng rt mng rt nh bn di b mt; , khng c tng trng; +N, b sung cc
NH4Cl(1 g1-1).
Ngun carbon
Herbaspirillum
seropedicae
Z67T
Herbaspirillum
rubrisubalbicans
LMG2286T
Herbaspirillum
frisingense
GSF30T
Herbaspirillum
frisingense 84B
Malate
+ + +*
+ + +*
+ + +*
+ + +*
D-Glucose
+++
+++
++
++
N-Acetyl-Lglucosamine
+ + +*
+ + +*
+ + +*
L-Arabinose
+++
+++
+++
+++
Mannitol
+++
+++
+++
+++
D-FructoseN
+++
+++
+++
meso-Inositol
++
+ +*
+++
+++
D-Fructose
L-Tartrate
L-Rhamnose
meso-Erythritol+N
+
+++
Kh nng c nh m
Loi Herbaspirillum frisingense sp. nov chng Mb11 c kh nng kh acethylene
thnh ethylene vi t l l c 108 t bo s to ra 130 nmol acethylen trong 1h
nhit 300C. S liu trn hon ton ph hp vi kh nng bin i kh acetylen ca
loi Herbaspirillum seropedicae 370C (Baldani et al., 1986).
Tp hp ca phn lp 8 loi Herbaspirillum (Mb1, 11, 13, 14, 16, 19, 20, 22) v kiu
chng LMG 2286T ca loi Herbaspirillum rubrisubalbican v LMG 6513T ca loi
Herbaspirillum seropedicae c khuych i gen c nh m bng PCR. Sau khi
in di s phn tch cc sn phm trong 1% gel agarose to thnh mt bng ring bit
vi 390bp quan st c, nh mong i (Stolzfus et al., 1997) ( khng th hin s
liu).
V tr pht sinh loi c phn tch bng s liu trnh t 16S rADN
Phn tch trnh t 16S rARN pht hin c rng Herbaspirillum seropedicae (LMG
6513T), Herbaspirillum rubrisubalbicans (LMG 2286T) v phn lp c mt nhm
c kiu chng gm Mb11, GSF30T v 75B l mt nhm c quan h gn gi vi phn
lp beta ca Proteobacteria (cng vi Oxalobacter formigenes chng minh bi
Sievers et al., 1998). Cy pht sinh loi c minh ha trong hnh 2 th hin v tr
10
11
Hnh 2. Cy pht sinh loi phn nh mi quan h ca Herbaspirillum frisingense sp. nov vi
nhng chng khc ca phn lp bta ca Proteobacteria . Cy c cu trc bi phn tch
hp l ti a da vo trnh t 70 16S rADN ca phn lp beta. Ch c trnh t sp xp c
tn tr li chia s ph bin t hn 50 chng ca phn lp beta ca vi khun chn hch
bao gm vic tnh ton ca thc vt ny. Vi li ch thy, ch c 1 la chn duy nht v
trnh t tham kho c hin th . Mt thanh cho thy c lng khong 10% nhng thay i
c bn. chng v s lng c nhp vo hin nay trong thc vt ny l Iodobacter fluviatlis
ATCC 3305T, M22501; Chromobacterium violaceum ATCC 12472T, M22510; Ralstonia
solanacearum CIp 238, U28226; Ralstonia pickettii PKO1, L37367; Pseudomonas lemoignei
LMG 2207T, C92555; Janthinobacterium lividum DSM 1522T, Y08846; Duganella
zoogloeoides IAM 12670, D14256; Telluria mixta ACM 17, X65589; Telluria chilinolytica
ACM 3522T, X65590; Ultramicrobacterium ND5, AB008506; Ultramicrobacterium D-6,
AB008504; Oxalobacter formigenes ATCC 35274T, V49757; Herbaspirillum
rubrisbalbicans LMG 2286T238356; H. frisingense Mb11, AJ238359; H. frisingense
GSF30T. AJ238357; Herbaspirillum seropedicae Z67T, AJ238361; Burkholderia cepacia
ATCC 25416T, M22518; Alcaligenes defrangrans 54Pin, AJ005447; Bordetella avium ATCC
35086T, U04947; Azoarcus tolulyticus Td15, L33688; Spirillum volants ATCC 19544T,
M34131; Comamonas testeronic ATCC 11996T, M11224.
12
100
91
99
96
100
105
100
93
110
100
96
93
76
94
100
70
95
93
110
ND
100
ND
94
73
80
86
84
100
73
82
ND
83
76
ND
ND
100
ND
66
ND
ND
81
62
91
100
110
60
65
72
63
102
66
100
88
100
100
21
31
11
29
19
34
ND
21
17
14
100
15
25
6
18
10
21
13
8
8
1
9
100
0
0
0
0
0
0
0
0
0
0
0
0
100
Vi khun*
Hsero
445
Hfris
445
(35% FA)
(35%
FA)
Hrubri
445
(60% FA)
(35%
FA)
(50%
FA)
Mb1
++
++
++
Mb10
++
++
++
Mb11
++
++
++
HERB
1432
HERB 68
14
Mb13
++
++
++
Mb14
++
++
++
Mb19
++
++
++
Mb20
++
++
++
Mb22
++
++
++
GSF23
++
++
++
GSF24
++
++
++
GSF25
++
++
++
GSF28
++
++
++
GSF30T
++
++
++
GSF34
++
++
++
13B
++
++
+ +
60B
++
++
++
75B
++
++
++
84B
++
++
++
103B
++
++
++
LMG 2286
++
++
++
LMG 1278
++
++
++
LMG 6415
++
++
++
LMG 6416
++
++
++
LMG 6420
++
++
++
LMG 6513
++
++
++
LMG 2284
++
++
++
++
++
++
++
++
++
LMG 5523
++
++
LMG 5321
++
++
LMG 2285
++
++
ND
ND
ND
ND
Herbaspirillum rubrisubalbicans
Herbaspirillum seropedicae
Herbaspirillum species 3
15
PCR fingerprinting
PCR du vn tay( PCR fingerprinting) l cng c ph bin nhn bit s khc nhau
mc ADN ca nhng dng (Rademaket & De Bruijn, 1997). Vi phng php
ny c th quyt nh mi quan h gia cc dng c phn lp v m t s a dng
di truyn ca chng (Versalovic et al., 1994). S thay i a dng ca nhng dng
phn lp c, c phn tch bi k thut PCR du vn tay bng vic s dng trc
tip trnh t oligonucleotide c ngun gc sinh vt chn hch LINEs v cho rng trnh
t oligonucleotide c bo ton tt c cc t bo (Smida et al., 1996; Kirchhof
et al., 1997). Vi cng ngh cao ny th nhng mu bng tng t ca sn phm PCR
ca Herbaspirillum c phn lp t Miscanthus hoc Pennisetum, tng t, c th
c tm thy (Kirchhof et al., 1997). Khi tip tc phn lp Miscanthus th phn lp
c (Mb1, 11, 16, 20) cng nh vi chng Herbaspirillum rubrisubalbicans v
Herbaspirillum seropedicae cng bao gm, nhng mu bng vi vi s thay i c
th c quan st (Hnh. 3). Da vo ngy phn lp v ngun gc thc vt nhng
nhm vi khun to thnh mt nhm nht nh. Tt c cc th h c phn lp t
Miscanthus (Mb1, 11, 16, 20 v GFS24, 28, 30T, 35) c ba bng c kch thc ging
nhau ngoi tr 8 hoc 9 nhm tng ng c nhn bit r rng cc sn phm
khuch i. Herbaspirilllum frisingense c phn lp t Pennisetum cho thy mu
khng c s bin i r rng, nhng nhng mu ca chng th khc vi mt s loi
phn lp c t Miscanthus (Hnh 3). Du vn tay ca Herbaspirillum
rubrisubalbicans v Herbaspirillum seropedicae chng minh nhng chng ny c s
khc bit r rng vi Herbaspirilllum frisingense phn lp c. Nhng tm kim xa
hn cho bit rng s a dng di truyn ca nhng chng vi khun c th tng ng
vi ngun gc thc vt ca chng. Nhng iu kin chn lc ca mi trng sng ni
sinh cng nh vic ti sn xut thng qua thn r ca thc vt cho si c th gy ra
nhng nh hng nht nh n s thay i di truyn trong s nhng chng ca
Herbaspirilllum frisingense c pht hin .
Hnh 3. Inter-Line PCR fingerprints ca chng Herbaspirillum ssp. Lanes: S, 123 bp ; t 112, nhng chng Herbaspirillum frisingense theo th t t Mb1, Mb11, Mb16, Mb20,
16
17