1

Herbaspirillum frisingense sp., mt loi vi khun c nh m mi c tm thy trong thc vt C4-cho si.

S phong ph ca vi khun c nh m t thc vt C4 x, m cc thc vt C4 bao

gm Spartina pectinata, Miscanthus sinensis, Miscanthus sacchariflorus v

Pennisetum purpureum, trong mi trng bn rn vi nit t do ta c th phn lp

c chng Herbaspirillum trong s cc chng vi khun c nh m trong khng kh

khc. Trn c s c tnh sinh l, s phn tch pht sinh ging loi bng s so snh

trnh t 16S rDNA v phng php lai DNA-DNA ca nhim sc th v DNA ca

loi mi phn lp, n c th c nhm li vi nhau trong mt loi mi vi tn

xut l Herbaspirilum frisingense sp. nov. c im hnh thi, hnh dng v kch

thc t bo, hnh dng khun lc, kh nng di ng v chuyn ng bng roi phn

ln ging vi cc loi bit nh Herbaspirillum rubrisubalbicans v Herbaspirillum

seropedicae. Trn c s s dng adipate (-), N-acetyl-D-glucosamine (+), meso-

erythrito (-), L-rhamnose (-) v meso-inositol (-), ta c th phn bit Herbaspirillum

frisingense sp. nov. vi cc dng Herbaspirillum spp bit khc. Kh nng c nh

nit c kim tra bi s khuch i gene nifD bng PCR v th nghim kh acetylen,

v cn c vo s hin din ca chng trong tt c cc kim tra phn lp. S ging

nhau ca trnh t 16S rDNA ca cc dng Herbaspirillum khc nhau l 98,5 99,1%.

Th nghim lai DNA-DNA trong b gene ca nhng dng Herbaspirillum frisingense

sp. nov. vi nhau to thnh mt nhm ng nht vi t l lai l 70 100 10%, v

c s khc bit r rng khi lai Herbaspirillum frisingense vi Herbaspirillum

seropedicae v Herbaspirillum rubribalbicans vi t l lai ch 1- 34% . on d

oligonucleotide 16S rRNA mc tiu, c trng cho c chi Herbaspirilllum v 3 loi

Herbaspirillum c thit k v rt ph hp cho s pht hunh quang trong phng

php lai lai vi vi sinh vt. Hm lng G + C ca Herbaspirillum frisingense sp. nov.

l 63 2mol %, tha mn vi gi tr ca 61 63% ca chi ny. PCR du vn tay (PCR

fingerprinting) cho thy nhng mu ph hp vi nhng nhm c phn lp t cng

mt cy, cho thy s a dng di truyn thp trong nhng vi khun sng trong m thc

vt C4 thn c. S a dng di truyn thp c v ph bin v c th xy ra gia cc

chng vi khun sng trong mt cy khc ca cng mt n v phn loi. Kiu ca

chng Herbaspirillum frisingense sp.nov. l GSF30T (=DSM 13128T).

GII THIU

Loi Herbaspirillum ln u tin c bo co bi Baldani et al.(1986) l mt loi c

lin quan n vi khun c nh m r ca thc vt la (Oryza sativa), bp (Zea

mays), v sorghum (Sorghum bicolor). giai on ny ch nghin cu c loi

Herbaspirillum seropedicae. Sau ny, th nghin cu cc tc nhn gy bnh nh

thc vt ma, v trc kia n c tn gi l Pseudomonas rubrisubalbicans, sau ny

c gi l Herbaspirillum rubrisubalbicans (Baldani et al., 1996). C hai loi ny

u c chng minh l c tm thy r v thn ca thc vt ma, sorghum v la,

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n c kh nng c inh m (Pimentel et al., 1991; Ureta et al., 1995). Loi

Herbaspirillum seropedicae cng c tm thy cc thc vt h c khc(Baldani et

al. 1992; Olivares et al. 1995). Loi th 3, loi Herbaspirillum th 3 c phn loi

da trn c s th nghim lai ADN-rARN v ADN-ADN (Gillis et al., 1990; Baldani

et al., 1996). C s phn lp rt khc nhau, ngun gc khng hon ton gy bnh v

khng th c nh m. Loi herbaspirillum rubrisubalbican c kh nng to khun

lc h thng mch ca thc vt sorghum v thc vt ma gy ra cc triu chng nh

ph thuc vo s a dng ca thc vt ch (Olivares et al., 1997; Jame et al., 1997).

Quan st c hai loi u khng th tn ti trong t khng trng trt (Baldani et

al., 1992), nhng tm thy b mt v trng m thc vt vi s lng ng k,

dn n kt lun loi Herbaspirillum spp. l vi khun ni sinh (Jame & Olivares,

1997).

Mt s ging ma ng gp 40-60% nit t vic c nh m sinh hc( Urquiaga et

al., 1992; Boddey et al., 1995; Yoneyama et al., 1997). i vi thc vt trng khc,

nh sorghum (Giller et al., 1986), bp (Boddey 1987) v la (Baldani et al 1995)

a vo 20- 30% nit t vic c nh m sinh hc. iu vn cha chng minh

c vi khun ni sinh gp phn c nh m nh th no, iu ny cn thit cho

s trao i cht dinh dng thc vt v do n s c li cho s tng trng ca

thc vt (Jame & Olivares, 1997). Cng c th, theo nhng c ch khc, n c th sn

xut hoocmon thc vt ( Bastian et al., 1998), c th chu trch nhim trong vic thc

y s pht trin thc vt, kt qu nng sut s cao hn nguyn liu thc vt

(Baldani et al., 1995). S sn xut sinh khi ng k ca thc vt C4 nh Miscanthus

spp., Pennisetum purpureum v Spartina pectinata, c trng sn xut si, v cc

nng lng ti to (Hotz,1993; Klug & Ofth, 1997), kt hp vi nhu cu m tng

i thp (Hotz et al., 1993) khuyn khch chng ti khm ph v nhng thc vt

ny kt hp vi vi khun c nh m. S dng mi trng bn c vi nit t do

(Dbereiner., 1995) dn n vic phn lp c nhng vi khun c nh m khc

nhau. Mt nhm th hin nhng c im ca Herbaspirillum (Kirchhof et al.,

1997). Nhng phn lp ny a ra c vn y mnh vic phn tch s phn

loi v a ra miu t mt loi spirillum mi, n khc bit vi nhng loi

Herbaspirillum c m t.

PHNG PHP S phn lp nhng chng. Ra r v b phn pha trn (thn, l) ca Miscanthus sinensis cv. Giganteus, Miscanthus sacchariflorus, Spartina pectinata trng Freising, c, v Pennisetum purpureum cv. Cameroon, Cana dAfrica, Gramafante, Guau, Merker, Merker x 239, Mineiro, Mott, Piracicaba, Roxo, s/pelo v Taiwan

c trng Brazil, c ngm t. Pha long ln lt trong dung dch sucrose 4% (w/v) xung 10-6 sau cy vo l nh c mi trng bn c c nit t do c cha NFB hoc JNFb, theo Dbereiner (1995). Sau 4-6 ngy 30oC lp mng lng ca vi khun c to thnh tip theo n tri qua cc bc lm sch bng cch cy thnh vch trn nhng a mi trng thch NFB v JNFb c cha mt thm 20 mg

3

chit xut nm men l-1 v cc khun lc n mt ln na cy chuyn mi trng bn rn c cha m t do thch hp. Xc nh nhng chng phn lp c v cc ngun m thc vt c lit k trong bng 1.

Bng 1. Ngun gc cc chng vi khun.

Spartinapectinata and Miscanthusspp. c trng Freising Germany, trong khi

Pennisetumpurpureum c trng Seropedica, Brazil. DSM, Deutsche Sammlungvon

Mikroorganismen und Zellkulturen, Braunschweig, Germany.

Phn lp Thc vt M

Herbaspirillum frisingensesp.nov.

GSF3 S.pectinata R c ra sch

GSF36 S.pectinata Thn c ra sch

GSF20 M.sinensiscv.Giganteus Thn c ra sch

GSF28 M.sacchariflorus R c ra sch

GSF22, 24, 27, 35

GSF23, 25, 30T

( =DSM13128T), 34

M.sacchariflorus

M.sacchariflorus

Thn c ra sch

L c ra sch

Mb1,9,10,11 (=DSM13130), 13, 14, 16, 19, 20, 22

M.sacchariflorus R c ra sch

70B ,74B ,96B 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

65H, 73B, 90B, 101H, 121B P.purpureumcv.Merker239 R c ra sch

11B P.purpureumcv.Mineiro 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

60B, 69B, 122B 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

84B ( = DSM13129), 103H, 113B P.purpureumcv.Taiwan Thn c ra sch

67B, 117B P.purpureumcv.Taiwan R c ra sch

Herbaspirillum seropedicae

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

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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, L-rhamnose, 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 oC 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.

5

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 oC. 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 (Gibco-BRL). 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 98.4

oC. 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

6

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 (LMG6513

T) 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,

7

cc c tnh ca vi khun c nh m c tm thy. C 4 k thut phn lp c th

nhn dng c Herbaspirillum seropedicae. Mt k thut phn lp khc c a ra

l da vo hnh thi mu trng c ca khun lc v trung tm l mu xanh lam trn

a mi trng thch NFb (vi 0,5 % dung dch bromthymolblue c cho vo).

Vic da vo mu sc trung tm ca khun lc khng c nh gi cao trong so

snh vi nhng ging Herbaspirillum bit. Di knh hin vi th c th kim tra v

pht hin c t bo vi khun gram m, di ng, hnh que mng. Vic kim tra bng

knh hin vi in t truyn sut (TEM) cho thy rng Herbaspirillum frisingense l

mt t bo c hnh dng xon c c t 1 n 3 chin mao, ch yu l 2 chin mao, c

chin mao mt u ca t bo (Hnh.1). ng knh t bo t 0.5-0.7m v chiu

di t 1.4 -1.8 m. m t bo ny c th c quan st a cha mi trng thch

mm NB.

Hnh 1. Hnh nh ca nhng t bo Herbaspirillum frisingense sp.nov. di knh hin

vi in t truyn sut c nui cy trong mi trng DYGS.

(a) T bo ang phn chia; (b) T bo c 2 chin mao.

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, L-

arabinose, D-fructose, L-fucose, galactose, D-glucose, glycerol, mannitol, mannose,

(a) (b)

8

ribose, sorbitol, D-xylose v xylitol b oxi ha. Di v trisaccharides, e.g cellobiose,

lactose, maltose, sucrose, melibiose, tre-hase and raffinose, th khng chuyn ha.

Acid hu c nh acetate, cis-aconitate, caprate, citrate, formate, galacturonate,

gluconate, -hydroxy-butyrate, p-hydroxyphenyacetate, itaconate, -keto-butyrate, -

ketoglutarate, DL-lactate, malate, malonate, propionate, phenylacetate, D-saccharate,

succinate v acid succinamic u c chuyn ha trong phn ln (>80%) trong cc

loi c kim tra. S oxi ha acid bo, c kim tra bng TWEEN 40 v 80, th cho

kt qu dng. Cc amino acid D-v L-alanine, L-asparagine, L-aspartate, L-

glutamate, L-leucine, L-proline, v L-pyroglutamate th cng c chuyn ha.

Phn ng c trng ca nhng loi lin quan- khc nhau v c cht (Baldani et al.,

1996) c kim tra bi API20NE (10 loi) v API50CHE (4 loi) c lit k

bng 2. Vi lu l meso-erythytiol, L-rhamnose, meso-inositol, N-acetyl-D-

glucosamine v adipate th c mt loi mi c mu khc bit so vi cc loi

Herbaspirillum khc.

Vi khun hiu kh sinh trng trong mi trng cht dinh dng khng c thnh phn

nit (ngoi tr cht dinh dng l N-acetyl-D-glucosamine) vi ngun carbon khc

nhau cho kt qu l mt mu hu ch duy nht. Tt c cc th nghim phn lp to

thnh lp mng mng vi L-arabinose, N-acetyl- D-glucosamine, D-fructose, D-

glucose, malate, manitol, v L-tartrate (ngoi t chng 84B). Chng khng th to

mng mng nu meso-inositol hoc L-rhamnose c s dng lm ngun carbon.

Trong nhng th nghim , s khc nhau trong chuyn ha ngun cacbon c th

c dng so snh kiu chng Herbaspirillum seropedicae (LGM 6513T ) v

Herbaspirillum rubrisubalbicans (LMG 2286T) c th quan st c. Trong s tng

quan ca cc thc vt cho si phn lp c v Herbaspirillum rubrisubalbicans

LMG 2286T, Herbaspirillum seropedicae LGM 6513

T to c mng mng sinh

trng vi meso-inositol v L-rhamnose. Loi Herbaspirillum rubrisubalbicans LMG

2286T

thiu cc thuc tnh nu N-acetyl- D-glucosamine c a ra lm ngun

carbon. Sinh trng vi meso-erythritol v cho thm NH4Cl th ch c tm thy

Herbaspirillum rubrisubalbicans LMG 2286T.

Nhit tng trng ti u c th nghim 25, 30, 37 v 420 C. Trong dy nhit

ta pht hin c cc loi sinh trng giai on ly tha l gia 30 v 370C ,

v sinh trng yu t 420C, iu ph hp vi cc loi khc ca chi. Trong th

nghim v iu kin pH, phn tch loi Herbaspirillum frisingense sp. nov. phn lp

c GSF3, GSF30T, 75B v 84B, cng nh loi Herbaspirillum rubrisubalbicans

LMG 2286T, a iu kin pH ti u kh acetylen l pH 6.0 v 7.0.

Bng 2. c im hu ch cho vic xc nh cc vi khun thuc chi Herbaspirillum v

phn bit Herbaspirillumfrisingense sp. nov. t cc loi bit khc.

Thng s chi tit cho Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans v

Herbaspirillum sp. 3 c m phng t Baldani et al. (1986, 1996) v Gillis et al. (1990). Tt

c cc loi dng tnh vi oxidase, catalase v s trao i cht ca cc loi ng, cn, axit

9

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.

c im Herbaspirillum

seropedicae

Herbaspirillm

rubrisubalbicans

Herbaspirillum

frisingense

Herbaspirillum

sp.

3a 3b

Hnh thi hc/di ng

Kch thc t bo(m) 15 500607 15500607 141805

07 ND ND

Roi ND ND

+ + + ND ND

c tnh sinh l

Nhit tng trng ti u (C)

34 30 3037 ND ND

pH tng trng ti u 53 80 57 68 6070 ND ND

C nh N2 + + +

Ngun Carbon s dng

N-Acetyl-D-

glucosamine + + +

meso-Inositol +

L-Rhamnose +

meso-Erythritol

Adipate + +

Hm lng G + C ADN (mol%)

64 65 62 63 61 65 ND 61

10

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-L- glucosamine + + +* + + +* + + +*

L-Arabinose + + + + + + + + + + + +

Mannitol + + + + + + + + + + + +

D-Fructose + + + +

D-FructoseN + + + + + + + + + + + +

meso-Inositol + +

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 2286

T) 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

11

nhng loi c mi quan h vi th h dng chnh trong phn lp beta ca

Proteobacteria. Cy c xy dng bi phng php hp l ti a phn tch 70

trnh t 16S rARN ca cc thnh vin trong phn lp beta. Cu trc lin kt ca cy

pht sinh c xc bng cch s dng phng php phn tch khong cch (distance

methods) v h tin ti a (maximum- parsimony). Cu trc lin kt ca cy c

nhn thy rng v tr ca vi khun oxalobacter formigenes khng b nh hng bi

nhng h thng biu hnh cy khc nhau.

Da vo gi tr tng th ca nhng trnh t ging nhau t 98.5 n 99.4% ca nhm

herbaspirilla v s khc bit r rng vi nhng loi c quan h gn nht, nh chng

Ultramicrobacterium D-6 v ND5 (Iizuka et al., 1998) c trnh t ging vi nhm

herbaspirilla l 95.8-97.3%. Loi c quan h xa hn, Janthinobacterium lividum v

Oxalobacter formigenes, c trnh t 16S rADN ging nhau chim 95.4-96.2% tng

t vi 94.6-96.4% vi trnh t nghin cu vi loi Herbaspirium spp. Trong trnh

t 16S rADN Herbaspirillum ca chng GSF30T, Mb11 v 75B, phn lp c

m si thc vt , 1 nhm kn c hnh thi ging nhau vi 98.9 v 99.4%. Nhng

chng ny cng c trnh t ging so vi Herbaspirillum seropedicae (LMG 6513T) l

98.7-99.1% . Mc trnh t ging nhau ny khng th a ra kt lun n l mt loi

mi ca chng Herbaspirillum (Strackebrandt v Goebel, 19994) nhng th nghim

lai ADN-ADN v kim tra sinh l cho thy rng chng GSF30T, Mb11 v 75B to

thnh mt loi mi ring bit c xut l Herbaspirillum frisingense sp. nov.

12

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 12472

T, M22510; Ralstonia

solanacearum CIp 238, U28226; Ralstonia pickettii PKO1, L37367; Pseudomonas lemoignei

LMG 2207T, C92555; Janthinobacterium lividum DSM 1522

T, 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 Z67

T, AJ238361; Burkholderia cepacia

ATCC 25416T, M22518; Alcaligenes defrangrans 54Pin, AJ005447; Bordetella avium ATCC

35086T, U04947; Azoarcus tolulyticus Td15, L33688; Spirillum volants ATCC 19544

T,

M34131; Comamonas testeronic ATCC 11996T, M11224.

Lai ADN-ADN v c s thnh phn ADN

chng minh chc chn v mi quan h tn ti trong nhng loi Herbaspirillum c

kh nng c nh m, ta thc hin th nghim lai ADN-ADN la chn 10 chng c

phn lp t Miscanthus v Pennisetum, kim tra cc kiu chng Herbaspirillum

seropedicae (LMG 6513T) v Herbaspirillum rubrisubalbicans (LMG 2286

T) v

Escherichia coli JM83. S bin i hon ton ca vic tnh ton gi tr lai b gen vi

phng php m t trn c c tnh l 10%. V vy, nhng sinh vt c ADN

ging nhau chim khong 60 n 110% c xem nh l thuc v 1 loi. Do , c

ba nhm r rng ( ngoi tr E.coli vi phng php lai hon ton chim 0%) c th

c nhn bit (bng 4). Nhim sc th ADN ca Herbaspirillum rubrisubalbicans

LMG 6513T cho thy 11-34% lai vi thc vt mi phn lp v vi Herbaspirillum

rubrisubalbicans LMG 2286T. Gi tr lai b gen ca nhim sc th ADN

Herbaspirillum rubrisubalbicans LMG2286T ging vi ADN ca cc cc chng khc

dao ng t 1-25%. Chng GSF23, 24, 25, 28, 30T v 35 c phn lp t Miscanthus

spp v 60B, 75B, 84B v 118B c phn lp t Penisetum purureum to thnh

mt nhm th 3 vi gi tr lai t 60 n 110 % v do c th c nhm thnh mt

loi mi.

ADN c hm lng G+C ca Herbaspirillum frisingense sp. nov c tnh ton t kt

qu bin tnh ADN bng nhit l 632mol%, ph hp vi gi tr ca 60-65% ca chi

Herbaspirillum.

13

Bng 4. DNA-DNA lai (t l gen lai)

ND, Khng xc nh

GSF23 GSF24 GSF25 GSF28 GSF30

T

GSF35 60B 75B 84B 118B H.ser.

LMG

H.rub.

LMG

E.coli

6513T 2286T

GSF23 100 91 96 100 96 70 ND 73 ND 60 21 15 0

GSF24 99 100 93 93 95 94 82 66 65 31 25 0

GSF25 105 110 76 93 73 ND ND 72 11 6 0

GSF28 100 94 110 80 83 ND 63 29 18 0

GSF30T 100 ND 86 76 81 102 19 10 0

GSF35 100 84 ND 62 66 34 21 0

60B 100 ND 91 100 ND 13 0

75B 100 100 88 21 8 0

84B 110 100 17 8 0

118B 100 14 1 0

H.ser. LMG 6513T 100 9 0

H.rub. LMG 2286T 100 0

E.coli 100

Thit k v ti u ha vic xc nh u d oligonucleotide.

Nhng u d oligonucleotide 23S rARN-ch c trng cho Herbaspirillum

rubrisubalbicans (HR) v Herbaspirillum seropedicae (HS) khng cho php xc nh

r rng v phn bit vi cc chng mi. Trong th nghim lai ARN thm tch im

(dot- blot), ch c mt phn nh Pennisetum c phn lp c xc nh vi cc loi

bit ca Herbaspirillum seropedicae (Kirchhof et al.,1997). Phn ln c phn lp t

r ca Misthuscans sacchariflorus (Mb1, 9, 11, 13, 14, 16, 19, 20, 22) cho thy nhng

tn hiu tch cc vi u d HR (Eckert et al.,1998). Vng trnh t 23S rADN ca

chng Mb11, n c s dng nh l u d mc tiu, th hin vic xc nh cc

thnh phn baz b sung nh u d HR. u d 23S rADN- ch mi, l beta 20,

phn ng vi rARN ca Herbaspirillum - c phn lp khng c lin quan n mt

trong s nhng loi bit, iu c trnh by bi Krichhof et al (1997).

Nhng khm ph su hn, bao gm c im sinh l v vic so snh trnh t 16S

rADN ( trnh by trn) cho bit mi quan h mt thit ca nhng loi c phn

lp t Misthuscans, rARN ca n lai vi u d ca Herbaspirillum rubisbalbicans v

nhng chng c phn lp t Misthuscans v Pennisetum m rARN ca n gn vi

u d beta 20. V th, s thay i qu mc ca u d HR mc tiu tri di trong

vng III ca 23S rADN l khng c trng loi Herbaspirillum rubrisubalbicans v

c thay i loi Herbaspirillum frisingense sp. nov. c phn lp.

Da trn cng c thit k u d ca gi phn mm ARB, s ko di nht nh ca

trnh t 16S rADN c kim tra cn c vo s ko di trong thnh phn ca baz

c trng cho chi Herbaspirillum, bao gm hai loi bit l Herbaspirillum

rubrisubalbicans v Herbaspirillum seropedicae v mt loi mi l Herbaspirilllum

14

frisingense. Hai chi v ba loi c u d oligonucleotide c trng cho hai loi

bit nh Herbaspirillum rubrisubalbicans v Herbaspirillum seropedicae, cng nh

i vi tt c cc loi mi c phn lp, Herbaspirillum frisingense, c thit k

v ti u ha trong th nghim lai ti ch(Amann et al., 1995; bng 5). S c trng v

tnh nhy ca u d c nh gi bng phng php lai vi hn hp

paraformaldehyde- c nh t bo ca herbaspirilla v cc c tnh sinh l c lin

quan n s phn loi pht sinh loi. Nhng iu kin cht ch ca phng php lai

c iu chnh bi s tng ln t t nng formamide trong dung dch m lai

trong khi phi gi nng ion (0,9M NaCl) v nhit lai khng i. Hai chi - vi

nhng u d c trng l HERB 68 v HERB 1432, c b sung vo nhng vng

khc nhau ca 16S rARN v lai trong iu kin lai nghim ngt vi tt c cc thnh

vin ca chi Herbaspirillum (Bng 6). Nhng loi-vi on d c trng, l Hsero

443, Hrubri 443 v Hfri 443, l mc tiu nhn bit vng ging nhau ca 16S rARN

(E. coli c v tr 445-462). N cng c xem nh l cng c nhn bit s khc

bit Herbaspirillum rubrisubalbicans, Herbaspirillum seropedicae v Herbaspirillum

frisingense sp. nov. mc loi. Mt nhm gm herbasppirilla th 3 khng c kh

nng c nh nit (Baldani et al., 1996) c th c xc nh vi nhng u d c

trng nh HERB 1432 v HERB 68, nhng khng phi bt k loi no cng c u

d c trng (Bng 6). Nhng khm ph su hn ny cung cp nhng gi nh cho

rng nhng thnh vin ca nhm ny to thnh mt loi nht nh, iu c ra

bi Gillis et al. (1990) v Baldani et al. (1996). Vic ng dng nhng u d mi ca

Herbaspirillum cho php xc nh mt cch nhanh chng v thun li nhn bit

nhng chng c lin quan v phn lp nhng nhm gn gi vi chi ny.

Bng 6. Kt qu ca ton b t bo lai ti ch vi nhng u d oligonucleotide 16S rARN-

ch trong iu kin lai nghim ngt.

Kt qu cc u d m tnh vi cc loi sau: Agrobacterium rhizogenes DSM 30148T,

Alcalignes faecalis LMG 1229T, Alcaligenes ruhlanddi DSM 653

T, Azospirillum

amazonense DSM 2784T, Brevundimonas diminuta DSM 1635

T, Burkholderia

andropgonis LMG 2129T, Burkholderia brasilense M130, Burkholria caryophylli

LMG 2155T, Burkholderia cepacia LMG 1222

T, Burkholderia glumae LMG 2196

T,

Burkholderia plantarii LMG 9035T, Janthinobacterium lividum DSM 1522

T,

Neisseria elongata LMG 5124T v Ralstonia eutropha LMG 1199

T.

Vi khun* ud

HERB

1432

(35% FA)

HERB 68

(35% FA)

Hrubri

445

(60% FA)

Hsero

445

(35% FA)

Hfris

445

(50% FA)

Herbaspirillum frisingense phn lp t Miscanthus spp.

Mb1 + + + + - - + +

Mb10 + + + + - - + +

Mb11 + + + + - - + +

15

Mb13 + + + + - - + +

Mb14 + + + + - - + +

Mb19 + + + + - - + +

Mb20 + + + + - - + +

Mb22 + + + + - - + +

GSF23 + + + + - - + +

GSF24 + + + + - - + +

GSF25 + + + + - - + +

GSF28 + + + + - - + +

GSF30T + + + + - - + +

GSF34 + + + + - - + +

Herbaspirillum frisingense phn lp t Pennisetum

purpureum

13B + + + + - - + +

60B + + + + - - + +

75B + + + + - - + +

84B + + ++ - - + +

103B + + + + - - + +

Herbaspirillum rubrisubalbicans

LMG 2286 + + + + + + - -

LMG 1278 + + + + + + - -

LMG 6415 + + + + + + - -

LMG 6416 + + + + + + - -

LMG 6420 + + + + + + - -

Herbaspirillum seropedicae

LMG 6513 + + + + - + + -

LMG 2284 + + + + - + + -

16B(isolated from P.purpureum) + + + + - + + -

80B(isolated from P.purpureum) + + + + - + + -

Herbaspirillum species 3

LMG 5523 + + + + - - -

LMG 5321 + + ++ - - -

LMG 2285 + + + + - - -

Azospirillum brasilense DSM1690T - - ND -

Burkholderia vietnamiensis LMG 10929T - - ND - ND

Chromobacterium violaceum LMG 1267T - - - - ND

16

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 1-

12, nhng chng Herbaspirillum frisingense theo th t t Mb1, Mb11, Mb16, Mb20,

17

GSF24,GSF28,GSF30T,GSF35, 60B, 75B, 84B v 118B; 13-16, l cc kiu chng ca

Herbaspirillum rubrisubalbicans theo th t t LMG 2286T, LMG 6415, LMG6420 v LMG

1278; 17-18, l cc kiu chng ca Herbaspirillum seropedicae theo th t t LMG 6513T v

LMG 2284.

M t Herbaspirilllum frisingense sp.nov.

Herbaspirilllum frisingense (fri.sin.gense. L. gen. n frisingense ca Frisinga, ngy

nay c bit n nh l Freising, mt th trn c l ni m Spartina pectinata v

Miscanthus spp. c trng m t cc cy ny nhng sinh vt ny c phn lp u

tin). Nhng c im c li cho vic xc nh chi Herbaspirillum cng nh nhng

loi mi ca Herbaspirilllum frisingense sp.nov., c tm tt trong bng 2.

Nhng t bo hi cong, roi xon v c kh nng di ng. Hu ht cc t bo thng c

hai, i khi c mt hoc ba roi n v c th t li trn mi trng thch dinh dng

mm. ng knh t bo l 0.5-0.7m v di 1.4-1.8m . Nhng chng c th c

nh nit trong khng kh v trong iu kin vi hiu kh n pht trin tt vi N2 nh l

ngun nit duy nht. Nhit tng trng tt nht khi c mt nit l 30-370C. pH

tt nht pht trin l 6.0 -7.0. Catalase, oxidase, urease hot ng tch cc v

gelatin th khng b thy phn. NO3- bin i thnh NO2

- nhng khng thnh N2. S

trao i in hnh ca qu trnh h hp, oxi ha mt lot cc phn t ng v ru.

ng i v ng a khng c chuyn ha. Cc acid hu c c s dng nh

ngun cacbon. S oxi ha hon ton acid bo. V amino acid cng c s dng. S

pht trin ca cc vi snh vt vi hiu kh c theo di vi L-arabinose, N-acetyl-D-

glucosamine, D-fructose, D-glucose, malate, mannitol v L- tartrate v khng xy ra

vi meso-erythritol, meso-inositol v L-rhamnose. S ging nhau v trnh t 16S

rADN trong cng mt loi l 98,8-99,4%. Hm lng G + C ca ADN l 632 mol%.

Kiu chng l GSF30T (=DSM13128T); hm lng G + C l 64 mol%. Nhng sinh vt

ny l s kt hp vi r, thn, l ca thc vt C4 thn c nh Spartina pectinata,

Miscanthus sinensis, Miscanthus sacchariflorus v Pennisetum purpureum.