XC NH GEN M HA FLAGELLIN ARCOBACTER SPP. Characterization of the gene(s) encoding flagellin(s) in Arcobacter spp.

H Th Kim Hoa1, Len J. A. Lipman2 and Wim Gaastra2 Khoa Chn Nui Th Y, i Hc Nng Lm, Tp H Ch Minh 2 Faculty of Veterinary Medicine, Utrecht University, The Netherlands
1

Summary The aim of this study was to characterize the gene(s) encoding flagellin protein in Arcobacter spp., which are known as bacterial virulence factors. Like Campylobacter and Helicobacter spp., Arcobacter species possess two flagellin genes (flaA and flaB) located adjacent to each other. With the exception of A. nitrofigilis, arcobacter flagellins are almost half the size of those in other epsilonproteobacteria. Mutational study revealed a similar transcription pattern for the two fla genes in A. butzleri as in related bacteria. However, in the expression of the genes in Arcobacter spp. different sigma factors than 28- or 54 which are employed in other related bacteria seem to play a role. These results underline that the genus Arcobacter is an unusual taxon in the epsilon subdivision of the Proteobacteria. (Most of this manuscript is based on a publication by Ho at el. in FEMS Immunol. Med. Microbiol. 50: 51-58) Key words: Arcobacter; flagellin; motility; mutant; qPCR Tm tt Nghin cu ny c tin hnh nhm xc nh gen m ha flagellin ca vi khun Arcobacter, mt ging mi thuc h Campylobacteriaceae. Kt qu cho thy ging vi cc vi khun c h hng gn thuc nhm epsilon-proteobacteria, Arcobacter spp. c 2 gen m ha flagellin (flaA v flaB). Tuy nhin, ngoi tr A. nitrofigilis, cc phn t flagellin ca cc loi Arcobacter khc ngn hn (khong phn na) flagellin ca cc vi khun h hng. c bit, cc on bo tn thng thy ca cc sigma factor iu kin s phin m gen flagellin (28- or 54) khng c tm thy Arcobacter. C l s phin m cc gen flagellin Arcobacter spp. c iu khin bng mt h thng khc. Cc chng t bin gen flaA v flaB c to thnh v sau qPCR c thc hin nhm tm hiu vai tr ca cc gen ny. Kt qu cho thy ch c sn phm ca gen flaA gi vai tr ch yu to thnh flagella v kh nng di ng ca vi khun. GII THIU Ging Arcobacter, c cng b vo nm 1991, thuc h Campylobacteriaceae, gm nhng trc khun Gram m, khng bo t, di ng nh mt flagellum mt hay hai u t bo (Vandamme, 2000). Hin ti, ging Arcobacter c 6 loi, c mi trng sng rt a dng. A. nitrofigilis, loi in hnh (type species), l vi khun c nh m r mt loi cy mc m ly (McClung et al., 1983) v A. halophilus c pht hin mt h nc mn (Donachie et al., 2005). A. cibarius c phn lp t cc quy tht g (Houf et al., 2005). Ba loi cn li, A. butzleri, A. skirrowii v A. cryaerophilus, c tm thy trn cc sn phm c ngun gc ng vt (Kabeya et al., 2004; Scullion et al., 2006), trong phn ngi v ng vt bnh cng nh khe mnh (Anderson et al., 1993; Kabeya et al., 2003; On et al., 2002; Vandamme et al., 1992; Vandenberg et al., 2005). Do s a dng sinh thi ca cc loi Arcobacter, ging ny c xem l khc thng so vi cc thnh vin khc trong phn nhm epsilonproteobacteria. 47

Nghin cu v flagella chim phn ln trong cc nghin cu v Campylobacter v Helicobacter spp., hai vi khun gy bnh c h hng gn vi Arcobacter spp.. Flagella ca vi khun l mt trong nhng yu t c lc, gip vi khun gy bnh bm trn cc t bo nim mc v xm nhp vo c th vt ch. Flagellin, n v phn t cu thnh flagella l khng nguyn c nghin cu nhiu nht trn Campylobacter spp. (Guerry, 1997). Flagellin v gen m ha protein ny cn c s dng trong nhiu phng php chn on v phn loi vi khun (Winstanley and Morgan, 1997). Nghin cu ny c thc hin nhm xc nh gen m ha flagellin ca vi khun Arcobacter v tm hiu c ch iu khin s th hin gen ny. VT LIU V PHNG PHP NGHIN CU Vi khun, plasmid, v iu kin nui cy vi khun Tt c cc chng vi khun v plasmid dng trong nghin cu c lit k trong Bng 1. Cc Arcobacter c nui cy trong mi trng canh BHI (Brain Heart Infusion, Oxoid) c lc, hay thch BHI c b sung 5% mu nga, 30oC, trong iu kin vi hiu kh (dng ti sinh mi trng vi hiu kh, BD CampyPak, Becton, Dickinson and Company). Cc chng Escherichia coli c nui cy trong canh LB (lysogeny broth) (Bertani, 1951) hay thch LB 37oC. Trong qu trnh to t bin, cc khng sinh ampicillin (100 g/ml) hay chloramphenicol (25 g/ml) c thm vo mi trng LB. Phn lp protein flagellin Flagellin ca cc chng Arcobacter c tch chit bng phng php kt ta trong mi trng acid (acid pH disaggregation method) (Ibrahim et al., 1985). Canh khun nui cy qua m c ly tm (5000 g, 20oC, 30 pht). Kt ta c lm tan trong dung dch NaCl 0,9%. Sau khi iu chnh pH ca huyn ph ny ti pH 2,0 bng HCl 1M, huyn ph c ly tm 8000 g 4C trong 15 pht. Sau , phn dch trong c siu ly tm (100000 g, 4oC, trong 1 gi). Phn nc trong c thu li v c iu chnh ti pH 7,2 bng dung dch NaOH 1M. Protein trong dch c kt ta vi ammonium sulphate; kt ta c ha tan trong nc ct v dung dch ny c thm tch (dialysis) qua m 4oC. Dch thm tch cha protein flagellin c ct 70C. Bng 1. Cc chng vi khun v plasmid dng trong nghin cu Vi khun v plasmids
Bacterial strains A. butzleri LMG 6620 A. butzleri UU-K32 A. skirrowii LMG 6621 A. cryaerophilus LMG 7537 A. cibarius LMG 21996 A nitrofigilis LMG 7604 E. coli DH5, NCCB 2955 Plasmids pGEM-T Easy pAV35
* **

Ngun gc/ chc nng

Mu ca 1 ph n b cellulites Quy tht g Phn cu tiu chy ko di Thai cu b xy Quy tht g Loi in hnh Competent cells Cloning vector Plasmid c gen khng chloramphenicol (Cmr)

Ngun cung cp
BCCM* Phn lp ti phng th nghim BCCM BCCM BCCM BCCM NCCB** Promega van Vliet et al. (1998)

BCCM: The Belgian Co-ordinated Collections of Microorganisms. NCCB: The Netherlands Culture Collection of Bacteria.

48

in di trn polyacrylamide-SDS gel, xc nh chui a-xt a-min u N ca flagellin Dch tch chit cha flagellin c in di trn polyacrylamide SDS gel 12 %. Sau , protein c chuyn sang mng nitrocellulose (Schleicher & Schuell, BioScience) v nhum vi Coomassie Brilliant Blue. Mu protein ca chng vi khun no cho kt qu r nht trn gel s c ct ra v gi n Sequence Centre Utrecht (Faculty of Chemistry, Utrecht University, The Netherlands) xc nh chui a-xt a-min u N (N-terminal) ca protein flagellin. Trnh t chui a-xt a-min c phn tch bng chng trnh BLAST (The Basic Local Alignment Search Tool) ca NCBI (National Center for Biotechnology Information). Xc nh gen m ha flagellin (gen fla) ca Arcobacter spp. Vic xc nh gen fla ca cc chng Arcobacter c thc hin qua 3 bc: (i) PCR suy bin (degenerate PCR): Mt mi xui suy bin (degenerate forward primer) c thit k da trn chui a-xt a-min u N ca flagellin c xc nh phn trn v 1 s mi ngc suy bin (degenerate reverse primer) thit k da vo so snh (alignment) cc gen m ha cc flagellin thit yu ca cc vi khun c h hng gn vi Arcobacter: FlaA ca Campylobacter v Helicobacter spp., FlaG ca Wolinella succinogenes. Cc mi suy bin ny c dng cho cc phn ng PCR nhm thu c 1 on DNA (amplicon) ca gen m ha flagellin. Trnh t nucleotide ca on DNA ny s c xc nh. Trnh t a-xt a-min c suy ra v so snh (align) vi cc chui a-xt a-min ca cc flagellin ca Campylobacter spp., Helicobacter spp., v W. succinogenes, thit k cc mi suy bin cho cc phn ng PCR, nhm thu 1 on DNA (amplicon) ca gen m ha flagellin cc chng Arcobacter cn li. (ii) Southern blot hybridization: Southern hybridization c tin hnh nhm xc nh s lng gen m ha flagellin ca cc loi Arcobacter. DNA b gen (genomic DNA) ca cc vi khun Arcobacter c tinh sch, v c vi cc enzyme EcoR I, Hind III hay Pst I (New England Biolabs). Cc mu DNA c in di trn agarose gel, sau c chuyn sang mng Hybond-N+ (Amersham Biosciences) v c c nh (immobilized) nh thit b Stratalinker UV Crosslinker (Stratagene). Qu trnh lai DNA vi on d (probe) gn DIG c thc hin 60oC. Cc on d ny c tng hp bng cc phn ng PCR, dng cc mi c thit k trn cc on DNA xc nh trong bc (i) (Bng 2), v s dng PCR DIG Probe Synthesis kit (Roche). Anti-DIG-alkaline phosphatase antibody v NPT/BCIP (Roche) c dng c kt qu. Bng 2. Cc mi (primer) dng tng hp cc on d (probe), to cc chng t bin gen fla (fla mutants, v qPCR
M i DNA (5- 3) M i DNA (5- 3)
Tng hp cc on d (Southern hybridization) Probe-F agcagcagatgatgcttctgg Probe-R-BUTZ ttgattctctaccatcagctg Probe-R-SKIR ctgctggtagagttgctattg Probe-R-CRY tcagctatttgtaaaagagcaactg Probe-R-CIBA tagcttctctaccatcagcag To cc chng t bin gen flaA 6620-MF1 tgagaattaatacaaacgtttcatctt 6620-MR1 gagaacctaaccttttgctaagac 6620-BamHIF1 cgaggatccagtacagcacatgttttcc* 6620-BamHIR1 cgaggatcctaccagaagttgcatcagc* * V tr chui BamH I c gch di Quantitative PCR Arco-gyrA-F gaaagaaccagcagttcttcctaca Arco-gyrA-R cgccaagattatgaggtggaat 6620-flaF1 gcagcaacaggacaaactgaa 6620-flaR1 agctgattcaacttggttttgagtag 6620-flaF2 tctttaacaggtttagcggtttca 6620-flaR2 atatccattaagtgttgtgattgcacta To cc chng t bin gen flab 6620-MF2 agcagggtcttatgcgattagcc 6620-MR2 tggagcatttcttaatggagcaactcc 6620-BamHIF2 cgaggatccgatctaatacagcaggtttggct* 6620-BamHIR2 cgaggatcccgaagcagtagaagcagcacc*

49

(iii) Xc nh gen m ha flagellin: Da trn cc on DNA c xc nh trong bc (i), DNA walking c thc hin vi TOPO Walker Kit (Invitrogen) v cc enzyme Pst I hay Nsi I (New England Biolabs), nhm xc nh nguyn gen m ha flagellin ca cc chng Arcobacter. Xc nh trnh t nucleotide bng phng php cloning Trnh t nucleotide ca cc on DNA trong nghin cu ny c xc nh qua phng php cloning. Cc on DNA c chn vo pGEM-T Easy vector (Promega). Plasmid c bin np (transform) vo E. coli DH5 alpha (Bng 1) bng sc in hay bng sc nhit ty vo di ca on DNA. Sau , cc plasmid c phn lp, tinh sch (Miniprep, QIAGEN) v gi i xc nh trnh t nucleotide (BaseClear, The Netherlands). To cc chng t bin gen fla Cc chng t bin gen fla ca vi khun A. butzleri LMG 6620 c to ra bng phng php hon v a-len (allelic exchange method) (Wosten et al., 2004). Cc on DNA ca gen fla c khuch i bng PCR (cc mi c lit k trong Bng 2), sau c chn vo pGEM-T Easy vector. Cc plasmid s c dui thng (linearized) nh cc phn ng PCR ngc (reverse amplification) vi cc mi c on BamH I. Sau , cc plasmid thng ny c vi enzyme BamH I (New England Biolabs) v c kh pht-pho (dephosphorylated) bng enzyme calf intestine alkaline phosphatase (Fermentas). Sau cng, cc plasmid ny c ng kn vi on DNA cha gen khng chloramphenicol (Cmr) (c to thnh bng cch ct plasmid pAV35 (van Vliet et al., 1998) bng BamH I). Plasmid cha Cmr c bin np vo A. butzleri LMG 6620 bng cch sc in. Sau khi n nh trong mi trng canh SOC, vi khun c cy trn cc da thch mu BHI c b sung chloramphenicol (25 g/ml) v 30oC, 2 ngy trong iu kin vi hiu kh. Cc gc vi khun t bin (c gen fla b chn bi on DNA cha gen Cmr) c xc nh bng PCR v kh nng di ng ca cc gc vi khun ny c kim tra trn mi trng thioglycolate 0,4 % (Fluka). Quantitative PCR (qPCR) v kho st s th hin (expression) cc gene fla S phin m (transcription) gen m ha flagellin ca cc chng A. butzleri LMG 6620 (chng nguyn thy v t bin) c kho st thng qua phn ng qPCR. Vi khun c nui cy trong canh BHI 30oC hay 37oC trong cc iu kin -xy khc nhau (vi hiu kh hay hiu kh) cho ti gia sau giai on tng sinh (mid-log phase) (khi OD = 0,6 - 0,8). Ton b RNA ca vi khun c ly trch bng dung dch TRIzol (Sigma). i vi mi mu RNA, 3 g RNA c x l vi 60 l DNase I (Invitrogen) v 4 l mu x l c dng cho mi phn ng qPCR (25 l). One-step RT qPCR MasterMix Plus for SYBR Green I kit (Eurogentec) c s dng v phn ng c thc hin trn h thng ABI Prism 7000 sequence detection system (Applied Biosystems). Gen gyrA (gyrase A) c dng nh gen ti gia (housekeeping gene) tnh ton s n b cho sai bit ca lng mRNA gia cc mu khc nhau (thng tin chui gen gyrA ca cc chng Arcobacter c ly t NCBI database). Kt qu c tnh ton v trnh by bng s ln khc nhau ca mRNA (mRNA-fold difference) gia cc chng (nguyn thy v t bin) hay gia cc iu kin kho st, theo phng php Ct (Pfaffl, 2001). Mi phn ng qPCR c lp li 3 ln. Thng tin v cc chui a-xt a-min v DNA, v phn tch d liu Cc chui a-xt a-min v DNA dng trong cc so snh s tng ng thit k cc on 50

mi c ly t NCBI (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/). Chng trnh BLAST (The Basic Local Alignment Search Tool) (http://www.ncbi.nlm.nih.gov/blast/) c dng so snh s tng ng gia cc chui a-xt amin v DNA tm c vi cc chui c trong NCBI database. So snh s tng ng gia cc chui chui a-xt a-min v DNA c thc hin nh chng trnh ClustalW (http://align.genome.jp/). Cy ph h (phylogenetic tree) c thit k nh chng trnh Mega 3.1 (Kumar et al., 2004). Cc mi PCR (primer) c thit k nh chng trnh FastPCR (Institute of Biotechnology, Univerversity of Helsinki). S hiu trong Genbank (nucleotide sequence accession numbers) ca cc chui DNA cha gen fla tm c trong nghin cu ny Cc chui DNA cha cc gen fla tm c trong nghin cu ny c chuyn n Genbank v c accession numbers t EF667144 n EF667148 v EU056204. KT QU V THO LUN Xc nh gen m ha flagellin ca Arcobacter Trong cc mu protein ly trch ca cc chng Arcobacter, ch c mu protein ca chng A. butzleri UU-K32 cho thy 1 vch r rng, c trng lng phn t khong 36-43 kDa, trn polyacrylamide-SDS gel. Phn tch 20 a-xt a-min u N ca protein ny cho kt qu: M R I N T N V S S L T A Q E A A V N T N K N I S S S L E K. Chui a-xt a-min ny gn ging chui a-xt amin u N ca cc flagellin ca Helicobacter spp. v W. succinogenes. T kt qu ny, phn ng PCR suy bin c thc hin thu c on DNA u tin ca gen fla chng A. butzleri UU-K32. i vi u 5 (u N ca protein), mi xui suy bin c thit k da theo chui A Q E A A V N T va thu c. i vi u 3, mi ngc suy bin c thit k theo chui A Q A(S) G S Y A(S), chui a-xt a-min ny kh bo tn u C (Cterminal) ca cc flagellin ca Campylobacter spp., Helicobacter spp., v W. succinogenes. Kt qu PCR cho 1 sn phm c 840 nucleotide. Phn tch bng TBLASTX (search translated nucleotide database using a translated nucleotide query, NCBI) khng nh s tng ng ca chui nucleotide ny vi cc chui thuc gen fla ca cc vi khun h hng k trn. Mt PCR suy bin khc c thc hin trn tt c cc chng Arcobacter (Bng 1), dng mi xui c thit k da trn kt qu PCR suy bin u tin v mi ngc ca phn ng PCR suy bin ny. i vi A. nitrofigilis, PCR cho 1 on DNA c kch thc 1332 bp. PCR trn cc chng thuc cc loi khc cho 1 on DNA c kch thc khong 678 - 786 bp. Phn tch TBLASTX mt ln na khng nh cc on DNA ny c s tng ng rt ln vi cc gen fla ca Campylobacter spp., Helicobacter spp., v W. succinogenes. T kt qu ny, cc on d cho Southern hybridization v cc on mi cho qu trnh gene walking xc nh ton b gen fla v c thit k cho mi chng Arcobacter. Kt qu Southern blot cc chng ch cho mt vch khi DNA ct vi EcoR I. Hybridization ca DNA ct bng Hind III cho hai vch i vi A. cryaerophilus v 1 vch vi cc chng cn li. Hybridization ca DNA ca A. butzleri v A. skirrowii ct bng Pst I cho 2 vch. Ring i vi A. cibarius, kt qu ch cho 1 vch khi DNA ct bng Hind III hay Pst I. Kt qu ny cho thy c t nht 2 gen m ha flagellin trong A. butzleri, A. skirrowii, v A. cryaerophilus. Nguyn gen fla ca cc chng A. nitrofigilis, A. butzleri, A. skirrowii, v A. cryaerophilus c xc nh nh cc phn ng s dng TOPO gene walker kit. Tng t Campylobacter spp., cc chng Arcobacter ny c 2 gen fla cng chiu nm k nhau, cch nhau bng mt on khng m di truyn (non-coding region) c 158 - 168 nucleotides, hay 217 nucleotides trong trng hp A. nitrofigilis (ging nh i vi Campylobacter spp., chng ti gi 2 gen ny ca 51

Arcobacter spp. ln lt l flaA v flaB). Ring i vi A. cibarius, qu trnh gene walking khng th ko di DNA u 3, do ch c mt gen fla c xc nh. Bng 3. S tng ng gia FlaA v FlaB trong cc Arcobacter Chng vi khun
A. butzleri LMG 6620 A. butzleri UU-K32 A. skirrowii LMG 6621 A. cryaerophilus LMG 7537 A. cibarius LMG 21996 A. nitrofigilis LMG 7604 C. jejuni RM1221 H. pylori 26695 W. succinogenes DSM 1740

S a-xt a-min
FlaA FlaB

Mc tng ng gia FlaA & FlaB

84 % 90 % 81 % 90 % 62 % 95 % 59 % 69 %

283 307 300 316 280 498 573 510 518

282 307 302 316 495 573 514 513

Ngoi tr loi in hnh A. nitrofigilis, phn t flagellin ca cc chng cn li c kch thc ch bng phn na kch thc flagellin ca Campylobacter spp., Helicobacter spp., v W. succinogenes (Bng 3). Cc nghin cu cho thy, mt flagellin vn duy tr chc nng c th c kch thc nh nht khong 250 a-xt a-min, vi cc u N v C bo tn (khong 140 a-xt a-min u N v 90 u C) (Beatson et al., 2006). Cc on bo tn ny nm trong phn li (the inner core) ca si flagellum, ng vai tr quan trng trong cu trc v kh nng di ng ca flagella (Yonekura et al., 2003). Thnh phn v cch sp xp cc a-xt a-min on gia cc phn t flagellin kh bin ng, mang tnh khng nguyn c th cho cc chng hay cc loi khc nhau. flagellin ca cc ca Arcobacter, on b ngn i so vi flagellin ca cc vi khun h hng l on nm gia phn t. iu ny c th c gii thch qua thuyt gt da gen (gene shortening v genomic shaping) trong qu trnh tin ha ca b gen vi khun (Batut et al., 2004; Mira et al., 2001; Schneider and Ebert, 2004). S rt ngn b gen c th em li hiu qu cao hn cho vi khun trong cc qu trnh chuyn ha v tng trng, cng nh gip vi khun trnh tch ly nhng t bin hy dit. iu ny phn no gii thch ti sao mc d tn s chuyn i gen gia cc chng (horizontal gene transfer) v nhn i gen (gene duplicate) cao, b gen vi khun vn duy tr kch thc n nh (Mira et al., 2001).
0.05

88 81 95 100 100 93 87 96 51

A. skirrowii-FlaA A. skirrowii-FlaB A. cryaerophilus-FlaA A. cryaerophilus-FlaB A. butzleri UU-K32-FlaA A. butzleri UU-K32-FlaB A. butzleri 6620-FlaA A. butzleri 6620-FlaB A. cibarius A. FlaA A. nitrofigilisnitrofigilisA. nitrofigilis-FlaB

100

100 100 100 100 99 56 100 53 100

C. jejuni-FlaA C. jejuni-FlaB C. coli-FlaA C. coli-FlaB H. pylori-FlaA H. hepaticus-FlaA W. succinogenes-FlaG W. succinogenes-FlaB H. pylori-FlaB H. hepaticus-FlaB E. coli-FliC

Phn tch chui a-xt a-min cho thy 2 phn t flagellin (FlaA v FlaB) ca cng mt chng A. butzleri, A. skirrowii, hay A. 52

Hnh 1. Cy ph h (Phylogenetic tree; Neighbor-Joining with boot strap value) ca flagellin ca Arcobacter spp., cc vi khun h hng (C. jejuni RM1221, C. coli VC167, H. pylori J99, H. hepaticus ATCC51449, W. succinogenes DSM 1740), v E. coli K12.

cryaerophilus c tng ng khong 80 - 90 %. S tng ng gia 2 phn t ny A. nitrofigilis kh thp (62%). So snh gia cc chng khc nhau cho thy s tng ng ca FlaA hay FlaB gia cc chng A. butzleri, A. skirrowii, v A. cryaerophilus l 73 - 79 %, v gia cc chng ny vi A. nitrofigilis l 61-72 %. Nh vy, c s khc bit ln v di truyn gia cc thnh vin trong ging Arcobacter, c bit gia vi khun mi trng A. nitrofigilis v cc Arcobacter tm thy ngi hay ng vt. Tuy nhin cy ph h (Hnh 1) cho thy FlaA ca cc chng Arcobacter to thnh mt nhm ring bit tch khi FlaA ca cc vi khun c h hng gn. S ng tn ti 2 gen m ha flagellin v c 2 u ngn, cng vic c 2 flagellin protein ca cng 1 chng cng hp thnh mt nhm ring trn cy ph h cho thy c th c mt s ng tin ha (concerted evolution) ca 2 gen fla ca cc Arcobacter. Hin tng ny cng c thy i vi C. jejuni (Meinersmann and Hiett, 2000). Cho ti nay, ngha ca vic ng tin ha ca cc gen nhn i (duplicated genes) vn cha c hiu r. Ngi ta cho rng, hin tng ny xy ra do s hon v gen khng cn i (unequal crossover) hay/v do s bin trng gen (gene conversion) xy ra trong qu trnh tin ha ca cc gen nhn i (Eickbush and Eickbush, 2007; Nei and Rooney, 2005), gip bo tn cc phn m ha chc nng ca gen ng thi cho php pht sinh s a dng di truyn ca vi khun (Santoyo and Romero, 2005). S ng tn ti 2 gen k nhau m ha cho cng 1 loi protein c cng chc nng, v s ng tin ha ca 2 gen ny cho thy mc d flaB khng ng vai tr thit yu trong s di chuyn ca vi khun, nhng bn thn n v protein do n m ha gi chc nng cung cp s a dng khng nguyn cho vi khun, iu ny c ngha quan trng trong mi tng tc gia vi khun gy bnh v c th vt ch, gp phn vo tnh sinh bnh ca chng. Phn tch on DNA pha trc cc gen fla ca cc chng Arcobacter cho thy mt khc bit quan trng trong c ch iu khin s th hin cc gen ny trong cc vi khun Arcobacter so vi cc vi khun khc. Trong Campylobacter v Helicobacter spp., flaA c phin m di s iu khin ca promoter 28 (28- type promoter) v s phin m flaB do promoter 54 (Guerry et al., 1990; Josenhans et al., 1999; Kostrzynska et al., 1991; Nuijten et al., 1990). Cc sigma factor ny c cc on c trnh t nucleotide kh bo tn (consensus promoter elements). Tuy nhin, cc on ny khng thy hin din trn on DNA pha trc cc gen fla ca Arcobacter, mc d v
H.pylori-J99-B H.pylori-26695-B H.pylori-26695-A W.succinogenes-G W.succinogenes-B H.hepaticus-A2 H.hepaticus-A1 H.hepaticus-B C.lari-B

C.lari-A
C.coli-A C.coli-B C.jejuni-TGH9011-A C.jejuni-11168-A C.jejuni-RM1221-A C.jejuni-RM1221-B C.jejuni-TGH9011-B C.jejuni-11168-B

A.nitrofigilisA.cibarius-A A.butzleri-UU-K32A A.butzleri-6620-A A.cryaerophilus-A A.skirrowii-A

A.butzleri-6620-B A.butzleri-UU-K32-B

A.cryaerophilus-B A.skirrowii-B A.nitrofigilis-A

0.05

Hnh 2. Cy ph h (phylogenetic tree; Neighbor-Joining) ca vng promoter gen fla (120 nucleotides, pha trc start codon ATG) ca Arcobacter spp. v cc vi khun h hng (A: flaA; B: flaB; G: flaG ca W. succinogenes; H. hepaticus ATCC 51449 c 2 gen flaA ging nhau, flaA-1 and flaA-2).

53

tr gn ribosome (ribosome-binding site) c xc nh r rng. Cy ph h (phylogenetic tree) thit lp da trn on DNA di 120 nucleotide pha trc b ba khi ng ATG (start codon) ca cc gen fla ca cc chng Arcobacter nghin cu v cc vi khun h hng cho thy cc on ny ca cc Arcobacter to Chng nguyn thy thnh 1 nhnh ring. Trong khi , cc on thuc gen fla c iu khin bi 28 phn b trn 1 nhnh v cc on thuc gen fla c iu khin bi 54 phn b trn mt nhnh khc (Hnh 2). Kho st chc nng ca gen flaA v flaB A. butzleri flaA flaB LMG 6620 v s th hin cc gen ny trong cc chng t bin Cc chng A. butzleri LMG 6620 t bin gen flaA hay flaB c to thnh bng cch chn gen khng chloramphenicol vo gia mi gen ny. Kh nng di ng Hnh 3. Kh nng di ng ca cc ca cc chng t bin c kho st trn mi trng thch chng A. butzleri LMG 6620 nguyn thioglycolate 0,4 %. Kt qu cho thy, chng t bin flaA thy, t bin flaA (flaA-) v t bin hon ton khng di ng, trong khi chng t bin flaB c flaB ) trn mi trng thioglycolate. mc di ng tng t chng nguyn thy (Hnh 3). Kho st di knh hin vi in t cho thy cc t bo ca chng t bin gen flaA khng c flagellum, trong khi 1 flagellum di c thy mt u cc t bo ca chng t bin flaB (Hnh 4).

Hnh 4. Hnh chp di knh hin vi in t t bo cc chng A. butzleri LMG 6620 nguyn thy (A), t bin flaA (B), v t bin flaB (C). Bar = 500 nm. kho st s phin bn (transcription) ca 2 gen fla, cc mc mRNA trong t bo vi khun c phn tch qua cc phn ng qPCR. Kt qu cho thy, chng nguyn thy, flaB c phin m nhng mc rt thp so vi flaA. S thay i iu kin nui cy, nhit khc nhau (30oC hay 37oC) hay iu kin -xy khc nhau, khng nh hng n mc phin m flaA, nhng mc phin m flaB 30oC cao hn 37oC t 3 n 6 ln v iu kin hiu kh cao hn iu kin vi hiu kh (Hnh 5A). iu ny cho thy s th hin 2 gen ny c iu khin bng cc c ch khc nhau. So snh s phin m ca mi gen gia chng nguyn thy v chng t bin tng ng cho thy, s t bin ca flaB khng nh hng n mc th hin flaA, nhng s t bin ca flaA dn n vic tng s phin m flaB chng t bin (Hnh 5B). Hin tng ny c th do hiu qu n b (compensation effect), c th gp i vi cc gen nhn i 54

(duplicated genes) khi 1 gen b t bin khng cn c phin m (Gu et al., 2003). c bit, mc d chng t bin flaA khng di ng v khng to thnh flagella, s phin bn flaB vn xy ra, v xy ra mc cao hn so vi chng nguyn thy. Hin tng ny cng c quan st Campylobacter spp.. Vai tr ca flaB v protein ca n trong Campylobacter spp. vn cha c lm sng t.
7

mRNA-fold Difference

5 4 3 2 1 0

mRNA-fold Difference

flaA flaB

12 10 8 6 4 2 0

flaA flaB

Vi hi u kh M icro

HiA u ir kh

1 o 30oC 37oC 30 C 37oC

Hnh 5A. So snh s th hin mi gen fla chng A. butzleri LMG 6620 nguyn thy nhit 30oC v 37oC bng qPCR; kt qu cho thy nhit khng nh hng n s th hin flaA (mc mRNA thay i <2); trong khi s th hin flaB 30oC cao hn 37oC (mc m-RNA thay i >3).

Hnh 5B. So snh s th hin tng i cc gen fla gia chng A. butzleri LMG 6620 nguyn thy v cc t bin bng qPCR; kt qu cho thy s th hin flaA khng khc bit gia chng nguyn thy v t bin flaB (mc m-RNA thay i <2), nhng s th hin flaB chng t bin flaA cao hn so vi chng nguyn thy (mc m-RNA thay i 4).

KT LUN Nghin cu xc nh c cc gen m ha flagellin trong cc loi Arcobacter. Tng t nh cc thnh vin ca nhm epsilon proteobacteria, vi khun Arcobacter c 2 gen m ha cho cc phn t flagellin. Tuy nhin, ch c 1 gen (flaA) ng vai tr thit yu cu to nn flagella v kh nng di ng ca t bo. Vai tr ca ca gen cn li (flaB) cha c hiu r. c bit, cc chui nucleotide bo tn cho php xc nh h thng iu khin s th hin gen m ha flagellin thng thy cc vi khun khc, cc sigma factor 28 v 54, khng hin din on DNA ngay trc cc gen fla ca cc vi khun Arcobacter kho st. C ch no chu trch nhim iu khin s th hin cc gen fla Arcobacter spp. s l ti cho cc nghin cu mi v vi khun ny. TI LIU THAM KHO
ANDERSON, K.F., KIEHLBAUCH, J.A., ANDERSON, D.C., MCCLURE, H.M., WACHSMUTH, I.K., 1993. Arcobacter (Campylobacter) Butzleri-Associated Diarrheal Illness In A Nonhuman Primate Population. Infect. Immun. 61, 2220-2223. BATUT, J., ANDERSSON, S.G., O'CALLAGHAN, D., 2004. The Evolution Of Chronic Infection Strategies In The AlphaProteobacteria. Nat. Rev. Microbiol. 2, 933-945. BEATSON, S.A., MINAMINO, T., PALLEN, M.J., 2006. Variation In Bacterial Flagellins: From Sequence To Structure. Trends Microbiol. 14, 151-155. BERTANI, G., 1951. Studies On Lysogenesis. I. The Mode Of Phage Liberation By Lysogenic Escherichia Coli. J. Bacteriol. 62, 293-300. DONACHIE, S.P., BOWMAN, J.P., ON, S.L., ALAM, M., 2005. Arcobacter Halophilus Sp. Nov., The First Obligate Halophile In The Genus Arcobacter. Int. J. Syst. Evol. Microbiol. 55, 1271-1277.

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