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ANALYS I S OF GENOMIC REARRANGEMENT AND PLASMID CONJUGAT I ON OF AN
INOCULANT S TRAIN OF RHIZOBIUM LEGUMINOSARUM BV TRIFOLII .
A T he s i s P re s e n t e d i n P a r t i a l F u l f i l lment o f t h e Degree o f Doctor o f Philosophy.
MICHAEL J . O'HARA
1989
Table o f Content s . List o f Tables.
List of D iagrams . List o f F igure s . Acknowledgement s . Abstract .
INTRODUCT ION
page : 5 5 5 8 9
1 . 1 The S o i l . 1 0
1 . 2 Life i n the S o i l 1 1
1 . 3 Conservat ion o f Identity and Adapt ation t o New Factors
in the S o i l . 1 2
1 . 4 Mechani sms o f Adapt ation . 1 3
1 . 5 P l asmids : their Role i n Bacteria and I nduct ion o f
Genomic Rearrangement . 2 1
1 . 6 Ident i f icat ion o f Rhi z obium Species and t he i r P l asmids . 2 6 1 . 7 The Mo lecular Anatomy o f Symbios i s . 3 2
1 . 8 Rhi z ob ium in the S o i l . 3 6
1 . 9 Competit ion and Interact ion : the fate o f Inoculant Rhi z obium .
MATERIALS AND METHODS
2 . 1 Bacte rial S t ra ins Used in this Inves t igat ion . 2 . 2 Media Used for Strain Maintenance .
2 . 3 Ant ibiot ic Cont aining Media . 2 . 4 Gene ra l Purpos e Solutions .
2 . 5 Con j ugat ion o f Bacterial S t ra ins .
2 . 6 I s o lat ion o f Rhi zobium S t rains f rom Nodule s . 2 . 7 Germinat ion o f Seeds , Inoculat ion o f Clove r P lant s
and Re- i s o lation f rom Nodules .
2 . 8 Chromatographic Analys i s o f Nit rogenase Act ivity . 2 . 9 P l a smid Characterizat ion by Elect rophore s i s .
4 1
4 5 5 2 5 4 5 5 5 6 5 7
5 8 5 9 6 1
2 . 1 0 Res t rict ion Endonuclease Ana lys is o f Tot a l DNA . 2 . 1 1 P reparation o f Gel s for Hybridi zat ion .
2 . 12 Recove ry o f DNA F ragments f rom Aga rose Gel s . 2 . 1 3 Rapid I s o lat ion o f Plasmid DNA .
2 . 1 4 P repa ration o f Radioact ively Label led DNA P robe s . 2 . 1 5 Hybridi zat ion of Radioact ively Label led P robe to
Nit roce llulose Bound DNA .
RESULTS AND D I SCUSS ION 3 . 0 P e r spect ive
3 . 1 S e lect ion of Strains
3 . 2 Ident i ficat ion of Soil Bact e ria
3 . 3 Ant ibiot ic Sens it ivity and Res istance 3 . 4 P lant Tes t s and I s o lation f rom Nodules
3 . 5 Rea rrangement o f a st rain o f R. leguminosarum biovar
page : 62 68 6 9 7 0 7 0
7 1
7 3 7 7 8 0 8 5 8 8
t ri f o l i i carrying RP 4 1 2 6
3 . 6 T ransfer o f the Symbiotic P lasmid of R . leguminosa rum biovar t ri f o l i i 2 6 6 8 to a range of rec ipient s and expres s i on o f symbiot ic genes
CONCLUS IONS
4 . 1 The genome of rhizobia can undergo r e a r r angement
4 . 2 Symb i ot ic informat ion is present in MO 1 0 3 / 1 0 4 and
1 4 5
175
can be exp re s sed under appropriate condit ions 1 7 7 4 . 3 The symbiotic plasmid o f 2 6 6 8 can t ransfer t o and
be exp re s s ed in a va riety of hosts 1 7 9
4 . 4 The s i ze o f the symbiotic plasmid and the number o f
bands showing homo logy to the symbiotic genes can va ry 1 8 1 4 . 5 How might the va rious DNA t ransactions a f fect the
bact e r ial genome? 1 8 4
4 . 6 What are the ecological consequences o f plasticity for
Rhi zobium ? 1 8 6
4 . 7 How can laborat ory s imulations help u s make predict ions
about the f ie ld environment ?
4 . 8 Future direct ions for invest igat ion .
5 . 0 B IBLIOGRAPHY
1 8 7 1 9 0
1 9 3
List o f Tables
1 . S t rains of Rhi z obia Used . 2 . St rains o f Non- rhi z obia Used . 3 . Re st rict ion Endonucleases Used .
4 . B iochemical Analys i s o f the S o i l S t ra ins NR 4 1 , NR 4 2 , NR 6 4 , OR 1 6 8 and the Inoculant 2 6 6 8 .
5 . Leve l s o f Ant ibiot ic Re s i stance and F requency o f mutat ion t o ant ibiot ic resistance
6a . Nodulat ion and Nit rogen F ixat ion S t atus of S t rains I s olated in P a rt I .
6b . Nodu l at ion and Nit rogen Fixation Status o f S t rains I s o lated in P a rt I I .
List o f Diagrams
1 . Nodulat ion and Nit rogen F ixat ion Gene Organ i zation . 2 . As sembly o f Apparatus for Southern Blott ing .
3 . Summa ry o f Experiment s Performed in P art I . 4 . S umma ry o f Expe riment s Performed in P a rt I I . 5 . Flow Chart Depict ing Steps Involved in Sampling
the S t re s s Expe riment
6 . Summary o f the Pla smids Obse rved in the Crosses
of P art I I List o f F igures
1 . Eckhardt plasmid profile o f iso lates f rom plant s inoculated with MO 1 0 3 , 1 0 4 , 1 0 5 , 1 0 7
2 . Light and e lect ron mic rographs o f nodule sect ions f rom a plant inoculated with 2 6 6 8 .
3 . L ight and elect ron micrographs o f nodule sect i on s f rom a plant inoculated with MO 1 0 5 .
4 . Light and e lect ron mic rographs o f nodule sections f rom a plant inoculated with MO 1 1 0 .
5 . Photographs o f c lover plant s inocu+ated with s t rains f rom the 2 6 6 8 / s o i l bacteria c ros s .
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page : 4 9 5 1 6 6
8 3
8 7
9 4
1 12
3 3 6 7 7 4 7 6
1 2 7
1 7 2
9 2
9 6
9 8
1 0 0
1 0 4
6 . Photographs o f c l over plants inoculated with st rains
f rom the 2 6 6 8 / so i l bacteria cross . 1 0 6 7 . Light and elect ron mic rographs o f nodule sect ions
f rom a plant inoculated with MO 1 2 1 . 1 0 8 8 . L ight and e lect ron mic rographs o f nodule sect ions
of a plant inoculated with MO 1 2 4 . 1 1 0
9 . Photographs o f c lover plants inoculated with s t rains
f rom the 120 serie s /PN 1 0 4 cross . 1 1 5 1 0 . Light and elect ron micrographs o f nodule sect ions
f rom a plant inoculated with MO 2 0 1 . 1 1 7 1 1 . Photographs o f c love r plants inoculated with pa rent
and excon j ugant s f rom the 1 2 1 / 2 6 6 8 cross . 1 2 1 1 2 . L ight and elect ron micrographs f rom nodule sect ions o f
plants inoculated with MO 8 1 0 . 1 2 3
1 3 . Light and e lect ron mic rographs f rom nodule sect ions o f
plant s inoculated with MO 8 1 2 . 1 2 5
1 4 . Bacte rial i s o lates f rom the s t re s s test 1 3 1 1 5 . Eckha rdt plasmid profile o f the f ive s t rains de rived in
P a rt I . 1 3 5
1 6 . Southern blot o f Eckha rdt ge l o f f ive st rains probed
with nod ABC . 1 3 5
1 7 . S outhern blot o f Eckha rdt ge l o f f ive s t ra ins probed
with nodABC 1 3 7
1 8 . S outhern blot o f Eckhardt gel o f five s t rains probed
with RP 4 . 1 3 7
1 9 . E lect ropho ret ic prof i le o f a n EcoRI digest o f the
t o t a l genomic DNA o f the five s t ra ins f rom part I . 1 4 0 2 0 . S outhern blot o f t o t a l genomic DNA digest probed with
nif KDH . 1 4 0
2 1 . Southern blot o f t o t a l genomic DNA dige st probed with
nod ABC 1 42
2 2 . S outhe rn blot o f t o t a l genomic DNA digest probed with
RP 4 . 1 42
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page : 2 3 . Eckha rdt plasmid pro f i le o f the parent s and ex
con j ugant s f rom the cross between 2 6 6 8 and the s o i l bacteria .
2 4 . S outhern blot o f an Eckhardt gel o f the 2 6 6 8 and s o i l bacteria cross probed with Tn5 . 2 5 . S outhe rn blot o f a n Eckha rdt ge l of the 2 6 6 8
and s o i l bacteria cross probed with n i f KDH . 2 6 . S outhern blot of an Eckhardt gel of the 2 6 6 8
and s o i l bacteria cross probed with nod ABC . 2 7 . E lect ropho ret ic pro fi le o f an EcoRI digest o f the
DNA f rom pa rent s and exconj ugant s of the 2 6 6 8 / so i l bacteria cross .
t o t a l
2 8 . S outhern B l o t o f t h e t o t a l genomic DNA from the 2 6 6 8 / s o i l bacteria cross probed with Tn5 .
2 9 . S outhern Blot o f the total genomic DNA from the 2 6 6 8 / s o i l bacteria cross probed with nod ABC and n i f KDH comb ined .
3 0 . E c khardt plasmid pro f i le o f the parent s and
exconjugants f rom the cross between the 1 2 0 series and PN 1 0 4 .
3 1 . S outhern blot o f Eckha rdt gel o f 1 2 0 s e ries / PN 1 0 4 c r o s s probed with TnS .
3 2 . S outhern blot o f Eckha rdt gel of 1 2 0 se ries / PN 1 0 4 cross probed with nod ABC .
3 3 . E c kha rdt plasmid pro f i le o f the parent s and excon j ugant s f rom the cross between the 1 2 0 series and
E . c o l i H B 1 0 1 .
3 4 . S outhern blot o f an Eckha rdt gel of the st rains f rom t he 1 2 0 s e ries / HB 1 0 1 cross probed with nod ABC . 3 5 . E c khardt plasmid pro f i le o f the parent s from the
c r o s s between MO 12 1 and 2 6 6 8 .
3 6 . S outhern blot o f an Eckha rdt gel o f the 1 2 1 / 2 6 6 8 cross p robed with nod ABC .
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1 5 1
1 5 1
1 5 3
1 5 3
1 5 5
1 5 5
1 5 7
1 6 1
1 6 1
1 6 3
1 67
1 6 7
1 7 0
170
ACKNOWLEDGEMENTS
I wish t o t hank the following people for a s s istance during the va rious st age s o f this pro j ect .
The Department o f Mic robi ology and Genet i c s , Mas sey Unive r s ity f o r t he space a n d t he equ ipment and the Ma s s e y U n i ve r s i t y P o s t graduate Fund f o r f inance .
My s upe rv i s o r s D r . E r i c T e r z a ghi and D r . B r i o n J a r v i s f o r keeping me o n the st raight and narrow (most ly) .
Prof . D . B . S cott , Drs . B . Mans field, M . Nulsen, E . Roc krnan, A . N . MacGregor and J . R . Rao and Mr . R . Cleave r , L . Wa rd, G . I on a s , Ms . J . C o l l in s , C . Young and P . M c L e n a c h a n f o r t e c hn ique s a n d he l p f u l adv i c e . T h a n k s a l s o t o t he T u e s day mo rning d i s c u s s i o n g roup f o r ins ight ful ( and otherwise!) discuss ion .
Thanks t o Mr . D . Hopc roft and R . Bennett o f D . S . I . R . f o r the masterful nodule sect ions and elect ron mic rographs .
T h a n k s t o D r s . R . E . R o w l a nd and C . J . O ' Ke l l y a n d Ms . E . M . Nickle s s o f Botany and Zoology for acce s s to and ins t ruct ion in l ight microscopy .
T h a n k s t o A n d r e w a n d t he C . P . U . f o r pe r s i s t e n c e w i t h t he photographs .
Thank s t o Judy and Laura f o r a s s i s t ance with p r o g r amming the diagrams .
T h a n k s t o D a v i d , f l a tma t e ext r a o rdina i re , f o r l o a n o f t he computer on which this the s i s was typed and checking f o r e r ro r s .
Thanks t o J . Veale for binding this thes i s .
Much g r a t i t ude t o my Z . D . K . c o l leagues Je f f , Jul ie , T a l it a , J a me s a n d Ma rt y a n d a l l o f my o t he r ma r t i a l a r t s f r i e nd s a n d s tudent s . Often t ra ining was a l l that kept me sane .
Many thanks t o a l l the great people I met at Mas sey dur ing my inca rce ration , f a r too numerous to name without print ing a companion volume , without whom this the s i s would have been pos s ible but ha rdly worth it .
Thanks to Zoe for smiles , hugs and support at a dif f icult t ime . TH I S THE S I S I S DED ICATED TO MY PARENTS WHO ALWAYS HELPED ME OUT AND MY FRI ENDS WHO WERE ALWAYS THERE .
8
ABSTRACT
Va r i a t i o n o f p l a s m i d p r o f i l e i s t he h a l lma r k o f Rh i z ob i um l egumi n o s a r u m b v t r i f o l i i s t r a i n s i s o l a t e d f rom the n o du l e s o f pasture p l a nt s . P revious at tempt s t o demonstrate that t hese variant t yp e s were de r ived f rom t he o r iginal inoculant we re inconclus ive . Subsequent l aboratory based s imulat ions revealed that the broad host range pla smid RP 4 was capable of generat ing stable alterat ions in the p l a smid a n d t o t a l genomic DNA pro f i le . This va riat ion invo lved a n apparent l o s s o f pSym, with concurrent loss o f t h e nod and n i f genes , but t he s e s t ra in s p r o du c e d n o du l e s o n w h i t e c l ove r p l an t s . T h e s t rains recove red f rom the nodule s , while not ident ical , were clearly de r ived f rom t he p S ym- s t ra in . A p l a smid c l o s e ly c o r re s ponding in s i ze t o p S ym wa s det ected in t he nodu l e re - i s o lates and repea t ed t r i a l s o f t h i s
( appa rent ly) s ym
e x p e r i m e n t i n v o l v i n g a n t i b i o t i c a l l y m a r k e d st rains c onf irmed this obse rvat ion . This left the c o n c l u s i o n t h a t t he DNA w a s s t i l l t he r e but in a f o rm which was dif f icult t o detect by convent i onal DNA hybridi zat ion procedure s , a result which i s not totally without precedent ( Downs and Rot h , 1 9 8 7 ) . The second portion o f this project involved an inve s t igation o f t h e t ransmi s sability o f pla smids from the inoculant strain 2 6 6 8 . The s t r a i n w a s ma r ked with T n S with t he e xpect at i o n that s ome of t he mo v a b l e D N A p i e c e s w o u l d c a r r y a T nS i n s e r t and c o u l d t h u s be selected f o r . A t ransmis s able symbiot ic plasmid was detected, as had been previous ly observed in other rhi zobia by Johnston et al ( 1 9 7 8 ) . The plasmid was s hown to be t ransferable , in an altered form, t o s o i l mi c r o o r g a n i s m s o f u n i de n t i f i e d gene r a , t o a sym s t r a in o f � legumin o s a rum bv t r i fo l i i , to its o riginal pa rent 2 6 6 8 and to � c o l i . I n a l l s t r a in s , with the except ion of E . c o l i the nodulat ion g e n e s we re f u nc t i o na l , p r o du c ing n o rma l l o o k ing n o du l e s ( on t he o u t s ide ) a n d i n many s t ra i n s n i t rogen was a l s o f ixed, t hough not gene rally a s we l l as by the pa rent 2 6 6 8 . The s igni ficance o f a s e l f t ransmi s s able broad h o s t range symbiot ic plasmid i s discussed in the context of the mic robial ecology o f rhi z obia .
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