A molecular and genetical analysis of symbiotic genes in Lotus rhizobia : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

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A MOLECULAR AND GENETICAL ANALYSIS OF SYMBIOTIC GENES IN LOTUS RHIZOBIA

By

KAW-YAN CHUA

A thesis presented in partial

fulfi lment of the requirements for the degree of Doctor of Phi losophy in Microbiology at

Massey Univers ity ,

Palmerston North , New Zealand

October , 1 984

ABSTRACT

Relat ive DNA homologies were determined among 24 strains of fast- growing rhizobia and 26 strains of s low- growing rhizobia with particular

reference to those which nodulate Lotus species . Two ma j or DNA homology groups were identified which shared less than 1 0 % relative DNA homology . Fast-growing Lotus rhizobia were grouped with related fast-growing

strains in DNA homology group I, and these strains are des i gnated as

Rhizobium loti . Slow-growing strains from Lotus , ( Bradyrhizobium spp .

( Lotus ) ) , G lycine max ( Bradyrhizobium j aponicum ) , Ornithopus and Lupinus formed DNA homology group

II

which was further divided into 4 DNA

homology subgroups . Bradyrhizobium spp . ( Lotus ) were shown to be genetical ly distinct from B . j aponicum.

Plasmi d profiles were determined for strains of R . loti and Bradyrhizobium spp . ( Lotus ) . A l l R. loti strains contained a single large indi genous plasmi d , whereas strains f rom Bradyrhizobium spp .

( Lotus ) carried mu ltiple plasmids of mol ecular wei ghts ranging from 1 3 0-

280 MDa l . A plasmid-cured derivative of R . loti NZP2 2 1 3 was isolated �

and found to still form effective nodules on Lotus tenuis , suggesting

that nodulation and nitrogen fixation genes are not plasmid-borne in this stra i n . The functions of the indigenous p lasmids carried in Lotus rhizobia are unknown .

Symbiotic mutants of R . loti strain NZP 2 0 3 7 were isolated by random

Tn5-muta gene s i s . Mutants included strains blocked in root hair curling

( Hac ) , nodule initiation ( Noi ) , bacterial release ( Bar ) and nitrogen

fixation ( Cof ) on Lotus pedunculatus .

The nodulation ( nod) gene region from R . loti strain NZP2 0 3 7 was isolated f rom a pLAFR1 -NZP2 0 3 7 gene library using the c loned Tn5

containing EcoRI fragment from the Nod- mutant as a probe . Two cosmids were isolated and were found to complement the NZP2 0 3 7 Nod- mutant . Hybridi sation and complementation experiments confirmed that a 7 . 1 kb EcoRI fragment present in both nod cosmids carried gene sequences

involved in nodulation .

An

EcoRI and Hind I I I restriction enzyme map of the nod gene region in R . loti NZP2 0 3 7 was constructed using nod cosmids pPN 3 0 5 and pPN 3 06 .

Using the cloned R . loti NZP2 0 3 7 nod gene region ( 7 . 1 kb EcoRI fragment f rom pPN3 05 ) as a hybridisation probe , hi ghly conserved DNA sequences f rom other strains of R. loti and Bradyrhizobium spp . ( Lotus ) were ident i f ied. The nod gene region from Bradyrhizobium spp . ( Lotus ) strain NZP2 3 0 9 was isolated by direct 'in planta' complementation of the R. loti Nod- mutant using a NZP2 3 0 9 pLAFR1 gene l ibrary .

Comparative physical and genetical studies showed that the R. l oti

NZP2 0 3 7 nod gene region isolated shared functional simi larities with

previous ly isolated nod gene regions from R . trifolii and R. mel iloti

despite the fact that only weak DNA homology was observed between the

corresponding regions . This suggested that at least some of the nod

gene sequences carried on the c loned R . loti nod gene region belong to

the highly conserved ' common' nod gene sequence category .

AC KNOWLE DGE ME NTS

I wi sh to thank my su pervis ors Dr B . D . W . Jarvis , D epar tment of Micr obi ol ogy a nd Geneti c s , Mas s ey U niver s i ty , f or his c ons ta nt i nter es t and advic e thr ough out this s tudy a nd D r D . B . Sc ott, A ppl i ed Bi och emi s try Divi s i on, D epar tment of Sci entific a nd Indus trial Res earch (D . S. I. R . ) f or his i ns pirati on a nd guidanc e wi th all as pec ts of this work a nd his gener osi ty in shari ng his lab ora tory s pace and r es earch res ourc es f or th e pas t two y ears .

I a l s o wish to tha nk Dr C . E . Pa nkhurs t of A ppl i ed Bi och emis try Divisi on, D . S. I. R . , f or the advi c e a nd ass is tanc e wi th the ligh t a nd el ec tr on micr osc opy s tudies a nd th e c ons truc ti on of Rhiz obium l oti genomic l ibrary , as wel l as f or his c onti nued i nter es t i n this work.

Special tha nks are als o due to th e f ol l owi ng me mb ers of D . s. I. R. , Pal mers ton N or th : Dr J. J. Pa te l , f or his assis ta nce wi th th e

bacteri ophage typi ng techni que ; Dr w. T. Jones , f or carryi ng out the ta nd em c r os s ed-i rnmunoel ec tr oph or esis ex peri ment; Mr D . H. Hopcrof t, f or his ass i s ta nc e wi th th e elec tr on mic r osc opy s tudies a nd Dr B . Terzaghi f or her advice wi th the tra ns pos on mu tagenes is experi me nt.

I w ould also like to tha nk the s taff me mbers of Rhiz obi um

laboratory, D . S. I . R . , f or th eir c ooperati on a nd enc ourage me nt over the pas t two y ears ; Mr C . B . C our t f or his ass i s tanc e i n thesis figures pr epara ti on a nd Mrs G. Shaw f or ty pi ng the th es i s . T o all the members of my f ami l y , I would like to show my gr eates t gra ti tude , f or their l ove and suppor t over the y ears .

During the per i od of my Ph . D . pr ogra mme I have been su ppor ted by a s tudy grant f r om o.s.r.R. which I grate fully acknowledge .

CONTE NTS

ABSTRA CT

ACKNOWLEDGE ME NTS CONTE NTS

INDE X OF TABLES INDE X OF FIGURE S ABB REVIATIONS

CHAPTER ONE . GENERAL INTRODUCTION

1 .1 SIG NIFICANCE OF BIOLOG ICA L NITROGEN FIXA TION

1 . 2 SIGNIFICA NCE OF LOTU S SPP . IN NEW ZEALA ND AGRICULTU RE

1 . 3 CHARA CTERISTICS OF LOTU S RHIZ OB IA

1 . 4 CLASSIFICA TION OF ROOT NODULE BACTERIA 1 . 5 NODULE DE VE LOP ME NT

1 . 6 NODULE FORMATION IN LOTU S 1 . 7 GE NE TICS OF NITROGE N FIXATION 1 . 8 AIMS OF THIS STUD Y

CHAPTER TWO . D NA HOMOLOGY STUD IE S OF LOTU S RHIZ OBIA 2. 1 INTRODUCTION

2. 2 ME THOD S A ND MATERIA LS 2 . 2 . 1

2. 2 . 2 2. 2 . 2 . 1 2. 2. 2 . 2 2. 2. 3 2. 2.4 2 . 2. 5 2. 2 . 6 2 . 2 . 7 2. 2 . 8 2. 2 . 9

2. 3 2. 3. 1

Bac terial s trains

Preparation of cul tur e media Li quid media

Solid media

Main tenanc e of cul tur es Gro wth of bac teria

Prepara tion of unlabel l ed D NA Pr epara tion of [32p]- label l ed DNA D NA r eassociation

Separation of s i ngl e and doubl e s tranded D NA on hydroxyapati te (HA )

Th er mal s tabi l i ty s tudy of r eassocia ted dupl ex es

RE SULTS

DNA-D NA r eassociation at 65 °C among rhizobia from Lo tus spp . and s trai ns fro m oth er plants

PAGE

1 1 1

2 3 5 7 7 1 3

1 4 1 4

1 4 1 4 1 4 1 4 1 5 1 5 1 5 1 5

�')..?

1 8 1 8 1 8 1 9 20 20

2.3.2 Th er mal s tabi lity of r ea s s ocia ted DNA dupl ex es 21

2.4 D ISCUSSION 22

2.5 SUMMARY 26

CHAP TER THREE . CHARACTERISA TION OF P LASMID S IN LOTU S RHIZ OB IA 27

3.2 3.2.1 3.2.2 3.2.2.1 3.2.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.7 3.2 .8 3.2.9 3.2.1 0 3.2.1 1 3.2.1 2 3.2.1 3 3.2.1 4 3.2.1 5 3. 2. 1 6 3.2.1 6.1 3.2.1 6.2 3.2.1 7 3.2.1 8 3.2.1 9

3.3 3.3.1

3.3.2 3.3.3

3.3.5 3.3.6

3.3.6.1 3.3.6.2

3.3.7

INTRODU CTION

ME THOD S AND MATERIA LS

Bac terial s trai ns a nd plas mi ds Pr epara ti on of media

Li quid media Solid media

Mai ntena nc e of cul tur es Growth of bac teria

U l travi olet ligh t mu tagenesis of Rhi z obi um l oti Plant tes t

Is ola ti on of bac teria fr om nodul es Hea t curi ng ex peri ment

Bac teri ophage susc eptibi li ty tes ts Ta ndem-c r os s ed i mmu noel ec tr oph or es i s Plas mid i s ola ti on by Eckhardt meth od

Is ola ti on of plas mi ds by th e Tai t proc edur e Pr epara ti on of R. l oti plas mi d D NA

Is ola ti on of genomic DNA

Diges ti on of DNA wi th r es tr i c ti on enzy mes Puri ficati on of D NA pr epara ti on

Purificati on by ph enol chl or of or m extrac ti on Des a l ti ng wi th Sephadex G - 5 0 c olumn

Horiz ontal agar os e gel el ec tr oph or esis of DNA Pr epara ti on of [32p]- lab el l ed DNA pr obes DNA tra ns f er a nd hybridi s a ti on

RE SU LTS

Physical charac terisati on of pla s mids i n Lotus rhiz obia

DNA h omol ogy b etween pRl o221 3a a nd pRl o20 37a A ttempt to is olate N od - d erivatives of NZP 221 3

by h ea t cur i ng

A ttempt to i s ol a te a N od- d eriva tive of NZP221 3 by UV mutagenes i s

Hybridisati on of pRl o221 3a to total DNA of R. l oti s trai ns NZP221 3 a nd PN225

Identificati on of s trai n PN225 as deriva ti v e of R. l oti NZP221 3

Strai n identi f icati on by phage ty ping Strai n i d enti f ica ti on by tandem cross ed-

i mmu noel ec tr oph or es is

27

28 28 28 28 29 29 29 30 30 31 31 31 32 32 33 35 37 38 38 38 39 39 4 0 4 1

4 2 42

42 4 3 4 3 44 44

44 44

Strai n i d entificati on by r es tr i c ti on enzyme diges t 45 pa tter ns

D NA h omol ogy betw een th e nod gene r egi on of 45 R. l oti NZP20 37 a nd th e plas mid-cur ed

deriva tive s trai n PN 225

3. 4 D ISCU SSION

3. 5 SUMMARY

CHAPTER F OUR . ISOLAT ION AND CHARA CTERISAT ION OF TN5- INDU CED SYMB IOT IC MUTANT S OF RHIZ OB IUM LOT!

4 ⁰ 1

4 . 2 4 ⁰ 2 ⁰ 1 4 . 2 . 2 4 . 2 . 2 . 1 4 . 2 . 2 . 2 4 . 2 . 3 4 . 2 . 4 4 . 2 . 5 4 . 2 . 6 4 . 2 . 7 4 . 2 . 8 4 . 2 . 9

4 . 2 . 1 0 4 . 2 . 1 1 4 . 2 . 1 2 4 . 2 . 1 3 4 . 2 . 1 4 4 . 2 . 1 5 4 . 2 . 1 6 4 . 2 . 1 7 4 . 2 . 1 8

4 . 3. 3

4 . 3. 5 4 . 3. 5 . 1

4 . 3. 5 . 2 4 . 3. 5 . 3 4 . 4 4 . 5

INTRODUCT ION

METHODS AND MATERIALS

Bac teria strai ns a nd plasmids Prepara tion of cul tur e media

Liquid media Solid media

Mai ntena nc e of cul tures Growth of bac teria Co njuga tio n

Scr eeni ng method for sy mbio tic mu ta nts Plasmid a nalysis by th e Eckhardt proc edur e Bac teriophage susc ep tibi l i ty tests

Pr epara tive isola tion of plasmid D NA from E . coli by c l ear lysa te method

Isola tion of genomic D NA

Digestion of D NA wi th r estric tion enzymes Purif i c a tion of D NA

Agarose gel el ec trophor esis of D NA

P r epar a tion of [32 p]-label l ed D NA -probes D NA tra nsf er a nd hybridisatio n

D eter mi na tion of mol ecular weights

Light a nd electron mi croscopy of nodul es Exami na tion of roo t hairs by ligh t microscopy

RE SULT S

Tra nsposo n mu tagenesis of Rhizobi um loti Physical a nalysis of pr esu mp tive T n5 -i nduc ed

symbio ti c muta nts by South er n hybridisa tion Hybridi sa tion of total D NA from R . loti T n5 -

muta nts wi th[32 p]-label l ed nif HDK probe

Light microscopic exa mi na tion of Lo tus p edu nculatu s roo t hairs i nocula ted wi th Nod- muta nt o f

R . loti

Ligh t a nd el ec tron microscopic exa mi na tion of nodul es i nduc ed by th e Fix - mutants

Morphology of nodul es i nduc ed by th e wil d- type R. loti strai n NZP 2 0 37

Morphology of tumour- like nodul es Morphology of i neff ec tive nodul es D ISCU SSION

SUMMARY

CHAPTER F IVE . MOLE CULAR CLONING AND GENET ICAL ANALYSIS OF NODULATION GENE S IN RHIZ OB IU M L OTI

5 . 1 INTRODUCT ION

45 49

50

5 0

5 2 5 2 5 2 5 2 5 2 5 2 5 3 5 3 5 3 5 4 5 4 5 4

5 6 56 5 6 5 6 5 6 5 6 56 5 6 5 7

5 7 5 7 58 59 59

60 6 0

6 1 6 1 64 6 7

69

69

5 .2 5 .2 .1 5 .2 .2 5 .2 .2 .1 5 .2 .2 .2 5 .2 .3 5 .2 .4 5 .2 .5 5 .2 .6 5 .2 .6 .1 5 . 2 .6 .2 5 .2 .6 .3 5 .2 .7 5 .2 .8 5 .2 .9 5 .2 .1 0 5 .2 .1 1 5 .2 .1 2 5 .2 .1 3 5 .2 .1 4 5 .2 .1 5 5 .2 .1 6 5 .2 .1 7

METHOD S AND MATE RIALS

5 .2 .1 8

5 .3 5 .3.1 5 .3.2 5 .3.3 5 .3.4

5 .3.5

5 .3.6 5 .3.7

5 .3.8

5 .4 . 5 .5

Bac ter ial s tra ins and plas mids Pr epara tion of cul tur e med ia

Liquid media Sol id media

Ma in tenanc e of cul tures Gr owth of bac ter ia

Is ola tion of gen omic DNA Is ola tion of pla s mid DNA Cl ear ed lysate method Alka l ine lys is meth od Ra pid boil ing meth od

E l ec tr oelu tion of DNA f r om a garos e DNA l iga tion

Transf orma tion of

�

^{ol i}

Col ony hybridisa tion

Pr epara tion of [32 p]-label l ed DNA pr ob e DNA transf er and hybridisa tion

Conjuga tion Plant tes t

Is ola tion of bac ter ia f r om n odul es

Light and el ec tr on microsc opy of n odul es

Construc tion of a pLAFR1 g en e bank to Bradyrhiz obium s pp ( Lotus ) s train NZP 2 30 9 ( CC8 1 4 s )

�n planta ' c ompl emen ta tion of Rhiz obium l oti N od­

mutant

RE SULTS

Cl oning of Tn5 ::n od r egion of NZP 2 0 37

Is ola tion of wid e ty pe n odula tion gen es by c ol ony hybridisa tion

Cons truc tion of a phys ical ma p in th e nodula tion gene r egion of R. l oti NZP 2 0 37

Compl ementa tion of N od- mutant of

�

l oti s train NZP2 0 37 by c os mi ds carry ing pr esu mptive wild ty pe nod DNA of NZP20 37

-

Light and el ec tr on mic r osc opy of n odul es f ormed on Lotus peduncul a tus by c ompl emented d er iva tiv es of th e N od- mu tant s train PN2 33

DNA h omol ogy b etween th e n od gen e r egion of R.l oti NZP2 0 37 and oth er Rhiz obium s trains

'In plan ta ' dir ec t c omplementa tion of R.l oti NZP2 0 37 N od- mutan t with a gen e l ibrary of Bradyrhiz ob ium s pp ( Lotus ) s train NZP2 30 9

Compl ementa tion of Rhiz obium n odula tion func tion us ing h eter ol ogous n od r egions

D ISCUSSION SUMMARY

CHAPTER SIX: GENE RAL DISCU SSION

RE FE REN CE S

7 1 7 1 7 1 7 1 7 1 7 1 7 1 7 1 7 1 7 1 7 1 72 7 3 74 74 75 76 76 76 76 76 76 76 7 8

78 78 79 79 8 1

8 2

8 3 84

86

86 9 3

94

99

Table Number

1 . 1

2 . 3

5 . 1 5 . 2

INDEX

OF

TABLES

Title

Proposed steps in Rhizobium-legume symbiosis .

Strains of fast and s low- growing rhizobia included in this study .

DNA-DNA reassociation at 65°c among rhizobia f rom Lotus spp . and strains from other plants .

Thermal stability of rhizobial DNA duplexes formed at 65°c .

Bacterial strains and plasmi ds . Plasmids in Lotus rhizobia . Bacterial strains and plasmids .

Characterisation of Tn5- induced mutants of R. loti NZP2 03 7 .

Bacterial strains and plasmids .

EcoRI restriction f ragments from Rhizobium loti , R . trifolii

and R. me liloti total DNA homologous to R. loti nod probe

and/or R. trifolii nod probe .

Fi gur e Numb er

3.1

3.3

3. 5

3. 6 3. 7

IND EX OF F IGURES

Tit l e

Ther mal elution prof i l es o f r eassoci at ed D NA dupl ex es formed at 65°c b etween B . japoni cum ATCC 1 0 324 r ef er en ce D NA and D NA from ot her s el ect ed str ains .

A Sep had ex G - 5 0 colu mn us ed for D NA purifi cation.

App ar atus for D NA tr ansf er by Sout hern blotting.

D NA ho mology b etween pRlo22 13a and pRlo 2 0 37a.

Agaros e gel of p l asmids in R . loti strains .

Hybridis ation of pRlo2 2 1 3a to Eco RI enzy me digests of R . loti tot al D NA .

I d entifi cation of R . loti str ains by p hage typing.

Tandem cros s ed -i mmuno el ectrop horesis patterns of R. loti strains .

3. 8 EcoRI r estri ction enzyme digest p att erns of tot al D NA f rom R . loti str ains .

3. 9 Hybridis ation of t he nod gen e r egion of NZP2 0 37 to EcoRI dig ests of D NA from str ains NZP2 2 13, PN2 2 5 and NZP2 0 37 . 4 .1 The v ector p l asmid pSUP10 11 us ed for Tn5 mut agen esis .

4 . 2 Lotus peduncul atus plants inocul at ed with R . loti wild typ e and Tn5 - indu ced symbiot i c mut ant str ains .

4 . 3 Physi cal analys i s of presumptive Tn5 mutants by South ern hybridis ation .

4 . 4 Hybri di s ation of Tn5 and n i f HDK s equ en ces to tot al D NA f rom R . loti Tn5 -indu ced mut ants .

4 . 5 Light mi crogr ap hs of root hairs of Lotus p edun cul atus p l ants .

4 . 6 Light and el ectron mi crogr aphs s howing t he structur e of root nodul es formed by R . loti strain NZP2 0 37 on L. p edun cul atus .

4 . 7 Light and el ectron mi crogr ap hs s howing t he morp hology of th e tumour -l i ke nodul e formed by t he Noi mutants P N2 34 , P N2 36 , P N�37, P N2 38 and P N244 on L . p edun cu l atus .

4 . 8 Light and el ectron microgr ap hs s howing t he structur e of th e nodul es formed by t he Bar mut ant P N2 39 on L . p eduncul atus . 4 .9 Light and el ectron micro grap hs s howing t he structur e of th e

nodu l es f ormed by t he Cof mut ants PN2 35 on L. p edun cu l atus .

4 .10

5 .1 5 .2

5 .3

5 .4

5 .5

5 .6

5 .7

5 .a

5 .9

5 .1 0

Light and el ectron microgr aphs s howing the structur e of nodu l es for med by Cof mut ant PN 246 on L. pedun cul atus . Agaros e gel of an EcoRI di gest of pPN 30 1.

Hybridis ation of Tn5 ::nod r egion from pPN 30 1 to cos mids carrying R. loti wi ld type DNA s equ en ces .

Hind III ( H) and EcoRI ( E) r estri ction en zyme map of t he nod gen e r egion of R. loti NZP 2 0 37.

Compl ernent ation of t he Tn5 -indu ced Nod- mut ant wit h R. loti NZP2 0 37 wild typ e gen e s equ en ces .

Light and el ectron microgr ap hs s howing t he stru ctur e of i n eff ective nodul es and tumour -li ke nodul es for med on

Lotus p edun cu l atus by a d erivative of t he Nod- mut ant PN2 33 carrying pPN 30 5 .

Hybridi s ation o f t he cloned nod gen e r egion of NZP 2 0 37 to R. loti and Br adyr hizobi um spp. ( Lotu s ) tot al DNA.

Hybridi s ation of t he clon ed nod gen e r egion of NZP 2 0 37 to R . loti , R. trifoli i and R. mel iloti tot al DNA.

Hybridis ation of clon ed R. trifolii nod DNA to R. loti , R . trifolii and R. mel iloti tot al DNA .

Hybridi s ation of R . trifolii nod DNA t o clon ed nod gene s equ en ces of

R .

loti NZP 2 0 37.

Light and e l ectron microgr ap hs s howing t he structur e of root nodul es formed on Lotus p eduncul atus by a derivative of t he Nod- mut ant PN2 33 cont aining pPN32 9 .

Nm Km Cb Tc Ap Sp Cm Str

ABB REVIATIONS

n eo mycin

kanamy cin carb enici l l in t etracycline ampi ci llin spectino mycin chlor amp heni col strepto mycin kilobas es

2 -(N-morp holino ) �t hanesul foni c acid sodium dodecyl sulp hat e

Rhi zobium p henotyp e char act eris ed by t he abi l ity to

induce visib l e nodul es on p l ant roots whi ch ar e cap abl e of nitrogen fixation .

Rhi zobi um p henotyp e char act er i s ed by t he abi lity to induce vis ibl e nodul es on pl ant roots whi ch ar e not cap ab l e of nitrogen f i x ation .

Abbr evi ations not d efined in t hi s l i st ar e "accept ed " abbr evi ations (Bio chemi cal Journal (1 983) 2 0 9 , 1 - 2 7) .