Stanley and Cervantes

8/10/2019 Stanley and Cervantes

1/11

Journal of Appl ied Bacteriology 1991, 70 %19

ADONIS002188479100003G

A R E V I E W

Biology and genetics

of the

broad host range hizobium sp

NGR234

J. Stanley and E. Cervantes

NCTC Plasmid Genetics Unit Lond on UK and IRNA-CSIC Salamanc a Spain

Accepted 7 Septembe r 1990

Paper nu mb er: 3237/11/89

1.

In t roduc t ion,

9 6.

7.

3.

Strain identi t ies and genetic techniques,

11

8.

9.

13 10.

2. Ecological an d ev olut ionary relat ionships, 9

4.

Organizat ion

of

t he ge nome ,

12

5.

Genetic analysis

of

broad host range nodulat ion,

Exopolysacchar ide of

NGR234, 14

Symbiotic ni trogen fixat ion and metaboli te

exchange in the nodule ,

15

Concluding remarks , 16

Acknowledgements, 16

References, 16

1. INTRODUCTION

The Leguminosae, the third largest family of higher plants,

possess genetic determinants for development of root

nodules and are able to enter into nitrogen-fixing symbiosis

with soil bacteria (rhizobia) possessing the appropriate sym-

biotic (Sym) genes. Legume plants differ in the nature of

the nitrogenous compounds they export from their no dules

for

systemic transport. Most tropical legumes employ the

ureides, allantoin and allantoic acid

;

temperate zone

legumes generally employ the amides, glutamine and

asparagine. Legume root nodule morphology is of two

types : indeterminate (nodules remain meristematic and

continue to elongate as the plant grows) and determinate

(nodules are spherical with no meristematic tissue at

maturity).

Among the symbiotic Rhizobiaceae, two genera differ

radically in growth rate, DNA homology, base ratios and

organization, capsular exopolysaccharide, carbohydrate

metabolism and their intrinsic resistance to antibiotics

(Jordan 1984). Th ey are the slow-growing Bradyrhizobium

which typically infect and fix nitrogen in tropical legumes,

and the fast-growing Rhizobium, typically symbiotic on

temperate zone legumes. The latter exhibit narrow or

specialized host range : for ,example, Rhizobium meliloti is

symbiotic with Medicago, Melilotus and Trigonella. In con-

trast, the bradyrhizobia exhibit a non-specialized host range

which has been regarded

as

evolutionarily primitive and

ancestral (Norris 1956).

Correspondence to: Dr

J .

Stanley, NC TC Plasmid Genetics Unit, Central

Public Health Laboratory, London N W 9 5HT , U K .

Trinick (1980) described the bacteriology and host range

relationships between fast-growing rhizobia isolated from

several tropical legumes which are normally nodulated by

bradyrhizobia. From

Lablab

he obtained only

a

single effec-

tive fast-growing strain. Although effective cross-infections

between other

of

the divergent plants were common, no

other Rhizobium in the study fixed nitrogen with this plant.

T h e Lablab strain was designated NGR (New Guinea

Rhizobium) 234. In this review we focus on the biology and

the molecular genetics of NGR234. The organism nodu-

lates a very wide range of legumes, including evolutionarily

divergent plants with fundamental differences of nodule

morphology and physiology. We have, where useful, con-

trasted NGR234 with R . meliloti, a narrow host range Rhi-

zobium with which

it

shares certain bacteriological and

genetic properties. Direct comparison is not always possible

since the m olecular genetics of R. meliloti

(a

subject beyond

the scope of the present review) is considerably more

advanced than th at of NGR234.

2 ECOLOGICAL AND EVOLUTIONARY

RELATIONSHIPS

Trinick (1980) reported that NGR234 exhibited over 80

of the symbiotic effectiveness on Vigna unguiculata as the

cowpea Bradyrhizobium inoculum strain CB756; a signifi-

cantly better performance than any other fast-growing

strain. The list

of

tropical legumes nodulated by NGR234

has been revised upwards several times as more plants are

tested. Table 1 lists the plant hosts currently known to be

nodulated by NGR234. This list

is

provisional since the


2/11

10 J . S T A N L E Y AND

E.

C E R V A N T E S

Table 1 Plant

host

range

of

nodulation

(Nod+)

nd nitrogen

fixation (F ix +) of

Rhizobium

strain NGR234

hizobium

m liloti

Reference

Nod Fix-

Acacia farnesiana

Arcachis hypogaea

Centrosema pubescens

Lotus pedunculatus

Medicago sativa

C D )

Sesbania rostrata

Stylosanthes hamata

Phaseolus coccineus

Parasponia andersonii

Nod Fix

Calopogonium caeruleum

Desmodium intortum

Flemingia rongerta

Glycine

max

C D )

unrinatum

canescens

tabacina

soJa

Neiinotonia) wrghtii

Lablab purpureus*

Leucaena leucocephala

Macroptilium atropurpureum

Pachyr hizus tuberosus

Psophocarpus tetragonolobus

Tephrosia candida

Vigna caracalla

lathyroides

palustris

vogelii

iuteola

radiata

sesquipedalis

umbellata

unguiculata

vexillata

wilmsii

hizobium

phaseoli

Trinick (1980)

Lewin

et

al. (1987)

Trinick (1980)

Broughton

et

al . (1986)

Trinick (1980)

Nayudu

&

Rolfe (1987)

Lewin et a l . (1987)

Pueppke (pers. comm .)

Nayudu & Rolfe (1987)

Trinick (1980)

Momson et al. (1986)

Lewin et al. (1987)

Trinick (1980)

Trinick (1980)

Pueppke (pers. comm.)

Morrison et

al.

(1986)

Bassam

et

a l . (1988)

Trinick (1980)

Trinick (1980)

Trinick (1980)

Trinick (1980)

Trinick (1980)

Pueppke (pers. comm .)

Broughton et al. (1986)


Trinick

(1980)





Trinick (1980)


Trinick (1980)



Original

host plant of

NGR234.

CD , Cultivardepen dent response.

num ber of described symbioses is l ikely to increase. Th e

present list indicates that NGR234 neffectively nodulates

( is No d+ Fi x- on) e ight legume genera and the non-legume

plant P a r a s p o n i a . It nodulates and fixes nitrogen with (is

NodFix on)

26

legume species (inclu ding different

genera). Paraspon ia anderson i i , a non-legume plant must be

regarded

as

in a separate category. T h e capacity of

NGR234 o give nitrogen-fixing nodules on plants forming

either indeterminate L e u c a e n a )

or

determinate (V i g n a ,

M a c r o p t i l l i u m , etc.) nodules is very useful for generalized

studies of nodule organogenesis and fu nction.

NGR234 does not conform to the classical cross-

inoculation group concept of Rhizobium speciation. In this

respect, the strain is not unique among rhizobia , but does

provide a striking example of the limitations of current

classification. Trinick

1980)

ostulated that

NGR234

ep-

resented an intermediate evolutionary form between the

ancestral promiscuous bradyrhizobia of tropical soils and

the specialized fast-growing rhizobia (advanced-degener-

ate type) typical of temperate zones such as

R.

melaloti.

A

fairly close evolutionary relationship of NGR234 and

R .

melaloti is indicated by analysis of nif gene sequences from

diverse rhizobia (see Fig.

1

and Badendoch-Jones et al .

1989). Fast-growing soybean rhizobia which share some

bacteriological and host range properties with NGR234

were subsequently isolated from soybean nodules in China.

These

PRC

trains (Keyser

et

al .

1982)

were termed

R .

f r e i i

by Scholla & Elkan 1984). entral and South Am er-

ican biotypes of

R. phaseoli

described by Quinto

e t

a l .

1982, 1985) also share certain host range properties with

NGR234,

and comparative analysis of the

nrfH

gene

sequences (Fig. 1) supports their evolutionary relationship

to NGR234.

Rhizobium st roin NGR234

I--

Rhizobium sesbonioe n if H

1

Rhizobium sesbanioe

n i f H 2

Azotobacter chroococcum

Azotobocter vtnelandti

Klebsiello pneumonioe

Flg. 1

Evolutionary relationships between NGR234, other

rhizobia and bradyrhizobia, and other Gram-negative diazotrophs.

Th e m aterial

is

abstracted from the dendrogram

of

Badendoch-Jones et al. (1989) based

on

comparison of nucleotide

sequences of the nitrogenase Fe-protein


3/11


4/11

12 J. S T A N L E Y A N D E. C E A V A N T E S

NGR234 genome along with the transposon (Bassam et al.

1986). Similarly, Badendoch -Jones et al. (1989) were unable

to obtain homogenotization of NGR234

nif

genes cloned in

vector pSUP102 (pA CYC184 replicon) and mutated with a

non-transposable K mR cassette. The y concluded that

inability to show double-reciprocal crossover in their

experiments was a property of NGR234.

The alternative homogenotization technique, defined by

Ruvkun

&

Ausubel (1981) for R. meliloti, uses IncPl RK2-

derived cloning vectors which replicate in both E.

coli

and

rhizobia and transfer the mutated fragment into the latter

using a helper plasmid which itself fails to replicate.

At

low frequency, reciprocal recombination occurs in the

merodiploids and this homogenotizes the mutated frag-

ment, generating

a

sitedirected mutation. It is selected by

eliminating the IncPl vector through incompatibility with a

chaser IncPI R plasmid, while maintaining selection for

the transposon or interposon marker. Bachem et al. (1985)

used this method to homogenotize three nodC: Tn 5 muta-

tions into pSym of MPIK3030, but observed insertion of

T n 5

a t

deviated positions in nine other cases. They attrib-

uted this to an unexpectedly high frequency of independent

T n 5 transposition in MPIK 3030. Stanley et al. (1988)

employed the method to generate site-directed mutants by

double crossover at the chromosomal hemA locus of

NGR234. Here the R K2 der ive d vector, pRK78 13 (Jones

& Gutterson 1987) was transferred to N GR 234 and R751-

pGM2 was used as chaser, while selecting

a

non-

transposable marker (SpR)cloned into the gene of interest:

a low frequency (4 ) of homogenotization was found. T h e

relative instability of pRK7813 in NGR234 was also

exploited by making serial subcultures of NGR234 contain-

ing

a

pRK7813 clone alone, and subsequently screening for

the low frequ ency of Tcs recomb inants retaining the inter-

poson marker. These were accurate sitedirected mutants a t

two chrom osom al loci (Stanley et al. 1988, 1989).

4.

O R G A N I Z A T I O N

O F THE

GENOME

A circular chromosomal map of NGR234 has been con-

structed (Osteras et al. 1989) using metho ds d escribed in

the preceding section. Th is is shown in Fig. 2. Similar gene

orders can be inferred by comparing the NGR234 chromo-

some map with data for

R .

meliloti (Kondorosi et al . 1977;

Meade & Signer 1977). Several sitedirected mutations

including kem A and rpoN have been mapped, the latter

being

a

useful point of comparison with the homologous

locus ntrA71 of R. meliloti (Finan et al. 1988).

Analytical techniques for extrachromosomal replicons of

various sizes have been widely used to show that most fast-

growing rhizobia have plasmid-coded nodulation (nod) and

nitrogen fixation n t f ) genes (Hooykaas et al. 1981; John-

ston et al. 1987). Rosenberg et al. (1981) demonstrated the

Fig. 2 Chromosome map

of

Rhizobium

NGR234

as described by

Osteras

et

al . 1989)

existence of a symbiotic megaplasmid ca

400

MD a) in R.

meliloti. Subsequent analysis revealed the presence of a

second megaplasmid in R. meliloti, which encodes exo-

polysaccharide

exo)

and dicarboxylate transport dc t ) loci

(Finan et al. 1986). Morrison et al. (1984) detected plas-

mids of 20, 25, 300 and 400 MDa in NGR234. Heat

curing of the 300 MDa plasmid was accomplished by

plating a marked derivative on YM agar with temperature

inhibition (37C) of growth for 7 d followed by shift to

ambient temperature. Two subsequently analysed strains,

ANU264 and ANU265, were confirmed to lack this

plasmid and had lost the capacity to nodulate Lablab,

Vigna, Macroptilium, Leucaena and Parasponia. T h e

nif

genes were located on this 300 MDa l plasmid (M orrison et

al.

1983). Pankhurst et

al.

(1983) sized pSym MPI K30 30 at

some 300 MDa ca 460 kb).

.4

cosmid library of pSym

MP IK30 30 contained

nif

and

nod

hybridizing cosmids and

these regions were reported as separated by about 20-25

kb. Neither pSym NGR234 nor pSym MPIK3030 were

shown to be self-transmissible, and conjugative transfer

methods have paraileled transfer of the R . meliloti mega-

plasmid to other rhizobia (Kondorosi et

al.

1982), using

pJB3JI to mobilize the pSym from an inserted RP4 mob

site. A pSym NGR234 co-integrate (Morrisson et al. 1984)

was transferred to the cured Nod derivative AN U265 ,

using the host plant to select co-transfer with pJB3JI.

Tra nsfe r restored Nod phenotype to a Nod- mutant of

R.

meliloti

but only with respect to the N GR2 34 host plant,

siratro. Similarly, Broughton et al. (1986) transferred pSym

MPIK3030 to a nif-nod deletion derivative of R . meliloti,

and found that transconjugants formed

Fix

nodules on

Vigna unguiculata. Thus the siratro and cowpea host range

of nodulation and Nif-Fix genes of NGR234/MPIK3030


5/11

M O L E C U L A R M I C R O B I O L O G Y O F R H l Z O B l U M SP. N G R 2 3 4 13

were expressed in the R. meliloti background. The nod

mutants of R . meliloti were not complemented for nodu-

lation of their own host, but instead exhibited extended

host range of nodulation to an N GR 234 host.

R-prime plasmids were constructed containing large

regions of pSym NGR234 (Nayudu

&

Rolfe 1987). The

largest, pMN49 (330 kb), conferred on the Sym plasmid-

cured strain ANU265 the whole tested spectrum of

NGR 234 nodulation host range

Lablab, Macroptilium,

Des-

modium, Vigna, Leucaena, Glycine max, Parasponia and Ses-

bania rostrata).

It contained restriction fragments

hybridizing to gene-specific DNA probes for nodA, B, C,

D , I

and

J and nif H , D and

K .

A transconjugant of

ANU265 containing pMN49 was more efficient in nodu-

lation of

Parasponia

and

Sesbania

than NGR234 itself, indi-

cating that some undescribed pSym NGR234 loci restrict

host range: pMN49 is equivalent to a 130 kb deletion of

pSym NGR234. That certain host-specific nodulation loci

are widely dispersed on the pSym is also suggested by the

transfer of a clone bank of pSym MPIK3030 (Pankhurst et

al. 1983) into R . loti, which permitted the detection of three

unlinked cosmids conferring ability to nodulate Vigna

unguiculuta. These cosmids were termed HsnI, HsnII and

Hs nII I (Broughton

et al.

1986; Lewin

et al.

1987).

5.

GENETIC ANALYSIS OF BROAD HOST

RANGE NODULATION

Strains of R. meliloti have provided one of the classical

models for the study of the organization of genes required

for nodulation of legume hosts nod genes). Given the rela-

tive suitability for genetics of fast-growing rhizobia

vis

li vis

bradyrhizobia, strain NGR234 was a natural choice for the

investigation of broad tropical legume host range. Fast-

growing rhizobia from temperate zone legumes possess

large symbiotic (Sym) plasmids containing nod genes,

nitrogenase n z f ) genes and j i x genes encoding associated

proteins for

in

planta nitrogen fixation (for reviews, see

Johnston

et al .

1987; Long 1989). Large Sym plasmids

have not been shown in bradyrhizobia, which have chromo-

somal Sym genes.

T h e nod genes are arranged in operons whose expression

during the infection process is activated by the nodD gene

product in response to flavonoids, compounds of plant

origin which act as inducers. Different R. meliloti nod genes

are required for root hair curling nodABC and H) of Medz-

cago sativa,

formation of the infection thread nodFE) and

induction of cortical cell division/nodule organogenesis

nodABC and H ; Debellk et al. 1987). The nodABC genes

form a contiguous operon in R. meliloti (Kondorosi et al.

1984) and in all rhizobial species where the locus has been

analysed except for NGR234/MPIK3030. The

nodA

and

nodB products are involved in the production of com-

pounds that stimulate mitosis in a variety of plant proto-

plasts (Schmidt et al. 1988), while the nodC product,

located in the membrane, is similar to eukaryotic hormone

receptors (John et al. 1988). Introduction of R. meliloti

nodABC genes together with nodD into Agrobacterium

tumefaciens,

allows transconjugants to curl root hairs of

clover. When in addition the

nodH

gene was introduced,

transconjugants could also curl root hairs of alfalfa. When

transferred to other rhizobia, the R.

meliloti

genes nodFE,

nodH, and nodQ behave like avirulence genes of phytopa-

thogenic bacteria ; .e. they suppress infectivity of the trans-

conjugants on some of their own host plants (Faucher et al.

1989). Following on from this genetic analysis, it has

become possible to identify molecular signals generated via

the activity of nod proteins. Such signal molecules are

essential to the complex process of coordinated infection

leading to nodule formation, and an important example is

the recently identified root hair deformation factor. This

nodABC gene product, modified by the activity of the

alfalfa host-specific gene nodH is a sulphated

1,4 tetra-

saccharide of wglucosamine which elicits root hair defor-

mation of this host plant (Lerouge et al. 1990). Expression

of all nod operons is precisely regulated via three copies of

nodD (Gyorgipal

et al.

1988; Honma & Ausubel 1988)

whose products respond differentially to distinct flavonoids

in the root exudates of R. meliloti host plants; Medicago,

Melilotus

or

Trigonella.

Th e products of

nodD l, nodD2

and

nodD3 bind to nod operon promoters at conserved regions

of

ca

40 bases which have been termed nod-noxes (Rostas

et

al . 1986). Each protein is activated or inhibited by different

flavonoids, and the variety of these molecules present in a

given plant exudate thereby results in overall activation or

repression of the nod operons.

In MPIK3030, the node gene was isolated from a

cosmid library of the Sym plasmid.

nodA

and

nodB

are

closely linked to, but not contiguous with, nodC in

MPIK3030. Although incomplete sequence data are avail-

able, the operon structure clearly differs from other rhizo-

bia, since there is no copy of nodD linked to nodABC, and

insertion mutants at either side of nodC retain nodulation

ability. T n 5 insertions in

nodC

were

N o d -

on

Macroptilium

and could be complemented by cloned nod genes from R .

meliloti (Bachem et al. 1985; Kondorosi et al. 1986).

NGR234 contains two

nodD

loci, one of which

nodD1)

is

currently known to be functional, and can be com-

plemented by the nodD3 gene of R . meliloti (Honma

et

al.

1990). Historically, the function of

nodDl

of NGR234 and

MPIK3030 has been established as a result of experiments

where diverse cloned DNA fragments were transferred to

other rhizobia, and the transconjugants tested for altered

host range of nodulation (Bachem et al. 1986; Bassam et al.

1986). A precisely delimited subclone of nodDl of NGR234


6/11

14

J. STANLEY AND E CERVANTES

conferred on R. trrfolii ability to nodulate Macroptilium,

Vigna, Glycine and the non-legume Parasponia andersonii

(Bender et al. 1988). Transconjugants containing the cloned

gene also induced cell division on Desmodium intortum,

callus-like structures on Leucaena leucocephala and root

swellings on Sesbania rostrata (Nayudu et al. 1988). The

nodD1 protein, interacting with plant flavonoids, regulates

the expression of nodA-lac2 fusions in M P I K 3 0 3 0 . Com-

parison of the DNA sequencederived nodD proteins from

R . meliloti and NGR234 showed that their amino terminal

region was highly conserved, while their carboxy terminal

regions were divergent. Construction of a chimaeric

(MPIK3030-R. meliloti) nodD gene confirmed that the

carboxy terminal domain of its product was responsible for

interaction with specific root exudate factors of alfalfa

or

siratro (Horvath

e t

al. 1987). In NGR 234,

nodDl is

constitu-

tively expressed (Bassam et

al.

1988). Nayud u et al. (1988)

cloned and sequenced the gene demonstrating also that th e

encoded protein activated nod gene expression in response

to a great variety of plant root exudates. These included all

flavonoid activators for R. meliloti and R. leguminosarum

(apigenin, luteolin snd dihydroxyflavone), antagonists of

these systems such as formononetin and umbelliferone, and

even non-legume root exudates such as those of cotton,

sunflower and Casuarina, a plant which is nodulated by the

actinomycete Frankia.

It

is

difficult to make further comparisons between nod

genes of NGR234 and

R.

meliloti,

since

nod

genetics in

NGR234 has its basis only in gain-of-function approaches,

v i z . transfer of broad host range. On the other hand, in R.

meliloti, long-term programmes have been based on random

T n S mutagenesis. leading to the identification of many nod

genes by loss-of-function (delay or absence of nodulation,

atypical physiological or cytological responses during root

hair infection

or

nodule organogenesis).

At

the time of

writing three nod genes, other than nodC and nodDl , have

been identified in NGR234/MPIK3030. A gene closely

linked to nodD, nod-81 (Bassam et al. 1988) has no defined

phenotype and lacks a nod-box in the 5 region, although

the NGR234 nodD protein activated its transcription in

response to the broad spectrum

of

flavonoids described

above. Two genes characterized in the MPIK3030 cosmid

HsnII (Lewin et al. 1987) were termed nodSiJ. These are

preceded by a nod-box promoter. n o d s was sequenced and

a site-directed mutant therein was unable to nodulate Leu-

caena

(Lewin, personal communication).

6.

EXOPOLYSACCHARIDE OF NGR234

Polysaccharides contribute greatly to the composition of the

rhizobial cell surface and are therefore likely to be involved

in recognition events and plant infection mechanisms. Rhi-

zobial extracellular polysaccharides (EPS) include charged

heteropolysaccharides, neutral glucans and lipopolysaccha-

rides (LPS). T h e precise role of EPS

in

the development of

nitrogen-fixing nodules in legumes

is

not completely resolv-

ed. Some Rhizobium exopolysaccharide mutants are unaf-

fected in symbiotic nitrogen fixation, others are uncoupled

for development of normal nodules (Finan et al . 1985);

others are defective in either nodulation or nitrogen fixation

(Chen

et al.

1985). Exopolysaccharide synthesis

exo)

genes

are defined by their effect on production of extracellular

or

capsular polysaccharide. They have been extensively char-

acterized in R . meliloti, which produces acidic hetero-

polysaccharide (see Leigh & Lee 1988). The major acidic

exopolysaccharide produced by

R .

meliloti

is succinoglycan,

which consists of repeating units of p-linked glucose and

galactose with acidic sidegroups. The precise structure of

the nonasaccharide repeat unit of the EPS of NGR234,

which lacks acidic substituents, has been determined by

13N MR spectroscopy (Djordjevic et al. 1987).

In R. rneliloti 12 loci have been described in the second

megaplasmid e x0 P , N , M ,

A ,

L ,

K , H

J

G, F

Q, and B)

and four in the chromosome exo

C ,

D , R and

S ) .

Several

exo mutants are pleiotropic, and also affect lipopolysaccha-

ride and 8-1-2 glucan synthesis. All exo mutants exhibit

altered synthesis of acidic exopolysaccharide (non-pro-

duction, over-production

or

non-succinylation of EPS). In

NGR234, Chen

et al.

(1985), isolated nine phenotypic

classes of EPS-defective T n 5 mutants differentiated by the

quantity and quality of the EPS produced, as well as by

infection phenotypes on various host plants. For instance,

the mutant ANU2885 produced normal nodules on deter-

minate nodule plants Macroptilium, Desmodium, and

Lablub), but only calli on Leucaena, an indeterminate

nodule plant host. The mutants ANU2811, ANU2820 and

ANU2840 produced no detectable oligosaccharides. In co-

inoculation experiments with the pSym-cured strain

ANU265, these mutants can form normal nodules on Leu-

caena. Addition of wild type EPS

or

its oligosaccharide

repeat unit mimicked the helper effect of ANU265, and

allowed the

exo

mutants to form normal indeterminate

nodules (Djordjevic et al. 1987). The pleiotropic mutant

AN U28 6 1, an EPS-over-producing adenine-requiring aux -

otroph, was Nod- on Macroptilium and Desmodium, and

induced localized pathogenic effects on the former plant

host (Djordjevic et al. 1988). In split-root experiments

ANU2861 could inhibit nodulation of Macroptilium by wild

type NGR234. By using R plasmids for complementation

analysis, Chen et al . (1988) located within a 15 kb region of

DNA, 26 mutants in five NGR234 loci encoding EPS syn-

thesis. Two regulatory genes, exoX and Y, have been

sequenced (G ray et al. 1990).


7/11

M O L E C U L A R M I C R O B I O L O G Y

OF

R H l Z O B l U M

SP.

N G R 2 3 4 15

7 SYMBIOTIC NITROGEN FIXATION A ND

METABOL ITE EXCHANGE IN THE

NODULE

T h e overall physiology of the root nodu le symbiosis may be

described in the broadest terms as an exchange, modulated

by oxygen, of plant-reduced carbon for bacterially-reduced

nitrogen. Rhizobial bacteroids must respire aerobically to

provide energy for nitrogen fixation, itself a process sensi-

tive to free oxygen. These seemingly contradictory physio-

logical constraints are resolved by facilitated diffusion of

oxygen, mediated by a nodule-specific plant protein

(nodulin) called leghaemoglobin (Lb). Evidence that the

apoprotein of L b is plant-encoded was initially provided

since a common globin was produced in Vigna sesquipedalis

nodules induced by either NGR234

or

the divergent

Bradyrhizobium

NGR46 (Broughton & Dilworth 1971).

Tracer experiments indicate that the haem prosthetic group

of Lb is synthesized by rhizobial bacteroids (Cutting &

Schulma n 1972) probably via the rhizobial C-4 haem-

biosynthetic pathway whose first committed step is cataly-

sed by 6-aminolaevulinic acid synthase ALAS (Nadler &

Avissar 1977). Molecular genetic analysis of the

hemA

gene

coding ALAS showed that, in R . meliloti, site-directed

hemA mutants lack ALAS and are Fix- on Medicago sativa

(Leong

et

al. 1982). However, a similar site-directed

hemA

mutant of

Bradyrhizobium japonicum

was Fix on

Glycine

max (Guerinot

&

Chelm 1986). These authors remarked

that either the considerable plant (indeterminate nodule

alfalfa vs determi nate nodule soyabean) or bacterial

R .

mel-

iloti

vs

B . japonicum) divergences might account for the

opposite Fix phenotypes of these

hemA

mutants. Stanley

et

al.

(1988) cloned the NGR234 hemA gene and constructed

a site-directed

hemA

mutant, which produced Fix- nodules

on either determinate Lablab,

Vigna,

Macroptiliurn)

or

indeterminate Leucaena) nodule plants. This result sup-

ports the general concept that bacteroid ALAS is required

for synthesis of the prosthetic group of nodule L b. Th e

B.

japonicum data might be explained by the presence in this

bacterium of tRNA-glu dependent C-5 haem synthesis.

This pathway, which exists in higher plant plastids, was

recently shown to also exist in E.

colt

(Li

et

al

1989).

T h e nitrogenase-catalysed reduction of dinitrogen rep-

resents one side of the metabolic exchange in the mature

nodule, while supply of reduced carbon from host photo-

synthate represents th e other. T h e three componen t poly-

peptides of nitrogenase and their

n i f

structural gene

sequences are strongly conserved among diazotrophic bac-

teria (Ruvkun & Ausubel 1980). Organization and expres-

sion of the pSym-coded rhizobial nif genes have been most

fully characterized in R .

meliloti,

where the nitrogenase

polypeptides are encoded by an operon, nrjHDK. This, and

the adjacent cluster of n i f and j x genes correspond to an

equivalent region in the chromosome of the free-living

diazotrophic bacterium Klebsiella pneumoniae. T he cluster

is positively regulated in symbiosis by the nifA gene

product and by RNA polymerase containing an alternative

Sigma factor. T h e latter is encoded by the rpoN gene

(Ronson et al. 1987). Expressio n of nifA itself is regulated,

not by combined nitrogen as in Klebsiella pneumoniae, but

by oxygen (Ditta et al. 1987). A second g roup of symbiotic

j x

enes are nifA-independent. The y, and

nifA,

depend for

expression on the key regulatory genes f ixL and

j x J .

Pro-

ducts of these genes form a two-component regulatory

system (as is the case with

d c t B / D ;

see Ronson

et

al. 1987

and description below). A summary of the R .

meliloti

n i f

genes and their cascade regulation via jixLJ may be found

in David

et

al.

(1988). Among all these genes only

nzjHDK

and rpoN have been fully characterized in N GR2 34.

Badendoch-Jones et al (1989) showed that a n i j H D K

operon exists in NGR234, characteristic of fast-growing

rhizobia, rather than

Bradyrhizobium,

where n i j H is

separated from ntfDK by some 20 kb. The operon is preci-

sely duplicated, as confirmed by D N A sequence analysis of

both copies. The existence of two EcoRI niffragments of

4.0 and 3.2 kb and 2

Hind111

niffragmen ts of 8 and 13 kb

is diagnostic for this

n f

gene duplication on pSym

NGR234. Within the coding region

of

the operon(s)

restriction sites are completely conserved. NGR234 shares

this nifgene duplication with Central and South American

biotypes of R.

phaseoli

(Quinto

et al.

1982, 1985) and with

R .

fredii (Prakash & Atherley 1984). The predicted amino

acid sequence of the nitrogenase Fe protein was compared

with that of other diazotropic bacteria, showing that

NGR234 is phylogenetically related both to R .

meliloti

and the Central/South American biotypes of R .

phaseoli

and divergent from

Bradyrhizobium japonicum

or

Bradyrhizobium parasponia. Both NGR234 nif operons

show typical regulatory elements for nif gene expression

as also found in R.

meldoti.

A consensus element was

found at positions 25/18 bp and 13/9 bp of the tran-

scription start point. This conserved promoter,

5-(C/T)TCG-Nl,-GC(A/T)-3

is the site of interaction

of RNA polymerase charged with Sigma factor RPON. One

phenotype of an rpoN mutant of NGR234 is, therefore,

inability to express the nif operons. An Upstream Activator

Sequence, 5-TGT-N4-T-N,-ACA-3, where n ~ A

product interacts was also found (for a review of these pro-

moters, see Dixon 1987). Fusions to a chloramphenicol

acetyltransferase gene were used to elucidate functionality

of the two,

nif

operons in planta. Both n i f promoters/

operons had similar activity in nodules of the original

NGR234 host plant,

Lablab purpureus.

There has been no

report of the expression of NG R23 4 nitrogenase activity ex

planta, as can be shown for some bradyrhizobia. However,


8/11

16 J. STANLEY AND

E.

CERVANTES

as in

R. meliloti

(Szeto

et al.

1987), microaerobic conditions

allow a low level of

nif

gene expression in NGR234 ex

planta, which can be detected with ap prop riate gene fusions

(Stanley

et al.

1989).

In R.

meliloti, rpoN

has been sequenced. The gene is

required for expression of the d c t A (dicarboxylate

permease) gene (Ronson et a l 1987)

A

sitedirected rpoN

mutant of NGR234 did not fix nitrogen in nodules of any

tested host plant, and ex planta failed to transport labelled

succinate, a C-4 dicarboxylic acid. The NGR234

rpoN

gene

was sequenced and showed strong similarity to its R. melil-

oti homologue. Furthermore, the NGR234 rpoN mutant

exhibited a delayed nodulation phenotype on its hosts,

Macroptilium and Vigna. Its phenotype was examined with

respect to determinate nodule organogenesis

:

microscopic

analysis of

Vigna

and

Macroptilium

nodules showed that

the mutant formed bacteroids, but that these were not

enclosed by host synthesized peribacteroid membrane

(pbm) . Thus

rpoN

is an important regulatory element

throughout the symbiotic life cycle of NGR234 (Stanley et

al. 1989; van Slooten et al. 1990).

It is established that both NG R234 and R. meliloti use a

broad range of hexoses, pentoses, disaccharides, trisaccha-

rides and organic acids. Both metabolize hexoses via the

Entner-Doudoroff and pentose phosphate pathways. With

respect to carbon metabolism in the nodule, the operation

of

the T C A cycle is essential for bacteroid metabolism

(Trinick 1980; Stowers 1985).

A

question of importance

is

the identity of the plant-supplied subs trate which drives

nitrogen fixation. Saroso et

al.

(1984) showed that NG R234

possesses a large number of inducible catabolic enzyme

systems. However, functional sugar transport systems are

not required for bacteroid nitrogen fixation, which instead

depends on C-4 dicarboxylic acids (succinate, malate),

transported via a common inducible (D ct) permease

(summarized in Stowers 1985). Saroso

et al.

(1986) found

that bacteroids

of

NGR234 isolated from

Vigna sesquipe-

dalis nodules contained very low activities of Enmer-

Doudoroff and other sugar-catabolic enzymes ; in this

respect they resembled succinate-grown vegetative cells of

NGR234. If vegetative cells were grown in a mixture of a

C4-dicarboxylate and sucrose, sugar-catabolic enzymes

were present. Therefore Vigna pbm was presumed to be

impermeable to sugars, dictating that C-4 dicarboxylates

are the carbon source for NGR234 bacteroids. Experimen-

tal evidence has shown that the pbm of Glycine max, an

NGR234 host, was not permeable

to

the amino acid gluta-

mate, but did contain a plant dicarboxylate transporter

(Udvardi et

al .

1988). The bacterial Dct regulon in R. mel-

i lo t i is composed of three genes dctA B and

D

(see Yarosh

et al.

1989). These encode a membrane-bound permease

(D CT A) and a sensor protein for C-4 dicarboxylates

(DCTR) which transduces a signal to a protein activator

( D C T D ) o f dctA transcription. T h e latter co-regulates

dctA

expression with RNA polymerase containing the alternative

sigma factor, RPON. DCTB and

DCTD

form a two-

component system, homologous to a number of similar

pairs

of

proteins such as the products of the

R.

meliloti

genes J x L ,

J

(Ronson et

al.

1987) The dctA gene of R.

meliloti (Engelke et a l 1989) and of NG R234 (our unpu b-

lished results) both contain in their

5

regulatory region, a

nrf-type consens us promo ter recognized by RN A

polymerase-RPON. Hen ce in both these rhizobia, the

uptake of plant-supplied reduced carbon substrate , and the

synthesis of nitrogenase depend on the same system

of

genetic regulation via th e rpoN encoded Sigm a factor.

8.

CONCLUDING REMARKS

Rhizobium NGR234 is a broad host range strain, resem-

bling R. meliloti bacteriologically, and with respect to

various analysed chromosomal and plasmid-borne genetic

loci. Determinants of nodulation, plant host-specific inter-

action, exopolysaccharide, nitrogenase and symbiotic

metabolism have been characterized. Structural

nifKDH)

and regulatory nodD1, rpoN, exoX, Y) genes have been

sequenced. The chromosome and Sym plasmid have been

partly map ped. T h e broad host range of NGR234 is largely

determined by its possession of

a

nodD gene apparently

non-specific in action, rather than host-specific as with

nodD

genes of narrow host range rhizobia. T hi s prope rty

has allowed studies of rhizobial gene expression in evolu-

tionarily divergent plant hosts. Our current knowledge of

NGR234 suggests that this organism should find wider use

in genetic, physiological and ecological studies of the

Rhizobium-legume symbiosis.

9 ACKNOWLEDGEMENTS

We wish to thank

S.

Pueppke for kindly communicating his

recent nodulation test data for NGR234 on various

legumes, which have been included in Table 1.

M.

Osteras

and

J.

van Slooten contributed unpublished results.

J . S

thanks

S.

Dawa for helpful discussions.

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