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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
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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)
Pueppke (pers. comm.)
Trinick
(1980)
Pueppke (pers. comm.)
Pueppke (pers. comm.)
Pueppke (pers. comm.)
Pueppke (pers. comm.)
Trinick (1980)
Pueppke (pers. comm.)
Trinick (1980)
Pueppke (pers. comm.)
Pueppke (pers. comm.)
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
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8/10/2019 Stanley and Cervantes
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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
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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
8/10/2019 Stanley and Cervantes
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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).
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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,
8/10/2019 Stanley and Cervantes
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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