Molecular Microbiology (1992) 6(2)

Molecular Microbiology (1992) 6(2), 231-238
Secretion of the Rhizobium leguminosarum nodulation
protein NodO by haemolysin-type systems
A. K. Scheu,^^ A. Economou,^^ G. F. Hong,^
S. Ghelani,^ A. W. B. Johnston^ and J. A. Downie^*
^John Innes Institute. John Innes Centre, Coiney Lane,
Norwich NR4, 7UH. UK.
^Institute of Biochemistry, Academy Sinica. Shanghai
20031. China.
^School of Biological Sciences. University of East Anglia.
Norwich NR4 7rj. UK.
Summary
The Rhizobium leguminosarum biovar viciae nodutation protein NodO is partially homologous to haemolysin of Escherichia coli and, like haemolysin, is
secreted into the growth medium. The NodO protein
can be secreted by a strain of E, coli carrying the
cloned nodO gene plus the haemolysin secretion
genes hlyBD, in a process that also requires the outer
membrane protein encoded by tolC. The related protease secretion genes, prtDEF, from Erwinia chrysanthemi also enable E. coli to secrete NodO. The
Rhizobium genes encoding the proteins required for
NodO secretion are unlinked to nodO and are unlike
other nod genes, since they do not require flavonoids
or NodO for their expression. Although proteins similar
to NodO were not found in rhizobia other than R.
leguminosarum bv. viciae, several rhizobia and an
Agrobacterium strain containing the cloned nodO
gene were found to have the ability to secrete NodO.
These observations indicate that a wide range of the
Rhizobiaceae have a protein secretion mechanism
analogous to that which secretes haemotysin and
related toxins and proteases in the Enterobacteriaceae.
Introduction
Rhizobium leguminosarum biovar viciae has 13 identified
nodulation (nod) genes that are located on the indigenous
'symbiotic' piasmid and are involved in the host-specific
nodulation of legumes such as peas and vetch (Downie
Received 1 August. 1991: revised 7 October, 1991. tBoth of these authors
contributed equally to this work. JPresent address: Centro Nacional de
Biotechnologia, Serrano 115, Madrid 28006, Spain. "For correspondence.
Tel. (603) 52571; Fax (603) 56844,
i
and Surin, 1990). Several of the nod gene products are
involved in the synthesis of a low molecular-weight signal
molecule that is secreted into the growth medium.
Lerouge et ai {1990) characterized the signal from Rhizobium meliloti as an acylated sulphated derivative of
tetraglucosamine. This glycoNpid signai is necessary for
initiating the programme of nodule organogenesis in
alfalfa and can induce empty nodular structures when
added to alfalfa roots even in the absence of rhizobia
(Truchetefa/., 1991). It is clear that different rhizobia make
structurally related but subtly different signal molecules
{Spaink et ai. 1991) that can determine specificity
between the Rhizobium strain and its host legumes.
Although the glycolipid signal is a prerequisite for all of
the steps in nodulation. one R. leguminosarum bv. viciae
nod gene {nodO) encodes a secreted protein (de Maagd et
ai., 1989; Economou et ai.. 1990) that plays an important
role in nodulation of peas and vetch. Whereas mutations in
nodO do not significantly affect nodulation (Economou et
ai, 1989). a deletion analysis revealed that, in the absence
of the nodFE genes, nodO is essential for nodulation of
vetch (Downie and Surin, 1990).
The NodO protein contains multiple repeats of a nineamino-acid glycine+aspartic acid-rich region that have
been proposed to be responsible for its ability to bind Ca^*
(Economou ef ai., 1990) and to be analogous to the
Ca^*-binding domain of haemolysin defined by Ludwig et
ai. (1988). The secretion of NodO into the grovrth medium
(Economou et ai.. 1990; de Maagd etai. 1989) does not
involve the general export pathway (Pugsley et ai, 1990)
that utilizes an W-terminal transit sequence since there is
no processing of the N-terminus of NodO. In these
respects NodO is similar to haemolysin which is secreted
by uropathogenic strains of Escherichia coii. These strains
have a specialized secretion mechanism that involves two
membrane proteins, encoded by the hiyBD genes
(Wagner et ai, 1983; Mackman et ai, 1986). An outer
membrane protein (encoded by tolC) is also necessary for
the secretion of haemolysin (Wandersman and
Delepelaire, 1990). The plant pathogen Erwinia chrysanthemi uses a similar secretion mechanism for the
secretion of the proteases PrtB and PrtC. In this case the
E. chrysanthemi prtDEF genes (analogous to hlyBD and
tolC) are involved in the secretion mechanism (Letoffe et
al.. 1990) and confer on £ coli the ability to secrete the
proteases. Here, we demonstrate that the NodO protein is
232 A K.Scheuetal.
a b e d
NodO*' —
e f g h
—
nodO + - + ' ' lnduc«r + + - -
•••
+
+ +
^
+^ -
Fig. 1. Protein gel and immunoblot showing secretion of NodO in the
absence of flavones or other nod genes. Polyacrylamide gels corrtaining
proteins concentrated from growth-medium supernatants were either
stained with Coomassie Brilliant Blue (lanes a, b, c, d) or electrobtotted
to a nitrocellulose filter which was then reacted with antiserum to NodO
(lanes e, f. g. h). Lanes (a) and (e) were from a sample prepared from the
control strain, A168(Sym' NodO*), grown with 1 M-M hasperetinto
induce nod genes. Lanes (b) and (f) were from a sample ot strain A169
(Sym' NodO ), grown in the presence of 1 ^M hesperetin showing the
absence of NodO (b) and the lack of antibody staining (f). Lanes (c) and
(g) were from Sym-plasmid-cured strain Al65 carrying plJ1790 (with
nodO expressed constitutively} grown jn the absence of nod gene
Inducars. showing that NodO is secreted in the absence of the symbiotic
piasmid or nod gene induction. Lanes (d) and (h) were from control
samples of Sym-plasmid-cured strain A165 carrying the vector (pKT230)
used to make plJI 790. The presence of riodO is indicated and 'c' refers
to the constitutively expressed rmdO gene on plJ1790.
signal with a similar fraction from a nodO mutant strain
(Fig. 1. lanes b and t) or with total cellular proteins from
either the mutant or wild-type strain {data not shown).
Strain A165 (which lacks a symbiotic piasmid) containing
the nodO gene expressed constitutively on plJ1790
secretes NodO protein in the absence of flavonoids {Fig. 1.
lanes c and g). Therefore it is evident that genes required
for NodO secretion are expressed independently of flavonoid induction and are not present on the symbiotic
piasmid.
NodO secretion mediated via the haemolysin secretion
system
No secretion of NodO protein was observed from a strain
of E. coli carrying the nodO gene cloned on piasmid
plJ1814 in which nodO is under the control of the lacZ
promoter {Fig. 2, lane c). Since NodO protein could be
identified within the cellular fraction {Fig. 3, lane d) of this
strain, the lack of secretion cannot be due to a problem of
expression of NodO protein.
secreted by a similar mechanism. {The term 'secretion' is
used here to signify release of a protein into the growth
medium, whereas 'export' is used more generally to
signify transiocation of a protein across the cytoplasmic
membrane.) It appears that although NodO is absent from
many other rhizobia, it can be secreted by a wide variety of
rhizobial strains.
Results
To study the secretion of the NodO protein, a polyclonal
antiserum was prepared to the purified NodO protein.
Piasmid plJ1790 is a broad host-range piasmid in which
the nodO gene is expressed constitutively (from a vector
promoter) and can also be expressed inducibly {by flavones such as hesperetin) at high levels from its own
promoter under the control of the nod gene transcriptional
regulator encoded by nodD. When R. leguminosarum bv.
viciae strain A168 carrying plJ1790 is grown in the
presence of hesperetin, a large amount of NodO protein is
secreted into the growth medium. NodO protein was
purified from the growth medium of such a culture and
used to raise an antiserum. As shown {Fig. 1, lanes a and e)
the antibody reacts with the NodO protein present in the
growth-medium supernatant of a strain of R. leguminosarum bv. viciae induced for nodO expression but gives no
nodO
hlyBD
toiC
prtDEF
hlyA"
-
Rg. 2. Stained protein gel showing secretion of NodO proteins by strains
of E. coli carrying the haemolysin or protease (PriB) secretion genes.
Strains of E. coli were inoculated from an ovemight cutture o( L-broth
and grown for 2~3h until an optical density of about 0.7 was reached,
and then for 5h in ths presence of isopropyl thiogolactoside (20M.gml '),
The proteins from 75ml of culture supernatant were concentrated on
nitrocellulose filters, resuspended in 400 iil of loading buffer and a
fraction (20p.! for each, except lane b which contained IOM-') was
electrophoresed on a 12% polyacrylamide gel, which was stainad with
Coomassie Brilliant Blue. The strains used were: a. MC4100 + pLG575
(WySD); b, MC4100+PLG575 (WySD)-* pLG609 {rt/yA'); c, fVIC4100
+p!J1814 (nodp); d, MC4100 + pLG575 (ft/yBCi) + plJ1814 {nodQ: e.
MC4100 (to;C::Tn)0)+pLG575 (h/yeCJ)+plJ1814 {nodQ: f, MC4100
((o/C::TnrO)+pRUW4 (prfD£fl+plJ1814 {nodQ: g. MC4100 + pRUW4
(prtD£f)+plJ1814 (nodQ: h, MC4100+pRUW4 ipnOEF). Strain MC4100
{tolCv.lniOi was a kind gift from Dr C. Wandsrsman. The identity o( the
NodO protein in lanes (d), (f) and (g] was confirmed by immunoblotting
and no NodO protein was detected in the other lar>es.
Secretion of NodO by haemolysin-type systems
abc d e
Fig. 3. Immunobiot of total cellular proteins showing that cellular NodO
protein is present within E. coli but not Rhizobium strains. The bacterial
cells were washed three times with lOmM Tris-HCI (pH7.0), sonicated,
and the released proteins solubilized by boiling in loading buffer. The
total cellular proteins (10M.g} were electrophoresed in a potyacrylaniide
gel and immunoblotted to nitrocellulose. NodO was detected using
antibody as described earlier (Fig. 1). Lane (a) contains secreted NodO
protein control from a semi-purified preparation. The other lanes contain
total cellular proteins from: b, cells of Rhizobium strain Al 68 induced for
NodO expression; c, cells ot E. co/'strain MC4100+pUCl8: d, cells of E.
coli, MC41D0+pU181't (nodQ\e, cells of E. co''MC4100-HplJi814
It was established previously (Economou etai, 1990; de
fyiaagd et a/.. 1989) that NodO is secreted by a mechanism
that does not involve an W-terminal transit sequence and
that NodO shows homology to the Ca^"" -binding domain of
haemotysin (Economou etai. 1990) and a group of other
proteins (including cyclolysin, leukotoxin and two metalloproteases) that are secreted by a system that does not
involve an W-terminal signal sequence. Instead, it appears
that these proteins are secreted by a mechanism that
involves an interaction between a C-terminal secretion
signal (Nicaud et ai.. 1986; Koronakis et ai, 1989;
Delepelaire and Wandersman, 1990) and a specialized
secretion system comprising the products of the hlyBD
and toiC genes (Wagner et ai.. 1983; Mackman ef ai,
1986; Wandersman and Delepelaire. 1990). In addition to
mediating the secretion of haemolysin, the hiyBD+tolC
genes can allow E. coii to secrete other proteins such as
ieukotoxin, cyclolysin and some proteases, and some
bacteriocins (see the Discussion).
To test if NodO is secreted via a similar mechanism, the
noc/Ogene cloned on plJ1814 was transferred to an E coli
strain cartying the hiyBD genes on a separate repiicon
(pLG575) and the wild-type toiC gene on the chromosome. As shown in Fig. 2 (lane d), a significant amount of
NodO protein was secreted by this strain. The amount of
NodO secreted was less than that observed with the
positive control, a truncated derivative of a-haemolysin
(Fig. 2, laneb). However, it is clear that NodO secretion can
be mediated via the haemolysin secretion system since no
secretion of NodO was seen in the absence of hiyBD
genes (Fig, 2, lane c). In a similar experiment it was found
233
that the E. chrysanthemi protease PrtB secretion genes,
prtDEF (cloned on pRUW4). could confer on E. coii the
ability to secrete the NodO protein (Fig. 2, lane g).
Figure 2 (lane e) shows that the secretion of NodO from
E coli is to/C-dependent, since the tolC mutant carrying
the hlyBD genes could not secrete NodO. The tolC mutant
strain carrying the prtDEF genes does secrete NodO (Fig.
2, lane f). Since PrtF is significantly homologous to TolC
and PrtD and E are homologous to HlyB and D (Letoffe et
ai, 1990), it is probable that in the presence of the prtDE
gene products. prtF complements the toiC mutant for the
ability to secrete NodO. The prtF and toiC gene products
normally confer specificity on the secretion system since
the E. coli TolC protein cannot substitute for PrtF in the
secretion of the E chrysanthemi protease. PrtB (Letoff6
et ai. 1990). In this respect, it appears that NodO can be
secreted via either system.
However, as shown in Fig. 3 (lane e), not all of the NodO
protein was secreted from the E coli strain carrying nodO
and hiyBD (or prtDEF. data not shown) since NodO could
be identified in the total cellular fraction using NodO
antibody. As shown (Fig. 3. lane b), NodO protein is not
normally found in the total cellular fraction of R leguminosarum bv. viciae induced for expression of nodO under the
control of its own promoter. Even in those situations where
high levels of nodO expression have been achieved in R.
ieguminosarum bv. viciae carrying the cloned nodO. no
cellular NodO protein has been detected (data not shown).
The NodO protein secreted from R. ieguminosarum bv,
viciae is of the same apparent molecular weight as the
protein found in the cellular fraction of E. coli, indicating
that there is no significant alteration in the size of NodO as
a result of its secretion (Fig. 3. lanes a and d). The
isoelectric points of the secreted and unsecreted proteins
were also identical (pi = 4.2 as judged by Western blotting
of two-dimensional gels), indicating that there is no
apparent alteration of NodO during secretion (data not
shown).
NodO is secreted by severai rhizobiai genera
Since NodO secretion genes are not on the symbiotic
plasmid of R. Ieguminosarum it seemed possible that
other strains of rhizobia might be able to secrete NodO,
although it was noted previously (de Maagd et ai. 1989)
that antibody against NodO did not react with other
rhizobiai strains induced for nod gene expression. Using
the antibody described here, we confirmed and extended
the observations of de Maagd etai (1989) in that the NodO
antibody prepared here did not cross-react with strains of
Azorhizobium caulinodans. Bradyrhizobium japonicum,
Rhizobium meiiloti. Rhizobium fredii, or even R. ieguminosarum biovar trifoiii grown under conditions in which their
nod genes were expressed. However, when ptasmids
234
A. K. Scheu et al.
a b c d 6 f
B g . 4. Secretion of NodO by rtiizobia and Agrobacterium. Proteins from
growth-medium supernatants of strains grown in TV in the presence of
flavonoids {to induce nod gene expression) were concentrated, electrophoresed, electroblottsd to nitrocellulose, and visualized with NodOantiserum as described in the legend to Fig. 1. The samples were
prepared from various representative strains of Rhizobiaceae carrying
plJI815 containing the cloned Ft. leguminosarum bv viciae nodO gone:
a, fl. leguminosarum (bv. trifolii): b, R. fredii\ c, R. meliloii\ d, S.
iaponicum: e. A. caulinodans: f, A. rhizogenes. in R melilotiXhe piasmid
carrying riodO was plJ1788. The amount of NodO secreted by the
different strains is approximately equivalent to that found with R. leguminosarum bv. viciae (Fig. 1).
carrying the nodO gene were transferred to a wide range of
bacteria in the Rhizobiaceae it was found that they could
secrete NodO protein into the growth medium. As expected, R. leguminosarum strains carrying the cloned nodO
gene secrete NodO and this is illustrated with R. leguminosarum bv. /r/fo//y{Fig. 4, lane a). NodO protein could also be
identified (Fig. 4) in the growth-medium supernatant of
strains of R. meiiioti. R. fredii. B. japonicum, A. cauiinodans and Agrobacterium rhizogenes carrying the cloned
nodO gene from R leguminosarum bv. viciae. The protein
profiles of the growth-medium supernatant from these
NodO-secreting strains did not show any other newly
appearing polypeptides when compared with those of the
control strains {data not shown). Similarly, using antibody
to transferase (encoded by the vector pKT230) and
neomycin phosphotransferase to probe the blots, no
'leakage' of the cytoplasm was observed. These observations indicate that secretion of NodO from these strains is
active and not the result of cell lysis. In conclusion, it is
evident that many representatives of the Rhizobiaceae do
have the capacity to secrete NodO, although it appears
that they do not normally secrete a NodO-homologous
protein.
Rhizobium ndvA is not required for NodO secretion
Genes such as hlyB and prtD that are involved in secretion
of NodO and related proteins belong to a large group of
genes that encode membrane-associated proteins
thought to be involved in ATP-dependent transport of a
wide variety of substrates (Blight and Holland. 1990). The
HlyB and PrtD proteins belong to a subgroup of these
proteins that are involved in export rather than import
across the bacteria! membrane. The R. meliloti noduledevelopment gene ndvA also belongs to this group and
shows extensive homology with HlyB. NdvA is necessary
for the secretion of a cyclic fi-1,2 glucan and is important
in nodule development. The ndvA gene is not on the
symbiotic piasmid and is widespread among the Rhizobiaceae (Stanfield et ai, 1988), Given these criteria, we
considered the possibility that NdvA could be involved in
the secretion of NodO and, possibly, related proteins.
However, as shown (Fig. 5), a nvdA mutant of R. meliloti \s
not blocked for the secretion of NodO. It is interesting to
note that the efficiency of NodO secretion is reduced,
since some NodO protein could be detected in the cellular
protein fraction of the mutant strain but not in that of the
control strain. However, mutation of nvdA in R. meliloti has
pleiotropic effects on cell-surface and membrane proteins
(Dylan et ai., 1990) and the lowered efficiency of NodO
secretion is probably a secondary effect of the nvdA
mutation.
Discussion
The NodO protein belongs to a small but growing group of
proteins that are recognized as being secreted by a
mechanism that does not involve an /V-terminal transit
a b
W.T. ndvA
LUTEOLIN: +
-
-
W.T. ndvA
+
SECRETED
NON-SECHETEO
Fig. 5. NodO secretion by an nvdA mutant. The Rhizobium strains were
grown as described in the legend to Fig. 1. The secreted proteins from
growth-medium supernatant (lanes a, b, c, d) were prepared as
described in Fig. 1, and the total cellular (non-secreted) proteins (lanes e,
f, g. h} were solubilized as described in Fig. 3. All samples were
electrophoresed in a 12% polyacrylamide gel, immunoblotted to
nitrocellulose, and the NodO proteins visualized by antibodies. Samples
used were: a and e. wild-type R. meliloti 102F34 carrying pU 1788 {nodQ
grown in the presence of 1 \LM luteolin to Induce nodO. b and f, R.
meliloti ^02fZA {O\\Xa et ai. 1980) carrying plJI 788 (nodQ grown in the
absence of luteolln; c and g. R meliloti LI1 {ndvA) (Stanfield et ai. 1988)
carrying plJt 788 {nodQ grown in the absence of luteolin; d and h, R
meliloti U^ (ndv^) carrying plJ1788 grown in the presence of 1 nM
luteolin. There is a low level of constitutive expression of NodO In these
strains grown in the absence of flavorralds (lanes b and c).
Secretion of NodO by haemotysin-type systems
sequence. Instead, these proteins, of which haemoiysin is
the best characterized, are secreted by a mechanism that
involves a C-terminal signal (Mackman et ai, 1987) and a
specialized secretion system (Blight and Holland. 1990).
For E. coli a-haemolysin this secretion system is encoded
by the hiyBD totC genes, and there are analogous genes
for secretion of haemolysin-like proteins from species of
Proteus, Morganella and Actinobacillus (Koronakis ef ai,
1987; Gygi et ai, 1990) as well as the secretion of the
related toxins cyctolysin (Masure et ai, 1990) and leukotoxin (Chang et ai. 1989). In addition to these protein
toxins, metalloproteases from species of Erwinia, Serratia
and Pseudomonas (Delepelaire and Wandersman, 1990;
Letoffeefa/.. 1991; Guzzo etai, 1991) are secreted by an
analogous system, as are the bacteriocins, microcin B17
and colicin V, made by F. co//(Garrido etai, 1988; Gitson
etai, 1990).
All of these bacterial genera belong to the -y-division of
the Proteobacteria, whereas the Rhizobiaceae belong to
an entirely different class, the a-division of the Proteobacteria (Woese. 1987). It is clear from the work presented
here that many genera of the Rhizobiaceae. including
Rhizobium, Azorhizobium, Bradyrhizobium and Agrobacterium. have a similar protein secretion system analogous
to that of haemoiysin secretion from E. co//and secretion
of the protease PrtB from £ chrysanthemi. There is
normally a considerable degree of selectivity in these
secretion systems since E. coli carrying the haemoiysin
secretion genes hiyBD and toiC secretes only very small
amounts of the E chrysanthemi protease PrtB (Delepelaire and Wandersman. 1990). although it efficiently
secretes the Pseudomonas aeruginosa alkaline protease,
AprA (Guzzo etai, 1991). Conversely, the E chrysanthemi
secretion genes, prtDEF, were unable to mediate the
secretion of haemoiysin A. However, it apears that the
NodO protein can be secreted from E coli by either the
a-haemolysin or PrtB secretion system.
In most of the cases described above, some (but not
necessarily all) of the genes encoding the secretion
apparatus are closely linked to the gene encoding the
secreted product. This does not appear to be the case for
nodO, which is present on the symbiotic plasmid (the
genes required for its secretion are not). Moreover, in most
of these other systems, expression of the export genes
{hiyB and its homologues) is under the same regulatory
control as the gene encoding the secreted protein. In this
respect. nodO is also different since the nodO gene is
expressed only after flavonoid-mediated induction
(requiring NodD) whilst those genes required for its
secretion appear to be expressed constitutively. This
situation is similar to that of the cyclolysin gene, which is
environmentally controlled white export genes are
expressed constitutively (Laoide and Ullman, 1990).
These observations, together with the apparent lack of
235
nodO in several rhizobia, suggest that nodO may be a
relativety recent acquisition as a nodulation gene.
However, a wide range of bacteria in the Rhizobiaceae
have the potential to secrete NodO (the first demonstration of a wide-ranging protein secretion system in
these bacteria). This secretion system is very efficient (no
NodO protein was detected in the cellular fractions) and
appears to be expressed constitutiveiy. Therefore this
secretion system is probably not a recent acquisition and
is likety to have a 'housekeeping' function, secreting other
proteins during normal (non-symbiotic) growth. Indeed,
other extracellular polypeptides have been identified in
rhizobial growth-medium supernatants (Katinakis et ai.
1988; Smit et ai. 1989, and our unpublished observations). Another constitutively expressed rhizobial transporter homologous to HlyB is that encoded by the ndvA
genes, but this transporter is required for the secretion of
pi-2 linked glucans (Dylan ef ai, 1990) and is not
necessary for NodO secretion.
It will be of interest to define the domain of NodO that
interacts with the secretion system. By analogy with
haemoiysin and the protease PrtB this domain is likely to
be C-terminal. Koronakis et ai, (1989) defined three
important domains within the 53 terminal amino acids of
haemoiysin; an amphiphilic helix, a cluster of charged
residues, and a terminal hydrophobic region rich in
hydroxylated residues. There is no obvious amino acid
sequence homology between these domains of haemoiysin and the C-terminal domain of NodO. Nevertheless, it
is evident that a signal domain within NodO must be
recognized by both the haemoiysin and protease
secretion systems. Previously, Economou ef ai (1990)
noted that the domain 60-100 residues from the C-terminus of NodO has characteristics similar to those found by
Koronakis ef ai (1989) to be important for haemoiysin
secretion. It may be of interest to note that removal of the
25 C-terminal amino acids of NodO resulted in a truncated
protein that could still be exported (our unpublished
observations).
It is not yet clear why this mode of protein secretion has
evolved in Gram-negative bacteria. It is possible that the
structure of some proteins (such as haemoiysin or NodO)
are such that they required dedicated haemolysin-type
secretion systems that can negotiate both membranes of
Gram-negative bacteria. Whatever the reasons, it appears
that the haemoiysin type of secretion system is very
widespread among Gram-negative bacteria.
Experimental procedures
Microbiological techniques
Media (Tf) and general growth conditions for rfiizobia were those
described by Beringer (1974). using appropriate antibiotics. The
236 A. K. Scheu et al.
Table 1. Bacterial strains and piasmids.
Strain/Plasmid
Charactenstics
Reference
S ^ R. leguminosarum
R. leguminosarum bv, viciae
8400 pflLlJI nod:i99 {nodF-nifH)
8400pssI::Tn5(exopolysaccharide-deficient)
8400pHL1JlpssI::Tn5
A168 notfO::Tn3 HoHoi
Lamb af a'. (1982)
Downieef a'. (1983)
Downia and Surin (1990)
This work
This work
This work
hlyBD
h//4 " derivative
prtDEF
nodABCOEFIJLMN TO
nodO In pLAFRl
nodO in pKT230
nodO in pUC 18
nodO nodD in pKT230
Mackman Gf a/. (1985)
Nicaud efa/. (1986)
L^toffe era'. (1990)
Downie efa/. (1983)
Economou ef a/. (1990)
This work
This work
Ttirs work
Strain
8400
8400 pRLI
A69
A165
A168
A169
Piasmid
PLG575
pLG609
pRUW4
pijiosg
PIJ1788
PIJ1790
PIJ1814
PIJ1S15
tiavone hesperetin was made up as a 1 tnM solution in ethanol and
added to a final concentration of 1 p.M. Strains of E. coii were
grown in L-broth (Maniatis et al.. 1982) at ST^C. Piasmids were
transferred to strains of E. coii by transformation and to rhizobial
strains by conjugation using a helper piasmid to mobilize piasmid
transfer in patch matings. Strains A l 65 and Al 68 were made by
transduction using a derivative of 8401 pRLI Jl pss1::Tri5 carrying
plJ1427 (Borthakur et ai, 1986) as a source ot the pss1::Jn5
allele.
Microbial strains and piasmids
The strains and piasmids used in this study are described in the
text or in Table 1. Routine DNA manipuiations were carried out as
described by Maniatis et al. (1982). Piasmid plJ1790 was
constructed by subcloning a 1.45kb EcoRi fragment from
plM236 (Economou et al.. 1990) into the EcoRI site of pKT230
(Bagdasarian e( ai. 1931), oriented such that the nodO gene is
expressed from the promoter that normally expresses the streptomycin-resistance gene on pKT230; the 1.45 kb EcoRi fragment
also carries the normal nodO promoter that is inducible by
flavonoids in the presence of the nodD gene product, Piasmid
plJ1814 was made by subcioning a l.5kbPsri-Hfndiil fragment
carrying nodO (Economou etai. 1990) into the Psfl-H/ndill sites
of pUC18 (Yanisch-Perron et ai. 1985), such that nodO was
expressed from the lacZ promoter.
Piasmid piJ1815 was derived by cioning the nodO gene on a
1.45kb EcoRI fragment into plJ15l8. a derivative of pKT230
carrying the R. leguminosarum bv. viciae nodD gene (Rossen ef
al., 1985). In this piasmid the nodO gene is expressed at a iow
ievel from a vector promoter and is strongly inducible by fiavones
under the control of NodD.
Protein preparation and electrophoresis
The strains of R. leguminasarum bv. viciae used for the analysis of
secreted proteins were exopotysaccharide-deficient (pss)
mutants, and this simplified the preparation of the proteins from
the growth-medium supernatant. Strains of Rhizobium were
grown for 48h at 28"C in tryptone-yeast (TY) medium. Proteins
from the growth-medium supernatant were concentrated on
0.22-micron nitrocellulose filters and solubilized as described by
Economou et al. (1990). This method was also found to be
suitable for concentration of the truncated HlyA protein encoded
on piasmid pLG609. The proteins were solubilized in sodium
dodecyl sulphate (SDS) loading buffer and eiectrophoresed in
12% acrylamide gels as described by Bradley ef ai. (1988). The
proteins in the gets were visualized with Coomassie Brilliant Blue
R.250, and the NodO protein electroblotted to nitrocellulose
filters was visualized immunologically using antiserum to NodO.
Antibody binding was detected with goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase (Sigma) using the
staining procedures described by Bradley ef a/. (1988).
Preparation of NodO antibody
The NodO protein was concentrated and purified from the
growth-medium supernatant of strain A168+plJ1790 essentially
as described by Economou etal. (1990). As a final step, the NodO
protein was electrophoresed on a preparative SDS-polyacrylamide gel and electroeluted from the acrylamide with a BioRad
model 422 electroeiuter using the conditions described by the
manufacturer. The protein was collected in 500 (tl of running
buffer, desalted using a Pharmacia PDIO (Sephadex G-25M)
column, and freeze-dried. The resulting purified protein (about
500 M.g) was redlssolved in phosphate-buffered saline, mixed with
complete adjuvant, and injected subcutaneousiy into a rabbit at
10 d intervals. After three such injections, the serum, which had
previously not reacted with Rhizobium or E. coli proteins, was
found to have a high titre of NodO antibody. The NodO antiserum
was collected and stored frozen at -20°C.
Acknowledgements
We are indebted to Dr C. Wandersman, Dr B. Holland. Dr G. Ditta,
Dr H. P. Spaink and Dr P. Young for the gifts of strains and
piasmids and for communicating results prior to publication. Dr T.
Kieser kindly made available antiserum to neomycin phosphotransferase and we thank DrV. Koronakis, DrC. Hughes and DrM.
Dow for helpful discussions. Mrs J. Peart and the staff of the
antibody facility at the Food Research Institute, Norwich. UK,
gave invaluable help in preparing NodO antiserum. We also thank
Ms A. Davies for help with strains and piasmids, and Professor D.
A. Hopwood for critically reading the manuscript.
Secretion of NodO by haemolysin-type systems
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