Protein and lipopolysaccharide pro®les of a salt-sensitive
Rhizobium
sp.
and its exopolysaccharide-de®cient mutant
S. Unni, K.K. Rao*
Department of Microbiology and Biotechnology Centre, Faculty of Science, M.S. University of Baroda, Baroda 390 002, Gujarat, India
Received 9 November 1999; received in revised form 1 May 2000; accepted 22 May 2000
Abstract
A fast-growing, salt-sensitive rhizobium (Rhizobiumsp. ST1) with a narrow host range of infectivity was isolated from the root nodules of locally grown pigeonpea (Cajanus cajan). One of the Tn5 mutants ofRhizobiumsp. ST1 was exopolysaccharide (EPS) de®cient (exo2) and showed a 50% growth inhibition (GI50) at 350 mM NaCl, compared to the GI50value of the wild type strain at 250 mM NaCl. Whole cell
protein pro®les of the wild type in the presence of NaCl showed an overall increase in the levels of several proteins (22, 38, 68,.97 kDa), whereas in its exo2mutant, certain low molecular weight outer membrane proteins (38 and 22 kDa) decreased. Other outer membrane proteins (22, 38, 40, 42,62 and 68 kDa) also markedly decreased in both the wild type and the exo2mutant in the presence of salt. Similarly, both the wild type and the exo2mutant showed decreased levels of both the lipopolysaccharide (LPS) components (LPS I and LPS II) in the presence of NaCl. These observations suggest the possible involvement of the outer membrane components, along with other factors, during growth under salt stress, in both salt-sensitive and relatively salt-tolerant strains of rhizobia.q2001 Elsevier Science Ltd. All rights reserved.
Keywords:Rhizobium; Salt-sensitive; Salt-stress proteins; Exopolysaccharides; Lipopolysaccharides
1. Introduction
Land degradation due to salts constitute about 7% of the world's land area occupying nearly 952 million hectares. In saline soils, organic matter application or inoculation of
crops and trees with tolerant, symbiotic strains ofRhizobium
or vescicular-arbuscular-mycorrhizae (VAM) help the plants in tolerating stress through improved nutrition (Rao, 1998). The best results are obtained if both the symbiotic partners and all the different steps in their inter-action resist such stress (Sprent and Zahran, 1988; Zahran, 1991). Among the nitrogen-®xing organisms, rhizobia have been shown to be more tolerant to salt stress than their symbiotic host (Singleton et al., 1982). The different
rhizo-bial species vary in their sensitivity to salt.Rhizobium
meli-lotistrains which nodulateMedicago sativaare salt tolerant
(Zhang et al., 1991) so alsoRhizobium fredii(Yelton et al.,
1983). In contrast, certainRhizobium leguminosarumstrains
are sensitive (Chein et al., 1992). The effect of salt-stress on rhizobial lipopolysaccharides (LPS) (Zahran et al., 1994; Lloret et al., 1995), protein pro®les (Saxena et
al., 1996) and exopolysaccharide (EPS) (Lloret et al., 1998) have been studied in highly halotolerant rhizobia. The mechanisms underlying salt tolerance have, however,
not been completely elucidated inRhizobiumand functional
aspects of salt-stress proteins (SSPs) have not been ascer-tained (Saxena et al., 1996). Proteins induced in response to stress may suggest that they have an important role in home-ostasis and maintenance of vital cellular functions (Wankhade et al., 1996). In the present investigation, a salt-sensitive wild type and its relatively salt-tolerant
exopo-lysaccharide de®cient (exo2) mutant were characterized
with respect to their whole cell, outer membrane proteins and LPS pro®les in the presence of NaCl.
2. Materials and methods
2.1. Bacterial strains
Rhizobiumsp. ST1 was isolated from the root nodules of
locally grown pigeonpea (Cajanus cajan) plants. The
culture was grown in Ashby's mannitol (AM) broth (Vincent, 1970) with the following composition, mannitol
1% (w/v), sodium glutamate 0.2% (w/v), K2HPO4 0.05%
(w/v), MgSO4´7H2O 0.02% (w/v), NaCl 0.01% (w/v), on a
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* Corresponding author. Tel.: 191-0265-794396; fax: 1 91-0265-792508.
rotary shaker (180 rev min21) and maintained on AM agar
with 2 drops of 1% (w/v) Congo red at 28^28C.
Escher-ichia coli was grown in LB medium (Sambrook et al., 1989).
Antibiotics were added at the following concentrations:
streptomycin (str) 100mg ml21, kanamycin (kan)
100mg ml21, chloramphenicol (cam)100mg ml21.
2.2. Tn5 mutagenesis
EPS-de®cient (exo2) mutants were generated by random
Tn5 mutagenesis ofRhizobiumsp. ST1 (str1, kan2, cam2)
with the suicide vector pGS 9 inE. coli(str2, kan1, cam1)
using ®lter mating (Saxena et al., 1989). One of the exo2
mutants (str1, kan1, cam2) was designated STxo2.
2.3. Salt stress
The salt tolerance of the wild type and the mutant was screened by their growth in liquid AM medium
supplemen-ted with 50±1000 mM NaCl at 28^28C:These tests were
done in triplicates. The GI50value, i.e. the concentration of
NaCl at which growth of the strain was inhibited by 50%, was calculated.
2.4. Isolation of whole cell proteins, membrane and LPS fractions
For the isolation of whole cell, membrane and LPS frac-tions, the methods described earlier by de Maagd et al. (1988) were followed.
2.5. Analysis of polypeptide and LPS patterns
Sodium dodecyl sulfate (SDS) polyacrylamide gel elec-trophoresis (PAGE) was performed as described previously (Lugtenberg et al., 1975). Samples were prepared by mixing suspensions of whole-cell and membrane fractions with concentrated sample buffer. All samples were routinely
heated at 958C for 10 min prior to electrophoresis. Proteins
were separated on 11% polyacrylamide gels containing 0.2% SDS and stained with Coomassie brilliant blue R-250. LPS was analyzed by PAGE on 15% acrylamide gels with 10% SDS and 0.5% deoxycholic acid as detergents (Parveen
S. Unni, K.K. Rao / Soil Biology & Biochemistry 33 (2001) 111±115
0 50 100 150 200 250 300 350 400
0 50 100 150 200 250 300 350 400
NaCl (mM)
Pr
otein
(ug
ml
)
Fig. 1. Salt tolerance of the wild-typeRhizobiumsp. ST1 proteinmg ml21[X]. and of the exo2mutant STxo2proteinmg ml21[O]. Arrows point to the GI50
value of NaCl forRhizobiumsp. ST1 [Ð] and of the exo2mutant STxo2[- - - -]. (Values represented are mean^s.e of three independent experiments,
p0:05).
Fig. 2. Whole-cell protein pro®les in 11% SDS-PAGE of the wild-type
Rhizobiumsp. ST1 and its exo2mutant STxo2in the presence of NaCl.
et al., 1997) and stained with Alcian blue/silver stain as described by Reuhs et al. (1993).
3. Results
3.1. Salt tolerance
The pigeonpea isolate,Rhizobiumsp. ST1 was found to
be salt-sensitive. The strain had a GI50value of 250 mM
NaCl (Fig. 1) and it showed only 2% growth at 400 mM NaCl as compared to the control.
The exo2mutant ofRhizobiumsp. ST1, STxo2had a GI50
value of 350 mM NaCl (Fig. 1), and it showed about 30% growth at 400 mM NaCl.
3.2. Effect of salt stress on protein patterns
Growth in the presence of NaCl generally altered the
whole-cell protein patterns in SDS-PAGE in both
Rhizo-bium sp. ST1 and its exo2 mutant STxo2. Some bands
disappeared and new bands appeared (Fig. 2). Under salt stress, in the wild type, two low molecular weight proteins
(,40 kDa) 38 and 22 kDa, and three high molecular weight
proteins (66, 68, .97 kDa) were enhanced. In the exo2
mutant, two low molecular weight proteins (36 and 22 kDa) decreased with no appreciable change in the other protein bands in the presence of NaCl. In both the wild type
and its exo2mutant, salt stress caused marked decrease in
concentration of several outer membrane proteins of mole-cular weights 22, 38, 40, 42, 62, 68 kDa (Fig. 3).
3.3. Effect of salt stress on LPS pro®les
In the presence of NaCl, both the wild typeRhizobiumsp.
ST1 and its exo2mutant STxo2showed less LPS
compo-nents than in the absence of NaCl (Fig. 4). The structures of both LPS I and LPS II were clearly modi®ed as observed and the bands present in the controls without salt were absent in the presence of salt.
4. Discussion
A composite stress like salinity, having both an ionic as well as osmotic component, can be extremely detrimental
for the growth of soil-inhabiting bacteria like Rhizobium
(Saxena et al., 1996). Strains ofRhizobiumand
Bradyrhizo-bium show marked variation in salt tolerance (Graham,
1992) and many strains can grow and survive at salt concen-trations, which are inhibitory to the growth, infection and nodulation of various legumes (Busse and Bottemley, 1989; Rao and Sharma, 1995). Most rhizobial strains such as
Rhizobiumsp.WR 1001 (Hua et al., 1982),Rhizobium
meli-loti (Bernard et al., 1986), chickpea and soybean rhizobia
(El Sheikh and Wood, 1990), Rhizobium sp. fromAcacia
senegalandProsopis chilensis(Zahran et al., 1994),
Rhizo-bium sp. ANG4 (Lal and Khanna, 1994) and pigeonpea
S. Unni, K.K. Rao / Soil Biology & Biochemistry 33 (2001) 111±115
Fig. 3. Envelope protein patterns in 11% SDS-PAGE of the wild-type
Rhizobiumsp. ST1 and its exo2mutant STxo2in the presence of NaCl. Lane 1 Ð wild-type control; lane 2 Ð wild-type1NaCl; lane 3 Ð mutant control; lane 4 Ð mutant1NaCl; lane 5 Ð molecular weight markers (kDa). Left arrowheads (Ã) indicate proteins whose concentration declined in the presence of NaCl, and right arrowheads (!) indicate the proteins whose concentration enhanced in the presence of NaCl. Numbers to the right of the gels are in kDa.
Fig. 4. LPS pro®les in 15% SDS-PAGE of the wild-typeRhizobiumsp. ST1 and its exo2mutant STxo2in the presence of NaCl. Lane 1 Ð wild-type
rhizobia (Subbarao et al., 1990) tolerated 0.34±1.7 M NaCl.
Reports on salt-sensitive wild-type Rhizobium sp. were
fewer (Chein et al., 1992; Zahran et al., 1994).
Rhizobiumsp. ST1 and its exo2mutant were able to grow at about the same rate, with respect to each other, both in the presence and the absence of NaCl, i.e. no change in the mean generation times were observed and stationary phases were reached within 24 h. However, studies on the growth
rate of another wild-type Rhizobium sp.(Ohwada et al.,
1998) showed that after the addition of NaCl, its salt-sensi-tive mutant was unable to grow for upto 13 h, though it grew at the same rate as the wild-type without the addition of NaCl.
Though whole-cell protein pro®les of salt-sensitive
Rhizobium sp. HAMBI 1505 from P. chilensis have been studied (Zahran et al., 1994), reports in pigeonpea rhizobia are not available. Saxena et al. (1996) reported that response to NaCl does not induce the synthesis of any unique protein and instead a modulation in the net synthesis of a number of proteins took place, certain proteins were clearly discernible
after NaCl stress. As the wild-typeRhizobiumsp. ST1 was
salt-sensitive, a larger number of proteins were observed in the presence of NaCl as compared to the relatively
salt-tolerant exo2mutant where negligible difference in whole
cell proteins were observed in the presence of NaCl as compared to the protein pro®le in the absence of NaCl. The number of proteins in the wild type may have increased in the presence of the stress condition as these proteins along with other factors may be responsible for the growth and hence survival of the wild type in those stress conditions. As
the exo2mutant was relatively salt-tolerant, fewer proteins
needed to be induced or increased under the stressed conditions.
The two membrane proteins of both ST1 and STxo2,
which had decreased in the presence of NaCl indicate that these outer membrane proteins coupled with several other factors may be essential in responding to the salt stress, the outer membrane proteins being the foremost to encounter the stress. The induction and enhanced synthesis of salinity-stress proteins (SSPs) at higher NaCl concentrations
(appar-ent molecular weights 98±15 kDa) in a salt-tolerant
Rhizo-biumsp. 4a has been reported previously (Wankhade et al.,
1996), where the induced SSPs were identical to osmotic stress proteins (OSPs) indicating that these proteins were induced by the osmotic and not the ionic component of NaCl as seen in other systems such as cyanobacteria (Iyer et al., 1994).
Our results indicate that under salt stress, predominant changes occurred in LPS I compared to LPS II both in the
wild type and its exo2 mutant. A direct effect on the
O-somatic antigen (a component of LPS I) by NaCl may be implicated. Many salt-tolerant rhizobia synthesize LPS with longer chain length in response to stress (Zahran et al., 1994), though no consistent LPS-type was produced as a result of the stress. Further they have reported a shift towards lower mobility bands of LPS I indicates synthesis
of LPS with longer chain lengths which may help protect the cells from the stress. This shift was observed in both the
wild-type Rhizobium sp. ST1 and its exo2 mutant in the
presence of salt.
The wild type, ST1 effectively nodulated its host legume,
pigeonpea. Its exo2mutant, STxo2also showed nodulation
on pigeonpea producing nitrogen ®xing nodules of the same histology as its wild type (data not shown). Hence, there existed no correlation with respect to the EPS production
and nodulation ef®ciency. When the wild-type Rhizobium
sp. ST1 was grown on solid AM medium supplemented with NaCl (250 mM), reduced mucoidy was observed than those grown on non supplemented media, though no difference in ¯uorescence was observed on TY-calco¯uor media in the
presence or absence of NaCl (data not shown). The exo2
mutant was calco¯uor `dark' on both NaCl supplemented and non-supplemented media with no differences in colony morphology. This could indicate that salt-induced structural alterations in the LPS and may be in EPS II (galactoglucan) component and not the EPS I (succinoglycan) of the EPS in the wild type, ST1 whereas in the mutant, EPS I was absent (due to a mutation), though reduced levels of EPS II existed. These small quantities of EPS II along with the unaltered LPS component in the mutant may be responsible for its unchanged nodulation properties. Similar reports of salt-regulated modi®cations on LPS (Lloret et al., 1995) and
EPS II (Lloret et al., 1998) are available in the strain
Sinor-hizobium melilotiEFB1.
More studies on the nature of the proteins affected by salt and the structural changes taking place in the EPS in
presence of NaCl in Rhizobium sp. ST1 and its mutant
could show other mechanisms involved in the bacteria under salt-stress.
Acknowledgements
One of the authors (S.U.) is grateful for the Senior Research Fellowship granted by the Council for Scienti®c and Industrial Research, New Delhi.
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