The Rhizobium-legume symbiosis is the most promising
plant bacterium association so far known. Inoculated Rhizobium
sp. strains often fail to compete with indigenous rhizobia and do not
increase nodulation (Hafeez et al., 2005). Thus the successful
use of rhizobial inoculants requires the knowledge of factors affecting
the effectiveness and competitive ability of the rhizobia. One of the
major factors reported to be affecting competition among rhizobia are
bacteriocins (Oresnik et al., 1999).
Bacteriocins constitute a heterogenous group comprising
protein complexes or peptides with antibiotic effect against closely related
species and strains (Tagg et al., 1976). Root nodule bacteria have
been shown to produce bacteriocins, which have been grouped as small,
medium and large based on their size and diffusion characteristics. The
small bacteriocins are heat tolerant, chloroform soluble and are less
than 2000 kDa molecular weight, medium is generally bactericidal, heat
labile and retained by cellophane and large are defective bacteriophages
Oresnik et al. (1999) found that the bacteriocins
appear to play a major role in determining competitiveness for nodulation
when assayed against some strains. So, the successful preparation of mixed
inoculum requires the knowledge of bacteriocin producing ability of inoculating
strains as well as their effect on related rhizobia. The present research
was taken up to study the bacteriocin producing ability of 20 Rhizobium
spp. isolated from root nodules of 20 legume hosts.
MATERIALS AND METHODS
Microorganism, Medium and Growth Conditions
Twenty Rhizobium spp. were isolated from root nodules of 20
different legume hosts (Arachis hypogaea, Cajanus cajan,
Cassia absus, Clitorea ternatea, Cowpea, Crotalaria
alata, C. juncea, C. laburnifolia, C. retusa,
C. verrucosa, Indigofera hirsuta, I. linnaei, I.
tinctorea, I. trita, Macrotyloma uniflorum, Sesbania
procumbens, S. rostrata, Vigna mungo, V. radiata
and V. trilobata) using Yeast Extract Mannitol Agar (YEMA) medium.
The isolated bacteria were identified as Rhizobium sp. by Bergey`s
Manual of Determinative Bacteriology (Jordan, 1984) and plant infection
test (Vincent, 1970).
Bacteriocin Activity Assay
The bacteriocin producing ability of the strains was bioassayed by
simultaneous (direct) antagonism method (Tagg et al., 1976). Bacteriocin
activity was examined by adding 1 mL of each diluted, sterile filtered
sample on to the wells made on Tryptone Yeast extract (TY) medium (0.6%
w/v agar) seeded with log phase indicator strains (0.5 μL/100 mL
of the medium). Activity was quantified by two fold serial dilution (Barefoot
and Klaenhammer, 1983) and is expressed in arbitrary units mL-1
Purification and Estimation of Bacteriocin Protein
Purification of bacteriocin was carried out by using procedure of Yang
et al. (1992) and Cell Free Supernatant (CFS) was used to carry
out protein extractions. Twenty percent chloroform was added to CFS in
a separatory funnel. The aqueous phase was saturated with cold ammonium
sulphate from 20-100% (w/v) saturation and was kept overnight at 4 °C.
The precipitate was collected by centrifugation at 15,000 g for 30 min.
The solid pellet dissolved in distilled water and dialyzed against distilled
water at room temperature for 24 h. The suspension obtained was designated
as proteinaceous fraction or crude bacteriocin fraction. All the different
dialyzed material of 0.01 g was added in 100 μL Tris HCl (pH 6.5)
buffer and tested for inhibitory activity. The quantity of protein concentration
was done by the Bradford method (Thimmaiah, 1999). Bovine Serum Albumin
(BSA) was used to construct the standard curve.
The concentrated bacteriocin was treated with protease, DNase and RNase
with a final concentration of 5, 10, 15, 20 and 25 μg mL-1
in 10 mM Tris HCl, pH 7.0 for 4 h at 37 °C and residual activity was
determined. Sensitivity to different temperatures (40, 50, 60, 70, 80
and 90 °C) was determined by incubating up to 30 min. After incubation,
the samples were cooled and residual activity was determined (Nirmala
et al., 2001).
Polyacrylamide Gel Electrophoresis
Polyacrylamide gel electrophoresis (PAGE) in the presence of 10% Sodium
Dodecyl Sulphate (SDS) was performed (Laemmli, 1970). Electrophoresis
was conducted at a constant current of 30 mA for 12 h at 30 °C. The
gel was stained with coomassie blue. A 10 kDa protein ladder was used
as protein standard.
RESULTS AND DISCUSSION
The 20 Rhizobium spp. isolated from 20 legume
hosts were fast growers (colony diameter greater than 2.0 mm) and produced
acid in YEM broth. Among the twenty spp., three species from C. alata,
C. juncea and C. laburnifolia produced bacteriocins.
Rhizobium sp. from C. alata produced bacteriocins against Rhizobium
spp. from A. hypogaea, C. ternatea, D. lablab,
I. linnaei and V. mungo. The Rhizobium sp. from C.
juncea produced bacteriocins against Rhizobium spp. from V.
mungo while the Rhizobium sp. from C. laburnifolia produced
bacteriocins against Rhizobium spp. from D. lablab and I.
linnaei. The Rhizobium sp. isolated from C. alata showed
highest activity than other species of
|| Activity spectrum of bacteriocin producing Rhizobium
|- : Ineffective; +: Less effective; ++: Moderately effective;
+++: Highly effective. All the results are means of triplicates; *I.Z:
Diameter of inhibition zone
|| Purification of isolated bacteriocin protein from Rhizobium
sp. from C. alata
|- : Ineffective; +: Less effective; ++: Moderately effective;
+++: Highly effective. All the results are means of triplicates
Rhizobium and it also showed the broad spectrum
of activity (Table 1). Thus the activity spectrum varied
from strain to strain was reported earlier in Rhizobium legumunosarum
bv. viciae (Hafeez et al., 2005). An auto-antagonism relationship
was not observed; no test strains inhibited its own growth, which is characteristic
of bacteriocin producers (Hardy, 1975; Nirmala and Gaur, 2000). As marked
bacteriocin production was observed in Rhizobium sp. from C.
alata, further studies were carried out for this strain.
The bacteriocin production was appeared after 48 h of
incubation and reached maximum after 96 h of incubation. Further incubation
does not affect the zone size, therefore 96 h of growth of the producer
strains at 30 °C was considered as optimum conditions for bacteriocin
production in this study. That the production of bacteriocin is closely
related with bacterial growth of producing organism and bacteriocin activity
decreases more or less sharply at the end of the growth phase as a result
of degradation by proteases was reported earlier in Micrococcus
sp. (Kim et al., 2006).
Present results showed that when the sample is successively
diluted, inhibition zone decreased until critical dilution was achieved,
where no inhibition of the sensitive organism was observed. When the purified
bacteriocin was tested against indicator strains, it showed highest activity
at 75% ammonium sulphate saturated pellet. The activity does not depend
on the quantity of the protein produced (Table 2).
When the concentrated bacteriocin was treated with protease,
DNase and RNase, it was observed that it is sensitive to protease at the
concentration of 25 μg mL-1 indicating its proteinaceous
nature. The activity gradually decreases, when the concentration of protease
increased (Fig. 1). That the bacteriocins from Cicer-Rhizobium
were also sensitive to proteases reported earlier (Nirmala et al.,
2001). The bacteriocin was insensitive to DNase and RNase indicating it
is not a nucleoprotein. This bacteriocin is like those of R. leguminosarum
bv. trifolii, which is insensitive to DNase and RNase (Schwinghamer,
Temperature also affects the activity of bacteriocins.
Optimum temperature range for bacteriocin activity was found to be 30-70
°C and above 70 °C, activity decreases slowly (Fig.
2). The activity
|| Effect of protease concentration on bacteriocin activity
|| Effect of temperature on bacteriocin activity
||SDS-PAGE of purified bacteriocin of Rhizobium
sp. from C. alata, Lanes; (A) Molecular weight markers; (B)
Purified bacteriocin of 29 kDa
remained at 80 °C for 15 min and at 90 °C, the
activity diminished. That the bacteriocin from Cicer Rhizobium
was found to be heat stable even after 5 min at 80 °C was reported
earlier (Nirmala et al., 2001).
SDS-PAGE analysis of protein isolated from the Rhizobium
sp. showed the presence of 29 kDa protein band (Fig.
3). This band was visible only in samples isolated from 30-75% ammonium
sulphate saturation of CFS. The molecular mass showed that this bacteriocin
to be much smaller than those reported in R. leguminosarum bv.
trifolii (Joseph et al., 1983).
The results of this study have shown that bacteriocin
production may play an important role in interspecific competition. In
this study the broad spectrum activity of Rhizobium sp. from C.
alata may help in the improvement of legume inoculants.
We thank Andhra Pradesh Council of Science and Technology
(APCOST), Hyderabad, India, for financial assistance in the form of Young
Scientist Fellowship (YSF) to M.S.