Bull. Hiroshima Inst. Tech. Research Vol.49（2015）27-33

ArticleInhibitory effect of cadmium on competitive nodulation ability of Bradyrhizobium japonicum
Takashi OZAWA* and Kenji IJIRI**（Received Oct. 23, 2014）

Abstract
Competitive nodulation abilities of Bradyrhizobium japonicum strains USDA110ET and A1017ET
were signiﬁcantly depressed by growing the strains in yeast extract-mannitol broth supplemented
with 2 μM CdCl2. Soybean seedlings were co-inoculated with each test strain and its competitor
strain 138NR, and the bacteria in nodules formed on 21 days old plant roots were identiﬁed by the
antibiotic resistant markers. Cell surface hydrophobicity of each test strain increased by growing thestrains with 2 μM CdCl2, though deﬁnite increase in bacterial attachment to plant root surface washindered by excessive secretion of exopolysaccharides of the strains. Polyacrylamide gel electrophoresis revealed that growing the test strains in the medium with 2 μM CdCl2 induced the production of lipopolysaccharides of small molecular sizes. The results in this study suggest that cadmiumof low concentration causes the weakening of competitive nodulation ability of rhizobia through inhibition of the lipopolysaccharide synthesis.
Key Words: cadmium, competitiveness, hydrophobicity, legume, lipopolysaccharide, rhizobium

manner affected markedly the CNA of the indigenous
Bradyrhizobium strains (Ozawa et al. 2000). These
results suggest that some environmental factors affect the expression of the genes involving the competitive nodulation.
Cadmium (Cd) is one of the heavy metals generally existing as a minor element in soils. Cd is ahighly toxic metal that causes deleterious effects at
higher concentrations on soil bacteria as well as
plants (Sanitá di Toppi and Gabbrielli 1999). Degreeof sensitivity of rhizobia to Cd differs from strain tostrain. Consequently, contamination with Cd at higher concentrations could change a population structure of rhizobia in the soil (Kinkle et al. 1987). Thus,
the strain more tolerant to Cd would have more opportunity to form nodules on the host plant. Effect of
Cd on the CNA of a rhizobial cell, however, has notbeen elucidated.
Cell surface hydrophobicity of a rhizobial strain

1. Introduction

Rhizobium is a soil bacterium that infects leguminous plants and produces nodules in the root to fix
atmospheric nitrogen symbiotically with their hostplant. There is a difference in the root nodule formation ability between strains of rhizobia (Dowling andBroughton 1986). A rhizobium strain should competewith other strains to form a root nodule on their hostplant and occupy it.
The competitive nodulation ability (CNA) of arhizobial strain is genetically determined, but notconstant. Environmental factors such as temperature,soil pH, and soil nitrate can affect the CNA of
Rhizobium and Bradyrhizobium (Triplett 1990). We
demonstrated that Bradyrhizobium japonicum
strains changed their CNA if the strains grown in anutrient broth were introduced into soil environment(Ozawa 1988). We have also reported that fertilization＊＊＊＊＊＊

Department of Food Sciences and Biotechnology, Hiroshima Institute of Technology
Department of Life and Environmental Sciences, Osaka Prefecture University― 27 ―

Takashi OZAWA and Kenji IJIRI

is one of the key characteristics that determine the
CNA of the strain (Araujo et al. 1994). We have demonstrated that Bradyrhizobium japonicum strains had
a positive correlation between the cell surface hydrophobicity and the CNA (Ozawa et al. 1991, Ozawa
et al. 1992). Cell surface hydrophobicity of Gramnegative bacteria was determined by the amountand nature of exopolysaccharide (EPS) and lipopolysaccharide (LPS) (Nikaido 1976 , Nikaido 1994 ,Triplett 1990). Schue et al. (2011) reported that extracellular Cd induces the over-production of EPS.
These results suggest that Cd could change the
CNA of a rhizobial strain by affecting bacterial
metabolic processes of polysaccharides. The objectiveof this study is to conﬁrm the possible role of extracellular Cd in controlling the cell surface hydrophobicity and the CNA of Bradyrhizobium japonicum
strains.

fold with distilled water, and then mixed with thecompetitor strain 138NR to prepare ﬁve varieties of
inoculum. Bacterial cell densities of the strains were
examined by the dilution plate count method. Densities of the test strains and the competitor in the inoculum were approximately as follows: 106 and 108, 107
and 108, 108 and 108, 108 and 107, 108 and 106 cfu mL-1,
respectively.
2.2. Bacterial inoculation to plants

Soybean (Glycine max cv. Tamanishiki) seeds surface sterilized with 5% calcium hypochlorite weresown on vermiculite (approximate particle size: 5mm) with a depth of 10 cm in test tubes (25 x 200mm), one seed per tube. Each tube was previously
supplied with 30 mL of Jensen＇s mineral solution(Gibson 1980), and sterilized by autoclaving (120°C,
20 min). After 3 days at 30°C in the dark, each seedling was inoculated with 1.0 mL of the mixed cellsuspension of a test strain and strain 138NR, which
was prepared as mentioned above.
The inoculated plants were grown for 21 days ina growth chamber ( 23°C; 110 μ mol m -2 s -1, 16 h
light-8 h dark). Forty to 50 nodules (> 1 mmφ) weredetached from 6 plants for each treatment. The nodules were surface sterilized with 5% calcium hypochlorite, and then nodule occupancy was determinedby examining the antibiotic resistance of bacteria ineach nodule as described elsewhere (Ozawa 1988).
N50 value, which is the inoculum ratio of a test
strain to the competitor strain when it yields 50%
nodule occupancy, was used to express the competitive nodulation ability of each strain. N50 was calculated in the same way for estimating ED50 (effective
dose 50%) by the Probit method (Finney 1952).

2. Materials and Methods
2.1. Bacterial strains

B. japonicum USDA 110 and USDA 138 were obtained from the U.S. Department of Agriculture,
Beltsville, Md. B. japonicum A1017 was obtained
from Dr. K. Minamisawa, Tohoku University, Japan.
Spontaneous mutants of these strains resistant to 100
mg L -1 nalidixic acid and 100 mg L -1 rifampicin(strain 138NR) or to 100 mg L-1 erythromycin and 10
mg L-1 tetracycline (strains 110ET and A1017ET)
were isolated in our laboratory. The parental strains
and the mutants were maintained on yeast extractmannitol (YEM) agar slants and stored at 4°C (MacGregor and Alexander 1971). Prior to inoculation tosoybean, the strains were grown on a shaker (110rpm) for 5 days at 30°C in 5 mL of YEM broth in a
glass tube (18 x 200 mm). One hundred μL of theculture was transferred to another YEM broth (50mL) in a 200-mL ﬂask, and grown for 7 days under
the same conditions as above. In case of examining
the effect of Cd, 10 μL of 10 mM CdCl2 was added to
the 50 mL of YEM just before the transfer. Concentration of CdCl2 in YEM was 2 μM.
We evaluated here competitive nodulation abilities of the strains USDA 110 , A 1017 , 110 ET, andA1017ET against the strain 138NR as a competitor.
Each culture of the test strains was diluted 10- to 103-2.3. Bacterial adhesion to plant root and vermiculite

Strains 110ET and A1017ET were examined on competitive adhesion to soybean root surface against
strain 138NR. Each strain was cultured for 7 days in
YEM broth with or without 2 μM CdCl2 at 30°C, andthen washed with distilled water as described above.
Each of the washed cell suspension of former strains
was mixed with the washed 138NR cell suspension.
Cell density of each strain in the mixture was adjusted to 1 x 108 cfu mL-1. Three days old soybean seed― 28 ―

Inhibitory effect of cadmium on competitive nodulation ability of Bradyrhizobium japonicum

where ODa and ODb are optical density at 660 nm of
the lower phase after and before agitation, respectively.

lings, which were aseptically germinated on paper ina petri dish at 30°C in the dark, were cut with a razor
blade at position about 3 cm above the root tip. Setsof 4 excised root segments were soaked in 50 mL ofthe mixed cell suspension to remove loosely boundbacteria. After 90 min of incubation at 30°C, the root
segments were washed 4 times with distilled water.
Bacterial cells attached to plant roots were removed
by ultrasonic cleaning with the Bronson Sonifiermodel 250 (output level 4, 2 min on ice). Bacteria released from the root segments were counted by the
dilution plate method with YEM agar containing theantibiotics.
Adhesion ability of strains USDA110, A1017,
110ET, A1017ET, and 138NR to vermiculite used inthis study for soybean cultivation was evaluated in
the following way using vermiculite columns. Optical density (OD) at 660 nm of cell suspension of eachstrain obtained as described above was adjusted to0.2, and then the cell suspension was passed througha column of vermiculite (1.5 x 20 cm) at room temperature. We used in this study the rate of decrease in
OD of cell suspension during the ﬁltration as an indication of adhesion ability of the bacterial strain tovermiculite particle.

2.5. Determination of EPS

Bacteria grown for 7 days at 30°C in 50 mL of YEM
broth were harvested by centrifugation at 14,000 rpm
for 3 min, and the precipitate was washed 4 times
with 50 mM potassium phosphate (pH 6.5). First two
washes were collected, and assayed for hexose by
the phenol-H2SO4 method (Dubois et al. 1956).
2.6. Polyacrylamide gel electrophoresis (PAGE) of LPS

The pelleted cells from 1 mL culture were washedonce with 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2,
10 mM 2-mercaptoethanol, and then suspended in 130
μL of sodium dodecyl sulfate (SDS) sample buffer (12mM Tris [pH 6.8], 4% SDS, 10% glcerol, 0.2% bromophenol blue, 4% 2-mercaptoethanol). The total
SDS extracts of bacterial strains were heated at 100°C
for 10 min followed by digestion with proteinase K(150 μg mL-1) at 60°C for 60 min (Cava et al. 1989).
LPS in the crude preparations was electrophoresed
through 4% stacking and 12.5% separating acrylamide gel with 0 . 4 % SDS. The gels were silver
stained by the method of Tsai and Frasch (1982).

2.4. Cell surface hydrophobicity

3. Results and Discussion

Cell surface hydrophobicity of bacterial cells wasmeasured by the method of Rosenberg et al. (1980).
This method is based on partitioning behavior ofbacterial cells between oil and water. Bacteria withmore hydrophobic surfaces tend to be attracted to theoil-water phase boundary (Marshal 1976). The rhizobial cells grown in the YEM broth for 7 days were
washed twice with distilled water or 50 mM potassium phosphate (pH 6.5). To 3.0 mL of the washedcell suspension of approximately 108 cfu mL-1 in atest tube (12 x 100 mm) with a screw cap, 0.3 mL noctane as the water-insoluble phase was added,
and then the tube was vertically agitated on a shaker(300 rpm, amplitude of 5 cm) for 2 min. After standingfor 2 min, optical density of the lower aqueous
phase was determined at 660 nm.
Cell surface hydrophobicity index (CHI) was
expressed as the following equation:
CHI = (ODb － ODa)/ODb

3.1. Cd tolerance

We assessed minimum inhibitory concentration(MIC) of Cd against the Bradyrhizobium strains inthis study by using YEM broth containing CdCl 2.
The strains from stock cultures were incubated for 7
days at 30°C in 5 mL of YEM supplemented with 0－ 100 μM CdCl2 on a shaker (110 rpm), then OD at
660 nm of each culture was measured. MIC of Cd
was 30 μ M against every strains tested. Two μ M
CdCl2 had no effect on growth rate of the strainsused in this study (data not shown).
3.2. Effect of Cd on CNA

A rhizobial strain competes with other strains to infect their host plant and multiply in nodules formedin the host roots. Figure 1 shows nodule-occupancy
rates of the test strains USDA110, A1017, 110ET, andA1017ET in competition with strain 138NR after the― 29 ―

Takashi OZAWA and Kenji IJIRI

co-inoculation to soybean. In case of 110 ET and
A1017ET, proportion of the nodules containing boththe test strain and the reference strain 138NR ranged
from 0 to 14%. The double-infected nodules were
excluded to calculate nodule occupancy rates in Fig.
1. The rate of nodules occupied by a strain increased
with the increase in the ratio of the strain to the competitor strain in an inoculum.

Table 1. Effect of cultivation with CdCl2 on the competitive

nodulation index (N50) of B. japonicum strains.
mU and mL are upper and lower limit of conﬁdence
interval at 95 %, respectively.

StrainUSDA110
A1017
110ET

A1017ET

1

Nodule occupancy rate

0.8

Cd in growth
medium
0 μM
2 μM
0 μM
2 μM
0 μM
2 μM
0 μM
2 μM

N50

(mU, mL)

7.6 × 10-5

(0.20, 2.9 × 10-8)(560, 3.1 × 10-3)(0.62, 0.11)(18, 3.1 × 10-2)(2.27, 0.80)(17.6, 3.58)(13.3, 4.11)(26.8, 8.92)

1.3
0.26
0.75
1.35
7.93
7.41
13.5

3.3. Effect of Cd on adhesion of bacterial cells to hostroots and vermiculite

0.6

0.4

As a rhizobial cell density on the host root surface is
one of the major factors for competitive nodule formation by the strain (Dowling and Broughton 1986),
higher ability to adhere to host roots and lower ability to adhere to vermiculite would both give higher
CNA to the rhizobial strain. Cultivation of A1017ET
strain in YEM broth with 2 μM CdCl2 caused significantly higher ratio of A1017ET cells to 138NR cells
that adhered to soybean root segments, but in case of
110ET there was no effect (Table 2). Adhesion abilityof 110ET to vermiculite particles declined by cultivating them in YEM with 2 μM CdCl2 (Table 3).

USDA11
0
110ET

0.2

A1017

0
-2

-1

0

1

2

log of test strain/138NR ratio in inoculum
Fig. 1. Nodule occupancy rates of B. japonicum strains USDA110,
110 ET, A 1017 , and A 1017 ET in competition with B.
japonicum strain 138NR.

From the dose-response curve, we can obtain a
N50 value, the proportion of a strain to its competingstrain in an inoculum when 50 % of nodules areformed by the strain (Table 1 ). A strain showing
smaller value of N50 is more competitive for nodule
formation. Growing in YEM supplemented with
2 μ M CdCl 2 significantly decreased the CNA ofstrains 110ET and A1017ET. Strains USDA110 and
A1017, showing more competitive than the antibiotic
resistant mutants, also showed a decreasing tendency
in CNA by growing with 2 μM CdCl2, though statistical signiﬁcance was low.
As both strains USDA110 and A1017 have noantibiotic marker, we were not able to distinguishnodules occupied by single strain from double-infected nodules. We did not use strains USDA110 and
A1017 in the following experiments.

Table 2. Effect of cultivation with CdCl2 on the adhesion of B.

japonicum strains to soybean root segments.

Strains
138NR
110ET
110ET/138NR ratio

Root-attached cells (cfu per root segment)
cultivated with
0 μM Cd
2 μM Cd
3.6×105±3.0×104 2.4 × 105 ± 3.8 × 104
2.4×105±4.5×104 1.6 × 105 ± 1.5 × 104
0.68
0.65

138NR

8.0×104±1.0×104

4.3×104±2.5×103

A1017ET
A1017/138NR ratio

1.0×104±5.0×103

7.3×104±2.5×103

0.13

1.71

Table 3. Effect of cultivation with CdCl2 on the adhesion of B.

japonicum strains to vermiculite. Values with asterisk
are significantly different (n = 3, p < 0.05) from thecontrol value.

Strains
110ET
A1017ET
138NR― 30 ―

Adhesion rate (%) of cells cultivated with
0 μM Cd (Control)
2 μM Cd
25
7*
28

23

1

7

Inhibitory effect of cadmium on competitive nodulation ability of Bradyrhizobium japonicum

Araujo et al. (1994) reported that a Tn-5 mutant
of Rhizobium etli produced EPS that was indistinguishable from that of its parent. However, the mutant was highly hydrophobic, while greatly reducedin competitive nodulation. Bittinger et al. (1997) established that a single gene homologous to a familyof transcriptional regulators affected both competitive nodulation and cell surface hydrophobicity ofthe strain. The results in this study also indicate thecorrelation between cell surface hydrophobicity andcompetitive nodulation.
LPS is one of the major components of the outermembrane of Gram-negative bacteria, and is a heterogeneous molecule that varies O-antigen length(Jann et al. 1975). LPS that has shorter length of thepolysaccharide chain would make more hydrophobicof bacterial cell surface (Nikaido 1976).
Growing in the YEM broth supplemented with2μM CdCl2 resulted in an alteration in LPS-band patterns of SDS-PAGE (Fig. 3). LPSs of small molecular size appeared in the cells of 110ET and A1017ET
when they were grown in YEM broth with 2 μ M
CdCl2. This explains the increase in cell surface hydrophobicity by growing with 2 μ M CdCl 2 as described above.

Table 4. Effect of cultivation with CdCl2 on the cell surfacehydrophobicity of B. japonicum strains.
Values (mean ± S.E., n = 3) with asterisk are signiﬁcantly
different from the control (0 μM CdCl2) value of each
washed cells (p < 0.05).

Strain

Wash#

110ET

DW
PB

A1017ETDWPB

CHI of rhizobium cultivated with
0 μM Cd
2 μM Cd
0.62 ± 0.01
0.49 ± 0.02*
0.60 ± 0.06
0.90 ± 0.02*
0.56 ± 0.06

0.39 ± 0.04*

0.69 ± 0.02

0.82 ± 0.03*# DW: distilled water, PB: 50 mM K-phosphate (pH 6.5)

Other strains tested did not show signiﬁcant change
in the adhesion ability after growing in YEM with
CdCl 2. These results indicate that 2μM CdCl2 in a
growth medium does not cause a decrease in rhizobial cell density on the host root surface to bring about
the decrease of CNA.
3.4. Effect of Cd on cell surface hydrophobicity

Adhesiveness of bacterial cells to solid surface
would be affected by the cell surface hydrophobicity(van Loosdrecht et al. 1987). Vesper et al. (1987) reported that transposon mutants of a Bradyrhizobium
japonicum strain reduced both attachment to soybean
roots and cell hydrophobicity. Surface hydrophobicity of strains 110 ET and A1017ET in this study thatwere washed with distilled water significantly decreased by cultivation in YEM broth with 2 μ M
CdCl2 (Table 4). On the contrary, the Cd-cultured
cells of both strains washed with phosphate buffer
increased hydrophobicity.
3.5. Effect of Cd on production of EPS and LPS

B. japonicum 110ET and A1017ET produced a large
amount of extracellular substances when grown in
YEM broth with 2 μM CdCl2 (Fig. 2). When bothstrains were grown in YEM supplemented with 2 μM
CdCl2, significantly larger amount of EPS was detected in the culture than in YEM without CdCl2 (Table 5). As EPS is a hydrophilic molecule, the large
amount production of EPS would be responsible forthe decrease in cell surface hydrophobicity as shownin Table 4. When the EPSs loosely bound to cell surface were washed out with 50 mM phosphate, theCd-cultured cells showed larger values of CHI thancontrol cells.

Fig. 2. Production of gel-like substances by B. japonicum strains.
Strain 110ET (a, b) and A1017ET (c, d) were grown for 7
days in YEM broth supplemented with 0μM (a, c) and2 μM CdCl 2 (b, d), then 1 . 5 mL of the culture wascentrifuged (10,000 rpm, 3 min).

Table 5. Effect of cultivation with CdCl2 on the production of

EPS by B. japonicum strains.
Values are means ± S.E. (n =3). Values with asterisk ofeach strain are signiﬁcantly different (p < 0.05) from thecontrol values.

Cd in YEM

― 31 ―EPS (μg Glc/mL)
110 ET

A1017ET0μM (Control)

97 ± 10216 ± 42μM

476 ± 56 *497 ± 57 *

Takashi OZAWA and Kenji IJIRI

nodulation of legumes. Annu. Rev. Microbiol., 40,
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Fig. 3. SDS-PAGE of LPS of B. japonicum 110ET and A1017ET.

Both strains were grown in the YEM supplemented with
0μM (Cd -) or 2μM (Cd +) CdCl2. The total SDS-extracts
from 10 mg (dry weight) of cells of each strain were
loaded on each well, and electrophoresed. Slanting arrows
indicate characteristically appeared bands in the cells
grown with Cd.

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LPS molecules have been thought to play various roles in establishing the symbiosis between rhizobia and their host plants (Stacey et al. 1982). O-antigen polysaccharide portion of a LPS moleculewould be responsible for successful infection of thehost plant (Cava et al. 1989). Noel et al. (1986) reported that reduction or defect of the polysaccharide
chains resulted in abortion of infection thread development. The observations in the present study provide a hypothesis that a very small amount (2μM) of
Cd causes the decrease in CNA of B. japonicum by
disturbing the synthesis of polysaccharide chains in
LPSs.

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