The cbbL gene is required for
thiosulfate-dependent autotrophic growth of
Bradyrhizobium japonicum
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MASUDA SACHIKO, EDA SHIMA, SUGAWARA
MITSUI HISAYUKI, MINAMISAWA KIWAMU
Microbes and environments
25
3
220-223
2010
http://hdl.handle.net/10097/52077
CHIAKI,
Microbes Environ. Vol. 25, No. 3, 220–223, 2010
http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME10124
Short Communication
The cbbL Gene is Required for Thiosulfate-Dependent Autotrophic Growth
of Bradyrhizobium japonicum
SACHIKO MASUDA1*, SHIMA EDA1, CHIAKI SUGAWARA1, HISAYUKI MITSUI1, and KIWAMU MINAMISAWA1
1
Graduate School of Life Sciences, Tohoku University, 2–1–1 Katahira, Aoba-ku, Sendai 980–8577, Japan
(Received April 7, 2010—Accepted April 16, 2010—Published online May 14, 2010)
Bradyrhizobium japonicum is a facultative chemolithoautotroph capable of using thiosulfate and H2 as an electron
donor and CO2 as a carbon source. In B. japonicum USDA110, the mutant of cbbL gene encoding a large subunit
of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was unable to grow using thiosulfate and H2 as an
electron donor. The cbbL deletion mutant was able to grow and oxidize thiosulfate in the presence of succinate. These
results showed that the major route of CO2 fixation for thiosulfate-dependent chemoautotrophic growth is the CalvinBenson-Bassham cycle involving RuBisCO in B. japonicum.
Key words: Bradyrhizobium japonicum, CO2 fixation, chemoautotroph, cbb
Bradyrhizobium japonicum, a nitrogen-fixing endosymbiont of soybean nodules (6), is a facultative chemoautotroph
utilizing thiosulfate (15), H2 (5, 8, 13) and CO (11, 14) as an
electron donor and CO2 as a carbon source. However, the
genes relevant to CO2 fixation during the chemoautotrophic
growth of B. japonicum have yet to be identified.
There are four major pathways for CO2 fixation; the
Calvin-Benson-Bassham (CBB) cycle, reductive tricarboxylic acid (rTCA) cycle, 3-hydroxypropionate (3-HP) cycle
and reductive acetyl coenzyme A (acetyl-CoA) pathway, in
bacteria and archaea (17). A search for the genes related to
CO2 fixation pathway on the genome of B. japonicum strain
USDA110 revealed the presence of structural genes for the
CBB cycle and rTCA cycle.
Activity of ribulose 1,5-bisphosphate carboxylase
(RuBisCO) was biochemically detected in a crude cell
extract of B. japonicum strain USDA122 grown chemoautotrophically with H2 as an electron donor under a gas
mixture (v/v) of 84% N2, 5% CO2, 1% O2 and 10% H2 (13).
RuBisCO was also purified from USDA122 cells (18). In
addition, the expression of cbbLS encoding RuBisCO was
enhanced with H2 as a sole electron donor, as compared with
that under heterotrophic conditions in B. japonicum strain
USDA110 (5).
Recently, Masuda et al. (15) found that B. japonicum
USDA110 is able to fix ambient CO2 during chemoautotrophic growth using thiosulfate at quite low concentrations of CO2 (0.03–0.07% [v/v]) in contrast with previous
reports of H2-dependent chemoautotrophic growth (5% [v/v]
CO2) (5, 8, 13). Generally, RuBisCO enzymes have low
affinity for CO2 and require higher CO2 concentrations (1).
Therefore, we examined whether cbb encoding RuBisCO is
required for thiosulfate-dependent chemoautotrophic growth
at ambient concentrations of CO2 in the air.
The bacterial strains and plasmids used in this study are
listed in Table 1. B. japonicum strains were cultured aerobi* Corresponding author. E-mail: [email protected];
Tel: +81–22–217–5687; Fax: +81–22–217–5684.
cally at 30°C in HM salt medium (2) supplemented with
0.1% (w/v) arabinose and 0.025% (w/v) DifcoTM yeast
extract (Becton Dickinson, Sparks, MD, USA), unless otherwise indicated. Antibiotics were added to the medium for
growing B. japonicum as follows: tetracycline (Tc) and
kanamycin (Km) at 100 μg mL−1, and polymyxin B at 50 μg
mL−1. For Escherichia coli, the concentrations were Tc, 15
μg mL−1 and Km, 50 μg mL−1.
For H2-dependent chemoautotrophic growth, B. japonicum
strains were grown on H2-uptake agar medium under a
gas mixture (v/v) composed of 84% N2, 5% CO2, 1% O2
and 10% H2 for 28 d at 25°C (5, 8, 13). H2-uptake medium
contained the following components dissolved in 1 liter of
distilled water (pH 6.8): 150 mg NaH2PO4·H2O, 150 mg
CaCl2·2H2O, 250 mg MgSO4·7H2O, 100 mg NH4Cl, 5 mg
FeCl3·6H2O, 10 mg MnSO4·H2O, 3 mg H3BO3, 2 mg
ZnSO4·7H2O, 0.25 mg NaMoO4·2H2O, 0.78 mg KI, 40 μg
CuSO4·5H2O, 25 μg CoCl2·6H2O, 25 μg NiCl2·H2O, 0.1 mg
thiamine·HCl, 0.1 mg biotin, 0.2 mg nicotinic acid, 0.1 mg
pyridoxine·HCl and 0.05 mg inositol (8).
For thiosulfate-dependent chemoautotrophic growth, B.
japonicum strains were grown in Taylor broth medium
containing 4 mM sodium thiosulfate (15) in the air for 28 d at
25°C. CFU counts were made as described previously (15).
For mixotrophic growth, the cells were grown in Taylor
broth medium with 4 mM sodium thiosulfate and 0.1%
(w/v) sodium succinate for 4 d at 25°C (15). The OD660 of
cultures was measured with a UV-1200 spectrophotometer
(Shimadzu, Kyoto, Japan). Thiosulfate concentrations in the
culture supernatants were measured by iodometric titration
(10).
We generated a cbbL mutant of B. japonicum as described
below. Isolation of plasmids, DNA ligation, and transformation of E. coli were performed as described by Sambrook et
al. (19). Total bacterial DNA was isolated from cultured cells
as reported previously (16). A 3.6-kb SphI DNA fragment
containing the cbbALSX genes was isolated from brc04278, a
cosmid clone from the genomic library of B. japonicum
USDA110 (9), and inserted into the SphI site of pK18mob,
CO2 Fixation of B. japonicum Chemoautotrophy
Table 1.
221
Bacterial strains and plasmids used in this study
Relevant characteristicsa
Strain or plasmid
Strains
Bradyrhizobium japonicum
USDA110
USDA110 ΔcbbL
Escherichia coli
DH5α
Plasmids
pSAC50
pSAC51
brc04278
p34S-Tc
pK18mob
pRK2013
a
b
Reference or source
wild type
USDA110 ΔcbbL::tet, Tcr
(9)
This study
cloning host strain
Toyobob
pK18mob carrying 3.6-kb cbbALSX fragment of brc04278; Kmr
pK18mob carrying cbbL::Tcr cassette; Tcr Kmr
pKS800 carrying cbb operon
Plasmid carrying 2.1-kb Tcr cassette; Tcr
integration vector; oriV, oriT, mob; Kmr
ColE1 replicon carrying RK2 transfer genes; Kmr
This study
This study
(9)
(3)
(21)
(4)
Tcr, tetracycline resistant; Kmr, kanamycin resistant.
Toyobo, Osaka, Japan.
Fig. 2. Cell growth of USDA110 (left) and the cbbL mutant (right) on
H2-uptake agar medium (A) and HM medium (B) for 28 d at 25°C
under a gas mixture (v/v) of 84% N2, 10% H2, 5% CO2 and 1% O2.
Fig. 1. Construction of the cbbL mutant of B. japonicum USDA110.
(A) The cbb genes clustered in the B. japonicum USDA110 genome.
The position of the Tcr cassette insertion is indicated. (B) Calculated
sizes of PCR products amplified by primers P1 and P2 (black arrows) in
the wild type, and single & double crossover mutants. (C) Agarose gel
electrophoresis of PCR products amplified with P1 and P2. Lane 1,
HyperLadder I (DNA marker); lane 2, USDA110; lane 3–6, transconjugants. The strains shown in lanes 5 and 6 were confirmed to have a correct genome structure of the cbbL mutant.
yielding pSAC50 (Table 1). The Tcr-cassette was isolated
from p34S-Tc at the SmaI site (3), and inserted into the AatII
site of pSAC50, yielding pSAC51. It was conjugated into B.
japonicum strain USDA110 by triparental mating using
pRK2013 as a helper plasmid (20) and Tcr Kmr transconjugants were selected. PCR was performed using primers
P1 (5'-ACTACACGCCAAAGGACACC-3') and P2 (5'-GAAGGTCACGTCCTTCCAGA-3'), and total DNA of the transconjugants as templates (Fig. 1B and C). One of the transconjugants, strain USDA110ΔcbbL, which showed the
expected gene replacement (Fig. 1C), was used as the cbbL
mutant throughout this study (Fig. 1A and C).
The wild-type strain USDA110 and the cbbL mutant were
subjected to growth experiments on the H2-uptake medium
under an atmosphere containing 5% CO2 and 10% H2 (5, 8,
13). The cbbL mutant showed markedly weak growth compared to the wild type for 28 d (Fig. 2A). The growth of
cbbL mutant was similar to that of strain USDA110 on HM
plates supplemented with arabinose (Fig. 2B). These results
suggested that the cbbL gene is required for H2-dependent
chemoautotrophic growth of B. japonicum USDA110, supporting the previous biochemical observation that RuBisCO
activity increased during chemoautotrophic growth (5, 8,
13).
Subsequently, the wild type and the cbbL mutant were
examined for chemoautotrophic growth using thiosulfate as
MASUDA et al.
222
Fig. 3. Cell growth and thiosulfate oxidation of USDA110 (A and C) and the cbbL mutant (B and D) under chemoautotrophic (A and B) and mixotrophic (C and D) growth conditions. Cell growth under chemoautotrophic conditions (A and B) was monitored using CFU counts. Cell growth
under mixotrophic conditions (C and D) was monitored at OD660. Open symbols; cell growth, closed symbols; thiosulfate concentration in culture.
an electron donor under an ambient CO2 level in the air. The
cbbL mutant was unable to grow and oxidize thiosulfate
under the condition over 27 d (Fig. 3B), whereas the wild
type was able to grow chemoautotrophically using thiosulfate as an electron donor (Fig. 3A). The cbbL mutant was
able to grow and oxidize thiosulfate like strain USDA110 in
the presence of succinate (Fig. 3C and D). These results indicate that the cbbL gene is required for chemoautotrophic
growth using thiosulfate under ambient CO2 levels in the air.
Therefore, we concluded that the major route of CO2 fixation
is the CBB cycle, not the rTCA cycle, during chemoautotrophic growth using thiosulfate and ambient CO2 in the air.
The cbbL gene was required for chemoautotrophic growth
using H2 (Fig. 2A) or thiosulfate (Fig. 3A and B). Thus, it is
possible that the CBB cycle plays a major role also in
chemoautotrophic growth using other inorganic electron
donors such as CO in B. japonicum strain USDA110. However, we cannot exclude the possibility that the rTCA cycle
makes a minor contribution to CO2 fixation, because the
cbbL mutant showed weak growth on the H2-uptake medium
(Fig. 2A).
A thiosulfate-oxidizing capability is frequently found
along with hydrogenase (H2 oxidation) activity in the
Bradyrhizobiaceae (15). A study of the genomic sequences
of Rhodopseudomonas palustris CGA009 (12), Bradyrhizobium sp. BTAi1 (7) and Bradyrhizobium sp. ORS278 (7),
which show thiosulfate-oxidizing activity, revealed that they
carried structural genes for RuBisCO (rpa1559–1560 for
CGA009; BBta0451–0452, BBta2641–2642, and BBta6396–
6397 for BTAi1; and BRADO1659–1650 and BRADO2274–2275 for ORS278). Thus, it is possible that the CBB
cycle is commonly required for chemoautotrophic growth in
other members of the Bradyrhizobiaceae.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific
Research on Priority Area “Comparative Genomics”, by Grants-inAid for Scientific Research (no. 17380046) and by Probrain.
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