Documentos de Académico
Documentos de Profesional
Documentos de Cultura
www.elsevier.com/locate/ijhydene
Received 29 March 2005; received in revised form 4 April 2005; accepted 14 April 2005
Available online 14 June 2005
Abstract
Increase in levels of photosynthetic spectral complexes by maintaining the plasmids harboring DNA encoding puhA, pufBA,
or pucBAC in trans in Rhodobacter sphaeroides resulted in decrease of the photoheterotrophic production of H2 . However,
removal of B875 or B800–850 light-harvesting (LH) complexes affected H2 production differently. Lack of B875 complex
following in-frame deletion of pufBA (mutant PUF1) not only slowed photoheterotrophic growth but also decreased H2
production, indicative of the essential requirement of the complex for LH process. However, the pucBA-deleted mutant, PUC1
lacking of B800–850 complex, increased H2 production in comparison with its parental cell by approximately twofold, given
irradiated with light (10 W/m2 ) saturating the growth of wild type. The H2 production of PUC1 did not increase in proportion
to the light intensity, which is also observed with wild type. Thus, we suggest that light is not limited for the H2 production
of the cells under the experimental conditions employed in this work, but the cellular energy to be used for the formation of
B800–850 complex may flow into the metabolism leading to the H2 production in PUC1.
䉷 2005 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
0360-3199/$30.00 䉷 2005 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2005.04.053
532 E.-J. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 531 – 538
the excitation energy to a photochemical reaction center E. coli and R. sphaeroides. Ampicillin (Ap) and tetracycline
(RC), the site of primary photochemistry. The pathway for (Tc) were used at 50 and 20 g/ml, respectively, for E. coli,
energy transduction is from B800–850 to B875 complex, whereas Tc was used at 1 g/ml for R. sphaeroides.
and to RC, in which B875 complex apparently serves as an
obligatory intermediate [11].
Previously, we isolated a purple photosynthetic bacterium 2.2. Construction of plasmids harboring puhA, pufBA, and
from mud samples collected at the seashore of Korea, which pucBAC of R. sphaeroides 2.4.1, and their maintenance in
showed higher production of H2 in comparison with the R. sphaeroides KCTC 12085
laboratory strain, Rhodobacter sphaeroides 2.4.1 [12]. The
photoproduction of H2 by the isolate, which was identified The nucleotide sequence of R. sphaeroides 2.4.1 DNA
and deposited as R. sphaeroides KCTC 12085, was further was obtained from a genome site at http://mmg.uth.tmc.edu/
increased in proportion to light intensity following the sup- sphaeroides/. A 903-bp HinfI-BamHI fragment (Fig. 1) ex-
pression of H2 uptake by mutation in hupSL coding for H2 - tending from 40-bp upstream from puhA (reaction center
uptake hydrogenase whose activity was regulated according H polypeptide) [19] to its 80-bp downstream DNA was
to the light intensity [12]. A fixed level of H2 production filled-in and cloned into SmaI site of pBS. The recombi-
was also elevated irrespective of the light intensities when nant HindIII-EcoRI fragment from the plasmid was cloned
poly--hydroxybutyrate (PHB) formation was blocked by into pRK415 [20] to generate pRKH100, in which puhA
mutation in phbC, reflecting the constitutive expression of was situated in the same direction as tet promoter. The
cellular PHB content under the conditions [12]. DNA containing pufBA (B875- and B875- polypeptides,
There have been reports of the improvement of H2 pro- respectively) [21] (Fig. 2) was provided with pRKF100, a
duction following changes in the level of spectral complexes recombinant pRK415 containing 1997-bp EcoRI-KpnI frag-
of R. sphaeroides [13,14]. In case of using solar energy, the ment extending from the 216th residue of bchZ to the 117th
strong light (ca. 1.0 kW/m2 ) needs to be dispersed to el- residue of pufL. The insert DNA of plasmid pRKF100 also
evate H2 production by R. sphaeroides [15]. However, self- contains orfQ and pufK [22]. A 3386-bp XhoI-SacI DNA
shading of cells due to their LH complexes could result in (Fig. 3) containing pucBAC (B800–850-, B800–850-
reduction of light penetration [13,16] and therefore lower polypeptides, and an assembly protein for the B800–850
the H2 production. In this work, we altered the level of LH complex, respectively) [23] as well as its 465-bp upstream
complexes of R. sphaeroides and examined the H2 produc- and 980-bp downstream DNA was cloned into the SalI and
tion with respect to light levels. The results in this work SacI sites of pRK415 to yield pRKC100.
suggest that the expenditure of cellular energy for the forma- Plasmids were mobilized from E. coli S17-1 into R.
tion of B800–850 complex, rather than the complex-caused sphaeroides through conjugation as described previously
shade on nearby cells in light path, appears to be a primary [24]. Mating mixtures were plated aerobically on Sistrom’s
reason to result in reduction of H2 production in wild type, minimal medium containing appropriate antibiotics to ob-
and B875 complex is obligatory for the efficient harvesting tain isolated transconjugants.
of light energy even at the light level saturating cell growth.
2.3. Spectrophotometric assay of spectral complexes and
protein determination
2. Materials and methods
Cell-free lysate of R. sphaeroides during exponential
2.1. Bacterial strains and growth conditions
growth was prepared as described previously [25] and
was used to examine the absorption spectra of photosyn-
R. sphaeroides KCTC 12085 (KCTC: the Korean Collec-
thetic complexes with UV 2041-PC spectrophotometer
tion for Type Cultures) [12] and R. sphaeroides 2.4.1 were
(Shimadzu, Japan). Amount of the B800–850 and B875
used as wild type strains. R. sphaeroides was grown aero-
complexes were measured as described previously [11].
bically or photoheterotrophically at 28 ◦ C in Sistrom’s suc-
The amount of B800–850 complex was determined from
cinate minimal medium [17] as previously described [18].
the spectral data by using A849.900 with an extinction
Light intensity for photoheterotrophic growth was measured
at the surface of culture vessels with a photometer (Li-Cor, coefficient () of 96 mM−1 cm−1 , normalized for three
Inc., USA) [18], and was adjusted to 100 or 10 W/m2 . Mod- molecules of bacteriochlorophyll a per complex, whereas
ified Sistrom’s medium containing DL-malate (30 mM) and the amount of B875 complex was calculated from A878.820
L-glutamate (7 mM) was used for H2 accumulation [12]. with of 73 mM−1 cm−1 , normalized for two molecules
Cell growth was monitored with a Klett–Summerson col- of bacteriochlorophyll a per complex. Protein content was
orimeter (Manostat, USA) equipped with a KS-66 filter. determined by the modified Lowry method [26]. The quan-
Escherichia coli was grown at 37 ◦ C in Luria–Bertani (LB) tification of LH complexes was repeated three separate
medium. Kanamycin (Km), streptomycin (Sm), and specti- times, and the data shown in this work were reproduced
nomycin (Sp) were used at 25, 50, and 50 g/ml for both within the standard deviations less than 10%.
E.-J. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 531 – 538 533
BamH I
BamH I
Hinf I
Bgl II
Xho I
Xho I
Xho I
Pst I
100 bp
Fig. 1. Restriction map of the R. sphaeroides 2.4.1 DNA containing puhA coding for RC-H polypeptide.
Hinc II
EcoR I
Sma I
Kpn I
Kpn I
Xho I
Acc I
Acc I
Acc I
Sma I
Pst I
Sal I
Mutant PUF1 ∆
200 bp
puf
bchZ orfQ K B A L M X
(A)
∆A = 0.2
WT
PUF1
Fig. 2. (A) Restriction map of the R. sphaeroides 2.4.1 DNA containing pufBA (B875- and B875- polypeptides, respectively) is shown
and pufBA is in-frame deleted in mutant PUF1. Genes bchZ (BchZ subunit of chlorophyllide a reductase), orfQ (spectral complex assembly
protein [22]), pufLM (RC-L and RC-M polypeptides, respectively), and pufX (intrinsic membrane protein) are shown. (B) Absorption spectral
profiles are provided with cell-free extracts from R. sphaeroides KCTC 12085 (solid line; WT) and its mutant PUF1 (dotted line) grown
photoheterotrophically at 10 W/m2 .
2.4. Construction of pufBA-deleted and pucBA-deleted the deletion region was PCR amplified with 5 primer F1
mutants of R. sphaeroides KCTC 12085 (5 -GTC AAC ACG CCG GTC CAT-3 ) and 3 primer R1
(5 -CTG CGC CTG CTG CAG TCC-3 , mutated sequence
Genes of pufBA and pucBA of R. sphaeroides KCTC underlined, unless otherwise noted) having the 14th and 15th
12085 were disrupted through homologous recombination codons of pufB changed into PstI site (shown in bold). The
using appropriate DNA of R. sphaeroides 2.4.1. The pufBA- 424-bp DNA fragment just next to the downstream from
deleted mutant PUF1 lacking of B875 complex was con- the deletion region was also amplified using 5 primer F2
structed as follows. The 220-bp DNA from the first nu- (5 -CCA CCA TGC ATC CTG CTG A-3́) having the 33rd
cleotide of the 15th residues of pufB to the first nucleotide of and 34th codons of pufA changed into NsiI site (shown in
the 33rd residue of pufA was in-frame deleted from the 0.9- bold), and 3 primer R2 (5 -CAA GGG CCG GCG GGT
kb AccI-KpnI DNA (Fig. 2A, thick line) of R. sphaeroides AGA C-3 ). The upstream DNA fragment was digested by
2.4.1. A 404-bp DNA fragment immediately upstream from AccI and PstI, and the downstream fragment was digested
534 E.-J. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 531 – 538
BamH I
Xma III
Pvu II
Xmn I
Xho I
Xho I
Xho I
Xho I
Sph I
Sac I
Pst I
Stu I
Pst I
Pst I
Pst I
Mutant PUC1 ∆
200 bp
puc
orf124 B A C nnr
(A)
∆A=0.2
WT
PUC1
Fig. 3. (A) Restriction map of the R. sphaeroides 2.4.1 DNA containing pucBA (B800–850- and B800–850- polypeptides, respectively)
is shown and pucBA is in-frame deleted in mutant PUC1. pucC (assembly protein for B800–850 complex [23]) and nnr (LuxR-type
transcriptional regulator) are shown. (B) Absorption spectral profiles are provided with cell-free extracts from R. sphaeroides KCTC 12085
(solid line; WT) and its mutant PUC1 (dotted line) grown photoheterotrophically at 10 W/m2 .
by NsiI and KpnI. The 329-bp AccI-PstI upstream fragment binant DNA fragment was cloned into pLO1 to generate
and 375-bp NsiI-KpnI downstream fragment were cloned pLO1-pucBA, which was subsequently mobilized from E.
into the AccI and KpnI sites of pBS to generate pBSFD. Se- coli S17-1 into R. sphaeroides KCTC 12085 through con-
quence analysis confirmed an in-frame deletion of pufBA as jugation as described previously [24]. Double-crossover re-
well as no other mutations in the amplified DNA. The insert combinations were selected as described above for PUF1.
DNA of pBSFD was then subcloned into SacI and SphI sites As a result, four of such recombinants devoid of B800–850
of a suicide plasmid pLO1 [27,28]. The resulting plasmid complex were obtained. The genomic DNA of the recombi-
pLO1-pufBA, which harbored aph (Kmr ) and sacB, was nants was examined for correct deletion of pucBA by South-
mobilized from E. coli S17-1 into R. sphaeroides KCTC ern hybridization analysis [29], and one recombinant PUC1
12085 through conjugation as described [24]. The Kmr ex- was analyzed further.
conjugant, which was generated from single crossover, was
isolated and subjected to segregation to double-crossover 2.5. Measurement of hydrogen gas
recombination showing Kms phenotype on sucrose (15%)-
containing Sistrom’s minimal medium. As a result, five of R. sphaeroides was inoculated into a modified Sistrom’s
such recombinants devoid of B875 complex were obtained. medium for H2 evolution in a homemade serum bottle
The chromosomal structures of the mutants were examined (60 ml) having a side arm (1.2 × 11; diameter × height in
for the correct deletion by genomic Southern hybridization centimeters), followed by gassing with argon as described
analysis [29], and one recombinant PUF1 was analyzed fur- previously [12]. Cells were contained in the side arm of the
ther. bottle, and the arm tube was stood in a tube rack in front of
In order to construct the pucBA-deleted mutant PUC1, light box. The gas phase (100–300 l) was withdrawn inter-
the 267-bp XmnI-XmaIII DNA spanning from pucB to pucA mittently during photoheterotrophic growth and analyzed
was removed from 1.34-kb PstI-PvuII fragment (Fig. 3A, for H2 by GC-17A gas chromatograph (Shimadzu, Japan)
thick line) of R. sphaeroides 2.4.1. The resulting recom- equipped with a thermal conductivity detector and a col-
E.-J. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 531 – 538 535
umn containing 60/80 mesh Molecular Sieve 5A (Supelco, and 100 W/m2 , and compared with those of the cells con-
USA). Argon was used as a carrier gas. The analyses of taining pRK415 as controls. As observed previously [12],
H2 accumulation in this work were performed at least three H2 production reached the maximum level at the stationary
separate times, and the data presented here were reproduced phase of cell growth (Fig. 4, A1 and A2). All the cells grew
within standard deviations of 10–15%. with doubling time between 5 and 6 h (Fig. 4, B1 and B2).
Therefore, the light level as much as 10 W/m2 is saturating
cell growth.
3. Results and discussion The cells containing the recombinant plasmids produced
less H2 compared with the cells containing pRK415 (Fig.
3.1. Photosynthetic spectral complexes are increased by 4, A1 and A2). Although it remains to be determined how
maintaining the plasmids harboring DNA encoding puhA, the H2 production is lowered in the order of cells con-
pufBA, or pucBAC in trans in R. sphaeroides taining plasmids pRKH100, pRKF100, and pRKC100, the
presence of additional spectral complexes appears to result
Plasmids pRKH100, pRKF100, and pRKC100 contain in reduction of the maximum level of H2 production by
the insert DNA encoding puhA (Fig. 1), pufBA (Fig. 2), approximately 10–50% in comparison with those of the
and pucBAC (Fig. 3), respectively, which are derived from control cells at the light intensities examined (Fig. 4, A1
R. sphaeroides 2.4.1. Each plasmid was mobilized into R. and A2). The same decrease was also observed with the H2
sphaeroides KCTC 12085, and the transconjugants were accumulation rates (data not shown), which were in parallel
grown photoheterotrophically at 10 and 100 W/m2 . The cel- with the maximum levels of H2 production as described
lular level of B875 and B800–850 complexes were measured previously [12]. The results suggest that the low level of
and compared with those of cells containing the vector plas- H2 production by maintaining the recombinant plasmids in
mid pRK415 (Table 1). The B875 complex was increased cells cannot be ascribed to light-limiting due to the shadow
by approximately 30–50% only when pRKF100 containing by higher level of spectral complexes, because neither the
pufBA was provided in trans. No such increase was observed H2 production nor cell growth were enhanced at the brighter
by either pRKH100 or pRKC100. However, all the transcon- light 100 W/m2 (Fig. 4, A2 and B2) that should have di-
jugants containing the recombinant plasmids showed an in- minished the shade if any. The energy-wasting metabolism
crease of the level of B800–850 complex by approximately leading to the formation of surplus spectral complexes
20–40%. rather appears to be a primary reason for the decreased H2
production.
3.2. Increase in cellular level of photosynthetic spectral
complexes decreased the photoheterotrophic production 3.3. Removal of B875 complex not only slowed cell
of H2 growth but also decreased H2 production even at light
( 10 W/m2 ) saturating the growth of wild type
R. sphaeroides KCTC 12085 containing pRKH100,
pRKF100, and pRKC100 were examined for the photo- Since an increase in cellular level of the photosynthetic
heterotrophic production of H2 at light intensities of 10 spectral complexes decreased the photoheterotrophic pro-
duction of H2 (Fig. 4), it was examined whether removal of
either B875 or B800–850 complex results in elevation of H2
Table 1 production. The DNA encoding pufBA was in-frame deleted
Relative level of LH complexes of the wild-type cells carry-
from the chromosome of R. sphaeroides KCTC 12085. The
ing pRKH100 (puhA), pRKF100 (pufBA), pRKC100 (pucBAC),
resulting mutant PUF1 (Fig. 2A) did not show any B875
and pRK415 (vector plasmid control), which were grown photo-
heterotrophically at 10 and 100 W/m2 complex under the photoheterotrophic conditions (Fig. 2B).
The level of B800–850 complex of PUF1 was not different
Light Strains Level of LH complex from the wild type although the carotenoid level appears to
intensity carrying (nmole/mg of protein) be lowered (Fig. 2B).
(W/m2) plasmids The H2 production and cell growth of wild type at
B875 B800–850 10 W/m2 were not much different from those observed at
10 KCTC12085 (pRK415) 11.9 ± 0.1 13.6 ± 0.6 100 W/m2 (Fig. 5, A and B). The growth of PUF1 under
KCTC12085 (pRKH100) 12.3 ± 0.6 16.4 ± 1.0 aerobic conditions is the same as that of wild type (data not
KCTC12085 (pRKF100) 17.8 ± 0.1 17.3 ± 0.8 shown). However, PUF1 grew with a doubling time of ap-
KCTC12085 (pRKC100) 10.7 ± 0.5 17.8 ± 0.1 proximately 13 h at 10 W/m2 and its maximum cell density
(KU) went up to approximately 80% of that of wild type
100 KCTC12085 (pRK415) 11.8 ± 0.5 9.1 ± 0.6 which doubled in every 5 h under the same conditions (Fig.
KCTC12085 (pRKH100) 11.7 ± 0.9 12.9 ± 0.1
5, B1). PUF1, though with 7-h doubling time, grew up to
KCTC12085 (pRKF100) 15.1 ± 0.6 11.4 ± 0.4
the same KU as that of wild type at 100 W/m2 (Fig. 5, B2).
KCTC12085 (pRKC100) 9.3 ± 0.2 12.2 ± 0.3
The results suggest that B875 complex is essential for the
536 E.-J. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 531 – 538
H2 (µl/ml culture)
H2 (µl/ml culture)
1500 pRK415 1500 pRK415
pRKH100 pRKH100
1000 pRKF100 1000 pRKF100
pRKC100
pRKC100
500 500
0 0
0 40 80 120 0 40 80 120
(A1) Time (h) (A2) Time (h)
1000 1000
KU
KU
100 100
10 10
0 40 80 120 0 40 80 120
(B1) Time (h) (B2) Time (h)
Fig. 4. H2 accumulation (A1, A2) and cell growth (B1, B2) of R. sphaeroides KCTC 12085 containing pRK415 (square), pRKH100
(diamond), pRKF100 (circle), and pRKC100 (triangle) under the photoheterotrophic conditions at 10 W/m2 (A1, B1) and 100 W/m2 (A2, B2).
H2 (µl/ml culture)
WT WT
1000 1000
PUF1
500 500
PUF1
0 0
0 50 100 150 0 50 100 150
(A1) Time (h) (A2) Time (h)
1000 1000
KU
KU
100 100
10 10
0 50 100 150 0 50 100 150
(B1) Time (h) (B2) Time (h)
Fig. 5. H2 accumulation (A1, A2) and cell growth (B1, B2) of R. sphaeroides KCTC 12085 (square; WT), and its two mutants, PUC1
(diamond) and PUF1 (triangle), under the photoheterotrophic conditions at 10 W/m2 (A1, B1) and 100 W/m2 (A2, B2).
efficient light harvesting and transfer of excitation energy the wild-type level (Fig. 5, A1). PUF1, however, produced
to RC. The growth of PUF1 was fully complemented with H2 up to the 80% level of wild type at 100 W/m2 (Fig. 5,
pRKF100 (data not shown), which suggests no additional A2), which is consistent with its growth restoration at the
mutation(s) in the mutant except for pufBA deletion. brighter light (Fig. 5, B). Thus, the B875 complex needs to
The H2 production of PUF1 during the photo- be present for efficient cell growth and H2 production of R.
heterotrophic growth at 10 W/m2 amounted to only 30% of sphaeroides.
E.-J. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 531 – 538 537
3.4. Removal of B800–850 complex increased the H2 Programs, funded by the Ministry of Science and Technol-
production at the light level saturating the growth of wild ogy of Korea.
type
[15] Wakayama T, Miyake J. Light shade bands for the [23] Lee JK, Kiley PJ, Kaplan S. Posttranscriptional control
improvement of solar hydrogen production efficiency by of puc operon expression of B800–850 light-harvesting
Rhodobacter sphaeroides RV. Int J Hydrogen Energy complex formation in Rhodobacter sphaeroides. J Bacteriol
2002;27:1495–500. 1989;171:3391–405.
[16] Kim NJ, Lee JK, Lee CG. Pigment reduction to improve [24] Davis J, Donohue TJ, Kaplan S. Construc-
photosynthetic productivity of Rhodobacter sphaeroides. tion, characterization, and complementation of a Puf-
J Microbiol Biotechnol 2004;14:442–9. mutant of Rhodobacter sphaeroides. J Bacteriol 1988;170:
[17] Sistrom WR. The kinetics of the synthesis of photopigments 320–9.
in Rhodopseudomonas sphaeroides. J Gen Microbiol [25] Chory J, Donohue TJ, Varga AR, Staehelin LA,
1962;28:607–16. Kaplan S. Induction of the photosynthetic membranes
[18] Donohue TJ, McEwan AG, Kaplan S. Cloning, DNA of Rhodopseudomonas sphaeroides: biochemical and
sequence, and expression of the Rhodobacter sphaeroides morphological studies. J Bacteriol 1984;159:540–54.
cytochrome c2 gene. J Bacteriol 1986;168:962–72. [26] Markwell MA, Haas SM, Bieber LL, Tolbert NE. A
[19] Donohue TJ, Hoger JH, Kaplan S. Cloning and expression modification of the Lowry procedure to simplify protein
of the Rhodobacter sphaeroides reaction center H gene. determination in membrane and lipoprotein samples. Anal
J Bacteriol 1986;168:953–61. Biochem 1978;87:206–10.
[20] Keen NT, Tamaki S, Kobayashi D, Trollinger D. Improved [27] Lenz O, Schwartz E, Dernedde J, Eitinger M, Friedrich B.
broad-host-range plasmids for DNA cloning in Gram-negative The Alcaligenes eutrophus H16 hoxX gene participates in
bacteria. Gene 1988;70:191–7. hydrogenase regulation. J Bacteriol 1994;176:4385–93.
[21] Kiley PJ, Donohue TJ, Havelka WA, Kaplan S. [28] Jeffke T, Gropp NH, Kaiser C, Grzesik C, Kusian B, Bowein
DNA sequence and in vitro expression of the B875 B. Mutational analysis of the cbb operon (CO2 assimilation)
light-harvesting polypeptides of Rhodobacter sphaeroides. promoter of Ralstonia eutropha. J Bacteriol 1999;181:
J Bacteriol 1987;169:742–50. 4374–80.
[22] Gong L, Lee JK, Kaplan S. The Q gene of Rhodobacter [29] Sambrook J, Russell DW. Molecular cloning: a laboratory
sphaeroides: its role in puf operon expression and spectral manual. 3rd ed., Cold Spring Harbor, NY: Cold Spring Harbor
complex assembly. J Bacteriol 1994;176:2946–61. Laboratory Press; 2001.