Growth curves of <i>C. tepidum</i> mutants after gene complementation experiments.

<p>Growth curves of <i>C. tepidum</i> mutants used as host strains (A), transconjugant strains of ∆<i>cycA</i> mutant (B), and ∆<i>soxB</i> mutant (C). Each strain was grown in a liquid CL medium at 40°C (for details, see Materials and Methods), and its optical density (O.D.) was monitored at 660 nm. In the transconjugant cultures, 1 µg/mL of Em was added for the stable maintenance of plasmids. The average values and standard deviations, which were obtained from at least three independent experiments, were plotted.</p

Restriction enzyme mappings of the plasmids in the <i>C. tepidum</i> and <i>C. limnaeum</i> transconjugants.

<p>(A, B) Physical maps of the plasmids from the <i>C. tepidum</i> transconjugants. The maps were constructed with restriction enzymes <i>Eco</i>RI (A) and <i>Bgl</i>II (B). The restriction fragments were separated by agarose gel (1%) electrophoresis. The control plasmids pDSK5191 (lane 1), pDSK5191-<i>cycA</i> (lane 2), and pDSK5191-<i>soxB</i> (lane 3) were obtained from the donor S17-1 cultures. Lanes 4-11 are the plasmid samples of the <i>C. tepidum</i> cultures. The genotype of <i>C. tepidum</i> is indicated above each lane. The bars and numbers at the left side of the panel indicate mobility and size of the <i>Sty</i>I digests of the λ-phage DNA. (C) Physical maps of the plasmids from <i>C. limnaeum</i> transconjugants. The maps were constructed with restriction enzymes <i>Eco</i>RI (lanes 1–3) and <i>Bam</i>HI (lanes 4–6). Lanes 1 and 4 are pDSK5192 plasmids prepared from the donor S17-1 cultures. Lanes 2-3 and 5-6 are the plasmid samples of the <i>C. limnaeum</i> cultures. The genotype of <i>C. limnaeum</i> is indicated above each lane. The bars and numbers at the left side of the panel indicate mobility and sizes of the <i>Sty</i>I digests of the λ-phage DNA, respectively. </p

Gene Expression System in Green Sulfur Bacteria by Conjugative Plasmid Transfer

<div><p>Gene transfer and expression systems in green sulfur bacteria were established by bacterial conjugation with <i>Escherichia coli</i>. Conjugative plasmid transfer from <i>E. coli</i> S17-1 to a thermophilic green sulfur bacterium, <i>Chlorobaculum tepidum</i> (formerly <i>Chlorobium tepidum</i>) WT2321, was executed with RSF1010-derivative broad-host-range plasmids, named pDSK5191 and pDSK5192, that confer erythromycin and streptomycin/spectinomycin resistance, respectively. The transconjugants harboring these plasmids were reproducibly obtained at a frequency of approximately 10^-5 by selection with erythromycin and a combination of streptomycin and spectinomycin, respectively. These plasmids were stably maintained in <i>C. tepidum</i> cells in the presence of these antibiotics. The plasmid transfer to another mesophilic green sulfur bacterium, <i>C. limnaeum</i> (formerly <i>Chlorobium phaeobacteroides</i>) RK-j-1, was also achieved with pDSK5192. The expression plasmid based on pDSK5191 was constructed by incorporating the upstream and downstream regions of the <i>pscAB</i> gene cluster on the <i>C. tepidum</i> genome, since these regions were considered to include a constitutive promoter and a ρ-independent terminator, respectively. Growth defections of the ∆<i>cycA</i> and ∆<i>soxB</i> mutants were completely rescued after introduction of pDSK5191-<i>cycA</i> and -<i>soxB</i> that were designed to express their complementary genes. On the other hand, pDSK5191-<i>6xhis-pscAB</i>, which incorporated the gene cluster of <i>6xhis-pscA</i> and <i>pscB</i>, produced approximately four times more of the photosynthetic reaction center complex with His-tagged PscA as compared with that expressed in the genome by the conventional natural transformation method. This expression system, based on conjugative plasmid, would be applicable to general molecular biological studies of green sulfur bacteria.</p> </div

Schematic genetic maps of pDSK5191 and its derivative expression plasmids.

<p>(A) Genetic map of the IncQ-group conjugation plasmid pDSK5191. Protein coding sequences are shown as block arrows. The pale gray rectangle represents the region of Ω-cassette in which T4-phage transcription and translation terminator sequences [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082345#B23" target="_blank">23</a>] are located at both ends. “Ori” represents the region containing the <i>oriV</i> and <i>oriT</i> sequences, which are derived from RSF1010 and are required for replication and mobilization of the plasmid, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082345#B15" target="_blank">15</a>]. (B) Expression constructs of the pDSK5191-derivative plasmids. Each construct was inserted into the unique <i>Eco</i>RI site of pDSK5191, as indicated by the arch-shaped arrow at the top of the panels. Protein-coding sequences are shown as block arrows. The pale gray rectangle represents the six-consecutive histidine-tag (6xhis) attached to the 5’ end of the <i>pscA</i> gene. “P_<i>pscA</i>” and “ter” are a putative constitutive promoter and a ρ-independent transcription terminator, respectively, of the <i>C. tepidum </i><i>pscAB</i> gene cluster.</p

Orientations of Iron–Sulfur Clusters F_A and F_B in the Homodimeric Type‑I Photosynthetic Reaction Center of <i>Heliobacterium modesticaldum</i>

Orientations of the F_A and F_B iron–sulfur (FeS) clusters in a structure-unknown type-I homodimeric heriobacterial reaction center (hRC) were studied in oriented membranes of the thermophilic anaerobic photosynthetic bacterium <i>Heliobacterium modesticaldum</i> by electron paramagnetic resonance (EPR), and compared with those in heterodimeric photosystem I (PS I). The Rieske-type FeS center in the cytochrome <i>b/c</i> complex showed a well-oriented EPR signal. Illumination at 14 K induced an F_B^– signal with <i>g</i>-axes of <i>g</i>_<i>z</i> = 2.066, <i>g</i>_<i>y</i> = 1.937, and <i>g</i>_<i>x</i> = 1.890, tilted at angles of 60°, 60°, and 45°, respectively, with respect to the membrane normal. Chemical reduction with dithionite produced an additional signal of F_A^–, which magnetically interacted with F_B^–, with <i>g</i>_<i>z</i> = 2.046, <i>g</i>_<i>y</i> = 1.942, and <i>g</i>_<i>x</i> = 1.911 at 30°, 60°, and 90°, respectively. The angles and redox properties of F_A^– and F_B^– in hRC resemble those of F_B^– and F_A^–, respectively, in PS I. Therefore, F_A and F_B in hRC, named after their <i>g</i>-value similarities, seem to be located like F_B and F_A, not like F_A and F_B, respectively, in PS I. The reducing side of hRC could resemble those in PS I, if the names of F_A and F_B are interchanged with each other

Menaquinone as the Secondary Electron Acceptor in the Type I Homodimeric Photosynthetic Reaction Center of <i>Heliobacterium modesticaldum</i>

The type I photosynthetic reaction center (RC) of heliobacteria (hRC) is a homodimer containing cofactors almost analogous to those in the plant photosystem I (PS I). However, its three-dimensional structure is not yet clear. PS I uses phylloquinone (PhyQ) as a secondary electron acceptor (A₁), while the available evidence has suggested that menaquinone (MQ) in hRC has no function as A₁. The present study identified a new transient electron spin-polarized electron paramagnetic resonance (ESP-EPR) signal, arising from the radical pair of the oxidized electron donor and the reduced electron acceptor (P800⁺MQ^–), in the hRC core complex and membranes from <i>Heliobacterium modesticaldum</i>. The ESP signal could be detected at 5–20 K upon flash excitation only after prereduction of the iron–sulfur center, F_X, and was selectively lost by extraction of MQ with diethyl ether. MQ was suggested to be located closer to F_X than PhyQ in PS I based on the simulation of the unique A/E (A, absorption; E, emission) ESP pattern, the reduction/oxidation rates of MQ, and the power saturation property of the static MQ^– signal. The result revealed the quinone usage as the secondary electron acceptor in hRC, as in the case of PS I

Experimental and Theoretical Mutation of Exciton States on the Smallest Type‑I Photosynthetic Reaction Center Complex of a Green Sulfur Bacterium Chlorobaclum tepidum

The exciton states on the smallest type-I photosynthetic reaction center complex of a green sulfur bacterium Chlorobaculum tepidum (GsbRC) consisting of 26 bacteriochlorophylls a (BChl a) and four chlorophylls a (Chl a) located on the homodimer of two PscA reaction center polypeptides were investigated. This analysis involved the study of exciton states through a combination of theoretical modeling and the genetic removal of BChl a pigments at eight sites. (1) A theoretical model of the pigment assembly exciton state on GsbRC was constructed using Poisson TrESP (P-TrESP) and charge density coupling (CDC) methods based on structural information. The model reproduced the experimentally obtained absorption spectrum, circular dichroism spectrum, and excitation transfer dynamics, as well as explained the effects of mutation. (2) Eight BChl a molecules at different locations on the GsbRC were selectively removed by genetic exchange of the His residue, which ligates the central Mg atom of BChl a, with the Leu residue on either one or two PscAs in the RC. His locations are conserved among all type-I RC plant polypeptide, cyanobacteria, and bacteria amino acid sequences. (3) Purified mutant-GsbRCs demonstrated distinct absorption and fluorescence spectra at 77 K, which were different from each other, suggesting successful pigment removal. (4) The same mutations were applied to the constructed theoretical model to analyze the outcomes of these mutations. (5) The combination of theoretical predictions and experimental mutations based on structural information is a new tool for studying the function and evolution of photosynthetic reaction centers

Light-Induced Electron Spin-Polarized (ESP) EPR Signal of the P800⁺ Menaquinone^– Radical Pair State in Oriented Membranes of Heliobacterium modesticaldum: Role/Location of Menaquinone in the Homodimeric Type I Reaction Center

Function/location of menaquinone (MQ) was studied in the photosynthetic reaction center of Heliobacterium (Hbt.) modesticaldum (hRC), which is one of the most primitive homodimeric type I RCs. The spin-polarized electron paramagnetic resonance signals of light-induced radical pair species, which are made of oxidized electron donor bacteriochlorophyll <i>g</i> (P800⁺) and reduced menaquinone (MQ^–) or iron–sulfur cluster (F_X^–), were measured in the oriented membranes of Hbt. modesticaldum at cryogenic temperature. The spectral shape of transient electron spin-polarized signal of P800⁺F_X^– radical pair state varied little with respect to the direction of the external magnetic field. It suggested a dominant contribution of the spin evolution on the precursor primary radical pair P800⁺A₀^– state with the larger isotropic magnetic exchange interaction <i>J</i> than the anisotropic dipole interaction <i>D</i>. The pure P800⁺MQ^– signal was simulated by subtracting the effects of spin evolution during the electron-transfer process. It was concluded that the <i>J</i> value of the P800⁺MQ^– radical pair is negative with an amplitude almost comparable to |<i>D</i>|. It is in contrast to a positive and small <i>J</i> value of the P700⁺PhyQ^– state in photosystem I (PS I). The results indicate similar but somewhat different locations/binding sites of quinones between hRC and PS I

Experimental and Theoretical Mutation of Exciton States on the Smallest Type‑I Photosynthetic Reaction Center Complex of a Green Sulfur Bacterium Chlorobaclum tepidum

The exciton states on the smallest type-I photosynthetic reaction center complex of a green sulfur bacterium Chlorobaculum tepidum (GsbRC) consisting of 26 bacteriochlorophylls a (BChl a) and four chlorophylls a (Chl a) located on the homodimer of two PscA reaction center polypeptides were investigated. This analysis involved the study of exciton states through a combination of theoretical modeling and the genetic removal of BChl a pigments at eight sites. (1) A theoretical model of the pigment assembly exciton state on GsbRC was constructed using Poisson TrESP (P-TrESP) and charge density coupling (CDC) methods based on structural information. The model reproduced the experimentally obtained absorption spectrum, circular dichroism spectrum, and excitation transfer dynamics, as well as explained the effects of mutation. (2) Eight BChl a molecules at different locations on the GsbRC were selectively removed by genetic exchange of the His residue, which ligates the central Mg atom of BChl a, with the Leu residue on either one or two PscAs in the RC. His locations are conserved among all type-I RC plant polypeptide, cyanobacteria, and bacteria amino acid sequences. (3) Purified mutant-GsbRCs demonstrated distinct absorption and fluorescence spectra at 77 K, which were different from each other, suggesting successful pigment removal. (4) The same mutations were applied to the constructed theoretical model to analyze the outcomes of these mutations. (5) The combination of theoretical predictions and experimental mutations based on structural information is a new tool for studying the function and evolution of photosynthetic reaction centers

Experimental and Theoretical Mutation of Exciton States on the Smallest Type‑I Photosynthetic Reaction Center Complex of a Green Sulfur Bacterium Chlorobaclum tepidum

The exciton states on the smallest type-I photosynthetic reaction center complex of a green sulfur bacterium Chlorobaculum tepidum (GsbRC) consisting of 26 bacteriochlorophylls a (BChl a) and four chlorophylls a (Chl a) located on the homodimer of two PscA reaction center polypeptides were investigated. This analysis involved the study of exciton states through a combination of theoretical modeling and the genetic removal of BChl a pigments at eight sites. (1) A theoretical model of the pigment assembly exciton state on GsbRC was constructed using Poisson TrESP (P-TrESP) and charge density coupling (CDC) methods based on structural information. The model reproduced the experimentally obtained absorption spectrum, circular dichroism spectrum, and excitation transfer dynamics, as well as explained the effects of mutation. (2) Eight BChl a molecules at different locations on the GsbRC were selectively removed by genetic exchange of the His residue, which ligates the central Mg atom of BChl a, with the Leu residue on either one or two PscAs in the RC. His locations are conserved among all type-I RC plant polypeptide, cyanobacteria, and bacteria amino acid sequences. (3) Purified mutant-GsbRCs demonstrated distinct absorption and fluorescence spectra at 77 K, which were different from each other, suggesting successful pigment removal. (4) The same mutations were applied to the constructed theoretical model to analyze the outcomes of these mutations. (5) The combination of theoretical predictions and experimental mutations based on structural information is a new tool for studying the function and evolution of photosynthetic reaction centers