Crystal structure of the electron carrier domain of the reaction center cytochrome c(z) subunit from green photosynthetic bacterium Chlorobium tepidum.

In green sulfur photosynthetic bacteria, the cytochrome c(z) (cyt c(z)) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt c(z) subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt c(z) subunit (C-cyt c(z)) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-A resolution. The overall fold of C-cyt c(z) consists of four alpha-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt c(z) supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt c(z)

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

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

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

Two molecules of cytochrome c function as the electron donors to P840 in the reaction center complex isolated from a green sulfur bacterium, Chlorobium tepidum

AbstractA photoactive reaction center complex was isolated from a thermophilic green sulfur bacterium, Chlorobium tepidum under anaerobic conditions. The electron transfer occurred from heme c to the photo-oxidized reaction center chlorophyll, P840+, with a half time (t12) of 110 or 340 μs at 24 or 12°C, respectively. Optical measurements under multiflash excitations indicated that two hemes function as the immediate electron donors to P840+. SDS-PAGE analysis of the RC complex in combination with the N-terminal amino acid sequence analyses revealed five subunit bands; a core protein (65 kDa), the light harvesting bacteriochlorophyll a protein (41 kDa), a protein with 2[4Fe4S] clusters (31 kDa), monoheme cytochrome c (22 kDa), and a 18-kDa protein whose function is unknown. The reaction center complex, thus, contains two molecules of cytochrome c per P840

Crystallization and preliminary X-ray diffraction study of BchU, a methyltransferase from Chlorobium tepidum involved in bacteriochlorophyll c biosynthesis

Recombinant BchU from C. tepidum has been crystallized. Crystals diffract to 2.27 Å using synchrotron radiation at SPring-8 and belong to space group P6122 or P6522, with unit-cell parameters a = b = 81.5, c = 250.7 Å