Photosynthetic Green Sulfur Bacteria Research Articles

In vitro self-assemblies of bacteriochlorophylls-c from Chlorobaculum tepidum and their supramolecular nanostructures

Chlorosomes are major light-harvesting antenna apparatuses in green photosynthetic bacteria. Chlorosomes contain a large amount of bacteriochlorophyll(BChl)-c, d, e and f molecules and the supramolecular nanostructures of their self-assemblies are suggested to be rods with 5-nm or 10-nm diameters and/or lamellas with approximately 2-nm spacing. BChls-c were extracted, isolated and purified from cultured cells of a green sulfur photosynthetic bacterium, Chlorobaculum (Cba.) tepidum. Self-assemblies of the natural composite BChls-c as well as each pure homolog/epimer were prepared in a hydrophobic hexane-based solution and the resulting self-assembled solids were investigated by spectroscopic and microscopic techniques. Visible and near-infrared absorption and circular dichroism spectra of the in vitro self-assemblies of the natural composite BChls-c on a quartz substrate were closely similar to those in cells of Cba. tepidum in an aqueous buffer solution. Self-assemblies of (31R)-epimerically pure BChl-c molecules showed a wider Qy absorption band with increase in the steric bulkiness of hydrocarbon substituents at the 8- and 12-positions, which were effective for capturing wider wavelength light. (31S)-BChls-c gave similar self-assembly bands, but more monomeric and/or dimeric species than (31R)-BChls-c. Atomic force microscopic images of in vitro self-assemblies of natural composite BChls-c showed a rod with a 5-nm height and bundles of fibrils with 1–2-nm heights. Spectroscopic and microscopic analysis suggested that the 82-methylation enhanced π–π stacking of self-assemblies by hydrophobic interaction, but the 121-methylation slightly weakened the intermolecular interactions due to its steric bulkiness.

Stereochemical conversion of C3-vinyl group to 1-hydroxyethyl group in bacteriochlorophyll c by the hydratases BchF and BchV: adaptation of green sulfur bacteria to limited-light environments.

Photosynthetic green sulfur bacteria inhabit anaerobic environments with very low-light conditions. To adapt to such environments, these bacteria have evolved efficient light-harvesting antenna complexes called as chlorosomes, which comprise self-aggregated bacteriochlorophyll c in the model green sulfur, bacterium Chlorobaculum tepidum. The pigment possess a hydroxy group at the C3(1) position that produces a chiral center with R- or S-stereochemistry and the C3(1) -hydroxy group serves as a connecting moiety for the self-aggregation. Chlorobaculum tepidum carries the two possible homologous genes for C3-vinyl hydratase, bchF and bchV. In the present study, we constructed deletion mutants of each of these genes. Pigment analyses of the bchF-inactivated mutant, which still has BchV as a sole hydratase, showed higher ratios of S-epimeric bacteriochlorophyll c than the wild-type strain. The heightened prevalence of S-stereoisomers in the mutant was more remarkable at lower light intensities and caused a red shift of the chlorosomal Qy absorption band leading to advantages for light-energy transfer. In contrast, the bchV-mutant possessing only BchF showed a significant decrease of the S-epimers and accumulations of C3-vinyl BChl c species. As trans- criptional level of bchV was upregulated at lower light intensity, the Chlorobaculum tepidum adapted to low-light environments by control of the bchV transcription.

In Vitro Enzymatic Activities of Bacteriochlorophyll a Synthase Derived from the Green Sulfur Photosynthetic Bacterium Chlorobaculum tepidum.

The activity of an enzyme encoded by the CT1610 gene in the green sulfur photosynthetic bacterium Chlorobaculum tepidum, which was annotated as bacteriochlorophyll (BChl) a synthase, BchG (denoted as tepBchG), was examined in vitro using the lysates of Escherichia coli containing the heterologously expressed enzyme. BChl a possessing a geranylgeranyl group at the 17-propionate residue (BChl aGG) was produced from bacteriochlorophyllide (BChlide) a and geranylgeranyl pyrophosphate in the presence of tepBchG. Surprisingly, tepBchG catalyzed the formation of BChl a bearing a farnesyl group (BChl aF) as in the enzymatic production of BChl aGG, indicating loose recognition of isoprenoid pyrophosphates in tepBchG. In contrast to such loose recognition of isoprenoid substrates, BChlide c and chlorophyllide a gave no esterifying product upon being incubated with geranylgeranyl or farnesyl pyrophosphate in the presence of tepBchG. These results confirm that tepBchG undoubtedly acts as the BChl a synthase in Cba. tepidum. The enzymatic activity of tepBchG was higher than that of BchG of Rhodobacter sphaeroides at 45 °C, although the former activity was lower than the latter below 35 °C.

Silver island film substrates for ultrasensitive fluorescence detection of (bio)molecules.

A silver island film (SIF) substrate was used to demonstrate that Metal-Enhanced Fluorescence (MEF) is a powerful tool to enable detection of emission from (bio)molecules at very low concentrations. The experiments were carried out with the Fenna-Matthews-Olson (FMO) pigment-protein complex from the photosynthetic green sulfur bacterium Chlorobaculum tepidum. FMO was diluted to a level, at which no emission was detectable on a glass substrate. In contrast, the fluorescence of FMO was readily observed on the SIF substrate, even though the emission wavelength of FMO is displaced by over 300nm from the maximum of the plasmon resonance of the SIF layer. Estimated enhancements of the fluorescence intensity of FMO on SIF are about 40-fold. The enhancement factor correlates with the improvement of the signal-to-noise ratio for FMO emission on SIF substrates.

Alternative Excitonic Structure in the Baseplate (BChl a-CsmA Complex) of the Chlorosome from Chlorobaculum tepidum.

In the photosynthetic green sulfur bacterium Chlorobaculum tepidum, the baseplate mediates excitation energy transfer from the light-harvesting chlorosome to the Fenna-Matthews-Olson (FMO) complex and subsequently toward the reaction center (RC). Literature data suggest that the baseplate is a 2D lattice of BChl a-CsmA dimers. However, recently, it has been proposed, using 2D electronic spectroscopy (2DES) at 77 K, that at least four excitonically coupled BChl a are in close contact within the baseplate structure [ Dostál , J. ; et al., J. Phys. Chem. Lett. 2014 , 5 , 1743 ]. This finding is tested via hole burning (HB) spectroscopy (5 K). Our results indicate that the four excitonic states identified by 2DES likely correspond to contamination of the baseplate with the FMO antenna and possibly the RC. In contrast, HB reveals a different excitonic structure of the baseplate chromophores, where excitation is transferred to a localized trap state near 818 nm via exciton hopping, which leads to emission near 826 nm.

Modification of the esterifying farnesyl chain in light-harvesting bacteriochlorophylls in green sulfur photosynthetic bacteria by supplementation of 9-decyn-1-ol, 9-decen-1-ol, and decan-1-ol

Abstract Green sulfur photosynthetic bacteria Chlorobaculum ( Cba .) tepidum and Cba . limnaeum were cultivated with the supplementation of 9-decyn-1-ol, 9-decen-1-ol, and decan-1-ol by two methods to biosynthesize unnatural chlorosomal bacteriochlorophyll (BChl) pigments possessing an (un)saturated bond at the terminus of the esterifying chain. The supplementation of the acetone solutions of the exogenous alcohols every 24 h to Cba . tepidum (denoted as Method A) successfully produced unnatural BChls c esterified with the alcohols in vivo . The ratios of unnatural BChls c esterified with 9-decyn-1-ol, 9-decen-1-ol, and decan-1-ol over the total BChls c in Cba . tepidum were not so different from each other, indicating that terminal unsaturated moieties in the exogenous alcohols barely affected the last reaction in the BChl c biosynthesis catalyzed by the BChl c synthase. Cba . tepidum grown in a liquid culture that initially contained 9-decyn-1-ol and 9-decen-1-ol (denoted as Method B) also accumulated BChls c esterified with these alcohols; the relative ratios of the unnatural BChls c were analogous to those obtained by Method A at the corresponding final concentrations. In contrast, Cba . limnaeum did not synthesize BChl e esterified with the three exogenous alcohols. The incorporation of unsaturated bonds into the esterifying moieties of BChl c by a biosynthetic reaction will be useful as structural probes for the investigation of pigment interactions and the biogenesis of chlorosomes as well as the scaffolds for bioorthogonal modifications.

Structural analysis of the homodimeric reaction center complex from the photosynthetic green sulfur bacterium Chlorobaculum tepidum.

The reaction center (RC) complex of the green sulfur bacterium Chlorobaculum tepidum is composed of the Fenna–Matthews–Olson antenna protein (FMO) and the reaction center core (RCC) complex. The RCC complex has four subunits: PscA, PscB, PscC, and PscD. We studied the FMO/RCC complex by chemically cross-linking the purified sample followed by biochemical and spectroscopic analysis. Blue-native gels showed that there were two types of FMO/RCC complexes, which are consistent with complexes with one copy of FMO per RCC and two copies of FMO per RCC. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the samples after cross-linking showed that all five subunits of the RC can be linked by three different cross-linkers: bissulfosuccinimidyl suberate, disuccinimidyl suberate, and 3,3-dithiobis-sulfosuccinimidyl propionate. The interaction sites of the cross-linked complex were also studied using liquid chromatography coupled to tandem mass spectrometry. The results indicated that FMO, PscB, PscD, and part of PscA are exposed on the cytoplasmic side of the membrane. PscD helps stabilize FMO to the reaction center and may facilitate transfer of the electron from the RC to ferredoxin. The soluble domain of the heme-containing cytochrome subunit PscC and part of the core subunit PscA are located on the periplasmic side of the membrane. There is a close relationship between the periplasmic portions of PscA and PscC, which is needed for the efficient transfer of the electron between PscC and P840.

The ultrastructure of Chlorobaculum tepidum revealed by cryo-electron tomography

Chlorobaculum (Cba) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. As other anoxygenic green photosynthetic bacteria, Cba tepidum synthesizes bacteriochlorophylls for the assembly of a large light-harvesting antenna structure, the chlorosome. Chlorosomes are sac-like structures that are connected to the reaction centers in the cytoplasmic membrane through the BChl α-containing Fenna–Matthews–Olson protein. Most components of the photosynthetic machinery are known on a biophysical level, however, the structural integration of light harvesting with charge separation is still not fully understood. Despite over two decades of research, gaps in our understanding of cellular architecture exist. Here we present an in-depth analysis of the cellular architecture of the thermophilic photosynthetic green sulfur bacterium of Cba tepidum by cryo-electron tomography. We examined whole hydrated cells grown under different electron donor conditions. Our results reveal the distribution of chlorosomes in 3D in an unperturbed cell, connecting elements between chlorosomes and the cytoplasmic membrane and the distribution of reaction centers in the cytoplasmic membrane.

Chromatic acclimation and population dynamics of green sulfur bacteria grown with spectrally tailored light.

Living organisms have to adjust to their surrounding in order to survive in stressful conditions. We study this mechanism in one of most primitive creatures – photosynthetic green sulfur bacteria. These bacteria absorb photons very efficiently using the chlorosome antenna complexes and perform photosynthesis in extreme low-light environments. How the chlorosomes in green sulfur bacteria are acclimated to the stressful light conditions, for instance, if the spectrum of light is not optimal for absorption, is unknown. Studying Chlorobaculum tepidum cultures with far-red to near-infrared light-emitting diodes, we found that these bacteria react to changes in energy flow by regulating the amount of light-absorbing pigments and the size of the chlorosomes. Surprisingly, our results indicate that the bacteria can survive in near-infrared lights capturing low-frequency photons by the intermediate units of the light-harvesting complex. The latter strategy may be used by the species recently found near hydrothermal vents in the Pacific Ocean.

In vitro self-assembly of bacteriochlorophyll c derivatives monoesterified with α,ω-diols isolated from the green sulfur photosynthetic bacterium Chlorobaculum tepidum

Esterifying chains of chlorophyllous pigments play important roles in the formation of photosynthetic supramolecules, but their effects have not thoroughly been unraveled yet. Substitution of the esterifying chains in these pigments will be one possible strategy to elucidate this enigma. Recently, unnatural bacteriochlorophylls (BChls) c possessing a hydroxy group at the terminus of the esterifying chains were successfully biosynthesised in the green sulfur bacterium Chlorobaculum (Cba.) tepidum grown by supplementation of α,ω-diols. In this paper, in vitro assembling behaviours of unnatural BChls c isolated from Cba. tepidum grown with 1,8-octanediol, 1,12-dodecanediol and 1,16-hexadecanediol were characterised in aqueous Triton X-100 micelles to investigate the effects of the terminal hydroxy group in the esterifying chains of BChls c on self-aggregates such as chlorosomes, major antenna complexes in green photosynthetic bacteria. The bacteriochlorophyll (BChl) c derivatives monoesterified with α,ω-diols formed chlorosomal self-aggregates, but their formations were much slower than those of natural BChl c. The Qy absorption bands of the residual monomers of these BChl c derivatives were larger than those of natural BChl c. These suggest that the esterifying α,ω-diols in these unnatural BChls c somewhat interfered with formation of chlorosome-like aggregates compared with the natural esterifying farnesol.

Biosynthesis of bacteriochlorophyll c derivatives possessing chlorine and bromine atoms at the terminus of esterifying chains in the green sulfur bacterium Chlorobaculum tepidum

The green sulfur photosynthetic bacterium Chlorobaculum tepidum newly produced BChl c derivatives possessing a chlorine or bromine atom at the terminus of the esterifying chain in the 17-propionate residue by cultivation with exogenous ω-halo-1-alkanols. The relative ratios of BChl c derivatives esterified with 8-chloro-1-octanol and 10-chloro-1-decanol were estimated to be 26.5% and 33.3% by cultivation with these ω-chloro-1-alkanols at the final concentrations of 300 and 150 μM, respectively. In contrast, smaller amounts of unnatural BChls c esterified with ω-bromo-1-alkanols were biosynthesized than those esterified with ω-chloro-1-alkanols: the ratios of BChl c derivatives esterified with 8-bromo-1-octanol and 10-bromo-1-decanol were 11.3% and 12.2% at the concentrations of 300 and 150 μM, respectively. These indicate that ω-chloro-1-alkanols can be incorporated into bacteriochlorophyllide c more than ω-bromo-1-alkanols in the BChl c biosynthetic pathway. The homolog compositions of the novel BChl c derivatives possessing a halogen atom were analogous to those of coexisting natural BChl c esterified with farnesol. These results demonstrate unique properties of BChl c synthase, BchK, which can utilize unnatural substrates containing halogen in the BChl c biosynthesis of Cba. tepidum.

Structure analysis and characterization of the cytochrome c-554 from thermophilic green sulfur photosynthetic bacterium Chlorobaculum tepidum

The cytochrome (Cyt) c-554 in thermophilic green photosynthetic bacterium Chlorobaculum tepidum serves as an intermediate electron carrier, transferring electrons to the membrane-bound Cyt c z from various enzymes involved in the oxidations of sulfide, thiosulfate, and sulfite compounds. Spectroscopically, this protein exhibits an asymmetric α-absorption band for the reduced form and particularly large paramagnetic (1)H NMR shifts for the heme methyl groups with an unusual shift pattern in the oxidized form. The crystal structure of the Cyt c-554 has been determined at high resolution. The overall fold consists of four α-helices and is characterized by a remarkably long and flexible loop between the α3 and α4 helices. The axial ligand methionine has S-chirality at the sulfur atom with its C(ε)H3 group pointing toward the heme pyrrole ring I. This configuration corresponds to an orientation of the lone-pair orbital of the sulfur atom directed at the pyrrole ring II and explains the lowest-field (1)H NMR shift arising from the 18(1) heme methyl protons. Differing from most other class I Cyts c, no hydrogen bond was formed between the methionine sulfur atom and polypeptide chain. Lack of this hydrogen bond may account for the observed large paramagnetic (1)H NMR shifts of the heme methyl protons. The surface-exposed heme pyrrole ring II edge is in a relatively hydrophobic environment surrounded by several electronically neutral residues. This portion is considered as an electron transfer gateway. The structure of the Cyt c-554 is compared with those of other Cyts c, and possible interactions of this protein with its electron transport partners are discussed.

Synthesis of biopterin and related pterin glycosides

Certain pterins having a hydroxyalkyl side chain at C-6 have been found as glycosidic forms in certain prokaryotes, such as 2'-O-(α-D-glucopyranosyl)biopterin from various kinds of cyanobacteria, and limipterin from a green sulfur photosynthetic bacterium. Synthetic studies on glycosides of biopterin and related pterins have been made in view of the structural proof as well as for closer examination of their biological activities and functions. The syntheses of these natural pterin glycosides have effectively been achieved, mostly through appropriately protected N(2) -(N,N-dimethylaminomethylene)-3-[2-(4-nitrophenyl)ethyl]pterin derivatives as glycosyl acceptors, and are reviewed here.

Fluorescence Excitation Spectra from Individual Chlorosomes of the Green Sulfur Bacterium Chlorobaculum tepidum.

We performed polarization-resolved fluorescence excitation spectroscopy on individual chlorosomes from the photosynthetic green sulfur bacterium Chlorobaculum tepidum. The experiments were conducted at room temperature and under cryogenic conditions. All spectra showed a strong intensity modulation as a function of the polarization of the incident radiation, and we determined the modulation ratio as a function of the excitation energy. Under ambient conditions this ratio shows only little variation across the absorption band, whereas the low-temperature experiments clearly revealed that the broad absorption band around 740 nm consists of several spectral contributions.

Excitation Energy Transfer in Isolated Chlorosomes from Chlorobaculum tepidum and Prosthecochloris aestuarii

Excitation energy transfer in chlorosomes from photosynthetic green sulfur bacteria, Chlorobaculum (Cba.) tepidum and Prosthecochloris (Pst.) aestuarii, have been studied at room temperature by time-resolved femtosecond transient absorption spectroscopy. Bleach rise times from 117 to 270 fs resolved for both chlorosomes reflect extremely efficient intrachlorosomal energy transfer. Bleach relaxation times, from 1 to 3 ps and 25 to 35 ps, probed at 758 nm were tentatively assigned to intrachlorosomal energy transfer based on amplitude changes of the global fits and model calculations. The anisotropy decay constant of about 1 ps resolved at 807 nm probe wavelength for the chlorosomes from Chloroflexus aurantiacus, Pst. aestuarii and Cba. tepidum was related to energy transfer between bacteriochlorophyll a molecules of the baseplate and partly to intrachlorosomal energy transfer. The longer anisotropy components 6.6, 8.8 and 12.1 ps resolved for the three chlorosomes, respectively, were assigned to chlorosome to baseplate energy transfer. Global fits of magic-angle data also revealed longer chlorosome to baseplate energy transfer components from 95 to 135 ps, in accord with results from simulations.

Isolation and Characterization of Homodimeric Type-I Reaction Center Complex from Candidatus Chloracidobacterium thermophilum, an Aerobic Chlorophototroph

The recently discovered thermophilic acidobacterium Candidatus Chloracidobacterium thermophilum is the first aerobic chlorophototroph that has a type-I, homodimeric reaction center (RC). This organism and its type-I RCs were initially detected by the occurrence of pscA gene sequences, which encode the core subunit of the RC complex, in metagenomic sequence data derived from hot spring microbial mats. Here, we report the isolation and initial biochemical characterization of the type-I RC from Ca. C. thermophilum. After removal of chlorosomes, crude membranes were solubilized with 0.1% (w/v) n-dodecyl β-D-maltoside, and the RC complex was purified by ion-exchange chromatography. The RC complex comprised only two polypeptides: the reaction center core protein PscA and a 22-kDa carotenoid-binding protein denoted CbpC. The absorption spectrum showed a large, broad absorbance band centered at ∼483 nm from carotenoids as well as smaller Q(y) absorption bands at 672 and 812 nm from chlorophyll a and bacteriochlorophyll a, respectively. The light-induced difference spectra of whole cells, membranes, and the isolated RC showed maximal bleaching at 840 nm, which is attributed to the special pair and which we denote as P840. Making it unique among homodimeric type-I RCs, the isolated RC was photoactive in the presence of oxygen. Analyses by optical spectroscopy, chromatography, and mass spectrometry revealed that the RC complex contained 10.3 bacteriochlorophyll a(P), 6.4 chlorophyll a(PD), and 1.6 Zn-bacteriochlorophyll a(P)' molecules per P840 (12.8:8.0:2.0). The possible functions of the Zn-bacteriochlorophyll a(P)' molecules and the carotenoid-binding protein are discussed.

緑色硫黄光合成細菌Chlorobium tepidumの糖脂質解析：培養時の温度・時間依存性とその機能

緑色硫黄光合成細菌Chlorobium tepidumの糖脂質解析：培養時の温度・時間依存性とその機能

Biosynthesis of Unnatural BacteriochlorophyllcDerivatives Esterified with α,ω-Diols in the Green Sulfur Photosynthetic BacteriumChlorobaculum tepidum

Unnatural bacteriochlorophyll (BChl) c derivatives possessing a hydroxy group at the terminus of a hydrocarbon chain at the 17-propionate were biosynthesized in the green sulfur photosynthetic bacterium Chlorobaculum tepidum. Addition of exogenous 1,8-octanediol, 1,12-dodecanediol, and 1,16-hexadecanediol in acetone to liquid cultures resulted in accumulation of BChl c monoesterified with the corresponding diols. The relative ratios of the novel BChl c derivatives esterified with 1,8-, 1,12-, and 1,16-diols to totally producing BChl c were 8.2, 50.2, and 57.6% in the cells grown with additive α,ω-diols at concentrations of 1.5, 0.06, and 0.06 mM, respectively, at the final concentration. The homologue composition of BChl c derivatives esterified with these α,ω-diols was similar to that of original, coexisting BChl c esterified with farnesol (BChl c(F)), suggesting that esterification of α,ω-diols occurred at the last step of the BChl c biosynthetic pathway by BChl c synthase, BchK, in the same manner as in BChl c(F). Chlorosomes, which were isolated from cells grown in the presence of exogenous α,ω-diols, contained a ratio and a composition of BChl c derivatives esterified with the diols similar to those in the whole cells, indicating that these BChl c derivatives were actually present in chlorosomes. Q(y) absorption bands of C. tepidum cells containing the novel BChl c derivatives were shifted to a shorter wavelength, although their bandwidths were analogous to those of cells obtained by normal cultivation. Circular dichroism spectra of cells that had BChl c derivatives esterified with α,ω-diols exhibited S-shaped signals in the Q(y) region, whose polarities were the reverse of those of cells grown in the normal medium and by supplementation with neat acetone as a control experiment. These spectral features of C. tepidum possessing BChl c derivatives esterified with α,ω-diols imply that the novel BChl c derivatives possessing a hydroxy group at the terminus of a hydrocarbon chain affect their self-assembly in chlorosomes.

Source-specific variability in post-depositional DNA preservation with potential implications for DNA based paleoecological records

Recent studies have shown that genotyping preserved plankton DNA in marine and lacustrine sediment records using ancient DNA methods is a promising approach for refining paleoenvironmental information. However, the extent to which the preservation of fossil plankton DNA differs between species is poorly understood. Using a continuous 2700 year sediment record from Watts Basin in Ellis Fjord (Antarctica), we compared the level of preservation of fossil DNA derived from important plankton members with varying cellular architecture. The amount of preserved small subunit ribosomal DNA (SSU rDNA; ca. 500 base pair fragments) of dinoflagellates (as extracellular DNA rather than as preserved cysts) that could be amplified by way of PCR declined up to five orders of magnitude with increasing sediment depth and age. In contrast, the amount of similar-sized, PCR-amplifiable, diatom SSU rDNA (predominantly from a cyst-forming Chaetoceros sp.) declined only up to tenfold over 2700 years of deposition. No obvious decline in copy numbers with increasing sediment age was observed for similar-sized SSU rDNA of past chemocline-associated photosynthetic green sulfur bacteria (GSB), which do not have a protective resting stage. In good agreement with the quantitative data, the extent of post-depositional natural degradation to fragments too small to serve as a template for the quantitative PCR assays was greatest for dinoflagellates and lowest for GSB. An increase in the ratio between GSB-derived DNA and intact carotenoids with sediment depth implies that short GSB DNA fragments were better preserved than intact carotenoids and provide a more accurate view into paleoproductivity and the sediment flux of GSB in Watts Basin. We discuss the possible causes behind the variation in the level of DNA preservation among the plankton groups investigated, as well as consequences for the use of using fossil DNA records in paleoecology studies.

Temperature- and Time-Dependent Changes in the Structure and Composition of Glycolipids during the Growth of the Green Sulfur Photosynthetic Bacterium Chlorobaculum tepidum

The green sulfur photosynthetic bacterium Chlorobaculum (Cba.) tepidum (previously known as Chlorobium tepidum), which grows at an optimal temperature of around 45 °C, biosynthesized unique disaccharide rhamnosylgalactosyldiacylglyceride (RGDG) having a methylene-bridged palmitoleyl (17:Cyc) and a palmitoyl group (16:0) as the two acyl chains in a molecule [RGDG(17:Cyc,16:0)], together with the corresponding monosaccharide monogalactosyldiacylglyceride (MGDG). Here, we report changes in the structure and composition of the glycolipids that are dependent upon the temperature and period of cultivation. With a decrease in temperature to 25 °C, the two major glycolipids were almost completely eliminated, and MGDG with a palmitoleyl (16:1) and a (16:0) group concomitantly became the major glycolipid. MGDG(16:1,16:0) corresponded to the removal of an α-rhamnosyl and a cyclopropyl methylene group from RGDG(17:Cyc,16:0) and the lack of the CH(2) group in MGDG(17:Cyc,16:0). The structural conversion was almost reversible when the Cba. tepidum adapted to low and high temperatures was cultured again at 45 and 25 °C, respectively. Moreover, during this cultivation, the structure and composition of glycolipids were sequentially changed: MGDG(16:1,16:0), MGDG(17:Cyc,16:0), and RGDG(17:Cyc,16:0) predominated in the exponential, stationary and late phases of the cultivation, respectively. On the basis of these time-dependent changes, the unique disaccharide RGDG(17:Cyc,16:0) was thought to be created by the site-specific transfer of an α-rhamnosyl group to MGDG(17:Cyc,16:0) after insertion of a methylene group into the precursor MGDG(16:1,16:0). These culturing temperature- and time-dependent changes in glycolipids at the molecular level allow us to discuss their biosynthesis as well as physiological function in green photosynthetic bacteria.