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The impact of different intensities of green light on the bacteriochlorophyll homologue composition of the Chlorobiaceae Prosthecochloris aestuarii and Chlorobium phaeobacteroides.

Members of the Chlorobiaceae and Chloroflexaceae are unique among the phototrophic micro-organisms in having a remarkably rich chlorophyll pigment diversity. The physiological regulation of this diversity and its ecological implications are still enigmatic. The bacteriochlorophyll composition of the chlorobiaceae Prosthecochloris aestuarii strain CE 2404 and Chlorobium phaeobacteroides strain UdG 6030 was therefore studied by both HPLC with photodiode array (PDA) detection and liquid chromatography-mass spectrometry (LC-MS). These strains were grown in liquid cultures under green light (480-615 nm) at different light intensities (0.2-55.7 micromol photons m(-2) s(-1)), simulating the irradiance regime at different depths of the water column of deep lakes. The specific growth rates of Ptc. aestuarii under green light achieved a maximum of 0.06 h(-1) at light intensities exceeding 6 micromol photons m(-2) s(-1), lower than the maximum observed under white light (approx. 0.1 h(-1)). The maximal growth rates of Chl. phaeobacteroides under green light were slightly higher (0.07 h(-1)) than observed for Ptc. aestuarii and were achieved at 3.5 and 4.3 micromol photons m(-2) s(-1). LC-MS/MS analysis of pigment extracts revealed most (>90 %) BChl c homologues of Ptc. aestuarii to be esterified with farnesol. The homologues differed in mass by multiples of 14 Da, reflecting different alkyl subsituents at positions C-8 and C-12 on the tetrapyrrole macrocycle. The relative proportions of the individual homologues varied only slightly among different light intensities. The specific content of BChl c was maximal at 3-5 micromol photons m(-2) s(-1) [400+/-150 nmol BChl c (mg protein)(-1)]. In the case of Chl. phaeobacteroides, the specific content of BChl e was maximal at 4.3 micromol photons m(-2) s(-1) [115 nmol BChl e (mg protein)(-1)], and this species was characterized by high carotenoid (isorenieratene) contents. The major BChl e forms were esterified with a range of isoprenoid and straight-chain alcohols. The major isoprenoid alcohols comprised mainly farnesol and to a lesser extent geranylgeraniol. The straight-chain alcohols included C(15), C(15 : 1), C(16), C(16 : 1) and C(17). Interestingly, the proportion of straight alkyl chains over isoprenoid esterified side chains shifted markedly with increasing light intensity: the isoprenoid side chains dominated at low light intensities, while the straight-chain alkyl substituents dominated at higher light intensities. The authors propose that this phenomenon may be explained as a result of changing availability of reducing power, i.e. the highly reduced straight-chain alcohols have a higher biosynthetic demand for NADPH(2) than the polyunsaturated isoprenoid with the same number of carbon atoms.

Bacteriochlorophylls↗

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

The S-adenosylmethionine-dependent methyltransferase BchU is an enzyme involved in the bacteriochlorophyll c biosynthetic pathway and catalyzes methylation at the C-20 position of the chlorin moiety. Recombinant Chlorobium tepidum BchU overproduced in Escherichia coli was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystals belonged to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 81.5, c = 250.7 A. A native data set was collected to 2.27 A resolution using synchrotron radiation at SPring-8.

Bacterial Proteins↗

Auracyanin A from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus represents an unusual class of small blue copper proteins.

The amino acid sequence of the small copper protein auracyanin A isolated from the thermophilic photosynthetic green bacterium Chloroflexus aurantiacus has been determined to be a polypeptide of 139 residues. His58, Cys123, His128, and Met132 are spaced in a way to be expected if they are the evolutionary conserved metal ligands as in the known small copper proteins plastocyanin and azurin. Secondary structure prediction also indicates that auracyanin has a general beta-barrel structure similar to that of azurin from Pseudomonas aeruginosa and plastocyanin from poplar leaves. However, auracyanin appears to have sequence characteristics of both small copper protein sequence classes. The overall similarity with a consensus sequence of azurin is roughly the same as that with a consensus sequence of plastocyanin, namely 30.5%. We suggest that auracyanin A, together with the B forms, is the first example of a new class of small copper proteins that may be descendants of an ancestral sequence to both the azurin proteins occurring in prokaryotic nonphotosynthetic bacteria and the plastocyanin proteins occurring in both prokaryotic cyanobacteria and eukaryotic algae and plants. The N-terminal sequence region 1-18 of auracyanin is remarkably rich in glycine and hydroxy amino acids, and required mass spectrometric analysis to be determined. The nature of the blocking group X is not yet known, although its mass has been determined to be 220 Da. The auracyanins are the first small blue copper proteins found and studied in anoxygenic photosynthetic bacteria and are likely to mediate electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center.

Amino Acid Sequence↗

Redox titration of two [4Fe-4S] clusters in the photosynthetic reaction center from the anaerobic green sulfur bacterium Chlorobium vibrioforme.

Anaerobic green sulfur bacteria contain photosynthetic reaction centers analogous to photosystem I (PS I) of plants and cyanobacteria. These reaction centers, termed type I, are characterized by the presence of bound iron-sulfur clusters as the terminal electron acceptors. In this work, the iron-sulfur clusters in Chlorobium vibrioforme were studied using selective light-induced reduction protocols, spin quantifications, and chemical redox titrations coupled with EPR detection. Illumination of a dark-frozen sample at 12 K results in the appearance of a spectrum termed signal I. Chemical reduction in darkness at solution potentials between -414 mV and -492 mV results in the appearance of a different spectrum termed signal II. Illumination of these chemically poised samples at 12 K results in the appearance of signal I such that the sum of the intensity of signal I + signal II is nearly constant for every ratio of signal I/signal II. As the solution potential is lowered to -545 mV, the spectrum shifts to yet a third set of resonances, termed signal III. Concomitant with this shift is a loss of low temperature light-induced reduction of signal I. Photoaccumulation of a sample poised at a solution potential of -50 mV results also in the appearance of signal III at nearly the same spin concentration as the chemically reduced sample. Spin quantifications imply that signals I and II are both derived from the reduction of one iron-sulfur cluster, termed center I; signal III is derived from simultaneous reduction of two iron-sulfur clusters, centers I and II. By measuring the EPR signal intensities over a range of solution potentials, centers I and II were shown to have Em (pH 10.0) values of -446 mV and -501 mV, respectively. The observations are consistent with a structural and functional analogy of centers I and II with F(A) and F(B) of PS I.

Anaerobiosis↗

Electron transfer towards the RCI-type photosystem in the green sulphur bacterium Chlorobium limicola forma thiosulphatophilum studied by time-resolved optical spectroscopy in vivo.

Flash-induced spectral changes in the wavelength region of the alpha-peaks of heme proteins and in the time domain from microseconds to seconds have been recorded on whole cells of the green sulphur bacterium Chlorobium limicola forma thiosulfatophilum. Extensive flash-excitation by trains of flashes resulted in oxidation of 7-8 c-type heme molecules/photosynthetic reaction centre. The complement of heme species was found to be spectrally heterogeneous allowing the study of electron transfer events induced by an isolated single-turnover flash. Under single-flash conditions, a c553 heme was seen to become oxidised with tau = 30 micros, concommitant with the reduction of the primary donor of the reaction centre. Subsequently, the alpha-peak of the photooxidised heme broadened and shifted towards longer wavelengths (tau = 70 micros) indicating equilibration of the positive charge over two differing heme species. In the time domain t > 1 ms, rereduction of c-type hemes was seen to be paralleled by a blue shift and further broadening of the alpha-peak. Concommitantly, b-type hemes were observed to first become reduced (within a few milliseconds), then over-oxidised (t > 200 ms) and eventually rereduced to their redox state prior to the flash. The results obtained are discussed with respect to the question of the identity of the immediate electron donor to the photosynthetic reaction centre and with respect to the involvement of a cytochrome bc complex in photo-induced electron transport of green sulphur bacteria.

Chlorobi↗

Site-specific variation in Antarctic marine biofilms established on artificial surfaces.

The community structure and composition of marine microbial biofilms established on glass surfaces was investigated across three differentially contaminated Antarctic sites within McMurdo Sound. Diverse microbial communities were revealed at all sites using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques. Sequencing of excised DGGE bands demonstrated close affiliation with known psychrophiles or undescribed bacteria also recovered from the Antarctic environment. The majority of bacterial sequences were affiliated to the Gammaproteobacteria, Cytophaga/Flavobacteria of Bacteroidetes (CFB), Verrucomicrobia and Planctomycetales. Principal components analysis of quantitative FISH data revealed distinct differences in community composition between sites. Each of the sites were dominated by different bacterial groups: Alphaproteobacteria, Gammaproteobacteria and CFB at the least impacted site, Cape Armitage; green sulfur and sulfate reducing bacteria near the semi-impacted Scott Base and Planctomycetales and sulfate reducing bacteria near the highly impacted McMurdo Station. The highest abundance of archaea was detected near Scott Base (2.5% of total bacteria). Multivariate analyses (non-metric multidimensional scaling and analysis of similarities) of DGGE patterns revealed greater variability in community composition between sites than within sites. This is the first investigation of Antarctic biofilm structure and FISH results suggest that anthropogenic impacts may influence the complex composition of microbial communities.

Actinobacteria↗

Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland).

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime.

Arctic Regions↗

217 000-year-old DNA sequences of green sulfur bacteria in Mediterranean sapropels and their implications for the reconstruction of the paleoenvironment.

Deep-sea sediments of the eastern Mediterranean harbour a series of dark, organic carbon-rich layers, so-called sapropels. Within these layers, the carotenoid isorenieratene was detected. Since it is specific for the obligately anaerobic phototrophic green sulfur bacteria, the presence of isorenieratene may suggest that extended water column anoxia occurred in the ancient Mediterranean Sea during periods of sapropel formation. Only three carotenoids (isorenieratene, beta-isorenieratene and chlorobactene) are typical for green sulfur bacteria and thus do not permit to differentiate between the approximately 80 known phylotypes. In order to reconstruct the paleoecological conditions in more detail, we searched for fossil 16S rRNA gene sequences of green sulfur bacteria employing ancient DNA methodology. 540 bp-long fossil sequences could indeed be amplified from up to 217 000-year-old sapropels. In addition, such sequences were also recovered from carbon-lean intermediate sediment layers deposited during times of an entirely oxic water column. Unexpectedly, however, all the recovered 16S rRNA gene sequences grouped with freshwater or brackish, rather than truly marine, types of green sulfur bacteria. It is therefore feasible that the molecular remains of green sulfur bacteria originated from populations which thrived in adjacent freshwater or estuarine coastal environments rather than from an indigenous pelagic population.

Benzopyrans↗

Phylogeny of the genus Chlorobium based on 16S rDNA sequence.

A comparative analysis of 16S ribosomal DNA sequences from 18 strains belonging to different species of the genus Chlorobium has been made in order to investigate their phylogenetic relationships. All studied strains with brown pigmentation, belonging to species C. phaeobacteroides and C. phaeovibrioides, were clustered together and separated from green species. Most of the C. limicola strains formed a consistent group and were clustered next to C. tepidum. But, despite their identical morphology, C. vibrioforme strain 6030 (= DSM 260T) and C. vibrioforme strain 8327 (= DSM 263) were clearly differentiated on the basis of their 16S rDNA sequence. On the other hand, morphologically distinctive strains of C. chlorovibrioides, C. vibrioforme and C. limicola presented sequences with a high degree of similarity. Since morphological characters do not seem to be sufficient to adequately classify this group, these findings help to set up the basis for a revision of the taxonomy of the genus Chlorobium.

Base Sequence↗

The phylogenetic relationships of Chlorobium tepidum and Chloroflexus aurantiacus based upon their RecA sequences.

Using RecA as the phylogenetic marker, the relationships of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus to other eubacteria were investigated. The recA genes of the two organisms were cloned, and the resulting protein sequences aligned with 86 other eubacterial RecA sequences. Cb. tepidum was placed as the nearest relative to the Cytophaga/Flexibacter/Bacteriodes group, a relationship supported by results obtained with several phylogenetic markers. Cf. aurantiacus was placed near Chlamydia trachomatis and the high-GC Gram-positives; however, this branching pattern was not strongly supported statistically by bootstrap analyses. Possible reasons for this ambiguity are discussed.

Amino Acid Sequence↗

Insertional inactivation studies of the csmA and csmC genes of the green sulfur bacterium Chlorobium vibrioforme 8327: the chlorosome protein CsmA is required for viability but CsmC is dispensable.

Targeted mutagenesis was used to investigate the roles of the CsmA and CsmC proteins of the chlorosomes of the green bacteria Chlorobium tepidum and Chlorobium vibrioforme 8327. Under the photoautotrophic growth conditions employed, CsmA is required for the viability of the cells but CsmC is dispensable. The absence of CsmC caused a small red shift in the near-infrared absorption maximum of bacteriochlorophyll d in whole cells and chlorosomes, but chlorosomes were assembled in and could be isolated from the csmC mutant. The doubling time of the csmC mutant was approximately twice that of the wild-type strain. Fluorescence emission measurements suggested that energy transfer from the bulk bacteriochlorophyll d to another pigment, perhaps bacteriochlorophyll a, emitting at 800-804 nm, was less efficient in the csmC mutant cells than in wild-type cells. These studies establish that transformation and homologous recombination can be employed in targeted mutagenesis of Chlorobium sp. and further demonstrate that chlorosome proteins play important roles in the structure and function of these light-harvesting organelles.

Bacterial Proteins↗

Genes involved in hydrogen and sulfur metabolism in phototrophic sulfur bacteria.

The dsr genes and the hydSL operon are present as separate entities in phototrophic sulfur oxidizers of the genera Allochromatium, Marichromatium, Thiocapsa and Thiocystis and are organized similarly as in Allochromatium vinosum and Thiocapsa roseopersicina, respectively. The dsrA gene, encoding the alpha subunit of 'reverse' siroheme sulfite reductase, is also present in two species of green sulfur bacteria pointing to an important and universal role of this enzyme and probably other proteins encoded in the dsr locus in the oxidation of stored sulfur by phototrophic bacteria. The hupSL genes are uniformly present in the members of the Chromatiaceae family tested. The two genes between hydS and hydL encode a membrane-bound b-type cytochrome and a soluble iron-sulfur protein, respectively, resembling subunits of heterodisulfide reductase from methanogenic archaea. These genes are similar but not identical to dsrM and dsrK, indicating that the derived proteins have distinct functions, the former in hydrogen metabolism and the latter in oxidative sulfur metabolism.

Bacteria↗

Active and energy-dependent rapid formation of cell aggregates in the thermophilic photosynthetic bacterium Chloroflexus aggregans.

The thermophilic filamentous phototroph Chloroflexus aggregans was able to form a bacterial mat-like dense cell aggregate rapidly. The aggregate formation, which was observed in growing cells in a liquid medium in a bottle, occurred every time within 20-30 min after the cells were dispersed by shaking. The aggregation depended on the energy supplied by photosynthesis or respiration. Cells aggregated most rapidly under temperature and pH conditions that support maximum growth. The aggregation was also accelerated by the addition of 3-isobutyl-1-methylxanthine that inhibits cyclic 3',5'-AMP phosphodiesterase. Microscopic observation revealed that the bacterium has a fast gliding mobility (1-3 microm s(-1)). The distinctive cell aggregation of C. aggregans was due to this rapid gliding movement.

1-Methyl-3-isobutylxanthine↗

Structure and organization of a 25 kbp region of the genome of the photosynthetic green sulfur bacterium Chlorobium vibrioforme containing Mg-chelatase encoding genes.

A region comprising approximately 25 kbp of the genome of the strictly anaerobic and obligate photosynthetic green sulfur bacterium Chlorobium vibrioforme has been mapped, subcloned and partly sequenced. Approximately 15 kbp have been sequenced in it's entirety and three genes with significant homology and feature similarity to the bchI, -D and -H genes and the chlI, -D and -H genes of Rhodobacter and Synechocystis strain PCC6803, respectively, which encode magnesium chelatase subunits, have been identified. Magnesium chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX, and is the first enzyme unique to the (bacterio)chlorophyll specific branch of the porphyrin biosynthetic pathway. The organization of the three Mg-chelatase encoding genes is unique to Chlorobium and suggests that the magnesium chelatase of C. vibrioforme is encoded by a single operon. The analyzed 25 kbp region contains five additional open reading frames, two of which display significant homology and feature similarity to genes encoding lipoamide dehydrogenase and genes with function in purine synthesis, and another three display significant homology to open reading frames with unknown function in distantly related bacteria. Putative E. coli sigma 70-like promoter sequences, ribosome binding sequences and rho-independent transcriptional stop signals within the sequenced 15 kbp region are related to the identified genes and orfs. Southern analysis, restriction mapping and partial sequencing of the remaining ca. 10 kbp of the analyzed 25 kbp region have shown that this part includes the hemA, -C, -D and -B genes (MOBERG and AVISSAR 1994), which encode enzymes with function in the early part of the biosynthetic pathway of porphyrins.

Amino Acid Sequence↗

Whole-genome analysis of photosynthetic prokaryotes.

The process of photosynthesis has had profound global-scale effects on Earth; however, its origin and evolution remain enigmatic. Here we report a whole-genome comparison of representatives from all five groups of photosynthetic prokaryotes and show that horizontal gene transfer has been pivotal in their evolution. Excluding a small number of orthologs that show congruent phylogenies, the genomes of these organisms represent mosaics of genes with very different evolutionary histories. We have also analyzed a subset of "photosynthesis-specific" genes that were elucidated through a differential genome comparison. Our results explain incoherencies in previous data-limited phylogenetic analyses of phototrophic bacteria and indicate that the core components of photosynthesis have been subject to lateral transfer.

Bacteria↗

Photic zone euxinia during the Permian-triassic superanoxic event.

Carbon and sulfur isotopic data, together with biomarker and iron speciation analyses of the Hovea-3 core that was drilled in the Perth Basin, Western Australia, indicate that euxinic conditions prevailed in the paleowater column during the Permian-Triassic superanoxic event. Biomarkers diagnostic for anoxygenic photosynthesis by Chlorobiaceae are particularly abundant at the boundary and into the Early Triassic. Similar conditions prevailed in the contemporaneous seas off South China. Our evidence for widespread photiczone euxinic conditions suggests that sulfide toxicity was a driver of the extinction and a factor in the protracted recovery.

Anaerobiosis↗

Biomarker evidence for a major preservation pathway of sedimentary organic carbon.

Hydrogenation processes leading from biomolecules to fossil biomarkers in anoxic sediments are crucial for the preservation of organic matter. However, these processes are still poorly understood. The present identification of several reduced carotenoids in recent sediments attests that these processes operate at the earliest stages of diagenesis without structural or stereochemical specificity, implying a nonbiological reduction pathway. Sulfur species (e.g., H2S) are the hydrogen donors involved in such reduction, as demonstrated with laboratory experiments. These reactions allow the preservation of abundant organic carbon in the rock record.

Anaerobiosis↗

Gram-positive bacteria: possible photosynthetic ancestry.

A 16S ribosomal RNA gene has been sequenced from Heliobacterium chlorum, the recently discovered photosynthetic bacterium that contains a novel form of chlorophyll. Comparisons with other 16S ribosomal RNA sequences show that the organism belongs to the Gram-positive bacteria (one of ten eubacterial "phyla")--more precisely to the so-called low G + C (G, guanine; C, cytosine) subdivision thereof. This brings to five the number of such phyla that contain photosynthetic species, the other four being the purple bacteria and relatives, the green sulfur bacteria, the green nonsulfur bacteria, and the cyanobacteria. The finding suggests that Gram-positive bacteria may be of photosynthetic ancestry, and it strengthens the case for a common photosynthetic ancestry for all eubacteria.

Bacteria↗