Katsumi Matsuura

Roseiflexus castenholzii gen. nov., sp. nov., a thermophilic, filamentous, photosynthetic bacterium that lacks chlorosomes

A novel thermophilic, photosynthetic bacterium, designated strain HLO8T, was isolated from a bacterial mat in a Japanese hot spring. Morphologically, the isolate was an unbranched multicellular filament with a cell diameter of 0.8-1.0 microm. The bacterium was red to reddish-brown in colour and formed a distinct red bacterial mat in the natural environment. It was able to grow photoheterotrophically under anaerobic light conditions and also chemoheterotrophically under aerobic dark conditions. Optimal growth occurred at 50 degrees C and pH 7.5-8.0. The cells contained bacteriochlorophyll (Bchl) a and gamma-carotene derivatives as photosynthetic pigments, but lacked Bchl c and chlorosomes. Cellular fatty acids in the isolate were mainly C16:0, C14:0 and C15:0. The major quinone was menaquinone-11. The DNA G+C content was 62.0 mol% (by HPLC). Phylogenetic analysis based on 16S rDNA sequencing suggested that the isolate belonged to the anoxygenic filamentous phototrophic bacteria represented by Chloroflexus aurantiacus, but was clearly distant from all members in this group (the sequence similarities between the isolate and its relatives were less than 83.8%). Based on genotypic and phenotypic data, the name Roseiflexus castenholzii gen. nov., sp. nov. is proposed for this isolate; the type strain is HLO8T (= DSM 13941T = JCM 11240T).

HORIZONTAL TRANSFER OF GENES CODING FOR THE PHOTOSYNTHETIC REACTION CENTERS OF PURPLE BACTERIA

Phylogenetic trees were drawn and analyzed based on the nucleotide sequences of the 1.5-kb gene fragment coding for the L and M subunits of the photochemical reaction center of various purple photo-synthetic bacteria. These trees are mostly consistent with phylogenetic trees based on 16S rRNA and soluble cy-tochrome c, but differ in some significant details. This inconsistency implies horizontal transfer of the genes that code for the photosynthetic apparatus in purple bacteria. Possibilities of similar transfers of photosynthesis genes during the evolution of photosynthesis are discussed especially for the establishment of oxygenic photosynthesis.

Horizontal Transfer of the Photosynthesis Gene Cluster and Operon Rearrangement in Purple Bacteria

Abstract. A 37-kb photosynthesis gene cluster was sequenced in a photosynthetic bacterium belonging to the β subclass of purple bacteria (Proteobacteria), Rubrivivax gelatinosus. The cluster contained 12 bacteriochlorophyll biosynthesis genes (bch), 7 carotenoid biosynthesis genes (crt), structural genes for photosynthetic apparatuses (puf and puh), and some other related genes. The gene arrangement was markedly different from those of other purple photosynthetic bacteria, while two superoperonal structures, crtEF–bchCXYZ–puf and bchFNBHLM–lhaA–puhA, were conserved. Molecular phylogenetic analyses of these photosynthesis genes showed that the photosynthesis gene cluster of Rvi. gelatinosus was originated from those of the species belonging to the α subclass of purple bacteria. It was concluded that a horizontal transfer of the photosynthesis gene cluster from an ancestral species belonging to the α subclass to that of the β subclass of purple bacteria had occurred and was followed by rearrangements of the operons in this cluster.

Quinones in chlorosomes of green sulfur bacteria and their role in the redox-dependent fluorescence studied in chlorosome-like bacteriochlorophyll c aggregates

Abstract The light-harvesting chlorosome antennae of anaerobic, photosynthetic green sulfur bacteria exhibit a highly redox-dependent fluorescence such that the fluorescence intensity decreases under oxidizing conditions. We found that chlorosomes from Chlorobium tepidum contain three isoprenoid quinone species (chlorobiumquinone, menaquinone-7, and an unidentified quinone that probably is a chlorobiumquinone derivative) at a total concentration of approximately 0.1 mol per mol bacteriochlorophyll c. Most of the cellular chlorobiumquinone was found in the chlorosomes and constituted about 70% of the total chlorosome quinone pool. When the quinones were added to artificial, chlorosome-like bacteriochlorophyll c aggregates in an aqueous solution, a high redox dependency of the fluorescence was observed. Chlorobiumquinones were most effective in this respect. A lesser redox dependency of the fluorescence was still observed in the absence of quinones, probably due to another unidentified redox-active component. These results suggest that quinones play a significant, but not exclusive role in controlling the fluorescence and in inhibiting energy transfer in chlorosomes under oxic conditions. Chlorosomes from Chloroflexus aurantiacus contained menaquinone in an amount similar to that of total quinone in Chlorobium tepdium chlorosomes, but did not contain chlorobiumquinones. This may explain the much lower redox-dependent fluorescence observed in Chloroflexus chlorosomes.

Chloroflexus aggregans sp. nov., a Filamentous Phototrophic Bacterium Which Forms Dense Cell Aggregates by Active Gliding Movement

Two strains of thermophilic photosynthetic bacteria, designated MD-66T (T = type strain) and YI-9, were isolated from bacterial mats in two separate hot springs in Japan. These new isolates were phenotypically similar to Chloroflexus aurantiacus in some respects. They were thermophilic filamentous photosynthetic bacteria that grew well at 55 degrees C either anaerobically as photoheterotrophs or aerobically as chemoheterotrophs. They exhibited gliding motility, produced bacteriochlorophylls a and c, contained chlorosomes, and required thiamine and folic acid as growth factors. However, isolates MD-66T and YI-9 had the ability to rapidly form mat-like dense aggregates of filaments, an ability which has not been observed in any C. aurantiacus strain. Carbon source utilization tests revealed that unlike C. aurantiacus, the new isolates did not utilize acetate, citrate, ethanol, or glycylglycine. An analysis of the carotenoid components revealed that isolates MD-66T and YI-9 contained mainly gamma-carotene and OH-gamma-carotene glucoside fatty acid esters. These isolates also contained only trace amounts of beta-carotene, which is a major carotenoid component (28.4% of the total carotenoids) in C. aurantiacus. The results of DNA hybridization studies suggested that the new strains were genetically distinct from C. aurantiacus (levels of similarity, 9 to 18%), and 16S rRNA sequence comparisons showed that strain MD-66T was related to C. aurantiacus at a similarity level of 92.8%. On the basis of our data, we propose that a new Chloroflexus species should be created for our new isolates; the name of this new species is Chloroflexus aggregans, and the type strain is strain MD-66 (= DSM 9485).

Phylogeny and photosynthetic features of Thiobacillus acidophilus and related acidophilic bacteria : its transfer to the genus Acidiphilium as Acidiphilium acidophilum comb. nov

Phylogenetic analyses based on 16S rDNA sequences and genomic DNA-DNA relatedness showed that the sulphur-oxidizing facultative chemolithotroph Thiobacillus acidophilus was closely related to members of the genus Acidiphilium, which is a group of strictly aerobic, heterotrophic acidophiles now categorized into aerobic photosynthetic bacteria. Lipophilic pigment analyses revealed that zinc-chelated bacteriochlorophyll a and carotenoids occurred in appreciable amounts in T. acidophilus and all established species of the genus Acidiphilium. PCR experiments showed that T. acidophilus as well as Acidiphilium species contained puf genes, encoding the photosynthetic reaction centre proteins and the core light-harvesting complex of the purple bacteria. There were high similarities between T. acidophilus and Acidiphilium species in the primary structure of their reaction centre proteins deduced from the nucleotide sequence data. The phylogenetic tree of the reaction centre proteins was in agreement with the 16S rDNA sequence-based phylogenetic tree in the relationship between T. acidophilus and Acidiphilium species and between the Acidiphilium cluster and other purple photosynthetic bacteria. Based on these results, together with previous phylogenetic and phenotypic information, it is proposed to reclassify T. acidophilus (Guay and Silver) Harrison 1983 as Acidiphilium acidophilum comb. nov. The type strain is ATCC 27807T (= DSM 700T).

SPECTRAL FORMS AND ORIENTATION OF BACTERIOCHLOROPHYLLS c AND α IN CHLOROSOMES OF THE GREEN PHOTOSYNTHETIC BACTERIUM Chloroflexus aurantiacus

Spectral forms of bacteriochlorophyll (Bchl) in chlorosomes were analyzed by linear dichroism, circular dichroism (CD), and deconvolution of these spectra. Isolated chlorosomes were embedded in polyacrylamide gels and compressed unidirectionally (along the x‐axis) while allowing the gel to stretch in another direction (along the z‐axis). The chlorosomes were aligned three‐dimensionally due to their flat oblong shape; the longest axis was presumed to parallel the z‐axis, its shortest axis was presumed to parallel the x‐axis, and the intermediate‐length axis was presumed to parallel the y‐axis. Degrees of polarization (AI− A1)/(AI+ A1) of Bchl c and a measured from the y‐axis with linearly polarized light were significantly different from those measured from the x‐axis. Deconvolution of spectra into components revealed the presence of two major forms of Bchl c with peaks at 744 nm and 727 nm. The degrees of polarization of the 744 and 727 nm spectral forms were 0.76 and 0.59 from the y‐axis and 0.48 and 0.39 from the x‐axis, respectively. The degrees of polarization of Bchl a794 were –0.21 from the y‐axis and 0.12 from the x‐axis. These values indicate that the direction of the Qy transition moment of Bchl c744 is almost completely parallel to the longest axis of chlorosomes and that of Bchl c727 is also nearly, but slightly less so, parallel to the longest axis of the chlorosomes. The Qy transition moment of the baseplate Bchl a peak at 794 nm is nearly perpendicular to the longest axis and parallel to the shortest axis: that is, it is perpendicular to the associated membrane plane in the cell. These alignments of Bchl transition moments in chlorosomes were lost after suspending the chlorosomes in a solution saturated with 1‐hexanol accompanying a shift in the peak position from 742 nm to 670 nm. The alignment recovered after the hexanol concentration was decreased. The presence of two major spectral forms of Bchl c was supported by the deconvolution of CD spectra and absorption spectra.

High degree of organization of bacteriochlorophyll c in chlorosome-like aggregates spontaneously assembled in aqueous solution

Pigment-lipid aggregates were formed in aqueous solution by diluting a chloroform/methanoll extract of chlorosomes of the green photosynthetic bacterium,3Chlorobium limicola. The aggregates showed absorption and fluorescence spectra very similar to those of intact chlorosomes. No proteins were detected in the aggregates. Electron micrographs showed that the pigment-lipid aggregates were ellipsoidal bodies with average size of 130 nm along the long axis and 86 nm along the short axis. The linear dichroism spectrum of bacteriochlorophyll c in the pigment-lipid aggregates oriented in a stretched polyacrylamide gel was as strong as that in chlorosomes. These results suggest that spontaneous assembly of the protein-free pigments and lipids extracted from chlorosomes restores not only direct chromophore-chromophore interactions of bacteriochlorophyll c molecules but also the chlorosome-like higher-order structures.

Association of bacteriochlorophyll a with the CsmA protein in chlorosomes of the photosynthetic green filamentous bacterium Chloroflexus aurantiacus

The protein assumed to be associated with bacteriochlorophyll (BChl) a in chlorosomes from the photosynthetic green filamentous bacterium Chloroflexus aurantiacus was investigated by alkaline treatment, proteolytic digestion and a new treatment using 1-hexanol, sodium cholate and Triton X-100. Upon alkaline treatment, only the 5.7 kDa CsmA protein was removed from the chlorosomes among six proteins detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, concomitantly with the disappearance of BChl a absorption at 795 nm. Trypsin treatment removed two proteins with molecular masses of 11 and 18 kDa (CsmN and CmsM), whereas the spectral properties of BChl a and BChl c were not changed. By the new hexanol-detergent (HD) treatment, most BChl c and all of the detected proteins except CsmA were removed from the chlorosomes without changing the BChl a spectral properties. Subsequent proteinase K treatment of these HD-treated chlorosomes caused digestion of CsmA and a simultaneous decrease of the BChl a absorption band. Based on these results, we suggest that CsmA is associated with BChl a in the chlorosomes. This suggestion was supported by the measured stoichiometric ratio of BChl a to CsmA in isolated chlorosomes, which was estimated to be between 1.2 and 2.7 by amino acid analysis of the SDS-PAGE-resolved protein bands.

Porphyrobacter tepidarius sp. nov., a moderately thermophilic aerobic photosynthetic bacterium isolated from a hot spring.

A new thermophilic bacterium, strain OT3T (T = type strain), was isolated from a brackish hot spring. Strain OT3T is an obligate aerobe that synthesizes bacteriochlorophyll a and has a photosynthetic apparatus. This isolate is a thermophilic bacterium with an optimal growth temperature of 40 to 48 degrees C. The cells are nonmotile, ovoid to short rods. An analysis of 16S rRNA sequences revealed that the new strain forms a coherent cluster with members of the alpha-4 group of the alpha subclass of the Proteobacteria, which contains the genera Erythrobacter, Erythromicrobium, and Porphyrobacter. The closest relative is Porphyrobacter neustonensis, with a 16S rRNA sequence similarity of 96.8%. The in vivo absorption spectrum has maxima at 460, 494, 596, 800, and 870 nm. The main carotenoids are OH-beta-carotene sulfate derivatives, nostoxanthin, and bacteriorubixanthinal. Growth occurs with glucose, acetate, glutamate, butyrate, Casamino Acids, and yeast extract as sole energy sources. The pigment composition and nutritional profile of the new isolate are similar to the pigment composition and nutritional profile of P. neustonensis. Although there are marked differences in cell morphology between the new isolate and the budding bacterium P. neustonensis, the results of phenotypic and genetic comparisons suggest that the new isolate is closely related to P. neustonensis. Consequently, we assign the new isolate to the genus Porphyrobacter and propose the name Porphyrobacter tepidarius sp. nov. for it; the type strain of P. tepidarius is strain OT3 (= DSM 10595).