Circular dichroism and resonance Raman spectroscopies of bacteriochlorophyll b-containing LH1-RC complexes.

The core light-harvesting complexes (LH1) in bacteriochlorophyll (BChl) b-containing purple phototrophic bacteria are characterized by a near-infrared absorption maximum around 1010 nm. The determinative cause for this ultra-redshift remains unclear. Here, we present results of circular dichroism (CD) and resonance Raman measurements on the purified LH1 complexes in a reaction center-associated form from a mesophilic and a thermophilic Blastochloris species. Both the LH1 complexes displayed purely positive CD signals for their Qy transitions, in contrast to those of BChl a-containing LH1 complexes. This may reflect differences in the conjugation system of the bacteriochlorin between BChl b and BChl a and/or the differences in the pigment organization between the BChl b- and BChl a-containing LH1 complexes. Resonance Raman spectroscopy revealed remarkably large redshifts of the Raman bands for the BChl b C3-acetyl group, indicating unusually strong hydrogen bonds formed with LH1 polypeptides, results that were verified by a published structure. A linear correlation was found between the redshift of the Raman band for the BChl C3-acetyl group and the change in LH1-Qy transition for all native BChl a- and BChl b-containing LH1 complexes examined. The strong hydrogen bonding and π-π interactions between BChl b and nearby aromatic residues in the LH1 polypeptides, along with the CD results, provide crucial insights into the spectral and structural origins for the ultra-redshift of the long-wavelength absorption maximum of BChl b-containing phototrophs.

'Candidatus Oscillochloris fontis': a novel mesophilic phototrophic Chloroflexota bacterium belonging to the ubiquitous Oscillochloris genus.

We present the results of a study of mesophilic anoxygenic phototrophic Chloroflexota bacteria from Mechigmen hot spring (the Chukotka Peninsula) and Siberia. According to 16S rRNA phylogenetic analysis, these bacteria belong to Oscillochloris trichoides. However, sequencing the draft genome of the bacterium from the Chukotka and analysis of the average nucleotide identity, as well as in silico DNA-DNA hybridization, reveal that this bacterium belongs to a novel species within the Oscillochloris genus. We, therefore, propose 'Candidatus Oscillochloris fontis' as a novel taxon to represent this mesophilic alkaliphilic anaerobic anoxygenic phototrophic bacterium. Spectrophotometry and high-performance liquid chromatography analysis show that the bacterium possesses bacteriochlorophylls c and a, as well as lycopene, ß-carotene and γ-carotene. In addition, transmission electron microscopy shows the presence of chlorosomes, polyhydroxyalkanoate- and polyphosphate-like granules. The genome of 'Ca. Oscillochloris fontis' and the Siberian strains of Oscillochloris sp. possess the key genes for nitrogenase complex (nifH) and ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbL), as previously described for O. trichoides DG-6. The results presented here, and previously published data, show that Oscillochloris bacteria from different aquatic environments have the potential for CO2 and N2 fixation. Additionally, we describe a new primer system for the detection of RuBisCo form I.

'Candidatus Viridilinea mediisalina', a novel phototrophic Chloroflexi bacterium from a Siberian soda lake.

In this article, we present the description of a novel mesophilic phototrophic Chloroflexi bacterium, 'Candidatus Viridilinea mediisalina' Kir15-3F. We have isolated an anaerobic, highly enriched culture of this bacterium from the Kiran soda lake (Siberia) and optimized its cultivation. Metagenomic sequencing revealed that 'Ca. Viridilinea mediisalina' Kir15-3F is a bacteriochlorophyll-containing Chloroflexi bacterium in the enrichment culture. Fluorescent in situ hybridisation demonstrated a link between the phenotype described here and the 'Ca. Viridilinea mediisalina' Kir15-3F genome. Spectrophotometry and high-performance liquid chromatography analyses showed the presence of bacteriochlorophylls d, c and a, as well as lycopene, γ-carotene and ß-carotene. Transmission electron microscopy showed chlorosomes, gas vesicles, polyhydroxyalkanoate-like and polyphosphate-like granules. Our results illustrated that 'Ca. Viridilinea mediisalina' Kir15-3F is an alkaliphilic, salt-tolerant, obligately mesophilic, anaerobic, phototrophic bacterium. The genome sequences lack genes of the Calvin cycle and a sulphide:quinone reductase gene for sulphide oxidation. Owing to the lack of an axenic culture and based on the genomic and phenotypic data, we have presented the description of the bacterium in the Candidatus category.

Chromium removal from solution by five photosynthetic bacteria isolates.

Biological method has been recognized as a low-cost and ecofriendly approach for removing heavy metals from aqueous wastes. In this study, the ability of five photosynthetic bacteria isolates (strains labeled SC01, HN02, SC05, JS01, and YN01) was examined for their ability to remove Cr from Cr-containing solutions. Furthermore, the possible removal mechanisms were elucidated by comparing chromium removal rates, antioxidant reaction, and accumulation of reactive oxygen species (ROS). Among the five bacteria, strains SC01 and SC05 presented the highest removal rates of chromium ions and the activity of cysteine desulfhydrase under Cr stress. They also showed lower levels of ROS and cell death than the other three bacteria strains under Cr stress. In addition, total bacteriochlorophyll content and activities of six antioxidant enzymes in SC01 were highest among these selected strains. On the contrary, strain HN02 presented the lowest level of Cr removal and the lowest activities of antioxidant enzymes. It also exhibited the highest level of ROS under Cr(VI) stress. Overall, these results show that the strains SC01 and SC05 have good Cr removal ability and could be used for removal of Cr in industrial effluents.

Bacteriochlorophyll homolog compositions in the bchU mutants of green sulfur bacteria.

Chlorosomes of the green sulfur bacterium Chlorobaculum limnaeum contain a large number of self-aggregated bacteriochlorophyll (BChl) e molecules. The ΔbchU mutant of this organism lacks BchU, a C20-methyltransferase, and therefore produces BChl f, which is the C20-unsubstituted form of BChl e. The BChl e homolog compositions, in terms of degrees of C8(2)-methylation, were not changed in the wild type during growth, while the BChl f homolog patterns in the mutant were significantly altered at various time periods of growth. BChl f with an isobutyl group at the C8 position was dominant at the early stage of growth, whereas the proportion of BChl f with the C8-ethyl group increased in the late exponential phase. We also constructed the ΔbchU mutant of C. tepidum which originally produces BChl c: the mutant therefore produces BChl d. BChl d homologs highly methylated at the C8(2) position also increased in the ΔbchU mutant of C. tedium compared to those in the wild type. These phenomena suggest that BchU interferes with the methylation ability of BchQ, a C8(2)-methyltransferase, and that the enzymes might compete in terms of obtaining S-adenosyl-methionine, the source of a methyl group. As a result, when grown to the late log phase, the ΔbchU mutant of C. limnaeum had similar heterogeneities of pigment homolog compositions compared to those in the wild type. Chlorosomes with a high proportion of C8-ethylated BChl homologs might be important for fine-tuning the light-harvesting or energy-transfer efficiency. Chlorosomes of the ΔbchU mutants at the various growth stages will be good materials for investigating effects of C8(2)-methylations on supramolecular structures of self-aggregated pigments.

Marichromatium bheemlicum sp. nov., a non-diazotrophic, photosynthetic gammaproteobacterium from a marine aquaculture pond.

A rod-shaped, phototrophic, purple sulfur bacterium, strain JA124(T), was isolated in pure culture from a marine aquaculture pond, located near Bhimunipatnam, in a medium that contained 3 % NaCl (w/v). Strain JA124(T) is a Gram-negative, motile rod with a single polar flagellum. Strain JA124(T) has a requirement for NaCl, with optimum growth at 1.5-8.5 %, and tolerates up to 11 % NaCl. Intracellular photosynthetic membranes are of the vesicular type. Bacteriochlorophyll a and probably carotenoids of the spirilloxanthin series are present as photosynthetic pigments. Strain JA124(T) was able to utilize sulfide, sulfate, thiosulfate, sulfite, thioglycollate and cysteine as sulfur sources. Strain JA124(T) was able to grow photolithoautotrophically, photolithoheterotrophically and photo-organoheterotrophically. Chemotrophic and fermentative growth could not be demonstrated. Strain JA124(T) lacks diazotrophic growth and acetylene reduction activity. Pyridoxal phosphate is required for growth. During growth on reduced sulfur sources as electron donors, sulfur is deposited intermediately as a number of small granules within the cell. Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that strain JA124(T) clusters with species of the genus Marichromatium belonging to the class Gammaproteobacteria. The highest sequence similarities of strain JA124(T) were found with the type strains of Marichromatium indicum (98 %), Marichromatium purpuratum (95 %) and Marichromatium gracile (93 %). However, DNA-DNA hybridization with Marichromatium indicum DSM 15907(T) revealed relatedness of only 65 % with strain JA124(T). The DNA base composition of strain JA124(T) was 67 mol% G+C (by HPLC). Based on 16S rRNA gene sequence analysis, morphological and physiological characteristics and DNA-DNA hybridization studies, strain JA124(T) (=ATCC BAA-1316(T)=JCM 13911(T)) is sufficiently different from other Marichromatium species to merit its description as the type strain of a novel species, Marichromatium bheemlicum sp. nov.

Characterization of the chlorosome antenna of the filamentous anoxygenic phototrophic bacterium Chloronema sp. strain UdG9001.

The absorption and fluorescence properties of chlorosomes of the filamentous anoxygenic phototrophic bacterium Chloronema sp. strain UdG9001 were analyzed. The chlorosome antenna of Chloronema consists of bacteriochlorophyll (BChl) d and BChl c together with gamma-carotene as the main carotenoid. HPLC analysis combined with APCI LC-MS/MS showed that the chlorosomal BChls comprise a highly diverse array of homologues that differ in both the degree of alkylation of the macrocycle at C-8 and/or C-12 and the alcohol moiety esterified to the propionic acid group at C-17. BChl c and BChl d from Chloronema were mainly esterified with geranylgeraniol (33% of the total), heptadecanol (24%), octadecenol (19%), octadecanol (14%), and hexadecenol (9%). Despite this pigment heterogeneity, fluorescence emission of the chlorosomes showed a single peak centered at 765 nm upon excitation at wavelengths ranging from 710 to 740 nm. This single emission, assigned to BChl c, indicates an energy transfer from BChl d to BChl c within the same chlorosome. Likewise, incubation of chlorosomes under reducing conditions caused a weak increase in fluorescence emission, which indicates a small redox-dependent fluorescence. Finally, protein analysis of Chloronema chlorosomes using SDS-PAGE and MALDI-TOF-MS revealed the presence of a chlorosomal polypeptide with a molecular mass of 5.7 kDa, resembling the CsmA protein found in Chloroflexus aurantiacus and Chlorobium tepidum chlorosomes. Several minor polypeptides were also detected but not identified. These results indicate that, compared with other members of filamentous anoxygenic phototrophic bacteria and green sulfur bacteria, Chloronema possesses an antenna system with novel features that may be of interest for further investigations.

A comparative study of bchG from green photosynthetic bacteria.

The gene bchG, coding for bacteriochlorophyll a synthase from a variety of green sulfur bacteria and the filamentous anoxygenic phototrophic bacteria, Chloroflexus aurantiacus, Chloronema sp., and Roseiflexus castenholzii HL08, was partially sequenced and compared. The deduced amino acid consensus sequences for green sulfur bacteria and green filamentous anoxygenic phototrophic bacteria were found to belong to the UbiA enzyme family of polyprenyltransferases with the most similar sequences being those of photosynthetic organisms. All deduced amino acid sequences showed a highly conserved region, which includes the motif DRXXD, characteristic of polyprenyltransferases, which was extended to DREVDAINEP for green sulfur bacteria. Neighbor-joining analysis of a protein similitude matrix displayed a relatively high distance between green sulfur bacteria and the other groups. Sequences from green sulfur bacteria were more closely related to those of purple bacteria than to those of filamentous anoxygenic phototrophic bacteria. In addition, internal grouping within green sulfur bacteria was congruent regarding taxonomic features including cell shape, presence of gas vacuoles and NaCl requirement. In addition to bchlG, another gene encoding for a second chlorophyll synthetase, previously tentatively identified as chlG, was also found in Chlorobium tepidum, showing the highest similarities with polyprenyltransferases from chlorophyll- a-containing organisms.

The light-harvesting complex II (B800-850) of Rhodobacter sulfidophilus: characterization and formation under different growth conditions.

The photosynthetic bacterium Rhodobacter sulfidophilus is able to grow chemotrophically and phototrophically at a broad range of light intensities. In contrast to other facultative phototrophs, R. sulfidophilus synthesizes reaction center and light-harvesting (LH) complexes, B870 (LHI) and B800-850 (LHII) even under full aerobic conditions in the dark. The content of bacteriochlorophyll (BChl) varied from 3.8 micrograms Bchl per mg cell protein when grown at high light intensity (20,000 lux) to 60 micrograms Bchl per mg cell protein when grown at low light intensities (6 lux). After a shift from high light to low light conditions, the size of the photosynthetic unit increased by a factor of 4. Chromatographic analysis of the LHII complex, isolated and purified from cells grown phototrophically (at high and low light intensities) and chemotrophically, could resolve only one type of alpha and one type of beta polypeptide in the purified complex, of which the N-terminal sequences have been determined.

Effects of light, oxygen, and substrates on steady-state levels of mRNA coding for ribulose-1,5-bisphosphate carboxylase and light-harvesting and reaction center polypeptides in Rhodopseudomonas sphaeroides.

The mRNA levels specific for ribulose-1,5-bisphosphate carboxylase, light-harvesting I polypeptides alpha and beta, and reaction center polypeptides L and M were assayed by use of a series of DNA probes specific for each cognate mRNA. Both the steady-state amounts and sizes of the specific mRNAs were measured as a function of the light intensity incident to the culture, the presence or absence of oxygen, and the type of substrate present in the growth medium. Northern hybridization revealed at least two and possibly three transcripts for ribulose-1,5-bisphosphate carboxylase. The cellular level of mRNA specific for ribulose-1,5-bisphosphate carboxylase increased in consort with enzyme activity as a function of both light intensity and reducing state of the substrate. Neither mRNA nor enzyme activity was detectable in aerobically grown cells. For the light-harvesting I and reaction center polypeptides there exist two transcripts, the larger of which appears to be a polycistronic mRNA possessing information for all four polypeptides and a smaller transcript specific for only the alpha and beta polypeptides of the light-harvesting I complex. The regulation of each of these mRNAs was affected by light and oxygen, but was not significantly affected by the oxidation-reduction state of the substrate.

Molecular-organization and biosynthesis of pigment-protein complexes of Rhodopseudomonas capsulata.

The photosynthetic apparatus of the facultative phototrophic bacterium Rhodopseudomonas capsulata contains three bacteriochlorophyll-carotenoid-protein complexes: the reaction center and the light-harvesting (LH) antenna complexes LHI (B870) and LHII (B800--850). In contrast to green anoxygenic phototrophic bacteria and the oxygenic cyanobacteria, the light-harvesting complexes of Rhodospirillaceae and Chromatiaceae are integral membrane particles. Variations in light fluxes induce membrane differentiation mainly expressed as variations in the size of the photosynthetic unit and in the area of intracytoplasmic membrane per cell. The B800--850 complex is the variable part of the photosynthetic apparatus. Synthesis of bacteriochlorophyll and of the polypeptides of the pigment complexes was found to be strongly coordinated. The synthesis of these polypeptides was followed immediately by the assembly of the complexes in the membrane. Bacteriochlorophyll or a signal substance triggered by bacteriochlorophyll synthesis regulated the synthesis of these polypeptides at the level of translation. The pigment-binding subunits of the B800--850 complex form oligomeric structures which interact with subunit H of the reaction center. A model of the topographical relationships of the pigment complexes is discussed.

Comparative studies of protein properties and bacteriochlorophyll contents of bacteriochlorophyll-protein complexes from spectrally different types of Rhodopseudomonas palustris.

The amino acid compositions, constituent polypeptides and bacteriochlorophyll (Bchl) contents of two kinds of Bchl-protein complexes isolated from Rhodopseudomonas palustris were examined. Spectrally dissimilar intracytoplasmic membranes obtained from cells cultured under different conditions were used as starting materials. The B870-reaction center complex was consistent in its amino acid composition, constituent polypeptides and Bchl content, as well as in its near-infrared absorption spectrum. B800-850 complexes from the different types of intracytoplasmic membrane varied in their absorption spectra, though they had similar amino acid compositions and were comprised of basically similar kinds of polypeptides with variations only in the levels of some minor constituent polypeptides. The B800-850 complex with a low absorption peak at 850 nm had a Bchl content 1.3 times greater than the B800-850 complex with a high absorption peak at 850 nm. These results indicate that the B800-850 complex from R. palustris contains more components (both polypeptides and Bchl molecules) than the B800-850 complexes from Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides.

Development and growth of photosynthetic membranes of Rhodospirillum rubrum.

In cell-free extracts from low-aeration suspensions of Rhodospirillum rubrum strain G-9, bacteriochlorophyll a was distributed in two bands after rate-zone sedimentation in sucrose density gradients. From the physicochemical properties of these fractions, it was concluded that the upper band consisted of small membrane fragments, whereas the major band was composed of fragmented vesicular intracytoplasmic membrane (chromatophores). After a pulse with L-[35S]methionine, apparent polypeptide subunits of the reaction center and light-harvesting complexes within the upper pigmented fraction were labeled more rapidly than those of chromatophores; after a chase with excess unlabeled L-methionine, radioactivity from these components within the upper band appeared to be chased into the corresponding polypeptides of chromatophores. These labeling patterns are interpreted to reflect growth initiation and maturation of the photosynthetic apparatus and may, in part, represent a general mechanism for the development of vesicular intracytoplasmic membranes.

Isolation and characterization of cytoplasmic membranes and chlorosomes from the green bacterium Chloroflexus aurantiacus.

A method was developed which allows the isolation and purification of cytoplasmic membranes and chlorosomes from cells of Chloroflexus aurantiacus grown under different light conditions. The dipolar ionic detergent Deriphat (0.08%) and a sodium iodide gradient centrifugation were used in isolating cytoplasmic membranes. Chlorosomes were prepared with 0.16% of the dipolar ionic detergent Miranol and purified by a sucrose gradient centrifugation. Cytoplasmic membrane fractions prepared from either high- (3,000 W m-2), medium-(200 W m-2) or low- (7 W m-2) light-grown cells had near infrared absorption bands at 866, 808, and 755 nm in a constant characteristic absorbance ratio of 6:3.8:1. In all cytoplasmic membrane preparations, the amount of bacteriochlorophyll a (Bchl a) per cytochrome, the amount of Bchl a per reaction center, and reaction center per milligram of cytoplasmic membrane protein was found to be constant. No Bchl c was present. Five respiratory enzyme activities have been measured in the cytoplasmic membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured cytoplasmic membrane showed many bands, but a major polypeptide with an apparent molecular weight of 8,000. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified chlorosomes did not contain the 8,000-molecular-weight band but revealed only three distinct protein bands with molecular weights of 15,000, 12,000, and 6,000. Isolated chlorosomes contained Bchl c and a small, yet constant, amount of Bchl a (absorbing at 790 nm) in a molar ratio of 25:1. The data indicated that the components of the photosynthetic apparatus in the cytoplasmic membrane of Chloroflexus aurantiacus remained constant and only the amount of antenna Bchl c varied with light conditions.

The transport of microbial biomass and suspended material in a high-marsh creek.

The transport of microbial biomass and suspended material in a high-marsh creek was investigated during four 40-h tidal studies throughout the year. Although considerable differences were noted between successive tidal cycles, overall the creek was found to be an exporting system and transported a mean concentration of ATP (-33g), chlorophyll a (-66g), particulate organic carbon (-31kg), total suspended material (-344kg), and fixed suspended material (-195kg) during each tidal cycle. This net outward flux of materials was associated with a net flow of water out of creek, while the net import of aerobic, heterotrophic bacteria (43 x 10(12)) and volatile suspended material (238kg) was generally due to higher mean concentrations of these materials per unit volume of water during the flooding tide. Also the latter generally were associated with increased amounts of suspended material suggesting an association between bacteria and suspended matter.

Membranes of Rhodospirillum rubrum: isolation and physicochemical properties of membranes from aerobically grown cells.

Highly purified preparations of cytoplasmic and outer membrane were isolated from aerobically grown Rhodospirillum rubrum lysed by sequential treatment with lysozyme, ethylenediaminetetraacetate, and Brij 58. The membranes were resolved and separated from other cellular constitutents by a combination of velocity and isopyknic sedimentation in sucrose density gradients. On the basis of their appearance in electron micrographs and their protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these preparations appear to be quite similar to those obtained from other gram-negative bacteria. The cytoplasmic membrane fraction contained the majority of the total membrane-bound succinic dehydrogenase activity and was 10-fold enriched in b- and c-type cytochrome with respect to the outer membrane. The latter fraction was characterized by a much greater carbohydrate content and the presence of arachidic acid, which is typical of R. rubrum lipopolysaccharide. Their protein fatty acid, and overall chemical compositions suggested that these preparations were freer from cross-contamination than those obtained from R. rubrum with currently available methods.