30] Purification and properties of cytochrome c-555 from phototrophic green sulfur bacteria

A green phototrophic bacterium was enriched with ferrous iron as sole electron donor and was isolated in defined coculture with a spirilloid chemoheterotrophic bacterium. The coculture oxidized ferrous iron to ferric iron with stoichiometric formation of cell mass from carbon dioxide. Sulfide, thiosulfate, or elemental sulfur was not used as electron donor in the light. Hydrogen or acetate in the presence of ferrous iron increased the cell yield of the phototrophic partner, and hydrogen could also be used as sole electron source. Complexed ferric iron was slowly reduced to ferrous iron in the dark, with hydrogen as electron source. Similar to Chlorobium limicola, the phototrophic bacterium contained bacteriochlorophyll c and chlorobactene as photosynthetic pigments, and also resembled representatives of this species morphologically. On the basis of 16S rRNA sequence comparisons, this organism clusters with Chlorobium, Prosthecochloris, and Pelodictyon species within the green sulfur bacteria phylum. Since the phototrophic partner in the coculture KoFox is only moderately related to the other members of the cluster, it is proposed as a new species, Chlorobium ferrooxidans. The chemoheterotrophic partner bacterium, strain KoFum, was isolated in pure culture with fumarate as sole substrate. The strain was identified as a member of the epsilon-subclass of the Proteobacteria closely related to "Geospirillum arsenophilum" on the basis of physiological properties and 16S rRNA sequence comparison. The "Geospirillum" strain was present in the coculture only in low numbers. It fermented fumarate, aspartate, malate, or pyruvate to acetate, succinate, and carbon dioxide, and could reduce nitrate to dinitrogen gas. It was not involved in ferrous iron oxidation but possibly provided a thus far unidentified growth factor to the phototrophic partner.

A novel selective enrichment method is described for phototrophic green sulfur bacteria even in the presence of purple sulfur and purple nonsulfur bacteria using sulfanilate, which was discovered during efforts to selectively isolate sulfanilate-metabolizing anoxygenic phototrophic bacteria from marine habitats. Samples for these experiments were obtained from beaches, saltpans, subsurface mangrove soils, fish and prawn aquaculture ponds and backwaters of the East and West coasts of India. Photoorganoheterotrophic and photolithoautotrophic enrichments in the absence of sulfanilate predominantly yielded purple bacterial enrichments. In contrast, photolithoautotrophic enrichments in the presence of sulfanilate yielded green-colored enrichments from the same samples. Whole cell absorption spectra of the enrichment cultures revealed the presence of bacteriochlorophyll c and thus green phototrophic bacteria. Microscopic observation demonstrated the presence of sulfur globules outside the bacterial cells and the presence of non-motile cells, some of which had prosthecae. 16S rDNA sequences obtained from green sulfur bacterial strains isolated from enrichment cultures confirmed the presence of representatives of the green sulfur bacterial genera Prosthecochloris and Chlorobaculum. The selective pressure of sulfanilate exerted through inhibition of phototrophic purple sulfur bacteria was demonstrated by inhibition studies using the purple sulfur bacteria Marichromatium indicum JA100 and Marichromatium sp. JA120 (JCM 13533) and the green sulfur bacterium Prosthecochloris sp. JAGS6 (JCM 13299).

Here we examine the basic biology of three major groups of “green” anoxygenic phototrophic bacteria: the green sulfur bacteria (Chlorobiaceae), the green nonsulfur bacteria (also called the filamentous anoxygenic phototrophs) (Chloroflexaceae), and the heliobacteria (Heliobacteriaceae). Only organisms that have been grown in laboratory culture are considered. Interestingly, the model organisms for each family are thermophiles: the green sulfur bacterium Chlorobaculum tepidum, the filamentous green nonsulfur bacterium Chloroflexus aurantiacus, and the hot spring heliobacterium species, Heliobacterium modesticaldum. All model green bacteria have had their genomes sequenced, and in the green sulfur bacteria, genome sequences of all recognized species have been completed and compared. Although species in each family are distinct from species in each of the other families in many ways, there are key properties that unite two families to the exclusion of the third. These include the presence of chlorosomes in the green sulfur and green nonsulfur bacteria and the structure of the reaction centers in the green sulfur bacteria and heliobacteria. However, the three families of green-colored bacteria are phylogenetically distinct and thus any similarities are likely the result of horizontal gene transfers.

In order to compare and contrast the structure and function of the light-harvesting antennae (i.e. chlorosomes) of green bacteria, a procedure for isolating and characterizing them from green sulfur bacteria was developed. The chlorosomes from Chlorobium species with bacteriochlorophyll (Bchl) c or e were isolated by a two step sucrose density centrifugation in the presence of 2% miranol, a mild detergent, and 2 M sodium thiocyanate (NaSCN). Purified chlorosomes from two green sulfur bacteria, Chlorobium phaeobacteroides and Chlorobium tepidum, and the filamentous green bacterium Chloroflexus aurantiacus were analysed by spectrophotometry, SDS-polyacrylamide gel electrophoresis, and immunological procedures. Isolated chlorosomes from both Chlorobium species contain only two electrophoretically separable protein components with approximate molecular masses of 5–7.5 and 34.5 kDa. In addition, they have a major light-harvesting antenna pigment (Bchl c or e), a minor Bchl a species, and carotenoids. Chloroflexus aurantiacus antisera for the three major chlorosome proteins (5.6, 11, and 18 kDa), and the reaction center proteins (24 and 24.5 kDa) did not cross react with any Chlorobium proteins analyzed in this study. Chlorobium limicola f. thiosulfatophilum antisera against the 7.5 kDa chlorosome protein cross reacted strongly with the 5–7.5 kDa protein from Cb. tepidum, weakly with the Cb. phaeobacteroides protein, but not at all to the 5.6 kDa chlorosome protein from Cf. aurantiacus. These results provide further evidence for the evolutionary divergence of the chlorosomes from green phototrophic bacteria (e.g., Chlorobium-type and Chloroflexus-type).

SummaryThe only common feature of the two families of green bacteria, Chlorobiaceae (green sulfur bacteria) and Chloroflexaceae (green gliding bacteria), is their type of light harvesting chlorophyll and the organization of these pigments into chlorosomes. In most other respects, including metabolism, photosynthetic apparatus and phylogeny, they are very different and far apart. Each of the two most studied genera possesses a unique pathway for autotrophic fixation of CO2: the reductive tricarboxylic acid cycle used by Chlorobium and the newly discovered 3-hydroxypropionate cycle used by Chloroflexus.The two pathways are described in detail and the experimental evidence for their existence is given. Likewise are the experimental approaches used to elucidate these pathways discussed and evaluated. The biochemical reactions participating in the pathways are considered in relation to the nature of the photosynthetic apparatus of the organisms and their ecology.The current knowledge of the metabolism of organic compounds in Chlorobium and Chloroflexus is reviewed, and the accumulation of glycogen and its breakdown in Chlorobium is discussed in detail.Assuming that life originated in a primordial sea of abiotically produced organic molecules, and taking into account phylogenetic relationships based on 16S rRNA analysis, the possible development by retroevolution of the metabolic pathways used by the green sulfur bacteria and the green gliding bacteria is discussed.

The structure of the communities present in both Lake Ciso and the microbial mats at the Ebro Delta, in Catalonia, Spain, as well as their changes with time and depth, were studied following a variety of approaches including taxonomic and physiological analyses, trophic state, population dynamics and structure of the food web, and competition among bacteria. Habitats with either an excess or a limitation of some natural condition harbour different kinds of microbial assemblages, which can be regarded as a means for self-regulation of the participating populations. Assemblages of phototrophic bacteria (cyanobacteria and purple and green sulphur bacteria) are among the most stable and balanced microbial communities. The population structure of a phototrophic community in Lake Ciso is determined by light penetration and by the abundance of sulphide. These two environmental factors are rarely found together in present day aquatic systems, except in habitats such as the metalimnion of some lakes (as in the case of Lake Ciso) and shallow water marine sediments (microbial mats). Simultaneous presence of green and purple sulphur bacteria has been observed in many occasions in karstic lakes. However, few studies have analysed which environmental factors are important in determining whether purple or green sulphur bacteria should be more abundant at a given time of the year. Light quantity and light quality have been implicated in the selection of purple or green sulphur bacteria in different lakes. This phenomenon has been studied in detail in Lake Ciso, where both groups of bacteria are present at different times of the year, thus providing a natural experiment where changes in environmental conditions change the relative abundance of the green and purple bacteria. Major factors determining the abundance of these two groups of bacteria are: solar radiation, temperature, accumulated rainfall, and the vertical distribution of both oxygen and sulphide.

Chapter Four - Green Bacteria: Insights into Green Bacterial Evolution through Genomic Analyses

The spatial distribution and composition of anoxygenic phototrophic bacteria in the enriched bacterial communities from different depths of karst lakes Kirkilai and Ramunelis was studied using spectrophotometric analysis, as well as microbiological and molecular methods. In Lake Kirkilai, the highest bacterial abundance was measured in the metalimnion and near the bottom (up to 10.7 × 106 cell/mL); in Lake Ramunelis it was in the anoxic hypolimnion (up to 22.4 × 106 cell/mL). Increased water mineralization (0.5–1.2 g/L) with the domination of SO 4 2− and Ca2+ ions created favorable conditions for the development of sulfate-reducing bacteria; hydrogen sulfide produced as a result of their life activity facilitated the development of sulfur-oxidizing bacteria. The pigment analysis of phototrophic green and purple sulfur bacteria showed the domination of green sulfur bacteria in the enrichment culture. The results of phylogenetic analysis showed that Chlorobium limicola dominated in the enrichment culture for the green sulfur bacteria, whereas purple nonsulfur bacteria of the genus Rhodopseudomonas dominated in the enrichment culture for the purple sulfur bacteria.

Based upon their photosynthetic nature and the presence of a unique light-harvesting antenna structure, the chlorosome, the photosynthetic green bacteria are defined as a distinctive group in the Bacteria. However, members of the two taxa that comprise this group, the green sulfur bacteria (Chlorobi) and the filamentous anoxygenic phototrophic bacteria ("Chloroflexales"), are otherwise quite different, both physiologically and phylogenetically. This review summarizes how genome sequence information facilitated studies of the biosynthesis and function of the photosynthetic apparatus and the oxidation of inorganic sulfur compounds in two model organisms that represent these taxa, Chlorobium tepidum and Chloroflexus aurantiacus. The genes involved in bacteriochlorophyll (BChl) c and carotenoid biosynthesis in these two organisms were identified by sequence homology with known BChl a and carotenoid biosynthesis enzymes, gene cluster analysis in Cfx. aurantiacus, and gene inactivation studies in Chl. tepidum. Based on these results, BChl a and BChl c biosynthesis is similar in the two organisms, whereas carotenoid biosynthesis differs significantly. In agreement with its facultative anaerobic nature, Cfx. aurantiacus in some cases apparently produces structurally different enzymes for heme and BChl biosynthesis, in which one enzyme functions under anoxic conditions and the other performs the same reaction under oxic conditions. The Chl. tepidum mutants produced with modified BChl c and carotenoid species also allow the functions of these pigments to be studied in vivo.

Anoxygenic phototrophic bacteria are able to oxidize and remove hydrogen sulfide from natural sources by using inorganic substrates and light. In the present study, anoxygenic phototrophic bacteria were isolated from Vartoon hot spring (east of Isfahan Province, Iran) and used for removal of sulfide compounds from natural gas in a semi-continuous photobioreactor. Mat samples that obtained from the depth of spring were introduced into Winogradsky columns and kept in the presence of sunlight for 3–4 months until green and purple layers were composed. The layers were then transferred into mineral medium and incubated under anaerobic condition. The sequence of amplified 16S rDNA in green bacteria and an amplified sequence among the gene encoding photosynthetic reaction center in purple bacteria were used for molecular identification of bacteria. The bacterial growth based on the contents of bacteriochlorophylls and the extent of elemental sulfur production by the cells were measured after ethanol extraction. Molecular identification validated the presence of Chlorobium phaeobacteroides and Prosthecochloris vibrioformis from green sulfur bacteria in the consortium. The results indicated the most increase in bacteriochlorophylls c, d, and e content of mineral medium after bacterial growth. In addition, by releasing 62.4 mmol elemental sulfur, 79.30% increase was detected in sulfur content of the medium during 3 weeks. The consortium of sulfur-oxidizing phototrophic bacteria was potentially used for natural gas desulfurization in the semi-closed photobioreactor. This system is suggested as a replacement for chemoautotrophic systems in areas where there is sufficient access to sunlight.

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.

Green Bacteria: Secondary Electron Donor (Cytochromes)

Green sulfur bacteria (GSB) are exquisitely adapted for growth at extraordinarily low light intensities. They are important primary producers of biomass in many anoxic environments, and they contribute significantly to the biogeochemical cycling of carbon, nitrogen, and sulfur on Earth. Green bacteria more generally share the property of using chlorosomes for light-harvesting, and these unusual organelles have many unique features. These include the presence of a monolayer lipid-protein envelope, self-assembling bacteriochlorophylls (BChl) in which pigment-pigment interactions predominate, and the presence of redox components ([2Fe-2S] ferredoxins and quinones) that play a role in regulating excitation energy transfer to the type-1 homodimeric reaction centers. The reaction centers of GSB are related to Photosystem I of cyanobacteria and higher plants but also exhibit several unique structural and functional features. The long-term objectives of this research program are to understand the structure, functions, and biogenesis of the chlorosomes, reaction centers, and electron transport chains that carry out the photochemical transduction of light energy into chemical energy in the model green sulfur bacterium, Chlorobaculum (formerly Chlorobium) tepidum. Over a period of 26 years, we characterized chlorosomes in detail. We identified the proteins in the chlorosome envelopes of diverse organisms, identified the nearest neighbors of those proteins, and characterized the chlorosomes of mutant strains lacking one to five of these proteins. After the genome sequence of Cba. tepidum became available, we identified all genes encoding enzymes for BChl a, BChl c/d/e/ƒ, and Chl a biosynthesis Cba. tepidum. We additionally identified all genes encoding enzymes for carotenoid biosynthesis. By constructing a bchQRU mutant strain that produces [Et, Me]-BChl d, together with collaborators who are specialists in solid-state NMR and cryo-electron microscopy, we solved the structure of the BChls in chlorosomes. Using similar methods and chlorosomes containing [Et, Me]-BChl c, we then showed that alternative structures were possible using the same syn-anti BChl dimer. The structures were refined by introducing spectroscopic data from single-chlorosome measurements. Over the course of this project, we sequenced the genomes of approximately twenty GSB strains, which provided important information for comparative analyses. Through analyses of metagenomic data from Mushroom and Octopus Springs in Yellowstone National Park, we identified a novel chlorophototroph belonging to the phylum Acidobacteriota. We successfully isolated an axenic culture of this organism. We characterized the photosynthetic apparatus of this bacterium, named Chloracidobacterium thermophilum, in significant detail, in particular its type-1, homodimeric reaction centers. Surprisingly, these reaction centers contain three types of Chls, BChl a, Chl a, and Zn-BChl a'. We showed that a dimer of Zn-BChl a' is the primary donor and Chl a the primary acceptor of electrons in this reaction center by using advanced spectroscopic techniques. We isolated eight additional strains of Chloracidobacterium spp. from Mushroom Spring and Rupite hot springs in Bulgaria. Comparative genomes showed that these represent three species, Cab. thermophilum, Cab. aggregatum, and Cab. validum. By applying comparative genomics, genetic, biochemical biophysical and physiological approaches to study green bacteria, we produced a wealth of new information about the remarkable light-harvesting and energy transduction capabilities of these poorly characterized microorganisms.

Pollutants of inorganic nature (acids, alkalis, mineral salts of different composition, metals) change the course of biological processes of environmental purification, but their influence on the physiological properties of phototrophic sulfur bacteria has not been studied enough. The usage of nitrite ions as an electron donor of anoxygenic photosynthesis by cells of phototrophic green and purple sulfur bacteria Chlorobium limicola IMV K-8, Thiocapsa sp. Ya-2003 and Lamprocystis sp. Ya-2003, isolated from Yavorivske Lake, under the influence of the most widespread inorganic pollutants – hydro- and dihydrophosphates, sulfates, chlorides and chlorates, has been studied. It is shown that KH2PO4, K2HPO4, Na2SO4, NaCl and KClO3, present in the van Niel medium with 4.2 mM NaNO2 at concentrations that are 0.5, 1.0, 2.0, 3.0, 4.0 times different from the maximum permissible concentrations (MPC), influenced the biomass accumulation and nitrite ions oxidation by phototrophic green and purple sulfur bacteria. In media with hydro- and dihydrophosphate ions at concentrations 4.0 times higher than the MPC, inhibition of bacterial growth was up to 1.7 times lower than in the control. The biomass accumulation by bacteria in media with chloride and chlorate ions at concentrations 3.0–4.0 times higher than MPC was 2.0–2.8 times lower compared to the control. In the medium with Na2SO4 at concentrations 2.0–4.0 times higher than MPC, the biomass was 2.0–4.0 times lower than in the control. Nitrites’ oxidation by all strains in the media with the studied pollutants was slowed down. The residual content of nitrite ions in media with hydro- and dihydrophosphate, chloride and chlorate ions at their concentrations 4.0 times higher than MPC, exceeded the NO2– content in the control variants up to 1.7 times. If in the medium without pollutants the cells of C. limicola IMV K-8, Thiocapsa sp. Ya-2003 and Lamprocystis sp. Ya-2003 strains oxidized 72.7%, 72.2% and 71.4%, respectively, of nitrite ions present in the medium, then in the medium with sulfate ions at concentration 4.0 times higher than the MPC, bacteria oxidized nitrite ions only at 39.6%, 34.4% and 27.0%, respectively. Oxidation of a lower quantity of nitrites by phototrophic bacteria in the media with inorganic pollutants led to the production by them of a lower quantity of nitrates. The content of NO3– in the media with hydro-, dihydrophosphate and chlorate ions at all concentrations was up to 1.9 times lower than in the control. In media with sulfate ions at concentrations 2.0–4.0 times higher than MPC and chloride at concentration 4.0 times higher than MPC, the content of nitrate ions was 2.1–4.3 and 2.0 times, respectively, lower than in the control variants. Inorganic pollutants stimulated the synthesis of intracellular carbohydrates in C. limicola IMV K-8. If the content of intracellular glucose in cells grown in the medium without pollutants was 10.3 mg/g dry cell weight, then in cells grown in media with K2HPO4, KH2PO4, Na2SO4, NaCl and KClO3 at concentrations 4.0 times higher than MPC, its content increased by 12.2%, 10.7%, 51.6%, 17.1% and 35.9%, respectively. The glycogen content in the cells grown in the medium without pollutants was 45.1 mg/g dry cell weight. Hydro- and dihydrophosphate, chloride and chlorate ions at concentrations 4.0 times higher than MPC stimulated glycogen synthesis in cells by 47.5%, 57.6%, 67.4% and 74.6%, respectively. The glycogen content in cells grown in the medium with Na2SO4 at concentrations 3.0 and 4.0 times higher than MPC increased by 102.9% and 107.5%, respectively. Therefore, it is established that pollutants of inorganic nature affect the physiological properties of photosynthetic sulfur bacteria and thus change the course of biological processes of environment purification, in particular, from nitrite ions.

The seasonal population dynamics of phototrophic sulphur bacteria and their vertical stratification in Lake La Cruz were studied over two consecutive summers. Development of purple sulphur bacteria occurred just below the oxic-anoxic boundary (the plate located at 14-15 m depth). The principal constituent was a species of Amoebobacter, containing okenone as its main carotenoid and which reached maximal densities at the end of summer (2.2 × 10 6 cells/ml and BChl.-a concentrations of 111 μg/l in September 1987). Green phototrophic bacteria developed below the Amoebobacter layer at a mean depth of 16 m. Pelodictyon clathratiforme was the dominant species, reaching maximal cell concentrations of 3.1 × 10 6 cells/ml and BChl.-d concentrations of 362 μg/l, also in September. Growth of these purple and green sulphur bacteria was light-limited, and sulphide was also present in very low concentrations owing to the low sulphate content of the lake. The two thermal stratification periods under study differed noticeably as a consequence of altered meteorological conditions and structural differences in the water column, with a shallower oxic-anoxic boundary and a major development of algal populations in the upper oxic layer during the second period. This reduced the light available to deep phototrophic bacteria, which were unable to attain the biomass achieved in the earlier period. The monimolimnion of Lake La Cruz acts as a refuge for phototrophic bacteria during the mixing period and this is considered to be a key factor in understanding the ecology of these microorganisms.