Anoxygenic phototrophic bacteria of the high-altitude meromictic Lake Gek-Gel,Azerbaijan

The anoxygenic phototrophic bacterial community of the high-altitude meromictic Lake Gek-Gel (Azerbaijan) was investigated in September 2003. The highest concentration of bacteriochlorophyll e (48 μg/l) was detected at a depth of 30 m; the peak of bacteriochlorophyll a (4.5 μg/l) occurred at 29 m. Phylogenetic analysis revealed that brown-colored green sulfur bacteria Chlorobium phaeobacteroides predominated in the lake. Nonsulfur purple bacteria phylogenetically close to Blastochloris sulfoviridis were found in insignificant amounts; these organisms have not been previously reported in Lake Gek-Gel.     

Production of elemental sulfur by green and purple sulfur bacteria

The utilization of sulfide by phototrophic sulfur bacteria temporarily results in the accumulation of elemental sulfur. In the green sulfur bacteria (Chlorobiaceae), the sulfur is deposited outside the cells, whereas in the purple sulfur bacteria (Chromatiaceae) sulfur is found intracellularly. Consequently, in the latter case, sulfur is unattainable for other individuals. Attempts were made to analyze the impact of the formation of extracellular elemental sulfur compared to the deposition of intracellular sulfur.According to the theory of the continuous cultivation of microorganisms, the steady-state concentration of the limiting substrate is unaffected by the reservoir concentration (S _R).It was observed in sulfide-limited continuous cultures ofChlorobium limicola f.thiosulfatophilum that higherS _R values not only resulted in higher steady-state population densities, but also in increased steady-state concentrations of elemental sulfur. Similar phenomena were observed in sulfide-limited cultures ofChromatium vinosum.It was concluded that the elemental sulfur produced byChlorobium, althouth being deposited extracellularly, is not easily available for other individuals, and apparently remains (in part) attached to the cells. The ecological significance of the data is discussed.Non-standard abbreviations RP reducing power - BChl bacteriochlorophyll - N_cell cell material - specific growth rate - {ie52-1} maximal specific growth rate - D dilution rate - K _s saturation constant - s concentration of limiting substrate - S _R same ass but in reservoir bottle - Y yield factor - iS^o intracellular elemental sulfur - eS^o extracellular elemental sulfur - PHB poly-beta-hydroxybutyric acid     

Population dynamics of phototrophic bacteria in three basins of Lake Banyoles (Spain)

The annual dynamics of the phototrophic bacterial populations developing in the anoxic layers has been monitored in three basins of the northern area of Lake Banyoles (Spain). Although two of the studied basins are meromictic and one is holomictic, chemical properties of the water are almost identical. Therefore, differences in both the spatial and temporal distribution, as well as in the composition of phototrophic bacterial communities, dominated by Chlorobium phaeobacteroides and Chromatium minus, are discussed on the basis of the structural and morphometric characteristics of each basin. Both species showed the same physiological adaptations to light intensity changes by modifying the carotenoid/bacteriochlorophyll ratio. Light reaching the oxic-anoxic boundary appears to be the most important factor controlling the growth of phototrophic bacteria in Lake Banyoles. The oxic-anoxic boundary becomes shallower as summer advances, until enough light is available for bacterial photosynthesis.     

Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria

1. Dry weight yields from mixed cultures ofProsthecochloris aestuarii orChlorobium limicola with the sulfur reducingDesulfuromonas acetoxidans were determined on different growth limiting amounts of acetate, ethanol or propanol. The obtained yields agreed well with values predicted from stoichiometric calculations. 2. From mixed cultures of twoChlorobium limicola strains withDesulfovibrio desulfuricans orD. gigas on ethanol as the growth limiting substrate, dry weight yields were obtained as calculated for the complete utilization of the ethanol by the mixed cultures. 3. Dry weight yield determinations for two pure cultures ofChlorobium limicola with different growth limiting amounts of sulfide in the absence and presence of excess acetate confirmed that acetate is incorporated byChlorobium in a fixed proportion to sulfide; compared to the yield in the absence of acetate the yield is increased two to threefold in the presence of acetate. 4. The lowest possible sulfide concentrations necessary for optimal growth of mixed cultures of eitherProsthecochloris orChlorobium withDesulfuromonas on acetate were 7–8 mg H₂S per liter of medium. 5. Doubling times at the growth rate limiting light intensities of 5, 10, 20, 50, 100 and 200 lux were determined under optimal growth conditions for the following phototrophic bacteria:Prosthecochloris aestuarii, Chlorobium phaeovibriodes, Chromatium vinosum andRhodopseudomonas capsulata. Reasonably good growth was still obtained withProsthecochloris at 10 and 5 lux light intensity at which no growth of the purple bacteria could be observed.     

Susceptibility of various purple and green sulfur bacteria to different antimicrobial agents

Abstract Several purple and green sulfur bacteria (genera Chromatium, Thiocapsa and Chlorobium ) were tested for their sensitivity to different antimicrobial agents by a disc diffusion assay, using thioacetamide as a source of hydrogen sulfide for plate growth. Chlorobium limicola strains were more sensitive to amoxicillin, erythromycin and nalidixic acid, whereas gentamicin and netilmicin were more active against the purple bacteria tested. None of the organisms were sensitive to oxacillin and trimethoprim + sulfamethoxazole. The critical concentrations at the edge of the inhibition zone were also calculated for three organisms and the antimicrobials colistin, mitomycin C, penicillin G, rifampicin, and streptomycin. The results obtained suggest that colistin, mitomycin C, penicillin G would provide selective conditions against the growth of Chlorobium limicola strains, while streptomycin and other aminoglycoside antibiotics would select against purple bacteria.     

Ammonia inhibition of nitrogenase activity in purple and green bacteria

Ammonia reversibly inhibits the nitrogenase activity not only in purple nonsulfur bacteria but in purple (Thiocapsa roseopersicina) and green (Chlorobium limicola forma thiosulfatophilum) sulfur bacteria as well.The complete inhibition of nitrogenase activity (acetylene reduction) is observed about 30 s after addition of NH ₄ ⁺ (2.5×10^-6 M) to cell suspensions. The pattern of ammonia inhibition of acetylene reduction in T. roseopersicina does not differ from the action of tetrabutylammonium and tetraphenylphosphonium (3 · 10^-6-5·10^-5 M) on nitrogenase activity of this bacterium.Simultaneously with the switch-off effect of NH ₄ ⁺ a considerable increase of ATP in cells of Rhodobacter sphaeroides and C. limicola f. thiosulphatophilum was observed.     

Chlorosomes of green sulfur bacteria: Pigment composition and energy transfer

The pigment composition and energy transfer pathways in isolated chlorosomes ofChlorobium phaeovibrioides andChlorobium vibrioforme were studied by means of high performance liquid chromatography (HPLC) and picosecond absorbance difference spectroscopy. Analysis of pigment extracts of the chlorosomes revealed that they contain small amounts of bacteriochlorophyll (BChl)a esterified with phytol, whereas the BChlsc, d ande are predominantly esterified with farnesol. The chlorosomal BChla content inC. phaeovibrioides andC. vibrioforme was found to be 1.5% and 0.9%, respectively. The time resolved absorbance difference spectra showed a bleaching shifted to longer wavelengths as compared to the Q_y absorption maxima and in chlorosomes ofC. vibrioforme also an absorbance increase at shorter wavelengths was observed. These spectral features were ascribed to excitation of oligomers of BChle and BChlc/d, respectively. One-color and two-color pump-probe kinetics ofC. phaeovibrioides showed rapid energy transfer to long-wavelength absorbing BChle oligomers, followed by trapping of excitations by BChla with a time constant of about 60 ps. Time resolved anisotropy measurements inC. vibrioforme showed randomization of excitations among BChla molecules with a time constant of about 20 ps, indicating that BChla in the baseplate is organized in clusters. One-color and two-color pump-probe measurements inC. vibrioforme showed rapid energy transfer from short-wavelength to long-wavelength absorbing oligomers with a time constant of about 11 ps. Trapping of excitations by BChla in this species could not be resolved unambiguously due to annihilation processes in the BChla clusters, but may occur with time constants of 15, 70 and 200 ps.     

Dominance of a clonal green sulfur bacterial population in a stratified lake

For many years, the chemocline of the meromictic Lake Cadagno, Switzerland, was dominated by purple sulfur bacteria. However, following a major community shift in recent years, green sulfur bacteria (GSB) have come to dominate. We investigated this community by performing microbial diversity surveys using FISH cell counting and population multilocus sequence typing [clone library sequence analysis of the small subunit (SSU) rRNA locus and two loci involved in photosynthesis in GSB: fmoA and csmCA ]. All bacterial populations clearly stratified according to water column chemistry. The GSB population peaked in the chemocline ( c . 8 × 10⁶ GSB cells mL⁻¹) and constituted about 50% of all cells in the anoxic zones of the water column. At least 99.5% of these GSB cells had SSU rRNA, fmoA , and csmCA sequences essentially identical to that of the previously isolated and genome-sequenced GSB Chlorobium clathratiforme strain BU-1 (DSM 5477). This ribotype was not detected in Lake Cadagno before the bloom of GSB. These observations suggest that the C. clathratiforme population that has stabilized in Lake Cadagno is clonal. We speculate that such a clonal bloom could be caused by environmental disturbance, mutational adaptation, or invasion.     

Selective enrichment of green sulfur bacteria in the presence of 4-aminobenzenesulfonate (sulfanilate)

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).     

Distribution and population dynamics of ostracodes (Crustacea,Ostracoda) in a karstic lake: Lake Banyoles (Catalonia,Spain)

Qualitative and quantitative benthic samples were collected monthly between December 1986 and December 1987 from several stations at various depths and basins in the karstic Lake Banyoles (Catalonia, Spain), to study the spatial distribution and seasonality of ostracodes. A total of eight ostracode species were found in the lake. Of these, Candona neglecta Sars, Isocypris beauchampi Paris and Cypria ophtalmica Jurine were the most common ostracodes at all depth stations. Darwinula stevensoni Brady & Robertson, Ilyocypris species (I. bradyi Sars and I. gibba (Ramdohr)), Cyprideis torosa (Jones) and Cyclocypris ovum Jurine, on the other hand, were confined to sublittoral and littoral areas of the lake.Substrate and organic matter content appeared to be relevant factors determining the distribution of ostracode species at the different stations. The highest abundance of ostracodes were in the sublittoral (Basin IV–7 m and Basin I-5 m) where the substrate was coarse and sediment had a high organic matter content (>35% LOI). Low oxygen levels in the hypolimnetic waters (i.e. < 1 mg l^–1) may explain the absence of ostracodes at deeper zones in stations where the anoxic period lasts more than four months. On the other hand, low temperatures seem to favour the development of C. neglecta and D. stevensoni, while with higher temperatures I. beauchampi, C. torosa and C. ovum increased their abundances. However, no clear pattern can be observed, so ostracode species in Lake Banyoles, apparently, have non-seasonal life histories.     

Phylogeny of the PscB reaction center protein from green sulfur bacteria

The reaction center (RC) of green sulfur bacteria has iron—sulfur clusters as terminal acceptors and is related to the Type I RC found in Heliobacter sp. and in Photosystem I (PS I) of green plants and cyanobacteria. Degenerate primers were used to retrieve the genes coding for one of the RC proteins, PscB, from 11 strains of green sulfur bacteria. PCR using the same primers gave no product with a second group of strains and the protein from these strains did not crossreact with antibodies raised against purified PscB from the first group, suggesting the presence of a high degree of variability. The sequences shared a high degree of similarity in the region coding for the binding motif for the 4Fe–4S centers. However, the N-terminal portion of the deduced protein sequences was highly variable and contained a highly positively charged, low-complexity region with repeated tetrapeptides with two alanines flanked by proline or lysine. The PscB sequences obtained fell into two major groups, and the results suggested a lack of correlation between the pigmentation of the chlorosome antenna system and the reaction center protein. There is also a lack of correlation between the grouping of the pscB sequences and the phylogeny deduced from 16S rRNA.This revised version was published online in October 2005 with corrections to the Cover Date.     

Photoproduction of H2 from acetate by syntrophic cocultures of green sulfur bacteria and sulfur-reducing bacteria

The marine green sulfur bacterium Chlorobium vibrioforme strain 1930 produced H₂ and elemental sulfur from sulfide or thiosulfate under N limitation in the light. H₂ production depended on nitrogenase and occurred only in the absence of ammonia. Methionine sulfoximine, an inhibitor of glutamine synthetase, prevented the switch-off by ammonia. In defined syntrophic cocultures of the acetate-oxidizing, sulfur-reducing bacterium Desulfuromonas acetoxidans with green sulfur bacteria, H₂ was produced from acetate via a light-driven sulfur cycle. The sulfur-reducing bacterium could not be replaced by sulfate-reducing bacteria in these experiments. In a coculture of the marine Chlorobium vibrioforme strain 1930 and the sulfur-reducing bacterium Desulfuromonas acetoxidans strain 5071, optimum long-term H₂ production from acetate was obtained with molecular nitrogen as N source, at low light intensity (110 mol · m^-2 · s^-1), in sulfide-reduced mineral medium (2 mM Na₂S) at pH 6.8. Traces of sulfide (10 M) were sufficient to keep the sulfur cycle running. The coculture formed no poly--hydroxyalkanoates (PHA), but 20%–40% polysaccharide per cell dry mass. Per mol acetate added, the coculture formed 3.1 mol of H₂ (78% of the theoretical maximum). Only 8% of the reducing equivalents was incorporated into biomass. The maximum rate of H₂ production was 1300 ml H₂ per day and g cell dry mass.Non-standard abbrevations MOPS 2-(N-morpholino) propane sulfonic acid - MSX Methionine sulfoximine - PHA poly--hydroxyalkanoates     

Phototrophic purple sulfur bacteria as heat engines in the South Andros Black Hole

Photosynthetic organisms normally endeavor to optimize the efficiency of their light-harvesting apparatus. However, here we describe two bacterial isolates belonging to the genera Allochromatium and Thiocapsa that demonstrate a novel adaptation by optimizing their external growth conditions at the expense of photosynthetic efficiency. In the South Andros Black Hole, Bahamas, a dense l-m thick layer of these anoxygenic purple sulfur bacteria is present at a depth of 17.8 m. In this layer the water temperature increases sharply to 36°C as a consequence of the low-energy transfer efficiency of their carotenoids (ca. 30%). These include spirilloxanthin, and related polyene molecules and a novel chiral carotenoid identified as spirilloxanthin-2-ol, not previously reported in purple bacteria. To our knowledge, this study presents the first evidence of such a bacterial mass significantly increasing the ambient water temperature. The transduction of light to heat energy to excess heat may provide these anoxygenic phototropic bacteria with a competitive advantage over non-thermotolerant species, which would account for their predominance within the microbial layer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phylogenic status of a purple sulfur bacterium and its bloom in Lake Kaiike

Two bacterial species at the upper boundary of the H₂S-containing lower layer of Lake Kaiike, a purple sulfur bacterium and Macromonas sp., markedly changed their population densities in a single year (maximum cell numbers ranged between 10⁶ and <10³ cells ml^–1), although neither species ever entirely disappeared from the lake over at least the past 30 years. Genetic characteristics based on the sequence of the 16S rDNA of the purple sulfur bacterium showed it to be a new species of the Chromatiaceae family. This bloom of purple sulfur bacterium occurred when the H₂S layer was disturbed by an external intrusion of seawater.     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Iron sulfur proteins of the purple sulfur bacterium Ectothiorhodospira shaposhnikovii

In addition to several cytochromes three iron sulfur proteins were detected in mixotrophically grown cells of Ectothiorhodospira shaposhnikovii, a member of the Chromatiaceae. They were identified as a bacterial ferredoxin and two high potential iron sulfur proteins (HIPIPs). The two HIPIPs were purified and characterized. They were named according to their differing retention times on a DEAE-cellulose column using a continuous NaCl gradient: early and late HIPIP. The HIPIPs contain 4 mol of non-heme iron and 4 mol of acid labile sulfur per mol protein. Under the conditions of purification the early HIPIP (E _{m, 7}+270 mV) was present in a semi-reduced state. Using ion-exchange chromatography the early HIPIP could be split into a reduced green-brown (pI=3.7) and an oxidized red-brown (pI=3.9) fraction. The late HIPIP (pI=3.8) showed a midpoint potential of only+155 mV, the lowest redox potential of a HIPIP described so far.Non-common abbreviations HIPIP high potential iron sulfur protein - MOPS 3(N-morpholino)propane sulfonate - SDS sodium dodecylsulfate     

<Emphasis Type="Italic">Thiocapsa imhoffii</Emphasis>, sp. nov., an alkaliphilic purple sulfur bacterium of the family <Emphasis Type="Italic">Chromatiaceae</Emphasis> from Soap Lake,Washington (USA)

An alkaliphilic purple sulfur bacterium, strain SC5, was isolated from Soap Lake, a soda lake located in east central Washington state (USA). Cells of strain SC5 were gram-negative, non-motile, and non-gas vesiculate cocci, often observed in pairs or tetrads. In the presence of sulfide, elemental sulfur was deposited internally. Liquid cultures were pink to rose red in color. Cells contained bacteriochlorophyll a and spirilloxanthin as major photosynthetic pigments. Internal photosynthetic membranes were of the vesicular type. Optimal growth of strain SC5 occurred in the absence of NaCl (range 0–4%), pH 8.5 (range pH 7.5–9.5), and 32°C. Photoheterotrophic growth occurred in the presence of sulfide or thiosulfate with only a limited number of organic carbon sources. Growth factors were not required, and cells could fix N₂. Dark, microaerobic growth occurred in the presence of both an organic carbon source and thiosulfate. Sulfide and thiosulfate served as electron donors for photoautotrophy, which required elevated levels of CO₂. Phylogenetic analysis placed strain SC5 basal to the clade of the genus Thiocapsa in the family Chromatiaceae with a 96.7% sequence similarity to its closest relative, Thiocapsa roseopersicina strain 1711^T (DSM217^T). The unique assemblage of physiological and phylogenetic properties of strain SC5 defines it as a new species of the genus Thiocapsa, and we describe strain SC5 herein as Tca. imhoffii, sp. nov.     

Low-temperature spectroscopy of isolated FMO-protein and a membrane-free reaction center complex from the green sulfur bacteriumChlorobium tepidum

We have isolated the water-soluble BChla-protein (FMO-protein) from the greer sulfur bacteriumChlorobium tepidum by a new procedure involving a salt-wash of isolated membranes at alkaline pH. The absorption spectrum of the isolated FMO-protein at 77 K was compared with that of a reaction-center complex containing the FMO-protein (FMO-RC complex) isolated fromC. tepidum following the procedure of Feiler U, Nitsche W and Michel H (1992) Biochemistry 31: 2608–2614. Oxidation or illumination of the FMO-RC complex caused bleaching of a component with a maximum at 836 nm which was not present in the purified FMO-protein.     

Colorless sulfur bacteria <Emphasis Type="Italic">Thioploca</Emphasis> from different sites in Lake Baikal

The colorless sulfur bacteria Thioploca spp. found in Lake Baikal are probably a marker for the influx of subterranean mineralized fluids. Bacteria act as a biological filter; by consuming sulfide in their metabolism, they detoxicate it and maintain the purity of Lake Baikal’s water. The bacteria were investigated by various techniques. According to analysis of the 16S rRNA gene fragment, Thioploca sp. from Frolikha Bay, Baikal belongs to the clade of freshwater species found in Lake Biwa and Lake Constance; it is most closely related to Thioploca ingrica.     

On the distribution patterns and population dynamics of sublittoral and profundal oligochaeta fauna from Lake Banyoles (Catalonia,NE Spain)

Oligochaeta assemblages from Lake Banyoles were composed of one species of Naididae and eight species of Tubificidae, Potamothrix heuscheri (8–1350 ind. m^–2) being the most abundant and widely distributed species, followed by Limnodrilus hoffmeisteri (25–858 ind. m^–2) and Psammoryctides barbatus (12–792 ind. m^–2). The remaining species were Potamothrix hammoniensis, Potamothrix bavaricus, Limnodrilus claparedeanus, Branchiura sowerbyi, Aulodrilus pigueti and Dero digitata, all of which occurred at lower densities and with restricted distributions.Regardless of sampling depth, maximum densities of oligochaetes were found in winter (up to 5,142 ind. m^–2 in December) and secondarily in June. Immature Tubificidae with hair setae and mature P. heuscheri were present and dominant throughout the year. Cestode parasites were recorded infesting L. hoffmeisteri and P. heuscheri populations.Oligochaeta densities increased with depth, but low oxygen concentrations (less than 1 mg l^–1) during a long period (4 months) in some basins of the lake acted as a key factor in reducing the density of worms and the species richness to one species, Potamothrix heuscheri.