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.     

Calcite-accumulating large sulfur bacteria of the genus Achromatium in Sippewissett Salt Marsh

Large sulfur bacteria of the genus Achromatium are exceptional among Bacteria and Archaea as they can accumulate high amounts of internal calcite. Although known for more than 100 years, they remain uncultured, and only freshwater populations have been studied so far. Here we investigate a marine population of calcite-accumulating bacteria that is primarily found at the sediment surface of tide pools in a salt marsh, where high sulfide concentrations meet oversaturated oxygen concentrations during the day. Dynamic sulfur cycling by phototrophic sulfide-oxidizing and heterotrophic sulfate-reducing bacteria co-occurring in these sediments creates a highly sulfidic environment that we propose induces behavioral differences in the Achromatium population compared with reported migration patterns in a low-sulfide environment. Fluctuating intracellular calcium/sulfur ratios at different depths and times of day indicate a biochemical reaction of the salt marsh Achromatium to diurnal changes in sedimentary redox conditions. We correlate this calcite dynamic with new evidence regarding its formation/mobilization and suggest general implications as well as a possible biological function of calcite accumulation in large bacteria in the sediment environment that is governed by gradients. Finally, we propose a new taxonomic classification of the salt marsh Achromatium based on their adaptation to a significantly different habitat than their freshwater relatives, as indicated by their differential behavior as well as phylogenetic distance on 16S ribosomal RNA gene level. In future studies, whole-genome characterization and additional ecophysiological factors could further support the distinctive position of salt marsh Achromatium.     

Development of mass blooms of Thiocapsa roseopersicina on sheltered beaches on the Orkney Islands

Abstract Mass developments of the purple sulfur bacterium Thiocapsa roseopersicina in the surface layers of sandy beaches on the Orkney Islands were examined with respect to microcolony formation on sand grains, vertical distribution of viable cells and the ability to colonize beach surfaces. It was observed that microcolonies of the non-motile phototrophic bacterium cemented individual sand grains to each other and that the resulting aggregates could withstand severe wave action and may play a decisive role in the stabilization of these sandy beaches.
After removal of the top layer similar population densities of T. roseopersicina were recorded within seven days. It was calculated that the net specific growth rate initially was 0.53 day⁻¹ (0.022 h⁻¹).
Laboratory studies strongly suggest that the populations of T. roseopersicina on sheltered beaches on the Orkney Islands were growing phototrophically in the light even when the microenvironment was oxic. Bacteriochlorophyll a synthesis was repressed by oxygen and occurred during periods with low light intensities when the microenvironment was anoxic and contained sulfide.     

Evidence of covalent modification of glutamine synthetase in the purple sulfur bacterium Thiocapsa roseopersicina

Abstract It was shown that glutamine synthetase of purple sulfur bacterium Thiocapsa roseopersicina is regulated by covalent modification. This conclusion is made on the basis of results showing that: (i) incubation of cells under conditions of nitrogen deprivation in the light lead to an increase of glutamine synthetase activity; (ii) addition of ammonium to nitrogen-starved cell suspensions caused a rapid decrease of glutamine synthetase activity; (iii) inhibition of glutamine synthetase by feedback modifiers was higher in ammonium-treated cells than in those starved for a nitrogen source; (iv) treatment of purified glutamine synthetase and cell-free extracts with phosphodiesterase was accompanied by an increase of glutamine synthetase activity, indicating the cleavage of modifying residues covalently bound to glutamine synthetase molecules.     

Candidatus "Thiodictyon syntrophicum", sp. nov., a new purple sulfur bacterium isolated from the chemocline of Lake Cadagno forming aggregates and specific associations with Desulfocapsa sp

Strain Cad16(T) is a small-celled purple sulfur bacterium (PSB) isolated from the chemocline of crenogenic meromictic Lake Cadagno, Switzerland. Long term in situ observations showed that Cad16(T) regularly grows in very compact clumps of cells in association with bacteria belonging to the genus Desulfocapsa in a cell-to-cell three dimensional structure. Previously assigned to the genus Lamprocystis, Cad16(T), was here reclassified and assigned to the genus Thiodictyon. Based on comparative 16S rRNA gene sequences analysis, isolate Cad16(T) was closely related to Thiodictyon bacillosum DSM234(T) and Thiodictyon elegans DSM232(T) with sequence similarities of 99.2% and 98.9%, respectively. Moreover, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis separated Cad16(T) from other PSB genera, Lamprocystis and Thiocystis. Major differences in cell morphology (oval-sphere compared to rod-shaped) and arrangement (no netlike cell aggregates), carotenoid group (presence of okenone instead of rhodopinal), chemolithotrophic growth as well as the ability to form syntrophic associations with a sulfate-reducing bacteria of the genus Desulfocapsa suggested a different species within the genus Thiodictyon. This isolate is therefore proposed and described as Candidatus "Thiodictyon syntrophicum" sp. nov., a provisionally novel species within the genus Thiodictyon.     

Convenient method to prepare neutral sulfide solution for cultivation of phototrophic sulfur bacteria

A device is described for the preparation and storage of sterile neutral sulfide solution which is required for the repeated addition of this substrate to growing cultures of phototrophic sulfur bacteria.     

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.     

Urease formation in purple sulfur bacteria (Chromatiaceae) grown on various nitrogen sources

Batch cultures of Thiocapsa roseopersicina strain 6311, Thiocystis violacea strain 2311 and Chromatium vinosum strain 1611, grown anaerobically in the light on sulfide with urea, ammonia, N₂ or casein hydrolysate as nitrogen source exhibited urease activity, while Chromatium vinosum strain D neither showed any degradation of urea nor urease activity on any of the nitrogen sources tested.In T. violacea and C. vinosum strain 1611 urease was little affected by the nitrogen source and seemed to be constitutive. In T. roseopersicina, however, the enzyme was repressed by ammonia (although a low basal level of activity remained) and, to a lesser degree, induced by urea: The presense of urea stimulated a temporary increase in urease activity in the early exponential growth phase. The highest activities, however, were found after growth on N₂, and especially on 0.1% casein hydrolysate (in the absence or after exhaustion of external ammonia), but not before the stationary growth phase was reached. Derepressed urease synthesis required an efficient external source of nitrogen.In cultures of T. roseopersicina urease activity showed a periodic oscillation which depended on the repeated feeding with sulfide and subsequent variation in the sulfur content of the cells. The possible reasons of this oscillation are discussed.     

Characterization of a new platelet-forming purple sulfur bacterium,Amoebobacter pedioformis sp. nov.

The dominant purple sulfur bacterium of a reddish-colored waste water pond near Taichung, Taiwan, was isolated in pure culture, strain CML2. Individual cells were nearly spherical, nonmotile, and contained in their peripheral parts was vacuoles that appeared like elongated, curved tubes. Four to sixteen cells formed platelet-like aggregates reminiscent of Thiopedia rosea. The intracellular photosynthetic membrane system of the cells was of vesicular type; the photosynthetic pigments consisted of bacteriochlorophyll a and spirilloxanthin as the major carotenoid. The color of cell suspensions was pink to rosered. Under anaerobic conditions photolithoautotrophic growth occurred with sulfide, elemental sulfur or thiosulfate; sulfur globules were stored as an intermediary oxidation product. In the presence of sulfide, acetate, lactate and pyruvate were photoassimilated; strain CML2 lacked assimilatory sulfate reduction. Fastest photoautotrophic growth (11 h doubling time) was obtained at pH 7.5, 35°C and a light intensity of about 1000 lux (tungsten lamp). Chemolithoautotrophic growth in the dark was possible under reduced oxygen partial pressure with reduced sulfur compounds as respiratory substrates. The DNA base composition of strain CML2 was 65.5 mol% G+C. Strain CML2 is described as type strain of a new species, Amoebobacter pedioformis sp. nov., in the family Chromatiaceae.     

A new purple sulfur bacterium from saline littoral sediments,Thiorhodovibrio winogradskyi gen. nov. and sp. nov.

Two strains of a new purple sulfur bacterium were isolated in pure culture from the littoral sediment of a saline lake (Mahoney Lake, Canada) and a marine microbial mat from the North Sea island of Mellum, respectively. Single cells were vibrioid-to spirilloid-shaped and motile by means of single polar flagella. Intracellular photosynthetic membranes were of the vesicular type. As photosynthetic pigments, bacteriochlorophyll a and the carotenoids lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin and spirilloxanthin were present.Hydrogen sulfide and elemental sulfur were used under anoxic conditions for phototrophic growth. In addition one strain (06511) used thiosulfate. Carbon dioxide, acetate and pyruvate were utilized by both strains as carbon sources. Depending on the strain propionate, succinate, fumarate, malate, tartrate, malonate, glycerol or peptone may additionally serve as carbon sources in the light. Optimum growth rates were obtained at pH 7.2, 33 °C, 50 mol m^-2 s^-1 intensity of daylight fluorescent tubes and a salinity of 2.2–3.2% NaCl. During growth on sulfide, up to ten small sulfur globules were formed inside the cells. The strains grew microaerophilic in the dark and exhibited high specific respiration rates. No vitamins were required for growth. The DNA base composition was 61.0–62.4 mol% G+C.The newly isolated bacterium belongs to the family chromatiaceae and is described as a member of a new genus and species, Thiorhodovibrio winogradskyi gen. nov. and sp. nov. with the type strain SSP1, DSM No. 6702.     

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.     

Diversity of purple nonsulfur bacteria in shrimp ponds with varying mercury levels

This research aimed to study the diversity of purple nonsulfur bacteria (PNSB) and to investigate the effect of Hg concentrations in shrimp ponds on PNSB diversity. Amplification of the pufM gene was detected in 13 and 10 samples of water and sediment collected from 16 shrimp ponds in Southern Thailand. In addition to PNSB, other anoxygenic phototrophic bacteria (APB) were also observed; purple sulfur bacteria (PSB) and aerobic anoxygenic phototrophic bacteria (AAPB) although most of them could not be identified. Among identified groups; AAPB, PSB and PNSB in the samples of water and sediment were 25.71, 11.43 and 8.57%; and 27.78, 11.11 and 22.22%, respectively. In both sample types, Roseobacter denitrificans (AAPB) was the most dominant species followed by Halorhodospira halophila (PSB). In addition two genera, observed most frequently in the sediment samples were a group of PNSB (Rhodovulum kholense, Rhodospirillum centenum and Rhodobium marinum). The UPGMA dendrograms showed 7 and 6 clustered groups in the water and sediment samples, respectively. There was no relationship between the clustered groups and the total Hg (Hg_T) concentrations in the water and sediment samples used (<0.002–0.03 μg/L and 35.40–391.60 μg/kg dry weight) for studying the biodiversity. It can be concluded that there was no effect of the various Hg levels on the diversity of detected APB species; particularly the PNSB in the shrimp ponds.     

Characterization of ammonium and methylamine transport systems in phototrophic sulfur bacteria

Abstract Transport of ammonium and methylamine into the cells of green sulfur bacterium Chlorobium limicola and purple sulfur bacterium Thiocapsa roseopersicina is carried out by a common transport system. This system has (for C. limicola and T. roseopersicina , respectively) pH optimum 7.0 and 7.5; V _max 0.6 and 4.2 nmol min⁻¹ (mg protein)⁻¹; K_m 5.9 × 10⁻⁵ M and 1.3 × 10⁻⁵ M, and is capable of forming 120- and 600-fold methylamine gradients. The methylamine transport can be energized by the artificially imposed transmembrane K⁺ diffusive potential and is inhibited by tetraphenylphosphonium or valinomycin and K⁺. The data presented indicate that methylamine transport in both studied species is exclusively driven by the membrane potential gradient (ΔΨ).     

On the quantitation of bacteriochlorophyll in chromatophore suspensions from purple bacteria

A direct photometric quantitation of bacteriochlorophyll (BChl) at 375 nm in aqueous chromatophore suspensions from various purple bacteria is described. The assay is rapid and reproducible. It is utilized easily for processing large numbers of samples and is as sensitive as extraction methods usually applied today. Drawbacks of extraction methods, particularly not quantitative extractions, photo- and autooxidation are avoided. There is good linearity up to 20 μg BChl/ml suspension, and no interference by buffers is observed.     

Discrepancy between experimental and theoretical excitation transfer rates in LH2 bacteriochlorophyll-protein complexes of purple bacteria

Discrepancy is revealed between the values of excitation transfer times measured experimentally, and those calculated, for the atomic structures of B800 → B850 bacteriochlorophylls within the LH2 light-harvesting pigment–protein complex of the purple bacterium Rhodopseudomonas acidophila. The value 2.9–3.2 ps for the B800 → B850 excitation transfer, calculated on the basis of atomic structure of LH2, is about 4-times longer than that measured for this bacterium (0.7 ps). This discrepancy appears common in at least two purple bacteria. Possible sources responsible for this discrepancy are discussed. It may either signify some drawback/s/ in our notions about the precise in vivo structure of LH2 complexes, for example, possible changes of LH2 structure during crystallization, or it may reflect our ignorance of some mechanisms involved in excitation migration.     

Distribution of iron in Lake Banyoles in relation to the ecology of purple and green sulfur bacteria

The distribution of iron both in suspended sediment and in the water column has been studied during summer stratification in Lake Banyoles. In this lake, near bottom springs, a very fine material suspended sediment remains in suspension. Dissolved Fe²⁺ in interstitial water of this suspended sediment, is related to redox potential and to the bottom water inflow. In the water column, soluble iron is present in the hypolimnion of the six different basins forming Lake Banyoles. Under those conditions Fe²⁺ is partially removed by sulfide produced in the anoxic sediment. In addition, a peak of Fe²⁺ found at the density gradient level in basins C-III, C-IV and C-VI. A three compartment model on the dynamics of the processes involving iron in Lake Banyoles is proposed. The bottom springs supply oxygen to the anoxic hypolimnion affecting chemical processes of the iron cycle. The presence of phototrophic sulfur bacteria in the anoxic monimolimnion of basins C-III and C-IV can be related to the kinetics of Fe²⁺ and sulfide. In C-III sulfide concentration exceeds Fe²⁺ concentration whereas in C-IV sulfide is not detectable and iron reached values up to 60 mM. The presence of phototrophic sulfur bacteria in iron-containing environments with no detectable sulfide is explained by the ability of such microorganisms to use FeS as electron donor instead of H₂S.     

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.     

Induction of purple sulfur bacterial growth in dairy wastewater lagoons by circulation

Aims:  To determine whether circulation of dairy wastewater induces the growth of phototrophic purple sulfur bacteria (PSB).
Methods and Results:  Two dairy wastewater lagoons that were similar in size, geographic location, number and type of cattle loading the lagoons were chosen. The only obvious visual difference between them was that one was stagnant and the water was brown in colour (Farm 1), and the other was circulated and the water was red in colour because of the presence of PSB that contained carotenoid pigments (Farm 2). Both wastewaters were sampled monthly for 3 months and assayed for PSB and extractable carotenoid pigments (ECP). After this point, circulators were placed in the wastewater lagoon on Farm 1, and samples were taken monthly for 9 months and assayed for PSB and ECP. Before the installation of circulators, no PSB-like 16S rRNA sequences or ECP were observed in the wastewater from Farm 1; however, both were observed in the wastewater from Farm 2. After the installation of circulators, statistically greater levels of PSB and extractable carotenoid pigments were observed in the wastewater from Farm 1.
Conclusions:  Circulation enhances the growth of PSB in dairy wastewater.
Significance and Impact of this Study:  Because PSB utilize H₂S and volatile organic acids (VOA) as an electron source for photosynthesis, and VOA and alcohols as a carbon source for growth, the increase in these bacteria should reduce H₂S, volatile organic compounds and alcohol emissions from the lagoons, enhancing the air quality in dairy farming areas.     

Development of a genetic system for a purple sulfur bacterium: Conjugative plasmid transfer inChromatium vinosum

Establishment of a system for manipulative genetics in phototrophic sulfur bacteria of the family Chromatiaceae has mainly been hampered by the lack of reliable methods for growth of these organisms on agar surfaces, techniques for streaking, growth on selective media, screening for antibiotic resistance markers, and most importantly by the lack of a system for DNA transfer. We, therefore, developed minimal and complex agar media for Chromatium vinosum strain D (DSM 180^T), a representative of the purple sulfur bacteria. Sensitivity of C. vinosum towards a broad range of antibiotics was tested in liquid cultures and solidified media, allowing us to select appropriate antibiotic resistance markers. Furthermore, a system for conjugative transfer of IncP-mobilizable plasmids from Escherichia coli to C. vinosum was established. Broad-host-range IncQ vectors were mobilized to C. vinosum with the aid of plasmid RP4 either present extrachromosomally or integrated in the chromosome of E. coli S17-1. Conjugation efficiencies of up to 1 were observed. Agarose gel electrophoretic analysis showed that transconjugants contained the transferred plasmids in addition to the two detectable plasmids of wild-type C. vinosum. All genetic markers tested (kanamycin, gentamicin, ampicillin, amikacin, tetracycline) were expressed in C. vinosum. Furthermore, high-frequency transfer of plasmid RP4 from C. vinosum to E. coli and to Rhodospirillum rubrum K100 was demonstrated. Received: 3 March 1995 / Accepted: 22 May 1995