Evidence for autotrophic growth of purple sulfur bacteria using pyrite as electron and sulfur source.

Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a range of anoxic environments. In this study, we show that Allochromatium vinosum (a PSB species) is capable of autotrophic growth using pyrite as the electron and sulfur source. Comparative growth profile, substrate characterization, and transcriptomic sequencing data provided valuable insight into the molecular mechanisms underlying the bacterial utilization of pyrite and autotrophic growth. Specifically, the pyrite-supported cell cultures ("py"') demonstrated robust but much slower growth rates and distinct patterns from their sodium sulfide-amended positive controls. Up to ~200-fold upregulation of genes encoding various c- and b-type cytochromes was observed in "py," pointing to the high relevance of these molecules in scavenging and relaying electrons from pyrite to cytoplasmic metabolisms. Conversely, extensive downregulation of genes related to LH and RC complex components indicates that the electron source may have direct control over the bacterial cells' photosynthetic activity. In terms of sulfur metabolism, genes encoding periplasmic or membrane-bound proteins (e.g., FccAB and SoxYZ) were largely upregulated, whereas those encoding cytoplasmic proteins (e.g., Dsr and Apr groups) are extensively suppressed. Other notable differentially expressed genes are related to flagella/fimbriae/pilin(+), metal efflux(+), ferrienterochelin(-), and [NiFe] hydrogenases(+). Characterization of the biologically reacted pyrite indicates the presence of polymeric sulfur. These results have, for the first time, put the interplay of PSB and transition metal sulfide chemistry under the spotlight, with the potential to advance multiple fields, including metal and sulfur biogeochemistry, bacterial extracellular electron transfer, and artificial photosynthesis. IMPORTANCE: Microbial utilization of solid-phase substrates constitutes a critical area of focus in environmental microbiology, offering valuable insights into microbial metabolic processes and adaptability. Recent advancements in this field have profoundly deepened our knowledge of microbial physiology pertinent to these scenarios and spurred innovations in biosynthesis and energy production. Furthermore, research into interactions between microbes and solid-phase substrates has directly linked microbial activities to the surrounding mineralogical environments, thereby enhancing our understanding of the relevant biogeochemical cycles. Our study represents a significant step forward in this field by demonstrating, for the first time, the autotrophic growth of purple sulfur bacteria using insoluble pyrite (FeS2) as both the electron and sulfur source. The presented comparative growth profiles, substrate characterizations, and transcriptomic sequencing data shed light on the relationships between electron donor types, photosynthetic reaction center activities, and potential extracellular electron transfer in these organisms capable of anoxygenic photosynthesis. Furthermore, the findings of our study may provide new insights into early-Earth biogeochemical evolutions, offering valuable constraints for understanding the environmental conditions and microbial processes that shaped our planet's history.

Microbial Community Composition during a Bloom of Purple Bacteria in Intertidal Sediments in Vigo (Northwest Spain).

In summer 2019, a large, bright pink microbial mat was visible on top of macroalgal deposits in muddy sediments of an urban beach (Playa do Adro, Vigo). In order to characterize the dominant organisms in these colored mats, results from microscopic observations, photosynthetic pigments, and molecular analysis were gathered. Light microscopy examination revealed pinkish microbial aggregates with minor contributions of larger protists and cyanobacteria. High-performance liquid chromatography (HPLC) pigment analysis documented the dominance of bacteriochlorophyll a and carotenoids whose spectra were compatible with those described in photosynthetic purple bacteria. 16S rRNA gene amplicon sequencing confirmed that the vast majority of reads belonged to Proteobacteria (73.5%), and among them, nearly 88% of those reads belonged to purple sulfur bacteria (Gammaproteobacteria). A single family, Chromatiaceae, constituted the bulk of this assemblage, including the genera Thiohalocapsa (32%), Marichromatium (12.5%), Phaeochromatium (5%), and Halocromatium (2%) as main contributors. Nonetheless, a considerable number of sequences could not be assigned to a particular genus, stressing the large biological diversity in these microbial mats and the potential presence of novel taxa of purple sulfur bacteria. IMPORTANCE Urban beaches are valuable recreational areas particularly vulnerable to human disturbance. In these areas, the intertidal sediments harbor a diverse community of microorganisms, including virus, bacteria, fungi, and protozoa. In this sense, pollution events can introduce pathogenic allochthonous microbes which may constitute a human health risk. Visual and sensory observations, such as a weird color or bad smell, are usually appreciated as a warning by beachgoers and authorities, as indeed was the case at do Adro beach in 2019. The observed proliferation seems to be common in summertime, but its dimension alerted beachgoers and media. The obtained results allowed for the identification of purple sulfur bacteria as responsible for the pink-violet top layer staining the intertidal zone. These blooms have never been associated with public health risks. Beyond solving the sanitary concern, other important findings were its diversity and large proportion of novel taxa, illustrating the complexity of these ecosystems.

Thermal stability tuning without affecting gas-binding function of Thermochromatium tepidum cytochrome c'.

Hydrogenophilus thermoluteolus, Thermochromatium tepidum, and Allochromatium vinosum, which grow optimally at 52, 49, and 25 °C, respectively, have homologous cytochromes c' (PHCP, TTCP, and AVCP, respectively) exhibiting at least 50% amino acid sequence identity. Here, the thermal stability of the recombinant TTCP protein was first confirmed to be between those of PHCP and AVCP. Structure comparison of the 3 proteins and a mutagenesis study on TTCP revealed that hydrogen bonds and hydrophobic interactions between the heme and amino acid residues were responsible for their stability differences. In addition, PHCP, TTCP, and AVCP and their variants with altered stability similarly bound nitric oxide and carbon oxide, but not oxygen. Therefore, the thermal stability of TTCP together with PHCP and AVCP can be tuned through specific interactions around the heme without affecting their gas-binding function. These cytochromes c' will be useful as specific gas sensor proteins exhibiting a wide thermal stability range.

Direct effects of organic pollutants on the growth and gene expression of the Baltic Sea model bacterium Rheinheimera sp. BAL341.

Organic pollutants (OPs) are critically toxic, bioaccumulative and globally widespread. Moreover, several OPs negatively influence aquatic wildlife. Although bacteria are major drivers of the ocean carbon cycle and the turnover of vital elements, there is limited knowledge of OP effects on heterotrophic bacterioplankton. We therefore investigated growth and gene expression responses of the Baltic Sea model bacterium Rheinheimera sp. BAL341 to environmentally relevant concentrations of distinct classes of OPs in 2-h incubation experiments. During exponential growth, exposure to a mix of polycyclic aromatic hydrocarbons, alkanes and organophosphate esters (denoted MIX) resulted in a significant decrease (between 9% and 18%) in bacterial abundance and production compared with controls. In contrast, combined exposure to perfluorooctanesulfonic acids and perfluorooctanoic acids (denoted PFAS) had no significant effect on growth. Nevertheless, MIX and PFAS exposures both induced significant shifts in gene expression profiles compared with controls in exponential growth. This involved several functional metabolism categories (e.g. stress response and fatty acids metabolism), some of which were pollutant-specific (e.g. phosphate acquisition and alkane-1 monooxygenase genes). In stationary phase, only two genes in the MIX treatment were significantly differentially expressed. The substantial direct influence of OPs on metabolism during bacterial growth suggests that widespread OPs could severely alter biogeochemical processes governed by bacterioplankton.

A Dual Role for Ca2+ in Expanding the Spectral Diversity and Stability of Light-Harvesting 1 Reaction Center Photocomplexes of Purple Phototrophic Bacteria.

The light-harvesting 1 reaction center (LH1-RC) complex in the purple sulfur bacterium Thiorhodovibrio ( Trv.) strain 970 cells exhibits its LH1 Q y transition at 973 nm, the lowest-energy Q y absorption among purple bacteria containing bacteriochlorophyll a (BChl a). Here we characterize the origin of this extremely red-shifted Q y transition. Growth of Trv. strain 970 did not occur in cultures free of Ca2+, and elemental analysis of Ca2+-grown cells confirmed that purified Trv. strain 970 LH1-RC complexes contained Ca2+. The LH1 Q y band of Trv. strain 970 was blue-shifted from 959 to 875 nm upon Ca2+ depletion, but the original spectral properties were restored upon Ca2+ reconstitution, which also occurs with the thermophilic purple bacterium Thermochromatium ( Tch.) tepidum. The amino acid sequences of the LH1 α- and ß-polypeptides from Trv. strain 970 closely resemble those of Tch. tepidum; however, Ca2+ binding in the Trv. strain 970 LH1-RC occurred more selectively than in Tch. tepidum LH1-RC and with a reduced affinity. Ultraviolet resonance Raman analysis indicated that the number of hydrogen-bonding interactions between BChl a and LH1 proteins of Trv. strain 970 was significantly greater than for Tch. tepidum and that Ca2+ was indispensable for maintaining these bonds. Furthermore, perfusion-induced Fourier transform infrared analyses detected Ca2+-induced conformational changes in the binding site closely related to the unique spectral properties of Trv. strain 970. Collectively, our results reveal an ecological strategy employed by Trv. strain 970 of integrating Ca2+ into its LH1-RC complex to extend its light-harvesting capacity to regions of the near-infrared spectrum unused by other purple bacteria.

Dark aerobic sulfide oxidation by anoxygenic phototrophs in anoxic waters.

Anoxygenic phototrophic sulfide oxidation by green and purple sulfur bacteria (PSB) plays a key role in sulfide removal from anoxic shallow sediments and stratified waters. Although some PSB can also oxidize sulfide with nitrate and oxygen, little is known about the prevalence of this chemolithotrophic lifestyle in the environment. In this study, we investigated the role of these phototrophs in light-independent sulfide removal in the chemocline of Lake Cadagno. Our temporally resolved, high-resolution chemical profiles indicated that dark sulfide oxidation was coupled to high oxygen consumption rates of ~9 µM O2 ·h-1 . Single-cell analyses of lake water incubated with 13 CO2 in the dark revealed that Chromatium okenii was to a large extent responsible for aerobic sulfide oxidation and it accounted for up to 40% of total dark carbon fixation. The genome of Chr. okenii reconstructed from the Lake Cadagno metagenome confirms its capacity for microaerophilic growth and provides further insights into its metabolic capabilities. Moreover, our genomic and single-cell data indicated that other PSB grow microaerobically in these apparently anoxic waters. Altogether, our observations suggest that aerobic respiration may not only play an underappreciated role in anoxic environments but also that organisms typically considered strict anaerobes may be involved.

Comparative genome analysis of marine purple sulfur bacterium Marichromatium gracile YL28 reveals the diverse nitrogen cycle mechanisms and habitat-specific traits.

Mangrove ecosystems are characteristic of the high salinity, limited nutrients and S-richness. Marichromatium gracile YL28 (YL28) isolated from mangrove tolerates the high concentrations of nitrite and sulfur compounds and efficiently eliminates them. However, the molecular mechanisms of nitrite and sulfur compounds utilization and the habitat adaptation remain unclear in YL28. We sequenced YL28 genome and further performed the comparative genome analysis in 36 purple bacteria including purple sulfur bacteria (PSB) and purple non-sulfur bacteria (PNSB). YL28 has 6 nitrogen cycle pathways (up to 40 genes), and possibly removes nitrite by denitrification, complete assimilation nitrate reduction and fermentative nitrate reduction (DNRA). Comparative genome analysis showed that more nitrogen utilization genes were detected in PNSB than those in PSB. The partial denitrification pathway and complete assimilation nitrate reduction were reported in PSB and DNRA was reported in purple bacteria for the first time. The three sulfur metabolism genes such as oxidation of sulfide, reversed dissimilatory sulfite reduction and sox system allowed to eliminate toxic sulfur compounds in the mangrove ecosystem. Several unique stress response genes facilitate to the tolerance of the high salinity environment. The CRISPR systems and the transposon components in genomic islands (GIs) likely contribute to the genome plasticity in purple bacteria.

Spatio-temporal dynamics of sulfur bacteria during oxic--anoxic regime shifts in a seasonally stratified lake.

Sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria drive major transformations in the sulfur cycle, and play vital roles in oxic--anoxic transitions in lakes and coastal waters. However, information on the succession of these sulfur bacteria in seasonally stratified lakes using molecular biological techniques is scarce. Here, we used 16S rRNA gene amplicon sequencing to study the spatio-temporal dynamics of sulfur bacteria during oxic--anoxic regime shifts in Lake Vechten. Oxygen and sulfate were mixed throughout the water column in winter and early spring. Meanwhile, SRB, green sulfur bacteria (GSB), purple sulfur bacteria (PSB), and colorless sulfur bacteria (CSB) exclusively inhabited the sediment. After the water column stratified, oxygen and nitrate concentrations decreased in the hypolimnion and various SRB species expanded into the anoxic hypolimnion. Consequently, sulfate was reduced to sulfide, stimulating the growth of PSB and GSB in the metalimnion and hypolimnion during summer stratification. When hypoxia spread throughout the water column during fall turnover, SRB and GSB vanished from the water column, whereas CSB (mainly Arcobacter) and PSB (Lamprocystis) became dominant and oxidized the accumulated sulfide under micro-aerobic conditions. Our results support the view that, once ecosystems have become anoxic and sulfidic, a large oxygen influx is needed to overcome the anaerobic sulfur cycle and bring the ecosystems back into their oxic state.

Formation of stromatolite lamina at the interface of oxygenic-anoxygenic photosynthesis.

In modern stromatolites, mineralization results from a complex interplay between microbial metabolisms, the organic matrix, and environmental parameters. Here, we combined biogeochemical, mineralogical, and microscopic analyses with measurements of metabolic activity to characterize the mineralization processes and products in an emergent (<18 months) hypersaline microbial mat. While the nucleation of Mg silicates is ubiquitous in the mat, the initial formation of a Ca-Mg carbonate lamina depends on (i) the creation of a high-pH interface combined with a major change in properties of the exopolymeric substances at the interface of the oxygenic and anoxygenic photoautotrophic layers and (ii) the synergy between two major players of sulfur cycle, purple sulfur bacteria, and sulfate-reducing bacteria. The repetition of this process over time combined with upward growth of the mat is a possible pathway leading to the formation of a stromatolite.

Probing structure-function relationships in early events in photosynthesis using a chimeric photocomplex.

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αß-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, ß-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.

Nitrogen transformation under different dissolved oxygen levels by the anoxygenic phototrophic bacterium Marichromatium gracile.

Marichromatium gracile: YL28 (M. gracile YL28) is an anoxygenic phototrophic bacterial strain that utilizes ammonia, nitrate, or nitrite as its sole nitrogen source during growth. In this study, we investigated the removal and transformation of ammonium, nitrate, and nitrite by M. gracile YL28 grown in a combinatorial culture system of sodium acetate-ammonium, sodium acetate-nitrate and sodium acetate-nitrite in response to different initial dissolved oxygen (DO) levels. In the sodium acetate-ammonium system under aerobic conditions (initial DO = 7.20-7.25 mg/L), we detected a continuous accumulation of nitrate and nitrite. However, under semi-anaerobic conditions (initial DO = 4.08-4.26 mg/L), we observed a temporary accumulation of nitrate and nitrite. Interestingly, under anaerobic conditions (initial DO = 0.36-0.67 mg/L), there was little accumulation of nitrate and nitrite, but an increase in nitrous oxide production. In the sodium acetate-nitrite system, nitrite levels declined slightly under aerobic conditions, and nitrite was completely removed under semi-anaerobic and anaerobic conditions. In addition, M. gracile YL28 was able to grow using nitrite as the sole nitrogen source in situations when nitrogen gas produced by denitrification was eliminated. Taken together, the data indicate that M. gracile YL28 performs simultaneous heterotrophic nitrification and denitrification at low-DO levels and uses nitrite as the sole nitrogen source for growth. Our study is the first to demonstrate that anoxygenic phototrophic bacteria perform heterotrophic ammonia-oxidization and denitrification under anaerobic conditions.

Crystallographic characterization of the high-potential iron-sulfur protein in the oxidized state at 0.8 Å resolution.

High-potential iron-sulfur protein (HiPIP) is a soluble electron carrier protein of photosynthetic bacteria with an Fe4S4 cluster. Although structural changes accompanying the electron transfer are important for understanding of the functional mechanism, the changes have not been clarified in sufficient detail. We previously reported the high-resolution crystal structures of HiPIP from a thermophilic purple bacterium Thermochromatium tepidum in the reduced state. In order to perform a detailed comparison between the structures in different redox states, the oxidized structure should also be revealed at high resolution. Therefore, in the present study we performed a crystallographic analysis of oxidized HiPIP and a structural comparison with the reduced form at a high resolution of 0.8 Å. The comparison highlighted small but significant contraction in the iron-sulfur cluster. The changes in Fe-S bond lengths were similar to that predicted by theoretical calculation, although some discrepancies were also found. Almost distances between the sulfur atoms of the iron-sulfur cluster and the protein environment are elongated upon the oxidation. Positional changes of hydrogen atoms in the protein environment, such as on the amide-hydrogen of Cys75 in the proximity of the iron-sulfur cluster, were also observed in the accurate analyses. None of the water molecules exhibited significant changes in position or anisotropy of atomic displacement parameter between the two states, while the orientations of some water molecules were different.

Geochemically distinct carbon isotope distributions in Allochromatium vinosum DSM 180T grown photoautotrophically and photoheterotrophically.

Anoxygenic, photosynthetic bacteria are common at redox boundaries. They are of interest in microbial ecology and geosciences through their role in linking the carbon, sulfur, and iron cycles, yet much remains unknown about how their flexible carbon metabolism-permitting either autotrophic or heterotrophic growth-is recorded in the bulk sedimentary and lipid biomarker records. Here, we investigated patterns of carbon isotope fractionation in a model photosynthetic sulfur-oxidizing bacterium, Allochromatium vinosum DSM180T . In one treatment, A. vinosum was grown with CO2 as the sole carbon source, while in a second treatment, it was grown on acetate. Different intracellular isotope patterns were observed for fatty acids, phytol, individual amino acids, intact proteins, and total RNA between the two experiments. Photoautotrophic CO2 fixation yielded typical isotopic ordering for the lipid biomarkers: δ13 C values of phytol > n-alkyl lipids. In contrast, growth on acetate greatly suppressed intracellular isotopic heterogeneity across all molecular classes, except for a marked 13 C-depletion in phytol. This caused isotopic "inversion" in the lipids (δ13 C values of phytol < n-alkyl lipids). The finding suggests that inverse δ13 C patterns of n-alkanes and pristane/phytane in the geologic record may be at least in part a signal for photoheterotrophy. In both experimental scenarios, the relative isotope distributions could be predicted from an isotope flux-balance model, demonstrating that microbial carbon metabolisms can be interrogated by combining compound-specific stable isotope analysis with metabolic modeling. Isotopic differences among molecular classes may be a means of fingerprinting microbial carbon metabolism, both in the modern environment and the geologic record.

Measurement of photosynthesis using PAM technology in a purple sulfur bacterium Thermochromatium tepidum (Chromatiaceae).

We demonstrate that Blue-diode-based pulse amplitude modulation (PAM) technology can be used to measure the photosynthetic electron transport rate (ETR) of purple sulfur bacteria (Thermochromatium tepidum, Chromatiaceae). Previous studies showed that PAM technology could be used to estimate photosynthesis in purple nonsulfur bacteria and so PAM technology can be used to estimate photosynthesis of both kinds of purple photosynthetic bacteria. The absorptance of Thermochromatium films on glass fiber disks was measured and used to calculate actual ETR. ETR vs Irradiance (P vs E) curves fitted the waiting-in-line model (ETR = (ETRmax × E/Eopt) × exp (1−E/Eopt)). Yield (Y) was only ≈ 0.3­0.4. Thermochromatium saturates at 325 ± 13.8 µmol photons m(−2) s(−1) or ≈15% sunlight and shows photoinhibition at high irradiances. A pond of Thermochromatium would exhibit classic surface inhibition. Photosynthesis is extremely low in the absence of an electron source: ETR increases in the presence of acetate (5 mol m(−3)) provided as an organic carbon source and also increases in the presence of sulfite (3 mol m(−3)) but not sulfide and is only marginally increased by the presence of Fe(2+). Nonphotochemical quenching does occur in Thermochromatium but at very low levels compared to oxygenic photo-organisms or Rhodopseudomonads.

Transformation of monothioarsenate by haloalkaliphilic, anoxygenic photosynthetic purple sulfur bacteria.

Thioarsenates are the dominant arsenic species in arsenic-rich, alkaline, and sulfidic waters, but bacterial interactions with these compounds have only recently been examined. Previous studies have shown that microorganisms play a role in the transformation of monothioarsenate to arsenate, including use of monothioarsenate as a chemolithotrophic electron donor coupled with oxygen as an electron acceptor. We obtained enrichment cultures from two saline, alkaline lakes (Mono Lake, CA and Big Soda Lake, NV) that are able to use monothioarsenate as the sole electron donor for anoxygenic photosynthesis. These anoxic cultures were able to convert a 1 mM mixture of thioarsenates completely to arsenate in c. 13 days and 4 mM monothioarsenate to arsenate in c. 17 days. This conversion was light dependent; thus, monothioarsenate can be used as the sole electron donor for anoxygenic photosynthesis. Both of the Mono Lake and Big Soda Lake enrichment cultures were dominated by an organism closely related to Ectothiorhodospira species. We tested additional strains of purple sulfur bacteria and found widespread ability to use monothioarsenate as an electron donor. The ability of bacteria to transform thioarsenates directly via anoxygenic photosynthesis adds a new perspective to the well-studied arsenic and sulfur cycles.

Protistan grazing in a meromictic freshwater lake with anoxic bottom water.

Phagotrophic protists are an important mortality factor of prokaryotes in most aquatic habitats. However, no study has assessed protistan grazing as loss factor of bacterial biomass across the stratification gradient of a temperate freshwater meromictic lake. Protistan grazing effect was quantified in the mixolimnion, the transition zone, and the sulfidic anoxic monimolimnion of Lake Alatsee (Germany). Grazing experiments were performed using prey analogues from the natural prokaryotic assemblage. Daily grazing effect declined from the mixolimnion to the monimolimnion. Heterotrophic flagellates were phagotrophically active in all three water horizons and the main grazers in the monimolimnion. Pigmented flagellates accounted for 70% of total grazing in the mixolimnion and ciliates only for a small fraction of grazing in each depth. Prokaryotic biomass removal peaked in the interface, but protistan impact on the respective prokaryotic abundance was low. Grazing in the anoxic monimolimnion was negligible, with prokaryotic turnover rate being only 0.4% of standing stock. Our results support the assumption that protistan predation in anoxic waters is lower than in oxygenated ones and identify the interface as a microhabitat that supports high grazer biomass, pinpointing the importance of purple sulfur bacteria as carbon source for the upper mixolimnion and the bottom monimolimnion.

Genome-wide transcriptional profiling of the purple sulfur bacterium Allochromatium vinosum DSM 180T during growth on different reduced sulfur compounds.

The purple sulfur bacterium Allochromatium vinosum DSM 180(T) is one of the best-studied sulfur-oxidizing anoxygenic phototrophic bacteria, and it has been developed into a model organism for laboratory-based studies of oxidative sulfur metabolism. Here, we took advantage of the organism's high metabolic versatility and performed whole-genome transcriptional profiling to investigate the response of A. vinosum cells upon exposure to sulfide, thiosulfate, elemental sulfur, or sulfite compared to photoorganoheterotrophic growth on malate. Differential expression of 1,178 genes was observed, corresponding to 30% of the A. vinosum genome. Relative transcription of 551 genes increased significantly during growth on one of the different sulfur sources, while the relative transcript abundance of 627 genes decreased. A significant number of genes that revealed strongly enhanced relative transcription levels have documented sulfur metabolism-related functions. Among these are the dsr genes, including dsrAB for dissimilatory sulfite reductase, and the sgp genes for the proteins of the sulfur globule envelope, thus confirming former results. In addition, we identified new genes encoding proteins with appropriate subcellular localization and properties to participate in oxidative dissimilatory sulfur metabolism. Those four genes for hypothetical proteins that exhibited the strongest increases of mRNA levels on sulfide and elemental sulfur, respectively, were chosen for inactivation and phenotypic analyses of the respective mutant strains. This approach verified the importance of the encoded proteins for sulfur globule formation during the oxidation of sulfide and thiosulfate and thereby also documented the suitability of comparative transcriptomics for the identification of new sulfur-related genes in anoxygenic phototrophic sulfur bacteria.

Light-harvesting complexes from purple sulfur bacteria Allochromatium minutissimum assembled without carotenoids.

Effect of carotenoid (Car) biosynthesis inhibitor diphenylamine (DPA) on purple sulfur bacteria Allochromatium (Alc) minutissimum cell growth has been investigated. Cell growth in the presence of maximum concentration of DPA results in practically complete suppression (∼99%) of carotenoids (Cars) according to the spectrophotometric, HPLC and CD data. Phytoene does not replace the colored carotenoids in these cells. Also Phytoene does not accumulate in large amounts in the cells treated with DPA. A new method for calculating the content of Cars in the complexes from the cells with inhibited Car synthesis including the number of empty Car's "pockets" has been used. Our results together with published data devoted to DPA action on the cell growth of purple bacteria revealed that Phytoene was not accumulated in the cells treated with DPA. We have concluded that (i) DPA completely inhibits or strongly reduces synthesis of the colored Cars in the cells of purple bacteria, (ii) Phytoene is the main one among the trace amounts of the other Cars in the case of significant inhibition of Car biosynthesis (80-90% or higher). The amount of the LH2 complexes presented in the membranes of Alc minutissimum was found to be little dependent on DPA. From DPA-grown cultures it was possible to isolate Car-less both the LH1 (as LH1-RC complex) and the LH2 complexes. Electronic absorption properties of BChl's were very similar to those isolated from the control cells. It is shown by HPLC data that the 100 LH2 complexes from cells of Alc minutissimum, in which the synthesis of Car was depressed, contained ∼9 Car molecules and 5 Phytoene molecules. Thus, only nine (with 1 Car molecule per a complex) or less (if more than one Car molecule per a complex) of the 100 LH2 complexes contain molecules of Cars. It means that 90 or more LH2 complexes from each 100 ones are assembled without any Cars. This is in strong contrast with the previous results obtained with purple non-sulfur bacterium Rhodobacter sphaeroides, where the amount of LH2 presented in the membrane was directly correlated to the amount of the carotenoids synthesized (H.P. Lang, C.N. Hunter, The relationship between carotenoid biosynthesis and the assembly of the light harvesting LH2 complex in Rhodobacter sphaeroides, Biochem. J. 298 (1994) 197-205). Our results show that although the presence of Car molecules is important for the stability of the LH2 complexes the overall native structure can be maintained without any Cars at least in the case of purple sulfur bacteria.

Communities of purple sulfur bacteria in a Baltic Sea coastal lagoon analyzed by puf LM gene libraries and the impact of temperature and NaCl concentration in experimental enrichment cultures.

Shallow coastal waters, where phototrophic purple sulfur bacteria (PSB) regularly form massive blooms, are subjected to massive diurnal and event-driven changes of physicochemical conditions including temperature and salinity. To analyze the ability of PSB to cope with these environmental factors and to compete in complex communities we have studied changes of the environmental community of PSB of a Baltic Sea lagoon under experimental enrichment conditions with controlled variation of temperature and NaCl concentration. For the first time, changes within a community of PSB were specifically analyzed using the photosynthetic reaction center genes pufL and M by RFLP and cloning experiments. The most abundant PSB phylotypes in the habitat were found along the NaCl gradient from freshwater conditions up to 7.5% NaCl. They were accompanied by smaller numbers of purple nonsulfur bacteria and aerobic anoxygenic phototrophic bacteria. Major components of the PSB community of the brackish lagoon were affiliated to PSB genera and species known as marine, halophilic or salt-tolerant, including species of M arichromatium, H alochromatium, T hiorhodococcus, A llochromatium, T hiocapsa, T hiorhodovibrio, and T hiohalocapsa. A dramatic shift occurred at elevated temperatures of 41 and 44°C when M arichromatium gracile became most prominent which was not detected at lower temperatures.

Tools providing new insight into coastal anoxygenic purple bacterial mats: review and perspectives.

Coastal photosynthetic microbial mats are highly structured microbial communities that populate a variety of shallow environments such as estuaries, sheltered sandy beaches, intertidal flats, salt marshes and hypersaline salterns. In soft sediments, most of these microbial mats are formed of vertically stratified, multicolored cohesive thin layers, of several functional groups of microorganisms, such as cyanobacteria, colorless sulfur bacteria, purple sulfur bacteria and sulfate-reducing bacteria, distributed along vertical microgradients of oxygen, sulfide and light. These microbial communities are highly productive and significant contributors to carbon, nitrogen and sulfur cycles and to sediment stability in shallow-water habitats. Many examples of these communities have been cited in the past, but comparatively few microbial mats have been presented for which mass developments of anoxygenic purple bacteria have been observed. Yet, application of molecular approaches has provided fresh insight into the ecology, diversity and evolution of microbial mats. In situ measurements using electrochemical and optical microprobes led to detailed characterization of their physical and chemical environment, whereas reflectance measurements revealed the spatial and temporal heterogeneity of microbial mat surfaces. We hereby report the main discoveries due to introduction of these powerful techniques and we point out the potential insight to be gained from the study of anoxygenic purple bacterial mats.