Exploring Viral Diversity in a Gypsum Karst Lake Ecosystem Using Targeted Single-Cell Genomics.

Little is known about the diversity and distribution of viruses infecting green sulfur bacteria (GSB) thriving in euxinic (sulfuric and anoxic) habitats, including gypsum karst lake ecosystems. In this study, we used targeted cell sorting combined with single-cell sequencing to gain insights into the gene content and genomic potential of viruses infecting sulfur-oxidizing bacteria Chlorobium clathratiforme, obtained from water samples collected during summer stratification in gypsum karst Lake Kirkilai (Lithuania). In total, 82 viral contigs were bioinformatically identified in 62 single amplified genomes (SAGs) of C. clathratiforme. The majority of viral gene and protein sequences showed little to no similarity with phage sequences in public databases, uncovering the vast diversity of previously undescribed GSB viruses. We observed a high level of lysogenization in the C. clathratiforme population, as 87% SAGs contained intact prophages. Among the thirty identified auxiliary metabolic genes (AMGs), two, thiosulfate sulfurtransferase (TST) and thioredoxin-dependent phosphoadenosine phosphosulfate (PAPS) reductase (cysH), were found to be involved in the oxidation of inorganic sulfur compounds, suggesting that viruses can influence the metabolism and cycling of this essential element. Finally, the analysis of CRISPR spacers retrieved from the consensus C. clathratiforme genome imply persistent and active virus-host interactions for several putative phages prevalent among C. clathratiforme SAGs. Overall, this study provides a glimpse into the diversity of phages associated with naturally occurring and highly abundant sulfur-oxidizing bacteria.

Structural Insight into a Membrane Intrinsic Acyltransferase from Chlorobium tepidum.

The Lipid A component of the outer membrane of Gram-negative bacteria is an integral part of the permeability barrier known as LPS, which actively prevents the uptake of bactericidal compounds. It is clinically very significant, as it is known to elicit a strong immune response in the humans, through the TLR4 complex. The Lipid A species are synthesized through a highly conserved multistep biosynthetic pathway. The final step is catalyzed by acyltransferases of the HtrB/MsbB family, which are members of a superfamily of enzymes, present in all domains of life with important roles to play in various biological processes. The investigation of a putative dual functioning enzyme which can add both laurate and myristate residues to the (Kdo)2-lipid IVA (precursor of Lipid A) would give a snapshot into the versatility of substrates that these enzymes catalyze. In this study we have cloned and purified to homogeneity, such a putative dual functional acyltransferase from Chlorobium tepidum, and attempted to study the enzyme in more details in terms of its sequence and structural aspects, as it lacks conserved residues with other enzymes of the same family.

In situ abundance and carbon fixation activity of distinct anoxygenic phototrophs in the stratified seawater lake Rogoznica.

Sulphide-driven anoxygenic photosynthesis is an ancient microbial metabolism that contributes significantly to inorganic carbon fixation in stratified, sulphidic water bodies. Methods commonly applied to quantify inorganic carbon fixation by anoxygenic phototrophs, however, cannot resolve the contributions of distinct microbial populations to the overall process. We implemented a straightforward workflow, consisting of radioisotope labelling and flow cytometric cell sorting based on the distinct autofluorescence of bacterial photopigments, to discriminate and quantify contributions of co-occurring anoxygenic phototrophic populations to in situ inorganic carbon fixation in environmental samples. This allowed us to assign 89.3% ± 7.6% of daytime inorganic carbon fixation by anoxygenic phototrophs in Lake Rogoznica (Croatia) to an abundant chemocline-dwelling population of green sulphur bacteria (dominated by Chlorobium phaeobacteroides), whereas the co-occurring purple sulphur bacteria (Halochromatium sp.) contributed only 1.8% ± 1.4%. Furthermore, we obtained two metagenome assembled genomes of green sulphur bacteria and one of a purple sulphur bacterium which provides the first genomic insights into the genus Halochromatium, confirming its high metabolic flexibility and physiological potential for mixo- and heterotrophic growth.

The importance of ergothioneine synthesis in ancient time by organisms living in oxygen free atmosphere.

The paper published by Ruczyszky and Liu (2017) reports on the biosynthesis of ergothioneine under both aerobic and anaerobic conditions. We would like to suggest a hypothesis as to what could be the reason that microorganisms on the Earth synthesized ergothioneine under anaerobic conditions.

Substrate and electron donor limitation induce phenotypic heterogeneity in different metabolic activities in a green sulphur bacterium.

Populations of genetically identical cells can display marked variation in phenotypic traits; such variation is termed phenotypic heterogeneity. Here, we investigate the effect of substrate and electron donor limitation on phenotypic heterogeneity in N2 and CO2 fixation in the green sulphur bacterium Chlorobium phaeobacteroides. We grew populations in chemostats and batch cultures and used stable isotope labelling combined with nanometer-scale secondary ion mass spectrometry (NanoSIMS) to quantify phenotypic heterogeneity. Experiments in H2 S (i.e. electron donor) limited chemostats show that varying levels of NH4+ limitation induce heterogeneity in N2 fixation. Comparison of phenotypic heterogeneity between chemostats and batch (unlimited for H2 S) populations indicates that electron donor limitation drives heterogeneity in N2 and CO2 fixation. Our results demonstrate that phenotypic heterogeneity in a certain metabolic activity can be driven by different modes of limitation and that heterogeneity can emerge in different metabolic processes upon the same mode of limitation. In conclusion, our data suggest that limitation is a general driver of phenotypic heterogeneity in microbial populations.

Microbial communities and organic biomarkers in a Proterozoic-analog sinkhole.

Little Salt Spring (Sarasota County, FL, USA) is a sinkhole with groundwater vents at ~77 m depth. The entire water column experiences sulfidic (~50 µM) conditions seasonally, resulting in a system poised between oxic and sulfidic conditions. Red pinnacle mats occupy the sediment-water interface in the sunlit upper basin of the sinkhole, and yielded 16S rRNA gene clones affiliated with Cyanobacteria, Chlorobi, and sulfate-reducing clades of Deltaproteobacteria. Nine bacteriochlorophyll e homologues and isorenieratene indicate contributions from Chlorobi, and abundant chlorophyll a and pheophytin a are consistent with the presence of Cyanobacteria. The red pinnacle mat contains hopanoids, including 2-methyl structures that have been interpreted as biomarkers for Cyanobacteria. A single sequence of hpnP, the gene required for methylation of hopanoids at the C-2 position, was recovered in both DNA and cDNA libraries from the red pinnacle mat. The hpnP sequence was most closely related to cyanobacterial hpnP sequences, implying that Cyanobacteria are a source of 2-methyl hopanoids present in the mat. The mats are capable of light-dependent primary productivity as evidenced by 13 C-bicarbonate photoassimilation. We also observed 13 C-bicarbonate photoassimilation in the presence of DCMU, an inhibitor of electron transfer to Photosystem II. Our results indicate that the mats carry out light-driven primary production in the absence of oxygen production-a mechanism that may have delayed the oxygenation of the Earth's oceans and atmosphere during the Proterozoic Eon. Furthermore, our observations of the production of 2-methyl hopanoids by Cyanobacteria under conditions of low oxygen and low light are consistent with the recovery of these structures from ancient black shales as well as their paucity in modern marine environments.

Anaerobic Origin of Ergothioneine.

Ergothioneine is a sulfur metabolite that occurs in microorganisms, fungi, plants, and animals. The physiological function of ergothioneine is not clear. In recent years broad scientific consensus has formed around the idea that cellular ergothioneine primarily protects against reactive oxygen species. Herein we provide evidence that this focus on oxygen chemistry may be too narrow. We describe two enzymes from the strictly anaerobic green sulfur bacterium Chlorobium limicola that mediate oxygen-independent biosynthesis of ergothioneine. This anoxic origin suggests that ergothioneine is also important for oxygen-independent life. Furthermore, one of the discovered ergothioneine biosynthetic enzymes provides the first example of a rhodanese-like enzyme that transfers sulfur to non-activated carbon.

Physiological characterization of a halotolerant anoxygenic phototrophic Fe(II)-oxidizing green-sulfur bacterium isolated from a marine sediment.

Anoxygenic photoautotrophic bacteria which use light energy and electrons from Fe(II) for growth, so-called photoferrotrophs, are suggested to have been amongst the first phototrophic microorganisms on Earth and to have contributed to the deposition of sedimentary iron mineral deposits, i.e. banded iron formations. To date only two isolates of marine photoferrotrophic bacteria exist, both of which are closely related purple non-sulfur bacteria. Here we present a novel green-sulfur photoautotrophic Fe(II) oxidizer isolated from a marine coastal sediment, Chlorobium sp. strain N1, which is closely related to the freshwater green-sulfur bacterium Chlorobium luteolum DSM273 that is incapable of Fe(II) oxidation. Besides Fe(II), our isolated strain grew phototrophically with other inorganic and organic substrates such as sulfide, hydrogen, lactate or yeast extract. Highest Fe(II) oxidation rates were measured at pH 7.0-7.3, the temperature optimum was 25°C. Mössbauer spectroscopy identified ferrihydrite as the main Fe(III) mineral and fluorescence and helium-ion microscopy revealed cell-mineral aggregates without obvious cell encrustation. In summary, our study showed that the new isolate is physiologically adapted to the conditions of its natural habitat but also to conditions as proposed for early Earth and is thus a suitable model organism for further studies addressing phototrophic Fe(II) oxidation on early Earth.

Speciation and ecological success in dimly lit waters: horizontal gene transfer in a green sulfur bacteria bloom unveiled by metagenomic assembly.

A natural planktonic bloom of a brown-pigmented photosynthetic green sulfur bacteria (GSB) from the disphotic zone of karstic Lake Banyoles (NE Spain) was studied as a natural enrichment culture from which a nearly complete genome was obtained after metagenomic assembly. We showed in situ a case where horizontal gene transfer (HGT) explained the ecological success of a natural population unveiling ecosystem-specific adaptations. The uncultured brown-pigmented GSB was 99.7% identical in the 16S rRNA gene sequence to its green-pigmented cultured counterpart Chlorobium luteolum DSM 273T. Several differences were detected for ferrous iron acquisition potential, ATP synthesis and gas vesicle formation, although the most striking trait was related to pigment biosynthesis strategy. Chl. luteolum DSM 273T synthesizes bacteriochlorophyll (BChl) c, whereas Chl. luteolum CIII incorporated by HGT a 18-kbp cluster with the genes needed for BChl e and specific carotenoids biosynthesis that provided ecophysiological advantages to successfully colonize the dimly lit waters. We also genomically characterized what we believe to be the first described GSB phage, which based on the metagenomic coverage was likely in an active state of lytic infection. Overall, we observed spread HGT and we unveiled clear evidence for virus-mediated HGT in a natural population of photosynthetic GSB.

Solution Binding and Structural Analyses Reveal Potential Multidrug Resistance Functions for SAV2435 and CTR107 and Other GyrI-like Proteins.

Multidrug resistance (MDR) refers to the acquired ability of cells to tolerate a broad range of toxic compounds. One mechanism cells employ is to increase the level of expression of efflux pumps for the expulsion of xenobiotics. A key feature uniting efflux-related mechanisms is multidrug (MD) recognition, either by efflux pumps themselves or by their transcriptional regulators. However, models describing MD binding by MDR effectors are incomplete, underscoring the importance of studies focused on the recognition elements and key motifs that dictate polyspecific binding. One such motif is the GyrI-like domain, which is found in several MDR proteins and is postulated to have been adapted for small-molecule binding and signaling. Here we report the solution binding properties and crystal structures of two proteins containing GyrI-like domains, SAV2435 and CTR107, bound to various ligands. Furthermore, we provide a comparison with deposited crystal structures of GyrI-like proteins, revealing key features of GyrI-like domains that not only support polyspecific binding but also are conserved among GyrI-like domains. Together, our studies suggest that GyrI-like domains perform evolutionarily conserved functions connected to multidrug binding and highlight the utility of these types of studies for elucidating mechanisms of MDR.

Kinetics of NADP+/NADPH reduction-oxidation catalyzed by the ferredoxin-NAD(P)+ reductase from the green sulfur bacterium Chlorobaculum tepidum.

Ferredoxin-NAD(P)+ oxidoreductase (FNR, [EC 1.18.1.2], [EC 1.18.1.3]) from the green sulfur bacterium Chlorobaculum tepidum (CtFNR) is a homodimeric flavoprotein with significant structural homology to bacterial NADPH-thioredoxin reductases. CtFNR homologs have been found in many bacteria, but only in green sulfur bacteria among photoautotrophs. In this work, we examined the reactions of CtFNR with NADP+, NADPH, and (4S-2H)-NADPD by stopped-flow spectrophotometry. Mixing CtFNRox with NADPH yielded a rapid decrease of the absorbance in flavin band I centered at 460 nm within 1 ms, and then the absorbance further decreased gradually. The magnitude of the decrease increased with increasing NADPH concentration, but even with ~50-fold molar excess NADPH, the absorbance change was only ~45 % of that expected for fully reduced protein. The absorbance in the charge transfer (CT) band centered around 600 nm increased rapidly within 1 ms, then slowly decreased to about 70 % of the maximum. When CtFNRred was mixed with excess NADP+, the absorbance in the flavin band I increased to about 70 % of that of CtFNRox with an apparent rate of ~4 s-1, whereas almost no absorption changes were observed in the CT band. Obtained data suggest that the reaction between CtFNR and NADP+/NADPH is reversible, in accordance with its physiological function.

The Path to Thioredoxin and Redox Regulation in Chloroplasts.

After a brief discussion of my graduate work at Duke University, I describe a series of investigations on redox proteins at the University of California, Berkeley. Starting with ferredoxin from fermentative bacteria, the Berkeley research fostered experiments that uncovered a pathway for fixing CO2 in bacterial photosynthesis. The carbon work, in turn, opened new vistas, including the discovery that thioredoxin functions universally in regulating the Calvin-Benson cycle in oxygenic photosynthesis. These experiments, which took place over a 50-year period, led to the formulation of a set of biological principles and set the stage for research demonstrating a role for redox in the regulation of previously unrecognized processes extending far beyond photosynthesis.

A comparison of the protein-coding genomes of two green sulphur bacteria, Chlorobium tepidum TLS and Pelodictyon phaeoclathratiforme BU-1.

BACKGROUND: Chlorobium tepidum and Pelodictyon phaeoclathratiforme are organisms within the green sulphur bacteria family, Chlorobiaceae, occupying very different habitats. It has recently been proposed that the genera Chlorobium and Pelodictyon are synonymous. RESULTS: To investigate generic boundaries for the two species, protein families were predicted computationally based on sequence similarity across the genome-wide protein sets of Chlorobium tepidum TLS and Pelodictyon phaeoclathratiforme BU-1. The distribution of the resulting protein families across the two species was summarized. The largest number of families exhibited 1:1 putative orthology between the two species (1468 families). Of families unique to one of the species, the largest number was unique to P. phaeoclathratiforme (113 families), of which the largest family contained pentapeptide repeat proteins (16 proteins). Families unique to P. phaeoclathratiforme also included a family of gas vesicle synthesis proteins (four proteins). Although only seven families were identified as containing paralogous proteins in both species (with two or more proteins in each species), this group included families of major biochemical importance. One such family, with three members in each species, contained magnesium chelatase, an enzyme involved in the chlorophyll biosynthetic pathway. CONCLUSION: The unique protein family groups in both C. tepidum and P. phaeoclathratiforme mirror the occupancy of different environments, while key shared family groups provide evidence for a common origin for the species, as previously suggested in the literature. The current study only uses sequence similarity-based protein families for the two species. This, alone, does not permit a firm conclusion to be drawn on the taxonomic question, of whether the two species belong in one genus or two.

Conformational heterogeneity of the Roc domains in C. tepidum Roc-COR and implications for human LRRK2 Parkinson mutations.

Ras of complex proteins (Roc) is a Ras-like GTP-binding domain that always occurs in tandem with the C-terminal of Roc (COR) domain and is found in bacteria, plants and animals. Recently, it has been shown that Roco proteins belong to the family of G-proteins activated by nucleotide (nt)-dependent dimerization (GADs). We investigated the RocCOR tandem from the bacteria Chlorobium tepidum with site-directed spin labelling and pulse EPR distance measurements to follow conformational changes during the Roco G-protein cycle. Our results confirm that the COR domains are a stable dimerization device serving as a scaffold for the Roc domains that, in contrast, are structurally heterogeneous and dynamic entities. Contrary to other GAD proteins, we observed only minor structural alterations upon binding and hydrolysis of GTP, indicating significant mechanistic variations within this protein class. Mutations in the most prominent member of the Roco family of proteins, leucine-rich repeat (LRR) kinase 2 (LRRK2), are the most frequent cause of late-onset Parkinson's disease (PD). Using a stable recombinant LRRK2 Roc-COR-kinase fragment we obtained detailed kinetic data for the G-protein cycle. Our data confirmed that dimerization is essential for efficient GTP hydrolysis and PD mutations in the Roc domain result in decreased GTPase activity. Previous data have shown that these LRRK2 PD-mutations are located in the interface between Roc and COR. Importantly, analogous mutations in the conserved C. tepidum Roc/COR interface significantly influence the structure and nt-induced conformational changes of the Roc domains.

Biological sulphide removal from anaerobically treated domestic sewage: reactor performance and microbial community dynamics.

We developed a biological sulphide oxidation system and evaluated two reactors (shaped similar to the settler compartment of an up-flow anaerobic sludge blanket [UASB] reactor) with different support materials for biomass retention: polypropylene rings and polyurethane foam. The start-up reaction was achieved using microorganisms naturally occurring on the open surface of UASB reactors treating domestic wastewater. Sulphide removal efficiencies of 65% and 90% were achieved with hydraulic retention times (HRTs) of 24 and 12 h, respectively, in both reactors. However, a higher amount of elemental sulphur was formed and accumulated in the biomass from reactor 1 (20 mg S(0) g(-1) VTS) than in that from reactor 2 (2.9 mg S(0) g(-1) VTS) with an HRT of 24 h. Denaturing gradient gel electrophoresis (DGGE) results revealed that the the pink and green biomass that developed in both reactors comprised a diverse bacterial community and had sequences related to phototrophic green and purple-sulphur bacteria such as Chlorobium sp., Chloronema giganteum, and Chromatiaceae. DGGE band patterns also demonstrated that bacterial community was dynamic over time within the same reactor and that different support materials selected for distinct bacterial communities. Taken together, these results indicated that sulphide concentrations of 1-6 mg L(-1) could be efficiently removed from the effluent of a pilot-scale UASB reactor in two sulphide biological oxidation reactors at HRTs of 12 and 24 h, showing the potential for sulphur recovery from anaerobically treated domestic wastewater.

Vibrational beatings conceal evidence of electronic coherence in the FMO light-harvesting complex.

In biological light harvesting, solar energy is captured by photosynthetic antennae for subsequent storage into chemical bonds. The remarkable efficiency reached in transferring the energy between the collection and storage events recently has been attributed to long-lived electronic coherence present in such antennae systems. We present numerical simulations indicating that the spectroscopic transients that supported this hypothesis are not induced by electronic coherence but instead are caused by vibrational (nuclear) motion in the electronic ground state potential. Besides emphasizing the significance of such nuclear modes, our findings stimulate a reconsideration of the role of electronic coherence in promoting energy transfer in natural photosynthesis. Furthermore, they require us to rethink how energy transfer efficiency is reflected in spectral signals.

The crystal structure of ferritin from Chlorobium tepidum reveals a new conformation of the 4-fold channel for this protein family.

Ferritins are ubiquitous iron-storage proteins found in all kingdoms of life. They share a common architecture made of 24 subunits of five α-helices. The recombinant Chlorobium tepidum ferritin (rCtFtn) is a structurally interesting protein since sequence alignments with other ferritins show that this protein has a significantly extended C-terminus, which possesses 12 histidine residues as well as several aspartate and glutamic acid residues that are potential metal ion binding residues. We show that the macromolecular assembly of rCtFtn exhibits a cage-like hollow shell consisting of 24 monomers that are related by 4-3-2 symmetry; similar to the assembly of other ferritins. In all ferritins of known structure the short fifth α-helix adopts an acute angle with respect to the four-helix bundle. However, the crystal structure of the rCtFtn presented here shows that this helix adopts a new conformation defining a new assembly of the 4-fold channel of rCtFtn. This conformation allows the arrangement of the C-terminal region into the inner cavity of the protein shell. Furthermore, two Fe(III) ions were found in each ferroxidase center of rCtFtn, with an average FeA-FeB distance of 3 Å; corresponding to a diferric µ-oxo/hydroxo species. This is the first ferritin crystal structure with an isolated di-iron center in an iron-storage ferritin. The crystal structure of rCtFtn and the biochemical results presented here, suggests that rCtFtn presents similar biochemical properties reported for other members of this protein family albeit with distinct structural plasticity.

Resonant coherence in photosynthetic electronic energy transfer by site-dependent pigment-protein interactions.

We have numerically examined the effect of site-dependent reorganization on the dynamics of coherent electronic excitation energy transfer in a donor and acceptor pair of photosynthetic pigment-protein complex. Using the quasi-adiabatic propagator path integral method (QUAPI), we have found that a specific proportionality between the site-energy mismatch ϵ and the site-reorganization energy mismatch λ simultaneously increases the length and robustness of the quantum coherence. This behavior is associated with a Rabi type resonance that is manifested in the amplitude and frequency in the coherent portion of the population dynamics. Impact of the resonance on robustness of the coherence under static disorder is also discussed.

Photophysical properties of the excited states of bacteriochlorophyll f in solvents and in chlorosomes.

Bacteriochlorophyll f (BChl f) is a photosynthetic pigment predicted nearly 40 years ago as a fourth potential member of the Chlorobium chlorophyll family (BChl c, d, and e). However, this pigment still has not been found in a naturally occurring organism. BChl c, d, and e are utilized by anoxygenic green photosynthetic bacteria for assembly of chlorosomes--large light-harvesting complexes that allow those organisms to survive in habitats with extremely low light intensities. Recently, using genetic methods on two different strains of Chlorobaculum limnaeum that naturally produce BChl e, two research groups produced mutants that synthesize BChl f and assemble it into chlorosomes. In this study, we present detailed investigations on spectral and dynamic characteristics of singlet excited and triplet states of BChl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The studies were performed on isolated BChl f in various solvents, at different temperatures, and on BChl f-containing chlorosomes in order to uncover any unusual or unfavorable properties that stand behind the lack of appearance of this pigment in natural environments.

Coupling dark metabolism to electricity generation using photosynthetic cocultures.

We investigated the role of green sulfur bacteria inlight-responsive electricity generation in microbial electrochemical cells (MXCs). We operated MXCs containing either monocultures or defined cocultures of previously enriched phototrophic Chlorobium and anode-respiring Geobacter under anaerobic conditions in the absence of electron donor. Monoculture control MXCs containing Geobacter or Chlorobium neither responded to light nor produced current, respectively. Instead, light-responsive current generation occurred only in coculture MXCs. Current increased above background levels only in the dark and declined slowly over 96 h. This pattern suggested that Chlorobium exhausted intracellular glycogen reserves via dark fermentation to supply an electron donor, presumably acetate, to Geobacter. With medium containing sulfide as the sole photosynthetic electron donor, current generation had a similar and reproducible negative light response. To investigate whether this metabolic interaction also occurred without an electrode, we performed coculture experiments in batch serum bottles. In this setup, sulfide served as the sole electron donor, whose oxidation by Chlorobium was required to provide S(0) as the electron acceptor to Geobacter. Copies of Geobacter 16S rDNA increased approximately 14-fold in batch bottle cocultures containing sulfide compared to those lacking sulfide, and did not decline after termination of sulfide feeding. These results suggest that products of both photosynthesis and dark fermentation by Chlorobium were sufficient both to yield an electrochemical response by Geobacter biofilms, and to promote Geobacter growthin batch cocultures. Our work expands upon the fusion of MXCs with coculture techniques and reinforces the utility of microbial electrochemistry for sensitive, real-time monitoring of microbial interactions in which a metabolic intermediate can be converted to electrical current.