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Light-absorbing chromophores in photoreceptors contain a π-electron system and are intrinsically planar molecules. However, within a protein environment these cofactors often become nonplanar and chiral in a manner that is widely believed to be functionally important. When the same chromophore is out-of-plane distorted in opposite directions in different members of a protein family, such conformers become a set of enantiomers. In techniques using chiral optical spectroscopy such as Raman optical activity (ROA), such proteins are expected to show opposite signs in their spectra. Here we use two microbial rhodopsins, Gloeobacter rhodopsin and sodium ion pump rhodopsin (NaR), to provide the first experimental and theoretical evidence that the twist direction of the retinal chromophore indeed determines the sign of the ROA spectrum. We disrupt the hydrogen bond responsible for the distortion of the retinal in NaR and show that the sign of the ROA signals of this nonfunctional mutant is flipped. The reported ROA spectra are monosignate, a property that has been seen for a variety of photoreceptors, which we attribute to an energetically favorable gradual curvature of the chromophore.

Clues to the evolutionary steps producing innovations in oxygenic photosynthesis may be preserved in the genomes of organisms phylogenetically placed between non-photosynthetic Vampirovibrionia (formerly Melainabacteria) and the thylakoid-containing Cyanobacteria. However, only two species with published genomes are known to occupy this phylogenetic space, both within the genus Gloeobacter. Here, we describe nearly complete, metagenome-assembled genomes (MAGs) of an uncultured organism phylogenetically placed near Gloeobacter, for which we propose the name Candidatus Aurora vandensis {Au’ro.ra. L. fem. n. aurora, the goddess of the dawn in Roman mythology; van.de’nsis. N.L. fem. adj. vandensis of Lake Vanda, Antarctica}. The MAG of A. vandensis contains homologs of most genes necessary for oxygenic photosynthesis including key reaction center proteins. Many accessory subunits associated with the photosystems in other species either are missing from the MAG or are poorly conserved. The MAG also lacks homologs of genes associated with the pigments phycocyanoerethrin, phycoeretherin and several structural parts of the phycobilisome. Additional characterization of this organism is expected to inform models of the evolution of oxygenic photosynthesis.

Photosystem I (PSI) is a multi-subunit pigment-protein complex that functions in light-harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO2 fixation in photosynthetic organisms. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the sites of low-energy Chls are still under debate. Here, we solved a 2.04-Å resolution structure of a PSI trimer by cryo-electron microscopy from a primordial cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure shows the absence of some subunits commonly found in other cyanobacteria, confirming the primordial nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 is missing in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. These findings provide insights into not only the identity of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls in oxyphototrophs.

The appearance of oxygenic photosynthesis in cyanobacteria is a major event in evolution. It had an irreversible impact on the Earth, promoting the Great Oxygenation Event (GOE) ~2.4 billion years ago. Ancient cyanobacteria predating the GOE were Gloeobacter-type cells lacking thylakoids, which hosted photosystems in their cytoplasmic membrane. The driver of the GOE was proposed to be the transition from unicellular to filamentous cyanobacteria. However, the appearance of thylakoids expanded the photosynthetic surface to such an extent that it introduced a multiplier effect, which would be more coherent with an impact on the atmosphere. Primitive thylakoids self-organize as concentric parietal uninterrupted multilayers. There is no robust evidence for an origin of thylakoids via a vesicular-based scenario. This review reports studies supporting that hexagonal II-forming glucolipids and galactolipids at the periphery of the cytosolic membrane could be turned, within nanoseconds and without any external source of energy, into membrane multilayers. Comparison of lipid biosynthetic pathways shows that ancient cyanobacteria contained only one anionic lamellar-forming lipid, phosphatidylglycerol. The acquisition of sulfoquinovosyldiacylglycerol biosynthesis correlates with thylakoid emergence, possibly enabling sufficient provision of anionic lipids to trigger a hexagonal II-to-lamellar phase transition. With this non-vesicular lipid-phase transition, a framework is also available to re-examine the role of companion proteins in thylakoid biogenesis.

Only two complete genomes of the cyanobacterial genus Gloeobacter from two very different regions of the world currently exist. Here, we present the complete genome sequence of a third member of the genus isolated from a waterfall cave in Mexico. Analysis of the average nucleotide identities (ANIs) between published Gloeobacter genomes revealed that the complete genome of this new member is only 92.7% similar to Gloeobacter violaceus and therefore we determined it to be a new species. We propose to name this new species Gloeobacter morelensis after the location in Mexico where it was isolated. The complete genome consists of one circular chromosome (4,921,229 bp), one linear plasmid (172,328 bp), and one circular plasmid (8,839 bp). Its genome is the largest of all completely sequenced genomes of Gloeobacter species. Pangenomic comparisons revealed that G. morelensis encodes 759 genes not shared with other Gloeobacter species. Despite being more closely related to G. violaceus, it features an extremely divergent psbA gene encoding an atypical D1 core subunit of Photosystem II previously only found within the genome of Gloeobacter kilaueensis. In addition, we detected evidence of concerted evolution of psbA genes encoding identical D1 in all three Gloeobacter genomes, a characteristic that seems widespread in cyanobacteria and may therefore be traced back to their last common ancestor.

Photosystem I (PSI) of photosynthetic organisms is a multi-subunit pigment-protein complex and functions in light harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO2 fixation. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the site of low-energy Chls is still under debate. Here, we solved a 2.04-Å resolution structure of a PSI trimer by cryo-electron microscopy from a primitive cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure showed absence of some subunits commonly found in other cyanobacteria, confirming the primitive nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 does not exist in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. Since Gloeobacter is susceptible to light, this indicates that Low1 serves as a main photoprotection site in most oxyphototrophs, whereas Low2 is involved in either energy transfer or energy quenching in some of the oxyphototrophs. Thus, these findings provide insights into not only the functional significance of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls responsible for the photoprotection machinery from photosynthetic prokaryotes to eukaryotes.

Photosynthetic Cyanobacteria and their descendants are the only known organisms capable of oxygenic photosynthesis. Their metabolism permanently changed the Earth's surface and the evolutionary trajectory of life, but little is known about their evolutionary history. Genomes of the Gloeobacterales, an order of deeply divergent photosynthetic Cyanobacteria, may hold clues about the evolutionary process. However, there are only three published genomes within this order, and it is difficult to make broad inferences based on such little data. Here, I describe five species within the Gloeobacterales retrieved from publicly available databases and examine their photosynthetic gene content and the environments in which Gloeobacterales genomes and 16S rRNA gene sequences are found. The Gloeobacterales contain reduced photosystems and inhabit cold, wet-rock, and low-light environments. They are likely present in low abundances due to their low growth rate. Future searches for Gloeobacterales should target these environments, and samples should be deeply sequenced to capture the low-abundance taxa. Publicly available databases contain undescribed taxa within the Gloeobacterales. However, searching through all available data with current methods is computationally expensive. Therefore, new methods must be developed to search for these and other evolutionarily important taxa. Once identified, these novel photosynthetic Cyanobacteria will help illuminate the origin and evolution of oxygenic photosynthesis. IMPORTANCE Early branching photosynthetic Cyanobacteria such as the Gloeobacterales may provide clues into the evolutionary history of oxygenic photosynthesis, but there are few genomes or cultured taxa from this order. Five new metagenome-assembled genomes suggest that members of the Gloeobacterales all contain reduced photosystems and lack genes associated with thylakoids and circadian rhythms. Their distribution suggests that they may thrive in environments that are marginal for other species, including wet-rock and cold environments. These traits may aid in the discovery and cultivation of novel species in this clade.

Photosynthetic Cyanobacteria and their descendants are the only known organisms capable of oxygenic photosynthesis. Their metabolism permanently changed the Earth’s surface and the evolutionary trajectory of life, but little is known about their evolutionary history. Genomes of the Gloeobacterales, an order of deeply divergent photosynthetic Cyanobacteria, may hold clues about the evolutionary process. However, there are only three published genomes within this order, and it is difficult to make broad inferences based on such little data. Here, I describe five species within the Gloeobacterales retrieved from publicly available databases and examine their photosynthetic gene content and the environments in which Gloeobacterales genomes and 16S rRNA gene sequences are found. The Gloeobacterales contain reduced photosystems and inhabit cold, wet-rock, and low-light environments. They are likely present in low abundances due to their low growth rate. Future searches for Gloeobacterales should target these environments, and samples should be deeply sequenced to capture the low-abundance taxa. Publicly available databases contain undescribed taxa within the Gloeobacterales. However, searching through all available data with current methods is computationally expensive. Therefore, new methods must be developed to search for these and other evolutionarily important taxa. Once identified, these novel photosynthetic Cyanobacteria will help illuminate the origin and evolution of oxygenic photosynthesis. IMPORTANCE Early branching photosynthetic Cyanobacteria such as the Gloeobacterales may provide clues into the evolutionary history of oxygenic photosynthesis, but there are few genomes or cultured taxa from this order. Five new metagenome-assembled genomes suggest that members of the Gloeobacterales all contain reduced photosystems and lack genes associated with thylakoids and circadian rhythms. Their distribution suggests that they may thrive in environments that are marginal for other species, including wet-rock and cold environments. These traits may aid in the discovery and cultivation of novel species in this clade.

The functionality of caspase homologs in prokaryotic cell execution has been perceived, yet the dimensions of their metabolic pertinence are still cryptic. Here, a detailed in silico study on putative cyanobacterial caspase homologs, termed orthocaspases, in a sequenced genome of 132 strains was performed. We observed that 473 putative orthocaspases were distributed among 62% cyanobacterial strains subsumed within all the taxonomical orders. However, high diversity among these orthocaspases was also evident as the conventional histidine–cysteine (HC) dyad was present only in 72.03% of orthocaspases (wild-type), whereas the rest 28.18% were pseudo-variants having substituted the catalytic dyad. Besides, the presence of various accessory functional domains with Peptidase C14 probably suggested the multifunctionality of the orthocaspases. Moreover, the early origin and emergence of wild-type orthocaspases were conferred by their presence in Gloeobacter; however, the complex phylogeny displayed by these caspase-homologs perhaps suggested horizontal a gene transfer for their acquisition. However, morpho-physiological advancements and larger genome size favored the acquisition of orthocaspases. Moreover, the conserved caspase hemoglobinase fold not only in the wild-type but also in the pseudo-orthocaspases in Nostoc sp. PCC 7120 ascertained the least effect of catalytic motifs in the protein tertiary structure. Further, the 100-ns molecular dynamic simulation and molecular mechanics/generalized born surface area exhibited stable binding of arginylarginine dipeptide with wild-type orthocaspase of Nostoc sp. PCC 7120, displaying arginine-P1 specificity of wild-type orthocaspases. This study deciphered the distribution, diversity, domain architecture, structure, and basic substrate specificity of putative cyanobacterial orthocaspases, which may aid in functional investigations in the future.

Rhodopsin and carotenoids are two molecules that certain bacteria use to absorb and utilize light. Type I rhodopsin, the simplest active proton transporter, converts light energy into an electrochemical potential. Light produces a proton gradient, which is known as the proton motive force across the cell membrane. Some carotenoids are involved in light absorbance and transfer of absorbed energy to chlorophyll during photosynthesis. A previous study in Salinibacter ruber has shown that carotenoids act as antennae to harvest light and transfer energy to retinal in xanthorhodopsin (XR). Here, we describe the role of canthaxanthin (CAN), a carotenoid, as an antenna for Gloeobacter rhodopsin (GR). The non-covalent complex formed by the interaction between CAN and GR doubled the proton pumping speed and improved the pumping capacity by 1.5-fold. The complex also tripled the proton pumping speed and improved the pumping capacity by 5-fold in the presence of strong and weak light, respectively. Interestingly, when canthaxanthin was bound to Gloeobacter rhodopsin, it showed a 126-fold increase in heat resistance, and it survived better under drought conditions than Gloeobacter rhodopsin. The results suggest direct complementation of Gloeobacter rhodopsin with a carotenoid for primitive solar energy harvesting in cyanobacteria.

Cyanobacteria have played pivotal roles in Earth's geological history, especially during the rise of atmospheric oxygen. However, our ability to infer the early transitions in Cyanobacteria evolution has been limited by their extremely lopsided tree of life-the vast majority of extant diversity belongs to Phycobacteria (or "crown Cyanobacteria"), while its sister lineage, Gloeobacteria, is depauperate and contains only two closely related species of Gloeobacter and a metagenome-assembled genome. Here, we describe a new cultured member of Gloeobacteria, Anthocerotibacter panamensis, isolated from a tropical hornwort. Anthocerotibacter diverged from Gloeobacter over 1.4 Ga ago and has low 16S rDNA identities with environmental samples. Our ultrastructural, physiological, and genomic analyses revealed that this species possesses a unique combination of traits that are exclusively shared with either Gloeobacteria or Phycobacteria. For example, similar to Gloeobacter, it lacks thylakoids and circadian clock genes, but the carotenoid biosynthesis pathway is typical of Phycobacteria. Furthermore, Anthocerotibacter has one of the most reduced gene sets for photosystems and phycobilisomes among Cyanobacteria. Despite this, Anthocerotibacter is capable of oxygenic photosynthesis under a wide range of light intensities, albeit with much less efficiency. Given its key phylogenetic position, distinct trait combination, and availability as a culture, Anthocerotibacter opens a new window to further illuminate the dawn of oxygenic photosynthesis.

SummaryCyanobacteria have played pivotal roles in Earth’s geological history especially during the rise of atmospheric oxygen. However, our ability to infer the early transitions in Cyanobacteria evolution has been limited by their extremely lopsided tree of life—the vast majority of extant diversity belongs to Phycobacteria (or “crown Cyanobacteria”), while its sister lineage, Gloeobacteria, is depauperate and contains only two closely related species of Gloeobacter and a metagenome-assembled genome. Here we describe a new culturable member of Gloeobacteria, Anthocerobacter panamensis, isolated from a tropical hornwort. Anthocerobacter diverged from Gloeobacter over 1.4 billion years ago and has low 16S identities with environmental samples. Our ultrastructural, physiological, and genomic analyses revealed that this species possesses a unique combination of traits that are exclusively shared with either Gloeobacteria or Phycobacteria. For example, similar to Gloeobacter, it lacks thylakoids and circadian clock genes, but the carotenoid biosynthesis pathway is typical of Phycobacteria. Furthermore, Anthocerobacter has one of the most reduced gene sets for photosystems and phycobilisomes among Cyanobacteria. Despite this, Anthocerobacter is capable of oxygenic photosynthesis under a wide range of light intensities, albeit with much less efficiency. Given its key phylogenetic position, distinct trait combination, and availability as a culture, Anthocerobacter opens a new window to further illuminate the dawn of oxygenic photosynthesis.

The earliest branching cyanobacterium, Gloeobacter, exhibits a number of ancestral traits including the lack of thylakoids. It occurs epilithically in microbial mats, both subaerially and submerged...

12 páginas, 6 figuras, Thioredoxin reductases control the redox state of thioredoxins (Trxs)—ubiquitous proteins that regulate a spectrum of enzymes by dithiol–disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeplyrooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here, we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox-sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments., This work was supported by the Spanish Ministry of Science and Innovation—State Research Agency (MICINN) Grants [PID2019-110900GB-I00 to M.B., PID2019-109671GB-I00 to R.M.B., BFU-2016-78232-P to A.V.-C., and PID2019-103901GB-I00 to M.M.], and the Government of Aragón FEDER [Grupo de Referencia Biología Estructural (E35_20R to M.M., and E45_17R to A.V.-C.)]. D.F.J. was supported by a predoctoral contract from the Junta de Castilla y León. A.G.- L. acknowledges support from CSIC Jae-Intro program.

Domination of certain aerophytic phototrophic group or specific taxon in biofilms is connected with biofilm features recognised in situ. Well-developed, gelatinous, olive to dark-green biofilms are composed mostly of coccoid cyanobacterial forms. The same features, characterised biofilms dominated by one coccoid taxon, except the latter were vividly coloured. Gloeobacter caused the appearance of purple, Gloeocapsa representatives yellow and Chroococcidiopsis black biofilm. The brown to the dark colour of heterocytous biofilms was mainly caused by Nostoc. Simple trichal Cyanobacteria were occasionally present in biofilm, except in one blue-coloured sample. According to the principal component analysis (PCA), well-developed and gelatinous biofilms were correlated with Cyanobacteria, while scanning electron microscopy (SEM) revealed richness of extracellular polymeric substances (EPS) in such biofilms. Biofilm with calcified cyanobacterium (Geitleria cf. calcarea) was also found. Chlorophyta-abundant biofilms (many rich in Desmococcus), thinner than cyanobacterial, were predominantly green and occasionally yellow and blue. Many were dry when observed in situ (confirmed with PCA), with few being moistened (i.e. Klebsormidium-dominant). Diatom biofilms were usually developed on sediment, mosses or near seeping water (demonstrated by PCA) and were also thinner than cyanobacterial ones. Compared to cyanobacterial biofilms, SEM showed less developed EPS in those rich in diatoms and green algae, where microorganisms are more exposed to the environment. The study demonstrates an easy method for biofilm assessment based on visual characterisation and provides encouragement for more frequent biofilm investigation in caves that can be important from an ecological, biological, biotechnological point of view and which assessment can have an irreplaceable role in potential monitoring and protection.

Photoreception requires amplification by mammalian rhodopsin through G protein activation, which requires a visual cycle. To achieve this in retinal gene therapy, we incorporated human rhodopsin cytoplasmic loops intoGloeobacterrhodopsin, thereby generatingGloeobacterand human chimeric rhodopsin (GHCR). In a murine model of inherited retinal degeneration, we induced retinal GHCR expression by intravitreal injection of a recombinant adeno-associated virus vector. Retinal explant and visual thalamus electrophysiological recordings, behavioral tests, and histological analysis showed that GHCR restored dim-environment vision and prevented the progression of retinal degeneration. Thus, GHCR may be a potent clinical tool for the treatment of retinal disorders.One Sentence SummaryOptogenetic therapy with Gloeobacter and human chimeric rhodopsin resulted in highly sensitive visual restoration and protection effects.

Gloeobacter rhodopsin (GR) is a cyanobacterial proton pump which can be potentially applied to optogenetics. We solved the crystal structure of GR and found that it has overall similarity to the homologous proton pump from Salinibacter ruber, xanthorhodopsin (XR). We identified distinct structural characteristics of GR’s hydrogen bonding network in the transmembrane domain as well as the displacement of extracellular sides of the transmembrane helices relative to those of XR. Employing Raman spectroscopy and flash-photolysis, we found that GR in the crystals exists in a state which displays retinal conformation and photochemical cycle similar to the functional form observed in lipids. Based on the crystal structure of GR, we selected a site for spin labeling to determine GR’s oligomerization state using double electron–electron resonance (DEER) spectroscopy and demonstrated the pH-dependent pentamer formation of GR. Determination of the structure of GR as well as its pentamerizing propensity enabled us to reveal the role of structural motifs (extended helices, 3-omega motif and flipped B-C loop) commonly found among light-driven bacterial pumps in oligomer formation. Here we propose a new concept to classify these pumps based on the relationship between their oligomerization propensities and these structural determinants.

Contains fulltext : 205161.pdf (Publisher’s version ) (Open Access) The approach of providing an oxygenic photosynthetic organism with a cyclic electron transfer system, i.e., a far-red light-driven proton pump, is widely proposed to maximize photosynthetic efficiency via expanding the absorption spectrum of photosynthetically active radiation. As a first step in this approach, Gloeobacter rhodopsin was expressed in a PSI-deletion strain of Synechocystis sp. PCC6803. Functional expression of Gloeobacter rhodopsin, in contrast to Proteorhodopsin, did not stimulate the rate of photoheterotrophic growth of this Synechocystis strain, analyzed with growth rate measurements and competition experiments. Nevertheless, analysis of oxygen uptake and-production rates of the Gloeobacter rhodopsin-expressing strains, relative to the DeltaPSI control strain, confirm that the proton-pumping Gloeobacter rhodopsin provides the cells with additional capacity to generate proton motive force. Significantly, expression of the Gloeobacter rhodopsin did modulate levels of pigment formation in the transgenic strain.

We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins.

Primary photochemical events in the unusually thermostable proton pumping rhodopsin of Thermus thermophilus bacterium (TR) are reported for the first time. Internal conversion in this protein is shown to be significantly faster than in bacteriorhodopsin (BR), making it the most rapidly isomerizing microbial proton pump known. Internal conversion (IC) dynamics of TR and BR were recorded from room temperature to the verge of thermal denaturation at 70 °C and found to be totally independent of temperature in this range. This included the well documented multiexponential nature of IC in BR, suggesting that assignment of this to ground state structural inhomogeneity needs revision. TR photodynamics were also compared with that of the phylogenetically more similar proton pump Gloeobacter rhodopsin (GR). Despite this similarity GR has poor thermal stability, and the excited state decays significantly more slowly and exhibits very prominent stretched exponential behavior. Coherent torsional wave-packets induced by impulsive photoexcitation of TR and GR show marked resemblance to each other in frequency and amplitude and differ strikingly from similar signatures in pump-probe data of BR and other microbial retinal proteins. Possible correlations between IC rates and thermal stability and the promise of using torsional coherence signatures for understanding chromophore protein binding in microbial retinal proteins are discussed.

The oxygenase reaction catalyzed by RuBisCO became an issue only after the evolution of the oxygenic photosynthesis in cyanobacteria. Several strategies were developed by autotrophic organisms as an evolutionary response to increase oxygen levels to help RuBisCO maximize its net carboxylation rate. One of the crucial advancements in this context was the development of more efficient inorganic carbon transporters which could help in increasing the influx of inorganic carbon (Ci) at the site of CO₂ fixation.We conducted a survey to find out the genes coding for cyanobacterial Ci transporters in 40 cyanobacterial phyla with respect to transporters present in Gloeobacter violaceous PCC 7421, an early-diverging cyanobacterium. An attempt was also made to correlate the prevalence of the kind of transporter present in the species with its habitat. Basically, two types of cyanobacterial inorganic carbon transporters exist, i.e. bicarbonate transporters and CO₂-uptake systems. The transporters also show variation in context to their structure as some exist as single subunit proteins (BicA and SbtA), while others exist as multisubunit proteins (namely BCT1, NdhI₃ and NdhI₄). The phylogeny and dist ribution of the former have been extensively studied and the present analysis provides an insight into the latter ones. The in silico analysis of the genes under study revealed that their distribution was greatly influenced by the habitat and major environmental changes such as the great oxidation event (GOE) in the course of their evolution.

International audience; Cyanobacteria are widely-diverse prokaryotes that colonize our planet. They use solar energy to assimilates huge amounts of atmospheric CO2 and produce a large part of the biomass and oxygen that sustain most life forms. Hence cyanobacteria are increasingly studied for basic research objective, as well as for the photosynthetic production of chemicals of industrial interests. One potential approach to reduce the cost of the future bioproduction processes will be to couple them with the treatment of wastewaters that are often polluted with urea, which in any case is cheaper than nitrate. As yet, however, research has mostly focused on a very small number of model cyanobacteria growing on nitrate. Thus, the genetic inventory of the cyanobacterial phylum is still insufficiently employed to meaningfully select the right host for the right purpose. This review reports what is known about urea transport and catabolism in cyanobacteria, and what can be inferred from the comparative analysis of the publicly available genome sequence of 308 cyanobacteria. We found that most cyanobacteria harbor the genes encoding the urea catabolytic enzymes urease (ureABCDEFG) mostly, but not systematically, together with the urea transport (urtABCDE). These findings are consistent with the capacity of the few tested cyanobacteria to grow on urea as the sole nitrogen source. They also indicate that urease is important for the detoxification of internally generated urea (re-cycling its carbon and nitrogen). In contrast, several cyanobacteria have urtABCDE but not ureABCDEFG, suggesting that urtABCDE could operate in the transport of not only urea but also of other nutrients. Only four cyanobacteria appeared to have the genes encoding the urea carboxylase (uc) and allophanate hydrolase (ah) enzymes that sequentially catabolize urea. Three of these cyanobacteria belongs to the genera Gloeobacter and Gloeomargarita that have likely diverged early from other cyanobacteria, suggesting that the urea carboxylase and allophanate hydrolase enzymes appeared in cyanobacteria before urease.

The oldest species of bacteria capable of oxygenic photosynthesis today are the freshwater Cyanobacteria Gloeobacter spp., belonging to the class Oxyphotobacteria. Several modern molecular evolutionary studies support the freshwater origin of cyanobacteria during the Archaean and their subsequent acquisition of salt tolerance mechanisms necessary for their expansion into the marine environment. This study investigated the effect of a sudden washout event from a freshwater location into either a brackish or marine environment on the photosynthetic efficiency of two unicellular freshwater cyanobacteria: the salt-tolerant Chroococcidiopsis thermalis PCC7203 and the cyanobacterial phylogenetic root species, Gloeobacter violaceus PCC7421. Strains were cultured under present atmospheric levels (PAL) of CO2 or an atmosphere containing elevated levels of CO2 and reduced O2 (eCO2 rO2 ) in simulated shallow water or terrestrial environmental conditions. Both strains exhibited a reduction in growth rates and gross photosynthesis, accompanied by significant reductions in chlorophyll a content, in brackish water, with only C. thermalis able to grow at marine salinity levels. While the experimental atmosphere caused a significant increase in gross photosynthesis rates in both strains, it did not increase their growth rates, nor the amount of O2 released. The differences in growth responses to increasing salinities could be attributed to genetic differences, with C. thermalis carrying additional genes for trehalose synthesis. This study demonstrates that, if cyanobacteria did evolve in a freshwater environment, they would have been capable of withstanding a sudden washout into increasingly saline environments. Both C. thermalis and G. violaceus continued to grow and photosynthesise, albeit at diminished rates, in brackish water, thereby providing a route for the evolution of open ocean-dwelling strains, necessary for the oxygenation of the Earth's atmosphere.

This study describes microbial community compositions, and various cold-responsive stress genes, encompassing cold-induced proteins (CIPs) and cold-associated general stress-responsive proteins (CASPs) in selected Antarctic lake water, sediment, and soil metagenomes. Overall, Proteobacteria and Bacteroidetes were the major taxa in all metagenomes. Prochlorococcus and Thiomicrospira were highly abundant in waters, while Myxococcus, Anaeromyxobacter, Haliangium, and Gloeobacter were dominant in the soil and lake sediment metagenomes. Among CIPs, genes necessary for DNA replication, translation initiation, and transcription termination were highly abundant in all metagenomes. However, genes for fatty acid desaturase (FAD) and trehalose synthase (TS) were common in the soil and lake sediment metagenomes. Interestingly, the Lake Untersee water and sediment metagenome samples contained histone-like nucleoid structuring protein (H-NS) and all genes for CIPs. As for the CASPs, high abundances of a wide range of genes for cryo- and osmo-protectants (glutamate, glycine, choline, and betaine) were identified in all metagenomes. However, genes for exopolysaccharide biosynthesis were dominant in Lake Untersee water, sediment, and other soil metagenomes. The results from this study indicate that although diverse microbial communities are present in various metagenomes, they share common cold-responsive stress genes necessary for their survival and sustenance in the extreme Antarctic conditions.

Atmospheric oxygen level rose dramatically around 2.4 billion years ago due to oxygenic photosynthesis by the Cyanobacteria. The oxidation of surface environments permanently changed the future of life on Earth, yet the evolutionary processes leading to oxygen production are poorly constrained. Partial records of these evolutionary steps are preserved in the genomes of organisms phylogenetically placed between non-photosynthetic Melainabacteria, crown-group Cyanobacteria, andGloeobacter, representing the earliest-branching Cyanobacteria capable of oxygenic photosynthesis. Here, we describe nearly complete, metagenome assembled genomes of an uncultured organism phylogenetically placed between the Melainabacteria and crown-group Cyanobacteria, for which we propose the name CandidatusAurora vandensis {au.roraLatin noundawnandvand.ensis, originating from Vanda}.The metagenome assembled genome ofA. vandensiscontains homologs of most genes necessary for oxygenic photosynthesis including key reaction center proteins. Many extrinsic proteins associated with the photosystems in other species are, however, missing or poorly conserved. The assembled genome also lacks homologs of genes associated with the pigments phycocyanoerethrin, phycoeretherin and several structural parts of the phycobilisome. Based on the content of the genome, we propose an evolutionary model for increasing efficiency of oxygenic photosynthesis through the evolution of extrinsic proteins to stabilize photosystem II and I reaction centers and improve photon capture. This model suggests that the evolution of oxygenic photosynthesis may have significantly preceded oxidation of Earth’s atmosphere due to low net oxygen production by early Cyanobacteria.

Photosystem II, the water oxidizing enzyme, altered the course of evolution by filling the atmosphere with oxygen. Here, we reconstruct the origin and evolution of water oxidation at an unprecedented level of detail by studying the phylogeny of all D1 subunits, the main protein coordinating the water oxidizing cluster (Mn4CaO5) of Photosystem II. We show that D1 exists in several forms making well-defined clades, some of which could have evolved before the origin of water oxidation and presenting many atypical characteristics. The most ancient form is found in the genome of Gloeobacter kilaueensis JS-1 and this has a C-terminus with a higher sequence identity to D2 than to any other D1. Two other groups of early evolving D1 correspond to those expressed under prolonged far-red illumination and in darkness. These atypical D1 forms are characterized by a dramatically different Mn4CaO5 binding site and a Photosystem II containing such a site may assemble an unconventional metal cluster. The first D1 forms with a full set of ligands to the Mn4CaO5 cluster are grouped with D1 proteins expressed only under low oxygen concentrations and the latest evolving form is the dominant type of D1 found in all cyanobacteria and plastids. In addition, we show that the plastid ancestor had a D1 more similar to those in early branching Synechococcus. We suggest each one of these forms of D1 originated from transitional forms at different stages towards the innovation and optimization of water oxidation before the last common ancestor of all known cyanobacteria.

Cys-loop receptors play important roles in signal transduction. The Gloeobacter ligand-gated ion channel (GLIC) pore binds similar compounds to Cys-loop receptor pores, but has the advantage of known structures in open and closed states. GLIC is activated by protons with a pEC50 of 5.4, and has a histidine residue (His 11') in its pore-forming α-helix (M2) which is involved in gating. Here we explore the role of this His and other M2 residues using two-electrode voltage clamp of mutant receptors expressed in oocytes. We show that 11'His is very sensitive to substitution; replacement with a range of amino acids ablates function. Similarly altering its location in M2 to the 8', 9', 10', 12', 13' or 14' positions ablated function. Most substitutions of Ser6' or Ile9' were also non-functional, although not Ile9'Leu and Ile9'Val. Unexpectedly, an Ile9'His substitution was constitutively active at pH 7, but closed as [H+] increased, with a pIC50 of 5.8. Substitution at 2', 5' and 7' had little effect on pEC50. Overall the data show Ser6' and His11' are critical for the function of the receptor, and thus distinguish the roles of these M2 residues from those of Cys-loop receptors, where substitutions are mostly well tolerated. These data suggest modellers should be aware of these atypical features when using the GLIC pore as a model for Cys-loop receptor pores.

Over the past few decades, the structure, functions, properties, and molecular mechanisms of retinal proteins have been studied extensively. The newly studied retinal protein Gloeobacter rhodopsin (gR) acts as a light-driven proton pump, transferring a proton from the cytoplasmic region to the extracellular region of a cell following light absorption. It was previously shown that gR can bind the carotenoid salinixanthin (sal). In the present study, we report the effect of pH on the binding of retinal to the apo-protein of gR, in the presence and absence of sal, to form the gR pigment. We found that binding at different pH levels reflects the titration of two different protein residues, one at the lower pKa 3.5 and another at the higher pKa 8.4, that affect the pigment’s formation. The maximum amount of pigment was formed at pH 5, both with and without the presence of sal. The introduction of sal accelerates the rate of pigment formation by a factor of 190. Furthermore, it is suggested that occupation of t...

Background The expression of the Gloeobacter rhodopsin (GR) in a chemotrophic Escherichia coli enables the light-driven phototrophic energy generation. Adaptive laboratory evolution has been used for acquiring desired phenotype of microbial cells and for the elucidation of basic mechanism of molecular evolution. To develop an optimized strain for the artificially acquired phototrophic metabolism, an ancestral E. coli expressing GR was adaptively evolved in a chemostat reactor with constant illumination and limited glucose conditions. This study was emphasized at an unexpected genomic mutation contributed to the improvement of microbial performance. Results During the chemostat culture, increase of cell size was observed, which were distinguished from that of the typical rod-shaped ancestral cells. A descendant ET5 strain was randomly isolated from the chemostat culture at 88-days. The phototrophic growth and the light-induced proton pumping of the ET5 strain were twofold and eightfold greater, respectively, than those of the ancestral E. coli strain. Single point mutation of C1082A at dgcQ gene (encoding diguanylate cyclase, also known as the yedQ gene) in the chromosome of ET5 strain was identified from whole genome sequencing analysis. An ancestral E. coli complemented with the same dgcQ mutation from the ET5 was repeated the subsequently enhancements of light-driven phototrophic growth and proton pumping. Intracellular c-di-GMP, the product of the diguanylate cyclase (dgcQ), of the descendant ET5 strain was suddenly increased while that of the ancestral strain was negligible. Conclusions Newly acquired phototrophic metabolism of E. coli was further improved via adaptive laboratory evolution by the rise of a point mutation on a transmembrane cell signaling protein followed by increase of signal molecule that eventually led an increase proton pumping and phototrophic growth. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0725-6) contains supplementary material, which is available to authorized users.

The retinal proton pump xanthorhodopsin (XR) was recently found to function with an attached carotenoid light harvesting antenna, salinixanthin (SX). It is intriguing to discover if this departure from single chromophore architecture is singular or if it has been adopted by other microbial rhodopsins. In search of other cases, retinal protein encoding genes in numerous bacteria have been identified containing sequences corresponding to carotenoid binding sites like that in XR. Gloeobacter rhodopsin (GR), exhibiting particularly close homology to XR, has been shown to attach SX, and fluorescence measurements suggest SX can function as a light harvesting (LH) antenna in GR as well. In this study, we test this suggestion in real time using ultrafast transient absorption. Results show that energy transfer indeed occurs from S2 of SX to retinal in the GR-SX composite with an efficiency of ∼40%, even higher than that in XR. This validates the earlier fluorescence study, and supports the notion that many microbial retinal proteins use carotenoid antennae to harvest light.

Many microbial rhodopsins self-oligomerize, but the functional consequences of oligomerization have not been well clarified. We examined the effects of oligomerization of a H+ pump, Gloeobacter rhodopsin (GR), by using nanodisc containing trimeric and monomeric GR. The monomerization did not appear to affect the unphotolyzed GR. However, we found a significant impact on the photoreaction: The monomeric GR showed faint M intermediate formation and negligible H+ transfer reactions. These changes reflected the elevated pKa of the Asp121 residue, whose deprotonation is a prerequisite for the functional photoreaction. Here, we focused on His87, which is a neighboring residue of Asp121 and conserved among eubacterial H+ pumps but replaced by Met in an archaeal H+ pump. We found that the H87M mutation removes the “monomerization effects”: Even in the monomeric state, H87M contained the deprotonated Asp121 and showed both M formation and distinct H+ transfer reactions. Thus, for wild-type GR, monomerization probably strengthens the Asp121-His87 interaction and thereby elevates the pKa of Asp121 residue. This strong interaction might occur due to the loosened protein structure and/or the disruption of the interprotomer interaction of His87. Thus, the trimeric assembly of GR enables light-induced H+ transfer reactions through adjusting the positions of key residues.

The genus name Gloeobacter with the single (= type) species Gloeobacter violaceus (Cyanophyta, Cyanoprokaryota, Cyanobacteria) was described by Rippka, Waterbury et Cohen-Bazire (Arch. Microbiol. 100: 419–436, 1974). However, this is not a validly published name and so it currently has no standing under the botanical International Code of Nomenclature (ICN, Mc Neil et al. 2012) or the International Code of Nomenclature of Prokaryotes (ICNB/ICNP, Lapage et al. 1992). The lack of valid publication of the genus name causes many problems in the taxonomy of this phylogenetically and experimentally important cyanophyte/cyanobacterium. The lack of thylakoids, a feature unique among all known cyanobacteria, as well as the phylogenetic position of the representative of this genus, warrant valid publication of this generic name. The type strain was deposited in the collection PCC in Paris under the number PCC 7421 and later introduced into numerous other strain collections; however, the dried specimens wer...