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Dinoflagellates are an abundant and diverse group of protists representing a wealth of unique biology and ecology. While many dinoflagellates are photosynthetic or mixotrophic, many taxa are heterotrophs, often with complex feeding strategies. Compared to their photosynthetic counterparts, heterotrophic dinoflagellates remain understudied, as they are difficult to culture. One exception, a long‐cultured isolate originally classified as Amphidinium but recently reclassified as Oxytoxum, has been the subject of a number of feeding, growth, and chemosensory studies. This lineage was recently determined to be closely related to Prorocentrum using phylogenetics of ribosomal RNA gene sequences, but the exact nature of this relationship remains unresolved. Using transcriptomes sequenced from culture and three single cells from the environment, we produce a robust phylogeny of 242 genes, revealing Oxytoxum is likely sister to the Prorocentrum clade, rather than nested within it. Molecular investigations uncover evidence of a reduced, nonphotosynthetic plastid and proteorhodopsin, a photoactive proton pump acquired horizontally from bacteria. We describe the ultrastructure of O. lohmannii, including densely packed trichocysts, and a new type of mucocyst. We observe that O. lohmannii feeds preferentially on cryptophytes using myzocytosis, but can also feed on various phytoflagellates using conventional phagocytosis. O. lohmannii is amenable to culture, providing an opportunity to better study heterotrophic dinoflagellate biology and feeding ecology. [ABSTRACT FROM AUTHOR]

Objective: To study the transport activity of bacterial rhodopsins, proteoliposomes with the same orientation of the protein molecules should be obtained. Methods: The genes of fusion proteins containing Exiguobacterium sibiricum proteorhodopsin (ESR) and various N-terminal soluble domains have been constructed. Results and Discussion: Effective synthesis in Escherichia coli cells was observed only in the case of fusion with chaperone Caf1M and maltose-binding protein MBP expressed as precursors with their own signal sequences. The study of the isolated MBP-ESR protein in micelles and proteoliposomes demonstrated formation and decay of the main photocycle intermediates at pH > 8. The photoelectric response of the fusion proteins Caf-ESR and MBP-ESR is comparable in amplitude to the wild-type ESR response, indicating their homogeneous orientation in the proteoliposome membrane. Conclusions: The obtained constructs can be used to create bacterial expression systems for various retinal proteins, ensuring their uniform incorporation into proteoliposomes. [ABSTRACT FROM AUTHOR]

Controlling the pH at the microliter scale can be useful for applications in research, medicine, and industry, and therefore represents a valuable application for synthetic biology and microfluidics. The presented vesicular system translates light of different colors into specific pH changes in the surrounding solution. It works with the two light‐driven proton pumps bacteriorhodopsin and blue light‐absorbing proteorhodopsin Med12, that are oriented in opposite directions in the lipid membrane. A computer‐controlled measuring device implements a feedback loop for automatic adjustment and maintenance of a selected pH value. A pH range spanning more than two units can be established, providing fine temporal and pH resolution. As an application example, a pH‐sensitive enzyme reaction is presented where the light color controls the reaction progress. In summary, light color‐controlled pH‐adjustment using engineered proteoliposomes opens new possibilities to control processes at the microliter scale in different contexts, such as in synthetic biology applications. [ABSTRACT FROM AUTHOR]

To elucidate the function of proteorhodopsin in Candidatus Puniceispirillum marinum strain IMCC1322, a cultivated representative of SAR116, we produced RNA-seq data under laboratory conditions. We examined the transcriptomes of six different cultures, including sets of expression changes under constant dark (DD), constant light (LL), and diel-cycled (LD; 14 h light: 10 h dark) conditions at the exponential and stationary/death phases. Prepared mRNA extracted from the six samples was analyzed on the Solexa Genome Analyzer with 36 cycles. Differentially expressed genes on the IMCC1322 genome were distinguished as four clusters by K-mean clustering and each CDS (n = 2546) was annotated based on the KEGG BRITE hierarchy. Cluster 0 (n = 1573) covered most constitutive genes including proteorhodopsin, retinoids, and glycolysis/TCA cycle. Cluster 1 genes (n = 754) were upregulated in stationary/death phase under constant dark conditions and included genes associated with bacterial defense, membrane transporters, nitrogen metabolism, and senescence signaling. Cluster 2 genes (n = 197) demonstrated upregulation in exponential phase cultures and included genes involved in genes for oxidative phosphorylation, translation factors, and transcription machinery. Cluster 3 (n = 22) contained light-stimulated upregulated genes expressed under stationary/phases. Stringent response genes belonged to cluster 2, but affected genes spanned various cellular processes such as amino acids, nucleotides, translation, transcription, glycolysis, fatty acids, and cell wall components. The coordinated expression of antagonistic stringent genes, including mazG, ppx/gppA, and spoT/relA may provide insight into the controlled cultural response observed between constant light and constant dark conditions in IMCC1322 cultures, regardless of cell numbers and biomass. [ABSTRACT FROM AUTHOR]

The possible influence of the continent on the microbiome of the Southwest Atlantic Ocean (SAO) is not yet fully known. The aim of the present study was to evaluate the metagenomic diversity of the SAO nearshore in different locations (e.g., rivers and upwellings) and offshore locations. The studied sites (n = 16) were clustered into four groups (G1-G4) according to the decadal sea surface temperature (SST) and levels of chlorophyll a (chl-a) (2007–2016). G1 (coastal upwelling) presented the highest chl-a values, G2 (shelf) had the highest SST, G3 (offshore north) had the lowest chl-a levels, and G4 (offshore south) was the coldest site. The higher nutrient levels may contribute to higher chl-a-based primary productivity and possibly higher bacterial productivity nearshore. The taxonomic and functional diversity of the total of 16 Illumina shotgun metagenomes were analysed (nearshore: 10, offshore: 6; mean ± standard deviation: 1.87 ± 0.41 million reads per sample). The SAO microbiomes were split into two groups: nearshore and offshore. The SAO coastal zone had higher abundance of picoplanktonic cyanobacteria (e.g., Prochlorococcus), copiotrophic bacteria (e.g., Alteromonas), chl-a photosynthesis, and metabolism of nitrogen and phosphorus. In clear contrast with offshore microbiomes, nearshore microbiomes were heterogenous as a possible effect of riverine runoff and the upwelling. G1 microbiomes had higher abundance of copiotrophic bacteria, while G2 had a higher abundance of Prochlorococcus, and G3 had a higher abundance of Pelagibacter. Offshore metagenomes (G3 and G4) had enrichment of proteorhodopsin light harvesting and sulfur metabolism, possible relevant processes in the SAO. Metagenome assembled genomes (MAGs) demonstrated significant novel microbiome diversity. The most abundant MAGs belong to Flavobacteria and Euryarchaeota. The discrimination of microbial populations and metabolisms in the four studied oceanographic groups provides a first glimpse on the microbial landscape in the SAO.

Background: The RCA (Roseobacter clade affiliated) cluster belongs to the family Roseobacteracea and represents a major Roseobacter lineage in temperate to polar oceans. Despite its prevalence and abundance, only a few genomes and one described species, Planktomarina temperata, exist. To gain more insights into our limited understanding of this cluster and its taxonomic and functional diversity and biogeography, we screened metagenomic datasets from the global oceans and reconstructed metagenome-assembled genomes (MAG) affiliated to this cluster. Results: The total of 82 MAGs, plus five genomes of isolates, reveal an unexpected diversity and novel insights into the genomic features, the functional diversity, and greatly refined biogeographic patterns of the RCA cluster. This cluster is subdivided into three genera: Planktomarina, Pseudoplanktomarina, and the most deeply branching Candidatus Paraplanktomarina. Six of the eight Planktomarina species have larger genome sizes (2.44–3.12 Mbp) and higher G + C contents (46.36–53.70%) than the four Pseudoplanktomarina species (2.26–2.72 Mbp, 42.22–43.72 G + C%). Cand. Paraplanktomarina is represented only by one species with a genome size of 2.40 Mbp and a G + C content of 45.85%. Three novel species of the genera Planktomarina and Pseudoplanktomarina are validly described according to the SeqCode nomenclature for prokaryotic genomes. Aerobic anoxygenic photosynthesis (AAP) is encoded in three Planktomarina species. Unexpectedly, proteorhodopsin (PR) is encoded in the other Planktomarina and all Pseudoplanktomarina species, suggesting that this light-driven proton pump is the most important mode of acquiring complementary energy of the RCA cluster. The Pseudoplanktomarina species exhibit differences in functional traits compared to Planktomarina species and adaptations to more resource-limited conditions. An assessment of the global biogeography of the different species greatly expands the range of occurrence and shows that the different species exhibit distinct biogeographic patterns. They partially reflect the genomic features of the species. Conclusions: Our detailed MAG-based analyses shed new light on the diversification, environmental adaptation, and global biogeography of a major lineage of pelagic bacteria. The taxonomic delineation and validation by the SeqCode nomenclature of prominent genera and species of the RCA cluster may be a promising way for a refined taxonomic identification of major prokaryotic lineages and sublineages in marine and other prokaryotic communities assessed by metagenomics approaches. 4nWA41D1uUohyPVze8XWvD Video Abstract [ABSTRACT FROM AUTHOR]

Retinal-containing light-sensitive proteins – rhodopsins – are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein of Exiguobacterium sibiricum. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct electrometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins. [ABSTRACT FROM AUTHOR]

Abstract Background Rhodopsin is a seven-transmembrane protein covalently linked with retinal chromophore that absorbs photons for energy conversion and intracellular signaling in eukaryotes, bacteria, and archaea. Haloarchaeal rhodopsins are Type-I microbial rhodopsin that elicits various light-driven functions like proton pumping, chloride pumping and Phototaxis behaviour. The industrial application of Ion-pumping Haloarchaeal rhodopsins is limited by the lack of full-length rhodopsin sequence-based classifications, which play an important role in Ion-pumping activity. The well-studied Haloarchaeal rhodopsin is a proton-pumping bacteriorhodopsin that shows promising applications in optogenetics, biosensitized solar cells, security ink, data storage, artificial retinal implant and biohydrogen generation. As a result, a low-cost computational approach is required to identify Ion-pumping Haloarchaeal rhodopsin sequences and its subtype. Results This study uses a support vector machine (SVM) technique to identify these ion-pumping Haloarchaeal rhodopsin proteins. The haloarchaeal ion pumping rhodopsins viz., bacteriorhodopsin, halorhodopsin, xanthorhodopsin, sensoryrhodopsin and marine prokaryotic Ion-pumping rhodopsins like actinorhodopsin, proteorhodopsin have been utilized to develop the methods that accurately identified the ion pumping haloarchaeal and other type I microbial rhodopsins. We achieved overall maximum accuracy of 97.78%, 97.84% and 97.60%, respectively, for amino acid composition, dipeptide composition and hybrid approach on tenfold cross validation using SVM. Predictive models for each class of rhodopsin performed equally well on an independent data set. In addition to this, similar results were achieved using another machine learning technique namely random forest. Simultaneously predictive models performed equally well during five-fold cross validation. Apart from this study, we also tested the own, blank, BLAST dataset and annotated whole-genome rhodopsin sequences of PWS haloarchaeal isolates in the developed methods. The developed web server ( https://bioinfo.imtech.res.in/servers/rhodopred ) can identify the Ion Pumping Haloarchaeal rhodopsin proteins and their subtypes. We expect this web tool would be useful for rhodopsin researchers. Conclusion The overall performance of the developed method results show that it accurately identifies the Ionpumping Haloarchaeal rhodopsin and their subtypes using known and unknown microbial rhodopsin sequences. We expect that this study would be useful for optogenetics, molecular biologists and rhodopsin researchers.

ABSTRACT Members of the genus Flavobacterium are widely distributed and produce various polysaccharide-degrading enzymes. Many species in the genus have been isolated and characterized. However, few studies have focused on marine isolates or fish pathogens, and in-depth genomic analyses, particularly comparative analyses of isolates from different habitat types, are lacking. Here, we isolated 20 strains of the genus from various environments in South Korea and sequenced their full-length genomes. Combined with published sequence data, we examined genomic traits, evolution, environmental adaptation, and putative metabolic functions in total 187 genomes of isolated species in Flavobacterium categorized as marine, host-associated, and terrestrial including freshwater. A pangenome analysis revealed a correlation between genome size and coding or noncoding density. Flavobacterium spp. had high levels of diversity, allowing for novel gene repertories via recombination events. Defense-related genes only accounted for approximately 3% of predicted genes in all Flavobacterium genomes. While genes involved in metabolic pathways did not differ with respect to isolation source, there was substantial variation in genomic traits; in particular, the abundances of tRNAs and rRNAs were higher in the host-associdated group than in other groups. One genome in the host-associated group contained a Microviridae prophage closely related to an enterobacteria phage. The proteorhodopsin gene was only identified in four terrestrial strains isolated for this study. Furthermore, recombination events clearly influenced genomic diversity and may contribute to the response to environmental stress. These findings shed light on the high genetic variation in Flavobacterium and functional roles in diverse ecosystems as a result of their metabolic versatility. IMPORTANCE The genus Flavobacterium is a diverse group of bacteria that are found in a variety of environments. While most species of this genus are harmless and utilize organic substrates such as proteins and polysaccharides, some members may play a significant role in the cycling for organic substances within their environments. Nevertheless, little is known about the genomic dynamics and/or metabolic capacity of Flavobacterium. Here, we found that Flavobacterium species may have an open pangenome, containing a variety of diverse and novel gene repertoires. Intriguingly, we discovered that one genome (classified into host-associated group) contained a Microviridae prophage closely related to that of enterobacteria. Proteorhodopsin may be expressed under conditions of light or oxygen pressure in some strains isolated for this study. Our findings significantly contribute to the understanding of the members of the genus Flavobacterium diversity exploration and will provide a framework for the way for future ecological characterizations.

Background: Rhodopsin is a seven-transmembrane protein covalently linked with retinal chromophore that absorbs photons for energy conversion and intracellular signaling in eukaryotes, bacteria, and archaea. Haloarchaeal rhodopsins are Type-I microbial rhodopsin that elicits various light-driven functions like proton pumping, chloride pumping and Phototaxis behaviour. The industrial application of Ion-pumping Haloarchaeal rhodopsins is limited by the lack of full-length rhodopsin sequence-based classifications, which play an important role in Ion-pumping activity. The well-studied Haloarchaeal rhodopsin is a proton-pumping bacteriorhodopsin that shows promising applications in optogenetics, biosensitized solar cells, security ink, data storage, artificial retinal implant and biohydrogen generation. As a result, a low-cost computational approach is required to identify Ion-pumping Haloarchaeal rhodopsin sequences and its subtype. Results: This study uses a support vector machine (SVM) technique to identify these ion-pumping Haloarchaeal rhodopsin proteins. The haloarchaeal ion pumping rhodopsins viz., bacteriorhodopsin, halorhodopsin, xanthorhodopsin, sensoryrhodopsin and marine prokaryotic Ion-pumping rhodopsins like actinorhodopsin, proteorhodopsin have been utilized to develop the methods that accurately identified the ion pumping haloarchaeal and other type I microbial rhodopsins. We achieved overall maximum accuracy of 97.78%, 97.84% and 97.60%, respectively, for amino acid composition, dipeptide composition and hybrid approach on tenfold cross validation using SVM. Predictive models for each class of rhodopsin performed equally well on an independent data set. In addition to this, similar results were achieved using another machine learning technique namely random forest. Simultaneously predictive models performed equally well during five-fold cross validation. Apart from this study, we also tested the own, blank, BLAST dataset and annotated whole-genome rhodopsin sequences of PWS haloarchaeal isolates in the developed methods. The developed web server (https://bioinfo.imtech.res.in/servers/rhodopred) can identify the Ion Pumping Haloarchaeal rhodopsin proteins and their subtypes. We expect this web tool would be useful for rhodopsin researchers. Conclusion: The overall performance of the developed method results show that it accurately identifies the Ionpumping Haloarchaeal rhodopsin and their subtypes using known and unknown microbial rhodopsin sequences. We expect that this study would be useful for optogenetics, molecular biologists and rhodopsin researchers. [ABSTRACT FROM AUTHOR]

The bacterial flagellar motor (BFM) is a rotary molecular machine that drives critical bacterial processes including motility, chemotaxis, biofilm formation, and infection. For over two decades, the bead assay, which measures the rotation of a microparticle attached to the flagellum of a surface-attached bacterium, has been instrumental in deciphering the motor's biophysical mechanisms. This technique has not only quantified the rotational speed and frequency of directional switching as a function of the viscous load on the flagellum but has also revealed the BFM's capacity for mechanosensitive speed modulation, adapting to environmental conditions. Yet, many fundamental mechanistic details of the BFM remain to be discovered, and recent breakthroughs in structural biology, providing atomic-level structures of many motor components, further fuel this active area of biophysical research. This chapter presents an in-depth protocol for the bead assay to measure BFM dynamics, emphasizing advanced methodologies for quantifying the intricate mechanics and rich behavior of this molecular machine., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Proteorhodopsin (PR) is a photoactive proton pump found in marine bacteria. There are two phenotypes of PR exhibiting an environmental adaptation to the ocean's depth which tunes their maximum absorption: blue‐absorbing proteorhodopsin (BPR) and green‐absorbing proteorhodopsin (GPR). This blue/green color‐shift is controlled by a glutamine to leucine substitution at position 105 which accounts for a 20 nm shift. Typically, spectral tuning in rhodopsins is rationalized by the external point charge model but the Q105L mutation is charge neutral. To study this tuning mechanism, we employed the hybrid QM/MM method with sampling from molecular dynamics. Our results reveal that the positive partial charge of glutamine near the C14−C15 bond of retinal shortens the effective conjugation length of the chromophore compared to the leucine residue. The derived mechanism can be applied to explain the color regulation in other retinal proteins and can serve as a guideline for rational design of spectral shifts. [ABSTRACT FROM AUTHOR]

ABSTRACT Photoheterotrophy is a widespread mode of microbial metabolism, notably in the oligotrophic surface ocean, where microbes experience chronic nutrient limitation. One especially widespread form of photoheterotrophy is based on proteorhodopsin (PR), which uses light to generate proton motive force that can drive ATP synthesis, flagellar movement, or nutrient uptake. To clarify the physiological benefits conferred by PR under nutrient stress conditions, we quantified protein-level gene expression of Vibrio campbellii CAIM 519 under both carbon and nitrogen limitation and under both light and dark conditions. Using a novel membrane proteomics strategy, we determined that PR expression is higher under C limitation than N limitation but is not light regulated. Despite expression of PR photosystems, V. campbellii does not exhibit any growth or survival advantages in the light and only a few proteins show significant expression differences between light and dark conditions. While protein-level proteorhodopsin expression in V. campbellii is clearly responsive to nutrient limitation, photoheterotrophy does not appear to play a central role in the survival physiology of this organism under these nutrient stress conditions. C limitation and N limitation, however, result in very different survival responses: under N-limited conditions, viability declines, cultivability is lost rapidly, central carbon flux through the Entner-Doudoroff pathway is increased, and ammonium is assimilated via the GS-GOGAT pathway. In contrast, C limitation drives cell dwarfing with maintenance of viability, as well as utilization of the glyoxylate shunt, glutamate dehydrogenase and anaplerotic C fixation, and a stringent response mediated by the Pho regulon. IMPORTANCE Understanding the nutrient stress responses of proteorhodopsin-bearing microbes like Vibrio campbellii yields insights into microbial contributions to nutrient cycling, lifestyles of emerging pathogens in aquatic environments, and protein-level adaptations implemented during times of nutrient limitation. In addition to its broad taxonomic and geographic prevalence, the physiological role of PR is diverse, so we developed a novel proteomics strategy to quantify its expression at the protein level. We found that proteorhodopsin expression levels in this wild-type photoheterotroph under these experimental conditions, while higher under C than under N limitation, do not afford measurable light-driven growth or survival advantages. Additionally, this work links differential protein expression patterns between C- and N-limited cultures to divergent stationary-phase survival phenotypes.

Previous studies showed no improvement in bacterial biomass for Candidatus Puniceispirillum marinum IMCC1322 under light regimes. Nevertheless, in nutrient-replete cultures with higher inoculating cell densities, strain IMCC1322 exhibited proteorhodopsin photoheterotrophy. Increasing both inoculum size and the amino acid pool can eliminate quorum sensing and starvation responses in strain IMCC1322. Light regimes affected IMCC1322 cultures in stationary/death phases, where cellular ATP levels ranged from 0.0331 to 1.74 mM, with ATP/cell ranging from 13.9 to 367 zeptomoles. In nutrient-depleted conditions, strain IMCC1322 may suffer from excessive protons generated by proteorhodopsin under light conditions. IMCC1322 may tolerate excessive periplasmic protons through ATP-dependent proton pumping and protonation of augmented amino acids. Meanwhile, acid stress could also be mitigated by refining membrane permeability through unsaturation and cyclopropanation of phospholipids. Oceanic bacteria such as IMCC1322 and SAR11 preferred anaplerotic TCA cycles over glycolysis and rely on the Entner-Doudoroff (ED) pathway for growth. Although ATP generation is less efficient in the ED pathway, it offers advantages during rapid growth owing to its strong thermodynamic driving force. The metabolism of IMCC1322 favors gluconeogenesis over glycolysis, aligning with the metabolism of SAR11 reported in previous studies. However, the additional light-driven, PR-dependent ATP synthesis in IMCC1322 is expected to be insufficient to support protein turnover after the log phase, as well as in nutrient-limited conditions. Stable isotope measurements showed no significant differences in the inorganic carbon assimilation between constant light and constant dark cultures in late log phase.

The need of new diagnostic methods satisfying, as an early detection, a low invasive procedure and a cost-efficient value, is orienting the technological research toward the use of bio-integrated devices, in particular, bio-sensors. The set of know-why necessary to achieve this goal is wide, from biochemistry to electronics and is summarized in an emerging branch of electronics, called proteotronics. Proteotronics is here applied to state a comparative analysis of the electrical responses coming from type-1 and type-2 opsins. In particular, the procedure is used as an early investigation of a recently discovered family of opsins, the proteorhodopsins activated by blue light, BPRs. The results reveal some interesting and unexpected similarities between proteins of the two families, suggesting the global electrical response are not strictly linked to the class identity. [ABSTRACT FROM AUTHOR]

Gillisia limnaea Van Trappen et al. 2004 is the type species of the genus Gillisia, which is a member of the well characterized family Flavobacteriaceae. The genome of G. limnea R-8282(T) is the first sequenced genome (permanent draft) from a type strain of the genus Gillisia. Here we describe the features of this organism, together with the permanent-draft genome sequence and annotation. The 3,966,857 bp long chromosome (two scaffolds) with its 3,569 protein-coding and 51 RNA genes is a part of the GenomicEncyclopedia of Bacteria and Archaea project.

At least 7 proteorhodopsin sequences of Oxyrrhis marina were recently proven in bands obtained by sucrose density gradient centrifugation, and MS analyses revealed that the bands consisted almost of pure, native proteorhodopsins (Rhiel et al. 2020). The proteorhodopsin fractions, i.e., bands B2, B3, and B4 were subjected to transmission electron microscopy. Negative staining revealed that band B2 consisted most likely of monomeric/oligomeric proteorhodopsins with particle dimensions of about 6 nm. Negative staining, freeze-fracture, and cryo-transmission electron microscopy revealed that bands B3 and B4 consisted of vesicular, sheet-like, and cup-shaped structures which all seemed to be composed of protein. Frequently, ring-like protein aggregates were registered at higher magnifications. They measured about 4 nm in diameter with a tiny hole of 1.5 nm in the middle. The bands B2, B3, and B4 were pooled and used to raise an antiserum. Immunoelectron microscopy resulted in intense labeling of the isolated structures. Immunofluorescence light microscopy of formaldehyde-fixed Oxyrrhis cells resulted in intense labeling of the cell periphery. Some cell internal structures became labeled, too. Immunoelectron microscopy of freeze-fractured cells revealed that most likely the membranes of the amphiesmal vesicles were labeled at the cell periphery, while the cell internal label seemed to originate from the food vacuoles. [ABSTRACT FROM AUTHOR]

Iron (Fe) limitation is known to affect heterotrophic bacteria within the respiratory electron transport chain, therefore strongly impacting the overall intracellular energy production. We investigated whether the gene expression pattern of the light-sensitive proton pump, proteorhodopsin (PR), is influenced by varying light, carbon and Fe concentrations in the marine bacterium Photobacterium angustum S14 and whether PR can alleviate the physiological processes associated with Fe starvation. Our results show that the gene expression of PR increases as cells enter the stationary phase, irrespective of Fe-replete or Fe-limiting conditions. This upregulation is coupled to a reduction in cell size, indicating that PR gene regulation is associated with a specific starvation-stress response. We provide experimental evidence that PR gene expression does not result in an increased growth rate, cell abundance, enhanced survival or ATP concentration within the cell in either Fe-replete or Fe-limiting conditions. However, independent of PR gene expression, the presence of light did influence bacterial growth rates and maximum cell abundances under varying Fe regimes. Our observations support previous results indicating that PR phototrophy seems to play an important role within the stationary phase for several members of the Vibrionaceae family, but that the exact role of PR in Fe limitation remains to be further explored. [ABSTRACT FROM AUTHOR]

For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X‐100. Subsequent sucrose density gradient centrifugation resulted in three or four red‐colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514–516 nm with shoulders toward shorter wavelengths (470–490 nm). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in‐gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified. [ABSTRACT FROM AUTHOR]

The green-light absorbing proteorhodopsin (GPR) is the prototype of bacterial light-driven proton pumps. It has been the focus of continuous research since its discovery 20 years ago and has sparked the development and application of various biophysical techniques. However, a certain controversy and ambiguity about the oligomeric assembly of GPR still remains. We present here the first tag-free purification of pentameric GPR. The combination of ion exchange and size exclusion chromatography yields homogeneous and highly pure untagged pentamers from GPR overexpressing Escherichia coli. The presented purification procedure provides native-like protein and excludes the need for affinity purification tags. Importantly, three-dimensional protein crystals of GPR were successfully grown and analyzed by X-ray crystallography. These results together with data from single particle cryo-electron microscopy provide direct evidence for the pentameric stoichiometry of purified GPR.

Abstract Planktonic Archaea have been detected in all the world's oceans and are found from surface waters to the deep sea. The two most common Archaea phyla are Thaumarchaeota and Euryarchaeota. Euryarchaeota are generally more common in surface waters, but very little is known about their ecology and their potential metabolisms. In this study, we explore the genomic ecology of the Marine Group II (MGII), the main marine planktonic Euryarchaeota, and test if it is composed of different ecologically relevant units. We re‐analyzed Tara Oceans metagenomes from the photic layer and the deep ocean by annotating sequences against a custom MGII database and by mapping gene co‐occurrences. Our data provide a global view of the distribution of Euryarchaeota, and more specifically of MGII subgroups, and reveal their association to a number of gene‐coding sequences. In particular, we show that MGII proteorhodopsins were detected in both the surface and the deep chlorophyll maximum layer and that different clusters of these light harvesting proteins were present. Our approach helped describing the set of genes found together with specific MGII subgroups. We could thus define genomic environments that could theoretically describe ecologically meaningful units and the ecological niche that they occupy.

Abstract The production and release of extracellular vesicles (EVs) is a common process occurring in various types of bacteria. However, little is known regarding the functions of EVs derived from marine bacteria. We observed that during cell growth, Sediminicola sp. YIK13, a proteorhodopsin (PR)‐containing marine flavobacterium, produces EVs (S13EVs). Transmission electron microscopy showed that Sediminicola sp. YIK13 released two spherical vesicle types, with mono‐ and/or bi‐layered membranes, in the culture. Interestingly, the S13EVs have an orange pigment, indicating the presence of putative carotenoid and PR pigments ascribed to the parental cells. The S13EVs demonstrated the same PR‐derived absorption peak spectrum and light‐induced proton pump activity as the parental cells. Western blot (immunoblot) analysis of the S13EVs revealed the presence of PR. We confirmed the 16S rRNA gene, pro gene, and genes required for chromophore retinal synthesis, namely blh and crtI, in the DNA packaged into these vesicles. In addition, by metagenomic sequencing, we found microbial rhodopsin‐related genes in vesicles derived from natural aquatic environments. Our results suggest that EVs as well potentially pursue horizontal gene transfer of diverse microbial rhodopsin genes in marine ecosystems.

Light plays a central role on primary productivity of aquatic systems. Yet, its potential impact on the degradation of photosynthetically produced biomass is not well understood. We investigated the patterns of light-induced particle breakdown and bacterial assimilation of detrital C and N using 13C and 15N labeled freeze-thawed diatom cells incubated in laboratory microcosms with a marine microbial community freshly collected from the Pacific Ocean. Particles incubated in the dark resulted in increased bacterial counts and dissolved organic carbon concentrations compared to those incubated in the light. Light also influenced the attached and free-living microbial community structure as detected by 16S rRNA gene amplicon sequencing. For example, Sphingobacteriia were enriched on dark-incubated particles and taxa from the family Flavobacteriaceae and the genus Pseudoalteromonas were numerically enriched on particles in the light. Isotope incorporation analysis by phylogenetic microarray and NanoSIMS (a method called Chip-SIP) identified free-living and attached microbial taxa able to incorporate N and C from the particles. Some taxa, including members of the Flavobacteriaceae and Cryomorphaceae, exhibited increased isotope incorporation in the light, suggesting the use of photoheterotrophic metabolisms. In contrast, some members of Oceanospirillales and Rhodospirillales showed decreased isotope incorporation in the light, suggesting that their heterotrophic metabolism, particularly when occurring on particles, might increase at night or may be inhibited by sunlight. These results show that light influences particle degradation and C and N incorporation by attached bacteria, suggesting that the transfer between particulate and free-living phases are likely affected by external factors that change with the light regime, such as time of day, water column depth and season.

Many experiments have been carried out to display different colors of Proteorhodopsin (PR) and its mutants, but the mechanism of color tuning of PR was not fully elucidated. In this study, we applied the Electrostatically Embedded Generalized Molecular Fractionation with Conjugate Caps (EE-GMFCC) method to the prediction of excitation energies of PRs. Excitation energies of 10 variants of Blue Proteorhodopsin (BPR-PR105Q) in residue 105GLN were calculated with the EE-GMFCC method at the TD-B3LYP/6-31G* level. The calculated results show good correlation with the experimental values of absorption wavelengths, although the experimental wavelength range among these systems is less than 50 nm. The ensemble-averaged electric fields along the polyene chain of retinal correlated well with EE-GMFCC calculated excitation energies for these 10 PRs, suggesting that electrostatic interactions from nearby residues are responsible for the color tuning. We also utilized the GMFCC method to decompose the excitation energy contribution per residue surrounding the chromophore. Our results show that residues ASP97 and ASP227 have the largest contribution to the absorption spectral shift of PR among the nearby residues of retinal. This work demonstrates that the EE-GMFCC method can be applied to accurately predict the absorption spectral shifts for biomacromolecules.

Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli. Quantitative mass spectrometry shows that E. coli, exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize -148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans. [ABSTRACT FROM AUTHOR]

Strain IMCC1322 was isolated from a surface water sample from the East Sea of Korea. Based on 16S rRNA analysis, IMCC1322 was found to belong to the OCS28 sub-clade of SAR116. The cells appeared as short vibrioids in logarithmic-phase culture, and elongated spirals during incubation with mitomycin or in aged culture. Growth characteristics of strain IMCC1322 were further evaluated based on genomic information; proteorhodopsin (PR), carbon monoxide dehydrogenase, and dimethylsulfoniopropionate (DMSP)-utilizing enzymes. IMCC1322 PR was characterized as a functional retinylidene protein that acts as a light-driven proton pump in the cytoplasmic membrane. However, the PR-dependent phototrophic potential of strain IMCC1322 was only observed under CO-inhibited and nutrient-limited culture conditions. A DMSP-enhanced growth response was observed in addition to cultures grown on C1 compounds like methanol, formate, and methane sulfonate. Strain IMCC1322 cultivation analysis revealed biogeochemical processes characteristic of the SAR116 group, a dominant member of the microbial community in euphotic regions of the ocean. The polyphasic taxonomy of strain IMCC1322 is given as Candidatus Puniceispirillum marinum, and was confirmed by chemotaxonomic tests, in addition to 16S rRNA phylogeny and cultivation analyses. [ABSTRACT FROM AUTHOR]

Light plays a central role on primary productivity of aquatic systems. Yet, its potential impact on the degradation of photosynthetically produced biomass is not well understood. We investigated the patterns of light-induced particle breakdown and bacterial assimilation of detrital C and N using 13C and 15N labeled freeze-thawed diatom cells incubated in laboratory microcosms with a marine microbial community freshly collected from the Pacific Ocean. Particles incubated in the dark resulted in increased bacterial counts and dissolved organic carbon concentrations compared to those incubated in the light. Light also influenced the attached and free-living microbial community structure as detected by 16S rRNA gene amplicon sequencing. For example, Sphingobacteriia were enriched on dark-incubated particles and taxa from the family Flavobacteriaceae and the genus Pseudoalteromonas were numerically enriched on particles in the light. Isotope incorporation analysis by phylogenetic microarray and NanoSIMS (a method called Chip-SIP) identified free-living and attached microbial taxa able to incorporate N and C from the particles. Some taxa, including members of the Flavobacteriaceae and Cryomorphaceae, exhibited increased isotope incorporation in the light, suggesting the use of photoheterotrophic metabolisms. In contrast, some members of Oceanospirillales and Rhodospirillales showed decreased isotope incorporation in the light, suggesting that their heterotrophic metabolism, particularly when occurring on particles, might increase at night or may be inhibited by sunlight. These results show that light influences particle degradation and C and N incorporation by attached bacteria, suggesting that the transfer between particulate and free-living phases are likely affected by external factors that change with the light regime, such as time of day, water column depth and season. [ABSTRACT FROM AUTHOR]

Proteorhodopsin (PR) is a highly abundant, pentameric, light-driven proton pump. Proton transfer is linked to a canonical photocycle typical for microbial ion pumps. Although the PR monomer is able to undergo a full photocycle, the question arises whether the pentameric complex formed in the membrane via specific cross-protomer interactions plays a role in its functional mechanism. Here, we use dynamic nuclear polarization (DNP)-enhanced solid-state magicangle spinning (MAS) NMR in combination with light-induced cryotrapping of photointermediates to address this topic. The highly conserved residue H75 is located at the protomer interface. We show that it switches from the (t)- to the (p)-tautomer and changes its ring orientation in the M state. It couples to W34 across the oligomerization interface based on specific His/Trp ring orientations while stabilizing the pKa of the primary proton acceptor D97 within the same protomer. We further show that specific W34 mutations have a drastic effect on D97 and proton transfer mediated through H75. The residue H75 defines a cross-protomer Asp-His-Trp triad, which potentially serves as a pH-dependent regulator for proton transfer. Our data represent light-dependent, functionally relevant cross talk between protomers of a microbial rhodopsin homo-oligomer. [ABSTRACT FROM AUTHOR]

Summary: Hymenobacter nivis P3T is a heterotrophic bacterium isolated from Antarctic red snow generated by algal blooms. Despite being non‐photosynthetic, H. nivis was dominantly found in the red snow environment that is exposed to high light and UV irradiation, suggesting that this species can flourish under such harsh conditions. In order to further understand the adaptive strategies on the snow surface environment of Antarctica, the genome of H. nivis P3T was sequenced and analyzed, which identified genes putatively encoding for light‐reactive proteins such as proteorhodopsin, phytochrome, photolyase and several copies of cryptochromes. Culture‐based experiments revealed that H. nivis P3T growth was significantly enhanced under light conditions, while dark conditions had increased extracellular polymeric substances. Furthermore, the expression of several putative light‐reactive proteins was determined by proteomic analysis. These results indicate that H. nivis P3T is able to potentially utilize light, which may explain its dominance on the red snow surface environment of Antarctica. Originality‐significance statement: The role of proteorhodopsin in heterotrophic bacteria is not well‐characterized, as only a handful of proteorhodopsin‐harbouring isolates were shown to have a light‐enhanced phenotype through culture‐based experiments to date. This is the first study that demonstrates light‐stimulated growth and protein expression evidence of photoactive proteins for a non‐marine psychrophile and for a member of the genus Hymenobacter. It is also the first study that provides comprehensive proteome information for this genus. This study presents significant results in understanding the adaptive mechanism of a heterotrophic non‐photosynthetic bacterium thriving on the snow surface environment of Antarctica as well as demonstrating the role of light‐utilization in promoting growth, possibly through proteorhodopsin. [ABSTRACT FROM AUTHOR]

Abstract To fill the "green absorption gap", a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (ΔPSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the ΔPSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of ΔPSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (ΔPSI/ΔSynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing ΔPSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP+-reduction. Highlights • Proteorhodopsin expression increases growth rate of a ∆PSI Synechocystis strain with at least 16 %. • Proteorhodopsin contributes to proton motive force generation in vivo in Synechocystis. • A transgenic Synechocystis strain has been generated that can absorb infrared light. • 'Reversed electron transfer' through NDH-1 could not be demonstrated in Synechocystis. [ABSTRACT FROM AUTHOR]

The most abundant and ubiquitous microbes in the surface ocean use light as an energy source, capturing it via complex chlorophyll-based photosystems or simple retinal-based rhodopsins. Studies in various ocean regimes compared the abundance of these mechanisms, but few investigated their expression. Here we present the first full seasonal study of abundance and expression of light-harvesting mechanisms (proteorhodopsin, PR; aerobic anoxygenic photosynthesis, AAnP; and oxygenic photosynthesis, PSI) from deep-sequenced metagenomes and metatranscriptomes of marine picoplankton (

Student microbial ecology laboratory courses are often conducted as condensed courses in which theory and wet lab work are combined in a very intensive short time period. In last decades, the study of marine microbial ecology is increasingly reliant on molecular-based methods, and as a result many of the research projects conducted in such courses require sequencing that is often not available on site and may take more time than a typical course allows. In this work, we describe a protocol combining molecular and functional methods for analyzing proteorhodopsins (PRs), with visible results in only 4–5 days that do not rely on sequencing. PRs were discovered in oceanic surface waters two decades ago, and have since been observed in different marine environments and diverse taxa, including the abundant alphaproteobacterial SAR11 group. PR subgroups are currently known to absorb green and blue light, and their distribution was previously explained by prevailing light conditions – green pigments at the surface and blue pigments in deeper waters, as blue light travels deeper in the water column. To detect PR in environmental samples, we created a chimeric plasmid suitable for direct expression of PRs using PCR amplification and functional analysis in Escherichia coli cells. Using this assay, we discovered several exceptional cases of PRs whose phenotypes differed from those predicted based on sequence only, including a previously undescribed yellow-light absorbing PRs. We applied this assay in two 10-days marine microbiology courses and found it to greatly enhance students’ laboratory experience, enabling them to gain rapid visual feedback and colorful reward for their work. Furthermore we expect this assay to promote the use of functional assays for the discovery of new rhodopsin variants.

SAR86 is one of the most abundant groups of bacteria in the global surface ocean. However, since its discovery over 30 years ago, it has remained recalcitrant to isolation and many details regarding this group are still unknown. Here, we report the cellular characteristics from the first SAR86 isolate brought into culture, Magnimaribacter mokuoloeensis strain HIMB1674, and use its closed genome in concert with over 700 environmental genomes to assess the phylogenomic and functional characteristics of this order-level lineage of marine Gammaproteobacteria. The SAR86 order Magnimaribacterales invests significant genomic resources into the capacity for $\beta$-oxidation, which is present in most genomes with high gene copy numbers. This cyclical set of reactions appears to be fed by components of cell membranes that include lipids such as phosphatidylcholine, phosphatidylethanolamine, glycolipids, and sulfolipids. In addition to the widespread capacity to degrade the side chain of steroidal compounds via $\beta$-oxidation, several SAR86 sublineages also appear able to fully degrade the steroid polycyclic ring structure as well as other aromatic, polycyclic, and heterocyclic molecules. Read recruitment from publicly available metagenomes reveals that the Magnimaribacterales compose up to 6% of the global surface ocean microbial community. Only a subset of genera drives these high relative abundances, with some more globally dominant and others restricted to specific oceanic regions. This study provides an unprecedented foundation through which to understand this highly abundant yet poorly understood lineage of marine bacteria and charts a path to bring more representatives of this order into laboratory culture., (© The Author(s) 2024. Published by Oxford University Press.)

Microbial rhodopsins have become an important tool in the field of optogenetics. However, effective in vivo optogenetics is in many cases severely limited due to the strong absorption and scattering of visible light by biological tissues. Recently, a combination of opsin site-directed mutagenesis and analog retinal substitution has produced variants of proteorhodopsin which absorb maximally in the near-infrared (NIR). In this study, UV-Visible-NIR absorption and resonance Raman spectroscopy were used to study the double mutant, D212N/F234S, of green absorbing proteorhodopsin (GPR) regenerated with MMAR, a retinal analog containing a methylamino modified β-ionone ring. Four distinct subcomponent absorption bands with peak maxima near 560, 620, 710 and 780 nm are detected with the NIR bands dominant at pH <7.3, and the visible bands dominant at pH 9.5. FT-Raman using 1064-nm excitation reveal two strong ethylenic bands at 1482 and 1498 cm-1 corresponding to the NIR subcomponent absorption bands based on an extended linear correlation between λmax and γC = C. This spectrum exhibits two intense bands in the fingerprint and HOOP mode regions that are highly characteristic of the O640 photointermediate from the light-adapted bacteriorhodopsin photocycle. In contrast, 532-nm excitation enhances the 560-nm component, which exhibits bands very similar to light-adapted bacteriorhodopsin and/or the acid-purple form of bacteriorhodopsin. Native GPR and its mutant D97N when regenerated with MMAR also exhibit similar absorption and Raman bands but with weaker contributions from the NIR absorbing components. Based on these results it is proposed that the NIR absorption in GPR-D212N/F234S with MMAR arises from an O-like chromophore, where the Schiff base counterion D97 is protonated and the MMAR adopts an all-trans configuration with a non-planar geometry due to twists in the conjugated polyene segment. This configuration is characterized by extensive charge delocalization, most likely involving nitrogens atoms in the MMAR chromophore. [ABSTRACT FROM AUTHOR]

Microbial rhodopsins are well-known seven-transmembrane proteins that have been extensively studied for their structure and function. These retinal-binding proteins can be divided into two types. Type I is microbial rhodopsin, and type II (visual pigment) is expressed mostly in mammalian eyes. The two primary functions of type I rhodopsin are ion pumping activity and sensory transduction. Anabaena sensory rhodopsin (ASR) is a microbial rhodopsin with a specific function of photosensory transduction. ASR is expressed at moderate levels in Escherichia coli , but its expression level is lower compared to the general green light absorbing proteorhodopsin (GPR). In this study, full-length ASR was used to test the influence of codon usage on expression E. coli . Seven amino acids at the N-terminal region of ASR after the Met start codon were changed randomly using designed primers, which allowed for 8192 different nucleotide combinations. The codon changes were screened for the preferable codons that resulted in higher expression yield. Among the 57 selected mutations, 24 color-enhanced E. coli colonies contained ASR proteins, and they expressed ASR at a higher level than the bacteria with wild-type ASR codon usage. This result strongly suggests that the specific codon usage of only the N-terminal portion of a protein can increase the expression level of the entire protein. [ABSTRACT FROM AUTHOR]

The most abundant and ubiquitous microbes in the surface ocean use light as an energy source, capturing it via complex chlorophyll-based photosystems or simple retinal-based rhodopsins. Studies in various ocean regimes compared the abundance of these mechanisms, but few investigated their expression. Here we present the first full seasonal study of abundance and expression of light-harvesting mechanisms (proteorhodopsin, PR; aerobic anoxygenic photosynthesis, AAnP; and oxygenic photosynthesis, PSI) from deep-sequenced metagenomes and metatranscriptomes of marine picoplankton (<1 µm) at three coastal stations of the San Pedro Channel in the Pacific Ocean. We show that, regardless of season or sampling location, the most common phototrophic mechanism in metagenomes of this dynamic region was PR (present in 65-104% of the genomes as estimated by single-copy recA), followed by PSI (5-104%) and AAnP (5-32%). Furthermore, the normalized expression (RNA to DNA ratio) of PR genes was higher than that of oxygenic photosynthesis (average ± standard deviation 26.2 ± 8.4 vs. 11 ± 9.7), and the expression of the AAnP marker gene was significantly lower than both mechanisms (0.013 ± 0.02). We demonstrate that PR expression was dominated by the SAR11-cluster year-round, followed by other Alphaproteobacteria, unknown-environmental clusters and Gammaproteobacteria. This highly dynamic system further allowed us to identify a trend for PR spectral tuning, in which blue-absorbing PR genes dominate in areas with low chlorophyll-a concentrations (<0.25 µgL-1). This suggests that PR phototrophy is not an accessory function but instead a central mechanism that can regulate photoheterotrophic population dynamics. [ABSTRACT FROM AUTHOR]

Proteorhodopsin (PR) is a microbial rhodopsin functioning as a light‐driven proton pump in aquatic bacteria. We performed low‐temperature Raman measurements of PR to obtain the structure of the primary photoproduct, the K intermediate (PRK). PRK showed the hydrogen‐out‐of‐plane modes that are much less intense than those of bacteriorhodopsin as the prototypical light‐driven proton pump from haloarchaea. The present results reveal the significantly relaxed chromophore structure in PRK, which can be coupled to the slow kinetics of the K intermediate. This structure suggests that PR transports protons using the small energy storage within the chromophore at the start of its photocycle. [ABSTRACT FROM AUTHOR]

The lipid environment plays a crucial role for the function of membrane proteins. We analyzed the influence of various lipid properties on the photoreceptors Bacteriorhodopsin (BR) and Proteorhodopsin (PR) by time-resolved step-scan FTIR spectroscopy. The photoreceptors were reconstituted into liposomes thereby providing an uniform biomimetic membrane, and the physical properties of the lipids were systematically varied. The lipid phase alters significantly the protonation dynamics of both, BR and PR. We also analyzed the conformational changes of the photoreceptors during the proton pumping mechanism and could demonstrate that a lipid-induced altered protonation dynamics is correlated to an altered conformational dynamics. Our results indicate that the fluidity of the membrane environment directly influences the structural-functional correlation. [ABSTRACT FROM AUTHOR]

Summary: It is of great significance to understand CO2 fixation in the oceans. Using single cell Raman spectra (SCRS) as biochemical profiles, Raman activated cell ejection (RACE) was able to link phenotypes and genotypes of cells. Here, we show that mini‐metagenomic sequences from RACE can be used as a reference to reconstruct nearly complete genomes of key functional bacteria by binning shotgun metagenomic sequencing data. By applying this approach to 13C bicarbonate spiked seawater from euphotic zone of the Yellow Sea of China, the dominant bacteria Synechococcus spp. and Pelagibacter spp. were revealed and both of them contain carotenoid and were able to incorporate 13C into the cells at the same time. Genetic analysis of the reconstructed genomes suggests that both Synechococcus spp. and Pelagibacter spp. contained all genes necessary for carotenoid synthesis, light energy harvesting and CO2 fixation. Interestingly, the reconstructed genome indicates that Pelagibacter spp. harbored intact sets of genes for β‐carotene (precursor of retional), proteorhodopsin synthesis and anaplerotic CO2 fixation. This novel approach shines light on the role of marine ‘microbial dark matter’ in global carbon cycling, by linking yet‐to‐be‐cultured Synechococcus spp. and Pelagibacter spp. to carbon fixation and flow activities in situ. [ABSTRACT FROM AUTHOR]

In many pro- and eukaryotes, a retinal-based proton pump equips the cell to drive ATP synthesis with (sun)light. Such pumps, therefore, have been proposed as a plug-in for cyanobacteria to artificially increase the efficiency of oxygenic photosynthesis. However, little information on the metabolism of retinal, their chromophore, is available for these organisms. We have studied the in vivo roles of five genes (sll1541, slr1648, slr0091, slr1192, and slr0574) potentially involved in retinal metabolism in Synechocystis sp. strain PCC 6803. With a gene deletion approach, we have shown that Synechocystis apo-carotenoid-15,15-oxygenase (SynACO), encoded by gene sll1541, is an indispensable enzyme for retinal synthesis in Synechocystis, presumably via asymmetric cleavage of β-apo-carotenal. The second carotenoid oxygenase (SynDiox2), encoded by gene slr1648, competes with SynACO for substrate(s) but only measurably contributes to retinal biosynthesis in stationary phase via an asyet- unknown mechanism. In vivo degradation of retinal may proceed through spontaneous chemical oxidation and via enzyme-catalyzed processes. Deletion of gene slr0574 (encoding CYP120A1), but not of slr0091 or of slr1192, causes an increase (relative to the level in wild-type Synechocystis) in the retinal content in both the linear and stationary growth phases. These results suggest that CYP120A1 does contribute to retinal degradation. Preliminary data obtained using 13C-labeled retinal suggest that conversion to retinol and retinoic acid and subsequent further oxidation also play a role. Deletion of sll1541 leads to deficiency in retinal synthesis and allows the in vivo reconstitution of far-red-absorbing holo-proteorhodopsin with exogenous retinal analogues, as demonstrated here for all-trans 3,4-dehydroretinal and 3-methylamino-16-nor-1,2,3,4-didehydroretinal. IMPORTANCE Retinal is formed by many cyanobacteria and has a critical role in most forms of life for processes such as photoreception, growth, and stress survival. However, the metabolic pathways in cyanobacteria for synthesis and degradation of retinal are poorly understood. In this paper we identify genes involved in its synthesis, characterize their role, and provide an initial characterization of the pathway of its degradation. This led to the identification of sll1541 (encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a lightdependent proton pump with an action spectrum extending beyond 700 nm. The availability of this strain will be important for further work aimed at increasing the overall efficiency of oxygenic photosynthesis. [ABSTRACT FROM AUTHOR]

A versatile synthetic protocol is reported that allows high concentrations of functionally active membrane proteins to be incorporated in mesostructured silica materials. Judicious selections of solvent, surfactant, silica precursor species, and synthesis conditions enable membrane proteins to be stabilized in solution and during subsequent coassembly into silica-surfactant composites with nano- and mesoscale order. This was demonstrated by using a combination of nonionic (n-dodecyl-β-D-maltoside or Pluronic P123), lipid-like (1,2-diheptanoyl-sn-glycero-3-phosphocholine), and perfluoro-octanoate surfactants under mild acidic conditions to coassemble the light-responsive transmembrane protein proteorhodopsin at concentrations up to 15 wt % into the hydrophobic regions of worm-like mesostructured silica materials in films. Small-angle X-ray scattering, electron paramagnetic resonance spectroscopy, and transient UV-visible spectroscopy analyses established that proteorhodopsin molecules in mesostructured silica films exhibited native-like function, as well as enhanced thermal stability compared to surfactant or lipid environments. The light absorbance properties and light-activated conformational changes of proteorhodopsin guests in mesostructured silica films are consistent with those associated with the native H+-pumping mechanism of these biomolecules. The synthetic protocol is expected to be general, as demonstrated also for the incorporation of functionally active cytochrome c, a peripheral membrane protein enzyme involved in electron transport, into mesostructured silica-cationic surfactant films. [ABSTRACT FROM AUTHOR]

Student microbial ecology laboratory courses are often conducted as condensed courses in which theory and wet lab work are combined in a very intensive short time period. In last decades, the study of marine microbial ecology is increasingly reliant on molecular-based methods, and as a result many of the research projects conducted in such courses require sequencing that is often not available on site and may take more time than a typical course allows. In this work, we describe a protocol combining molecular and functional methods for analyzing proteorhodopsins (PRs), with visible results in only 4-5 days that do not rely on sequencing. PRs were discovered in oceanic surface waters two decades ago, and have since been observed in different marine environments and diverse taxa, including the abundant alphaproteobacterial SAR11 group. PR subgroups are currently known to absorb green and blue light, and their distribution was previously explained by prevailing light conditions - green pigments at the surface and blue pigments in deeper waters, as blue light travels deeper in the water column. To detect PR in environmental samples, we created a chimeric plasmid suitable for direct expression of PRs using PCR amplification and functional analysis in Escherichia coli cells. Using this assay, we discovered several exceptional cases of PRs whose phenotypes differed from those predicted based on sequence only, including a previously undescribed yellow-light absorbing PRs. We applied this assay in two 10-days marine microbiology courses and found it to greatly enhance students' laboratory experience, enabling them to gain rapid visual feedback and colorful reward for their work. Furthermore we expect this assay to promote the use of functional assays for the discovery of new rhodopsin variants. [ABSTRACT FROM AUTHOR]

Marine Bacteroidetes constitute a very abundant bacterioplankton group in the oceans that plays a key role in recycling particulate organic matter and includes several photoheterotrophic members containing proteorhodopsin. Relatively few marine Bacteroidetes species have been described and, moreover, they correspond to cultured isolates, which in most cases do not represent the actual abundant or ecologically relevant microorganisms in the natural environment. In this study, we explored the microdiversity of 98 Single Amplified Genomes (SAGs) retrieved from the surface waters of the underexplored North Indian Ocean, whose most closely related isolate is Kordia algicida OT-1. Using Multi Locus Sequencing Analysis (MLSA) we found no microdiversity in the tested conserved phylogenetic markers (16S rRNA and 23S rRNA genes), the fast-evolving Internal Transcribed Spacer and the functional markers proteorhodopsin and the beta-subunit of RNA polymerase. Furthermore, we carried out a Fragment Recruitment Analysis (FRA) with marine metagenomes to learn about the distribution and dynamics of this microorganism in different locations, depths and size fractions. This analysis indicated that this taxon belongs to the rare biosphere, showing its highest abundance after upwelling-induced phytoplankton blooms and sinking to the deep ocean with large organic matter particles. This uncultured Kordia lineage likely represents a novel Kordia species (Kordia sp. CFSAG39SUR) that contains the proteorhodopsin gene and has a widespread spatial and vertical distribution. The combination of SAGs and MLSA makes a valuable approach to infer putative ecological roles of uncultured abundant microorganisms.