Ecological diversification of a cyanobacterium through divergence of its novel chlorophyll d-based light-harvesting system.

The evolution of novel traits can have important consequences for biological diversification. Novelties such as new structures are associated with changes in both genotype and phenotype that often lead to changes in ecological function.1,2 New ecological opportunities provided by a novel trait can trigger subsequent trait modification or niche partitioning3; however, the underlying mechanisms of novel trait diversification are still poorly understood. Here, we report that the innovation of a new chlorophyll (Chl) pigment, Chl d, by the cyanobacterium Acaryochloris marina was followed by the functional divergence of its light-harvesting complex. We identified three major photosynthetic spectral types based on Chl fluorescence properties for a collection of A. marina laboratory strains for which genome sequence data are available,4,5 with shorter- and longer-wavelength types more recently derived from an ancestral intermediate phenotype. Members of the different spectral types exhibited extensive variation in the Chl-binding proteins as well as the Chl energy levels of their photosynthetic complexes. This spectral-type divergence is associated with differences in the wavelength dependence of both growth rate and photosynthetic oxygen evolution. We conclude that the divergence of the light-harvesting apparatus has consequently impacted A. marina ecological diversification through specialization on different far-red photons for photosynthesis.

Femtosecond optical studies of the primary charge separation reactions in far-red photosystem II from Synechococcus sp. PCC 7335.

Primary processes of light energy conversion by Photosystem II (PSII) were studied using femtosecond broadband pump-probe absorption difference spectroscopy. Transient absorption changes of core complexes isolated from the cyanobacterium Synechococcus sp. PCC 7335 grown under far-red light (FRL-PSII) were compared with the canonical Chl a containing spinach PSII core complexes upon excitation into the red edge of the Qy band. Absorption changes of FRL-PSII were monitored at 278 K in the 400-800 nm spectral range on a timescale of 0.1-500 ps upon selective excitation at 740 nm of four chlorophyll (Chl) f molecules in the light harvesting antenna, or of one Chl d molecule at the ChlD1 position in the reaction center (RC) upon pumping at 710 nm. Numerical analysis of absorption changes and assessment of the energy levels of the presumed ion-radical states made it possible to identify PD1+ChlD1- as the predominant primary charge-separated radical pair, the formation of which upon selective excitation of Chl d has an apparent time of ∼1.6 ps. Electron transfer to the secondary acceptor pheophytin PheoD1 has an apparent time of ∼7 ps with a variety of excitation wavelengths. The energy redistribution between Chl a and Chl f in the antenna occurs within 1 ps, whereas the energy migration from Chl f to the RC occurs mostly with lifetimes of 60 and 400 ps. Potentiometric analysis suggests that in canonical PSII, PD1+ChlD1- can be partially formed from the excited (PD1ChlD1)* state.

The structural basis for light harvesting in organisms producing phycobiliproteins.

Cyanobacteria, red algae, and cryptophytes produce two classes of proteins for light-harvesting: water-soluble phycobiliproteins and membrane-intrinsic proteins that bind chlorophylls and carotenoids. In cyanobacteria, red algae, and glaucophytes, phycobilisomes (PBS) are complexes of brightly colored phycobiliproteins and linker (assembly) proteins. To date, six structural classes of phycobilisomes have been described: hemiellipsoidal, block-shaped, hemidiscoidal, bundle-shaped, paddle-shaped, and far-red-light bicylindrical. Two additional antenna complexes containing single types of phycobiliproteins have also been described. Since 2017, structures have been reported for examples of all of these complexes except bundle-shaped phycobilisomes by cryogenic electron microscopy. Phycobilisomes range in size from about 4.6 to 18 MDa and can include ∼900 polypeptides and bind >2000 chromophores. Cyanobacteria additionally produce membrane-associated proteins of the PsbC/CP43 superfamily of Chl a/b/d-binding proteins, including the iron-stress protein IsiA and other paralogous chlorophyll-binding proteins that can form antenna complexes with Photosystem I and/or Photosystem II. Red and cryptophyte algae also produce chlorophyll-binding proteins associated with Photosystem I but which belong to the chlorophyll a/b-binding (CAB) protein superfamily and which are unrelated to the chlorophyll-binding proteins (CBP) of cyanobacteria. This review describes recent progress in structure determination for phycobilisomes and the chlorophyll proteins of cyanobacteria, red algae, and cryptophytan algae.

An integrated approach towards extracting structural characteristics of chlorosomes from a bchQ mutant of Chlorobaculum tepidum.

Chlorosomes, the photosynthetic antenna complexes of green sulfur bacteria, are paradigms for light-harvesting elements in artificial designs, owing to their efficient energy transfer without protein participation. We combined magic angle spinning (MAS) NMR, optical spectroscopy and cryogenic electron microscopy (cryo-EM) to characterize the structure of chlorosomes from a bchQ mutant of Chlorobaculum tepidum. The chlorosomes of this mutant have a more uniform composition of bacteriochlorophyll (BChl) with a predominant homolog, [8Ethyl, 12Ethyl] BChl c, compared to the wild type (WT). Nearly complete 13C chemical shift assignments were obtained from well-resolved homonuclear 13C-13C RFDR data. For proton assignments heteronuclear 13C-1H (hCH) data sets were collected at 1.2 GHz spinning at 60 kHz. The CHHC experiments revealed intermolecular correlations between 132/31, 132/32, and 121/31, with distance constraints of less than 5 Å. These constraints indicate the syn-anti parallel stacking motif for the aggregates. Fourier transform cryo-EM data reveal an axial repeat of 1.49 nm for the helical tubular aggregates, perpendicular to the inter-tube separation of 2.1 nm. This axial repeat is different from WT and is in line with BChl syn-anti stacks running essentially parallel to the tube axis. Such a packing mode is in agreement with the signature of the Qy band in circular dichroism (CD). Combining the experimental data with computational insight suggests that the packing for the light-harvesting function is similar between WT and bchQ, while the chirality within the chlorosomes is modestly but detectably affected by the reduced compositional heterogeneity in bchQ.

Listeria adhesion protein orchestrates caveolae-mediated apical junctional remodeling of epithelial barrier for Listeria monocytogenes translocation.

The cellular junctional architecture remodeling by Listeria adhesion protein-heat shock protein 60 (LAP-Hsp60) interaction for Listeria monocytogenes (Lm) passage through the epithelial barrier is incompletely understood. Here, using the gerbil model, permissive to internalin (Inl) A/B-mediated pathways like in humans, we demonstrate that Lm crosses the intestinal villi at 48 h post-infection. In contrast, the single isogenic (lap- or ΔinlA) or double (lap-ΔinlA) mutant strains show significant defects. LAP promotes Lm translocation via endocytosis of cell-cell junctional complex in enterocytes that do not display luminal E-cadherin. In comparison, InlA facilitates Lm translocation at cells displaying apical E-cadherin during cell extrusion and mucus expulsion from goblet cells. LAP hijacks caveolar endocytosis to traffic integral junctional proteins to the early and recycling endosomes. Pharmacological inhibition in a cell line and genetic knockout of caveolin-1 in mice prevents LAP-induced intestinal permeability, junctional endocytosis, and Lm translocation. Furthermore, LAP-Hsp60-dependent tight junction remodeling is also necessary for InlA access to E-cadherin for Lm intestinal barrier crossing in InlA-permissive hosts. IMPORTANCE: Listeria monocytogenes (Lm) is a foodborne pathogen with high mortality (20%-30%) and hospitalization rates (94%), particularly affecting vulnerable groups such as pregnant women, fetuses, newborns, seniors, and immunocompromised individuals. Invasive listeriosis involves Lm's internalin (InlA) protein binding to E-cadherin to breach the intestinal barrier. However, non-functional InlA variants have been identified in Lm isolates, suggesting InlA-independent pathways for translocation. Our study reveals that Listeria adhesion protein (LAP) and InlA cooperatively assist Lm entry into the gut lamina propria in a gerbil model, mimicking human listeriosis in early infection stages. LAP triggers caveolin-1-mediated endocytosis of critical junctional proteins, transporting them to early and recycling endosomes, facilitating Lm passage through enterocytes. Furthermore, LAP-Hsp60-mediated junctional protein endocytosis precedes InlA's interaction with basolateral E-cadherin, emphasizing LAP and InlA's cooperation in enhancing Lm intestinal translocation. This understanding is vital in combating the severe consequences of Lm infection, including sepsis, meningitis, encephalitis, and brain abscess.

Structure of the antenna complex expressed during far-red light photoacclimation in Synechococcus sp. PCC 7335.

Far-red light photoacclimation, or FaRLiP, is a facultative response exhibited by some cyanobacteria that allows them to absorb and utilize lower energy light (700-800 nm) than the wavelengths typically used for oxygenic photosynthesis (400-700 nm). During this process, three essential components of the photosynthetic apparatus are altered: photosystem I, photosystem II, and the phycobilisome. In all three cases, at least some of the chromophores found in these pigment-protein complexes are replaced by chromophores that have red-shifted absorbance relative to the analogous complexes produced in visible light. Recent structural and spectroscopic studies have elucidated important features of the two photosystems when altered to absorb and utilize far-red light, but much less is understood about the modified phycobiliproteins made during FaRLiP. We used single-particle, cryo-EM to determine the molecular structure of a phycobiliprotein core complex comprising allophycocyanin variants that absorb far-red light during FaRLiP in the marine cyanobacterium Synechococcus sp. PCC 7335. The structure reveals the arrangement of the numerous red-shifted allophycocyanin variants and the probable locations of the chromophores that serve as the terminal emitters in this complex. It also suggests how energy is transferred to the photosystem II complexes produced during FaRLiP. The structure additionally allows comparisons with other previously studied allophycocyanins to gain insights into how phycocyanobilin chromophores can be tuned to absorb far-red light. These studies provide new insights into how far-red light is harvested and utilized during FaRLiP, a widespread cyanobacterial photoacclimation mechanism.

Molecular diversity and evolution of far-red light-acclimated photosystem I.

The need to acclimate to different environmental conditions is central to the evolution of cyanobacteria. Far-red light (FRL) photoacclimation, or FaRLiP, is an acclimation mechanism that enables certain cyanobacteria to use FRL to drive photosynthesis. During this process, a well-defined gene cluster is upregulated, resulting in changes to the photosystems that allow them to absorb FRL to perform photochemistry. Because FaRLiP is widespread, and because it exemplifies cyanobacterial adaptation mechanisms in nature, it is of interest to understand its molecular evolution. Here, we performed a phylogenetic analysis of the photosystem I subunits encoded in the FaRLiP gene cluster and analyzed the available structural data to predict ancestral characteristics of FRL-absorbing photosystem I. The analysis suggests that FRL-specific photosystem I subunits arose relatively late during the evolution of cyanobacteria when compared with some of the FRL-specific subunits of photosystem II, and that the order Nodosilineales, which include strains like Halomicronema hongdechloris and Synechococcus sp. PCC 7335, could have obtained FaRLiP via horizontal gene transfer. We show that the ancestral form of FRL-absorbing photosystem I contained three chlorophyll f-binding sites in the PsaB2 subunit, and a rotated chlorophyll a molecule in the A0B site of the electron transfer chain. Along with our previous study of photosystem II expressed during FaRLiP, these studies describe the molecular evolution of the photosystem complexes encoded by the FaRLiP gene cluster.

A structure of the relict phycobilisome from a thylakoid-free cyanobacterium.

Phycobilisomes (PBS) are antenna megacomplexes that transfer energy to photosystems II and I in thylakoids. PBS likely evolved from a basic, inefficient form into the predominant hemidiscoidal shape with radiating peripheral rods. However, it has been challenging to test this hypothesis because ancestral species are generally inaccessible. Here we use spectroscopy and cryo-electron microscopy to reveal a structure of a "paddle-shaped" PBS from a thylakoid-free cyanobacterium that likely retains ancestral traits. This PBS lacks rods and specialized ApcD and ApcF subunits, indicating relict characteristics. Other features include linkers connecting two chains of five phycocyanin hexamers (CpcN) and two core subdomains (ApcH), resulting in a paddle-shaped configuration. Energy transfer calculations demonstrate that chains are less efficient than rods. These features may nevertheless have increased light absorption by elongating PBS before multilayered thylakoids with hemidiscoidal PBS evolved. Our results provide insights into the evolution and diversification of light-harvesting strategies before the origin of thylakoids.

Two-Dimensional Electronic Spectroscopy of the Far-Red-Light Photosystem II Reaction Center.

Understanding the role of specific pigments in primary energy conversion in the photosystem II (PSII) reaction center has been impeded by the spectral overlap of its constituent pigments. When grown in far-red light, some cyanobacteria incorporate chlorophyll-f and chlorophyll-d into PSII, relieving the spectral congestion. We employ two-dimensional electronic spectroscopy to study PSII at 77 K from Synechococcus sp. PCC 7335 cells that were grown in far-red light (FRL-PSII). We observe the formation of a radical pair within ∼3 ps that we assign to ChlD1•-PD1•+. While PheoD1 is thought to act as the primary electron acceptor in PSII from cells grown in visible light, we see no evidence of its involvement, which we attribute to its reduction by dithionite treatment in our samples. Our work demonstrates that primary charge separation occurs between ChlD1 and PD1 in FRL-PSII, suggesting that PD1/PD2 may play an underappreciated role in PSII's charge separation mechanism.

Loss of Biliverdin Reductase Increases Oxidative Stress in the Cyanobacterium Synechococcus sp. PCC 7002.

Oxygenic photosynthesis requires metal-rich cofactors and electron-transfer components that can produce reactive oxygen species (ROS) that are highly toxic to cyanobacterial cells. Biliverdin reductase (BvdR) reduces biliverdin IXα to bilirubin, which is a potent scavenger of radicals and ROS. The enzyme is widespread in mammals but is also found in many cyanobacteria. We show that a previously described bvdR mutant of Synechocystis sp. PCC 6803 contained a secondary deletion mutation in the cpcB gene. The bvdR gene from Synechococcus sp. PCC 7002 was expressed in Escherichia coli, and recombinant BvdR was purified and shown to reduce biliverdin to bilirubin. The bvdR gene was successfully inactivated in Synechococcus sp. PCC 7002, a strain that is naturally much more tolerant of high light and ROS than Synechocystis sp. PCC 6803. The bvdR mutant strain, BR2, had lower total phycobiliprotein and chlorophyll levels than wild-type cells. As determined using whole-cell fluorescence at 77 K, the photosystem I levels were also lower than those in wild-type cells. The BR2 mutant had significantly higher ROS levels compared to wild-type cells after exposure to high light for 30 min. Together, these results suggest that bilirubin plays an important role as a scavenger for ROS in Synechococcus sp. PCC 7002. The oxidation of bilirubin by ROS could convert bilirubin to biliverdin IXα, and thus BvdR might be important for regenerating bilirubin. These results further suggest that BvdR is a key component of a scavenging cycle by which cyanobacteria protect themselves from the toxic ROS byproducts generated during oxygenic photosynthesis.

A quantitative assessment of (bacterio)chlorophyll assignments in the cryo-EM structure of the Chloracidobacterium thermophilum reaction center.

Chlorophylls and bacteriochlorophylls are the primary pigments used by photosynthetic organisms for light harvesting, energy transfer, and electron transfer. Many molecular structures of (bacterio)chlorophyll-containing protein complexes are available, some of which contain mixtures of different (bacterio)chlorophyll types. Differentiating these, which sometimes are structurally similar, is challenging but is required for leveraging structural data to gain functional insight. The reaction center complex from Chloroacidobacterium thermophilum has a hybrid (bacterio)chlorophyll antenna system containing both chlorophyll a and bacteriochlorophyll a molecules. The recent availability of its cryogenic electron microscopy (cryo-EM) structure provides an opportunity for a quantitative analysis of their identities and chemical environments. Here, we describe a theoretical basis for differentiating chlorophyll a and bacteriochlorophyll a in a cryo-EM map, and apply the approach to the experimental cryo-EM maps of the (bacterio)chlorophyll sites of the chloroacidobacterial reaction center. The comparison reveals that at ~ 2.2-Å resolution, chlorophyll a and bacteriochlorophyll a are easily distinguishable, but the orientation of the bacteriochlorophyll a acetyl moiety is not; however, the latter can confidently be assigned by identifying a hydrogen bond donor from the protein environment. This study reveals the opportunities and challenges in assigning (bacterio)chlorophyll types in structural biology, the accuracy of which is vital for downstream investigations.

Structural comparison of allophycocyanin variants reveals the molecular basis for their spectral differences.

Allophycocyanins are phycobiliproteins that absorb red light and transfer the energy to the reaction centers of oxygenic photosynthesis in cyanobacteria and red algae. Recently, it was shown that some allophycocyanins absorb far-red light and that one subset of these allophycocyanins, comprising subunits from the ApcD4 and ApcB3 subfamilies (FRL-AP), form helical nanotubes. The lowest energy absorbance maximum of the oligomeric ApcD4-ApcB3 complexes occurs at 709 nm, which is unlike allophycocyanin (AP; ApcA-ApcB) and allophycocyanin B (AP-B; ApcD-ApcB) trimers that absorb maximally at ~ 650 nm and ~ 670 nm, respectively. The molecular bases of the different spectra of AP variants are presently unclear. To address this, we structurally compared FRL-AP with AP and AP-B, performed spectroscopic analyses on FRL-AP, and leveraged computational approaches. We show that among AP variants, the α-subunit constrains pyrrole ring A of its phycocyanobilin chromophore to different extents, and the coplanarity of ring A with rings B and C sets a baseline for the absorbance maximum of the chromophore. Upon oligomerization, the α-chromophores of all AP variants exhibit a red shift of the absorbance maximum of ~ 25 to 30 nm and band narrowing. We exclude excitonic coupling in FRL-AP as the basis for this red shift and extend the results to discuss AP and AP-B. Instead, we attribute these spectral changes to a conformational alteration of pyrrole ring D, which becomes more coplanar with rings B and C upon oligomerization. This study expands the molecular understanding of light-harvesting attributes of phycobiliproteins and will aid in designing phycobiliproteins for biotechnological applications.

Optically detected magnetic resonance and mutational analysis reveal significant differences in the photochemistry and structure of chlorophyll f synthase and photosystem II.

In cyanobacteria that undergo far red light photoacclimation (FaRLiP), chlorophyll (Chl) f is produced by the ChlF synthase enzyme, probably by photo-oxidation of Chl a. The enzyme forms homodimeric complexes and the primary amino acid sequence of ChlF shows a high degree of homology with the D1 subunit of photosystem II (PSII). However, few details of the photochemistry of ChlF are known. The results of a mutational analysis and optically detected magnetic resonance (ODMR) data from ChlF are presented. Both sets of data show that there are significant differences in the photochemistry of ChlF and PSII. Mutation of residues that would disrupt the donor side primary electron transfer pathway in PSII do not inhibit the production of Chl f, while alteration of the putative ChlZ, P680 and QA binding sites rendered ChlF non-functional. Together with previously published transient EPR and flash photolysis data, the ODMR data show that in untreated ChlF samples, the triplet state of P680 formed by intersystem crossing is the primary species generated by light excitation. This is in contrast to PSII, in which 3P680 is only formed by charge recombination when the quinone acceptors are removed or chemically reduced. The triplet states of a carotenoid (3Car) and a small amount of 3Chl f are also observed by ODMR. The polarization pattern of 3Car is consistent with its formation by triplet energy transfer from ChlZ if the carotenoid molecule is rotated by 15° about its long axis compared to the orientation in PSII. It is proposed that the singlet oxygen formed by the interaction between molecular oxygen and 3P680 might be involved in the oxidation of Chl a to Chl f.

Helical allophycocyanin nanotubes absorb far-red light in a thermophilic cyanobacterium.

To compete in certain low-light environments, some cyanobacteria express a paralog of the light-harvesting phycobiliprotein, allophycocyanin (AP), that strongly absorbs far-red light (FRL). Using cryo-electron microscopy and time-resolved absorption spectroscopy, we reveal the structure-function relationship of this FRL-absorbing AP complex (FRL-AP) that is expressed during acclimation to low light and that likely associates with chlorophyll a-containing photosystem I. FRL-AP assembles as helical nanotubes rather than typical toroids due to alterations of the domain geometry within each subunit. Spectroscopic characterization suggests that FRL-AP nanotubes are somewhat inefficient antenna; however, the enhanced ability to harvest FRL when visible light is severely attenuated represents a beneficial trade-off. The results expand the known diversity of light-harvesting proteins in nature and exemplify how biological plasticity is achieved by balancing resource accessibility with efficiency.

Identification of a Far-Red Light-Inducible Promoter that Exhibits Light Intensity Dependency and Reversibility in a Cyanobacterium.

As the demand for sustainable energy has increased, photoautotrophic cyanobacteria have become a popular platform for developing tools in synthetic biology. Although genetic tools are generally available for several model cyanobacteria, such tools have not yet been developed for many other strains potentially suitable for industrial applications. Additionally, most inducible promoters in cyanobacteria are controlled by chemical compounds, but adding chemicals into growth media on an industrial scale is neither cost-effective nor environmentally friendly. Although using light-controlled promoters is an alternative approach, only a cyanobacterial expression system inducible by green light has so far been described and employed for such applications. In this study, we have established a conjugation-based technique to express a reporter gene (eyfp) in the nonmodel cyanobacterium, Chlorogloeopsis fritschii PCC 9212. We also identified a promoter specifically activated by far-red light from the Far-Red Light Photoacclimation gene cluster of Leptolyngbya sp. JSC-1. This promoter, PchlFJSC1, was successfully used to drive eyfp expression. PchlFJSC1 is tightly regulated by light quality (i.e., wavelength) and leads to an approximately 30-fold increase in EYFP production when cells were exposed to far-red light. The induction level was controlled by the far-red light intensity, and induction stopped when cells were returned to visible light. This system has the potential for further applications in cyanobacteria by providing an additional choice of light wavelength to control gene expression. Collectively, this study developed a functional gene-expression system for C. fritschii PCC 9212 that can be regulated by exposing cells to far-red light.

Identification of a Functionally Efficient and Thermally Stable Outward Sodium-Pumping Rhodopsin (BeNaR) from a Thermophilic Bacterium.

Rhodopsins are transmembrane proteins with retinal chromophores that are involved in photo-energy conversion and photo-signal transduction in diverse organisms. In this study, we newly identified and characterized a rhodopsin from a thermophilic bacterium, Bellilinea sp. Recombinant Escherichia coli cells expressing the rhodopsin showed light-induced alkalization of the medium only in the presence of sodium ions (Na+), and the alkalization signal was enhanced by addition of a protonophore, indicating an outward Na+ pump function across the cellular membrane. Thus, we named the protein Bellilinea Na+-pumping rhodopsin, BeNaR. Of note, its Na+-pumping activity is significantly greater than that of the known Na+-pumping rhodopsin, KR2. We further characterized its photochemical properties as follows: (i) Visible spectroscopy and HPLC revealed that BeNaR has an absorption maximum at 524 nm with predominantly (>96%) the all-trans retinal conformer. (ii) Time-dependent thermal denaturation experiments revealed that BeNaR showed high thermal stability. (iii) The time-resolved flash-photolysis in the nanosecond to millisecond time domains revealed the presence of four kinetically distinctive photointermediates, K, L, M and O. (iv) Mutational analysis revealed that Asp101, which acts as a counterion, and Asp230 around the retinal were essential for the Na+-pumping activity. From the results, we propose a model for the outward Na+-pumping mechanism of BeNaR. The efficient Na+-pumping activity of BeNaR and its high stability make it a useful model both for ion transporters and optogenetics tools.

Structure of a dimeric photosystem II complex from a cyanobacterium acclimated to far-red light.

Photosystem II (PSII) is the water-splitting enzyme central to oxygenic photosynthesis. To drive water oxidation, light is harvested by accessory pigments, mostly chlorophyll (Chl) a molecules, which absorb visible light (400-700 nm). Some cyanobacteria facultatively acclimate to shaded environments by altering their photosynthetic machinery to additionally absorb far-red light (FRL, 700-800 nm), a process termed far-red light photoacclimation or FaRLiP. During far-red light photoacclimation, FRL-PSII is assembled with FRL-specific isoforms of the subunits PsbA, PsbB, PsbC, PsbD, and PsbH, and some Chl-binding sites contain Chls d or f instead of the usual Chl a. The structure of an apo-FRL-PSII monomer lacking the FRL-specific PsbH subunit has previously been determined, but visualization of the dimeric complex has remained elusive. Here, we report the cryo-EM structure of a dimeric FRL-PSII complex. The site assignments for Chls d and f are consistent with those assigned in the previous apo-FRL-PSII monomeric structure. All sites that bind Chl d or Chl f at high occupancy exhibit a FRL-specific interaction of the formyl moiety of the Chl d or Chl f with the protein environment, which in some cases involves a phenylalanine sidechain. The structure retains the FRL-specific PsbH2 subunit, which appears to alter the energetic landscape of FRL-PSII, redirecting energy transfer from the phycobiliprotein complex to a Chl f molecule bound by PsbB2 that acts as a bridge for energy transfer to the electron transfer chain. Collectively, these observations extend our previous understanding of the structure-function relationship that allows PSII to function using lower energy FRL.

Expansion of the global RNA virome reveals diverse clades of bacteriophages.

High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.

Molecular Evolution of Far-Red Light-Acclimated Photosystem II.

Cyanobacteria are major contributors to global carbon fixation and primarily use visible light (400-700 nm) to drive oxygenic photosynthesis. When shifted into environments where visible light is attenuated, a small, but highly diverse and widespread number of cyanobacteria can express modified pigments and paralogous versions of photosystem subunits and phycobiliproteins that confer far-red light (FRL) absorbance (700-800 nm), a process termed far-red light photoacclimation, or FaRLiP. During FaRLiP, alternate photosystem II (PSII) subunits enable the complex to bind chlorophylls d and f, which absorb at lower energy than chlorophyll a but still support water oxidation. How the FaRLiP response arose remains poorly studied. Here, we report ancestral sequence reconstruction and structure-based molecular evolutionary studies of the FRL-specific subunits of FRL-PSII. We show that the duplications leading to the origin of two PsbA (D1) paralogs required to make chlorophyll f and to bind chlorophyll d in water-splitting FRL-PSII are likely the first to have occurred prior to the diversification of extant cyanobacteria. These duplications were followed by those leading to alternative PsbC (CP43) and PsbD (D2) subunits, occurring early during the diversification of cyanobacteria, and culminating with those leading to PsbB (CP47) and PsbH paralogs coincident with the radiation of the major groups. We show that the origin of FRL-PSII required the accumulation of a relatively small number of amino acid changes and that the ancestral FRL-PSII likely contained a chlorophyll d molecule in the electron transfer chain, two chlorophyll f molecules in the antenna subunits at equivalent positions, and three chlorophyll a molecules whose site energies were altered. The results suggest a minimal model for engineering far-red light absorbance into plant PSII for biotechnological applications.