Low-CO2-inducible bestrophins outside the pyrenoid sustain high photosynthetic efficacy in diatoms.

Anion transporters sustain a variety of physiological states in cells. Bestrophins (BSTs) belong to a Cl- and/or HCO3- transporter family conserved in bacteria, animals, algae, and plants. Recently, putative BSTs were found in the green alga Chlamydomonas reinhardtii, where they are upregulated under low CO2 (LC) conditions and play an essential role in the CO2-concentrating mechanism (CCM). The putative BST orthologs are also conserved in diatoms, secondary endosymbiotic algae harboring red-type plastids, but their physiological functions are unknown. Here, we characterized the subcellular localization and expression profile of BSTs in the marine diatoms Phaeodactylum tricornutum (PtBST1 to 4) and Thalassiosira pseudonana (TpBST1 and 2). PtBST1, PtBST2, and PtBST4 were localized at the stroma thylakoid membrane outside of the pyrenoid, and PtBST3 was localized in the pyrenoid. Contrarily, TpBST1 and TpBST2 were both localized in the pyrenoid. These BST proteins accumulated in cells grown in LC but not in 1% CO2 (high CO2 [HC]). To assess the physiological functions, we generated knockout mutants for the PtBST1 gene by genome editing. The lack of PtBST1 decreased photosynthetic affinity for dissolved inorganic carbon to the level comparable with the HC-grown wild type. Furthermore, non-photochemical quenching in LC-grown cells was 1.5 to 2.0 times higher in the mutants than in the wild type. These data suggest that HCO3- transport at the stroma thylakoid membranes by PtBST1 is a critical part of the CO2-evolving machinery of the pyrenoid in the fully induced CCM and that PtBST1 may modulate photoprotection under CO2-limited environments in P. tricornutum.

Coordinated phosphate uptake by extracellular alkaline phosphatase and solute carrier transporters in marine diatoms.

Marine diatoms express genes encoding potential phosphate transporter and alkaline phosphatase (APase) under phosphate-limited (-P) condition. This indicates that diatoms use high-affinity phosphate uptake system with organic phosphate hydration. The function of molecules playing roles for Pi uptake was determined in this study. Pi uptake and APase activity of two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, were monitored during acclimation to -P condition. The transcript levels of Pi transporter were analyzed, and Pi transporters were localized with GFP tagging in diatom cells. KO mutants of plasma membrane solute carrier proteins (SLC34s) or APase were established, and their phenotype was evaluated. Some Na+ /Pi transporter candidates, SLC34s in P. tricornutum and T. pseudonana, increased transcript under -P condition. Whole-cell Pi transport was specifically stimulated by sodium ion but independent of potassium, lithium, or proton. Genome-editing KO of PtSLC34-5 and APase (Pt49678) in P. tricornutum was highly inhibitory for Pi uptake, and KO of TpSLC34-2 was also highly inhibitory for Pi uptake in T. pseudonana. SLC34s and APase were co-expressed under -P conditions in marine diatoms. SLC34s play a major role in the initial acclimation stage of diatom cells to -P condition and APase plays an increasing role in the prolonged Pi-starved condition.

Pyrenoid-core CO2-evolving machinery is essential for diatom photosynthesis in elevated CO2.

Marine diatoms are responsible for up to 20% of the annual global primary production by performing photosynthesis in seawater where CO2 availability is limited while HCO3- is abundant. Our previous studies have demonstrated that solute carrier 4 proteins at the plasma membrane of the diatom Phaeodactylum tricornutum facilitate the use of the abundant seawater HCO3-. There has been an unconcluded debate as to whether such HCO3- use capacity may itself supply enough dissolved inorganic carbon (DIC) to saturate the enzyme Rubisco. Here, we show that the θ-type carbonic anhydrase, Ptθ-CA1, a luminal factor of the pyrenoid-penetrating thylakoid membranes, plays an essential role in saturating photosynthesis of P. tricornutum. We isolated and analyzed genome-edited mutants of P. tricornutum defective in Ptθ-CA1. The mutants showed impaired growth in seawater aerated with a broad range of CO2 levels, from atmospheric to 1%. Independently of growth CO2 conditions, the photosynthetic affinity measured as K0.5 for DIC in mutants reached around 2 mm, which is about 10 times higher than K0.5[DIC] of high-CO2-grown wild-type cells that have repressed CO2-concentrating mechanism levels. The results clearly indicate that diatom photosynthesis is not saturated with either seawater-level DIC or even under a highly elevated CO2 environment unless the CO2-evolving machinery is at the core of the pyrenoid.

Extra O2 evolution reveals an O2-independent alternative electron sink in photosynthesis of marine diatoms.

Following the principle of oxygenic photosynthesis, electron transport in the thylakoid membranes (i.e., light reaction) generates ATP and NADPH from light energy, which is subsequently utilized for CO2 fixation in the Calvin-Benson-Bassham cycle (i.e., dark reaction). However, light and dark reactions could discord when an alternative electron flow occurs with a rate comparable to the linear electron flow. Here, we quantitatively monitored O2 and total dissolved inorganic carbon (DIC) during photosynthesis in the pennate diatom Phaeodactylum tricornutum, and found that evolved O2 was larger than the consumption of DIC, which was consistent with 14CO2 measurements in literature. In our measurements, the stoichiometry of O2 evolution to DIC consumption was always around 1.5 during photosynthesis at different DIC concentrations. The same stoichiometry was observed in the cells grown under different CO2 concentrations and nitrogen sources except for the nitrogen-starved cells showing O2 evolution 2.5 times larger than DIC consumption. An inhibitor to nitrogen assimilation did not affect the extra O2 evolution. Further, the same physiological phenomenon was observed in the centric diatom Thalassiosira pseudonana. Based on the present dataset, we propose that the marine diatoms possess the metabolic pathway(s) functioning as the O2-independent electron sink under steady state photosynthesis that reaches nearly half of electron flux of the Calvin-Benson-Bassham cycle.

Clinical and molecular significance of flow cytometric analysis for reactive oxygen species production and residual p67phox expression in p67phox-deficient chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by molecular defects in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. p67phox-CGD is an autosomal recessive CGD, which is caused by a defect in the cytosolic components of NADPH oxidase, p67phox, encoded by NCF2. We previously established a flow cytometric analysis for p67phox expression, which allows accurate assessment of residual protein expression in p67phox-CGD. We evaluated the correlation between oxidase function and p67phox expression, and assessed the relevancy to genotypes and clinical phenotypes in 11 patients with p67phox-CGD. Reactive oxygen species (ROS) production by granulocytes was evaluated using dihydrorhodamine-1,2,3 (DHR) assays. p67phox expression was evaluated in the monocyte population. DHR activity and p67phox expression were significantly correlated (r = 0.718, p < 0.0162). Additionally, DHR activity and p67phox expression were significantly higher in patients carrying one missense variant in combination with one nonsense or frameshift variant in the NCF2 gene than in patients with only null variants. The available clinical parameters of our patients (i.e., age at disease onset, number of infectious episodes, and each infection complication) were not linked with DHR activity or p67phox expression levels. In summary, our flow cytometric analysis revealed a significant correlation between residual ROS production and p67phox expression. More deleterious NCF2 genotypes were associated with lower levels of DHR activity and p67phox expression. DHR assays and protein expression analysis by using flow cytometry may be relevant strategies for predicting the genotypes of p67phox-CGD.

Stramenopile-Type Lipid Droplet Protein Functions as a Lipid Droplet Scaffold Protein in the Marine Diatom Phaeodactylum tricornutum.

Oleaginous microalgae are gaining great attention as feedstock for biofuels because of their substantial accumulation capacity for neutral lipids in the cytosolic compartment called the lipid droplet (LD). Understanding the regulatory mechanism of neutral lipid accumulation and degradation, which is mediated by LD-associated proteins, is an important issue in improving lipid productivity. However, LD-associated proteins vary among species and are waiting to be characterized in many microalgae. Stramenopile-type LD protein (StLDP) was previously identified as a primary LD protein in the marine diatom Phaeodactylum tricornutum. We produced a knockout mutant of StLDP by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing. Also, we tried to complement this mutant by expressing recognition site-modified StLDP (RSM-StLDP), which is designed to avoid an attack by Cas9 nuclease expressing in the mutant. The RSM-StLDP:enhanced green fluorescent protein was localized to both LDs and the outer chloroplast-endoplasmic reticulum. The decrease in the LD number per cell, increase in LD size and no alteration of neutral lipid content in the mutant under nitrogen deficiency clearly indicate that StLDP acts as an LD scaffold protein. The number of LDs per cell increased in the complemented strain compared to wild-type (WT) cells. The LD morphology in the mutant is probably over-rescued in the complemented strain by the strong function of the nitrate reductase promoter, which is also supported by high neutral lipid content in the complemented strain. The growth of stldp mutant showed a long lag phase relative to WT cells, suggesting that the low surface-to-volume ratio of fused LD decreased the efficiency of LD hydrolysis during the initial growth phase.

CD169 expression on monocytes as a marker for assessing type I interferon status in pediatric inflammatory diseases.

BACKGROUND: Evaluation of type I interferons (IFNs) in inflammatory or autoimmune diseases is challenging because of their rapid clearance in peripheral blood. The IFN gene expression signature has recently been used to evaluate the IFN status; however, this is often a labor-intensive and time-consuming procedure. Therefore, we assessed the feasibility of measuring expression of an IFN-inducible protein, CD169 (Siglec-1), on monocytes and circulating levels of soluble CD169 as alternative markers for type I IFN status in various pediatric inflammatory diseases. METHODS: Data from flow cytometric analysis of surface CD169 on monocytes and an enzyme-linked immunosorbent assay of soluble CD169 in peripheral blood were compared with serum IFN-α levels in 8 patients with viral infections, 5 with bacterial infections, 10 with systemic lupus erythematosus (SLE), 5 with Kikuchi-Fujimoto disease (KFD), 7 with Kawasaki disease (KD), and 8 with inflammatory bowel disease (IBD), and in 8 healthy controls. RESULTS: Surface CD169 expression was detected mainly on CD14+ monocytes and was significantly increased in patients with viral infections, SLE, and KFD, but not in patients with bacterial infections, KD, and IBD. There were similar trends for circulating soluble CD169; however, there was a significant increase only in patients with viral infections. Surface CD169 levels were significantly correlated with serum levels of IFN-α and soluble CD169. CONCLUSION: Analysis of CD169 expression on CD14+ monocytes may be useful for rapid assessment of type I IFN status for differentiation of pediatric inflammatory diseases from type 1 IFN-mediated diseases.

Novel STAT1 Variants in Japanese Patients with Isolated Mendelian Susceptibility to Mycobacterial Diseases.

PURPOSE: Heterozygous dominant-negative (DN) STAT1 variants are responsible for autosomal dominant (AD) Mendelian susceptibility to mycobacterial disease (MSMD). In this paper, we describe eight MSMD cases from four kindreds in Japan. METHODS: An inborn error of immunity-related gene panel sequencing was performed using genomic DNA extracted from whole blood samples. The identified variants were validated using Sanger sequencing. Functional analysis was evaluated with a luciferase reporter assay and co-transfection assay in STAT1-deficient cells. RESULTS: Patient 1.1 was a 20-month-old boy with multifocal osteomyelitis and paravertebral abscesses caused by Mycobacterium bovis bacillus Calmette-Guérin (BCG). Although the paravertebral abscess was refractory to antimycobacterial drugs, the addition of IFN-γ and drainage of the abscess were effective. Intriguingly, his mother (patient 1.2) showed an uneventful clinical course except for treatment-responsive tuberculous spondylitis during adulthood. Patient 2.1 was an 8-month-old boy with lymphadenopathy and lung nodules caused by BCG. He responded well to antimycobacterial drugs. His mother (patient 2.2) was healthy. Patient 3.1 was a 11-year-old girl with suspected skin tuberculosis. Her brother (patient 3.2) had BCG-osis, but their mother (patient 3.3) was healthy. Patient 4 was an 8-month-old girl with left axillary and supraclavicular lymphadenopathy associated with BCG vaccination. Kindreds 1, 2, and 3 were shown to have novel heterozygous variants (V642F, R588C, and R649G) in STAT1, respectively. Kindred 4 had previously reported heterozygous variants (Q463H). A luciferase reporter assay in STAT1-deficient cells followed by IFN-γ stimulation confirmed that these variants are loss-of-function. In addition, with co-transfection assay, we confirmed all of these variants had DN effect on WT STAT1. CONCLUSION: Four kindred MSMD subjects with 3 novel variants and 1 known variant in STAT1 were identified in this study. AD STAT1 deficiency might be prevalent in Japanese patients with BCG-associated MSMD.

Localization and characterization θ carbonic anhydrases in Thalassiosira pseudonana.

Carbonic anhydrase (CA) is a crucial component for the operation of CO2-concentrating mechanisms (CCMs) in the majority of aquatic photoautotrophs that maintain the global primary production. In the genome of the centric marine diatom, Thalassiosira pseudonana, there are four putative gene sequences that encode θ-type CA, which was a type of CA recently identified in marine diatoms and green algae. In the present study, specific subcellular locations of four θCAs, TpθCA1, TpθCA2, TpθCA3, and TpθCA4 were determined by expressing GFP-fused proteins of these TpθCAs in T. pseudonana. As a result, C-terminal GFP fusion proteins of TpθCA1, TpθCA2, and TpθCA3 were all localized in the chloroplast; TpθCA2 was at the central chloroplast area, and the other two TpθCAs were throughout the chloroplast. Immunogold-labeling transmission electron microscopy was further performed for the transformants expressing TpθCA1:GFP and TpθCA2:GFP with anti-GFP-monoclonal antibody. TpθCA1:GFP was localized in the free stroma area, including the peripheral pyrenoid area. TpθCA2:GFP was clearly located as a lined distribution at the central part of the pyrenoid structure, which was most likely the pyrenoid-penetrating thylakoid. Considering the presence of the sequence encoding the N-terminal thylakoid-targeting domain in the TpθCA2 gene, this localization was likely the lumen of the pyrenoid-penetrating thylakoid. On the other hand, TpθCA4:GFP was localized in the cytoplasm. Transcript analysis of these TpθCAs revealed that TpθCA2 and TpθCA3 were upregulated in atmospheric CO2 (0.04% CO2, LC) levels, while TpθCA1 and TpθCA4 were highly induced under 1% CO2 (HC) condition. The genome-editing knockout (KO) of TpθCA1, by CRISPR/Cas9 nickase, gave a silent phenotype in T. pseudonana under LC-HC conditions, which was in sharp agreement with the case of the previously reported TpθCA3 KO. In sharp contrast, TpθCA2 KO is so far unsuccessful, suggesting a housekeeping role of TpθCA2. The silent phenotype of KO strains of stromal CAs suggests that TpαCA1, TpθCA1, and TpθCA3 may have functional redundancy, but different transcript regulations in response to CO2 of these stromal CAs suggest in part their independent roles.

Multiple plasma membrane SLC4s contribute to external HCO3- acquisition during CO2 starvation in the marine diatom Phaeodactylum tricornutum.

The availability of CO2 is one of the restrictions on aquatic photosynthesis. Solute carrier (SLC) 4-2, a plasma membrane HCO3- transporter has previously been identified in the marine diatom Phaeodactylum tricornutum. In this study, we discovered two paralogs, PtSLC4-1 and PtSLC4-4, that are both localized at the plasma membrane. Their overexpression stimulated HCO3- uptake, and this was inhibited by the anion channel blocker 4,4´-diisothiocyanostilbene-2,2´-disulfonic (DIDS). Similarly to SLC4-2, PtSLC4-1 specifically required Na+ of ~100 mM for its maximum HCO3- transport activity. Unlike PtSLC4-1 and PtSLC4-2, the HCO3- transport of PtSLC4-4 depended equally on Na+, K+, or Li+, suggesting its broad selectivity for cations. Transcript analyses indicated that PtSLC4-1 was the most abundant HCO3- transporter under CO2 concentrations below atmospheric levels, while PtSLC4-4 showed little transcript induction under atmospheric CO2 but transient induction to comparable levels to PtSLC4-1 during the initial acclimation stage from high CO2 (1%) to very low CO2 (<0.002%). Our results strongly suggest a major HCO3- transport role of PtSLC4-1 with a relatively minor role of PtSLC4-2, and that PtSLC4-4 operates under severe CO2 limitation unselectively to cations when the other SLC4s do not function to support HCO3- uptake.

Mapping of subcellular local pH in the marine diatom Phaeodactylum tricornutum.

Diatoms are one of the most important phytoplankton on Earth. They comprise at least ten thousand species and contribute to up to 20% of the global primary production. Because of serial endosymbiotic events and horizontal gene transfers, diatoms have developed a "secondary plastid" bounded by four membranes containing a large phase-separated compartment, termed the pyrenoid. However, the physiological significance of this unique chloroplast morphology is poorly understood. Characterization of fundamental physiological parameters such as local pH in various subcellular compartments should facilitate a greater understanding of the physiological roles of the unique structure of the secondary plastid. A promising method to estimate local pH is the in situ expression of the pH-sensitive green fluorescent protein. Here, we first developed the molecular tool for the mapping of in situ local pH in the diatom Phaeodactylum tricornutum by heterologously expressing pHluorin2 in the cytosol, periplastidal compartment (PPC; the space in between two sets of outer and inner chloroplast envelopes), chloroplast stroma, and the pyrenoid matrix. Our data suggested that PPC and the pyrenoid matrix are more acidic than the adjacent areas, the cytosol and the chloroplast stroma. Finally, absolute pH values at each compartment were estimated from the ratiometric fluorescence of a recombinant pHluorin2 protein, giving pH values of approximately 7.9, 6.8, 8.0, and 7.5 respectively, for the cytosol, PPC, stroma, and pyrenoid of the P. tricornutum cells, indicating the occurrence of pH gradients and the associated electrochemical potentials at their boundary.

Immobilization of a Broad Range of Polypeptides on the Frustule of the Diatom Thalassiosira pseudonana.

Proteins immobilized on biosilica which have superior reactivity and specificity and are innocuous to natural environments could be useful biological materials in industrial processes. One recently developed technique, living diatom silica immobilization (LiDSI), has made it possible to immobilize proteins, including multimeric and redox enzymes, via a cellular excretion system onto the silica frustule of the marine diatom Thalassiosira pseudonana. However, the number of application examples so far is limited, and the type of proteins appropriate for the technique is still enigmatic. Here, we applied LiDSI to six industrially relevant polypeptides, including protamine, metallothionein, phosphotriesterase, choline oxidase, laccase, and polyamine synthase. Protamine and metallothionein were successfully immobilized on the frustule as protein fusions with green fluorescent protein (GFP) at the N terminus, indicating that LiDSI can be used for polypeptides which are rich in arginine and cysteine. In contrast, we obtained mutants for the latter four enzymes in forms without green fluorescent protein. Immobilized phosphotriesterase, choline oxidase, and laccase showed enzyme activities even after the purification of frustule in the presence of 1% (wt/vol) octylphenoxy poly(ethyleneoxy)ethanol. An immobilized branched-chain polyamine synthase changed the intracellular polyamine composition and silica nanomorphology. These results illustrate the possibility of LiDSI for industrial applications. IMPORTANCE Proteins immobilized on biosilica which have superior reactivity and specificity and are innocuous to natural environments could be useful biological materials in industrial processes. Living diatom silica immobilization (LiDSI) is a recently developed technique for in vivo protein immobilization on the diatom frustule. We aimed to explore the possibility of using LiDSI for industrial applications by successfully immobilizing six polypeptides: (i) protamine (Oncorhynchus keta), a stable antibacterial agent; (ii) metallothionein (Saccharomyces cerevisiae), a metal adsorption molecule useful for bioremediation; (iii) phosphotriesterase (Sulfolobus solfataricus), a scavenger for toxic organic phosphates; (iv) choline oxidase (Arthrobacter globiformis), an enhancer for photosynthetic activity and yield of plants; (v) laccase (Bacillus subtilis), a phenol oxidase utilized for delignification of lignocellulosic materials; and (vi) branched-chain polyamine synthase (Thermococcus kodakarensis), which produces branched-chain polyamines important for DNA and RNA stabilization at high temperatures. This study provides new insights into the field of applied biological materials.

Mitochondrial phosphoenolpyruvate carboxylase contributes to carbon fixation in the diatom Phaeodactylum tricornutum at low inorganic carbon concentrations.

Photosynthetic carbon fixation is often limited by CO2 availability, which led to the evolution of CO2 concentrating mechanisms (CCMs). Some diatoms possess CCMs that employ biochemical fixation of bicarbonate, similar to C4 plants, but whether biochemical CCMs are commonly found in diatoms is a subject of debate. In the diatom Phaeodactylum tricornutum, phosphoenolpyruvate carboxylase (PEPC) is present in two isoforms, PEPC1 in the plastids and PEPC2 in the mitochondria. We used real-time quantitative polymerase chain reaction, Western blots, and enzymatic assays to examine PEPC expression and PEPC activity, under low and high concentrations of dissolved inorganic carbon (DIC). We generated and analyzed individual knockout cell lines of PEPC1 and PEPC2, as well as a PEPC1/2 double-knockout strain. While we could not detect an altered phenotype in the PEPC1 knockout strains at ambient, low or high DIC concentrations, PEPC2 and the double-knockout strains grown under ambient air or lower DIC availability conditions showed reduced growth and photosynthetic affinity for DIC while behaving similarly to wild-type (WT) cells at high DIC concentrations. These mutants furthermore exhibited significantly lower 13 C/12 C ratios compared to the WT. Our data imply that in P. tricornutum at least parts of the CCM rely on biochemical bicarbonate fixation catalyzed by the mitochondrial PEPC2.

Effects of advanced glycation end products on dental pulp calcification.

OBJECTIVE: The main aim of this study was to elucidate the effects of advanced glycation end products (AGEs) on the calcification of cultured rat dental pulp cells (RDPCs) and to investigate the crystallisation ability of glycated collagen. MATERIALS AND METHODS: AGEs were prepared via non-enzymatic glycation of a dish coated with type I collagen using dl-glyceraldehyde. To investigate the effects of AGEs on RDPCs, we performed WST-1 and lactate dehydrogenase assays; alkaline phosphatase, Alizarin Red S and immunohistochemical staining; and real-time quantitative reverse transcription PCR. In addition, we performed crystallisation experiments on glycated collagen. All microstructures were analysed using scanning electron microscopy/energy-dispersive X-ray spectroscopy and transmission electron microscopy/diffraction pattern analysis. RESULTS: AGEs did not affect the proliferation or differentiation of RDPCs, but enhanced the calcification rate and cytotoxicity. No major calcification-related genes or proteins were involved in these calcifications, and glycated collagen was found to exhibit a negative polarity and form calcium phosphate crystals. Cytotoxicity due to drastic changes in the concentration of pericellular ions led to dystrophic calcification, assumed to represent an aspect of diabetic pulp calcifications. CONCLUSION: Glycated collagen-containing AGEs provide a nurturing environment for crystallisation and have a significant effect on the early calcification of RDPCs.

Role of E148Q in familial Mediterranean fever with an exon 10 mutation in MEFV.

BACKGROUND: Familial Mediterranean fever (FMF) is an autosomal recessive disease caused by mutations in the MEFV gene. Mutations in exon 10 are associated with typical FMF. Most Japanese patients with typical FMF are compound heterozygotes of M694I in exon 10 and E148Q in exon 2. However, the pathogenic role of E148Q remains controversial. METHODS: We assessed symptoms and serum cytokines among patients with FMF and their family members. They were divided into three subgroups, based on MEFV mutations: individuals carrying M694I and E148Q (group A, n = 14), individuals carrying M694I, but not E148Q (group B, n = 10), and individuals carrying E148Q, but not M694I (group C, n = 11). RESULTS: All but one individual in group A had typical FMF phenotypes, whereas no individual in groups B and C exhibited any episodes of fever or serositis. The serum levels of interleukin-18 during the afebrile phase were significantly elevated in group A (2,806 ± 2,107 pg/mL), compared to those in groups B (499 ± 369 pg/mL) and C (427 ± 410 pg/mL). No difference in interleukin-6 levels was observed among the three groups. CONCLUSIONS: These findings indicated that E148Q may contribute to disease development of FMF in Japanese patients carrying the heterozygous M694I mutation in MEFV and that genetic testing of both parents would lead to better counseling for their children.

Half of 'Micrococcus spp.' cases identified by conventional methods are revealed as other life-threatening bacteria with different drug susceptibility patterns by 16S ribosomal RNA gene sequencing.

Bacterial infection during chemotherapy is a fatal complication, therefore precise identification of the pathogenic microorganism is required for treatment. We report that 2 of 4 pediatric patients with malignancy who were diagnosed with Micrococcus spp. infection by conventional methods were finally revealed to have Kytococcus schroeteri and Kocuria marina infection by 16S ribosomal RNA gene sequence analysis (16S rRNA analysis). Although K. schroeteri is morphologically similar to Micrococcus spp., its drug susceptibility profile is quite different from that of Micrococcus spp. K. schroeteri is resistant to penicillin and cephalosporin, which are effective for Micrococcus spp. In fact, penicillin-resistant lethal pneumonia caused by K. schroeteri has been reported in compromised hosts. Based on our results, Micrococcus spp. determined by conventional methods could contain other life-threatening bacteria with different drug susceptibility patterns from Micrococcus spp. To develop an effective empirical treatment for immunocompromised hosts, accumulation of pathogen data by 16S rRNA analysis is required.

Cross-compartment metabolic coupling enables flexible photoprotective mechanisms in the diatom Phaeodactylum tricornutum.

Photoacclimation consists of short- and long-term strategies used by photosynthetic organisms to adapt to dynamic light environments. Observable photophysiology changes resulting from these strategies have been used in coarse-grained models to predict light-dependent growth and photosynthetic rates. However, the contribution of the broader metabolic network, relevant to species-specific strategies and fitness, is not accounted for in these simple models. We incorporated photophysiology experimental data with genome-scale modeling to characterize organism-level, light-dependent metabolic changes in the model diatom Phaeodactylum tricornutum. Oxygen evolution and photon absorption rates were combined with condition-specific biomass compositions to predict metabolic pathway usage for cells acclimated to four different light intensities. Photorespiration, an ornithine-glutamine shunt, and branched-chain amino acid metabolism were hypothesized as the primary intercompartment reductant shuttles for mediating excess light energy dissipation. Additionally, simulations suggested that carbon shunted through photorespiration is recycled back to the chloroplast as pyruvate, a mechanism distinct from known strategies in photosynthetic organisms. Our results suggest a flexible metabolic network in P. tricornutum that tunes intercompartment metabolism to optimize energy transport between the organelles, consuming excess energy as needed. Characterization of these intercompartment reductant shuttles broadens our understanding of energy partitioning strategies in this clade of ecologically important primary producers.

Plasma Membrane-Type Aquaporins from Marine Diatoms Function as CO2/NH3 Channels and Provide Photoprotection.

Aquaporins (AQPs) are ubiquitous water channels that facilitate the transport of many small molecules and may play multiple vital roles in aquatic environments. In particular, mechanisms to maintain transmembrane fluxes of important small molecules have yet to be studied in marine photoautotrophic organisms. Here, we report the occurrence of multiple AQPs with differential cellular localizations in marine diatoms, an important group of oceanic primary producers. The AQPs play a role in mediating the permeability of membranes to CO2 and NH3 In silico surveys revealed the presence of five AQP orthologs in the pennate diatom Phaeodactylum tricornutum and two in the centric diatom Thalassiosira pseudonana GFP fusions of putative AQPs displayed clear localization to the plasma membrane (PtAGP1 and PtAQP2), the chloroplast endoplasmic reticulum (CER; PtAGP1 and PtAQP3), and the tonoplast (PtAQP5) in P. tricornutum In T. pseudonana, GFP-AQP fusion proteins were found on the vacuole membrane (TpAQP1) and CER (TpAQP2). Transcript levels of both PtAQP1 and PtAQP2 were highly induced by ammonia, while only PtAQP2 was induced by high (1%[v/v]) CO2 Constitutive overexpression of GFP-tagged PtAQP1 and PtAQP2 significantly increased CO2 and NH3 permeability in P. tricornutum, strongly indicating that these AQPs function in regulating CO2/NH3 permeability in the plasma membrane and/or CER. Cells carrying GFP-tagged PtAQP1 and PtAQP2 had higher nonphotochemical quenching under high light relative to that of wild-type cells, suggesting that these AQPs are involved in photoprotection. These AQPs may facilitate the efflux of NH3, preventing the uncoupling effect of high intracellular ammonia concentrations.

Comparative analysis of strategies to prepare electron sinks in aquatic photoautotrophs.

While subject to illumination, photosystem I (PSI) has the potential to produce reactive oxygen species (ROS) that can cause photo-oxidative damage in oxygenic photoautotrophs. The reaction center chlorophyll in PSI (P700) is kept oxidized in excess light conditions to limit over-excitation of PSI and alleviate the production of ROS. Oxidation of P700 requires a sufficient electron sink for PSI, which is responsible for flavodiiron proteins (FLV) safely dissipating electrons to O2 in cyanobacteria, green algae, and land plants except for angiosperms during short-pulse light (SP) illumination under which photosynthesis and photorespiration do not occur. This fact implies that O2 usage is essential for P700 oxidation but also raises the question why angiosperms lost FLV. Here, we first found that aquatic photoautotrophs in red plastid lineage, in which no gene for FLV has been found, could keep P700 oxidized during SP illumination alleviating the photo-oxidative damage in PSI even without O2 usage. We comprehensively assessed P700 oxidation during SP illumination in the presence and absence of O2 in cyanobacteria (Cyanophyta), green algae (Chlorophyta), angiosperms (Streptophyta), red algae (Rhodophyta), and secondary algae (Cryptophyta, Haptophyta, and Heterokontophyta). A variety of dependencies of P700 oxidation on O2 among these photoautotrophs clearly suggest that O2 usage and FLV are not universally required to oxidize P700 for protecting PSI against ROS damage. Our results expand the understanding of the diverse strategies taken by oxygenic photoautotrophs to oxidize P700 and mitigate the risks of ROS.