A thylakoid biogenesis BtpA protein is required for the initial step of tetrapyrrole biosynthesis in cyanobacteria.

Biogenesis of the photosynthetic apparatus requires complicated molecular machinery, individual components of which are either poorly characterized or unknown. The BtpA protein has been described as a factor required for the stability of photosystem I (PSI) in cyanobacteria; however, how the BtpA stabilized PSI remains unexplained. To clarify the role of BtpA, we constructed and characterized the btpA-null mutant (ΔbtpA) in the cyanobacterium Synechocystis sp. PCC 6803. The mutant contained only c. 1% of chlorophyll and nearly no thylakoid membranes. However, this strain, growing only in the presence of glucose, was genetically unstable and readily generated suppressor mutations that restore the photoautotrophy. Two suppressor mutations were mapped into the hemA gene encoding glutamyl-tRNA reductase (GluTR) - the first enzyme of tetrapyrrole biosynthesis. Indeed, the GluTR was not detectable in the ΔbtpA mutant and the suppressor mutations restored biosynthesis of tetrapyrroles and photoautotrophy by increased GluTR expression or by improved GluTR stability/processivity. We further demonstrated that GluTR associates with a large BtpA oligomer and that BtpA is required for the stability of GluTR. Our results show that the BtpA protein is involved in the biogenesis of photosystems at the level of regulation of tetrapyrrole biosynthesis.

The cyanobacterial FtsH4 protease controls accumulation of protein factors involved in the biogenesis of photosystem I.

Membrane-bound FtsH proteases are universally present in prokaryotes and in mitochondria and chloroplasts of eukaryotic cells. These metalloproteases are often critical for viability and play both protease and chaperone roles to maintain cellular homeostasis. In contrast to most bacteria bearing a single ftsH gene, cyanobacteria typically possess four FtsH proteases (FtsH1-4) forming heteromeric (FtsH1/3 and FtsH2/3) and homomeric (FtsH4) complexes. The functions and substrate repertoire of each complex are however poorly understood. To identify substrates of the FtsH4 protease complex we established a trapping assay in the cyanobacterium Synechocystis PCC 6803 utilizing a proteolytically inactivated trapFtsH4-His. Around 40 proteins were specifically enriched in trapFtsH4 pulldown when compared with the active FtsH4. As the list of putative FtsH4 substrates contained Ycf4 and Ycf37 assembly factors of Photosystem I (PSI), its core PsaB subunit and the IsiA chlorophyll-binding protein that associates with PSI during iron stress, we focused on these PSI-related proteins. Therefore, we analysed their degradation by FtsH4 in vivo in Synechocystis mutants and in vitro using purified substrates. The data confirmed that FtsH4 degrades Ycf4, Ycf37, IsiA, and also the individual PsaA and PsaB subunits in the unassembled state but not when assembled within the PSI complexes. A possible role of FtsH4 in the PSI life-cycle is discussed.

Efficacy of switches within the class of IL-17 inhibitors: An analysis of data from the Czech nationwide registry of psoriatic patients receiving biological/targeted therapy (BIOREP).

The IL-17 cytokine family encompasses six different homodimers and heterodimers referred to as IL-17A-F. Due to some differences in the mechanism of IL-17 inhibition, aninsufficient effect of one IL-17 inhibitor does not necessarily imply lack of efficacy of the other agent of the same class. Aim of study was analysis of the success rate of switches among IL-17 inhibitors (secukinumab, ixekizumab, and brodalumab) in patients treated in the Czech Republic. Data were obtained from the Czech nationwide registry of psoriatic patients receiving biological/targeted therapy (BIOREP). Our analysis involved data of a total of 90 patients with severe chronic plaque psoriasis and baseline PASI scores >10 both prior to first-line biological therapy initiation and after switch to another agent of the class of IL-17 inhibitors. The most effective switch was that from secukinumab to brodalumab, with PASI 90 reached by 64.7% and 73.3% of patients at weeks 12 and 24. Among patients switched from secukinumab to ixekizumab target PASI 90 responses were achieved (at weeks 12 and 24) by 41.2% and 55.2% of patients. Among patients switched from ixekizumab to brodalumab target PASI 90 responses were achieved, at the above time points, by 30.8% and 38.5% of patients. Our analysis showed a high success rate of switches from secukinumab to ixekizumab and brodalumab, followed by the ixekizumab-to-brodalumab switch. Importantly, the therapeutic response and success rates of individual switches are independent of the patient's body weight and presence of psoriatis arthritis.

Research on Low-Cycle Fatigue Engineered Hybrid Sandwich Ski Construction.

This research is aimed at evaluating the effect of low-cycle fatigue on a newly designed hybrid sandwich ski structure to determine the changes that may occur due to cyclic loading and thus affect its use. This is primarily concerned with the fatigue behavior of the tested ski over different time intervals simulating its seasonal use and its effect on the mechanical properties of the ski, i.e., the durability and integrity of the individual layers of the sandwich ski structure. The ski was subjected to 70,000 deflections by moving the crossbar by 60 mm according to the ski deflection calculation in the arch. The results of the cyclic tests of the engineered ski design showed no significant changes in the ski during loading. The average force required to achieve deflection in the first 10,000 cycles was 514.0 ± 4.2 N. Thereafter, a secondary hardening of the structure occurred during relaxation and the force required increased slightly to 543.6 ± 1.7 N. The required force fluctuated slightly during the measurements and in the last series the value was 540.4 ± 0.8 N. Low-cycle fatigue did not have a significant effect on the mechanical properties of the ski; there was no change in shape or visual delamination of the individual layers of the structure. From the cross-section, local delamination was demonstrated by image analysis, especially between the Wood core and the composite layers E-Glass biaxial and Carbon triaxial.

Efficacy, safety, and drug survival of patients with psoriasis treated with IL-17 inhibitors - brodalumab, ixekizumab, and secukinumab: real-world data from the Czech Republic BIOREP registry.

BACKGROUND: Real-world data on the use of interleukin-17 (IL-17) inhibitors for the treatment of psoriasis are limited. OBJECTIVE: To evaluate and compare the efficacy, safety, and drug survival of IL-17 inhibitors. METHODS: This retrospective study analyzed the BIOREP registry data of patients treated with at least one IL-17 inhibitor (secukinumab, ixekizumab, and brodalumab). RESULTS: In total, 949 patients were included. The improvement in PASI score was significant for all drugs, and the proportion of patients achieving PASI 75, 90, and 100 after both 3 and 24 months of therapy was highest for brodalumab, followed by ixekizumab and secukinumab. The Dermatology Life Quality Index score decreased to ˂3 after 3 months and to ˂2 after 24 months of therapy for all inhibitors. Loss of effectiveness was the major reason for discontinuation in 17.2% of patients, followed by adverse events in 3.2% of patients. The drug survival probability was the highest for brodalumab, followed by ixekizumab and secukinumab. Negative predictors for treatment discontinuation were obesity and the number of treatment lines, whereas a positive predictor was the presence of concomitant psoriatic arthritis; sex had no influence. CONCLUSION: This real-life study demonstrated the effectiveness and good safety profile of all currently available IL-17 inhibitors.

Mutations Suppressing the Lack of Prepilin Peptidase Provide Insights Into the Maturation of the Major Pilin Protein in Cyanobacteria.

Type IV pili are bacterial surface-exposed filaments that are built up by small monomers called pilin proteins. Pilins are synthesized as longer precursors (prepilins), the N-terminal signal peptide of which must be removed by the processing protease PilD. A mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the PilD protease is not capable of photoautotrophic growth because of the impaired function of Sec translocons. Here, we isolated phototrophic suppressor strains of the original ΔpilD mutant and, by sequencing their genomes, identified secondary mutations in the SigF sigma factor, the γ subunit of RNA polymerase, the signal peptide of major pilin PilA1, and in the pilA1-pilA2 intergenic region. Characterization of suppressor strains suggests that, rather than the total prepilin level in the cell, the presence of non-glycosylated PilA1 prepilin is specifically harmful. We propose that the restricted lateral mobility of the non-glycosylated PilA1 prepilin causes its accumulation in the translocon-rich membrane domains, which attenuates the synthesis of membrane proteins.

Design, Development, and Characterization of Advanced Textile Structural Hollow Composites.

The research is focused on the design and development of woven textile-based structural hollow composites. E-Glass and high tenacity polyester multifilament yarns were used to produce various woven constructions. Yarn produced from cotton shoddy (fibers extracted from waste textiles) was used to develop hybrid preforms. In this study, unidirectional (UD), two-dimensional (2D), and three-dimensional (3D) fabric preforms were designed and developed. Further, 3D woven spacer fabric preforms with single-layer woven cross-links having four different geometrical shapes were produced. The performance of the woven cross-linked spacer structure was compared with the sandwich structure connected with the core pile yarns (SPY). Furthermore, three different types of cotton shoddy yarn-based fabric structures were developed. The first is unidirectional (UD), the second is 2D all-waste cotton fabric, and the third is a 2D hybrid fabric with waste cotton yarn in the warp and glass multifilament yarn in the weft. The UD, 2D, and 3D woven fabric-reinforced composites were produced using the vacuum-assisted resin infusion technique. The spacer woven structures were converted to composites by inserting wooden blocks with an appropriate size and wrapped with a Teflon sheet into the hollow space before resin application. A vacuum-assisted resin infusion technique was used to produce spacer woven composites. While changing the reinforcement from chopped fibers to 3D fabric, its modulus and ductility increase substantially. It was established that the number of crossover points in the weave structures offered excellent association with the impact energy absorption and formability behavior, which are important for many applications including automobiles, wind energy, marine and aerospace. Mechanical characterization of honeycomb composites with different cell sizes, opening angles and wall lengths revealed that the specific compression energy is higher for regular honeycomb structures with smaller cell sizes and a higher number of layers, keeping constant thickness.

Experimental Investigation of Wavy-Lap Bonds with Natural Cotton Fabric Reinforcement under Cyclic Loading.

This study is focused on the mechanical properties and service life (safety) evaluation of hybrid adhesive bonds with shaped overlapping geometry (wavy-lap) and 100% natural cotton fabric used as reinforcement under cyclic loading using various intensities. Cyclic loading were implemented between 5-50% (267-2674 N) and 5-70% (267-3743 N) from the maximum strength (5347 N) measured by static tensile test. The adhesive bonds were loaded by 1000 cycles. The test results demonstrated a positive influence of the used reinforcement on the mechanical properties, especially during the cyclic loading. The adhesive bonds Tera-Flat withstood the cyclic load intensity from 5-70% (267-3743 N). The shaped overlapping geometry (wavy-lap bond) did not have any positive influence on the mechanical performance, and only the composite adhesive bonds Erik-WH1 and Tera-WH1 withstood the complete 1000 cycles with cyclic loading values between 5-50% (267-2674 N). The SEM analysis results demonstrated a positive influence on the fabric surface by treatment with 10% NaOH aqueous solution. The unwanted compounds (lignin) were removed. Furthermore, a good wettability has been demonstrated by the bonded matrix material. The SEM analysis also demonstrated micro-cracks formation, with subsequent delamination of the matrix/reinforcement interface caused by cyclic loading. The experimental research was conducted for the analysis of hybrid adhesive bonds using curved/wavy overlapping during both static and cyclic loading.

Mechanical Performance of Knitted Hollow Composites from Recycled Cotton and Glass Fibers for Packaging Applications.

This research deals with the development of knitted hollow composites from recycled cotton fibers (RCF) and glass fibers (GF). These knitted hollow composites can be used for packaging of heavy weight products and components in aircrafts, marine crafts, automobiles, civil infrastructure, etc. They can also be used in medical prosthesis or in sports equipment. Glass fiber-based hollow composites can be used as an alternative to steel or wooden construction materials for interior applications. Developed composite samples were subjected to hardness, compression, flexural, and impact testing. Recycled cotton fiber, which is a waste material from industrial processes, was chosen as an ecofriendly alternative to cardboard-based packaging material. The desired mechanical performance of knitted hollow composites was achieved by changing the tube diameter and/or thickness. Glass fiber-reinforced knitted hollow composites were compared with RC fiber composites. They exhibited substantially higher compression strength as compared to cotton fiber-reinforced composites based on the fiber tensile strength. However, RC fiber-reinforced hollow composites showed higher compression modulus as compared to glass fiber-based composites due to much lower deformation during compression loading. Compression strength of both RCF- and GF-reinforced hollow composites decreases with increasing tube diameter. The RCF-based hollow composites were further compared with double-layered cardboard packaging material of similar thickness. It was observed that cotton-fiber-reinforced composites show higher compression strength, as well as compression modulus, as compared to the cardboard material of similar thickness. No brittle failure was observed during the flexural test, and samples with smaller tube diameter exhibited higher stiffness. The flexural properties of glass fiber-reinforced composites were compared with RCF composites. It was observed that GF composites exhibit superior flexural properties as compared to the cotton fiber-based samples. Flexural strength of RC fiber-reinforced hollow composites was also compared to that of cardboard packaging material. The composites from recycled cotton fibers showed substantially higher flexural stiffness as compared to double-layered cardboard material. Impact energy absorption was measured for GF and RCF composites, as well as cardboard material. All GF-reinforced composites exhibited higher absorption of impact energy as compared to RCF-based samples. Significant increase in absorption of impact energy was achieved by the specimens with higher tube thickness in the case of both types of reinforcing fibers. By comparing the impact performance of cotton fiber-based composites with cardboard packaging material, it was observed that the RC fiber-based hollow composites absorb much higher impact energy as compared to the cardboard-based packaging material. The current paper summarizes a comparative analysis of mechanical performance in the case of glass fiber-reinforced hollow composites vis-à-vis recycled cotton fiber-reinforced hollow composites. The use of recycled fibers is a positive step in the direction of ecofriendly materials and waste utilization. Their performance is compared with commercial packaging material for a possible replacement and reducing burden on the environment.

Characterization of Hybrid Composites with Polyester Waste Fibers, Olive Root Fibers and Coir Pith Micro-Particles Using Mixture Design Analysis for Structural Applications.

In the present work, hybrid composites were developed by using polyester waste fibers along with natural origin materials: olive root fibers and coir pitch filler. Such composite panels can be used as a potential alternative for fiber glass sunshade panels and room dividers in buildings. Hybrid composites were fabricated by mixing polyester waste fibers and olive root fibers in different ratios (0:100, 33:67, 67:33 and 100:0). Coir pith micro-particles with an average size of 312 d.nm were used as filler in the polyester matrix at three different levels (0%, 5%, and 10%) of the overall matrix weight. Mechanical properties, e.g., tensile strength, flexural strength and impact strength, thermal properties, e.g., coefficient of linear thermal expansion, thermo-gravimetric analysis (TGA) and environmental properties, e.g., water absorption, loss of density after exposure to weathering were characterized. For comparison purposes, a commercially available fiber glass sunshades sample was also investigated. Mixture design analysis was used to optimize the ratio of all components in the composite. Graphical comparison of experimental results using regression models showed a high degree of correlation. An optimized formulation of composite with an objective of maximization of tensile strength, flexural strength, impact strength and minimization of water absorption, density loss, as well as coefficient of linear thermal expansion, was determined at 70.83 wt%, 15.15 wt%, and 14.01 wt% of polyester waste fibers, olive root fibers and coir pith micro-fillers, respectively. Overall, it can be concluded that the developed hybrid composites from waste fibrous materials can be used as a promising alternative and a value-added application in buildings and construction purposes.

Evolution of Ycf54-independent chlorophyll biosynthesis in cyanobacteria.

Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light-adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.

A Manifestation of Ulcerative Colitis During Treatment for Severe Plaque Psoriasis with Ixekizumab - A Report of Two Cases and Review of the Literature.

L-17 inhibitors belong to the group of the most effective and highly safe biological preparations intended for the treatment of psoriasis, and in the case of secukinumab and ixekizumab, also for the treatment of some immune-mediated inflammatory diseases of the joints. Despite initial expectations, they did not prove to be effective for the treatment of non-specific bowel inflammations (IBD). On the contrary, IBD worsening was reported in some cases where IL-17 inhibitors were used, and registration studies were terminated for this indication. In clinical studies, extensive meta-analyses of IL-17 inhibitor use for psoriasis and joint inflammation indications generally did not demonstrate any statistically significant increase in the risk of de-novo IBD with this type of treatment. Data from real-time practice are mostly similar. The literature describes individual cases with an obvious relation of de novo IBD development subsequent to treatment with IL-17 inhibitors in registered indications. The activation of latent, thus far clinically asymptomatic bowel inflammation is usually expected. Therefore, a careful review of medical history focused on bowel problems in personal and family history is necessary before starting therapy with IL-17 inhibitors. We present a similar experience with de novo onset of ulcerative colitis in two patients treated for psoriasis with ixekizumab, with associated psoriatic arthritis in one case.

Infrared thermography and capillaroscopy in the diagnosis of Raynaud's phenomenon.

AIMS: Raynaud's phenomenon (RP) is a relatively common disease. There are two distinct forms of RP - primary (PRP), where no other associated diseases are present, and secondary (SRP), where RP is associated with other diseases. It can be challenging to differentiate between RP and other diseases through medical history alone, due to the episodic nature of RP. Objective analysis of anamnestic data was performed in our study using infrared thermography (IRT) and a cold pressor test (CPT). Capillaroscopy was performed to assess morphological changes in the acral circulation. METHODS: Patients with a history of cold hands were included in the study. IRT was performed before and after the CPT, and then capillaroscopy was performed. The results (including epidemiologic data) were statistically evaluated. RESULTS: A total of 150 patients were included in the study. Summarisation of the results from the IRT and capillaroscopy determined the final diagnosis - 4.7% acrocyanosis, 10.7% physiologic findings, 31.3% PRP, 29.3% borderline SRP and 24% SRP. The coldest fingers following the CPT were, in most patients, the 2nd and 3rd fingers. The correlation between the presence of connective tissue disease and the diagnosis of borderline SRP and SRP was significant (P=0.0001). CONCLUSIONS: Using the combination of the IRT and capillaroscopy in the diagnostic algorithm for RP has its justification. IRT distinguishes healthy patients from patients with RP, and capillaroscopy can then be used to differentiate PRP from SRP. IRT can also detect which fingers are more affected, and then these can direct the focus of capillaroscopy.

Simultaneous Presence of Bacteriochlorophyll and Xanthorhodopsin Genes in a Freshwater Bacterium.

Photoheterotrophic bacteria represent an important part of aquatic microbial communities. There exist two fundamentally different light-harvesting systems: bacteriochlorophyll-containing reaction centers or rhodopsins. Here, we report a photoheterotrophic Sphingomonas strain isolated from an oligotrophic lake, which contains complete sets of genes for both rhodopsin-based and bacteriochlorophyll-based phototrophy. Interestingly, the identified genes were not expressed when cultured in liquid organic media. Using reverse transcription quantitative PCR (RT-qPCR), RNA sequencing, and bacteriochlorophyll a quantification, we document that bacteriochlorophyll synthesis was repressed by high concentrations of glucose or galactose in the medium. Coactivation of photosynthesis genes together with genes for TonB-dependent transporters suggests the utilization of light energy for nutrient import. The photosynthetic units were formed by ring-shaped light-harvesting complex 1 and reaction centers with bacteriochlorophyll a and spirilloxanthin as the main light-harvesting pigments. The identified rhodopsin gene belonged to the xanthorhodopsin family, but it lacks salinixanthin antenna. In contrast to bacteriochlorophyll, the expression of xanthorhodopsin remained minimal under all experimental conditions tested. Since the gene was found in the same operon as a histidine kinase, we propose that it might serve as a light sensor. Our results document that photoheterotrophic Sphingomonas bacteria use the energy of light under carbon-limited conditions, while under carbon-replete conditions, they cover all their metabolic needs through oxidative phosphorylation.IMPORTANCE Phototrophic organisms are key components of many natural environments. There exist two main phototrophic groups: species that collect light energy using various kinds of (bacterio)chlorophylls and species that utilize rhodopsins. Here, we present a freshwater bacterium Sphingomonas sp. strain AAP5 which contains genes for both light-harvesting systems. We show that bacteriochlorophyll-based reaction centers are repressed by light and/or glucose. On the other hand, the rhodopsin gene was not expressed significantly under any of the experimental conditions. This may indicate that rhodopsin in Sphingomonas may have other functions not linked to bioenergetics.

Quasi-Static Shear Test of Hybrid Adhesive Bonds Based on Treated Cotton-Epoxy Resin Layer.

This research evaluates the mechanical properties of hybrid adhesive bonds with various 100% cotton fabrics in static and quasi-static conditions and the influence of alkali surface treatment (NaOH) of the cotton fabrics on the mechanical properties. Biological fibers in polymers are characterized by low wettability with the matrix, which decreases mechanical properties. Adhesive bonds usually operate in cyclic stress, which causes irreversible failure before maximal strength. In this paper, a quasi-static test was used to load the adhesive bonds in 5-50% (192-1951 N) and 5-70% (192-2732 N) intervals with 1000 cycles. The results of SEM analysis showed good wettability of alkali treated cotton fabric with NaOH solution in hybrid adhesive bonds. The static test proved the influence of reinforcing cotton fabrics on shear tensile strength against pure resin, i.e., sample Erik up to 19% on 14.90 ± 1.15 MPa and sample Tera up to 21% on 15.28 ± 1.05 MPa. The adhesive bonds with pure resin did not resist either quasi-static tests. Reinforcing cotton fabrics resisted both quasi-static tests, even shear tensile strength increases up to 10% on 16.34 ± 1.24 MPa for the fabric Erik. The results of strain difference of adhesive bonds with Tera and Erik confirmed that a lower value of the difference during cyclic loading positively influenced the ultimate shear tensile strength.

Quasi-Static Tests of Hybrid Adhesive Bonds Based on Biological Reinforcement in the Form of Eggshell Microparticles.

The paper is focused on the research of the cyclic loading of hybrid adhesive bonds based on eggshell microparticles in polymer composite. The aim of the research was to characterize the behavior of hybrid adhesive bonds with composite adhesive layer in quasi-static tests. An epoxy resin was used as the matrix and microparticles of eggshells were used as the filler. The adhesive bonds were exposed to cyclic loading and their service life and mechanical properties were evaluated. Testing was performed by 1000 cycles at 5-30% (165-989 N) and 5-70% (165-2307 N) of the maximum load of the filler-free bond in the static test. The results of the research show the importance of cyclic loading on the service life and mechanical properties of adhesive bonds. Quasi-static tests demonstrated significant differences between measured intervals of cyclic loading. All adhesive bonds resisted 1000 cycles of the quasi-static test with an interval loading 5-30%. The number of completed quasi-static tests with the interval loading 5-70% was significantly lower. The filler positively influenced the service life of adhesive bonds at a higher amount of quasi-static tests, i.e., the safety of adhesive bonds increased. The filler had a positive effect on adhesive bonds ABF2, where the strength significantly increased up to 20.26% at the loading of 5-30% against adhesive bonds ABF0. A viscoelasticity characteristic (creep) of the adhesive layer occurred at higher values of loading, i.e., between loading 5-70%. The viscoelasticity behavior did not occur at lower values of loading, i.e., between loading 5-30%.

The cyanobacterial protoporphyrinogen oxidase HemJ is a new b-type heme protein functionally coupled with coproporphyrinogen III oxidase.

Protoporphyrinogen IX oxidase (PPO), the last enzyme that is common to both chlorophyll and heme biosynthesis pathways, catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. PPO has several isoforms, including the oxygen-dependent HemY and an oxygen-independent enzyme, HemG. However, most cyanobacteria encode HemJ, the least characterized PPO form. We have characterized HemJ from the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) as a bona fide PPO; HemJ down-regulation resulted in accumulation of tetrapyrrole precursors and in the depletion of chlorophyll precursors. The expression of FLAG-tagged Synechocystis 6803 HemJ protein (HemJ.f) and affinity isolation of HemJ.f under native conditions revealed that it binds heme b The most stable HemJ.f form was a dimer, and higher oligomeric forms were also observed. Using both oxygen and artificial electron acceptors, we detected no enzymatic activity with the purified HemJ.f, consistent with the hypothesis that the enzymatic mechanism for HemJ is distinct from those of other PPO isoforms. The heme absorption spectra and distant HemJ homology to several membrane oxidases indicated that the heme in HemJ is redox-active and involved in electron transfer. HemJ was conditionally complemented by another PPO, HemG from Escherichia coli. If grown photoautotrophically, the complemented strain accumulated tripropionic tetrapyrrole harderoporphyrin, suggesting a defect in enzymatic conversion of coproporphyrinogen III to protoporphyrinogen IX, catalyzed by coproporphyrinogen III oxidase (CPO). This observation supports the hypothesis that HemJ is functionally coupled with CPO and that this coupling is disrupted after replacement of HemJ by HemG.

Binding of pigments to the cyanobacterial high-light-inducible protein HliC.

Cyanobacteria possess a family of one-helix high-light-inducible proteins (HLIPs) that are widely viewed as ancestors of the light-harvesting antenna of plants and algae. HLIPs are essential for viability under various stress conditions, although their exact role is not fully understood. The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains four HLIPs named HliA-D, and HliD has recently been isolated in a small protein complex and shown to bind chlorophyll and ß-carotene. However, no HLIP has been isolated and characterized in a pure form up to now. We have developed a protocol to purify large quantities of His-tagged HliC from an engineered Synechocystis strain. Purified His-HliC is a pigmented homo-oligomer and is associated with chlorophyll and ß-carotene with a 2:1 ratio. This differs from the 3:1 ratio reported for HliD. Comparison of these two HLIPs by resonance Raman spectroscopy revealed a similar conformation for their bound ß-carotenes, but clear differences in their chlorophylls. We present and discuss a structural model of HliC, in which a dimeric protein binds four chlorophyll molecules and two ß-carotenes.

Genomic Analysis of the Evolution of Phototrophy among Haloalkaliphilic Rhodobacterales.

A characteristic feature of the order Rhodobacterales is the presence of a large number of photoautotrophic and photoheterotrophic species containing bacteriochlorophyll. Interestingly, these phototrophic species are phylogenetically mixed with chemotrophs. To better understand the origin of such variability, we sequenced the genomes of three closely related haloalkaliphilic species, differing in their phototrophic capacity and oxygen preference: the photoheterotrophic and facultatively anaerobic bacterium Rhodobaca barguzinensis, aerobic photoheterotroph Roseinatronobacter thiooxidans, and aerobic heterotrophic bacterium Natronohydrobacter thiooxidans. These three haloalcaliphilic species are phylogenetically related and share many common characteristics with the Rhodobacter species, forming together the Rhodobacter-Rhodobaca (RR) group. A comparative genomic analysis showed close homology of photosynthetic proteins and similarity in photosynthesis gene organization among the investigated phototrophic RR species. On the other hand, Rhodobaca barguzinensis and Roseinatronobacter thiooxidans lack an inorganic carbon fixation pathway and outer light-harvesting genes. This documents the reduction of their photosynthetic machinery towards a mostly photoheterotrophic lifestyle. Moreover, both phototrophic species contain 5-aminolevulinate synthase (encoded by the hemA gene) incorporated into their photosynthesis gene clusters, which seems to be a common feature of all aerobic anoxygenic phototrophic Alphaproteobacteria. Interestingly, the chrR-rpoE (sigma24) operon, which is part of singlet oxygen defense in phototrophic species, was found in the heterotrophic strain Natronohydrobacter thiooxidans. This suggests that this organism evolved from a photoheterotrophic ancestor through the loss of its photosynthesis genes. The overall evolution of phototrophy among the haloalkaliphilic members of the RR group is discussed.