The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis.

Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.

Characterisation of Waterborne Psychrophilic Massilia Isolates with Violacein Production and Description of Massilia antarctica sp. nov.

A group of seven bacterial strains producing blue-purple pigmented colonies on R2A agar was isolated from freshwater samples collected in a deglaciated part of James Ross Island and Eagle Island, Antarctica, from 2017-2019. The isolates were psychrophilic, oligotrophic, resistant to chloramphenicol, and exhibited strong hydrolytic activities. To clarify the taxonomic position of these isolates, a polyphasic taxonomic approach was applied based on sequencing of the 16S rRNA, gyrB and lepA genes, whole-genome sequencing, rep-PCR, MALDI-TOF MS, chemotaxonomy analyses and biotyping. Phylogenetic analysis of the 16S rRNA gene sequences revealed that the entire group are representatives of the genus Massilia. The closest relatives of the reference strain P8398T were Massilia atriviolacea, Massilia violaceinigra, Massilia rubra, Massilia mucilaginosa, Massilia aquatica, Massilia frigida, Massilia glaciei and Massilia eurypsychrophila with a pairwise similarity of 98.6-100% in the 16S rRNA. The subsequent gyrB and lepA sequencing results showed the novelty of the analysed group, and the average nucleotide identity and digital DNA-DNA hybridisation values clearly proved that P8398T represents a distinct Massilia species. After all these results, we nominate a new species with the proposed name Massilia antarctica sp. nov. The type strain is P8398T (= CCM 8941T = LMG 32108T).

High-light-inducible proteins HliA and HliB: pigment binding and protein-protein interactions.

High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and ß-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a 'generalist' that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of 'specialists' (HliA, B or D) dictate interactions with proteins other than Hlips.

2.4-Å structure of the double-ring Gemmatimonas phototrophica photosystem.

Phototrophic Gemmatimonadetes evolved the ability to use solar energy following horizontal transfer of photosynthesis-related genes from an ancient phototrophic proteobacterium. The electron cryo-microscopy structure of the Gemmatimonas phototrophica photosystem at 2.4 Å reveals a unique, double-ring complex. Two unique membrane-extrinsic polypeptides, RC-S and RC-U, hold the central type 2 reaction center (RC) within an inner 16-subunit light-harvesting 1 (LH1) ring, which is encircled by an outer 24-subunit antenna ring (LHh) that adds light-gathering capacity. Femtosecond kinetics reveal the flow of energy within the RC-dLH complex, from the outer LHh ring to LH1 and then to the RC. This structural and functional study shows that G. phototrophica has independently evolved its own compact, robust, and highly effective architecture for harvesting and trapping solar energy.

Classification of a Violacein-Producing Psychrophilic Group of Isolates Associated with Freshwater in Antarctica and Description of Rugamonas violacea sp. nov.

A group of 11 bacterial strains was isolated from streams and lakes located in a deglaciated northern part of James Ross Island, Antarctica. They were rod-shaped, Gram-stain-negative, motile, and catalase-positive and produced blue-violet-pigmented colonies on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, whole-genome sequencing, automated ribotyping, repetitive element sequence-based PCR (rep-PCR), MALDI-TOF MS, fatty acid profile, chemotaxonomy analyses, and extensive biotyping was applied in order to clarify the taxonomic position of these isolates. Phylogenetic analysis based on the 16S rRNA gene indicated that all the isolates constituted a coherent group belonging to the genus Rugamonas. The closest relatives to the representative isolate P5900T were Rugamonas rubra CCM 3730T, Rugamonas rivuli FT103WT, and Rugamonas aquatica FT29WT, exhibiting 99.2%, 99.1%, and 98.6% 16S rRNA pairwise similarity, respectively. The average nucleotide identity and digital DNA-DNA hybridization values calculated from the whole-genome sequencing data clearly proved that P5900T represents a distinct Rugamonas species. The G+C content of genomic DNAs was 66.1 mol%. The major components in fatty acid profiles were summed feature 3 (C16:1ω7c/C16:1ω6c), C 16:0, and C12:0. The cellular quinone content contained exclusively ubiquinone Q-8. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The polyamine pattern was composed of putrescine, 2-hydroxputrescine, and spermidine. IMPORTANCE Our polyphasic approach provides a new understanding of the taxonomy of novel pigmented Rugamonas species isolated from freshwater samples in Antarctica. The isolates showed considerable extracellular bactericidal secretions. The antagonistic activity of studied isolates against selected pathogens was proved by this study and implied the importance of such compounds' production among aquatic bacteria. The psychrophilic and violacein-producing species Roseomonas violacea may play a role in the diverse consortium among pigmented bacteria in the Antarctic water environment. Based on all the obtained results, we propose a novel species for which the name Rugamonas violacea sp. nov. is suggested, with the type strain P5900T (CCM 8940T; LMG 32105T). Isolates of R. violacea were obtained from different aquatic localities, and they represent the autochthonous part of the water microbiome in Antarctica.

Psb35 Protein Stabilizes the CP47 Assembly Module and Associated High-Light Inducible Proteins during the Biogenesis of Photosystem II in the Cyanobacterium Synechocystis sp. PCC6803.

Photosystem II (PSII) is a large membrane protein complex performing primary charge separation in oxygenic photosynthesis. The biogenesis of PSII is a complicated process that involves a coordinated linking of assembly modules in a precise order. Each such module consists of one large chlorophyll (Chl)-binding protein, number of small membrane polypeptides, pigments and other cofactors. We isolated the CP47 antenna module from the cyanobacterium Synechocystis sp. PCC 6803 and found that it contains a 11-kDa protein encoded by the ssl2148 gene. This protein was named Psb35 and its presence in the CP47 module was confirmed by the isolation of FLAG-tagged version of Psb35. Using this pulldown assay, we showed that the Psb35 remains attached to CP47 after the integration of CP47 into PSII complexes. However, the isolated Psb35-PSIIs were enriched with auxiliary PSII assembly factors like Psb27, Psb28-1, Psb28-2 and RubA while they lacked the lumenal proteins stabilizing the PSII oxygen-evolving complex. In addition, the Psb35 co-purified with a large unique complex of CP47 and photosystem I trimer. The absence of Psb35 led to a lower accumulation and decreased stability of the CP47 antenna module and associated high-light-inducible proteins but did not change the growth rate of the cyanobacterium under the variety of light regimes. Nevertheless, in comparison with WT, the Psb35-less mutant showed an accelerated pigment bleaching during prolonged dark incubation. The results suggest an involvement of Psb35 in the life cycle of cyanobacterial Chl-binding proteins, especially CP47.

Plant and algal chlorophyll synthases function in Synechocystis and interact with the YidC/Alb3 membrane insertase.

In the model cyanobacterium Synechocystis sp. PCC 6803, the terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), forms a complex with high light-inducible proteins, the photosystem II assembly factor Ycf39 and the YidC/Alb3/OxaI membrane insertase, co-ordinating chlorophyll delivery with cotranslational insertion of nascent photosystem polypeptides into the membrane. To gain insight into the ubiquity of this assembly complex in higher photosynthetic organisms, we produced functional foreign chlorophyll synthases in a cyanobacterial host. Synthesis of algal and plant chlorophyll synthases allowed deletion of the otherwise essential native cyanobacterial gene. Analysis of purified protein complexes shows that the interaction with YidC is maintained for both eukaryotic enzymes, indicating that a ChlG-YidC/Alb3 complex may be evolutionarily conserved in algae and plants.

Chlorophyll-Mediated Changes in the Redox Status of Pancreatic Cancer Cells Are Associated with Its Anticancer Effects.

Nutritional factors which exhibit antioxidant properties, such as those contained in green plants, may be protective against cancer. Chlorophyll and other tetrapyrrolic compounds which are structurally related to heme and bilirubin (a bile pigment with antioxidant activity) are among those molecules which are purportedly responsible for these effects. Therefore, the aim of our study was to assess both the antiproliferative and antioxidative effects of chlorophylls (chlorophyll a/b, chlorophyllin, and pheophytin a) in experimental pancreatic cancer. Chlorophylls have been shown to produce antiproliferative effects in pancreatic cancer cell lines (PaTu-8902, MiaPaCa-2, and BxPC-3) in a dose-dependent manner (10-125 µmol/L). Chlorophylls also have been observed to inhibit heme oxygenase (HMOX) mRNA expression and HMOX enzymatic activity, substantially affecting the redox environment of pancreatic cancer cells, including the production of mitochondrial/whole-cell reactive oxygen species, and alter the ratio of reduced-to-oxidized glutathione. Importantly, chlorophyll-mediated suppression of pancreatic cancer cell viability has been replicated in in vivo experiments, where the administration of chlorophyll a resulted in the significant reduction of pancreatic tumor size in xenotransplanted nude mice. In conclusion, this data suggests that chlorophyll-mediated changes on the redox status of pancreatic cancer cells might be responsible for their antiproliferative and anticancer effects and thus contribute to the decreased incidence of cancer among individuals who consume green vegetables.

The Ribosome-Bound Protein Pam68 Promotes Insertion of Chlorophyll into the CP47 Subunit of Photosystem II.

Photosystem II (PSII) is a large enzyme complex embedded in the thylakoid membrane of oxygenic phototrophs. The biogenesis of PSII requires the assembly of more than 30 subunits, with the assistance of a number of auxiliary proteins. In plants and cyanobacteria, the photosynthesis-affected mutant 68 (Pam68) is important for PSII assembly. However, its mechanisms of action remain unknown. Using a Synechocystis PCC 6803 strain expressing Flag-tagged Pam68, we purified a large protein complex containing ribosomes, SecY translocase, and the chlorophyll-binding PSII inner antenna CP47. Using 2D gel electrophoresis, we identified a pigmented Pam68-CP47 subcomplex and found Pam68 bound to ribosomes. Our results show that Pam68 binds to ribosomes even in the absence of CP47 translation. Furthermore, Pam68 associates with CP47 at an early phase of its biogenesis and promotes the synthesis of this chlorophyll-binding polypeptide until the attachment of the small PSII subunit PsbH. Deletion of both Pam68 and PsbH nearly abolishes the synthesis of CP47, which can be restored by enhancing chlorophyll biosynthesis. These results strongly suggest that ribosome-bound Pam68 stabilizes membrane segments of CP47 and facilitates the insertion of chlorophyll molecules into the translated CP47 polypeptide chain.

Placental Growth Factor in Bladder Cancer Compared to the Diagnostic Accuracy and Prognostic Performance of Vascular Endothelial Growth Factor A.

BACKGROUND/AIM: To evaluate the diagnostic accuracy and prognostic performance of urinary and plasma levels of placental growth factor (PLGF) and provide their comparison with the results of vascular endothelial growth factor A (VEGF-A) in patients with primary and recurrent urinary bladder cancer. MATERIALS AND METHODS: The enzyme-linked immunosorbent assay (ELISA) was used to assess urinary and plasma concentrations of PLGF and VEGF-A in 240 individuals. RESULTS: PLGF levels in urine and plasma were significantly higher in patients with primary bladder cancer than in healthy individuals (p=0.003, p=0.005, respectively). Area under the curve (AUC) of urinary PLGF was 0.68; AUC of plasma PLGF levels was 0.65. Patients with the urine levels of PLGF higher than 82.33 pg/ml had three times higher risk of recurrence. In patients with recurrent bladder cancer, the urinary concentrations of PLGF did not significantly differ from the concentrations in patients without current disease (p=0.61). However, plasma PLGF levels were significantly higher in patients diagnosed with tumor recurrence (p=0.001); AUC of plasma PLGF levels was 0.69. Moreover, patients with plasma levels higher than 10.09 pg/ml had a five-times higher risk of future tumor recurrence. The diagnostic accuracy of PLGF was comparable with VEGF-A. CONCLUSION: From a clinical point of view, PLGF could be considered a valid diagnostic test for the detection of primary and recurrent bladder cancer. In patients with recurrent bladder cancer, plasma PLGF levels can differentiate individuals at risk of tumor recurrence.

Mucilaginibacter terrae sp. nov., isolated from Antarctic soil.

A bacterial strain designated CCM 8645T was isolated from a soil sample collected nearby a mummified seal carcass in the northern part of James Ross Island, Antarctica. The cells were short rods, Gram-stain-negative, non-motile, catalase and oxidase positive, and produced a red-pink pigment on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, extensive biotyping using conventional tests and commercial identification kits and chemotaxonomic analyses were applied to clarify its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene placed strain CCM 8645T in the genus Mucilaginibacter with the closest relative being Mucilaginibacter daejeonensis Jip 10T, exhibiting 96.5 % 16S rRNA pairwise similarity which was clearly below the 97 % threshold value recommended for species demarcation. The major components in fatty acid profiles were Summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C15 : 0 iso and C17 : 0 iso 3OH. The cellular quinone content was exclusively menaquinone MK-7. The major polyamine was sym-homospermidine and predominant polar lipids were phosphatidylethanolamine and phosphatidylserine. Based on presented results, we propose a novel species for which the name Mucilaginibacter terrae sp. nov. is suggested, with the type strain CCM 8645T (=LMG 29437T).

Prognostic Importance of Vitamins A, E and Retinol-binding Protein 4 in Renal Cell Carcinoma Patients.

AIM: To assess the prognostic importance of serum levels of retinol, retinol-binding protein 4 (RBP4) and vitamin E at the time of diagnosis in patients with renal cell carcinoma (RCC). PATIENTS AND METHODS: In this prospective study, in a cohort of 102 renal cell carcinoma patients, relationships between serum levels of the aforementioned markers and recurrence-free survival (RFS), overall survival (OS), as well as cancer-specific survival (CSS), were evaluated. The vitamin A and vitamin E levels were determined by high-performance liquid chromatography (HPLC), while the RBP4 level by enzyme-linked immunosorbent assay (ELISA). RESULTS: The median follow-up period was 39 months. Renal cell carcinoma recurred in 9 patients; 23 patients died with 12 of them from RCC. The preoperative vitamin E level was associated to RFS (p=0.02). We found a significant relationship between OS and the level of RBP4 (p=0.002), retinol (p=0.037) and vitamin E (p=0.007). The CSS period was significantly associated with the level of RBP4 (p=0.0001) and retinol (p=0.0003). Patients with an RBP4 level less than 21.0 mg/l at the time of diagnosis had a 13.5-times higher risk of death due to RCC progression; this risk was up to 7.7-times higher with vitamin A levels under 0.52 mg/l. CONCLUSION: Low levels of vitamin A, E and RBP4 at the time of RCC diagnosis are associated with a poorer prognosis after surgery.

Awakening of a Dormant Cyanobacterium from Nitrogen Chlorosis Reveals a Genetically Determined Program.

The molecular and physiological mechanisms involved in the transition of microbial cells from a resting state to the active vegetative state are critically relevant for solving problems in fields ranging from microbial ecology to infection microbiology. Cyanobacteria that cannot fix nitrogen are able to survive prolonged periods of nitrogen starvation as chlorotic cells in a dormant state. When provided with a usable nitrogen source, these cells re-green within 48 hr and return to vegetative growth. Here we investigated the resuscitation of chlorotic Synechocystis sp. PCC 6803 cells at the physiological and molecular levels with the aim of understanding the awakening process of a dormant bacterium. Almost immediately upon nitrate addition, the cells initiated a highly organized resuscitation program. In the first phase, they suppressed any residual photosynthetic activity and activated respiration to gain energy from glycogen catabolism. Concomitantly, they restored the entire translational apparatus, ATP synthesis, and nitrate assimilation. After only 12-16 hr, the cells re-activated the synthesis of the photosynthetic apparatus and prepared for metabolic re-wiring toward photosynthesis. When the cells reached full photosynthetic capacity after ∼48 hr, they resumed cell division and entered the vegetative cell cycle. An analysis of the transcriptional dynamics during the resuscitation process revealed a perfect match to the observed physiological processes, and it suggested that non-coding RNAs play a major regulatory role during the lifestyle switch in awakening cells. This genetically encoded program ensures rapid colonization of habitats in which nitrogen starvation imposes a recurring growth limitation.

Prognosis of Castration-resistant Prostate Cancer Patients - Use of the AdnaTest® System for Detection of Circulating Tumor Cells.

BACKGROUND: Detection of circulating tumor cells (CTC) in patients with castration-resistant prostate cancer (CRPC) may improve the estimate of chemotherapy response. We evaluated the AdnaTest® system in patients receiving docetaxel. PATIENTS AND METHODS: CTC analysis was carried out in 37 patients by immunomagnetic separation. Correlation between serum prostate-specific antigen (sPSA) change and CTC presence and the influence of each parameter on the overall survival (OS) were evaluated. RESULTS: We detected CTCs in 32 and 16 patients before and after three docetaxel cycles, respectively. The sPSA level correlated with CTC positivity during docetaxel therapy (p=0.0031). The longest OS was in patients negative for CTCs in both samples (p=0.0228). Change in sPSA levels was associated with treatment response (p=0.033). CONCLUSION: We detected CTCs in a considerable number of patients with CRPC. The absolute change of sPSA level correlated with OS. CTC presence during docetaxel therapy was associated with shorter OS.

The meaning of sampling density in multiple repeat prostate biopsies.

INTRODUCTION: Extended transrectal ultrasound-guided prostate biopsy is a state-of-the-art tool for prostate cancer detection. Nevertheless, approximately 1/3 of cancers are missed when using this method and repeat biopsy sessions are often required. The aim of this study was to investigate how sampling density (a compound variable reflecting the number of biopsy cores and prostate volume) impacts on detection rate in multiple repeat TRUS-biopsies. MATERIAL AND METHODS: A total of 1007 consecutive patients undergoing their 1st, 2nd, 3rd and any further repeat prostate biopsies were included. The relationship between sampling density and other clinical variables (age, prostate-specific antigen level, free/total PSA ratio, digital rectal examination, number of previous biopsies) and cancer detection rate were assessed by interaction analysis. RESULTS: There were 562 primary re-biopsies, 267 second re-biopsies and 178 third and further re-biopsies included in the study. Detection rate was 25.4%, 25.8% and 25.3%, respectively. Interaction of sampling density with age was demonstrated in patients undergoing their first repeat biopsy (but not further re-biopsies). No interaction was observed with other variables investigated. CONCLUSIONS: A more extensive prostate sampling leads to a higher cancer detection rate on repeat prostate biopsies, as shown previously. However, this effect seems to be particularly pronounced in men younger than 65 years undergoing their first repeat prostate biopsy.

Diagnostic Importance of Selected Protein Serum Markers in the Primary Diagnostics of Prostate Cancer.

OBJECTIVE: To determine a predictive model for the primary diagnosis of prostate cancer (PC) based on a multiple serum biomarker assay. MATERIAL AND METHODS: Between August 2011 and February 2013, a total of 387 prostate biopsies were performed. Serum or plasma concentrations of 22 biomarkers (neopterin, IGF-1, IGFBP-2, IGFBP-3, sarcosine, endoglin, TGF-ß1, periostin, sPLA2-IIa, chromogranin A, ZAG2, clusterin, PSP94, PSP94bp, leptin, cathepsin D, hepsin, KLK11, PSMA, AMACR, CRISP3 and A1AT) were determined. Biomarker levels were correlated with the prostate biopsy results. Several statistical models for PC detection were created. RESULTS: A total of 167 of the 373 evaluated patients (44.8%) were diagnosed with PC. None of the tested biomarkers reached statistical significance using the univariate analysis. However, the level of serum clusterin was not associated with any other tested parameter. Several basic models showed a higher positive predictive value than individual parameters. Addition of serum clusterin to the base model with prostate-specific antigen, digital rectal exam and prostate size significantly improved the area under curve value (0.723 vs. 0.716). CONCLUSION: Our findings suggested that multiple serum assays based on some promising markers may only have a limited practical benefit for the prediction of PC in the prostate biopsy.

Panel of Urinary Diagnostic Markers for Non-Invasive Detection of Primary and Recurrent Urothelial Urinary Bladder Carcinoma.

OBJECTIVES: To determine the combination of urinary protein markers for noninvasive detection of primary and recurrent urothelial bladder carcinomas. METHODS: Urinary concentrations of 27 biomarkers (NSE, ATT, AFABP, Resistin, Midkine, Clusterin, Uromodulin, ZAG2, HSP27, HSP 60, NCAM1/CD56, Angiogenin, Calreticulin, Chromogranin A, CEACAM1, CXCL1, IL13Ra2, Progranulin, VEGFA, CarbAnhydIX, Annexin-V, TIM4, Galectin1, Cystatin B, Synuclein G, ApoA1 and ApoA2) were assessed by enzyme-linked immunosorbent assay or by electrochemiluminiscence immunoassay. RESULTS: During the primary diagnostics, a group of 70 patients with primary occurrence of bladder cancer and 49 healthy control subjects were compared. For this clinical situation, the most accurate combination proved to be the combination of cytology with markers Midkine and Synuclein G (sensitivity 91.8%, specificity 97.5%). During the monitoring of patients with non-muscle invasive bladder cancer (NMIBC), a group of 44 patients with cancer recurrence was compared with the group of 61 patients with a history of NMIBC without current disease. For this clinical situation, the most accurate combination proved to be the combination of cytology and erythrocytes count in urine sediment with markers Midkine, ZAG2, CEACAM1, and Synuclein G (sensitivity 92.68%, specificity 90.16%). A lower accuracy of the diagnostic panel and the necessity to use more markers in the case of recurrence was connected with a different structure of patients. CONCLUSIONS: Multi-marker test can significantly improve the bladder cancer detection both during the primary diagnostics and monitoring of patients with NMIBC. This outcome should result in other, larger studies.

A cyanobacterial chlorophyll synthase-HliD complex associates with the Ycf39 protein and the YidC/Alb3 insertase.

Macromolecular membrane assemblies of chlorophyll-protein complexes efficiently harvest and trap light energy for photosynthesis. To investigate the delivery of chlorophylls to the newly synthesized photosystem apoproteins, a terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), was tagged in the cyanobacterium Synechocystis PCC 6803 (Synechocystis) and used as bait in pull-down experiments. We retrieved an enzymatically active complex comprising ChlG and the high-light-inducible protein HliD, which associates with the Ycf39 protein, a putative assembly factor for photosystem II, and with the YidC/Alb3 insertase. 2D electrophoresis and immunoblotting also provided evidence for the presence of SecY and ribosome subunits. The isolated complex contained chlorophyll, chlorophyllide, and carotenoid pigments. Deletion of hliD elevated the level of the ChlG substrate, chlorophyllide, more than 6-fold; HliD is apparently required for assembly of FLAG-ChlG into larger complexes with other proteins such as Ycf39. These data reveal a link between chlorophyll biosynthesis and the Sec/YidC-dependent cotranslational insertion of nascent photosystem polypeptides into membranes. We expect that this close physical linkage coordinates the arrival of pigments and nascent apoproteins to produce photosynthetic pigment-protein complexes with minimal risk of accumulating phototoxic unbound chlorophylls.

Making proteins green; biosynthesis of chlorophyll-binding proteins in cyanobacteria.

Chlorophyll (Chl) is an essential component of the photosynthetic apparatus. Embedded into Chl-binding proteins, Chl molecules play a central role in light harvesting and charge separation within the photosystems. It is critical for the photosynthetic cell to not only ensure the synthesis of a sufficient amount of new Chl-binding proteins but also avoids any misbalance between apoprotein synthesis and the formation of potentially phototoxic Chl molecules. According to the available data, Chl-binding proteins are translated on membrane bound ribosomes and their integration into the membrane is provided by the SecYEG/Alb3 translocon machinery. It appears that the insertion of Chl molecules into growing polypeptide is a prerequisite for the correct folding and finishing of Chl-binding protein synthesis. Although the Chl biosynthetic pathway is fairly well-described on the level of enzymatic steps, a link between Chl biosynthesis and the synthesis of apoproteins remains elusive. In this review, I summarize the current knowledge about this issue putting emphasis on protein-protein interactions. I present a model of the Chl biosynthetic pathway organized into a multi-enzymatic complex and physically attached to the SecYEG/Alb3 translocon. Localization of this hypothetical large biosynthetic centre in the cyanobacterial cell is also discussed as well as regulatory mechanisms coordinating the rate of Chl and apoprotein synthesis.

Split photosystem protein, linear-mapping topology, and growth of structural complexity in the plastid genome of Chromera velia.

The canonical photosynthetic plastid genomes consist of a single circular-mapping chromosome that encodes a highly conserved protein core, involved in photosynthesis and ATP generation. Here, we demonstrate that the plastid genome of the photosynthetic relative of apicomplexans, Chromera velia, departs from this view in several unique ways. Core photosynthesis proteins PsaA and AtpB have been broken into two fragments, which we show are independently transcribed, oligoU-tailed, translated, and assembled into functional photosystem I and ATP synthase complexes. Genome-wide transcription profiles support expression of many other highly modified proteins, including several that contain extensions amounting to hundreds of amino acids in length. Canonical gene clusters and operons have been fragmented and reshuffled into novel putative transcriptional units. Massive genomic coverage by paired-end reads, coupled with pulsed-field gel electrophoresis and polymerase chain reaction, consistently indicate that the C. velia plastid genome is linear-mapping, a unique state among all plastids. Abundant intragenomic duplication probably mediated by recombination can explain protein splits, extensions, and genome linearization and is perhaps the key driving force behind the many features that defy the conventional ways of plastid genome architecture and function.