Detection of outer membrane vesicles in Synechocystis PCC 6803.
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ABSTRACT:
SUBMITTER: Pardo YA
PROVIDER: S-EPMC4809984 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cyanobacteria are foundational drivers of global nutrient cycling, with high intracellular iron (Fe) requirements. Fe is found at extremely low concentrations in aquatic systems, however, and the ways in which cyanobacteria take up Fe are largely unknown, especially the initial step in Fe transport across the outer membrane. Here, we identified one TonB protein and four TonB-dependent transporters (TBDTs) of the energy-requiring Fe acquisition system and six porins of the passive diffusion Fe uptake system in the model cyanobacterium Synechocystis sp. strain PCC 6803. The results experimentally demonstrated that TBDTs not only participated in organic ferri-siderophore uptake but also in inorganic free Fe (Fe') acquisition. 55Fe uptake rate measurements showed that a TBDT quadruple mutant acquired Fe at a lower rate than the wild type and lost nearly all ability to take up ferri-siderophores, indicating that TBDTs are critical for siderophore uptake. However, the mutant retained the ability to take up Fe' at 42% of the wild-type Fe' uptake rate, suggesting additional pathways of Fe' acquisition besides TBDTs, likely by porins. Mutations in four of the six porin-encoding genes produced a low-Fe-sensitive phenotype, while a mutation in all six genes was lethal to cell survival. These diverse outer membrane Fe uptake pathways reflect cyanobacterial evolution and adaptation under a range of Fe regimes across aquatic systems.IMPORTANCE Cyanobacteria are globally important primary producers and contribute about 25% of global CO2 fixation. Low Fe bioavailability in surface waters is thought to limit the primary productivity in as much as 40% of the global ocean. The Fe acquisition strategies that cyanobacteria have evolved to overcome Fe deficiency remain poorly characterized. We experimentally characterized the key players and the cooperative work mode of two Fe uptake pathways, including an active uptake pathway and a passive diffusion pathway in the model cyanobacterium Synechocystis sp. PCC 6803. Our finding proved that cyanobacteria use ferri-siderophore transporters to take up Fe', and they shed light on the adaptive mechanisms of cyanobacteria to cope with widespread Fe deficiency across aquatic environments.
| S-EPMC6146993 | biostudies-literature
Project description:Cyanobacteria are a group of photosynthetic prokaryotes that contribute to primary production on a global scale. These microorganisms release vesicles to the extracellular environment, spherical nanosized structures, derived essentially from the outer membrane. Even though earlier works in model Gram-negative bacteria have hypothesized that outer membrane stability is crucial in vesicle formation, the mechanisms determining vesicle biogenesis in cyanobacteria remain unknown. Here, we report on the identification of six candidate genes encoding outer membrane proteins harboring SLH/OprB-domains in the genome of the model cyanobacterium Synechocystis sp. PCC 6803. Using a genetics-based approach, one gene was found to encode an essential protein (Slr1841), while the remaining five are not essential for growth under standard conditions. Vesicle production was monitored, and it was found that a mutant in the gene encoding the second most abundant SLH/OprB protein in Synechocystis sp. PCC 6803 outer membrane (Slr1908) produces more vesicles than any of the other tested strains. Moreover, the Slr1908-protein was also found to be important for iron uptake. Altogether, our results suggest that proteins containing the SLH/OprB-domains may have dual biological role, related to micronutrient uptake and to outer membrane stability, which, together or alone, seem to be involved in cyanobacterial vesicle biogenesis.
| S-EPMC8707739 | biostudies-literature
Project description:BackgroundThe membranes of Synechocystis sp. PCC 6803 play a central role in photosynthesis, respiration and other important metabolic pathways. Comprehensive identification of the membrane proteins is of importance for a better understanding of the diverse functions of its unique membrane structures. Up to date, approximately 900 known or predicted membrane proteins, consisting 24.5% of Synechocystis sp. PCC 6803 proteome, have been indentified by large-scale proteomic studies.ResultsTo resolve more membrane proteins on 2-D gels for mass spectrometry identification, we separated integral proteins from membrane associated proteins and collected them as the integral and peripheral fractions, respectively. In total, 95 proteins in the peripheral fraction and 29 proteins in the integral fraction were identified, including the 5 unique proteins that were not identified by any previous studies. Bioinformatic analysis revealed that the identified proteins can be functionally classified into 14 distinct groups according to the cellular functions annotated by Cyanobase, including the two largest groups hypothetical and unknown, and photosynthesis and respiration. Homology analysis indicates that the identified membrane proteins are more conserved than the rest of the proteome.ConclusionThe proteins identified in this study combined with other published proteomic data provide the most comprehensive Synechocystis proteome catalog, which will serve as a useful reference for further detailed studies to address protein functions through both traditional gene-by-gene and systems biology approaches.
| S-EPMC2666656 | biostudies-literature
Project description:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750(-1) L(-1). We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.
| S-EPMC3953072 | biostudies-literature
Project description:We designed and constructed a controllable inducing lysis system in Synechocystis sp. PCC 6803 to facilitate extracting lipids for biofuel production. Several bacteriophage-derived lysis genes were integrated into the genome and placed downstream of a nickel-inducible signal transduction system. We applied 3 strategies: (i) directly using the phage lysis cassette, (ii) constitutively expressing endolysin genes while restricting holin genes, and (iii) combining lysis genes from different phages. Significant autolysis was induced in the Synechocystis sp. PCC 6803 cells with this system by the addition of NiSO(4). Our inducible cyanobacterial lysing system eliminates the need for mechanical or chemical cell breakage and could facilitate recovery of biofuel from cyanobacteria.
| S-EPMC2799798 | biostudies-literature
Project description:BackgroundMetabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene.ResultsThis study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background.ConclusionMaking use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.
| S-EPMC5324202 | biostudies-literature
Project description:BACKGROUND:There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated. This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria. RESULTS:In this work, we focused on the production of two different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids, and chlorophylls. This allowed us to identify metabolic crosstalk between the native and the introduced metabolic pathways. Most results and simulations highlight the metabolic robustness of cyanobacteria, suggesting that the host organism tends to keep metabolic fluxes and metabolite concentrations steady, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites. CONCLUSIONS:There is a wealth of information that can be derived by combining targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to 'traditional' metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies.
| S-EPMC5556357 | biostudies-literature
Project description:The biosynthesis pathway of carotenoids in cyanobacteria is partly described. However, the subcellular localization of individual steps is so far unknown. Carotenoid analysis of different membrane subfractions in Synechocystis sp. PCC6803 shows that "light" plasma membranes have a high carotenoid/protein ratio, when compared to "heavier" plasma membranes or thylakoids. The localization of CrtQ and CrtO, two well-defined carotenoid synthesis pathway enzymes in Synechocystis, was studied by epitope tagging and western blots. Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of ?-carotene precursor molecules and echinenone.
| S-EPMC4470828 | biostudies-literature
Project description:In the light-harvesting antenna of the Synechocystis PCC 6803 phycobilisome (PB), the core consists of three cylinders, each composed of four disks, whereas each of the six rods consists of up to three hexamers (Arteni et al., Biochim Biophys Acta 1787(4):272-279, 2009). The rods and core contain phycocyanin and allophycocyanin pigments, respectively. Together these pigments absorb light between 400 and 650 nm. Time-resolved difference absorption spectra from wild-type PB and rod mutants have been measured in different quenching and annihilation conditions. Based upon a global analysis of these data and of published time-resolved emission spectra, a functional compartmental model of the phycobilisome is proposed. The model describes all experiments with a common set of parameters. Three annihilation time constants are estimated, 3, 25, and 147 ps, which represent, respectively, intradisk, interdisk/intracylinder, and intercylinder annihilation. The species-associated difference absorption and emission spectra of two phycocyanin and two allophycocyanin pigments are consistently estimated, as well as all the excitation energy transfer rates. Thus, the wild-type PB containing 396 pigments can be described by a functional compartmental model of 22 compartments. When the interhexamer equilibration within a rod is not taken into account, this can be further simplified to ten compartments, which is the minimal model. In this model, the slowest excitation energy transfer rates are between the core cylinders (time constants 115-145 ps), and between the rods and the core (time constants 68-115 ps).
| S-EPMC5784004 | biostudies-literature
Project description:The ribulose-1,5-bisphosphate (RuBP) oxygenation reaction catalyzed by Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is competing with carboxylation, being negative for both energy and carbon balances in photoautotrophic organisms. This makes RuBisCO one of the bottlenecks for oxygenic photosynthesis and carbon fixation. In this study, RuBisCO was overexpressed in the unicellular cyanobacterium Synechocystis PCC 6803. Relative RuBisCO levels in the engineered strains FL50 and FL52 increased 2.1 times and 1.4 times, respectively, and both strains showed increased growth, photosynthesis and in vitro RuBisCO activity. The oxygen evolution rate increased by 54% and 42% on per chlorophyll basis, while the in vitro RuBisCO activity increased by 52% and 8.6%, respectively. The overexpressed RuBisCO were tagged with a FLAG tag, in strain FL50 on the N terminus of the large subunit while in strain FL52 on the C terminus of the small subunit. The presence of a FLAG tag enhanced transcription of the genes encoding RuBisCO, and, with high possibility, also enhanced the initiation of translation or stability of the enzyme. However, when using a streptavidin-binding tag II (strep-tag II), we did not observe a similar effect. Tagged RuBisCO offers an opportunity for further studying RuBisCO expression and stability. Increased levels of RuBisCO can further improve photosynthesis and growth in the cyanobacterium Synechocystis PCC 6803 under certain growth conditions.
| S-EPMC5779733 | biostudies-literature