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Detection of outer membrane vesicles in Synechocystis PCC 6803.

ABSTRACT: It has been well established that many species of Gram-negative bacteria release nanoscale outer membrane vesicles (OMVs) during normal growth. Furthermore, the roles of these structures in heterotrophic bacteria have been extensively characterized. However, little is known about the existence or function of OMVs in photoautotrophs. In the present study, we report for the first time the production of OMVs by the model photosynthetic organism Synechocystis sp. PCC 6803, a species of biotechnological importance. We detected extracellular proteins and lipids in cell-free supernatants derived from Synechocystis culture, yet the cytoplasmic and thylakoid membrane markers NADH oxidase and chlorophyll were absent. This indicated that the extracellular proteins and lipids derived from the outer membrane, and not from cell lysis. Furthermore, we identified spherical structures within the expected size range of OMVs in Synechocystis culture using scanning electron microscopy. Taken together, these results suggest that the repertoire of Gram-negative bacteria that are known to produce OMVs may be expanded to include Synechocystis PCC6803. Because of the considerable genetic characterization of Synechocystis in particular, our discovery has the potential to support novel biotechnological applications as well.

SUBMITTER: Pardo YA

PROVIDER: S-EPMC4809984 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Detection of outer membrane vesicles in Synechocystis PCC 6803.

Pardo Yehudah A YA Florez Catalina C Baker Kristopher M KM Schertzer Jeffrey W JW Mahler Gretchen J GJ

FEMS microbiology letters 20150910 20

It has been well established that many species of Gram-negative bacteria release nanoscale outer membrane vesicles (OMVs) during normal growth. Furthermore, the roles of these structures in heterotrophic bacteria have been extensively characterized. However, little is known about the existence or function of OMVs in photoautotrophs. In the present study, we report for the first time the production of OMVs by the model photosynthetic organism Synechocystis sp. PCC 6803, a species of biotechnologi ...[more]

PMID: 26363014

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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.

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.

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.

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Detection of outer membrane vesicles in Synechocystis PCC 6803.

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Detection of outer membrane vesicles in Synechocystis PCC 6803.

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