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Himadri B. Pakrasi - One of the best experts on this subject based on the ideXlab platform.
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engineering natural competence into the fast growing cyanobacterium Synechococcus elongatus utex 2973
Applied and Environmental Microbiology, 2021Co-Authors: Kristen E Wendt, Justin Ungerer, Annesha Sengupta, Patricia Walker, Himadri B. PakrasiAbstract:Natural transformation is the process by which bacteria actively take up and integrate extracellular DNA into their genomes. In cyanobacteria, natural transformation has only been experimentally demonstrated in a handful of species. Although, cyanobacteria are important model systems for studying photosynthesis and circadian cycling, natural transformation in cyanobacteria has not been characterized to the degree that the process has been studied in other gram-negative bacteria. Two cyanobacterial species that are 99.8% genetically identical provide a unique opportunity to better understand the nuances of natural transformation in cyanobacteria: Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 7942 and Synechococcus 2973 respectively). Synechococcus 7942 is a naturally transformable model system, while Synechococcus 2973 is a recently discovered species that is not naturally competent. Taking only 1.5 hours to replicate, Synechococcus 2973 is the fastest growing cyanobacterial species known, and thus is a strong candidate for serving as a model organism. However, the organism's inability to undergo natural transformation has prevented it from becoming a widely used model system. By substituting polymorphic alleles from Synechococcus 7942 for native Synechococcus 2973 alleles, natural transformation was introduced into Synechococcus 2973. Two genetic loci were found to be involved in differential natural competence between the two organisms: transformation pilus component pilN and circadian transcriptional master regulator rpaA. By using targeting genome editing and enrichment outgrowth, a strain that was both naturally transformable and fast-growing was created. This new Synechococcus 2973-T strain will serve as a valuable resource to the cyanobacterial research community. Importance Certain bacterial species have the ability to take up naked extracellular DNA and integrate it into their genomes. This process is known as natural transformation and is widely considered to play a major role in bacterial evolution. Because of the ease of introducing new genes into naturally transformable organisms, this capacity is also highly valued in the laboratory. Cyanobacteria are photosynthetic and can therefore serve as model systems for some important aspects of plant physiology. Here, we describe the creation of a modified cyanobacterial strain (Synechococcus 2973-T) that is capable of undergoing natural transformation and has a replication time that is on par with the fastest-growing cyanobacterium that has been discovered to date. This new cyanobacterium has the potential to serve as a new model organism for the cyanobacterial research community and will allow experiments to be completed in a fraction of the time that it took to complete previous assays.
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photosynthetic co production of succinate and ethylene in a fast growing cyanobacterium Synechococcus elongatus pcc 11801
Metabolites, 2020Co-Authors: Annesha Sengupta, Himadri B. Pakrasi, Damini Jaiswal, Prem Pritam, Anindita Bandyopadhyay, Pramod P WangikarAbstract:Cyanobacteria are emerging as hosts for photoautotrophic production of chemicals. Recent studies have attempted to stretch the limits of photosynthetic production, typically focusing on one product at a time, possibly to minimise the additional burden of product separation. Here, we explore the simultaneous production of two products that can be easily separated: ethylene, a gaseous product, and succinate, an organic acid that accumulates in the culture medium. This was achieved by expressing a single copy of the ethylene forming enzyme (efe) under the control of PcpcB, the inducer-free super-strong promoter of phycocyanin β subunit. We chose the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801, as the host strain. A stable recombinant strain was constructed using CRISPR-Cpf1 in a first report of markerless genome editing of this cyanobacterium. Under photoautotrophic conditions, the recombinant strain shows specific productivities of 338.26 and 1044.18 μmole/g dry cell weight/h for ethylene and succinate, respectively. These results compare favourably with the reported productivities for individual products in cyanobacteria that are highly engineered. Metabolome profiling and 13C labelling studies indicate carbon flux redistribution and suggest avenues for further improvement. Our results show that S. elongatus PCC 11801 is a promising candidate for metabolic engineering.
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enhanced production of sucrose in the fast growing cyanobacterium Synechococcus elongatus utex 2973
Scientific Reports, 2020Co-Authors: Po-cheng Lin, Fuzhong Zhang, Himadri B. PakrasiAbstract:Cyanobacteria are attractive microbial hosts for production of chemicals using light and CO2. However, their low productivity of chemicals is a major challenge for commercial applications. This is mostly due to their relatively slow growth rate and carbon partitioning toward biomass rather than products. Many cyanobacterial strains synthesize sucrose as an osmoprotectant to cope with salt stress environments. In this study, we harnessed the photosynthetic machinery of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce sucrose under salt stress conditions and investigated if the high efficiency of photosynthesis can enhance the productivity of sucrose. By expressing the sucrose transporter CscB, Synechococcus 2973 produced 8 g L−1 of sucrose with a highest productivity of 1.9 g L−1 day−1 under salt stress conditions. The salt stress activated the sucrose biosynthetic pathway mostly via upregulating the sps gene, which encodes the rate-limiting sucrose-phosphate synthase enzyme. To alleviate the demand on high concentrations of salt for sucrose production, we further overexpressed the sucrose synthesis genes in Synechococcus 2973. The engineered strain produced sucrose with a productivity of 1.1 g L−1 day−1 without the need of salt induction. The engineered Synechococcus 2973 in this study demonstrated the highest productivity of sucrose in cyanobacteria.
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reply to zhou and li plasticity of the genomic haplotype of Synechococcus elongatus leads to rapid strain adaptation under laboratory conditions
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Justin Ungerer, John I. Hendry, Costas D. Maranas, Kristen E Wendt, Himadri B. PakrasiAbstract:Zhou and Li (1) describe a classic phenomenon in microbiology in which the genotypes of bacteria rapidly evolve to optimize growth under selective conditions. In the original paper describing the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973, Yu et al. (2) described the genome sequence that defines the strain. Since 2015, several colleagues who obtained the strain directly from the original Pakrasi laboratory stock successfully replicated the 2-h doubling time of the strain. Seemingly, specific loci affecting growth rate and light tolerance rapidly interconvert between alternative haplotypes based on the growth conditions. This is confirmed by the sequencing results of Zhou and Li (1) who report that the sample in … [↵][1]1To whom correspondence should be addressed. Email: pakrasi{at}wustl.edu. [1]: #xref-corresp-1-1
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Genome-Scale Fluxome of Synechococcus elongatus UTEX 2973 Using Transient 13C-Labeling Data.
Plant physiology, 2018Co-Authors: John I. Hendry, Saratram Gopalakrishnan, Justin Ungerer, Himadri B. Pakrasi, Yinjie J. Tang, Costas D. MaranasAbstract:Synechococcus elongatus UTEX 2973 (Synechococcus 2973) has the shortest reported doubling time (2.1 h) among cyanobacteria, making it a promising platform for the solar-based production of biochemicals. In this meta-analysis, its intracellular flux distribution was recomputed using genome-scale isotopic nonstationary 13C-metabolic flux analysis given the labeling dynamics of 13 metabolites reported in an earlier study. To achieve this, a genome-scale mapping model, namely imSyu593, was constructed using the imSyn617 mapping model for Synechocystis sp. PCC 6803 (Synechocystis 6803) as the starting point encompassing 593 reactions. The flux elucidation revealed nearly complete conversion (greater than 96%) of the assimilated carbon into biomass in Synechococcus 2973. In contrast, Synechocystis 6803 achieves complete conversion of only 86% of the assimilated carbon. This high biomass yield was enabled by the reincorporation of the fixed carbons lost in anabolic and photorespiratory pathways in conjunction with flux rerouting through a nondecarboxylating reaction such as phosphoketolase. This reincorporation of lost CO2 sustains a higher flux through the photorespiratory C2 cycle that fully meets the glycine and serine demands for growth. In accordance with the high carbon efficiency drive, acetyl-coenzyme A was entirely produced using the carbon-efficient phosphoketolase pathway. Comparison of the Synechococcus 2973 flux map with that of Synechocystis 6803 revealed differences in the use of Calvin cycle and photorespiratory pathway reactions. The two species used different reactions for the synthesis of metabolites such as fructose-6-phosphate, glycine, sedoheptulose-7-phosphate, and Ser. These findings allude to a highly carbon-efficient metabolism alongside the fast carbon uptake rate in Synechococcus 2973, which explains its faster growth rate.
Justin Ungerer - One of the best experts on this subject based on the ideXlab platform.
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engineering natural competence into the fast growing cyanobacterium Synechococcus elongatus utex 2973
Applied and Environmental Microbiology, 2021Co-Authors: Kristen E Wendt, Justin Ungerer, Annesha Sengupta, Patricia Walker, Himadri B. PakrasiAbstract:Natural transformation is the process by which bacteria actively take up and integrate extracellular DNA into their genomes. In cyanobacteria, natural transformation has only been experimentally demonstrated in a handful of species. Although, cyanobacteria are important model systems for studying photosynthesis and circadian cycling, natural transformation in cyanobacteria has not been characterized to the degree that the process has been studied in other gram-negative bacteria. Two cyanobacterial species that are 99.8% genetically identical provide a unique opportunity to better understand the nuances of natural transformation in cyanobacteria: Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 7942 and Synechococcus 2973 respectively). Synechococcus 7942 is a naturally transformable model system, while Synechococcus 2973 is a recently discovered species that is not naturally competent. Taking only 1.5 hours to replicate, Synechococcus 2973 is the fastest growing cyanobacterial species known, and thus is a strong candidate for serving as a model organism. However, the organism's inability to undergo natural transformation has prevented it from becoming a widely used model system. By substituting polymorphic alleles from Synechococcus 7942 for native Synechococcus 2973 alleles, natural transformation was introduced into Synechococcus 2973. Two genetic loci were found to be involved in differential natural competence between the two organisms: transformation pilus component pilN and circadian transcriptional master regulator rpaA. By using targeting genome editing and enrichment outgrowth, a strain that was both naturally transformable and fast-growing was created. This new Synechococcus 2973-T strain will serve as a valuable resource to the cyanobacterial research community. Importance Certain bacterial species have the ability to take up naked extracellular DNA and integrate it into their genomes. This process is known as natural transformation and is widely considered to play a major role in bacterial evolution. Because of the ease of introducing new genes into naturally transformable organisms, this capacity is also highly valued in the laboratory. Cyanobacteria are photosynthetic and can therefore serve as model systems for some important aspects of plant physiology. Here, we describe the creation of a modified cyanobacterial strain (Synechococcus 2973-T) that is capable of undergoing natural transformation and has a replication time that is on par with the fastest-growing cyanobacterium that has been discovered to date. This new cyanobacterium has the potential to serve as a new model organism for the cyanobacterial research community and will allow experiments to be completed in a fraction of the time that it took to complete previous assays.
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reply to zhou and li plasticity of the genomic haplotype of Synechococcus elongatus leads to rapid strain adaptation under laboratory conditions
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Justin Ungerer, John I. Hendry, Costas D. Maranas, Kristen E Wendt, Himadri B. PakrasiAbstract:Zhou and Li (1) describe a classic phenomenon in microbiology in which the genotypes of bacteria rapidly evolve to optimize growth under selective conditions. In the original paper describing the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973, Yu et al. (2) described the genome sequence that defines the strain. Since 2015, several colleagues who obtained the strain directly from the original Pakrasi laboratory stock successfully replicated the 2-h doubling time of the strain. Seemingly, specific loci affecting growth rate and light tolerance rapidly interconvert between alternative haplotypes based on the growth conditions. This is confirmed by the sequencing results of Zhou and Li (1) who report that the sample in … [↵][1]1To whom correspondence should be addressed. Email: pakrasi{at}wustl.edu. [1]: #xref-corresp-1-1
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Genome-Scale Fluxome of Synechococcus elongatus UTEX 2973 Using Transient 13C-Labeling Data.
Plant physiology, 2018Co-Authors: John I. Hendry, Saratram Gopalakrishnan, Justin Ungerer, Himadri B. Pakrasi, Yinjie J. Tang, Costas D. MaranasAbstract:Synechococcus elongatus UTEX 2973 (Synechococcus 2973) has the shortest reported doubling time (2.1 h) among cyanobacteria, making it a promising platform for the solar-based production of biochemicals. In this meta-analysis, its intracellular flux distribution was recomputed using genome-scale isotopic nonstationary 13C-metabolic flux analysis given the labeling dynamics of 13 metabolites reported in an earlier study. To achieve this, a genome-scale mapping model, namely imSyu593, was constructed using the imSyn617 mapping model for Synechocystis sp. PCC 6803 (Synechocystis 6803) as the starting point encompassing 593 reactions. The flux elucidation revealed nearly complete conversion (greater than 96%) of the assimilated carbon into biomass in Synechococcus 2973. In contrast, Synechocystis 6803 achieves complete conversion of only 86% of the assimilated carbon. This high biomass yield was enabled by the reincorporation of the fixed carbons lost in anabolic and photorespiratory pathways in conjunction with flux rerouting through a nondecarboxylating reaction such as phosphoketolase. This reincorporation of lost CO2 sustains a higher flux through the photorespiratory C2 cycle that fully meets the glycine and serine demands for growth. In accordance with the high carbon efficiency drive, acetyl-coenzyme A was entirely produced using the carbon-efficient phosphoketolase pathway. Comparison of the Synechococcus 2973 flux map with that of Synechocystis 6803 revealed differences in the use of Calvin cycle and photorespiratory pathway reactions. The two species used different reactions for the synthesis of metabolites such as fructose-6-phosphate, glycine, sedoheptulose-7-phosphate, and Ser. These findings allude to a highly carbon-efficient metabolism alongside the fast carbon uptake rate in Synechococcus 2973, which explains its faster growth rate.
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comparative genomics reveals the molecular determinants of rapid growth of the cyanobacterium Synechococcus elongatus utex 2973
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Justin Ungerer, John I. Hendry, Costas D. Maranas, Kristen E Wendt, Himadri B. PakrasiAbstract:Cyanobacteria are emerging as attractive organisms for sustainable bioproduction. We previously described Synechococcus elongatus UTEX 2973 as the fastest growing cyanobacterium known. Synechococcus 2973 exhibits high light tolerance and an increased photosynthetic rate and produces biomass at three times the rate of its close relative, the model strain Synechococcus elongatus 7942. The two strains differ at 55 genetic loci, and some of these loci must contain the genetic determinants of rapid photoautotrophic growth and improved photosynthetic rate. Using CRISPR/Cpf1, we performed a comprehensive mutational analysis of Synechococcus 2973 and identified three specific genes, atpA, ppnK, and rpaA, with SNPs that confer rapid growth. The fast-growth–associated allele of each gene was then used to replace the wild-type alleles in Synechococcus 7942. Upon incorporation, each allele successively increased the growth rate of Synechococcus 7942; remarkably, inclusion of all three alleles drastically reduced the doubling time from 6.8 to 2.3 hours. Further analysis revealed that our engineering effort doubled the photosynthetic productivity of Synechococcus 7942. We also determined that the fast-growth–associated allele of atpA yielded an ATP synthase with higher specific activity, while that of ppnK encoded a NAD+ kinase with significantly improved kinetics. The rpaA SNPs cause broad changes in the transcriptional profile, as this gene is the master output regulator of the circadian clock. This pioneering study has revealed the molecular basis for rapid growth, demonstrating that limited genetic changes can dramatically improve the growth rate of a microbe by as much as threefold.
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adjustments to photosystem stoichiometry and electron transfer proteins are key to the remarkably fast growth of the cyanobacterium Synechococcus elongatus utex 2973
Mbio, 2018Co-Authors: Justin Ungerer, Po-cheng Lin, Hui-yuan Chen, Himadri B. PakrasiAbstract:At the genome level, Synechococcus elongatus UTEX 2973 (Synechococcus 2973) is nearly identical to the model cyanobacterium Synechococcus elongatus PCC 7942 (Synechococcus 7942) with only 55 single nucleotide differences separating the two strains. Despite the high similarity between the two strains, Synechococcus 2973 grows three times faster, accumulates significantly more glycogen, is tolerant to extremely high light intensities, and displays higher photosynthetic rates. The high homology between the two strains provides a unique opportunity to examine the factors that lead to increased photosynthetic rates. We compared the photophysiology of the two strains and determined the differences in Synechococcus 2973 that lead to increased photosynthetic rates and the concomitant increase in biomass production. In this study, we identified inefficiencies in the electron transport chain of Synechococcus 7942 that have been alleviated in Synechococcus 2973. Photosystem II (PSII) capacity is the same in both strains. However, Synechococcus 2973 exhibits a 1.6-fold increase in PSI content, a 1.5-fold increase in cytochrome b6f content, and a 2.4-fold increase in plastocyanin content on a per cell basis. The increased content of electron carriers allows a higher flux of electrons through the photosynthetic electron transport chain, while the increased PSI content provides more oxidizing power to maintain upstream carriers ready to accept electrons. These changes serve to increase the photosynthetic efficiency of Synechococcus 2973, the fastest growing cyanobacterium known.IMPORTANCE As the global population increases, the amount of arable land continues to decrease. To prevent a looming food crisis, crop productivity per acre must increase. A promising target for improving crop productivity is increasing the photosynthetic rates in crop plants. Cyanobacteria serve as models for higher plant photosynthetic systems and are an important test bed for improvements in photosynthetic productivity. In this study, we identified key factors that lead to improved photosynthetic efficiency and increased production of biomass of a cyanobacterium. We suggest that the findings presented herein will give direction to improvements that may be made in other photosynthetic organisms to improve photosynthetic efficiency.
Susan S. Golden - One of the best experts on this subject based on the ideXlab platform.
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predicting the metabolic capabilities of Synechococcus elongatus pcc 7942 adapted to different light regimes
Metabolic Engineering, 2019Co-Authors: Jared T Broddrick, Susan S. Golden, David G Welkie, Denis Jallet, Graham Peers, Bernhard O PalssonAbstract:There is great interest in engineering photoautotrophic metabolism to generate bioproducts of societal importance. Despite the success in employing genome-scale modeling coupled with flux balance analysis to engineer heterotrophic metabolism, the lack of proper constraints necessary to generate biologically realistic predictions has hindered broad application of this methodology to phototrophic metabolism. Here we describe a methodology for constraining genome-scale models of photoautotrophy in the cyanobacteria Synechococcus elongatus PCC 7942. Experimental photophysiology parameters coupled to genome-scale flux balance analysis resulted in accurate predictions of growth rates and metabolic reaction fluxes at low and high light conditions. Additionally, by constraining photon uptake fluxes, we characterized the metabolic cost of excess excitation energy. The predicted energy fluxes were consistent with known light-adapted phenotypes in cyanobacteria. Finally, we leveraged the modeling framework to characterize existing photoautotrophic and photomixtotrophic engineering strategies for 2,3-butanediol production in S. elongatus. This methodology, applicable to genome-scale modeling of all phototrophic microorganisms, can facilitate the use of flux balance analysis in the engineering of light-driven metabolism.
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the international journeys and aliases of Synechococcus elongatus
New Zealand Journal of Botany, 2019Co-Authors: Susan S. GoldenAbstract:This perspective provides a historical account of the isolation and nomenclature of the cyanobacterial strains currently known as Synechococcus elongatus. The story focuses on an isolate from the San Francisco Bay area of California (Pasteur Culture Collection PCC 7942) that has, for decades, been the genetic model for this species, and its close relative isolated from Waller Creek in Texas (PCC 6301, also known as the University of Texas at Austin Culture Collection of Algae UTEX 625). Until recently, these strains have been the only representatives of the species. A new wild isolate, UTEX 3055, is distinctly different from the prior reference strains. S. elongatus strains have been widely used by labs around the world to discover fundamental cellular processes and to engineer cyanobacteria to generate useful products. The review clarifies relationships among strains that carry different names, and explains how names that appear in the literature have changed over the years.
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quantification of chlorophyll as a proxy for biofilm formation in the cyanobacterium Synechococcus elongatus
Bio-protocol, 2017Co-Authors: Eleonora Sendersky, Ryan Simkovsky, Susan S. Golden, Rakefet SchwarzAbstract:A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation in sessile and planktonic cells of biofilm-forming strains. These measurements allow quantification of the total biomass as estimated by chlorophyll level and representation of the extent of biofilm formation by depicting the relative fraction of chlorophyll in planktonic cells.
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type 4 pili are dispensable for biofilm development in the cyanobacterium Synechococcus elongatus
Environmental Microbiology, 2017Co-Authors: Elad Nagar, Ryan Simkovsky, Eleonora Sendersky, Susan S. Golden, Shaul Zilberman, Eyal Shimoni, Diana Gershtein, Moshe Herzberg, Rakefet SchwarzAbstract:Summary The hair-like cell appendages denoted as type IV pili are crucial for biofilm formation in diverse eubacteria. The protein complex responsible for type IV pilus assembly is homologous with the type II protein secretion complex. In the cyanobacterium Synechococcus elongatus PCC 7942, the gene Synpcc7942_2071 encodes an ATPase homologue of type II/type IV systems. Here we report that inactivation of Synpcc7942_2071 strongly affected the suite of proteins present in the extracellular milieu (exo-proteome) and eliminated pili observable by electron microscopy. These results support a role for this gene product in protein secretion as well as in pili formation. As we previously reported, inactivation of Synpcc7942_2071 enables biofilm formation and suppresses the planktonic growth of S. elongatus. Thus, pili are dispensable for biofilm development in this cyanobacterium, in contrast to their biofilm-promoting function in type IV pili-producing heterotrophic bacteria. Nevertheless, pili removal is not required for biofilm formation as evident by a piliated mutant of S. elongatus that develops biofilms. We show that adhesion and timing of biofilm development differ between the piliated and non-piliated strains. The study demonstrates key differences in the process of biofilm formation between cyanobacteria and well-studied type IV pili-producing heterotrophic bacteria. This article is protected by copyright. All rights reserved.
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Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus
Scientific reports, 2016Co-Authors: Rami Parnasa, Ryan Simkovsky, Elad Nagar, Eleonora Sendersky, Ziv Reich, Susan S. Golden, Rakefet SchwarzAbstract:Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms.
Kristen E Wendt - One of the best experts on this subject based on the ideXlab platform.
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engineering natural competence into the fast growing cyanobacterium Synechococcus elongatus utex 2973
Applied and Environmental Microbiology, 2021Co-Authors: Kristen E Wendt, Justin Ungerer, Annesha Sengupta, Patricia Walker, Himadri B. PakrasiAbstract:Natural transformation is the process by which bacteria actively take up and integrate extracellular DNA into their genomes. In cyanobacteria, natural transformation has only been experimentally demonstrated in a handful of species. Although, cyanobacteria are important model systems for studying photosynthesis and circadian cycling, natural transformation in cyanobacteria has not been characterized to the degree that the process has been studied in other gram-negative bacteria. Two cyanobacterial species that are 99.8% genetically identical provide a unique opportunity to better understand the nuances of natural transformation in cyanobacteria: Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 7942 and Synechococcus 2973 respectively). Synechococcus 7942 is a naturally transformable model system, while Synechococcus 2973 is a recently discovered species that is not naturally competent. Taking only 1.5 hours to replicate, Synechococcus 2973 is the fastest growing cyanobacterial species known, and thus is a strong candidate for serving as a model organism. However, the organism's inability to undergo natural transformation has prevented it from becoming a widely used model system. By substituting polymorphic alleles from Synechococcus 7942 for native Synechococcus 2973 alleles, natural transformation was introduced into Synechococcus 2973. Two genetic loci were found to be involved in differential natural competence between the two organisms: transformation pilus component pilN and circadian transcriptional master regulator rpaA. By using targeting genome editing and enrichment outgrowth, a strain that was both naturally transformable and fast-growing was created. This new Synechococcus 2973-T strain will serve as a valuable resource to the cyanobacterial research community. Importance Certain bacterial species have the ability to take up naked extracellular DNA and integrate it into their genomes. This process is known as natural transformation and is widely considered to play a major role in bacterial evolution. Because of the ease of introducing new genes into naturally transformable organisms, this capacity is also highly valued in the laboratory. Cyanobacteria are photosynthetic and can therefore serve as model systems for some important aspects of plant physiology. Here, we describe the creation of a modified cyanobacterial strain (Synechococcus 2973-T) that is capable of undergoing natural transformation and has a replication time that is on par with the fastest-growing cyanobacterium that has been discovered to date. This new cyanobacterium has the potential to serve as a new model organism for the cyanobacterial research community and will allow experiments to be completed in a fraction of the time that it took to complete previous assays.
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reply to zhou and li plasticity of the genomic haplotype of Synechococcus elongatus leads to rapid strain adaptation under laboratory conditions
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Justin Ungerer, John I. Hendry, Costas D. Maranas, Kristen E Wendt, Himadri B. PakrasiAbstract:Zhou and Li (1) describe a classic phenomenon in microbiology in which the genotypes of bacteria rapidly evolve to optimize growth under selective conditions. In the original paper describing the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973, Yu et al. (2) described the genome sequence that defines the strain. Since 2015, several colleagues who obtained the strain directly from the original Pakrasi laboratory stock successfully replicated the 2-h doubling time of the strain. Seemingly, specific loci affecting growth rate and light tolerance rapidly interconvert between alternative haplotypes based on the growth conditions. This is confirmed by the sequencing results of Zhou and Li (1) who report that the sample in … [↵][1]1To whom correspondence should be addressed. Email: pakrasi{at}wustl.edu. [1]: #xref-corresp-1-1
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comparative genomics reveals the molecular determinants of rapid growth of the cyanobacterium Synechococcus elongatus utex 2973
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Justin Ungerer, John I. Hendry, Costas D. Maranas, Kristen E Wendt, Himadri B. PakrasiAbstract:Cyanobacteria are emerging as attractive organisms for sustainable bioproduction. We previously described Synechococcus elongatus UTEX 2973 as the fastest growing cyanobacterium known. Synechococcus 2973 exhibits high light tolerance and an increased photosynthetic rate and produces biomass at three times the rate of its close relative, the model strain Synechococcus elongatus 7942. The two strains differ at 55 genetic loci, and some of these loci must contain the genetic determinants of rapid photoautotrophic growth and improved photosynthetic rate. Using CRISPR/Cpf1, we performed a comprehensive mutational analysis of Synechococcus 2973 and identified three specific genes, atpA, ppnK, and rpaA, with SNPs that confer rapid growth. The fast-growth–associated allele of each gene was then used to replace the wild-type alleles in Synechococcus 7942. Upon incorporation, each allele successively increased the growth rate of Synechococcus 7942; remarkably, inclusion of all three alleles drastically reduced the doubling time from 6.8 to 2.3 hours. Further analysis revealed that our engineering effort doubled the photosynthetic productivity of Synechococcus 7942. We also determined that the fast-growth–associated allele of atpA yielded an ATP synthase with higher specific activity, while that of ppnK encoded a NAD+ kinase with significantly improved kinetics. The rpaA SNPs cause broad changes in the transcriptional profile, as this gene is the master output regulator of the circadian clock. This pioneering study has revealed the molecular basis for rapid growth, demonstrating that limited genetic changes can dramatically improve the growth rate of a microbe by as much as threefold.
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crispr cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus utex 2973
Microbial Cell Factories, 2016Co-Authors: Kristen E Wendt, Justin Ungerer, Ryan E Cobb, Huimin Zhao, Himadri B. PakrasiAbstract:As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions. First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructs containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain. High expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.
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crispr cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus utex 2973
Microbial Cell Factories, 2016Co-Authors: Kristen E Wendt, Justin Ungerer, Ryan E Cobb, Huimin Zhao, Himadri B. PakrasiAbstract:As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions. First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructs containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain. High expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.
Xiaoming Tan - One of the best experts on this subject based on the ideXlab platform.
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a specific single nucleotide polymorphism in the atp synthase gene significantly improves environmental stress tolerance of Synechococcus elongatus pcc 7942
Applied and Environmental Microbiology, 2018Co-Authors: Wenjing Lou, Kuo Song, Xiaoming Tan, Shanshan Zhang, Guodong LuanAbstract:In response to a broad range of habitats and environmental stresses, cyanobacteria have evolved various effective acclimation strategies, which will be helpful for improving the stress tolerances of photosynthetic organisms, including higher plants. Synechococcus elongatus UTEX 2973 and PCC 7942 possess genomes that are 99.8% identical but exhibit significant differences in cell growth and stress tolerance. In this study, we found that a single amino acid substitution at FoF1 ATP synthase subunit α (AtpA), C252Y, is the primary contributor to the improved stress tolerance of S. elongatus UTEX 2973. Site-saturation mutagenesis experiments showed that point mutations of cysteine 252 to any of the four conjugated amino acids could significantly improve the stress tolerance of S. elongatus PCC 7942. We further confirmed that the C252Y mutation increases AtpA protein levels, intracellular ATP synthase activity, intracellular ATP abundance, transcription of psbA genes (especially psbA2), photosystem II activity, and glycogen accumulation in S. elongatus PCC 7942. This work highlights the importance of AtpA in improving the stress tolerance of cyanobacteria and provides insight into how cyanobacteria evolve via point mutations in the face of environmental selection pressures.IMPORTANCE Two closely related Synechococcus strains showed significantly different tolerances to high light and high temperature but limited genomic differences, providing us opportunities to identify key genes responsible for stress acclimation by a gene complementation approach. In this study, we confirmed that a single point mutation in the α subunit of FoF1 ATP synthase (AtpA) contributes mainly to the improved stress tolerance of Synechococcus elongatus UTEX 2973. The point mutation of AtpA, the important ATP-generating complex of photosynthesis, increases AtpA protein levels, intracellular ATP synthase activity, and ATP concentrations under heat stress, as well as photosystem II activity. This work proves the importance of ATP synthase in cyanobacterial stress acclimation and provides a good target for future improvement of cyanobacterial stress tolerance by metabolic engineering.
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the primary transcriptome of the fast growing cyanobacterium Synechococcus elongatus utex 2973
Biotechnology for Biofuels, 2018Co-Authors: Kuo Song, Xiaoming Tan, Shengwei Hou, Jens Georg, Stephan Klähn, Wolfgang R. HessAbstract:Cyanobacteria have shown promising potential for the production of various biofuels and chemical feedstocks. Synechococcus elongatus UTEX 2973 is a fast-growing strain with pronounced tolerance to high temperatures and illumination. Hence, this strain appears to be ideal for the development of photosynthetic biotechnology. However, molecular insights on how this strain can rapidly accumulate biomass and carbohydrates under high-light and high-temperature conditions are lacking. Differential RNA-Sequencing (dRNA-Seq) enabled the genome-wide identification of 4808 transcription start sites (TSSs) in S. elongatus UTEX 2973 using a background reduction algorithm. High light promoted the transcription of genes associated with central metabolic pathways, whereas the highly induced small RNA (sRNA) PsrR1 likely contributed to the repression of phycobilisome genes and the accelerated glycogen accumulation rates measured under this condition. Darkness caused transcriptome remodeling with a decline in the expression of genes for carbon fixation and other major metabolic pathways and an increase in the expression of genes for glycogen catabolism and Calvin cycle inhibitor CP12. Two of the identified TSSs drive the transcription of highly abundant sRNAs in darkness. One of them is widely conserved throughout the cyanobacterial phylum. Its gene is fused to a protein-coding gene in some species, illustrating the evolutionary origin of sRNAs from an mRNA 3′-end. Our comprehensive set of genome-wide mapped TSSs, sRNAs and promoter activities will be valuable for projects requiring precise information about the control of transcription aimed at metabolic engineering and the elucidation of stress acclimation mechanisms in this promising strain.
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The primary transcriptome of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973
BMC, 2018Co-Authors: Xiaoming Tan, Kuo Song, Shengwei Hou, Jens Georg, Stephan Klähn, Wolfgang R. HessAbstract:Abstract Background Cyanobacteria have shown promising potential for the production of various biofuels and chemical feedstocks. Synechococcus elongatus UTEX 2973 is a fast-growing strain with pronounced tolerance to high temperatures and illumination. Hence, this strain appears to be ideal for the development of photosynthetic biotechnology. However, molecular insights on how this strain can rapidly accumulate biomass and carbohydrates under high-light and high-temperature conditions are lacking. Results Differential RNA-Sequencing (dRNA-Seq) enabled the genome-wide identification of 4808 transcription start sites (TSSs) in S. elongatus UTEX 2973 using a background reduction algorithm. High light promoted the transcription of genes associated with central metabolic pathways, whereas the highly induced small RNA (sRNA) PsrR1 likely contributed to the repression of phycobilisome genes and the accelerated glycogen accumulation rates measured under this condition. Darkness caused transcriptome remodeling with a decline in the expression of genes for carbon fixation and other major metabolic pathways and an increase in the expression of genes for glycogen catabolism and Calvin cycle inhibitor CP12. Two of the identified TSSs drive the transcription of highly abundant sRNAs in darkness. One of them is widely conserved throughout the cyanobacterial phylum. Its gene is fused to a protein-coding gene in some species, illustrating the evolutionary origin of sRNAs from an mRNA 3′-end. Conclusions Our comprehensive set of genome-wide mapped TSSs, sRNAs and promoter activities will be valuable for projects requiring precise information about the control of transcription aimed at metabolic engineering and the elucidation of stress acclimation mechanisms in this promising strain
