CRISPR/Cas Technologies and Their Applications in Escherichia coli

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have revolutionized genome editing and greatly promoted the development of biotechnology. However, these systems unfortunately have not been developed and applied in bacteria as extensively as in eukaryotic organism. Here, the research progress on the most widely used CRISPR/Cas tools and their applications in Escherichia coli is summarized. Genome editing based on homologous recombination, non-homologous DNA end-joining, transposons, and base editors are discussed. Finally, the state of the art of transcriptional regulation using CRISPRi is briefly reviewed. This review provides a useful reference for the application of CRISPR/Cas systems in other bacterial species.

Translational control of lipogenesis links protein synthesis and phosphoinositide signaling with nuclear division in Saccharomyces cerevisiae

Continuously dividing cells coordinate their growth and division. How fast cells grow in mass determines how fast they will multiply. Yet, there are few, if any, examples of a metabolic pathway that actively drives a cell cycle event instead of just being required for it. Here, we show that translational upregulation of lipogenic enzymes in Saccharomyces cerevisiae increased the abundance of lipids and promoted nuclear elongation and division. De-repressing translation of acetyl CoA carboxylase and fatty acid synthase also suppressed cell cycle-related phenotypes, including delayed nuclear division, associated with Sec14p phosphatidylinositol transfer protein deficiencies, and the irregular nuclear morphologies of mutants defective in phosphatidylinositol 4-OH kinase activities. Our results show that increased lipogenesis drives a critical cell cycle landmark and report a phosphoinositide signaling axis in control of nuclear division. The broad conservation of these lipid metabolic and signaling pathways raises the possibility these activities similarly govern nuclear division in other eukaryotes. In this report, the authors show that increasing lipid synthesis promotes the division of the nucleus in yeast cells, a model eukaryotic organism. They also implicate phosphoinositide signaling in the control of nuclear division. Because lipid metabolic and signaling pathways are highly conserved, it is possible that these activities also control nuclear division in other organisms. AUTHOR SUMMARY In this report, the authors show that increasing lipid synthesis promotes the division of the nucleus in yeast cells, a model eukaryotic organism. They also implicate phosphoinositide signaling in the control of nuclear division. Because lipid metabolic and signaling pathways are highly conserved, it is possible that these activities also control nuclear division in other organisms.

Giardia duodenalis: Biology and Pathogenesis

Giardia duodenalis captured the attention of Leeuwenhoek in 1681 while he was examining his own diarrheal stool, but, ironically, it did not really gain attention as a human pathogen until the 1960s, when outbreaks were reported. Key technological advances, including in vitro cultivation, genomic and proteomic databases, and advances in microscopic and molecular approaches, have led to understand that this is a eukaryotic organism with a reduced genome rather than a truly premitochondriate eukaryote.

Identification and Characterization of Genes Encoding the Hydroxypyruvate Reductases in Chlamydomonas Reveal Their Distinct Roles in Photorespiration

Photorespiration plays an important role in maintaining normal physiological metabolism in higher plants and other oxygenic organisms, such as algae. The unicellular eukaryotic organism Chlamydomonas is reported to have a photorespiration system different from that in higher plants, and only two out of nine genes encoding photorespiratory enzymes have been experimentally characterized. Hydroxypyruvate reductase (HPR), which is responsible for the conversion of hydroxypyruvate into glycerate, is poorly understood and not yet explored in Chlamydomonas. To identify the candidate genes encoding hydroxypyruvate reductases in Chlamydomonas (CrHPR) and uncover their elusive functions, we performed sequence comparison, enzyme activity measurement, subcellular localization, and analysis of knockout/knockdown strains. Together, we identify five proteins to be good candidates for CrHPRs, all of which are detected with the activity of hydroxypyruvate reductase. CrHPR1, a nicotinamide adenine dinucleotide (NADH)-dependent enzyme in mitochondria, may function as the major component of photorespiration. Its deletion causes severe photorespiratory defects. CrHPR2 takes part in the cytosolic bypass of photorespiration as the compensatory pathway of CrHPR1 for the reduction of hydroxypyruvate. CrHPR4, with NADH as the cofactor, may participate in photorespiration by acting as the chloroplastidial glyoxylate reductase in glycolate-quinone oxidoreductase system. Therefore, the results reveal that CrHPRs are far more complex than previously recognized and provide a greatly expanded knowledge base for studies to understand how CrHPRs perform their functions in photorespiration. These will facilitate both modification of photorespiration and genetic engineering for crop improvement by synthetic biology.

Integrative analysis of the hydroxypyruvate reductases revealing their distinct roles in photorespiration of Chlamydomonas

Photorespiration plays an important role in maintaining normal physiological metabolism in higher plants and other oxygenic organisms such as algae. The unicellular eukaryotic organism Chlamydomonas is reported to have a different photorespiration system from that in higher plants, and only two out of nine genes encoding photorespiratory enzymes have been experimentally characterized. Hydroxypyruvate reductase (HPR), which is responsible for the conversion of hydroxypyruvate into glycerate, is poorly understood and not yet explored in Chlamydomonas. To identify the candidate genes encoding hydroxypyruvate reductase in Chlamydomonas (CrHPR) and uncover their elusive functions, we performed sequence comparison, enzyme activity measurement, subcellular localization, and analysis of knockout/knockdown strains. Together we identify five proteins to be good candidates as CrHPRs, all of which are detected with the activity of hydroxypyruvate reductase. CrHPR1, a NADH-dependent enzyme in mitochondria, may function as the major component of photorespiration, and deletion of CrHPR1 causes severe photorespiratory defects. CrHPR2 takes parts in the cytosolic bypass of photorespiration as the compensatory pathway of CrHPR1 for the reduction of hydroxypyruvate. CrHPR4, with NADH as the cofactor, may participate in photorespiration by acting as the chloroplastidial glyoxylate reductase in glycolate-quinone oxidoreductase system. Therefore, our results reveal that the CrHPRs are far more complex than previously recognized, and provide a greatly expanded knowledge base for studies to understand how CrHPRs perform their functions in photorespiration. These will facilitate the genetic engineering for crop improvement by synthetic biology.

Saccharomyces: Is a Necessary Organism or a Biological Warrior?

Saccharomyces is a eukaryotic organism that possesses approximately 6,000 known genes since 1996. It has long been used for food, bakeries, drinks, and therapeutics due to its many ingredients and its role in several mechanisms. Saccharomyces can be used as an experimental organism for medicinal products in the pharmaceutical industry. Particularly in public health, the use of Saccharomyces in the production of vaccines is remarkable. It has been alleviated that this yeast helps clarify the function of individual proteins in pathogenic viruses. To clarify virus life and host interactions, virus replication systems in Saccharomyces were interested in scientists. The new antiviral strategies with yeasts suggest the biological mechanism of a pathogen virus. Due to the variety of diseases and current epidemic conditions, these organisms play an essential role in prevention and treatment. This chapter will try to update Saccharomyces’ scientific discoveries with the most recent and up-to-date literature.

The Power of Yeast in Modelling Human Nuclear Mutations Associated with Mitochondrial Diseases

The increasing application of next generation sequencing approaches to the analysis of human exome and whole genome data has enabled the identification of novel variants and new genes involved in mitochondrial diseases. The ability of surviving in the absence of oxidative phosphorylation (OXPHOS) and mitochondrial genome makes the yeast Saccharomyces cerevisiae an excellent model system for investigating the role of these new variants in mitochondrial-related conditions and dissecting the molecular mechanisms associated with these diseases. The aim of this review was to highlight the main advantages offered by this model for the study of mitochondrial diseases, from the validation and characterisation of novel mutations to the dissection of the role played by genes in mitochondrial functionality and the discovery of potential therapeutic molecules. The review also provides a summary of the main contributions to the understanding of mitochondrial diseases emerged from the study of this simple eukaryotic organism.

Gas vesicle protein expression for reconstitution of synthetic gas vesicles in yeast

Given the potential applications of gas vesicles (GVs) in multiple fields including antigen-displaying and imaging, heterologous reconstitution of synthetic GVs is an attractive and interesting study that has translational potential. Here, we attempted to express and assemble GV proteins (GVPs) into GVs using the model eukaryotic organism Saccharomyces cerevisiae. We first selected and expressed two core structural proteins, GvpA and GvpC from cyanobacteria Anabaena flos-aquae and Planktothrix rubescens, respectively. We then optimized the protein expression conditions and validated GV assembly in the context of cell flotation and GV shapes. We found that when two copies of AnaA were integrated into the genome, it resulted in cell floatation and GV production regardless of GvpC expression. Next, we co-expressed chaperone-RFP with the GFP-AnaA to aid the AnaA aggregation. The co-expression of individual chaperones (Hsp42, Sis1, Hsp104, and GvpN) with AnaA led to the formation of larger inclusions and enhanced the sequestration of AnaA into the perivacuolar site. To our knowledge, this represents the first study on reconstitution of GVs in S. cerevisiae. Our results could provide insights into optimizing conditions for heterologous protein expressions as well as the reconstitution of other synthetic microcompartments in yeast.

Plants with self-sustained luminescence

In contrast to fluorescent proteins, light emission from luciferase reporters requires exogenous addition of a luciferin substrate. Bacterial bioluminescence has been the single exception, where an operon of five genes is sufficient to produce light autonomously. Although commonly used in prokaryotic hosts, toxicity of the aldehyde substrate has limited its use in eukaryotes1. Here we demonstrate autonomous luminescence in a multicellular eukaryotic organism by incorporating a recently discovered fungal bioluminescent system2 into tobacco plants. We monitored these light-emitting plants from germination to flowering, observing temporal and spatial patterns of luminescence across time scales from seconds to months. The dynamic patterns of luminescence reflected progression through developmental stages, circadian oscillations, transport, and response to injuries. As with other fluorescent and luminescent reporters, we anticipate that this system will be further engineered for varied purposes, especially where exogenous addition of substrate is undesirable.