Functional diversification within the heme-binding split-barrel family.

Due to neofunctionalization, a single fold can be identified in multiple proteins that have distinct molecular functions. Depending on the time that has passed since gene duplication and the number of mutations, the sequence similarity between functionally divergent proteins can be relatively high, eroding the value of sequence similarity as the sole tool for accurately annotating the function of uncharacterized homologs. Here, we combine bioinformatic approaches with targeted experimentation to reveal a large multifunctional family of putative enzymatic and nonenzymatic proteins involved in heme metabolism.

Biochemical characterization of Fsa16295Glu from the first hyperthermophilic GH50 with β-1,3-endoglucanase activity and founding member of the subfamily GH50_3.

The aerobic hyperthermophile catabolizes diverse polysaccharides and is the only cultivated member of the class within the phylum . It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu.

A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site.

A predicted CRISPR-mediated symbiosis between uncultivated archaea.

CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca.

Structural characterization of a soil viral auxiliary metabolic gene product - a functional chitosanase.

Metagenomics is unearthing the previously hidden world of soil viruses. Many soil viral sequences in metagenomes contain putative auxiliary metabolic genes (AMGs) that are not associated with viral replication. Here, we establish that AMGs on soil viruses actually produce functional, active proteins.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1).

Building a custom high-throughput platform at the Joint Genome Institute for DNA construct design and assembly-present and future challenges.

The rapid design and assembly of synthetic DNA constructs have become a crucial component of biological engineering projects via iterative design-build-test-learn cycles. In this perspective, we provide an overview of the workflows used to generate the thousands of constructs and libraries produced each year at the U.S.

Plant single-cell solutions for energy and the environment.

Progress in sequencing, microfluidics, and analysis strategies has revolutionized the granularity at which multicellular organisms can be studied. In particular, single-cell transcriptomics has led to fundamental new insights into animal biology, such as the discovery of new cell types and cell type-specific disease processes. However, the application of single-cell approaches to plants, fungi, algae, or bacteria (environmental organisms) has been far more limited, largely due to the challenges posed by polysaccharide walls surrounding these species' cells.

Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems.

The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.

ManiNetCluster: a novel manifold learning approach to reveal the functional links between gene networks.

Background: The coordination of genomic functions is a critical and complex process across biological systems such as phenotypes or states (e.g., time, disease, organism, environmental perturbation).

Gene Expression Analysis by Arylsulfatase Assays in the Green Alga Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a single-celled green alga, is a powerful microbial experimental system for understanding gene function. As a consequence of a high-quality genome sequence, community-wide efforts for gene model refinement and annotation, resources for strain collections and robust molecular techniques, research with this organism has significantly expanded in the past few decades. In two companion chapters, we outline colorimetric and fluorescence-based methodologies for genetic reporter systems in Chlamydomonas, which can be used to investigate and delineate gene expression and regulatory mechanisms.

Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea.

Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in in minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea.

Deep Learning in Label-free Cell Classification.

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput.

Genome-wide analysis on Chlamydomonas reinhardtii reveals the impact of hydrogen peroxide on protein stress responses and overlap with other stress transcriptomes.

Reactive oxygen species (ROS) are produced by and have the potential to be damaging to all aerobic organisms. In photosynthetic organisms, they are an unavoidable byproduct of electron transfer in both the chloroplast and mitochondrion. Here, we employ the reference unicellular green alga Chlamydomonas reinhardtii to identify the effect of H2O2 on gene expression by monitoring the changes in the transcriptome in a time-course experiment.

High-Resolution Profiling of a Synchronized Diurnal Transcriptome from Chlamydomonas reinhardtii Reveals Continuous Cell and Metabolic Differentiation.

The green alga Chlamydomonas reinhardtii is a useful model organism for investigating diverse biological processes, such as photosynthesis and chloroplast biogenesis, flagella and basal body structure/function, cell growth and division, and many others. We combined a highly synchronous photobioreactor culture system with frequent temporal sampling to characterize genome-wide diurnal gene expression in Chlamydomonas. Over 80% of the measured transcriptome was expressed with strong periodicity, forming 18 major clusters.

Activation of Autophagy by Metals in Chlamydomonas reinhardtii.

Autophagy is an intracellular self-degradation pathway by which eukaryotic cells recycle their own material in response to specific stress conditions. Exposure to high concentrations of metals causes cell damage, although the effect of metal stress on autophagy has not been explored in photosynthetic organisms. In this study, we investigated the effect of metal excess on autophagy in the model unicellular green alga Chlamydomonas reinhardtii.

Exploiting algal NADPH oxidase for biophotovoltaic energy.

Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii.

The Chlamydomonas genome project: a decade on.

The green alga Chlamydomonas reinhardtii is a popular unicellular organism for studying photosynthesis, cilia biogenesis, and micronutrient homeostasis. Ten years since its genome project was initiated an iterative process of improvements to the genome and gene predictions has propelled this organism to the forefront of the omics era. Housed at Phytozome, the plant genomics portal of the Joint Genome Institute (JGI), the most up-to-date genomic data include a genome arranged on chromosomes and high-quality gene models with alternative splice forms supported by an abundance of whole transcriptome sequencing (RNA-Seq) data.

Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism.

Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration.

Systems-level analysis of nitrogen starvation-induced modifications of carbon metabolism in a Chlamydomonas reinhardtii starchless mutant.

To understand the molecular basis underlying increased triacylglycerol (TAG) accumulation in starchless (sta) Chlamydomonas reinhardtii mutants, we undertook comparative time-course transcriptomics of strains CC-4348 (sta6 mutant), CC-4349, a cell wall-deficient (cw) strain purported to represent the parental STA6 strain, and three independent STA6 strains generated by complementation of sta6 (CC-4565/STA6-C2, CC-4566/STA6-C4, and CC-4567/STA6-C6) in the context of N deprivation. Despite N starvation-induced dramatic remodeling of the transcriptome, there were relatively few differences (5 × 10(2)) observed between sta6 and STA6, the most dramatic of which were increased abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically isocitrate lyase, malate synthase, transaldolase, fructose bisphosphatase and phosphoenolpyruvate carboxykinase (encoded by ICL1, MAS1, TAL1, FBP1, and PCK1 respectively), suggestive of increased carbon movement toward hexose-phosphate in sta6 by upregulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities.

Quinolinate salvage and insights for targeting NAD biosynthesis in group A streptococci.

The essential coenzyme NAD plays important roles in metabolic reactions and cell regulation in all organisms. As such, NAD synthesis has been investigated as a source for novel antibacterial targets. Cross-species genomics-based reconstructions of NAD metabolism in group A streptococci (GAS), combined with focused experimental testing in Streptococcus pyogenes, led to a better understanding of NAD metabolism in the pathogen.

Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas.

Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression.

The archaeal COG1901/DUF358 SPOUT-methyltransferase members, together with pseudouridine synthase Pus10, catalyze the formation of 1-methylpseudouridine at position 54 of tRNA.

The methylation of pseudouridine (Ψ) at position 54 of tRNA, producing m(1)Ψ, is a hallmark of many archaeal species, but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome.