scholarly journals Thiamine metabolism genes in diatoms are not regulated by thiamine despite the presence of predicted riboswitches

2022 ◽  
Author(s):  
Marcel Llavero-Pasquina ◽  
Katrin Geisler ◽  
Andre Holzer ◽  
Payam Mehrshahi ◽  
Gonzalo I Mendoza-Ochoa ◽  
...  
Keyword(s):  
Primary Production ◽  
Microbial Communities ◽  
Phaeodactylum Tricornutum ◽  
Gene Editing ◽  
Targeted Mutagenesis ◽  
Thiamine Pyrophosphate ◽  
Thiamine Metabolism ◽  
Targeted Mutation ◽  
Marine Microbial Communities ◽  
Thiamine Biosynthesis

Thiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for approximately 20% of global primary production. They have been predicted to contain TPP aptamers in the 3'UTR of some thiamine metabolism-related genes, but little is known about their function and regulation. We used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum. We found that thiamine-related genes with putative TPP aptamers are not responsive to thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and the targeted mutation of the TPP aptamer in the HMP-P synthase (THIC) does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtSSSP is necessary for thiamine uptake and that PtTHIC is essential for thiamine biosynthesis. Our results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance.

scholarly journals A Novel Transcriptional Regulator Related to Thiamine Phosphate Synthase Controls Thiamine Metabolism Genes in Archaea

2016 ◽  
Vol 199 (4) ◽  
Author(s):  
Dmitry A. Rodionov ◽  
Semen A. Leyn ◽  
Xiaoqing Li ◽  
Irina A. Rodionova
Keyword(s):  
Comparative Genomics ◽  
Dna Binding ◽  
Transcriptional Control ◽  
Binding Domain ◽  
Thiamine Pyrophosphate ◽  
Vitamin B ◽  
Binding Assays ◽  
Content Type ◽  
Thiamine Metabolism ◽  
Thiamine Biosynthesis

ABSTRACT Thiamine (vitamin B1) is a precursor of thiamine pyrophosphate (TPP), an essential coenzyme in the central metabolism of all living organisms. Bacterial thiamine biosynthesis and salvage genes are controlled at the RNA level by TPP-responsive riboswitches. In Archaea, TPP riboswitches are restricted to the Thermoplasmatales order. Mechanisms of transcriptional control of thiamine genes in other archaeal lineages remain unknown. Using the comparative genomics approach, we identified a novel family of transcriptional regulators (named ThiR) controlling thiamine biosynthesis and transport genes in diverse lineages in the Crenarchaeota phylum as well as in the Halobacteria and Thermococci classes of the Euryarchaeota. ThiR regulators are composed of an N-terminal DNA-binding domain and a C-terminal ligand-binding domain, which is similar to the archaeal thiamine phosphate synthase ThiN. By using comparative genomics, we predicted ThiR-binding DNA motifs and reconstructed ThiR regulons in 67 genomes representing all above-mentioned lineages. The predicted ThiR-binding motifs are characterized by palindromic symmetry with several distinct lineage-specific consensus sequences. In addition to thiamine biosynthesis genes, the reconstructed ThiR regulons include various transporters for thiamine and its precursors. Bioinformatics predictions were experimentally validated by in vitro DNA-binding assays with the recombinant ThiR protein from the hyperthermophilic archaeon Metallosphaera yellowstonensis MK1. Thiamine phosphate and, to some extent, TPP and hydroxyethylthiazole phosphate were required for the binding of ThiR to its DNA targets, suggesting that ThiR is derepressed by limitation of thiamine phosphates. The thiamine phosphate-binding residues previously identified in ThiN are highly conserved in ThiR regulators, suggesting a conserved mechanism for effector recognition. IMPORTANCE Thiamine pyrophosphate is a cofactor for many essential enzymes for glucose and energy metabolism. Thiamine or vitamin B1 biosynthesis and its transcriptional regulation in Archaea are poorly understood. We applied the comparative genomics approach to identify a novel family of regulators for the transcriptional control of thiamine metabolism genes in Archaea and reconstructed the respective regulons. The predicted ThiR regulons in archaeal genomes control the majority of thiamine biosynthesis genes. The reconstructed regulon content suggests that numerous uptake transporters for thiamine and/or its precursors are encoded in archaeal genomes. The ThiR regulon was experimentally validated by DNA-binding assays with Metallosphaera spp. These discoveries contribute to our understanding of metabolic and regulatory networks involved in vitamin homeostasis in diverse lineages of Archaea.

Enhanced CO2 concentrations change the structure of Antarctic marine microbial communities

2016 ◽  
Vol 552 ◽  
pp. 93-113 ◽  
Author(s):  
AT Davidson ◽  
J McKinlay ◽  
K Westwood ◽  
PG Thomson ◽  
R van den Enden ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Co2 Concentrations ◽  
Antarctic Marine ◽  
Marine Microbial Communities

scholarly journals Development of a time-series shotgun metagenomics database for monitoring microbial communities at the Pacific coast of Japan

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kazutoshi Yoshitake ◽  
Gaku Kimura ◽  
Tomoko Sakami ◽  
Tsuyoshi Watanabe ◽  
Yukiko Taniuchi ◽  
...  
Keyword(s):  
Time Series ◽  
Microbial Communities ◽  
Pacific Coast ◽  
Three Dimensional ◽  
Amplicon Sequencing ◽  
Metagenomic Data ◽  
Shotgun Metagenomics ◽  
Functional Features ◽  
Pacific Coast Of Japan ◽  
Marine Microbial Communities

AbstractAlthough numerous metagenome, amplicon sequencing-based studies have been conducted to date to characterize marine microbial communities, relatively few have employed full metagenome shotgun sequencing to obtain a broader picture of the functional features of these marine microbial communities. Moreover, most of these studies only performed sporadic sampling, which is insufficient to understand an ecosystem comprehensively. In this study, we regularly conducted seawater sampling along the northeastern Pacific coast of Japan between March 2012 and May 2016. We collected 213 seawater samples and prepared size-based fractions to generate 454 subsets of samples for shotgun metagenome sequencing and analysis. We also determined the sequences of 16S rRNA (n = 111) and 18S rRNA (n = 47) gene amplicons from smaller sample subsets. We thereafter developed the Ocean Monitoring Database for time-series metagenomic data (http://marine-meta.healthscience.sci.waseda.ac.jp/omd/), which provides a three-dimensional bird’s-eye view of the data. This database includes results of digital DNA chip analysis, a novel method for estimating ocean characteristics such as water temperature from metagenomic data. Furthermore, we developed a novel classification method that includes more information about viruses than that acquired using BLAST. We further report the discovery of a large number of previously overlooked (TAG)n repeat sequences in the genomes of marine microbes. We predict that the availability of this time-series database will lead to major discoveries in marine microbiome research.

scholarly journals Identification and functional analysis of novel SLC25A19 variants causing thiamine metabolism dysfunction syndrome 4

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Yuanying Chen ◽  
Boliang Fang ◽  
Xuyun Hu ◽  
Ruolan Guo ◽  
Jun Guo ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Fever Of Unknown Origin ◽  
Clinical Decision Making ◽  
Unknown Origin ◽  
Thiamine Pyrophosphate ◽  
Molecular Evidence ◽  
Sequencing Analysis ◽  
Acute Necrotizing Encephalopathy ◽  
Functional Studies ◽  
Thiamine Metabolism

Abstract Background Thiamine metabolism dysfunction syndrome 4 (THMD4, OMIM #613710) is an autosomal recessive inherited disease caused by the deficiency of SLC25A19 that encodes the mitochondrial thiamine pyrophosphate (TPP) transporter. This disorder is characterized by bilateral striatal degradation and progressive polyneuropathy with the onset of fever of unknown origin. The limited number of reported cases and lack of functional annotation of related gene variants continue to limit diagnosis. Results We report three cases of encephalopathy from two unrelated pedigrees with basal ganglia signal changes after fever of unknown origin. To distinguish this from other types of encephalopathy, such as acute necrotizing encephalopathy, exome sequencing was performed, and four novel heterozygous variations, namely, c.169G>A (p.Ala57Thr), c.383C>T (p.Ala128Val), c.76G>A (p.Gly26Arg), and c.745T>A (p.Phe249Ile), were identified in SLC25A19. All variants were confirmed using Sanger sequencing. To determine the pathogenicity of these variants, functional studies were performed. We found that mitochondrial TPP levels were significantly decreased in the presence of SLC25A19 variants, indicating that TPP transport activities of mutated SLC25A19 proteins were impaired. Thus, combining clinical phenotype, genetic analysis, and functional studies, these variants were deemed as likely pathogenic. Conclusions Exome sequencing analysis enables molecular diagnosis as well as provides potential etiology. Further studies will enable the elucidation of SLC25A19 protein function. Our investigation supplied key molecular evidence for the precise diagnosis of and clinical decision-making for a rare disease.

scholarly journals The Fate of Marine Bacterial Exopolysaccharide in Natural Marine Microbial Communities

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0142690 ◽  
Author(s):  
Zilian Zhang ◽  
Yi Chen ◽  
Rui Wang ◽  
Ruanhong Cai ◽  
Yingnan Fu ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Bacterial Exopolysaccharide ◽  
Marine Microbial Communities

Gene silencing or gene editing: the pros and cons.

2021 ◽  
pp. 47-53
Author(s):  
Huw D. Jones
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Gene Function ◽  
Gene Editing ◽  
Mutation Breeding ◽  
Gene Transcript ◽  
Commercial Activities ◽  
Targeted Mutation ◽  
Pros And Cons ◽  
Or Gene

Abstract Research into plant genetics often requires the suppression or complete knockout of gene expression to scientifically validate gene function. In addition, the phenotypes obtained from gene suppression can occasionally have commercial value for plant breeders. Until recently, the methodological choices to achieve these goals fell into two broad types: either some form of RNA-based gene silencing; or the screening of large numbers of natural or induced random genomic mutations. The more recent invention of gene editing as a tool for targeted mutation potentially gives researchers and plant breeders another route to block gene function. RNAi is widely used in animal and plant research and functions to silence gene expression by degrading the target gene transcript. Although RNAi offers unique advantages over genomic mutations, it often leads to the formation of a genetically modified organism (GMO), which for commercial activities has major regulatory and acceptance issues in some regions of the world. Traditional methods of generating genomic mutations are more laborious and uncertain to achieve the desired goals but possess a distinct advantage of not being governed by GMO regulations. Gene editing (GE) technologies have some of the advantages of both RNAi and classical mutation breeding in that they can be designed to give simple knockouts or to modulate gene expression more subtly. GE also has a more complex regulatory position, with some countries treating it as another conventional breeding method whilst the EU defines GE as a technique of genetic modification and applies the normal GMO authorization procedures. This chapter explores the pros and cons of RNAi alongside other methods of modulating gene function.

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