scholarly journals Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

2021 ◽  
Author(s):  
Heidi A Arjes ◽  
Jiawei Sun ◽  
Hualan Liu ◽  
Taylor H Nguyen ◽  
Rebecca N Culver ◽  
...  
Keyword(s):  
Growth Dynamics ◽  
Bacteroides Thetaiotaomicron ◽  
Growth Defects ◽  
Transposon Insertion ◽  
A Genome ◽  
Altered Cell ◽  
Genome Scale ◽  
Insertion Mutants ◽  
Mutant Collection

Genomic analyses have revealed how the gut microbiota impacts human health. However, knowledge about the physiology of most gut commensals is largely lacking. Here, we sorted cells from a pooled library to construct an ordered collection of transposon-insertion mutants in the model commensal Bacteroides thetaiotaomicron. We applied a pooling strategy with barcode sequencing to locate mutants and created a condensed collection with single insertions in 2,565 genes. This effort enabled the development of an accurate model for progenitor-collection assembly, which identified strain-abundance biases and multi-insertion strains as important factors that limit coverage. To demonstrate the potential for phenotypic screening, we analyzed growth dynamics and morphology of the condensed collection and identified growth defects and altered cell shape in the sphingolipid-synthesis gene BT0870 and the thiamine-scavenging gene BT2397. Analyses of this collection and utilization of the platform described herein to construct future ordered libraries will increase understanding of gut commensal physiology and colonization strategies.

scholarly journals A Genome-Wide Collection of Mos1 Transposon Insertion Mutants for the C. elegans Research Community

PLoS ONE ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. e30482 ◽  
Author(s):  
Elodie Vallin ◽  
Joseph Gallagher ◽  
Laure Granger ◽  
Edwige Martin ◽  
Jérôme Belougne ◽  
...  
Keyword(s):  
Research Community ◽  
C Elegans ◽  
Transposon Insertion ◽  
Genome Wide ◽  
A Genome ◽  
Insertion Mutants

Screening and characterization of transposon-insertion mutants in a pseudohyphal strain ofSaccharomyces cerevisiae

Yeast ◽  
2003 ◽  
Vol 20 (5) ◽  
pp. 407-415 ◽  
Author(s):  
Chise Suzuki ◽  
Yukiko Hori ◽  
Yutaka Kashiwagi
Keyword(s):  
Transposon Insertion ◽  
Insertion Mutants

scholarly journals Genome Characterization of Lipid-Containing Marine Bacteriophage PM2 by Transposon Insertion Mutagenesis

2006 ◽  
Vol 80 (18) ◽  
pp. 9270-9278 ◽  
Author(s):  
Mart Krupovič ◽  
Heikki Vilen ◽  
Jaana K. H. Bamford ◽  
Hanna M. Kivelä ◽  
Juha-Matti Aalto ◽  
...  
Keyword(s):  
Lipid Vesicle ◽  
Insertion Mutagenesis ◽  
Transposon Insertion ◽  
New Genes ◽  
Double Stranded Dna ◽  
Marine Phage ◽  
Protein Rich ◽  
Insertion Mutants

ABSTRACT Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed- and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.

scholarly journals Identification and Characterization of Small Noncoding RNAs in Genome Sequences of the Edible FungusPleurotus ostreatus

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Jibin Qu ◽  
Mengran Zhao ◽  
Tom Hsiang ◽  
Xiaoxing Feng ◽  
Jinxia Zhang ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Noncoding Rnas ◽  
Genome Sequences ◽  
Small Noncoding Rnas ◽  
Basidiomycetous Fungus ◽  
A Genome ◽  
Genome Assemblies ◽  
Genome Scale ◽  
Identification And Characterization

Noncoding RNAs (ncRNAs) have been identified in many fungi. However, no genome-scale identification of ncRNAs has been inventoried for basidiomycetes. In this research, we detected 254 small noncoding RNAs (sncRNAs) in a genome assembly of an isolate (CCEF00389) ofPleurotus ostreatus, which is a widely cultivated edible basidiomycetous fungus worldwide. The identified sncRNAs include snRNAs, snoRNAs, tRNAs, and miRNAs. SnRNA U1 was not found in CCEF00389 genome assembly and some other basidiomycetous genomes by BLASTn. This implies that if snRNA U1 of basidiomycetes exists, it has a sequence that varies significantly from other organisms. By analyzing the distribution of sncRNA loci, we found that snRNAs and most tRNAs (88.6%) were located in pseudo-UTR regions, while miRNAs are commonly found in introns. To analyze the evolutionary conservation of the sncRNAs inP. ostreatus, we aligned all 254 sncRNAs to the genome assemblies of some other Agaricomycotina fungi. The results suggest that most sncRNAs (77.56%) were highly conserved inP. ostreatus, and 20% were conserved in Agaricomycotina fungi. These findings indicate that most sncRNAs ofP. ostreatuswere not conserved across Agaricomycotina fungi.

scholarly journals Isolation and Characterization of Xenorhabdus nematophila Transposon Insertion Mutants Defective in Lipase Activity against Tween

2009 ◽  
Vol 191 (16) ◽  
pp. 5325-5331 ◽  
Author(s):  
Gregory R. Richards ◽  
Eugenio I. Vivas ◽  
Aaron W. Andersen ◽  
Delmarie Rivera-Santos ◽  
Sara Gilmore ◽  
...  
Keyword(s):  
Lipase Activity ◽  
Metabolic Pathways ◽  
Lipase Production ◽  
Xenorhabdus Nematophila ◽  
Transposon Insertion ◽  
Isolation And Characterization ◽  
Transposon Mutants ◽  
Conserved Gene ◽  
Insertion Mutants

ABSTRACT We identified Xenorhabdus nematophila transposon mutants with defects in lipase activity. One of the mutations, in yigL, a conserved gene of unknown function, resulted in attenuated virulence against Manduca sexta insects. We discuss possible connections between lipase production, YigL, and specific metabolic pathways.

scholarly journals Reconstruction and analysis of genome-scale metabolic model of weak Crabtree positive yeast Lachancea kluyveri

2020 ◽  
Author(s):  
Piyush Nanda ◽  
Pradipta Patra ◽  
Manali Das ◽  
Amit Ghosh
Keyword(s):  
Ethyl Acetate ◽  
Metabolic Flux ◽  
Carbon Sources ◽  
Growth Dynamics ◽  
Dry Weight ◽  
Metabolic Model ◽  
Flux Analysis ◽  
Specific Rate ◽  
A Genome ◽  
Genome Scale

Abstract Background Lachancea kluyveri, a weak Crabtree positive yeast, has been extensively studied for its unique URC pyrimidine catabolism pathway. It produces more biomass than Saccharomyces cerevisiae due to the underlying weak Crabtree effect and resorts to optimal fermentation only in oxygen limiting conditions that render it a suitable host for industrial-scale protein production. Ethyl acetate, an important industrial chemical, has been demonstrated to be a major overflow metabolite during aerobic batch cultivation with a specific rate of 0.12 g per g dry weight per hour. Here, we reconstruct a genome-scale metabolic model of the yeast to better explain the observed phenotypes and aid further hypothesis generation. Results We report the first genome-scale metabolic model, iPN730, using Build Fungal Model in KBase workspace. The inconsistencies in the draft model were semi-automatically corrected using literature and published datasets. The curated model comprises of 1235 reactions, 1179 metabolites, and 730 genes distributed in 8 compartments (organelles). The in silico viability in different media conditions and the growth characteristics in various carbon sources show good agreement with experimental data. Dynamic flux balance analysis describes the growth dynamics, substrate utilization and product formation kinetics in various oxygen-limited conditions. The URC pyrimidine degradation pathway incorporated into the model enables it to grow on uracil or urea as the sole nitrogen source. Conclusion The genome-scale metabolic construction of L. kluyveri will provide a better understanding of metabolism, particularly that of pyrimidine metabolism and ethyl acetate production. Metabolic flux analysis using the model will enable hypotheses generation to gain a deeper understanding of metabolism in weakly Crabtree positive yeast and in fungal biodiversity in general.

scholarly journals A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

2020 ◽  
Author(s):  
Nicholas J. McGlincy ◽  
Zuriah A. Meacham ◽  
Kendra Swain ◽  
Ryan Muller ◽  
Rachel Baum ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Essential Genes ◽  
Competitive Growth ◽  
Loss Of Function ◽  
Design Rules ◽  
Relative Growth ◽  
Growth Defects ◽  
A Genome ◽  
Genome Scale

CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding interpretable loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, but no genome-scale guide libraries have been reported. We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription. Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.

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