A Gateway platform for functional genomics inHaloferax volcanii: deletion of three tRNA modification genes

In part due to the existence of simple methods for its cultivation and genetic manipulation,Haloferax volcaniiis a major archaeal model organism. It is the only archaeon for which the whole set of post-transcriptionally modified tRNAs has been sequenced, allowing for anin silicoprediction of all RNA modification genes present in the organism. One approach to check these predictions experimentally is via the construction of targeted gene deletion mutants. Toward this goal, an integrative “Gateway vector” that allows gene deletion inH. volcaniiuracil auxotrophs was constructed. The vector was used to delete three predicted tRNA modification genes: HVO_2001 (encoding an archaeal transglycosyl tranferase or arcTGT), which is involved in archeosine biosynthesis; HVO_2348 (encoding a newly discovered GTP cyclohydrolase I), which catalyzes the first step common to archaeosine and folate biosynthesis; and HVO_2736 (encoding a member of the COG1444 family), which is involved inN4-acetylcytidine (ac4C) formation. Preliminary phenotypic analysis of the deletion mutants was conducted, and confirmed all three predictions.

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Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

Journal of Fungi ◽

10.3390/jof7020149 ◽

2021 ◽

Vol 7 (2) ◽

pp. 149

Author(s):

Sarah-Maria Wege ◽

Katharina Gejer ◽

Fabienne Becker ◽

Michael Bölker ◽

Johannes Freitag ◽

...

Keyword(s):

Genome Editing ◽

Cell Biology ◽

Gene Deletion ◽

Fluorescent Protein ◽

Point Mutations ◽

Model Organism ◽

Ustilago Maydis ◽

Genomic Locus ◽

Targeted Gene Deletion ◽

Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

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Towards a Systems Approach in the Genetic Analysis of Archaea: Accelerating Mutant Construction and Phenotypic Analysis inHaloferax volcanii

Archaea ◽

10.1155/2010/426239 ◽

2010 ◽

Vol 2010 ◽

pp. 1-11 ◽

Cited By ~ 22

Author(s):

Ian K. Blaby ◽

Gabriela Phillips ◽

Crysten E. Blaby-Haas ◽

Kevin S. Gulig ◽

Basma El Yacoubi ◽

...

Keyword(s):

High Throughput ◽

Gene Deletion ◽

Systems Approach ◽

Essential Gene ◽

Gene Knockout ◽

Deletion Mutants ◽

Mutant Library ◽

Phenotypic Analysis ◽

Genome Wide ◽

A Genome

With the availability of a genome sequence and increasingly sophisticated genetic tools,Haloferax volcaniiis becoming a model for both Archaea and halophiles. In order forH. volcaniito reach a status equivalent toEscherichia coli, Bacillus subtilis, orSaccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22H. volcaniideletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of anH. volcaniimutant library and suggest that they should form the basis of a larger genome-wide experiment.

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Targeted Gene Deletion and Phenotypic Analysis of the Drosophila melanogaster Seminal Fluid Protease Inhibitor Acp62F

Genetics ◽

10.1534/genetics.107.083766 ◽

2008 ◽

Vol 178 (3) ◽

pp. 1605-1614 ◽

Cited By ~ 50

Author(s):

Jacob L. Mueller ◽

Jon R. Linklater ◽

Kristipati Ravi Ram ◽

Tracey Chapman ◽

Mariana F. Wolfner

Keyword(s):

Drosophila Melanogaster ◽

Protease Inhibitor ◽

Gene Deletion ◽

Seminal Fluid ◽

Phenotypic Analysis ◽

Targeted Gene Deletion

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MoGT2 Is Essential for Morphogenesis and Pathogenicity of Magnaporthe oryzae

mSphere ◽

10.1128/msphere.00309-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Shuzhen Deng ◽

Wenda Sun ◽

Lihong Dong ◽

Guobing Cui ◽

Yi Zhen Deng

Keyword(s):

Magnaporthe Oryzae ◽

Vegetative Growth ◽

Rice Blast ◽

Gene Deletion ◽

Blast Disease ◽

Deletion Mutants ◽

Biological Functions ◽

Cultivated Rice ◽

Targeted Gene Deletion

ABSTRACT Magnaporthe oryzae causes the rice blast disease, which is one of the most serious diseases of cultivated rice worldwide. Glycosylation is an important posttranslational modification of secretory and membrane proteins in all eukaryotes, catalyzed by glycosyltransferases (GTs). In this study, we identified and characterized a type 2 glycosyltransferase, MoGt2, in M. oryzae. Targeted gene deletion mutants of MoGT2 (mogt2Δ strains) were nonpathogenic and were impaired in vegetative growth, conidiation, and appressorium formation at hyphal tips. Moreover, MoGT2 plays an important role in stress tolerance and hydrophobin function of M. oryzae. Site-directed mutagenesis analysis showed that conserved glycosyltransferase domains (DxD and QxxRW) are critical for biological functions of MoGt2. MoGT2 deletion led to altered glycoproteins during M. oryzae conidiation. By liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified several candidate proteins as potential substrates of MoGt2, including several heat shock proteins, two coiled-coil domain-containing proteins, aminopeptidase 2, and nuclease domain-containing protein 1. On the other hand, we found that a conidiation-related gene, genes involved in various metabolism pathways, and genes involved in cell wall integrity and/or osmotic response were differentially regulated in the mogt2Δ mutant, which may potentially contribute to its condiation defects. Taken together, our results show that MoGt2 is important for infection-related morphogenesis and pathogenesis in M. oryzae. IMPORTANCE The ascomycete fungus Magnapothe oryzae is the causal agent of rice blast disease, leading to severe loss in cultivated rice production worldwide. In this study, we identified a conserved type 2 glycosyltransferase named MoGt2 in M. oryzae. The mogt2Δ targeted gene deletion mutants exhibited pleiotropic defects in vegetative growth, conidiation, stress response, hyphal appressorium-mediated penetration, and pathogenicity. Furthermore, conserved glycosyltransferase domains are critical for MoGt2 function. The comparative transcriptome analysis revealed potential target genes under MoGt2 regulation in M. oryzae conidiation. Identification of potential glycoproteins modified by MoGt2 provided information on its regulatory mechanism of gene expression and biological functions. Overall, our study represents the first report of type 2 glycosyltransferase function in M. oryzae infection-related morphogenesis and pathogenesis.

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Optimization of a Lambda-RED Recombination Method for Rapid Gene Deletion in Human Cytomegalovirus

International Journal of Molecular Sciences ◽

10.3390/ijms221910558 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10558

Author(s):

Estéfani García-Ríos ◽

Julia Gata-de-Benito ◽

Mireia López-Siles ◽

Michael J. McConnell ◽

Pilar Pérez-Romero

Keyword(s):

Human Cytomegalovirus ◽

Gene Function ◽

Gene Deletion ◽

Genetic Manipulation ◽

Artificial Chromosome ◽

Deletion Mutants ◽

Transplant Patients ◽

Recombination System ◽

Rapid Generation ◽

Red Recombination

Human cytomegalovirus (HCMV) continues to be a major cause of morbidity in transplant patients and newborns. However, the functions of many of the more than 282 genes encoded in the HCMV genome remain unknown. The development of bacterial artificial chromosome (BAC) technology contributes to the genetic manipulation of several organisms including HCMV. The maintenance of the HCMV BAC in E. coli cells permits the rapid generation of recombinant viral genomes that can be used to produce viral progeny in cell cultures for the study of gene function. We optimized the Lambda-Red Recombination system to construct HCMV gene deletion mutants rapidly in the complete set of tested genes. This method constitutes a useful tool that allows for the quick generation of a high number of gene deletion mutants, allowing for the analysis of the whole genome to improve our understanding of HCMV gene function. This may also facilitate the development of novel vaccines and therapeutics.

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Mutability of demographic noise in microbial range expansions

The ISME Journal ◽

10.1038/s41396-021-00951-9 ◽

2021 ◽

Author(s):

QinQin Yu ◽

Matti Gralka ◽

Marie-Cécilia Duvernoy ◽

Megan Sousa ◽

Arbel Harpak ◽

...

Keyword(s):

Gene Deletion ◽

Genetic Manipulation ◽

Single Gene ◽

Population Level ◽

Growth Conditions ◽

Establishment Probability ◽

In The Wild ◽

Order Of Magnitude ◽

Demographic Noise ◽

Single Gene Deletion

AbstractDemographic noise, the change in the composition of a population due to random birth and death events, is an important driving force in evolution because it reduces the efficacy of natural selection. Demographic noise is typically thought to be set by the population size and the environment, but recent experiments with microbial range expansions have revealed substantial strain-level differences in demographic noise under the same growth conditions. Many genetic and phenotypic differences exist between strains; to what extent do single mutations change the strength of demographic noise? To investigate this question, we developed a high-throughput method for measuring demographic noise in colonies without the need for genetic manipulation. By applying this method to 191 randomly-selected single gene deletion strains from the E. coli Keio collection, we find that a typical single gene deletion mutation decreases demographic noise by 8% (maximal decrease: 81%). We find that the strength of demographic noise is an emergent trait at the population level that can be predicted by colony-level traits but not cell-level traits. The observed differences in demographic noise from single gene deletions can increase the establishment probability of beneficial mutations by almost an order of magnitude (compared to in the wild type). Our results show that single mutations can substantially alter adaptation through their effects on demographic noise and suggest that demographic noise can be an evolvable trait of a population.

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Targeted gene deletion in Brettanomyces bruxellensis with an expression-free CRISPR-Cas9 system

Applied Microbiology and Biotechnology ◽

10.1007/s00253-020-10750-5 ◽

2020 ◽

Vol 104 (16) ◽

pp. 7105-7115

Author(s):

Cristian Varela ◽

Caroline Bartel ◽

Cristobal Onetto ◽

Anthony Borneman

Keyword(s):

Gene Deletion ◽

Brettanomyces Bruxellensis ◽

Targeted Gene Deletion

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Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

Applied and Environmental Microbiology ◽

10.1128/aem.03666-13 ◽

2014 ◽

Vol 80 (8) ◽

pp. 2410-2416 ◽

Cited By ~ 65

Author(s):

Areen Banerjee ◽

Ching Leang ◽

Toshiyuki Ueki ◽

Kelly P. Nevin ◽

Derek R. Lovley

Keyword(s):

Ethanol Production ◽

Genetic Manipulation ◽

Electron Flow ◽

Model Organism ◽

Inducible Expression ◽

Carbon Flow ◽

Wild Type ◽

Content Type ◽

Microbial Electrosynthesis ◽

Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

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Targeted gene deletion of Leishmania major genes encoding developmental stage-specific leishmanolysin (GP63)

Molecular Microbiology ◽

10.1046/j.1365-2958.1998.00689.x ◽

1998 ◽

Vol 27 (3) ◽

pp. 519-530 ◽

Cited By ~ 84

Author(s):

Phalgun B. Joshi ◽

David L. Sacks ◽

Govind Modi ◽

W. Robert McMaster

Keyword(s):

Developmental Stage ◽

Gene Deletion ◽

Leishmania Major ◽

Major Genes ◽

Targeted Gene Deletion ◽

Genes Encoding

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Pleiotropic drug-resistance attenuated genomic library improves elucidation of drug mechanisms

Molecular BioSystems ◽

10.1039/c5mb00406c ◽

2015 ◽

Vol 11 (11) ◽

pp. 3129-3136 ◽

Cited By ~ 12

Author(s):

Namal V. C. Coorey ◽

James H. Matthews ◽

David S. Bellows ◽

Paul H. Atkinson

Keyword(s):

Saccharomyces Cerevisiae ◽

Drug Resistance ◽

Mechanism Of Action ◽

Gene Deletion ◽

Genomic Library ◽

Deletion Mutants ◽

Pleiotropic Drug Resistance ◽

Genome Wide

Identifying Saccharomyces cerevisiae genome-wide gene deletion mutants that confer hypersensitivity to a xenobiotic aids the elucidation of its mechanism of action (MoA).

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