Reconstructing a wild-type Pseudomonas aeruginosa reference strain PAO1

The P. aeruginosa reference strain PAO1 has been used to delineate much of the physiology, metabolism and fundamental biology of the species. The wild-type parent of PAO1 was lost, and PAO1 carries a regulatory mutation introduced for positive genetic selection that affects antibiotic resistance, virulence, quorum sensing and other traits. The mutation is a loss-of-function change in an oxidoreductase gene (mexS), which constitutively activates a stress response controlled by a positive regulator (MexT). Fitness defects associated with the constitutive response have led to the inadvertent selection of mexT– suppressor mutations, creating genetic heterogeneity in PAO1 sublines studied in different laboratories. To help circumvent complications due to the mexS–minus phenotypes, we created a wild-type version of PAO1 (called LPAO) by “reverting” its mexS to the functional allele likely to have been in its parent. Phenotypic analysis revealed that the mexS– allele in PAO1 makes growth sensitive to salt (NaCl) and is lethal when combined with mutations inactivating the major sodium antiporter (ShaABCDEF). The salt sensitivity of PAO1 may underlie some complex mexS– phenotypes and help explain the selection of mexT– suppressor mutations. To facilitate genetic comparisons of PAO1, LPAO and other P. aeruginosa strains, we developed a transformation procedure to transfer selectable alleles, such as transposon insertion alleles, between strains. Overall, the study helps explain phenotypic heterogeneity of PAO1-derived strains and provides resources to help recognize and eliminate difficulties due to it. IMPORTANCE The P. aeruginosa reference strain PAO1 carries a regulatory mutation that may affect processes characterized in it. To eliminate complications due to the mutation, we constructed a version of the missing wild-type parent strain and developed methods to transfer mutations between PAO1 and the new strain. The methods are likely to be applicable to other isolates of P. aeruginosa as well.

Download Full-text

16. Physiological effects of IDH1 loss-of-function on Chondrocyte Differentiation through Hedgehog Pathway upregulation

Inquiry@Queen's Undergraduate Research Conference Proceedings ◽

10.24908/iqurcp.9897 ◽

2018 ◽

Author(s):

Cassie Tyson

Keyword(s):

Developmental Stages ◽

Hedgehog Pathway ◽

Chondrocyte Differentiation ◽

Loss Of Function ◽

Wild Type ◽

Phenotypic Analysis ◽

Growth Plate Chondrocytes ◽

Idh Mutations ◽

Cartilage Tumors ◽

Deficient Mice

Cartilage tumors are the most common and terminal primary neoplasms in bone. Physiologically, bones formed through endochondral ossification are regulated by the Hedgehog pathway and Parathyroid hormone-like hormone feedback loop. The upregulation of the infamous Hedgehog pathway has been demonstrated in several non-cartilaginous neoplasms. Recently, frequent mutational events of isocitrate dehydrogenase1 (IDH1) were identified in cartilage tumors. In other neoplasms, IDH mutations produces an oncometabolite that can promote HIF1a activation, contributing to tumorigenesis. Currently, the role of IDH1 mutations in cartilage tumors remain unknown. Investigating the physiological aspect of IDH1proves useful in identifying novel therapeutic targets for cartilage tumors. IDH1 deficient and wild-type littermates, were harvested for forelimbs and hindlimbs at various developmental stages for phenotypic analysis via hematoxylin and eosin staining. Histological analysis demonstrated IDH1 homozygous deficient mice at embryonic stages exhibited dwarfism and an elongated layer of hypertrophic chondrocytes. This was verified via immunohistochemistry Type 10 Collagen staining and Quantitative PCR (qPCR) using the chondrocyte terminal differentiation marker Col10a1. Whole skeletons of IDH1 deficient mice were subjected to skeletal double staining which demonstrated delayed mineralization of underdeveloped IDH1 deficient mice contrasted with wild-type littermates. qPCR was performed to examine the status of chondrocyte differentiation through the Hedgehog pathway in cultured primarymouse growth plate chondrocytes. Interestingly, IDH1 deficient non-neoplastic cells revealed significant upregulation of Hedgehog target molecules in IDH1 deficient chondrocytes. As a result, the loss-offunction of IDH1 was identified as a potential impairment of chondrocyte differentiation and a factor towards chondrocyte tumorgenisis.

Download Full-text

Hxt-Carrier-Mediated Glucose Efflux upon Exposure of Saccharomyces cerevisiae to Excess Maltose

Applied and Environmental Microbiology ◽

10.1128/aem.68.9.4259-4265.2002 ◽

2002 ◽

Vol 68 (9) ◽

pp. 4259-4265 ◽

Cited By ~ 26

Author(s):

Mickel L. A. Jansen ◽

Johannes H. De Winde ◽

Jack T. Pronk

Keyword(s):

Saccharomyces Cerevisiae ◽

Reference Strain ◽

External Medium ◽

Hexose Transport ◽

Hexose Transporter ◽

Wild Type ◽

Experimental Proof ◽

Chemostat Cultures ◽

Selection Of ◽

Intracellular Glucose

ABSTRACT When wild-type Saccharomyces cerevisiae strains pregrown in maltose-limited chemostat cultures were exposed to excess maltose, release of glucose into the external medium was observed. Control experiments confirmed that glucose release was not caused by cell lysis or extracellular maltose hydrolysis. To test the hypothesis that glucose efflux involved plasma membrane glucose transporters, experiments were performed with an S. cerevisiae strain in which all members of the hexose transporter (HXT) gene family had been eliminated and with an isogenic reference strain. Glucose efflux was virtually eliminated in the hexose-transport-deficient strain. This constitutes experimental proof that Hxt transporters facilitate export of glucose from S. cerevisiae cells. After exposure of the hexose-transport-deficient strain to excess maltose, an increase in the intracellular glucose level was observed, while the concentrations of glucose 6-phosphate and ATP remained relatively low. These results demonstrate that glucose efflux can occur as a result of uncoordinated expression of the initial steps of maltose metabolism and the subsequent reactions in glucose dissimilation. This is a relevant phenomenon for selection of maltose-constitutive strains for baking and brewing.

Download Full-text

Molecular and Phenotypic Analysis of 25 Recessive, Homozygous-Viable Alleles at the Mouse agouti Locus

Genetics ◽

10.1093/genetics/160.2.659 ◽

2002 ◽

Vol 160 (2) ◽

pp. 659-674

Author(s):

Rosalynn J Miltenberger ◽

Kazumasa Wakamatsu ◽

Shosuke Ito ◽

Richard P Woychik ◽

Liane B Russell ◽

...

Keyword(s):

Loss Of Function ◽

Wild Type ◽

Allelic Series ◽

Phenotypic Analysis ◽

Protein Coding ◽

Agouti Locus ◽

Expression Control ◽

Gene Expression Control ◽

Specific Locus

Abstract Agouti is a paracrine-acting, transient antagonist of melanocortin 1 receptors that specifies the subapical band of yellow on otherwise black hairs of the wild-type coat. To better understand both agouti structure/function and the germline damage caused by chemicals and radiation, an allelic series of 25 recessive, homozygous-viable agouti mutations generated in specific-locus tests were characterized. Visual inspection of fur, augmented by quantifiable chemical analysis of hair melanins, suggested four phenotypic categories (mild, moderate, umbrous-like, severe) for the 18 hypomorphs and a single category for the 7 amorphs (null). Molecular analysis indicated protein-coding alterations in 8 hypomorphs and 6 amorphs, with mild-moderate phenotypes correlating with signal peptide or basic domain mutations, and more devastating phenotypes resulting from C-terminal lesions. Ten hypomorphs and one null demonstrated wild-type coding potential, suggesting that they contain mutations elsewhere in the ≥125-kb agouti locus that either reduce the level or alter the temporal/spatial distribution of agouti transcripts. Beyond the notable contributions to the field of mouse germ cell mutagenesis, analysis of this allelic series illustrates that complete abrogation of agouti function in vivo occurs most often through protein-coding lesions, whereas partial loss of function occurs slightly more frequently at the level of gene expression control.

Download Full-text

Biological Control of Bacterial Blight of Geranium with H-Mutant Bacteriophages

HortScience ◽

10.21273/hortsci.33.3.519c ◽

1998 ◽

Vol 33 (3) ◽

pp. 519c-519 ◽

Cited By ~ 1

Author(s):

Brent K. Harbaugh ◽

Jeffrey B. Jones ◽

Lee E. Jackson ◽

Gail Somodi ◽

Joseph E. Flaherty

Keyword(s):

Biological Control ◽

Bacterial Blight ◽

Xanthomonas Campestris ◽

Wild Type ◽

Novel Approach ◽

Bacterial Mutants ◽

Spraying Plants ◽

Serious Disease ◽

Wild Type Parent ◽

Selection Of

Bacterial blight, caused by Xanthomonas campestris pv. pelargonii (XCP), is considered the most serious disease of geraniums (Pelargonium × hortorum). A novel approach that uses bacteriophages (phages, viruses that kill bacteria) for the biological control of geranium blight will be presented. Phages were once abandoned as biological control agents due to the emergence of bacterial mutants resistant to the phages employed. However, our approach utilizes a mixture of three to eight different phages including host-range mutants (H-mutants). H-mutants are spontaneously derived from their wild-type parent phages and lyse not only parent wild-type bacteria, but also phage-resistant mutants originating from parent bacteria. Two phages specific for XCP initially were isolated from soil samples from Florida and California. These phages produced virulent reactions in six of 30 XCP strains, and lysogenic reactions in 22 strains. After selection of these phages for increased virulence and additional phages were isolated from MN and UT, 17 phages were evaluated for sensitivity to 21 XCP strains from around the world. Four to 14 phages produced virulent reactions in the 21 XCP strains. Five phages produced virulent reactions in at least 17 XCP strains. A mixture of five phages tested against the 21 XCP strains produced virulent reactions for all 21 XCP strains. Geraniums in 10-cm pots were inoculated with XCP and placed on a greenhouse bench in the middle of 5 non-inoculated plants. After 2 weeks of daily spraying plants with a phage solution (109 pfu phage/ml) or water, there was a 71% reduction in the number of bacterial lesions on phage-treated plants.

Download Full-text

A method for rapid selection of randomly induced mutations in a gene of interest using CRISPR/Cas9 mediated activation of gene expression

10.1101/788372 ◽

2019 ◽

Author(s):

William A. Ng ◽

Andrew Ma ◽

Molly Chen ◽

Bruce H. Reed

Keyword(s):

Gene Expression ◽

Screening Strategy ◽

Loss Of Function ◽

Wild Type ◽

Induced Mutations ◽

Allelic Series ◽

Lethal Mutations ◽

F1 Progeny ◽

Genetic Cross ◽

Selection Of

AbstractWe have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.

Download Full-text

Increased biosynthesis of acetyl-CoA in the yeast Saccharomyces cerevisiae by overexpression of a deregulated pantothenate kinase gene

10.1101/2021.05.25.445608 ◽

2021 ◽

Author(s):

Judith Olzhausen ◽

Mathias Grigat ◽

Larissa Seifert ◽

Tom Ulbricht ◽

Hans-Joachim Schueller

Keyword(s):

Reference Strain ◽

Coenzyme A ◽

Genetic Selection ◽

Fold Increase ◽

Temperature Sensitive ◽

Acetyl Coa ◽

Kinase Gene ◽

Yeast Saccharomyces Cerevisiae ◽

Pantothenate Kinase ◽

Selection Of

Coenzyme A (CoA) and its derivatives such as acetyl-CoA are essential metabolites for several biosynthetic reactions. In the yeast S. cerevisiae, five enzymes (encoded by essential genes CAB1-CAB5; coenzyme A biosynthesis) are required to perform CoA biosynthesis from pantothenate, cysteine and ATP. Similar to enzymes from other eukaryotes, yeast pantothenate kinase (PanK, encoded by CAB1) turned out to be inhibited by acetyl-CoA. By genetic selection of intragenic suppressors of a temperature-sensitive cab1 mutant combined with rationale mutagenesis of the presumed acetyl-CoA binding site within PanK, we were able to identify the variant CAB1 W331R, encoding a hyperactive PanK completely insensitive to inhibition by acetyl-CoA. Using a versatile gene integration cassette containing the TPI1 promoter, we constructed strains overexpressing CAB1 W331R in combination with additional genes of CoA biosynthesis (CAB2, CAB3, HAL3, CAB4 and CAB5). In these strains, the level of CoA nucleotides was 15-fold increased, compared to a reference strain without additional CAB genes. Overexpression of wild-type CAB1 instead of CAB1 W331R turned out as substantially less effective (4-fold increase of CoA nucleotides). Supplementation of overproducing strains with additional pantothenate could further elevate the level of CoA (2.3-fold). Minor increases were observed after overexpression of FEN2 (encoding a pantothenate permease) and deletion of PCD1 (CoA-specific phosphatase). We conclude that the strategy described in this work may improve the efficiency of biotechnological applications depending on acetyl-CoA.

Download Full-text

Mechanistic insights into global suppressors of protein folding defects

10.1101/2021.11.18.469098 ◽

2021 ◽

Author(s):

Gopinath Chattopadhyay ◽

Jayantika Bhowmick ◽

Kavyashree Manjunath ◽

Shahbaz Ahmed ◽

Parveen Goyal ◽

...

Keyword(s):

Bacterial Toxin ◽

Amino Acid Substitutions ◽

Receptor Binding Domain ◽

Loss Of Function ◽

Wild Type ◽

Partial Loss ◽

Suppressor Mutations ◽

Key Drivers

Most amino acid substitutions in a protein either lead to partial loss of function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss of function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined five global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. The suppressors both alone, and in conjunction with inactive mutants, stabilise the protein both thermodynamically and kinetically in-vitro, predominantly through acceleration of the refolding rate parameters. When coupled to inactive mutants they promote increased in-vivo solubilities as well as regain-of-function phenotypes. Our study also demonstrates that the global suppressor approach can be used to consistently stabilise wild-type proteins, including for downstream translational applications.

Download Full-text

Mutations in sfdA and sfdB Suppress Multiple Developmental Mutations in Aspergillus nidulans

Genetics ◽

10.1093/genetics/160.1.159 ◽

2002 ◽

Vol 160 (1) ◽

pp. 159-168

Author(s):

Ellen M Kellner ◽

Thomas H Adams

Keyword(s):

Aspergillus Nidulans ◽

Null Allele ◽

Hyphal Growth ◽

Loss Of Function ◽

Wild Type ◽

Regulatory Pathway ◽

Submerged Cultures ◽

Developmental Defects ◽

Suppressor Mutations ◽

Its Gene

Abstract Conidiophore morphogenesis in Aspergillus nidulans occurs in response to developmental signals that result in the activation of brlA, a well-characterized gene that encodes a transcription factor that is central to asexual development. Loss-of-function mutations in flbD and other fluffy loci have previously been shown to result in delayed development and reduced expression of brlA. flbD message is detectable during both hyphal growth and conidiation, and its gene product is similar to the Myb family of transcription factors. To further understand the regulatory pathway to brlA activation and conidiation, we isolated suppressor mutations that rescued development in strains with a flbD null allele. We describe here two new loci, designated sfdA and sfdB for suppressors of flbD, that bypass the requirement of flbD for development. sfd mutant alleles were found to restore developmental timing and brlA expression to strains with flbD deletions. In addition, sfd mutations suppress the developmental defects in strains harboring loss-of-function mutations in fluG, flbA, flbB, flbC, and flbE. All alleles of sfdA and sfdB that we have isolated are recessive to their wild-type alleles in diploids. Strains with mutant sfd alleles in otherwise developmentally wild-type backgrounds have reduced growth phenotypes and develop conidiophores in submerged cultures.

Download Full-text

A Method for Rapid Selection of Randomly Induced Mutations in a Gene of Interest Using CRISPR/Cas9 Mediated Activation of Gene Expression

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401299 ◽

2020 ◽

Vol 10 (6) ◽

pp. 1893-1901

Author(s):

William A. Ng ◽

Andrew Ma ◽

Molly Chen ◽

Bruce H. Reed

Keyword(s):

Gene Expression ◽

Screening Strategy ◽

Loss Of Function ◽

Wild Type ◽

Induced Mutations ◽

Allelic Series ◽

Lethal Mutations ◽

F1 Progeny ◽

Genetic Cross ◽

Selection Of

We have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.

Download Full-text