scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2020 ◽  
Vol 48 (8) ◽  
pp. 4585-4600
Author(s):  
Gabriel A Suárez ◽  
Kyle R Dugan ◽  
Brian A Renda ◽  
Sean P Leonard ◽  
Lakshmi Suryateja Gangavarapu ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Single Gene ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Transposon Insertion ◽  
A Genome ◽  
Acinetobacter Baylyi Adp1 ◽  
Transposon Insertion Sequencing ◽  
Improved Methods

Abstract One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2019 ◽  
Author(s):  
Gabriel A. Suárez ◽  
Kyle R. Dugan ◽  
Brian A. Renda ◽  
Sean P. Leonard ◽  
Lakshmi S. Gangavarapu ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Single Gene ◽  
Golden Gate ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
A Genome ◽  
Strain Collection ◽  
Acinetobacter Baylyi Adp1 ◽  
Improved Methods

ABSTRACTOne goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 19 successful multiple-gene deletions ranged in size from 21 to 183 kilobases and collectively accounted for 24.6% of its genome. Deletion success could only be partially predicted on the basis of a single-gene knockout strain collection and a new Tn-Seq experiment. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals A complete collection of single‐gene deletion mutants of Acinetobacter baylyi ADP1

2008 ◽  
Vol 4 (1) ◽  
pp. 174 ◽  
Author(s):  
Véronique de Berardinis ◽  
David Vallenet ◽  
Vanina Castelli ◽  
Marielle Besnard ◽  
Agnès Pinet ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Single Gene ◽  
Deletion Mutants ◽  
Acinetobacter Baylyi ◽  
Complete Collection ◽  
Acinetobacter Baylyi Adp1 ◽  
Single Gene Deletion

scholarly journals Markerless Multiple-Gene-Deletion System for Streptococcus mutans

2008 ◽  
Vol 74 (7) ◽  
pp. 2037-2042 ◽  
Author(s):  
Anirban Banerjee ◽  
Indranil Biswas
Keyword(s):  
Dental Caries ◽  
Streptococcus Mutans ◽  
Genetic Background ◽  
Double Mutant ◽  
Gene Deletion ◽  
Low Ph ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Modified Method ◽  
Oxidative Agents

ABSTRACT Inactivation or selective modification is essential to elucidate the putative function of a gene. The present study describes an improved Cre-loxP-based method for markerless multiple gene deletion in Streptococcus mutans, the principal etiological agent of dental caries. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. The effectiveness of this modified strategy was demonstrated by the construction of both single and double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans. HtrA and ClpP play key roles in the processing and maturation of several important proteins, including many virulence factors. Deletion of these genes resulted in reducing the organism's ability to withstand exposure to low pH and oxidative agents. The method described here is simple and efficient and can be easily implemented for multiple gene deletions with S. mutans and other streptococci.

scholarly journals A genome-scale antibiotic screen in Serratia marcescens identifies YdgH as a conserved modifier of cephalosporin and detergent susceptibility

2021 ◽  
Author(s):  
Jacob E. Lazarus ◽  
Alyson R. Warr ◽  
Kathleen A. Westervelt ◽  
David C. Hooper ◽  
Matthew K. Waldor
Keyword(s):  
Antibiotic Resistance ◽  
Serratia Marcescens ◽  
Cell Envelope ◽  
Single Gene ◽  
Intrinsic Resistance ◽  
Minimal Inhibitory Concentrations ◽  
Transposon Insertion ◽  
A Genome ◽  
Before And After ◽  
Invasive Infections

Serratia marcescens , a member of the order Enterobacterales, is adept at colonizing healthcare environments and an important cause of invasive infections. Antibiotic resistance is a daunting problem in S. marcescens because in addition to plasmid-mediated mechanisms, most isolates have considerable intrinsic resistance to multiple antibiotic classes. To discover endogenous modifiers of antibiotic susceptibility in S. marcescens , a high-density transposon insertion library was subjected to sub-minimal inhibitory concentrations of two cephalosporins, cefoxitin and cefepime, as well as the fluoroquinolone ciprofloxacin. Comparisons of transposon insertion abundance before and after antibiotic exposure identified hundreds of potential modifiers of susceptibility to these agents. Using single gene deletions, we validated several candidate modifiers of cefoxitin susceptibility and chose ydgH , a gene of unknown function, for further characterization. In addition to cefoxitin, deletion of y dgH in S. marcescens resulted in decreased susceptibility to multiple 3 rd generation cephalosporins, and in contrast, to increased susceptibility to both cationic and anionic detergents. YdgH is highly conserved throughout the Enterobacterales, and we observed similar phenotypes in Escherichia coli O157:H7 and Enterobacter cloacae mutants. YdgH is predicted to localize to the periplasm and we speculate that it may be involved there in cell envelope homeostasis. Collectively, our findings provide insight into chromosomal mediators of antibiotic resistance in S. marcescens and will serve as a resource for further investigations of this important pathogen.

scholarly journals A genome-scale antibiotic screen in Serratia marcescens identifies YdgH as a conserved modifier of cephalosporin and detergent susceptibility

2021 ◽  
Author(s):  
Jacob E Lazarus ◽  
Alyson R Warr ◽  
Kathleen A Westervelt ◽  
David C. Hooper ◽  
Matthew K. Waldor
Keyword(s):  
Antibiotic Resistance ◽  
Serratia Marcescens ◽  
Cell Envelope ◽  
Single Gene ◽  
Intrinsic Resistance ◽  
Minimal Inhibitory Concentrations ◽  
Transposon Insertion ◽  
A Genome ◽  
Before And After ◽  
Invasive Infections

Serratia marcescens, a member of the order Enterobacterales, is adept at colonizing healthcare environments and an important cause of invasive infections. Antibiotic resistance is a daunting problem in S. marcescens because in addition to plasmid-mediated mechanisms, most isolates have considerable intrinsic resistance to multiple antibiotic classes. To discover endogenous modifiers of antibiotic susceptibility in S. marcescens, a high-density transposon insertion library was subjected to sub-minimal inhibitory concentrations of two cephalosporins, cefoxitin and cefepime, as well as the fluoroquinolone ciprofloxacin. Comparisons of transposon insertion abundance before and after antibiotic exposure identified hundreds of potential modifiers of susceptibility to these agents. Using single gene deletions, we validated several candidate modifiers of cefoxitin susceptibility and chose ydgH, a gene of unknown function, for further characterization. In addition to cefoxitin, deletion of ydgH in S. marcescens resulted in decreased susceptibility to multiple 3rd generation cephalosporins, and in contrast, to increased susceptibility to both cationic and anionic detergents. YdgH is highly conserved throughout the Enterobacterales, and we observed similar phenotypes in Escherichia coli O157:H7 and Enterobacter cloacae mutants. YdgH is predicted to localize to the periplasm and we speculate that it may be involved there in cell envelope homeostasis. Collectively, our findings provide insight into chromosomal mediators of antibiotic resistance in S. marcescens and will serve as a resource for further investigations of this important pathogen.

scholarly journals Suboptimal Global Transcriptional Response Increases the Harmful Effects of Loss-of-Function Mutations

2020 ◽  
Author(s):  
Károly Kovács ◽  
Zoltán Farkas ◽  
Djordje Bajić ◽  
Dorottya Kalapis ◽  
Andreea Daraba ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Single Gene ◽  
Transcriptional Response ◽  
Gene Inactivation ◽  
Loss Of Function ◽  
Gene Deletions ◽  
Genomic Expression ◽  
Deleterious Gene ◽  
Functionally Diverse ◽  
Harmful Effects

Abstract The fitness impact of loss-of-function mutations is generally assumed to reflect the loss of specific molecular functions associated with the perturbed gene. Here, we propose that rewiring of the transcriptome upon deleterious gene inactivation is frequently nonspecific and mimics stereotypic responses to external environmental change. Consequently, transcriptional response to gene deletion could be suboptimal and incur an extra fitness cost. Analysis of the transcriptomes of ∼1,500 single-gene deletion Saccharomyces cerevisiae strains supported this scenario. First, most transcriptomic changes are not specific to the deleted gene but are rather triggered by perturbations in functionally diverse genes. Second, gene deletions that alter the expression of dosage-sensitive genes are especially harmful. Third, by elevating the expression level of downregulated genes, we could experimentally mitigate the fitness defect of gene deletions. Our work shows that rewiring of genomic expression upon gene inactivation shapes the harmful effects of mutations.

scholarly journals Metabolic modeling ofStreptococcus mutansreveals complex nutrient requirements of an oral pathogen

2018 ◽  
Author(s):  
Kenan Jijakli ◽  
Paul A. Jensen
Keyword(s):  
Single Gene ◽  
Metabolic Model ◽  
Tooth Decay ◽  
Nutrient Requirements ◽  
Positive Bacterium ◽  
Gene Deletions ◽  
A Genome ◽  
Removal Experiments ◽  
Acidic Conditions ◽  
Genome Scale

AbstractStreptococcus mutansis a Gram positive bacterium that thrives under acidic conditions and is a primary cause of tooth decay (dental caries). To better understand the metabolism ofS. mutanson a systematic level, we manually constructed a genome-scale metabolic model of theS. mutanstype strain UA159. The model, called iSMU, contains 656 reactions involving 514 metabolites and the products of 488 genes.We interrogatedS. mutans’ nutrient requirements using model simulations and nutrient removal experiments in defined media. The iSMU model matched experimental results in greater than 90% of the conditions tested. We also simulated effects of single gene deletions. The model’s predictions agreed with 78.1% and 84.4% of the gene essentiality predictions from two experimental datasets. Our manually curated model is more accurate thanS. mutansmodels generated from automated reconstruction pipelines. We believe the iSMU model is an important resource for understanding how metabolism enables the cariogenicity ofS. mutans.

scholarly journals Overexpression of a single ORF can extend chronological lifespan in yeast if retrograde signaling and stress response are stimulated

2021 ◽  
Author(s):  
Elzbieta Pogoda ◽  
Hanna Tutaj ◽  
Adrian Pirog ◽  
Katarzyna Tomala ◽  
Ryszard Korona
Keyword(s):  
Single Gene ◽  
Membrane Integrity ◽  
Complex Iii ◽  
Retrograde Signaling ◽  
Inner Mitochondrial Membrane ◽  
Gene Deletions ◽  
Chronological Lifespan ◽  
Genome Wide ◽  
A Genome ◽  
Resistance To Stress

AbstractSystematic collections of single-gene deletions have been invaluable in uncovering determinants of lifespan in yeast. Overexpression of a single gene does not have such a clear outcome as cancellation of its function but it can lead to a variety of imbalances, deregulations and compensations, and some of them could be important for longevity. We report an experiment in which a genome-wide collection of strains overexpressing a single gene was assayed for chronological lifespan (CLS). Only one group of proteins, those locating to the inner membrane and matrix of mitochondria, tended to extend CLS when abundantly overproduced. We selected two such strains—one overexpressing Qcr7 of the respiratory complex III, the other overexpressing Mrps28 of the small mitoribosomal subunit—and analyzed their transcriptomes. The uncovered shifts in RNA abundance in the two strains were nearly identical and highly suggestive. They implied a distortion in the co-translational assembly of respiratory complexes followed by retrograde signaling to the nucleus. The consequent reprogramming of the entire cellular metabolism towards the resistance to stress resulted in an enhanced ability to persist in a non-proliferating state. Our results show that surveillance of the inner mitochondrial membrane integrity is of outstanding importance for the cell. They also demonstrate that overexpression of single genes could be used effectively to elucidate the mitochondrion-nucleus crosstalk.

scholarly journals Protein Moonlighting Revealed by Non-Catalytic Phenotypes of Yeast Enzymes

2017 ◽  
Author(s):  
Adriana Espinosa-Cantú ◽  
Diana Ascencio ◽  
Selene Herrera-Basurto ◽  
Jiewei Xu ◽  
Assen Roguev ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Gene Loss ◽  
Genetic Interaction ◽  
Single Gene ◽  
Chromatin Modification ◽  
Biological Processes ◽  
Gene Deletions ◽  
Multifunctional Protein ◽  
Regulatory Processes ◽  
Catalytic Function

ABSTRACTA single gene can partake in several biological processes, and therefore gene deletions can lead to different—sometimes unexpected—phenotypes. However, it is not always clear whether such pleiotropy reflects the loss of a unique molecular activity involved in different processes or the loss of a multifunctional protein. Here, usingSaccharomyces cerevisiaemetabolism as a model, we systematically test the null hypothesis that enzyme phenotypes depend on a single annotated molecular function, namely their catalysis. We screened a set of carefully selected genes by quantifying the contribution of catalysis to gene-deletion phenotypes under different environmental conditions. While most phenotypes were explained by loss of catalysis, 30% could be readily complemented by a catalytically-inactive enzyme. Such non-catalytic phenotypes were frequent in the Alt1 and Bat2 transaminases and in the isoleucine/valine-biosynthetic enzymes Ilv1 and Ilv2, suggesting novel "moonlighting" activities in these proteins. Furthermore, differential genetic-interaction profiles of gene-deletion and catalytic mutants indicated thatILV1is functionally associated to regulatory processes, specifically to chromatin modification. Our systematic study shows that gene-loss phenotypes and their genetic interactions are frequently not driven by the loss of an annotated catalytic function, underscoring the moonlighting nature of cellular metabolism.

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