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 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 A genome-wide CRISPR/Cas9 screen to identify phagocytosis modulators in monocytic THP-1 cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin Lindner ◽  
Eva Martin ◽  
Monika Steininger ◽  
Aleksandra Bundalo ◽  
Martin Lenter ◽  
...  
Keyword(s):  
Translational Control ◽  
Molecular Mechanisms ◽  
Gene Knockout ◽  
Single Gene ◽  
Microbial Pathogens ◽  
Protein Accumulation ◽  
Cellular Mechanisms ◽  
Developmental Defects ◽  
Genome Wide ◽  
A Genome

AbstractPhagocytosis of microbial pathogens, dying or dead cells, and cell debris is essential to maintain tissue homeostasis. Impairment of these processes is associated with autoimmunity, developmental defects and toxic protein accumulation. However, the underlying molecular mechanisms of phagocytosis remain incompletely understood. Here, we performed a genome-wide CRISPR knockout screen to systematically identify regulators involved in phagocytosis of Staphylococcus (S.) aureus by human monocytic THP-1 cells. The screen identified 75 hits including known regulators of phagocytosis, e.g. members of the actin cytoskeleton regulation Arp2/3 and WAVE complexes, as well as genes previously not associated with phagocytosis. These novel genes are involved in translational control (EIF5A and DHPS) and the UDP glycosylation pathway (SLC35A2, SLC35A3, UGCG and UXS1) and were further validated by single gene knockout experiments. Whereas the knockout of EIF5A and DHPS impaired phagocytosis, knocking out SLC35A2, SLC35A3, UGCG and UXS1 resulted in increased phagocytosis. In addition to S. aureus phagocytosis, the above described genes also modulate phagocytosis of Escherichia coli and yeast-derived zymosan A. In summary, we identified both known and unknown genetic regulators of phagocytosis, the latter providing a valuable resource for future studies dissecting the underlying molecular and cellular mechanisms and their role in human disease.

scholarly journals Plasma-sensitive Escherichia coli mutants reveal plasma resistance mechanisms

2019 ◽  
Vol 16 (152) ◽  
pp. 20180846 ◽  
Author(s):  
Marco Krewing ◽  
Fabian Jarzina ◽  
Tim Dirks ◽  
Britta Schubert ◽  
Jan Benedikt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Atmospheric Pressure ◽  
Bacterial Infections ◽  
Bacterial Resistance ◽  
Gene Knockout ◽  
Single Gene ◽  
Complex Mixture ◽  
Plasma Resistance ◽  
A Genome

Non-thermal atmospheric pressure plasmas are investigated as augmenting therapy to combat bacterial infections. The strong antibacterial effects of plasmas are attributed to the complex mixture of reactive species, (V)UV radiation and electric fields. The experience with antibiotics is that upon their introduction as medicines, resistance occurs in pathogens and spreads. To assess the possibility of bacterial resistance developing against plasma, we investigated intrinsic protective mechanisms that allow Escherichia coli to survive plasma stress. We performed a genome-wide screening of single-gene knockout mutants of E. coli and identified 87 mutants that are hypersensitive to the effluent of a microscale atmospheric pressure plasma jet. For selected genes ( cysB , mntH , rep and iscS ) we showed in complementation studies that plasma resistance can be restored and increased above wild-type levels upon over-expression. To identify plasma-derived components that the 87 genes confer resistance against, mutants were tested for hypersensitivity against individual stressors (hydrogen peroxide, superoxide, hydroxyl radicals, ozone, HOCl, peroxynitrite, NO•, nitrite, nitrate, HNO 3 , acid stress, diamide, heat stress and detergents). k-means++ clustering revealed that most genes protect from hydrogen peroxide, superoxide and/or nitric oxide. In conclusion, individual bacterial genes confer resistance against plasma providing insights into the antibacterial mechanisms of plasma.

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.

ENVIRONMENTAL DEPENDENCY OF GENE KNOCKOUTS ON PHENOTYPE MICROARRAY ANALYSIS IN ESCHERICHIA COLI

2010 ◽  
Vol 08 (supp01) ◽  
pp. 83-99 ◽  
Author(s):  
YUKAKO TOHSATO ◽  
TOMOYA BABA ◽  
YUSAKU MAZAKI ◽  
MASAHIRO ITO ◽  
BARRY L. WANNER ◽  
...  
Keyword(s):  
Target Genes ◽  
Gene Knockout ◽  
Single Gene ◽  
Phenotype Microarray ◽  
Experimental Conditions ◽  
Gene Deletions ◽  
Alternative Pathways ◽  
Knockout Mutants ◽  
Phenotype Screening ◽  
Quantitative Screening

Systematic studies have revealed that single gene deletions often display little phenotypic effects under laboratory conditions and that in many cases gene dispensability depends on the experimental conditions. To elucidate the environmental dependency of genes, we analyzed the effects of gene deletions by Phenotype MicroArray™ (PM), a system for quantitative screening of thousands of phenotypes in a high-throughput manner. Here, we proposed a new statistical approach to minimize error inherent in measurements of low respiration rates and find which mutants showed significant phenotypic changes in comparison to the wild-type. We show analyzing results from comprehensive PM assays of 298 single-gene knockout mutants in the Keio collection and two additional mutants under 1,920 different conditions. We focused on isozymes of these genes as simple duplications and analyzed correlations between phenotype changes and protein expression levels. Our results revealed divergence of the environmental dependency of the gene among the knockout genes and have also given some insights into possibilities of alternative pathways and availabilities of information on protein synthesis patterns to classify or predict functions of target genes from systematic phenotype screening.

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 Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin monomers

2021 ◽  
pp. e00173
Author(s):  
Erika Arvay ◽  
Bradley W. Biggs ◽  
Laura Guerrero ◽  
Virginia Jiang ◽  
Keith Tyo
Keyword(s):  
Acinetobacter Baylyi ◽  
Acinetobacter Baylyi Adp1

scholarly journals Systematic Detection of Large-Scale Multi-Gene Horizontal Transfer in Prokaryotes

2021 ◽  
Author(s):  
Lina Kloub ◽  
Sean Gosselin ◽  
Matthew Fullmer ◽  
Joerg Graf ◽  
J Peter Gogarten ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Large Scale ◽  
Single Gene ◽  
Gene Families ◽  
Microbial Evolution ◽  
Phylogenetic Distance ◽  
Secretion Systems ◽  
Type Iii Secretion Systems ◽  
A Genome ◽  
Conserved Gene

Abstract Horizontal gene transfer (HGT) is central to prokaryotic evolution. However, little is known about the “scale” of individual HGT events. In this work, we introduce the first computational framework to help answer the following fundamental question: How often does more than one gene get horizontally transferred in a single HGT event? Our method, called HoMer, uses phylogenetic reconciliation to infer single-gene HGT events across a given set of species/strains, employs several techniques to account for inference error and uncertainty, combines that information with gene order information from extant genomes, and uses statistical analysis to identify candidate horizontal multi-gene transfers (HMGTs) in both extant and ancestral species/strains. HoMer is highly scalable and can be easily used to infer HMGTs across hundreds of genomes. We apply HoMer to a genome-scale dataset of over 22000 gene families from 103 Aeromonas genomes and identify a large number of plausible HMGTs of various scales at both small and large phylogenetic distances. Analysis of these HMGTs reveals interesting relationships between gene function, phylogenetic distance, and frequency of multi-gene transfer. Among other insights, we find that (i) the observed relative frequency of HMGT increases as divergence between genomes increases, (ii) HMGTs often have conserved gene functions, and (iii) rare genes are frequently acquired through HMGT. We also analyze in detail HMGTs involving the zonula occludens toxin and type III secretion systems. By enabling the systematic inference of HMGTs on a large scale, HoMer will facilitate a more accurate and more complete understanding of HGT and microbial evolution.

scholarly journals Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

2015 ◽  
Vol 81 (20) ◽  
pp. 6953-6963 ◽  
Author(s):  
Zhe Zhao ◽  
Lauren J. Eberhart ◽  
Lisa H. Orfe ◽  
Shao-Yeh Lu ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Disulfide Bonds ◽  
Gene Knockout ◽  
Single Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

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