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 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 Update on the Keio collection of Escherichia coli single‐gene deletion mutants

2009 ◽  
Vol 5 (1) ◽  
pp. 335 ◽  
Author(s):  
Natsuko Yamamoto ◽  
Kenji Nakahigashi ◽  
Tomoko Nakamichi ◽  
Mihoko Yoshino ◽  
Yuki Takai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Deletion ◽  
Single Gene ◽  
Deletion Mutants ◽  
Keio Collection ◽  
Single Gene Deletion

scholarly journals Sulfate import in Salmonella Typhimurium impacts bacterial aggregation and the respiratory burst in human neutrophils

2021 ◽  
Author(s):  
T. L. Westerman ◽  
M. K. Sheats ◽  
J. R. Elfenbein
Keyword(s):  
Respiratory Burst ◽  
Gene Deletion ◽  
Single Gene ◽  
Protein A ◽  
Human Neutrophils ◽  
Deletion Mutants ◽  
Flagellar Motility ◽  
Neutrophil Respiratory Burst ◽  
Single Gene Deletion

During enteric salmonellosis, neutrophil generated reactive oxygen species alter the gut microenvironment favoring survival of Salmonella Typhimurium. While the type-3 secretion system-1 (T3SS-1) and flagellar motility are potent Salmonella Typhimurium agonists of the neutrophil respiratory burst in vitro, neither of these pathways alone are responsible for stimulation of a maximal respiratory burst. In order to identify Salmonella Typhimurium genes that impact the magnitude of the neutrophil respiratory burst, we performed a two-step screen of defined mutant libraries in co-culture with human neutrophils. We first screened Salmonella Typhimurium mutants lacking defined genomic regions and then tested single gene deletion mutants representing particular regions under selection. A subset of single gene deletion mutants were selected for further investigation. Mutants in four genes, STM1696 (sapF), STM2201 (yeiE), STM2112 (wcaD), and STM2441 (cysA), induced an attenuated respiratory burst. We linked the altered respiratory burst to reduced T3SS-1 expression and/or altered flagellar motility for two mutants (ΔSTM1696 and ΔSTM2201). The ΔSTM2441 mutant, defective for sulfate transport, formed aggregates in minimal media and adhered to surfaces in rich media, suggesting a role for sulfur homeostasis in regulation of aggregation/adherence. We linked the aggregation/adherence phenotype of the ΔSTM2441 mutant to biofilm-associated protein A and flagellins and hypothesize that aggregation caused the observed reduction in the magnitude of the neutrophil respiratory burst. Our data demonstrate that Salmonella Typhimurium has numerous mechanisms to limit the magnitude of the neutrophil respiratory burst. These data further inform our understanding of how Salmonella may alter human neutrophil antimicrobial defenses.

Contribution of hypermutation to fosfomycin heteroresistance in Escherichia coli

2020 ◽  
Author(s):  
Inés Portillo-Calderón ◽  
Miriam Ortiz-Padilla ◽  
Jose Manuel Rodríguez-Martínez ◽  
Belen de Gregorio-Iaria ◽  
Jesús Blázquez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Gene Deletion ◽  
Single Gene ◽  
Clinical Isolates ◽  
Deletion Mutants ◽  
Growth Monitoring ◽  
Fosfomycin Resistance ◽  
Time Kill ◽  
Single Gene Deletion

Abstract Objectives To explore the effect of combining defects in DNA repair systems with the presence of fosfomycin-resistant mechanisms to explain the mechanisms underlying fosfomycin heteroresistance phenotypes in Enterobacteriaceae. Materials and methods We used 11 clinical Escherichia coli isolates together with isogenic single-gene deletion mutants in the E. coli DNA repair system or associated with fosfomycin resistance, combined with double-gene deletion mutants. Fosfomycin MICs were determined by gradient strip assay (GSA) and broth microdilution (BMD). Mutant frequencies for rifampicin (100 mg/L) and fosfomycin (50 and 200 mg/L) were determined. Using two starting inocula, in vitro fosfomycin activity was assessed over 24 h in growth (0.5–512 mg/L) and time–kill assays (64 and 307 mg/L). Results Strong and weak mutator clinical isolates and single-gene deletion mutants, except for ΔuhpT and ΔdnaQ, were susceptible by GSA. By BMD, the percentage of resistant clinical isolates reached 36%. Single-gene deletion mutants showed BMD MICs similar to those for subpopulations by GSA. Strong mutators showed a higher probability of selecting fosfomycin mutants at higher concentrations. By combining the two mechanisms of mutation, MICs and ranges of resistant subpopulations increased, enabling strains to survive at higher fosfomycin concentrations in growth monitoring assays. In time–kill assays, high inocula increased survival by 37.5% at 64 mg/L fosfomycin, compared with low starting inocula. Conclusions The origin and variability of the fosfomycin heteroresistance phenotype can be partially explained by high mutation frequencies together with mechanisms of fosfomycin resistance. Subpopulations should be considered until clinical meaning is established.

Behavioural phenotyping of C. elegans on UV-killed E. coli mutants v1

2021 ◽  
Author(s):  
Saul Moore
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Deletion ◽  
Uv Light ◽  
Single Gene ◽  
Deletion Mutants ◽  
E Coli ◽  
C Elegans ◽  
Behavioural Phenotyping ◽  
Keio Collection ◽  
Single Gene Deletion

Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on Caenorhabditis elegans behaviour when killed by ultraviolet (UV) light

Antioxidant rescue of C. elegans behaviour on Keio E. coli mutants v1

2021 ◽  
Author(s):  
Saul Moore
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Deletion ◽  
Single Gene ◽  
Deletion Mutants ◽  
E Coli ◽  
C Elegans ◽  
Keio Collection ◽  
Single Gene Deletion

Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on Caenorhabditis elegans behaviour in the presence of antioxidants.

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