scholarly journals Identification of Escherichia coli Host Genes That Influence the Bacteriophage Lambda (λ) T4rII Exclusion (Rex) Phenotype

Genetics ◽  
2020 ◽  
Vol 216 (4) ◽  
pp. 1087-1102
Author(s):  
Hibah Alattas ◽  
Shirley Wong ◽  
Roderick A. Slavcev
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Bacteriophage Lambda ◽  
Single Gene ◽  
Cellular Environment ◽  
Osmotic Balance ◽  
One Step ◽  
Gene Knockouts ◽  
First Time ◽  
Host Genes

The T4rII exclusion (Rex) phenotype is the inability of T4rII mutant bacteriophage to propagate in hosts (Escherichia coli) lysogenized by bacteriophage lambda (λ). The Rex phenotype, triggered by T4rII infection of a rex+ λ lysogen, results in rapid membrane depolarization imposing a harsh cellular environment that resembles stationary phase. Rex “activation” has been proposed as an altruistic cell death system to protect the λ prophage and its host from T4rII superinfection. Although well studied for over 60 years, the mechanism behind Rex still remains unclear. We have identified key nonessential genes involved in this enigmatic exclusion system by examining T4rII infection across a collection of rex+ single-gene knockouts. We further developed a system for rapid, one-step isolation of host mutations that could attenuate/abrogate the Rex phenotype. For the first time, we identified host mutations that influence Rex activity and rex+ host sensitivity to T4rII infection. Among others, notable genes include tolA, ompA, ompF, ompW, ompX, ompT, lpp, mglC, and rpoS. They are critical players in cellular osmotic balance and are part of the stationary phase and/or membrane distress regulons. Based on these findings, we propose a new model that connects Rex to the σS, σE regulons and key membrane proteins.

scholarly journals MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Karthik R. Chamakura ◽  
Jennifer S. Tran ◽  
Ry Young
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Single Gene ◽  
Specific Protein ◽  
Full Length ◽  
Missense Change ◽  
Content Type ◽  
Terminal Domain ◽  
Mutant Host ◽  
Host Genes

ABSTRACT The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to L lysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJ P330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJ P330Q mutant was recessive and could be suppressed by L overcomes d na J (Lodj ) alleles selected for restoration of lysis. All three Lodj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ∼20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the Lodj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of ϕX174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions. IMPORTANCE Small single-stranded nucleic acid lytic phages (Microviridae and Leviviridae) lyse their host by expressing a single “protein antibiotic.” The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.

Extending homologous sequence based on the single gene mutants by one-step PCR for efficient multiple gene knockouts

2012 ◽  
Vol 57 (3) ◽  
pp. 209-214 ◽  
Author(s):  
Mingji Li ◽  
Pengfei Gu ◽  
Junhua Kang ◽  
Yang Wang ◽  
Qian Wang ◽  
...  
Keyword(s):  
Single Gene ◽  
Homologous Sequence ◽  
Multiple Gene ◽  
One Step ◽  
Gene Knockouts

Nanogold hybrid silica gel and its 1-octadecanethiol self-assembled modified composite as a stationary phase for liquid chromatography

The Analyst ◽  
2019 ◽  
Vol 144 (9) ◽  
pp. 3072-3079 ◽  
Author(s):  
Yuhuan Wang ◽  
Ruyan Wang ◽  
Lei Wang ◽  
Licheng Wang ◽  
Yong Guo ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Stationary Phase ◽  
Silica Gel ◽  
Au Nanoparticle ◽  
Hybrid Silica ◽  
One Step ◽  
Self Assembled ◽  
Liquid Chromatographic ◽  
First Time

Au nanoparticle-hybridized silica (Au@sil) spheres were synthesized in one step as a liquid chromatographic stationary phase for the first time.

scholarly journals Determination of Antibiotic Hypersensitivity among 4,000 Single-Gene-Knockout Mutants of Escherichia coli

2008 ◽  
Vol 190 (17) ◽  
pp. 5981-5988 ◽  
Author(s):  
Cindy Tamae ◽  
Anne Liu ◽  
Katherine Kim ◽  
Daniel Sitz ◽  
Jeeyoon Hong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput Screening ◽  
Gene Knockout ◽  
Single Gene ◽  
E Coli ◽  
Knockout Mutants ◽  
Data Points ◽  
Increased Sensitivity ◽  
Gene Knockouts

ABSTRACT We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of “codrugs” that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's “intrinsic resistome,” which provides innate resistance to antibiotics.

Genetic analysis of tellurate reduction reveals the selenate/tellurate reductase genes ynfEF and the transcriptional regulation of moeA by NsrR in Escherichia coli

2020 ◽  
Author(s):  
Daiki Fujita ◽  
Ryuta Tobe ◽  
Hirotaka Tajima ◽  
Yukari Anma ◽  
Ryo Nishida ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Single Gene ◽  
E Coli ◽  
Twin Arginine Translocation ◽  
Iron Sulfur ◽  
Nitric Oxide Sensor ◽  
Biosynthesis Gene ◽  
Gene Knockouts ◽  
Translocation Pathway ◽  
Keio Collection

Abstract Several bacteria can reduce tellurate into the less toxic elemental tellurium, but the genes responsible for this process have not yet been identified. In this study, we screened the Keio collection of single-gene knockouts of Escherichia coli responsible for decreased tellurate reduction and found that deletions of 29 genes, including those for molybdenum cofactor (Moco) biosynthesis, iron-sulfur biosynthesis, and the twin-arginine translocation pathway resulted in decreased tellurate reduction. Among the gene knockouts, deletions of nsrR, moeA, yjbB, ynbA, ydaS, and yidH affected tellurate reduction more severely than those of other genes. Based on our findings, we determined that the ynfEF genes, which code for the components of the selenate reductase YnfEFGH, are responsible for tellurate reduction. Assays of several molybdoenzymes in the knockouts suggested that nsrR, yjbB, ynbA, ydaS, and yidH are essential for the activities of molybdoenzymes in E. coli. Furthermore, we found that the nitric oxide sensor NsrR positively regulated the transcription of the Moco biosynthesis gene moeA. These findings provided new insights into the complexity and regulation of Moco biosynthesis in E. coli.

scholarly journals Biosynthesis of dihydroquercetin in Escherichia coli from glycerol

2020 ◽  
Author(s):  
Seon Young Park ◽  
Dongsoo Yang ◽  
Shin Hee Ha ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Biosynthetic Pathway ◽  
Carbon Sources ◽  
Aromatic Amino Acids ◽  
Engineering Model ◽  
Producer Strain ◽  
E Coli ◽  
One Step ◽  
First Time

AbstractPhenylpropanoids are a group of diverse natural products derived from aromatic amino acids. Although their demands are high both as drugs and nutraceuticals, their production mainly depends on inefficient extraction from plants. To achieve sustainable production of phenylpropanoids, engineering model microorganisms such as Escherichia coli has been sought, but most strains require supplementation of expensive precursors. Here, we report one-step production of a representative phenylpropanoid, dihydroquercetin (DHQ), from simple carbon sources in E. coli for the first time. The best DHQ producer strain capable of producing 239.4 mg/L of DHQ from glycerol was obtained by optimizing the biosynthetic pathway and engineering the signal peptide of cytochrome P450 (TT7) from Arabidopsis thaliana. The engineered plant P450 could produce a significantly higher titer of DHQ than a bacterial monooxygenase, showing the potential of employing plant P450s for the production of diverse natural products that has been previously difficult in bacterial hosts. This study will serve as a guideline for industrial production of pharmaceutically important yet complex natural products.

Iso-octenidine: Promising Octenidine Analogue with Improved Solubility

2020 ◽  
Vol 17 ◽  
Author(s):  
Igor K. Yakuschenko ◽  
Nataliya N. Pozdeeva ◽  
Viktoriya A. Mumyatova ◽  
Alexey A. Terentiev ◽  
Svyatoslav Ya. Gadomsky
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Micrococcus Luteus ◽  
Octenidine Dihydrochloride ◽  
First Time

: Iso-octenidine, an isomer of octenidine dihydrochloride, was synthesized and studied for the first time. Isooctenidine was demonstrated to be 3-fold more soluble in water in comparison to original octenidine, and both substances had remarkably similar antibacterial activity (tested on Escherichia Coli and Micrococcus luteus).

scholarly journals Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Charles R. Midgett ◽  
Kacey Marie Talbot ◽  
Jessica L. Day ◽  
George P. Munson ◽  
F. Jon Kull
Keyword(s):  
Escherichia Coli ◽  
Small Molecules ◽  
Family Member ◽  
Shigella Flexneri ◽  
Decanoic Acid ◽  
Enterotoxigenic Escherichia Coli ◽  
Gram Negative Bacteria ◽  
Enteric Infections ◽  
First Time ◽  
Arac Family

AbstractEnteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

scholarly journals Electrochromic Performance and Capacitor Performance of α-MoO3 Nanorods Fabricated by a One-Step Procedure

Coatings ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 783
Author(s):  
Ying Duan ◽  
Chen Wang ◽  
Jian Hao ◽  
Yang Jiao ◽  
Yanchao Xu ◽  
...  
Keyword(s):  
Growth Parameters ◽  
Average Diameter ◽  
Fast Response ◽  
Cycle Stability ◽  
Electrochromic Devices ◽  
Step Procedure ◽  
One Step ◽  
Supercapacitor Performance ◽  
First Time ◽  
Prepared Nanorods

In this paper, we propose for the first time the synthesis of α-MoO3 nanorods in a one-step procedure at mild temperatures. By changing the growth parameters, the microstructure and controllable morphology of the resulting products can be customized. The average diameter of the as-prepared nanorods is about 200 nm. The electrochromic and capacitance properties of the synthesized products were studied. The results show that the electrochromic properties of α-MoO3 nanorods at 550 nm have 67% high transmission contrast, good cycle stability and fast response time. The MoO3 nanorods also exhibit a stable supercapacitor performance with 98.5% capacitance retention after 10,000 cycles. Although current density varies sequentially, the nanostructure always exhibits a stable capacitor to maintain 100%. These results indicate the as-prepared MoO3 nanorods may be good candidates for applications in electrochromic devices and supercapacitors.

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