Topoisomerase I function during Escherichia coli response to antibiotics and stress enhances cell killing from stabilization of its cleavage complex

Liu, I-Fen; Sutherland, Jeanette; Cheng, Bokun; Tse‐Dinh, Yuk‐Ching

doi:10.1093/jac/dkr150

Cited by 16 publications

(15 citation statements)

References 46 publications

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“…Furthermore, the recA mutation resulted in a pDNA supercoiled fraction of 68 ± 8% (Figure 1C). It has been shown that RecA protein participates in the regulation on toposiomerase A gene ( topA ) [31]. Therefore, it is possible that recA mutants display a higher topoisomerase activity, which helps to explain the effect observed in VH33 Δ recA .…”

Section: Resultsmentioning

confidence: 99%

Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode

Borja¹,

Mora

Barrón

et al. 2012

Microb Cell Fact

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BackgroundPlasmid DNA (pDNA) is a promising molecule for therapeutic applications. pDNA is produced by Escherichia coli in high cell-density cultivations (HCDC) using fed-batch mode. The typical limitations of such cultivations, including metabolic deviations like aerobic acetate production due to the existence of substrate gradients in large-scale bioreactors, remain as serious challenges for fast and effective pDNA production. We have previously demonstrated that the substitution of the phosphotransferase system by the over-expressed galactose permease for glucose uptake in E. coli (strain VH33) allows efficient growth, while strongly decreases acetate production. In the present work, additional genetic modifications were made to VH33 to further improve pDNA production. Several genes were deleted from strain VH33: the recA, deoR, nupG and endA genes were inactivated independently and in combination. The performance of the mutant strains was evaluated in shake flasks for the production of a 6.1 kb plasmid bearing an antigen gene against mumps. The best producer strain was cultivated in lab-scale bioreactors using 100 g/L of glucose to achieve HCDC in batch mode. For comparison, the widely used commercial strain DH5α, carrying the same plasmid, was also cultivated under the same conditions.ResultsThe various mutations tested had different effects on the specific growth rate, glucose uptake rate, and pDNA yields (YP/X). The triple mutant VH33 Δ (recA deoR nupG) accumulated low amounts of acetate and resulted in the best YP/X (4.22 mg/g), whereas YP/X of strain VH33 only reached 1.16 mg/g. When cultivated at high glucose concentrations, the triple mutant strain produced 186 mg/L of pDNA, 40 g/L of biomass and only 2.2 g/L of acetate. In contrast, DH5α produced only 70 mg/L of pDNA and accumulated 9.5 g/L of acetate. Furthermore, the supercoiled fraction of the pDNA produced by the triple mutant was nearly constant throughout the cultivation.ConclusionThe pDNA concentration obtained with the engineered strain VH33 Δ (recA deoR nupG) is, to the best of our knowledge, the highest reported for a batch cultivation, and its supercoiled fraction remained close to 80%. Strain VH33 Δ (recA deoR nupG) and its cultivation using elevated glucose concentrations represent an attractive technology for fast and efficient pDNA production and a valuable alternative to fed-batch cultivations of commercial strains.

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Section: Resultsmentioning

confidence: 99%

Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode

Borja¹,

Mora

Barrón

et al. 2012

Microb Cell Fact

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“…Recently, it has been identified in Salmonella spp. recovered from chickens in Japan, and in E. coli of poultry origin in Spain or of pig origin in China (Liu et al, 2011; Soufi et al, 2011; Du et al, 2012). Since this gene seems to be geographically widespread, stable over the time, and equally prevalent in ciprofloxacin-susceptible and -resistant strains (Park et al, 2006), its significance remains debatable.…”

Section: Plasmid-mediated Resistancementioning

confidence: 99%

Plasmid-Mediated Quinolone Resistance; Interactions between Human, Animal, and Environmental Ecologies

Poirel¹,

Cattoir²,

Nordmann³

2012

Front. Microbio.

232

159

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Resistance to quinolones and fluoroquinolones is being increasingly reported among human but also veterinary isolates during the last two to three decades, very likely as a consequence of the large clinical usage of those antibiotics. Even if the principle mechanisms of resistance to quinolones are chromosome-encoded, due to modifications of molecular targets (DNA gyrase and topoisomerase IV), decreased outer-membrane permeability (porin defect), and overexpression of naturally occurring efflux, the emergence of plasmid-mediated quinolone resistance (PMQR) has been reported since 1998. Although these PMQR determinants confer low-level resistance to quinolones and/or fluoroquinolones, they are a favorable background for selection of additional chromosome-encoded quinolone resistance mechanisms. Different transferable mechanisms have been identified, corresponding to the production of Qnr proteins, of the aminoglycoside acetyltransferase AAC(6′)-Ib-cr, or of the QepA-type or OqxAB-type efflux pumps. Qnr proteins protect target enzymes (DNA gyrase and type IV topoisomerase) from quinolone inhibition. The AAC(6′)-Ib-cr determinant acetylates several fluoroquinolones, such as norfloxacin and ciprofloxacin. Finally, the QepA and OqxAB efflux pumps extrude fluoroquinolones from the bacterial cell. A series of studies have identified the environment to be a reservoir of PMQR genes, with farm animals and aquatic habitats being significantly involved. In addition, the origin of the qnr genes has been identified, corresponding to the waterborne species Shewanella sp. Altogether, the recent observations suggest that the aquatic environment might constitute the original source of PMQR genes, that would secondly spread among animal or human isolates.

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“…More recently, a role of topoisomerase I was observed in E. coli SOS response (Liu et al, 2011; Yang et al, 2015), which prompted us to verify the possibility of a direct physical interaction between these proteins. Purified His-EcTOP1 and RecA were incubated together in the presence of ATP, and pulled-down with Cobalt agarose resin.…”

Section: Resultsmentioning

confidence: 99%

Investigating direct interaction between Escherichia coli topoisomerase I and RecA

Banda

Tiwari

Darici

et al. 2016

Gene

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Protein-protein interactions are of special importance in cellular processes, including replication, transcription, recombination, and repair. Escherichia coli topoisomerase I (EcTOP1) is primarily involved in the relaxation of negative DNA supercoiling. E. coli RecA, the key protein for homologous recombination and SOS DNA-damage response, has been shown to stimulate the relaxation activity of EcTOP1. The evidence for their direct protein-protein interaction has not been previously established. We report here the direct physical interaction between E. coli RecA and topoisomerase I. We demonstrated the RecA-topoisomerase I interaction via pull-down assays, and surface plasmon resonance measurements. Molecular docking supports the observation that the interaction involves the topoisomerase I N-terminal domains that form the active site. Our results from pull-down assays showed that ATP, although not required, enhances the RecA-EcTOP1 interaction. We propose that E. coli RecA physically interacts with topoisomerase I to modulate the chromosomal DNA supercoiling.

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Topoisomerase I function during Escherichia coli response to antibiotics and stress enhances cell killing from stabilization of its cleavage complex

Cited by 16 publications

References 46 publications

Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode

Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode

Plasmid-Mediated Quinolone Resistance; Interactions between Human, Animal, and Environmental Ecologies

Investigating direct interaction between Escherichia coli topoisomerase I and RecA

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