Study of the degradation of a multidrug transporter using a non-radioactive pulse chase method

Chai, Qian; Webb, Stacy R.; Wang, Zhaoshuai; Dutch, Rebecca Ellis; Wei, Yinan

doi:10.1007/s00216-016-9871-7

Cited by 10 publications

(8 citation statements)

References 33 publications

(31 reference statements)

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“…Taken together, the low accumulation and similar rate of efflux of EtBr across time in SL1344 suggests that although acrAB transcription peaks in mid-exponential phase, activity of the assembled AcrAB-TolC complex remains constant. The AcrB protein is known to be very stable once made, with a predicted half-life of 6 days ( 26 ). To measure AcrB protein level at different points during growth, a strain was constructed in which the AcrB protein was tagged with GFP at the C terminus as previously described ( 27 ).…”

Section: Resultsmentioning

confidence: 99%

Efflux Impacts Intracellular Accumulation Only in Actively Growing Bacterial Cells

Whittle

McNeil

Trampari

et al. 2021

mBio

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This study shows that efflux is important for maintaining low intracellular accumulation only in actively growing cells and that envelope permeability is the predominant factor in stationary-phase cells. This conclusion means that (i) antibiotics with intracellular targets may be less effective in complex infections with nongrowing or slow-growing bacteria, where intracellular accumulation may be low; (ii) efflux inhibitors may be successful in potentiating the activity of existing antibiotics, but potentially only for bacterial infections where cells are actively growing; and (iii) the remodeling of the cell envelope prior to stationary phase could provide novel drug targets.

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Section: Resultsmentioning

confidence: 99%

Efflux Impacts Intracellular Accumulation Only in Actively Growing Bacterial Cells

Whittle

McNeil

Trampari

et al. 2021

mBio

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“…Active degradation should be negligible compared to the timescale of the search process. The majority of proteins in E. coli are not subject to rapid degradation [ 46 ] and individual membrane proteins that have been studied in detail displayed life-times as long as 6 days [ 47 ]. Spontaneous separation of CadC dimers from the membrane (or proteolytic cleavage) also do not appear to play a role, since CadC is deactivated by external cadaverine [ 25 ].…”

Section: Discussionmentioning

confidence: 99%

Dynamics of chromosomal target search by a membrane-integrated one-component receptor

et al. 2021

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Membrane proteins account for about one third of the cellular proteome, but it is still unclear how dynamic they are and how they establish functional contacts with cytoplasmic interaction partners. Here, we consider a membrane-integrated one-component receptor that also acts as a transcriptional activator, and analyze how it kinetically locates its specific binding site on the genome. We focus on the case of CadC, the pH receptor of the acid stress response Cad system in E. coli. CadC is a prime example of a one-component signaling protein that directly binds to its cognate target site on the chromosome to regulate transcription. We combined fluorescence microscopy experiments, mathematical analysis, and kinetic Monte Carlo simulations to probe this target search process. Using fluorescently labeled CadC, we measured the time from activation of the receptor until successful binding to the DNA in single cells, exploiting that stable receptor-DNA complexes are visible as fluorescent spots. Our experimental data indicate that CadC is highly mobile in the membrane and finds its target by a 2D diffusion and capture mechanism. DNA mobility is constrained due to the overall chromosome organization, but a labeled DNA locus in the vicinity of the target site appears sufficiently mobile to randomly come close to the membrane. Relocation of the DNA target site to a distant position on the chromosome had almost no effect on the mean search time, which was between four and five minutes in either case. However, a mutant strain with two binding sites displayed a mean search time that was reduced by about a factor of two. This behavior is consistent with simulations of a coarse-grained lattice model for the coupled dynamics of DNA within a cell volume and proteins on its surface. The model also rationalizes the experimentally determined distribution of search times. Overall our findings reveal that DNA target search does not present a much bigger kinetic challenge for membrane-integrated proteins than for cytoplasmic proteins. More generally, diffusion and capture mechanisms may be sufficient for bacterial membrane proteins to establish functional contacts with cytoplasmic targets.

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“…Finally, in E. coli it has also been observed that AcrA and AcrB lasted for approximately six days (Chai et al. 2016 ).…”

Section: Model Formulationmentioning

confidence: 96%

Time dependent asymptotic analysis of the gene regulatory network of the AcrAB-TolC efflux pump system in gram-negative bacteria

et al. 2021

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Efflux pumps are a mechanism of intrinsic and evolved resistance in bacteria. If an efflux pump can expel an antibiotic so that its concentration within the cell is below a killing threshold the bacteria are resistant to the antibiotic. Efflux pumps may be specific or they may pump various different substances. This is why many efflux pumps confer multi drug resistance (MDR). In particular over expression of the AcrAB−TolC efflux pump system confers MDR in both Salmonella and Escherichia coli. We consider the complex gene regulation network that controls expression of genes central to controlling the efflux associated genes acrAB and acrEF in Salmonella. We present the first mathematical model of this gene regulatory network in the form of a system of ordinary differential equations. Using a time dependent asymptotic analysis, we examine in detail the behaviour of the efflux system on various different timescales. Asymptotic approximations of the steady states provide an analytical comparison of targets for efflux inhibition.

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Study of the degradation of a multidrug transporter using a non-radioactive pulse chase method

Cited by 10 publications

References 33 publications

Efflux Impacts Intracellular Accumulation Only in Actively Growing Bacterial Cells

Efflux Impacts Intracellular Accumulation Only in Actively Growing Bacterial Cells

Dynamics of chromosomal target search by a membrane-integrated one-component receptor

Time dependent asymptotic analysis of the gene regulatory network of the AcrAB-TolC efflux pump system in gram-negative bacteria

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