Single‐cell characterization of metabolic switching in the sugar phosphotransferase system of<i>Escherichia coli</i>

Westermayer, Sonja A.; Fritz, Georg; Gutiérrez, Joaquín; Megerle, Judith A.; Weißl, Mira P. S.; Schnetz, Karin; Gerland, Ulrich; Rädler, Joachim O.

doi:10.1111/mmi.13329

Cited by 16 publications

(11 citation statements)

References 53 publications

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“…For this purpose, we constructed a strain that contains an EI-mCherry fusion, expressed from the native chromosomal locus, as well as mCerulean gene under the regulation of N-acetyl glucosamine (NAG) promoter ( Pnag ). To induce expression from Pnag promoter, phosphorylated NAG needs to be present inside the cell (Yamada and Saier, 1988 ; Westermayer et al, 2016 ). Since EI is the first protein in the phosphorylation cascade that enables uptake and phosphorylation of PTS sugars, including NAG (Yamada and Saier, 1988 ), expression of mCerulean implies that EI is active.…”

Section: Resultsmentioning

confidence: 99%

“…SUT201, which expresses EI-mCherry and ZapA-GFP from the chromosome, was constructed by P1 transduction of zapA-gfp(::cat) from HC261 (Peters et al, 2011 ) to MG1655Φ( ptsI -mCherry). NTS102, which expresses EI-mCherry, Psrl -mVenus and Pnag -mCerulean from the chromosome, was constructed by P1 transduction of MG1655Φ( ptsI -mCherry), linked to KanR, to T1683 (Westermayer et al, 2016 ).…”

Section: Methodsmentioning

confidence: 99%

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Phenotypic Heterogeneity in Sugar Utilization by E. coli Is Generated by Stochastic Dispersal of the General PTS Protein EI from Polar Clusters

et al. 2018

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Although the list of proteins that localize to the bacterial cell poles is constantly growing, little is known about their temporal behavior. EI, a major protein of the phosphotransferase system (PTS) that regulates sugar uptake and metabolism in bacteria, was shown to form clusters at the Escherichia coli cell poles. We monitored the localization of EI clusters, as well as diffuse molecules, in space and time during the lifetime of E. coli cells. We show that EI distribution and cluster dynamics varies among cells in a population, and that the cluster speed inversely correlates with cluster size. In growing cells, EI is not assembled into clusters in almost 40% of the cells, and the clusters in most remaining cells dynamically relocate within the pole region or between the poles. In non-growing cells, the fraction of cells that contain EI clusters is significantly higher, and dispersal of these clusters is often observed shortly after exiting quiescence. Later, during growth, EI clusters stochastically re-form by assembly of pre-existing dispersed molecules at random time points. Using a fluorescent glucose analog, we found that EI function inversely correlates with clustering and with cluster size. Thus, activity is exerted by dispersed EI molecules, whereas the polar clusters serve as a reservoir of molecules ready to act when needed. Taken together our findings highlight the spatiotemporal distribution of EI as a novel layer of regulation that contributes to the population phenotypic heterogeneity with regard to sugar metabolism, seemingly conferring a survival benefit.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Phenotypic Heterogeneity in Sugar Utilization by E. coli Is Generated by Stochastic Dispersal of the General PTS Protein EI from Polar Clusters

et al. 2018

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“…Gene ASU28_RS14105 was down-regulated and ASU28_RS08220 did not expressed in luxS -pMG76e-L-ZS9. PTS is a sugar-phosphorylating system and a complex protein kinase system that regulates a wide variety of metabolic processes and controls the expression of numerous genes (Khajanchi et al, 2015 ; Somavanshi et al, 2016 ; Westermayer et al, 2016 ). It has been reported that AI-2 could be imported through the PTS (Quan et al, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

Overexpression of luxS Promotes Stress Resistance and Biofilm Formation of Lactobacillus paraplantarum L-ZS9 by Regulating the Expression of Multiple Genes

Liu

Zhang

et al. 2018

Front. Microbiol.

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Probiotics have evoked great interest in the past years for their beneficial effects. The aim of this study was to investigate whether luxS overexpression promotes the stress resistance of Lactobacillus paraplantarum L-ZS9. Here we show that overexpression of luxS gene increased the production of autoinducer-2 (AI-2, quorum sensing signal molecule) by L. paraplantarum L-ZS9. At the same time, overexpression of luxS promoted heat-, bile salt-resistance and biofilm formation of the strain. RNAseq results indicated that multiple genes encoding transporters, membrane proteins, and transcriptional regulator were regulated by luxS. These results reveal a new role for LuxS in promoting stress resistance and biofilm formation of probiotic starter.

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“…An additional assay that we used to compare the effect of TmaR subcellular level on PTS sugars uptake relied on the use of a strain expressing mCerulean, a cyan fluorescent protein (CFP), from the N-acetyl glucosamine (NAG) promoter (P NAG ) and EI-mCherry (31). NAG has been used in the past as a representative of PTS sugars, whose utilization is positively regulated by EI, since induction of P NAG by NAG is consistent with the positive feedback mechanism suggested for promoters of PTS sugar utilization genes (31) (15). Hence, production of mCerulean from P NAG implies that EI is active and enables estimation of EI activity by fluorescence microscopy.…”

Section: Tmar-mediated Ei Localization Affects Sugar Uptake By the Ptmentioning

confidence: 99%

Tyrosine phosphorylation-dependent localization of TmaR, a novelE. colipolar protein that controls activity of the major sugar regulator by polar sequestration

Szoke

Albocher

Govindarajan

et al. 2020

Preprint

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The poles of E. coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a novel protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization of the general PTS protein Enzyme I (EI) by preventing it from exerting its activity by binding and polar sequestration, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results argue that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release.SIGNIFICANCEIn recent years, we have learnt that bacterial cells have intricate spatial organization despite the lack of membrane-bounded organelles. The endcaps of rod-shaped bacteria, termed poles, are emerging as hubs for sensing and responding, but the underlying mechanisms for positioning macromolecules there are largely unknown. We discovered a novel protein, TmaR, whose polar localization depends on a phospho-tyrosine modification. We show that TmaR controls the activity of EI, the major regulator of sugar metabolism in most bacteria, by polar sequestration and release. Notably, TmaR is essential for survival in conditions that E. coli often encounters in nature. Hence, TmaR is a key regulator that connects tyrosine phosphorylation, spatial regulation, sugar metabolism and survival in bacteria and the first protein reported to recruit proteins to the E. coli cell poles.

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Single‐cell characterization of metabolic switching in the sugar phosphotransferase system ofEscherichia coli

Cited by 16 publications

References 53 publications

Phenotypic Heterogeneity in Sugar Utilization by E. coli Is Generated by Stochastic Dispersal of the General PTS Protein EI from Polar Clusters

Phenotypic Heterogeneity in Sugar Utilization by E. coli Is Generated by Stochastic Dispersal of the General PTS Protein EI from Polar Clusters

Overexpression of luxS Promotes Stress Resistance and Biofilm Formation of Lactobacillus paraplantarum L-ZS9 by Regulating the Expression of Multiple Genes

Tyrosine phosphorylation-dependent localization of TmaR, a novelE. colipolar protein that controls activity of the major sugar regulator by polar sequestration

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