Chaperone–substrate interactions monitored via a robust TEM-1 β-lactamase fragment complementation assay

Bai, Lijun; He, Wei; Li, Tianpeng; Yang, Cuiting; Zhuang, Yingping; Qin, Shu

doi:10.1007/s10529-017-2347-9

Cited by 4 publications

(5 citation statements)

References 30 publications

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“…TEM-1 ␤-lactamase complementation assay. The assay was performed as described previously (48), with the following modifications. Cells were grown to stationary phase in LB medium supplemented with spectinomycin (50 g/ml).…”

Section: Methodsmentioning

confidence: 99%

The Lipoprotein NlpE Is a Cpx Sensor That Serves as a Sentinel for Protein Sorting and Folding Defects in the Escherichia coli Envelope

2019

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The envelope of Gram-negative bacteria is a complex compartment that is essential for viability. To ensure survival of the bacterial cells in fluctuating environments, several signal transduction systems, called envelope stress response systems (ESRSs), exist to monitor envelope biogenesis and homeostasis. The Cpx twocomponent system is an extensively studied ESRS in Escherichia coli that is active during exposure to a vast array of stresses and protects the envelope under those harmful circumstances. Overproduction of NlpE, a two-domain outer membrane lipoprotein of unclear function, has been used in numerous studies as a molecular trigger to turn on the system artificially. However, the mechanism of Cpx activation by NlpE, as well as its physiological relevance, awaited further investigation. In this paper, we provide novel insights into the role played by NlpE in the Cpx system. We found that, among all outer membrane lipoproteins in E. coli, NlpE is sufficient to induce Cpx when lipoprotein trafficking is perturbed. Under such conditions, fitness is increased by the presence of NlpE. Moreover, we show that NlpE, through its N-terminal domain, physically interacts with the Cpx sensor kinase CpxA. Our data suggest that NlpE also serves to activate the Cpx system during oxidative folding defects in the periplasm and that its C-terminal domain is involved in the sensing mechanism. Overall, our data demonstrate that NlpE acts as a sentinel for two important envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding, and they further establish NlpE as a bona fide member of the Cpx twocomponent system. IMPORTANCE Bacteria rely on a sophisticated envelope to shield them against challenging environmental conditions and therefore need to ensure correct envelope assembly and integrity. A major signaling pathway that performs this role in Gramnegative species is the Cpx system. An outer membrane lipoprotein of unclear function, NlpE, has long been exploited as a research tool to study Cpx in E. coli, since it triggers this system when overproduced or mislocalized; however, the mechanism and physiological relevance of the NlpE-Cpx connection have awaited further investigation. We elucidate a new function for NlpE by showing that it physically interacts with the Cpx sensor CpxA and acts as a sentinel that specifically monitors two essential envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding.

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Section: Methodsmentioning

confidence: 99%

The Lipoprotein NlpE Is a Cpx Sensor That Serves as a Sentinel for Protein Sorting and Folding Defects in the Escherichia coli Envelope

2019

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“…and the pH was maintained at 6.8 using NaOH. For shake-flask cultivation, strains were From Prof. Quan (Bai et al, 2017) E. coli DH5α F + endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(r…”

Section: Medium and Culture Conditionsmentioning

confidence: 99%

“…Additionally, membrane porins exhibit specificity for various carbohydrates and may promote the efflux of oligosaccharides such as 2′‐FL (Saier, 2000). Considering the significance of cell membrane properties, recent attention has been given to a novel E. coli K12 derivative named BL27, which lacks the ampC gene and has a chromosomal integration of the T7 polymerase gene at the lacZ locus (Bai et al, 2017). The ampC gene has been found to be linked to membrane protein profiles, including efflux pump or porin (Jameel et al 2014; Martínez‐Martínez et al, 2000), which are crucial for substrate uptake and 2′‐FL efflux (Vergalli et al, 2020; Zhu et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

De novo 2′‐fucosyllactose bioproduction through modular engineering of a novel Escherichia coli K12‐derived strain

Zhang

Fan

et al. 2023

Food Bioengineering

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The most abundant human milk oligosaccharide 2′-fucosyllactose (2′-FL) is a valuable component that has gained significant attention from the food industry. To biosynthesize 2′-FL, various Escherichia coli K12 derivatives have been genetically modified. To further enhance the application performance of E. coli K12, a novel E. coli K12 derivative BL27 was used as a chassis cell in this study, and modular pathway enhancement was performed to achieve de novo synthesis of 2′-FL. The futC gene encoding α-1,2-fucosyltransferase was introduced, and the wcaJ gene was knocked out to prevent the conversion of GDP-L-fucose to colanic acid. Next, the effects of overexpressing transcriptional regulators rcsA and rcsB and knocking out transcriptional regulators mcbR and waaF were evaluated to optimize the colanic acid pathway. The expression level, solubility, and activity of FutC were improved through genomic integration, TrxA-tag fusion, and double mutation in F40S/Q237S. Fermentation conditions were optimized to achieve maximum 2′-FL titers of 3.86 and 23.56 g/L in shakeflask and fed-batch cultivation, respectively. Over 85% of the products were successfully excreted into extracellular and almost no byproduct 2′,3difucosyllactose was generated. This study has explored a new microbial platform and modification strategies for the synthesis of 2′-FL and provides opportunities for its commercial production.

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“…E. coli BL21 (DE3) was employed for protein expression. E. coli MG1655 (DE3), donated by Dr. Shu Quan at East China University of Science and Technology (ECUST), 28 was used as the host strain for succinic acid production. Luria broth (LB) medium was used for general E. coli cultures.…”

Section: Strains and Growthmentioning

confidence: 99%

“…E. coli MG1655(DE3) was used as the host for metabolic engineering. 28 Various genes affected the carbon flux flow to succinate production, including the tricarboxylic acid cycle, glyoxylate shunt pathway, and byproduct pathways (Figure 3). To further increase the metabolic flux toward succinate, isocitrate lyase regulator (iclR), succinate dehydrogenase (sdhAB), and malic enzymes (maeB) were deleted.…”

Section: Cooperative Metabolic Engineering Strategy To Produce Succin...mentioning

confidence: 99%

Metabolic Engineering for the Comprehensive Utilization of N,N-Dimethylformamide-Containing Wastewater

Chen

Wang

et al. 2022

J. Agric. Food Chem.

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N, N-dimethylformamide is frequently present in industrial wastewater and is environmentally detrimental. The current study aims to assess the utilization and biodegradation of N, N-dimethylformamide-containing wastewater to lessen the associated environmental load. Results show that addition of wastewater containing N, N-dimethylformamide to Trichoderma reesei fermentation media enhances cellulase production and facilitates cellulose hydrolysis. However, N, N-dimethylformamide is a cellulase enhancer that is not degraded during cellulase production in T. reesei fermentation and is retained in the N, Ndimethylformamide-enhanced cellulase solution. Indeed, the cellulosic sugar solution generated via lignocellulose hydrolysis with N, N-dimethylformamide-enhanced cellulase retains N, N-dimethylformamide. We further identified three core enzyme modules�N, N-dimethylformamidase, dimethylamine dehydrogenase, and methylamine dehydrogenase enzyme�which were inserted into Escherichia coli to develop metabolically engineered strains. These strains degraded N, N-dimethylformamide and produced succinate using N, N-dimethylformamide-enhanced cellulosic sugar as the substrate. The platform described here can be applied to effectively convert waste into valuable bioproducts.

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Chaperone–substrate interactions monitored via a robust TEM-1 β-lactamase fragment complementation assay

Abstract: The β-lactamase PCA system faithfully reports chaperone-substrate interactions in the bacterial cell envelope, and therefore this system has the potential to map the complex protein homeostasis network under a fluctuating environment.

Cited by 4 publications

References 30 publications

The Lipoprotein NlpE Is a Cpx Sensor That Serves as a Sentinel for Protein Sorting and Folding Defects in the Escherichia coli Envelope

The Lipoprotein NlpE Is a Cpx Sensor That Serves as a Sentinel for Protein Sorting and Folding Defects in the Escherichia coli Envelope

De novo 2′‐fucosyllactose bioproduction through modular engineering of a novel Escherichia coli K12‐derived strain

Metabolic Engineering for the Comprehensive Utilization of N,N-Dimethylformamide-Containing Wastewater

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