Far-reaching cellular consequences of tat deletion in Escherichia coli revealed by comprehensive proteome analyses

Dolata, Katarzyna Magdalena; Montero, Isabel Guerrero; Miller, Wayne H.; Sievers, Susanne; Sura, Thomas; Wolff, Christian; Schlüter, Rabea; Riedel, Katharina; Robinson, Colin

doi:10.1016/j.micres.2018.10.008

Cited by 8 publications

(6 citation statements)

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“…Quantification of pure ASNase when supplemented into some typical biochemistry laboratory solutions was next assessed. These solutions were: Tris–HCl buffer, which is commonly used to suspend and store purified ASNase; MgCl 2 solution, frequently used to stabilise E. coli spheroplasts during purification of ASNase by osmotic shock; 21 sucrose buffer, also used during the purification of ASNase from E. coli by the lysozyme method; 17 and CD medium, which is a chemically defined microbial fermentation medium used for high‐cell‐density cultivation of E. coli 16 . Less deviation in ASNase activity was calculated using the method developed herein that used stabilisers, compared to the standard Nessler method, regardless of the buffer tested (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The suitability of the new method was verified by quantification of pure recombinant Escherichia coli ASNase (EC 3.5.1.1) with a specific activity of 269 IU mg −1 (ProsPec‐Tany, Israel) supplemented to a chemically defined medium (CD medium) 16 containing: 5.6 mmol L −1 glucose, 19.5 mmol L −1 KH 2 PO 4 , 6.1 mmol L −1 (NH 4 ) 2 HPO 4 , 1.8 mmol L −1 citric acid, 1.0 mmol L −1 MgSO 4 .7H 2 O, 49.0 μmol L −1 Fe(III) citrate, 15.2 μmol L −1 MnCl 2 , 9.7 μmol L −1 H 3 BO 3 , 7.3 μmol L −1 Zn(CH 3 COO) 2 .2H 2 O, 5.0 μmol L −1 EDTA, 2.7 μmol L −1 thiamine–HCl, 2.1 μmol L −1 CoCl 2 .6H 2 O, 2.1 μmol L −1 Na 2 MoO 4 .2H 2 O and 1.8 μmol L −1 CuCl 2 .2H 2 O. The pure enzyme was also added to a sucrose buffer commonly used in enzymology research, which contained 250 mmol L −1 sucrose, 50 mmol L −1 Tris–acetate pH 8.2 and 18 mmol L −1 MgSO 4 17 . Finally, extracellular ASNase was determined from the fermentation broth of a recombinant strain of Pichia pastoris 18 grown in a buffered glycerol‐complex medium (BMGY) that contained (g L −1 ): 10.0 glycerol, 10.0 yeast extract, 20.0 peptone, 100 mmol L −1 KH 2 PO 4 pH 6.0, 3.4 yeast nitrogen base medium, 10.0 (NH 4 ) 2 SO 4 and 4 mg L −1 biotin.…”

Section: Methodsmentioning

confidence: 99%

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An improved method for simple and accurate colorimetric determination of l‐asparaginase enzyme activity using Nessler's reagent

Simas

Kleingesinds

Pessoa

et al. 2021

J of Chemical Tech & Biotech

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BACKGROUND Colorimetric determination of l‐asparaginase enzyme activity most commonly uses Nessler's reagent, but often yields unreliable results due to precipitation of tetraiodomercurate salts. Interference caused by some commonly used inorganic and organic compounds of biochemical buffers, reagents and microbiology fermentation media was determined. The stabilising effects of tartrate and polyvinyl alcohol upon the impact of these salts was next evaluated using the following two‐step assay in 96‐well microtitre plates: 160 μL of 50 mmol L−1 Tris–HCl pH 8.6, 32 μL of 100 mmol L−1 l‐asparagine, 16 μL analyte and 128 μL water were added to each well. After incubation at 37 °C for 1 h, 16 μL of 1.5 mol L−1 trichloroacetic acid was added to stop the reaction. A 35 μL volume was next transferred to a fresh well containing 35 μL Nessler's reagent, 35 μL stabiliser solution (4 mmol L−1 disodium tartrate and 10 mg L−1 polyvinyl alcohol) and 245 μL water. After incubation at room temperature for 10 min, absorbance was recorded at 436 nm and the enzyme activity calculated by extrapolation of a (NH4)2SO4 calibrated standard curve. RESULTS Addition of tartrate and polyvinyl alcohol proved to be effective in reducing interference caused by precipitation of salts, allowing determination of l‐asparaginase activity in a range from 0.050 to 0.796 IU mL−1, ideal for monitoring enzyme production in fermentations. CONCLUSION A method is offered for rapid and reliable determination of l‐asparaginase activity, particularly in complex samples such as microbial fermentation broths where existing methodologies do not account for interferences that frequently yield overvalues. © 2020 Society of Chemical Industry

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

An improved method for simple and accurate colorimetric determination of l‐asparaginase enzyme activity using Nessler's reagent

Simas

Kleingesinds

Pessoa

et al. 2021

J of Chemical Tech & Biotech

View full text Add to dashboard Cite

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“…Several studies provide evidence that membrane disturbance and oxidative stress are associated with biofilm formation. First, a loss of tat, encoding a twin-arginine translocation (52) pathway with important physiological functions for the bacterial envelope, impairs outer membrane integrity and triggers biofilm formation (52). Second, CpxAR can respond to a cell envelope stress caused by sub-MICs of penicillin G by significantly upregulating the production of matrix components to induce biofilm formation (53).…”

Section: Discussionmentioning

confidence: 99%

A CpxR-Regulated zapD Gene Involved in Biofilm Formation of Uropathogenic Proteus mirabilis

et al. 2020

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Proteus mirabilis, a frequent uropathogen, forms extensive biofilms on catheters that are infamously difficult to treat. To explore the mechanisms of biofilm formation by P. mirabilis, we performed in vivo transposon mutagenesis. A mutant with impaired biofilm formation was isolated. The mutant was found to have Tn5 inserted in the zapD gene, encoding an outer membrane protein of the putative type 1 secretion system ZapBCD. zapBCD and its upstream zapA gene, encoding a protease, constitute an operon under the control of CpxR, a two-component regulator. The cpxR mutant and zapA mutant strains also had a biofilm-forming defect. CpxR positively regulates the promoter activities of zapABCD, cpxP, and cpxR. An electrophoretic mobility shift assay revealed that CpxR binds zapA promoter DNA. The loss of zapD reduced CpxR-regulated gene expression of cpxR, zapA, cpxP, and mrpA, the mannose-resistant Proteus-like (MR/P) fimbrial major subunit gene. The restoration of biofilm formation in the zapD mutant with a CpxR-expressing plasmid reinforces the idea that CpxR-mediated gene expression contributes to zapD-involved biofilm formation. In trans expression of zapBCD from a zapBCD-expressing plasmid also reestablished the biofilm formation ability of the cpxR mutant to a certain level. The zapD and cpxR mutants had significantly lower protease activity, adhesion, and autoaggregation ability and production of exopolysaccharides and extracellular DNA (eDNA) than did the wild type. Finally, we identified copper as a signal for CpxR to increase biofilm formation. The loss of cpxR or zapD abolished the copper-mediated biofilm upshift. CpxR was required for copper-induced expression of zapA and cpxR. Taken together, these data highlight the important role of CpxR-regulated zapD in biofilm formation and the underlying mechanisms in P. mirabilis.

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“…Vancomycin treated cells displayed an altered cell wall without the typical tripartite-layer structure (red arrow), in contrast to control cells that had a normal cell structure (black arrow). Additionally, inclusion bodies structures and areas [59], were observed in the majority of intact cells (white arrows). This suggests that protein aggregation was likely associated with the formation of these vancomycin persisters.…”

Section: Morphological Characterizationmentioning

confidence: 99%

Isolation and characterization of persisters of the pathogenic microorganismStaphylococcus aureus

Liu,

Laman,

Jensen

et al. 2023

Preprint

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Staphylococcus aureusis a notorious pathogen responsible for significant morbidity and mortality in both human society and animal husbandry. The presence ofS. aureuspersisters is also one of the leading causes of recurrent and chronic diseases. Persisters are a subset of growth-arrested bacteria within a susceptible bacterial population that are able to tolerate antibiotic treatment and resuscitate after stress removal. Consequently, investigating their formation and characteristics is of crucial importance to provide mechanism-based options for their eradication. However, one challenge in mechanistic research on persisters is the enrichment of pure persisters. In this work, we validated a proposed method to isolate persisters from vancomycin and enrofloxacin generated persistent populations. With this, we analyzed the proteome profile of pure persisters and revealed the distinct mechanisms associated with vancomycin and enrofloxacin induced persisters. Furthermore, morphological and metabolic characterizations were performed, indicating further differences between these two persister populations. Finally, we assessed the effect of ATP repression, protein synthesis inhibition and reactive oxygen species (ROS) level on persister formation. In conclusion, this work provides a comprehensive understanding ofS. aureusvancomycin and enrofloxacin induced persisters at the molecular, single cell and population levels, facilitating a better understanding of persisters and the development of effective strategies to combat them.

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Far-reaching cellular consequences of tat deletion in Escherichia coli revealed by comprehensive proteome analyses

Cited by 8 publications

References 88 publications

An improved method for simple and accurate colorimetric determination of l‐asparaginase enzyme activity using Nessler's reagent

An improved method for simple and accurate colorimetric determination of l‐asparaginase enzyme activity using Nessler's reagent

A CpxR-Regulated zapD Gene Involved in Biofilm Formation of Uropathogenic Proteus mirabilis

Isolation and characterization of persisters of the pathogenic microorganismStaphylococcus aureus

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