Stabilization of Homoserine-O-Succinyltransferase (MetA) Decreases the Frequency of Persisters in <i>Escherichia coli</i> under Stressful Conditions

<div><p>Bacterial persisters are a small subpopulation of cells that exhibit multi-drug tolerance without genetic changes. Generally, persistence is associated with a dormant state in which the microbial cells are metabolically inactive. The bacterial response to unfavorable environmental conditions (heat, oxidative, acidic stress) induces the accumulation of aggregated proteins and enhances formation of persister cells in <i>Escherichia coli</i> cultures. We have found that methionine supplementation reduced the frequency of persisters at mild (37°C) and elevated (42°C) temperatures, as well as in the presence of acetate. Homoserine-<i>o</i>-succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway, is prone to aggregation under many stress conditions, resulting in a methionine limitation in <i>E. coli</i> growth. Overexpression of MetA induced the greatest number of persisters at 42°C, which is correlated to an increased level of aggregated MetA. Substitution of the native <i>metA</i> gene on the <i>E. coli</i> K-12 WE chromosome by a mutant gene encoding the stabilized MetA led to reduction in persisters at the elevated temperature and in the presence of acetate, as well as lower aggregation of the mutated MetA. Decreased persister formation at 42°C was confirmed also in <i>E. coli</i> K-12 W3110 and a fast-growing WErph+ mutant harboring the stabilized MetA. Thus, this is the first study to demonstrate manipulation of persister frequency under stressful conditions by stabilization of a single aggregation-prone protein, MetA.</p></div

Stabilized MetA decreases the frequencies of persisters in different <i>E. coli</i> strains at elevated temperature.

<p>Cells of the strains W3110 and W3110-LYD (A), WErph⁺ and WErph⁺-LYD (B), grown overnight for 16 h in M9 glucose medium at 37 or 42°C, were diluted to an OD₆₀₀ of 0.1 in fresh M9 glucose medium supplemented with ampicillin and incubated at 37°C for 10 hours. Samples were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110504#s2" target="_blank">Materials and Methods</a>.</p

Influence of stabilized MetA protein on the <i>E.coli</i> WE strain growth under stressful conditions.

<p>The WE and WE-LYD strains were incubated in M9 glucose medium at 44°C for 10 h (A) or in M9 glucose medium (pH 6.0) supplemented with 20 mM sodium acetate at 37°C for 28 h (B) in an automatic growth-measuring incubator. The average of two independent experiments is presented.</p

Effect of L-methionine on the frequency of persisters at different temperatures.

<p>The 16-h cultures of the strains WE (A, B) and JW0195 (C) grown in M9 glucose medium with or without L-methionine (50 µg/ml) at 37 or 42°C were diluted to an OD₆₀₀ of 0.1 in fresh M9 glucose medium supplemented with ampicillin (A, C) or ofloxacin (B) and incubated at 37°C for 10 hours. Samples were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110504#s2" target="_blank">Materials and Methods</a>.</p

Dependence of persister formation on stabilized MetA protein.

<p>Overnight cultures of the strains WE and WE-LYD grown for 16 h in M9 glucose medium at 37 or 42°C were diluted to an OD₆₀₀ of 0.1 in fresh M9 glucose medium supplemented with ampicillin (A) or ofloxacin (B) and incubated at 37°C for 10 hours. Samples were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110504#s2" target="_blank">Materials and Methods</a>. Soluble and insoluble protein fractions were purified from the cultures grown in M9 glucose medium at 37 or 42°C to an OD₆₀₀ = 1.0, subjected to 12% SDS-PAGE followed by Western blotting using rabbit anti-MetA antibody (C). The MetA in the samples was quantified through densitometry using WCIF ImageJ software. The MetA amount from the WE cells grown at 37°C was set to 1 (D). The data are presented as the average of two independent experiments.</p

Effect of the stabilized MetA on the persister cell frequency under acidic conditions.

<p>Cultures of WE and WE-LYD grown for 16 h in M9 glucose medium (pH 6.0) at 37°C with or without sodium acetate (20 mM; A); with or without L-methionine (50 µg/ml) and in the presence of sodium acetate (20 mM; B) were diluted in fresh M9 glucose medium to an OD₆₀₀ of 0.1, supplemented with ampicillin and incubated at 37°C for 10 hours. Samples were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110504#s2" target="_blank">Materials and Methods</a>. Soluble and insoluble protein fractions were purified from the 16 h-cultures grown in M9 glucose medium (pH 6.0) with or without sodium acetate (20 mM), and subjected to 12% SDS-PAGE, followed by Western blotting using rabbit anti-MetA antibody (C). The MetA in the samples was quantified through densitometry using WCIF ImageJ software. The amount of MetA in the WE cells grown without sodium acetate was set to 1 (D). The data are presented as the average of two independent experiments.</p

Strains and plasmids used in this study.

<p>Ap^r, ampicillin resistance; <i>kan</i>, kanamycin resistance gene.</p><p>Strains and plasmids used in this study.</p

Effect of the MetA overexpression on persister formation at different temperatures.

<p>Strain WE harboring the <i>metA</i> gene under pBAD promoter was grown in LB medium at 37 or 42°C with or without arabinose (10 mM) for 24 h, diluted to an OD₆₀₀ of 0.1 in fresh LB medium supplemented with ampicillin and incubated at 37°C for 10 hours. Samples were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110504#s2" target="_blank">Materials and Methods</a> (A). Soluble and insoluble protein fractions were isolated from the late-stationary phase cultures (24 h) grown in LB medium, subjected to 12% SDS-PAGE followed by Western blotting using rabbit anti-MetA antibody (B). The MetA in the samples was quantified through densitometry using WCIF ImageJ software. The MetA amount from the cells grown at 37°C without arabinose was set to 1 (C). The error bars represent the standard deviations of duplicate independent cultures.</p

Stability of lipase displayed on the surface of GM730/pJHC13 cells (■) and free form lipase (▲) during incubation at 37°C in a two-phase water-isooctane reaction system

<p><b>Copyright information:</b></p><p>Taken from "Display of a thermostable lipase on the surface of a solvent-resistant bacterium, GM730, and its applications in whole-cell biocatalysis"</p><p>BMC Biotechnology 2006;6():23-23.</p><p>Published online 19 Apr 2006</p><p>PMCID:PMC1459859.</p><p>Copyright © 2006 Jung et al; licensee BioMed Central Ltd.</p> Residual lipase activities were measured spectrophotometrically with pNPP as a substrate and calculated by assuming the initial activity was 100%

Synthesis of triacylglycerol by lipase displayed on the GM730/pJHC13 cell surfaces in the isooctane solvent reaction system at 37°C

<p><b>Copyright information:</b></p><p>Taken from "Display of a thermostable lipase on the surface of a solvent-resistant bacterium, GM730, and its applications in whole-cell biocatalysis"</p><p>BMC Biotechnology 2006;6():23-23.</p><p>Published online 19 Apr 2006</p><p>PMCID:PMC1459859.</p><p>Copyright © 2006 Jung et al; licensee BioMed Central Ltd.</p> Conversion yield during incubation is shown