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

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

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

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

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

Improved Thermostability and Acetic Acid Tolerance of Escherichia coli via Directed Evolution of Homoserine o-Succinyltransferase▿ †

In Escherichia coli, growth is limited at elevated temperatures mainly because of the instability of a single enzyme, homoserine o-succinyltransferase (MetA), the first enzyme in the methionine biosynthesis pathway. The metA gene from the thermophile Geobacillus kaustophilus cloned into the E. coli chromosome was found to enhance the growth of the host strain at elevated temperature (44°C), thus confirming the limited growth of E. coli due to MetA instability. In order to improve E. coli growth at higher temperatures, we used random mutagenesis to obtain a thermostable MetAE. coli protein. Sequencing of the thermotolerant mutant showed five amino acid substitutions: S61T, E213V, I229T, N267D, and N271K. An E. coli strain with the mutated metA gene chromosomally inserted showed accelerated growth over a temperature range of 34 to 44°C. We used the site-directed metA mutants to identify two amino acid residues responsible for the sensitivity of MetAE. coli to both heat and acids. Replacement of isoleucine 229 with threonine and asparagine 267 with aspartic acid stabilized the protein. The thermostable MetAE. coli enzymes showed less aggregation in vivo at higher temperature, as well as upon acetic acid treatment. The data presented here are the first to show improved E. coli growth at higher temperatures solely due to MetA stabilization and provide new knowledge for designing E. coli strains that grow at higher temperatures, thus reducing the cooling cost of bioprocesses

Chromosome Condensation in the Absence of the Non-SMC Subunits of MukBEF

MukBEF is a bacterial SMC (structural maintenance of chromosome) complex required for chromosome partitioning in Escherichia coli. We report that overproduction of MukBEF results in marked chromosome condensation. This condensation is rapid and precedes the effects of overproduction on macromolecular synthesis. Condensed nucleoids are often mispositioned; however, cell viability is only mildly affected. The overproduction of MukB leads to a similar chromosome condensation, even in the absence of MukE and MukF. Thus, the non-SMC subunits of MukBEF play only an auxiliary role in chromosome condensation. MukBEF, however, was often a better condensin than MukB. Furthermore, the chromosome condensation by MukB did not rescue the temperature sensitivity of MukEF-deficient cells, nor did it suppress the high frequency of anucleate cell formation. We infer that the role of MukBEF in stabilizing chromatin architecture is more versatile than its role in controlling chromosome size. We further propose that MukBEF could be directly involved in chromosome segregation