Abstract
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 Escherichia coli 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-o-succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway, is prone to aggregation under many stress conditions, resulting in a methionine limitation in E. coli 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 metA gene on the E. coli 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 E. coli 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.
Highlights
A small subpopulation of bacterial cells, designated persisters, which are able to survive lethal antibiotic treatment and produce a new population of antibiotic-sensitive cells genetically identical to the originals was first described by Joseph W
Exogenous methionine decreased frequency of persisters in the E. coli cells at mild and elevated temperatures Previous findings have revealed that E. coli growth in the defined medium was impaired at elevated temperatures due to methionine limitation resulting from the extreme inherent instability of the first enzyme in the methionine biosynthetic pathway, MetA [20,30]
Because the MetA was completely aggregated at 44uC [21], we studied the effect of temperature and methionine supplementation on persister formation in E. coli K-12 WE cells
Summary
A small subpopulation of bacterial cells, designated persisters, which are able to survive lethal antibiotic treatment and produce a new population of antibiotic-sensitive cells genetically identical to the originals was first described by Joseph W. Persistence arises from the dormant state when the bacterial cells are metabolically inactive [3]; the level of translation is greatly reduced [9], resulting in arrested protein biosynthesis [10]. E. coli cells exposed to thermal stress accumulated a large number of aggregated proteins [18]. Leszczynska et al showed that an increased level of protein aggregates in E. coli stationary-phase cells was strongly correlated with a higher frequency of persister formation [19]. In this context, we asked whether the inherently unstable MetA affects the formation of E. coli persisters under normal or stressful conditions. MetA was found to be extremely sensitive to many stress conditions (e.g., thermal, oxidative or weak-organic-acid stress) [22,23]
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