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Survival of Escherichia coli cells on solid copper surfaces is increased by glutathione.

ABSTRACT: Bacteria are rapidly killed on solid copper surfaces, so this material could be useful to limit the spread of multiple-drug-resistant bacteria in hospitals. In Escherichia coli, the DNA-protecting Dps protein and the NADH:ubiquinone oxidoreductase II Ndh were not involved in tolerance to copper ions or survival on solid copper surfaces. Decreased copper tolerance under anaerobic growth conditions in the presence of ascorbate and with melibiose as the carbon source indicated that sodium-dependent symport systems may provide an import route for Cu(I) into the cytoplasm. Glutathione-free ?copA ?gshA double mutants of E. coli were more rapidly inactivated on solid copper surfaces than glutathione-containing wild-type cells. Therefore, while DNA protection by Dps was not required, glutathione was needed to protect the cytoplasm and the DNA against damage mediated by solid copper surfaces, which may explain the differences in the molecular mechanisms of killing between glutathione-containing Gram-negative and glutathione-free Gram-positive bacteria.

SUBMITTER: Große C

PROVIDER: S-EPMC4249004 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Survival of Escherichia coli cells on solid copper surfaces is increased by glutathione.

Große Cornelia C Schleuder Grit G Schmole Christin C Nies Dietrich H DH

Applied and environmental microbiology 20140905 22

Bacteria are rapidly killed on solid copper surfaces, so this material could be useful to limit the spread of multiple-drug-resistant bacteria in hospitals. In Escherichia coli, the DNA-protecting Dps protein and the NADH:ubiquinone oxidoreductase II Ndh were not involved in tolerance to copper ions or survival on solid copper surfaces. Decreased copper tolerance under anaerobic growth conditions in the presence of ascorbate and with melibiose as the carbon source indicated that sodium-dependent ...[more]

PMID: 25192999

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Project description:Artificial laboratory evolution was used to produce mutant strains of Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) able to survive on antimicrobial metallic copper surfaces. These mutants were 12- and 60-fold less susceptible to the copper-mediated contact killing process than their respective parent strains. Growth levels of the mutant and its parent in complex growth medium were similar. Tolerance to copper ions of the mutants was unchanged. The mutant phenotype remained stable over about 250 generations under nonstress conditions. The mutants and their respective parental strains accumulated copper released from the metallic surfaces to similar extents. Nevertheless, only the parental strains succumbed to copper stress when challenged on metallic copper surfaces, suffering complete destruction of the cell structure. Whole-genome sequencing and global transcriptome analysis were used to decipher the genetic alterations in the mutant strains; however, these results did not explain the copper-tolerance phenotypes on the systemic level. Instead, the mutants shared features with those of stressed bacterial subpopulations entering the early or "shallow" persister state. In contrast to the canonical persister state, however, the ability to survive on solid copper surfaces was adopted by the majority of the mutant strain population. This indicated that application of solid copper surfaces in hospitals and elsewhere has to be accompanied by strict cleaning regimens to keep the copper surfaces active and prevent evolution of tolerant mutant strains.IMPORTANCE Microbes are rapidly killed on solid copper surfaces by contact killing. Copper surfaces thus have an important role to play in preventing the spread of nosocomial infections. Bacteria adapt to challenging natural and clinical environments through evolutionary processes, for instance, by acquisition of beneficial spontaneous mutations. We wish to address the question of whether mutants can be selected that have evolved to survive contact killing on solid copper surfaces. We isolated such mutants from Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) by artificial laboratory evolution. The ability to survive on solid copper surfaces was a stable phenotype of the mutant population and not restricted to a small subpopulation. As a consequence, standard operation procedures with strict hygienic measures are extremely important to prevent the emergence and spread of copper-surface-tolerant persister-like bacterial strains if copper surfaces are to be sustainably used to limit the spread of pathogenic bacteria, e.g., to curb nosocomial infections.

Project description:Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population called persister cells to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically repressed state. We occasionally noticed a high variation in persister levels, and to explore this, we tested clonal populations starting from a single cell using a modified Luria-Delbrück fluctuation test. Although we kept the conditions same, the diversity in persistence level among clones was relatively consistent: varying from ~60- to 100- and ~40- to 70-fold for ampicillin and apramycin, respectively. Then, we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for approximately five generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (>seven generations). Our result demonstrates (1) the existence of a small population prepared for stress ("primed cells") resulting in higher persister numbers; (2) the primed memory state is reproducible and transient, passed down for generations but eventually lost; and (3) a heterogeneous persister population is a result of a transiently primed reversible cell state and not due to a pre-existing genetic mutation. IMPORTANCE Antibiotics have been highly effective in treating lethal infectious diseases for almost a century. However, the increasing threat of antibiotic resistance is again causing these diseases to become life-threatening. The longer a bacteria can survive antibiotics, the more likely it is to develop resistance. Complicating matters is that non-genetic factors can allow bacterial cells with identical DNA to gain transient resistance (also known as persistence). Here, we show that a small fraction of the bacterial population called primed cells can pass down non-genetic information ("memory") to their offspring, enabling them to survive lethal antibiotics for a long time. However, this memory is eventually lost. These results demonstrate how bacteria can leverage differences among genetically identical cells formed through non-genetic factors to form primed cells with a selective advantage to survive antibiotics.

Project description:UnlabelledTo survive environmental challenges, biological systems rely on proteins that were selected by evolution to function in particular cellular and conditional settings. With the advent of protein design and synthetic biology, it is now possible to construct novel proteins that are not biased by eons of selection in natural hosts. The availability of these sequences prompts us to ask whether natural biological organisms can use naïve-non-biological-proteins to enhance fitness in stressful environments. To address this question, we transformed a library of DNA sequences encoding ?1.5 × 10(6) binary patterned de novo proteins into E. coli, and selected for sequences that enable growth in concentrations of copper that would otherwise be toxic. Several novel sequences were discovered, and one of them, called Construct K (ConK), was studied in detail. Cells expressing ConK accumulate approximately 50% less copper than control cells. The function of ConK does not involve an oxidase, nor does it require two of the best characterized copper efflux systems. However, the ability of ConK to rescue cells from toxic concentrations of copper does require an active proton motive force. Further selections for growth in higher concentrations of copper led to the laboratory evolution of variants of ConK with enhanced levels of activity in vivo. These studies demonstrate that novel proteins, unbiased by evolutionary history in the natural world, can enhance the fitness of biological systems.SynopsisLiving systems evolve to adapt to potentially lethal environmental changes. This normally involves repurposing existing genetic information (i.e. sequences that were selected by billions of years of evolution). Here we show that a completely de novo protein, not derived from nature, can enable E. coli cells to grow in otherwise toxic concentrations of copper, demonstrating that living systems also have the capacity to incorporate and protopurpose entirely novel genetic information.

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Survival of Escherichia coli cells on solid copper surfaces is increased by glutathione.

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Survival of Escherichia coli cells on solid copper surfaces is increased by glutathione.

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