Home » Key Scientific Articles » Putrescine controls the formation of Escherichia coli persister cells tolerant to aminoglycoside netilmicin.

Putrescine controls the formation of Escherichia coli persister cells tolerant to aminoglycoside netilmicin.

Tkachenko AG, Kashevarova NM, Karavaeva EA, Shumkov MS.

FEMS Microbiol Lett. 2014 Oct 3.

Laboratory of Microbial Adaptation, Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, Russia.

 

Abstract

Persisters are suggested to be the products of a phenotypic variability that are quasi-dormant forms of regular bacterial cells highly tolerant to antibiotics. Our previous investigations revealed that a decrease in antibiotic tolerance of Escherichia coli cells could be reached through the inhibition of key enzymes of polyamine synthesis (putrescine, spermidine). We therefore assumed that polyamines could be involved in persister cellformation. Data obtained in our experiments with the polyamine-deficient E. coli strain demonstrate that the formation of persisters tolerant tonetilmicin is highly upregulated by putrescine in a concentration-dependent manner when cells enter the stationary phase. This period is also accompanied by dissociation of initially homogenous subpopulation of persister cells to some fractions differing in their levels of tolerance tonetilmicin. With three independent experimental approaches, we demonstrate that putrescine-dependent upregulation of persister cell formation is mediated by stimulation of rpoS expression. Complementary activity of putrescine and RpoS results in ~ 1000-fold positive effect on persister cellformation.

© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

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Significance  statement

Polyamines (putrescine, spermidine and spermine) are essential for normal cell growth in both prokaryotic and eukaryotic cells. Data obtained in our experiments with the polyamine-deficient E. coli strain revealed that the formation of persisters tolerant to netilmicin was highly upregulated by polyamines in a concentration-dependent manner when cells entered the stationary phase. Transition of polyamine-supplemented cells to stationary phase was accompanied by dissociation of initially homogenous subpopulation of persister cells to some fractions widely differing in their tolerance to netilmicin. Formation of more tolerant persister fractions was enhanced at later stages of stationary phase.

Nowadays, a set of genes whose expression is specifically stimulated by polyamines at the level of translation is defined as a “polyamine modulon”. Some of these genes, including rpoS, are only active in the stationary phase and so can apparently be considered as polyamine targets implicated in the formation of persister cells. While in this aspect rpoS is of great interest, especially in terms of the implication in general stress response, the question of RpoS involvement in cell survival to antibiotics or persister cell formation is under debate. With the polyamine-deficient E. coli mutant we have shown that rpoS expression was upregulated by polyamines during stationary phase. As a result, an increase in cell RpoS protein enhanced persister cell formation. These data were obtained with three independent experimental approaches: (i) supplementation of polyamine-deficient mutant harboring rpoS::lacZ fusion with putrescine, (ii) an ectopic expression of rpoS overexpressing plasmid and (iii) reciprocal experiments with the pair of isogenic E. coli K12 rpoSand rpoS+ strains. Therefore, complementary activity of RpoS and putrescine resulted in almost 1000-fold positive effect on persister cell formation.

Polyamine modulon includes undoubtedly only one part of a number of genes that can be implicated in persister cell formation. Cooperative activity of all these genes in their multifarious combinations is rather responsible for the formation and heterogeneity of persister cells.

 

Figure Legend

 

Persister cell formation as a function of putrescine-modulated rpoS expression during E. coli transition to stationary phase. E. coli SHT03 cultures were grown in accordance with the standard procedure. At zero time (A600≈0.5), one of them was supplemented with 5.0 mM putrescine (Pt+), whereas the other one continued to grow without putrescine (Pt).  Cells taken from both cultures (as designated by arrows) were tested for levels of rpoS expression (blue lines) and cell RpoS protein with Western blot (photo on the top). Moreover, samples removed from (Pt+) and (Pt) -cultures were also tested for the frequency of persister cells tolerant to 2.8 µg mL-1 netilmicin (red lines).

Putrescine controls the formation of Escherichia coli persister cells tolerant to aminoglycoside netilmicin