Molecular and functional characterization of the HipBA toxin/antitoxin module of Shewanella oneidensis and its possible involvement in persistence

Bacteria are an essential part of human life and the environment; well established examples include the human enteric flora and the role of bacteria in the biogeochemical cycling of elements. Nevertheless, we mainly regard them as undesired guests, as e.g. pathogens causing disease, food spoilage and contamination of water supplies. For years, researchers have tried to find ways to control and eradicate bacteria, but despite numerous efforts the mechanisms behind their astounding capacity to survive are still not fully understood. About three decades ago research took an interesting turn, as it was recognized that bacteria are rarely living as free-living, planktonic organisms, but rather are organized, attached to a surface, in complex bacterial communities called U+2018biofilmsU+2019. Bacteria in these biofilms proved to be even harsher to eradicate. While research has mainly focused on the resistance mechanisms of the majority of a bacterial biofilm population, recently, the existence of a small subpopulation was discovered, one that is not resistant, but rather tolerant to a wide variety of stresses. These bacteria were called U+2018persistersU+2019 and they were proposed to be accountable for the chronic nature of infections and the ineradicable nature of bacteria living in a biofilm. In Part I of this thesis (U+2018LiteratureU+2019), a comprehensive background is given that aims to provide insight in the phenomenon of persistence. It gives an introduction about how bacteria adapt to an environment that can be both unpredictable and unfavourable. Further it is discussed what characterizes the persistence state and which genes are possibly involved. Then, the focus is on toxin-antitoxin (TA) systems, as they are proposed to play a major role in the formation of persister cells. In particular, attention is given to the hipBA system of E. coli, as it constitutes the first and best characterized U+2018persisterU+2019 operon. The role of HipBA in persistence in E. coli and the lack of information about the role of HipBA in other organisms prompted further investigation. The aim of this work was to obtain insights into the function of HipBA in another organism, S. oneidensis. This organism is regarded as a model organism, mainly for its properties in processes of dissimilatory metal reduction, redox processes in general, and biogeochemical cycling processes. However, detailed information about the phenomenon of persistence in this organism is lacking. Interestingly, in a screening of a series of transposon mutants for altered biofilm behaviour, carried out in our laboratory (L-ProBE), hipA was identified as a mutant that produced less biofilm. Part II (U+2018ResultsU+2019), Chapter 1 reports on the molecular characterization of the HipBA module in S. oneidensis. Here, we describe the HipBAS.O. operon organization, the HipA kinase activity and discuss the physiological implications of loss of the HipBA module. We also report on HipBsS.O. binding to the regulatory region of the hipBA operon and the involvement of cooperativity in this process. In contrast to the E. coli HipBA module, we find no evidence for the role of the ribosomal elongation factor Ef-Tu as HipA target. Hence the functional role of HipA in S. oneidensis remains to be determined. Chapter 2 of this section describes the development of a tandem affinity purification (TAP) strategy for the identification of protein interaction partners in S. oneidensis. This method was applied for the elucidation of the functional context of HipAS.O. in order to gain insight in its cellular function. Because the classical TAP tag is a rather large tag and might interfere with proper expression and functioning of the target protein, we developed a new TAP method based on fusion to a new tag, the Strep-6His tag. Chapter 3 presents directions for future research unraveling the functional role of HipBA in S. oneidenis. It includes preliminary results on the crystal and solution structural analysis of HipB, and the implications on HipB binding to the hipBA promoter region.

Publication year:2013

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