The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence

Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin-antitoxin proteins HipA(SO) and HipB(SO) from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin-antitoxin protein module. Here we show that phosphorylated HipA(SO) can engage in an unexpected ternary complex with HipB(SO) and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipB(SO) in complex with operator DNA reveals a flexible C-terminus that is sequestered by HipA(SO) in the ternary complex, indicative of its role in binding HipA(SO) to abolish its function in persistence. The structure of HipA(SO) in complex with a non-hydrolyzable ATP analogue shows that HipA(SO) autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipA(SO) is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought

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 ‘biofilms’. 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 ‘persisters’ 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 (‘Literature’), 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 ‘persister’ 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 (‘Results’), 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

High-Level Production of Recombinant Eukaryotic Proteins from Mammalian Cells Using Lentivirus

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Toxin–antitoxin systems: their role in persistence, biofilm formation, and pathogenicity

One of the most pertinent recent outcomes of molecular microbiology efforts to understand bacterial behavior is the discovery of a wide range of toxin–antitoxin (TA) systems that are tightly controlling bacterial persistence. While TA systems were originally linked to control over the genetic material, for example plasmid maintenance, it is now clear that they are involved in essential cellular processes like replication, gene expression, and cell wall synthesis. Toxin activity is induced stochastically or after environmental stimuli, resulting in silencing of the above-mentioned biological processes and entry in a dormant state. In this minireview, we highlight the recent developments in research on these intriguing systems with a focus on their role in biofilms and in bacterial virulence. We discuss their potential as targets in antimicrobial drug discovery

Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO2, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAAR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3–4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection