Dissecting the structural basis of controlled gene expression involved in antibiotic and solvent resistance in P.putida

Antibiotics have played major roles in combating infectious diseases. However, many of the widely used antibiotics are becoming less effective due to the multidrug resistance phenomena developed in bacteria. One of the major methods utilised in bacterial multidrug resistance is the active extrusion of numerous toxic compounds by proteins embedded in the bacterial cell membranes known as multidrug-efflux transporters or efflux pumps. Proteins carry out the majority of the work inside a cell and have well defined 3-dimensional structures. Protein structures determine how they work. The structural information on proteins involved in antibiotical resistance would therefore potentially help us design better antibiotics to combat the bacterial antibiotic resistance. Proteins are extremely small and we need to use a special technique called X-ray crystallography to determine their structures. X-ray crystallography is a challenging technique requiring the production of large amount of proteins and protein crystals. Membrane proteins are often difficult to produce and therefore impose significant challenge to study using X-ray crystallography. Transcription regulators control genes to be expressed and proteins to be produced and they are not often easier to produce in large quantity. Fortunately, some of the transcription regulators for the efflux pumps respond to the same range of toxic compounds as the transporters therefore studying the regulators offer alternative avenue to understand multidrug recognition and resistance.

The proposed study here tries to obtain the structures of the gene regulators that control the efflux pumps expression in the bacterium P. putida. P. putida has the unusual ability of tolerating high concentrations of toxic compounds and is a close relative of P. flourescens and P. aeruginosa, the later commonly found in humans and animals. Three sets of efflux pumps have been identified and four gene regulators were shown to control their gene expressions. Two of the regulators, called TtgR and TtgV, are shown to play important roles on the cell s ability to survive in toxic environment. We plan to study TtgR and TtgV structurally in order to understand how a single protein could recognise a wide range of different molecules and how the binding of the molecules allows the gene expression of the efflux pumps. This structural information obtained here should help us understand how P. putida and other pathogenic bacteria achieve multidrug resistance and could be exploited for developing potential antimicrobials and novel drugs.

Antibiotic resistance is a widely used phenomena in bacteria and is becoming a health problem worldwide. One of the major mechanisms underlying multidrug resistance in both prokaryotes and eukaryotes is the active extrusion of numerous structurally unrelated toxic compounds by membrane proteins known as multidrug-efflux transporters. The broad substrate specificity displayed by these transporters contrasts dramatically with the narrow chemical specificity of the vast majority of ligand-binding proteins. Despite extensive studies, the structural mechanisms that multidrug transporters use to recognize dissimilar compounds remain obscure, primarily because of the lack of structural information on these membrane proteins. Fortunately, the phenomenon of multidrug recognition is not exclusively confined to multidrug transporters, several transcription regulators have been demonstrated to promote transporter expression in response to structurally dissimilar toxic compounds. Therefore the elucidation of the regulator proteins offers alternative avenues for understanding the mechanism of bacterial multidrug resistance and potential antimicrobials and novel drug development.
The proposed study here tries to characterise structurally the set of regulators that control the efflux pumps in the best characterised P.putida DOT-T1E strain. P.Putida has the unusual property of tolerating high concentration of toxic compounds including antibiotics and organic solvents and is a close relative to P. Florescens and P. Aeruginosa, the latter being highly pathogenic to humans and animals. Therefore P. putida could serve as a model system to study antibiotic resistance and help to understand the multidrug resistant phenomena in other pathogens. Three sets of efflux pumps have been identified and four regulators were shown to control their gene expressions in P.Putida DOT-T1E. Two of the regulators, TtgR and TtgV, are shown to have a significant impact on the cell s ability to survive in toxic environment. We plan to use biochemical and structural techniques, primarily X-ray crystallography, to characterise the binding properties of TtgR and TtgV to antibiotics/organic solvent and their binding to DNA operators in order to understand how a single protein could recognise wide range of substrates and how the binding of the substrates allows the gene expression of the efflux pumps. TtgR and the corresponding efflux pumps is one of the few that have been shown to respond to both antibiotics and organic solvents. The structural information obtained and mechanisms derived should help us understand how P.Putida and other bacteria achieve multidrug and solvent resistance and provide avenues for future antimicrobials and drug design.