CONICET | Buscador de Institutos y Recursos Humanos

"Developing strategies to assess the distribution and bioremediation potential of chemotactic vs non chemotactic strains" Andrea Pedetta, Karina Herrera Seitz and Claudia Studdert Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata The ability of bacteria to follow gradients of chemicals and concentrate themselves near nutrients or away from toxic substances is called chemotaxis and represents a powerful strategy for survival. On the other hand, it has been shown that some contaminant-degrading bacteria display chemotaxis towards the specific contaminants in laboratory studies. The chemotactic ability might represent a crucial factor in the efficiency of bioremediation in natural environments. The general aim of our work is to establish how the chemotactic behavior affects the rate of hydrocarbon degradation in environment-like setups, and to that end we intend to develop methods to assess the distribution of different strains in those setups. Our model system is Pseudomonas putida KT2440 (che+) and a generally non-chemotactic derivative (che -), both transformed with a plasmid that confers the ability to grow using short-chain alkanes as their only carbon source. Preliminary experiments were performed using other carbon sources for growth and as chemoattractants since they were handled more easily than the highly volatile octane. In order to analyze the distribution pattern of che+ and che- strains in co-inoculation experiments, we used two variants of the plasmid named pFULL and pDEL, the latter of which carried a specific deletion. Two pairs of primers were used to amplify fragments from the plasmids, one pair whose target is missing in pDEL and the other one targeted to both templates. Petri dishes containing different solid or semi-solid supports on which the carbon sources were evenly distributed were inoculated at the center with mixtures of the strains che+/pDEL and che-/pFULL. Samples were taken at different times and at several distances from the point of inoculation and amplification reactions were performed with these samples using both pairs of primers. The ratio che+/total cells was calculated for each sample in order to see the population composition for each time and space point. In soft agar plates, with citrate as the sole carbon source, the population became enriched in che+ cells as they followed the self-generated gradient of nutrients. We discuss the relative distribution of che+ and che- strains in setups intending to mimics soil environment and that are more comparable to real environmental situations than the traditional soft agar plates. As a first approach to compare the degradative efficiency of both strains, parallel experiments were performed in which they were inoculated separately, and the carbon source exhaustion along the whole available space was determined. These methods represent a useful tool that will be used in future microcosms assays to analyze the distribution of inoculated cells in order to evaluate the relevance of chemotaxis in hydrocarbon degradation.