Physiological and molecular responses to water stress of a polycyclic aromatic hydrocarbons degrading bacteria isolated from Patagonian soil

MADUEÑO L.; COPPOTELLI BM; ALVAREZ HM; MORELLI I

Copenhagen

Simposio; 14th International Symposium on Mycrobial Ecology “The Power of the small”; 2012

14th International Symposium on Mycrobial Ecology “The Power of the small”

The oil industry is the main economical activity in Patagonia (Argentina). During crude oil extraction, transportation and storage, some places result contaminated with hydrocarbons. One of the strategies used for pollutant removal from soil is the inoculation with degrading bacteria, which increase the mineralization process. The extreme conditions of soil of semiarid Patagonia; which is poor in inorganic and organic nutrients, and in water content, may limit the bacterial survival and activity during bioremediation. For this reason, the use of indigenous microorganisms adapted to environmental conditions of the region is advisable. We isolated a bacterial strain (22B) from Patagonian soil identified as Sphingobium sp., with the ability to degrade polycyclic aromatic hydrocarbons. Moreover, strain 22B showed high tolerance to the main stresses occurring in Patagonian soil (desiccation and carbon and nitrogen starvation). The aim of this work was to analyze 22B proteome under desiccation in order to improve our understanding of the strain responses to the critical stress found in Patagonian soil. For this purpose, two cell cultures grown for 48 hours (OD600 2) in Liquid Mineral Mediun (LMM) with 0.1g/l of SO4(NH4)2 and 1% of Glucose was filtered (0.45 m) in order to collect the cells. Filters of one culture were placed in a desiccator at 18% Relative Humidity for 4 days, and filters of the other culture were resuspended in LMM without stress. Cells in two conditions were lysed and soluble cellular protein fractions were analyzed by two dimensional gel electrophoresis and mass spectrometry (MALDI-TOF/TOF MS), within the window of isoelectric point (pI) 4-7 and a molecular mass range of 6.5 to 200 kDa. Counts of heterotrophic populations in R2 Agar and gravimetric assays were performed in order to determine water loss and cell survival. Cell physiology was analyzed by optical microscopy using staining with Sudan Black B. In spite of that filters lost 93.8 ± 0.1% of water after 4 days of incubation under desiccation conditions, cell survival accounted up to 80.6± 2.6%. Comparative proteomic analyses to identify intracellular proteins involved in the response of 22B to desiccation showed 4 spots overexpressed in drying. Identified up-regulated proteins included an acetyl-CoA C-acetyltransferase (AtoB) probably involved in the metabolism of polyhydroxybutyrate (PHB) (spot A); an hypothetical protein with homology to an osmotically inducible sensory protein of a marine bacterium and with a glycine-rich protein which is expressed in response to drying by several microorganisms (spot B); a glyceraldehyde-3-phosphate dehydrogenase, which may be involved in carbohydrate metabolism probably related to the biosynthesis of compatible solutes or an extracellular polymeric substance (EPS) (spot C); and a protein with homology to channel protein OmpA (OOP-family protein) which are usually involved in the response to stress in several bacteria. In addition, physiological studies demonstrated the ability of strain 22B to synthesize PHB and an EPS during nutritional stress and desiccation. The proteins identified in this study may be part of an integral program response of cells to desiccation, including mechanism to withstand osmotic stress, carbon starvation, and the production of resistance structures like EPS.