(152c) Quantitative Proteomic Analysis Of A Soil Bacterium Under Different Levels Of Cadmium Stress

In a previous study, metaproteomics was used to obtain functional information about the response of soil microbial communities to cadmium stress. In that study, we were able to separate and identify a number of proteins that were differentially expressed due to the exposure to toxic levels of cadmium.

In an effort to better understand the roles played by proteins in cadmium resistance mechanisms, we isolated the bacterium most resistant to cadmium. A defined minimal medium was designed to avoid cadmium precipitation, and the microbial consortium was grown in increasing cadmium. During these isolation experiments, only a very small number of distinct colonies were capable of growing in high cadmium-level environments. The bacterium used for this study was selected for its ability to grow in the presence of 100 mg/L cadmium. Its 16S rDNA was sequenced, and the organism was identified as a strain of Burkholderia cepacia.

The specific objective of this investigation was to assess the mechanisms used by this bacterium to adapt to high levels of cadmium. To accomplish this, we used stable isotope metabolic labeling, in which either N-14 or N-15 was supplied as the only nitrogen source in the medium. Two cadmium concentrations were used: 10 and 100 mg/L of cadmium. Cultures were harvested at an OD600 of approximately 0.6, and equal amounts of labeled and unlabeled cultures were combined. Proteins were extracted and analyzed by 2DE and shotgun proteomics. The use of metabolic labeling yielded quantitative information about differential protein expression and increased the confidence level of protein identifications by adding an extra constraint that confirmed the presence of up- or down-regulation of two or more peptides per protein, also in agreement with gel images. A number of those proteins were identified from both workflows after database searching and de novo sequencing. There was a clear difference in the proteomes of the cultures grown on high and low level of cadmium, including proteins involved in energy metabolism, defense and transport. This approach represents one of the first applications of stable isotopic labeling to environmental proteomics of soil isolates.