iTRAQ-based quantitative proteomic analysis of Pseudomonas aeruginosa SJTD-1: A global response to n-octadecane induced stress.

N-octadecane, the shortest solid-state alkane, was efficiently consumed by Pseudomonas aeruginosa SJTD-1. To reveal its mechanism, the iTRAQ-LC-MS/MS strategy was applied for quantification of proteins in response to alkane. As a result, 383 alkane-responsive proteins were identified and these proteins could be linked to multiple biochemical pathways. Above all, the level of alkane hydroxylase AlkB2 has been significantly higher in alkane condition. Also, the presence of a putative novel AlmA-like monooxygenase and its role on alkane hydroxylation were firstly proposed in Pseudomonas. In addition, other proteins for chemotaxic, ß-oxidation, glyoxylate bypass, alkane uptake, cross membrane transport, enzymatic steps and the carbon flow may have important roles in the cellular response to alkane. Most of those differently expressed proteins were functionally mapped into pathways of alkane degradation or metabolism thereof. In this sense, findings in this study provide critical clues to reveal biodegradation of long chain n-alkanes and rationally be important for potent biocatalyst for bioremediation in future. BIOLOGICAL SIGNIFICANCE: We use iTRAQ strategy firstly to compare the proteomes of Pseudomonas SJTD-1 degrading alkane. Changes in protein clearly provide a comprehensive overview on alkane hydroxylation of SJTD-1, including those proteins for chemotaxis, alkane uptake, cross membrane transport, enzymatic steps and the carbon flow. AlkB2 and a putative novel AlmA-like monooxygenase have been highlighted for their outstanding contribution to alkane use. We found that several chemotaxic proteins were altered in abundance in alkane-grown cells. These results may be helpful for understanding alkane use for Pseudomonas.

Expression and characterization of thymine-DNA glycosylase from Aeropyrum pernix.

The recombinant thymine-DNA glycosylase (TDG) from Aeropyrum pernix (A. pernix) was expressed in Escherichia coli. The enzymatic activity of recombinant A. pernix TDG (ApeTDG) was characterized using oligonucleotides containing a thymine/uracil base as substrate. ApeTDG had distinct glycosylase activity on T/G mismatch. The optimal temperature and pH for thymine removal were 65-70 degrees C and pH 7.0-8.5, respectively. High concentration of NaCl inhibited the thymine removal. Divalent ions had different influence on the thymine removal by ApeTDG. Ca(2+) and Mg(2+) had no inhibition on the enzymic activity, but Ni(2+), Co(2+), Cu(2+), Mn(2+), and Zn(2+) completely inhibited the excision reaction. As derived from a hyperthermophilic archaea, ApeTDG protein was heat-resistant at 75 degrees C. ApeTDG also had a relatively weak DNA glycosylase activity on uracil base, with the following order: U/C>U/G approximately U/T>U/U approximately U/I approximately U/AP approximately U/->U/A. Additional mismatch located at 3' of T/G had less inhibition on the thymine removal than that located at 5' of T/G, and two additional mismatches located at each side of T/G completely inhibited the excision of thymine. Together, these data suggest that ApeTDG is a TDG protein with weak UDG activity.