Expression And Characterization Of Isocitrate Lyase Of Shewanella Piezotolerans WP3 In Various Environment

Isocitrate lyase, the key enzyme of glyoxylate cycle of a deep sea bacterium- Shewanella piezotolerans WP3, is unique in genome. ICL competes with isocitrate dehydrogenase of TCA cycle for its common substrate, isocitrate.The bacterium used in this study was isolated from deep-sea sediments of west Pacific at the depth of 1914 meter. The phylogenetic analysis and molecular study shows that the bacterium is a psychrophilic and piezotolerant microbe which may be ideal to study the adaptation mechanism of extremophiles. And the result of microarray of WP3 under various environment factors displays significant changes in the gene expression related with cold and pressure shock, adaptation. The aim of present study is to find the adaptation mechanism of extremophiles and possible role of ICL gene involved in, under the extreme condition of temperature and pressure invitro.The ICL gene of WP3 contrains an openreading frame of 1581bp, encoding a 526aa residues protein with molecular mass of 58kDa. Sequence analysis of this gene revealved that it is highly homologous to the ICL genes of Shewanella species and also has homology to ICL gene of cold adapted Colwellia maris corresponding to some other mesophilic microorganisms. We cloned the ICL gene from WP3 and purified the enzyme to investigate specificity of the enzyme and the metabolic regulation. Then we studied the ICL gene at both transcriptional and translational level using real-time PCR and western blotting, respectively. Furthermore the ICL gene was knocked out to identify the role and function of ICL in isocitrate metabolism by WP3.The ICL of WP3 exhibits many characteristic features of cold-adapted, thermolabile enzymes revealed by enzyme acivity assays and has maximum enzyme activity at 25?C. But it is not a cold-inducible enzyme like that of C.maris. It efficiently responses to varying environment by changing its transcription and translation level. At sharply decreases its expression level with cold, heat shock and increase for the pressuring shock, adaptation. And it suggests that ICL is important for acetate metabolism and is induced transcriptionally and translationally by acetate similar to the ICLs from other organisms. In icl mutant we unexpectly observe the idh gene expression that IDH-II notability reduce the RNA level expression while IDH-I has no change, which means that idh expression does not increase to supply the glyoxylate cycle. The growing conditions at 20?C and 4?C differ relatively little from wide type while ICL is the key enzyme in glyoxylate cycle. These results show that the proportion of glyoxylate cycle in WP3 metabolism is very tiny even at low temperature and TCA cycle is always the main path at kinds of growth conditions. Probably the glyoxylate cycle and TCA cycle in WP3 cooperate for intermedia products instead of competing for substrate.