Gene Expression under High Hydrostatic Pressure in Escherichia coli
Takako SATO*, Chiaki KATO, and Koki HORIKOSHI
The DEEPSTAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima- cho, Yokosuka, 237 Japan
High hydrostatic pressure is one of the most characteristic features of the deep- sea extreme environment. In our laboratory, several barotolerant and barophilic bacteria have been isolated from deep-sea samples (1). According to the phylogenetic analyses of 16S rDNA sequences from these bacteria, they belong to the Proteobacteria )γ-subgroup. Escherichia coli is a terresterial bacterium, though it also belongs to this phylogenetic group and grows under moderate hydrostatic pressure. To study the adaptive mechanism of gene expression to high pressure, E. coli can be used as a simplified model, as it has been well characterized by molecular genetics.
First, we examined the effect of pressure on gene expression controlled by various promoters in E. coli (2). To simplify the analysis of gene expression, several recombinant plasmids were constructed. Chloramphenicol acetyltransferase (CAT) was used as a reporter gene for measurement of gene expression, and positioned downstream of the promoter region. The CAT activities of E. coli transformed by these plasmids and grown under different pressures were determined. As a result, the CAT activities (the gene expression by lac and tac promoters coded on the plasmid pUC13 and pKK233-3, respectively) were increased by more than 90 times at 30 MPa and 50 MPa respectively, compared with at 0.1 MPa without any inducers like isopropy-β-D-thiogalactoside (IPTG) (3). However, gene expression by the other promoters (antitet, tet, amp and cat) were not affected under high pressures. Using primer extension analysis, we found that the highest abundance of mRNAs were transcribed from lac andtac promoters at high hydrostatic pressures. This illustrated an increase of mRNAs from both promoters at different pressures whereas the transcriptional start point was not changed by elevated pressure.
Next, we observed that the formation of plaques by a λ, phage in E. coli was prevented by elevated pressure (4). The numbers of phage plaques were compared from 0.1 MPa up to 30 MPa, no plaques were detected at 30 MPa. To check the details of this phenomenon, gene expression of the malB operon under high pressure was examined. We used CAT as a reporter gene placed downstream of two promoters (malK-lamB and malEGF). The CAT activities derived from both promoters were reduced by elevated pressure in the existence of an inducer, maltose. This phenomenon of gene expression being controlled by elevated pressure may be
