Development of a two-dimensional electrophoresis method to study soil bacterial diversity

Abstract

In recent years, culture-independent methods based on polymerase chain reaction (PCR) have been developed for the study of environmental bacteria. Denaturing gradient gel electrophoresis (DGGE) is one of the most useful of these methods. However, in the DGGE method, the low resolution of gel electrophoresis compared with the high diversity of soil bacterial communities can be a problem. Soil communities contain several thousand species of bacteria, but the resolution of more than 20–50 bands on a gel is difficult. To overcome these problems, we designed a two-dimensional electrophoresis (2-DE) method that combines size separation and the DGGE methods. In the present study, bacterial DNA was first used to design a protocol for 2-DE using PCR products of bacterial 16S rDNA. After examining different 2-DE approaches, the bacterial 16S rDNA could be separated successfully using the 2-DE described in this study. This protocol was applied to an environmental sample (i.e. soil) and the results showed that one band of size separation or DGGE produced two or more spots after 2-DE, thereby providing additional useful information about the bacterial population in the environment. Our results suggest that the new 2-DE method may be useful to monitor soil microbial populations in detail.

Key words:

• bacterial diversity
• DGGE
• size separation
• 2-D electrophoresis

ACKNOWLEDGMENTS

This work was supported by the 21st Century Center of Excellence (COE) program of the Ministry Education, Culture, Sports, Science and Technology of Japan, ‘Bio-Eco Environmental Risk Management.’