Phylogenic analysis of bacteria passed through 0.45-mu m-pore-size filters in the rhizosphere

Molecular analyses of 16S ribosomal RNA genes (rDNA) revealed that microbial communities in the rhizosphere are highly complex. To systematically characterize the cell size of the bacteria in the rhizosphere, we selected bacteria that potentially could be passed through 0.22- or 0.45-mu m-pore-size filters and then PCR amplified the 16S rDNA genes using the universal primer pairs 27F/1492R and 63F/1387R. The PCR-amplified rDNAs extracted from bacteria that had been passed through 0.45-mu m-pore-size filters could be detected in agarose gels after electrophoresis; whereas after filtration of the bacteria through 0.22-mu m-pore-size filters no PCR-amplified rDNAs were found. Comparison of random cloning and sequencing of the libraries of the PCR-amplified rDNAs with or without cell size selection showed that bacteria belonging to the candidate phylogenic divisions of OD1 (OP11-derived 1), OP11, TM7, and OP5 can be concentrated by cell size selection using 0.45-mu m-pore-size filters, but not by using 0.22-mu m-pore-size filters. OD1, OP11, TM7 and OP5 bacteria have yet to be cultivated; therefore, our concentration method may be used as an initial step in studies to analyze the structural properties of 001, OP11, and TM7 bacteria.