Molecular characterization of the bacterial predator Bdellovibrio

Abstract

Bdellovibrios (Bd) are unique, predatory bacteria which prey upon a wide variety of susceptible Grain-negative bacteria. Microorganisms exhibiting this trait have been included in the genus Bdellovibrio despite their diverse habitats. Currently, identification of bdellovibrios relies on culture-based methodology which can be non-specific, time-consuming and labor intensive. The lack of rapid, specific tools for identification and differentiation of Bdellovibrio strains has made studies examining the ecological impact of these predators difficult, leading investigators to suggest only limited roles for the predators in aquatic environments. In this study, three molecular fingerprinting techniques (arbitrarily-primed PCR [AP-PCR], pulsed field gel electrophoresis [PFGE], and ribotyping) were selected for the identification and characterization of different Bdellovibrio isolates. In the AP-PCR procedure, genomic DNA was amplified with 10--25 mer primers. For the PFGE procedure, total unsheared genomic DNA was digested with the restriction enzymes NotI, AscI, SmaI and SacII. The strains were characterized by ribotyping with probes complementary to E. coli 16S and 23S rRNA genes; hybridization patterns were analyzed with a fingerprinting software package. AP-PCR, PFGE and ribotyping readily differentiated between different Bdellovibrio isolates, demonstrating that B. bacteriovorus strains show little similarity to the other Bdellovibrio species. Finally, the evolutionary relationship between Bdellovibrio isolates was examined by 16S rDNA sequence analysis and DNA-DNA homology studies. The 16S rRNA gene from 14 Bdellovibrio strains was amplified with rDNA-targeted oligonucleotide primers and sequenced. 16S rDNA sequence analysis indicated that the B. bacteriovorus strains clustered together as a single group. B. starrii , B. stolpii, and the environmental terrestrial isolate Gunpowder clustered with the marine strains, with this group showing only a distant relationship to the B. bacteriovorus cluster. These results demonstrate that the genus Bdellovibrio consists of a very heterogeneous group of microorganisms. Based on the low homology values (<16%) and <85% 16S rDNA sequence similarity, these results support the re-classification of B. stolpii, B. starrii, Gunpowder and the marine isolates into a separate genus.