Design and Performance of a 16 S rRNA-Targeted Oligonucleotide Probe for Detection of Members of the Genus Bdellovibrio by Fluorescence In Situ Hybridization

Bdellovibrio and like organisms (BALOs) are predatory gram-negative bacteria that are ubiquitous in terrestrial and aquatic environments. Prey include free-living bacteria as well as plant, animal, and human pathogens (12, 13, 17). The life cycle of Bdellovibrio has two major stages: a motile, nonreplicative stage spent searching for prey (the attack phase) and a stage spent inside the periplasm of the gram-negative prey cell after the formation of an osmotically stable body termed the bdelloplast (the growth phase) (15, 27). Until recently, all bacteria that exhibited these life cycle traits were included in the genus Bdellovibrio. Phylogenetic studies based on 16S rRNA sequences have demonstrated extensive diversity among the organisms in this genus, and two new genera have been established: Bacteriovorax (4) and Peredibacter (8). To date, the genus Bdellovibrio comprises only one species, Bdellovibrio bacteriovorus. However, a Bdellovibrio-like organism (BLO) with a different life cycle was isolated from sewage on lawns of Caulobacter crescentus cells. This epibiotic strain, JSS, does not enter the periplasmic space of the prey cell (20, 26). Phylogenetic studies show that it belongs to the genus Bdellovibrio (8) and is a new species (S. F. Koval, unpublished data). Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has become a commonly used technique for the direct identification of prokaryotes and is widely used to determine bacterial community composition (2, 10). Previously designed BALO-targeted oligonucleotides for use in FISH were not specific enough for the identification of the genus Bdellovibrio, as the targets detected included non-BALOs (8, 16). However, Bdellovibrio-specific primers were used to detect Bdellovibrio sequences in soil (9). In this study, an oligonucleotide probe designated BDE525, targeting a region of the 16S rRNA gene sequence of Bdellovibrio, was designed. The probe was used in a FISH procedure with B. bacteriovorus strains, other BLO isolates, and environmental samples. Cultures. B. bacteriovorus strain 109J was cultured on Escherichia coli ML35 according to the method of Flannagan et al. (11). B. bacteriovorus 6-5-S was cultured on Aquaspirillum serpens VHL and BLO strains JSS and KL1 were cultured on C. crescentus CB2A as described previously by Koval and Hynes (20). BLO strains DM7C, DM8A, and DM11A were cultured on Burkholderia cenocepacia strains grown in Lennox LuriaBertani medium (Difco) for 24 h at 30°C. For the maintenance of predators, 3 ml of prey cells and 1 ml of predators were mixed in 20 ml of HM buffer (3 mM HEPES [Sigma], pH 7.6, with 1 mM CaCl2 and 0.1 mM MgSO4). Cocultures were incubated at 30°C for 18 to 24 h with vigorous aeration until prey cells were lysed. The prey-independent strain B. bacteriovorus 109JA and the facultative predator Bacteriovorax stolpii UKi2 were grown on PY medium (peptone [Bacto] at 10 g liter , yeast extract at 3 g liter , pH 6.8 to 7.2) (27) for 24 h at 30°C. Shewanella baltica 63 (NCTC 10735; obtained from T. J. Beveridge, University of Guelph) was grown aerobically in Trypticase soy medium for 24 h at 30°C. Cells were harvested for fixation when the coculture was composed of a mixture of bdelloplasts and attack-phase cells of predators. B. bacteriovorus 109JA, Bacteriovorax stolpii UKi2, and S. baltica 63 were harvested in log phase after 24 h of incubation. Cells were concentrated by centrifugation of 20 ml of each culture at 10,000 g for 15 min at 4°C. Each pellet was resuspended in 2 ml of phosphate-buffered saline (PBS; pH 7.4) and fixed in a 1:3 dilution of neutral 10% buffered formalin (pH 7.0; 10 ml of 36 to 38% formaldehyde [Sigma], 0.65 g of sodium phosphate dibasic [anhydrous], 0.4 g of sodium phosphate monobasic, 90 ml of double-distilled water) on ice at 4°C for 3 h. Cells were then centrifuged, washed twice in 20 ml of PBS, resuspended in 2 ml of PBS–96% ethanol solution (1:1), and stored at 20°C prior to hybridization. Probe design. An oligonucleotide probe was designed using the ARB probe tool (http://www.arb-home.de). The design of the oligonucleotide probe was based on the phylogeny of Bdellovibrio inferred by 16S rRNA sequence analysis. A search for a region that would allow the recognition of all nine clusters of the Bdellovibrio clade established by Davidov and Jurkevitch (8) did not reveal a suitable probe, which indicates the broad phylogenetic diversity of BALOs. The sequence of an oligonucleotide probe (E. coli positions 525 to 542) designated BDE525 (Table 1), or S-G-Bde-0525-a-A-18 according to the Oligonucleotide Database Project (1), was chosen, as it per* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1. Phone: (519) 661-3439. Fax: (519) 661-3499. E-mail: skoval@uwo.ca. Published ahead of print on 28 September 2007.