A conjugation procedure for Bdellovibrio bacteriovorus and its use to identify DNA sequences that enhance the plaque-forming ability of a spontaneous host-independent mutant

Abstract

Wild-type bdellovibrios are obligate intraperiplasmic parasites of other gram-negative bacteria. However, spontaneous mutants that can be cultured in the absence of host cells occur at a frequency of 10(-6) to 10(-7). Such host-independent (H-I) mutants generally display diminished intraperiplasmic-growth capabilities and form plaques that are smaller and more turbid than those formed by wild-type strains on lawns of host cells. An analysis of the gene(s) responsible for the H-I phenotype should provide significant insight into the nature of Bdellovibrio host dependence. Toward this end, a conjugation procedure to transfer both IncQ and IncP vectors from Escherichia coli to Bdellovibrio bacteriovorus was developed. It was found that IncQ-type plasmids were capable of autonomous replication in B. bacteriovorus, while IncP derivatives were not. However, IncP plasmids could be maintained in B. bacteriovorus via homologous recombination through cloned B. bacteriovorus DNA sequences. It was also found that genomic libraries of wild-type B. bacteriovorus 109J DNA constructed in the IncP cosmid pVK100 were stably maintained in E. coli; those constructed in the IncQ cosmid pBM33 were unstable. Finally, we used the conjugation procedure and the B. bacteriovorus libraries to identify a 5.6-kb BamHI fragment of wild-type B. bacteriovorus DNA that significantly enhanced the plaque-forming ability of an H-I mutant, B. bacteriovorus BB5.

Identification of a Bdellovibrio bacteriovorus genetic locus, hit, associated with the host-independent phenotype

“…All of the recipients were therefore merodiploid for the cloned sequences. Southern blot analysis has shown that integration of B. bacteriovorus DNA-containing constructs occurs via homologous recombination between the cloned B. bacteriovorus insert and the equivalent region of the recipient genome (3).…”

“…The conjugal transfer of plasmids from E. coli to B. bacteriovorus was done as described elsewhere (3). All of the genetic experiments described here involved the conjugal transfer of IncP plasmids containing B. bacteriovorus DNA and their integration into the recipient B. bacteriovorus genome via homologous recombination.…”

“…All bdellovibrio strains were singleplaque or single-colony purified and stored in 15% glycerol at -80°C. Media and culture conditions used for the propagation of all E. coli and B. bacteriovorus strains were as described elsewhere (3).…”

The dual probiotic and antibiotic nature of Bdellovibrio bacteriovorus

“…This changed in 1992 when Cotter and Tomashow tried to determine the genetic locus (hit locus) associated with the shifting to host independent mode of life (12,13) …”

Bdellovibrio exovorus sp. nov., a novel predator of Caulobacter crescentus

“…HM buffer [3 mM HEPES (pH 7.6) with 1 mM CaCl 2 and 0.1 mM MgSO 4 ] was used for further studies, to correlate with other studies on Bdellovibrio bacteriovorus 109J (Thomashow & Rittenberg, 1978;Flannagan et al, 2004). Dilute nutrient broth medium (Cotter & Thomashow, 1992) may also be used for co-cultures. For maintenance, co-cultures containing 1 ml strain JSS T and 4 ml of a 24 h peptone-yeast extract (PYE) broth culture (Johnson & Ely, 1977;Koval & Hynes, 1991) of C. crescentus CB2A were incubated in 125 ml flasks with 20 ml HM buffer.…”