Isolation and Characterization of Temperature-Sensitive Mutants of Host-Dependent Bdellovibrio bacteriovorus 109D

Abstract

A variety of temperature-sensitive mutants of host-dependent Bdellovibrio bacteriovorus 109D were selected after ethyl methane sulfonate mutagenesis. Mutants that demonstrated plaque-forming ability reversion frequencies of 10 −8 to 10 −9 were chosen for further study. Representatives of these mutants were then characterized by phase-contrast and electron microscopy, temperature-shifted one-step growth experiments, attachment kinetics, and macromolecular capabilities. Representative mutants demonstrate various types of blockage corresponding to the previously described morphological stages of Bdellovibrio predatious life cycle, i.e., attachment blockage (109D153), penetration blockage (109D3 and 109D48), and blockage of intracellular growth (109D4 and 109D152). The time of release from temperature repression for the mutant classes was found to correspond to the apparent morphological stage of blockage via temperature-shifted, one-step growth experiments. Mutants characterized as exhibiting blockage in the penetration or intracellular stages of the infection cycle exhibited, at the permissive and nonpermissive temperatures, kinetics of attachment to Escherichia coli WP2 similar to those of the wild type. One mutant, 109D153, exhibited depressed attachment at the restrictive temperature even though the Bdellovibrio cells were motile. The extent of 38.5 C attachment of 109D153 to E. coli is at the same level as that of wild-type 109D to Bacillus subtilis , a gram-positive, non-host organism. Subsequent detachments were revealed in the wild-type 109D- B. subtilis or mutant 109D153- Escherichia coli (38.5 C) cultures. These studies reveal a biphasic attachment phenomenon in the early interaction of Bdellovibrio with its host. It appears that, at the restrictive temperature, 109D153 is capable only of the initial, nonspecific type of attachment.