Predation by Bdellovibrio bacteriovorus HD100 requires type IV pili

Abstract

Early electron microscopy and more recent studies in our laboratory of Bdellovibrio bacteriovorus cells indicated the presence of narrow fibers at the nonflagellar pole of this unusual predatory bacterium. Analysis of the B. bacteriovorus HD100 genome showed a complete set of genes potentially encoding type IV pili and an incomplete gene set for Flp pili; therefore, the role of type IV pili in the predatory life cycle of B. bacteriovorus HD100 was investigated. Alignment of the predicted PilA protein with known type IV pilins showed the characteristic conserved N terminus common to type IVa pilins. The pilA gene, encoding the type IV pilus fiber protein, was insertionally inactivated in multiple Bdellovibrio replicate cultures, and the effect upon the expression of other pilus genes was monitored by reverse transcriptase PCR. Interruption of pilA in replicate isolates abolished Bdellovibrio predatory capability in liquid prey cultures and on immobilized yellow fluorescent protein-labeled prey, but the mutants could be cultured prey independently. Expression patterns of pil genes involved in the formation of type IV pili were profiled across the predatory life cycle from attack phase predatory Bdellovibrio throughout the intraperiplasmic bdelloplast stages to prey lysis and in prey-independent growth. Taken together, the data show that type IV pili play a critical role in Bdellovibrio predation.

FIG. 1.

Transmission electron micrographs of Bdellovibrio invasion and anterior fibers. (A and B) Wild-type HD100 Bdellovibrio invading E. coli S17-1 prey; prey invasion is accomplished through polar entry of the Bdellovibrio into the prey periplasm. (C) Anterior pilus-like fibers on HD100. The circle indicates the position of fiber attachment to the cell. Pilus-like fibers were seen on approximately 30% of cells examined (n > 1,500). (D) Anterior pilus-like fibers on B. bacteriovorus strain 109J. The circle indicates the position of fiber attachment to the cell pole. Pilus-like fibers were seen at roughly the same frequency as in HD100 cultures (n > 1,000). Bar, 0.2 μm.

FIG. 2.

Alignment of type IVa pilin proteins (8) showing Bdellovibrio bacteriovorus pilA to have the highly conserved N-terminal sequence homology common among type IVa pilins and phenylalanine that forms the N-terminal residue in the mature protein (indicated by the arrow). The processed Bdellovibrio peptide is predicted to be 179 amino acids in length. N. gonorrhoeae, Neisseria gonorrhoeae; D. nodosus, Dichelobacter nodosus. Black shading indicates identity of conserved residues; grey shading indicates conservatively substituted homologous residues. The N terminus of the mature protein is indicated by the arrow.

FIG. 3.

(A and B) Predation tests of pilA_HI::Km and pilA_HI/pilA::Km on immobilized YFP-labeled E. coli S17-1 prey cells on plates of PY medium supplemented with 50 μg ml⁻¹ kanamycin (17). The pilA_HI::Km mutant was seen to be nonpredatory, while the merodiploid pilA_HI/pilA::Km strain showed rounded bdelloplasts containing Bdellovibrio and thus normal predation. (C) HID2, a wild-type HD100-derived HI strain, predating on immobilized YFP host grown with no kanamycin and given the same number of bdelloplasts as the merodiploid pilA_HI/pilA::Km strain (n > 2,500) E. coli cells observed per strain after incubation for 24 h. (D) Transmission electron microscopy of pilA_HI/pilA::Km (frame i). Polar pilus fibers were seen at a frequency of approximately 20 to 30%, matching the frequency seen in wild-type pilA⁺ HID2 (n > 700). Transmission electron micrographs of pilA_HI::Km strains (frames ii and iii). No polar fibers were seen in the samples examined (n > 1,500). All samples were stained with 1% uranyl acetate. Bar, 0.2 μm.

FIG. 4.

Comparison of the operon structure of pil genes in M. xanthus and Bdellovibrio. Some cognate genes are highlighted in different colors and shapes to facilitate comparison. (A) Organization of Myxococcus pil genes, taken from Touhami et al. (33). (B) pil genes are scattered around the Bdellovibrio genome, but a Myxococcus-like, possibly ancestral, organization can be seen with the gene order being conserved. The starred annotated pilT within the operon is a good homologue, but Bd3852 on the left is a better homologue, suggesting a duplication event. Bdellovibrio does not have significant homologues of pilR2/S2. (C) RT-PCR on RNA isolated from different time points during the HD100 life cycle and HI growth using primers designed to amplify internal fragments of HD100 pil genes. Lanes are as labeled; markers are NEB 100-bp ladder. Taq PCR controls were performed for each template and were negative for DNA contamination of RNA (data not shown). (D) qRT-PCR on RNA isolated from attack phase and 30 min post-prey infection using primers designed to amplify an internal fragment of the pilA gene.