Type IV pilus biogenesis, twitching motility, and DNA uptake in Thermus thermophilus: discrete roles of antagonistic ATPases PilF, PilT1, and PilT2

Abstract

Natural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species. Thermus thermophilus HB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whether T. thermophilus has any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that in T. thermophilus, T4P and natural transformation are linked but distinct systems.

FIG 1

(A) Domain organization and kanamycin cassette insertion site in PilF; (B) organization of the pilF wild-type (wt) and mutant loci.

FIG 2

Electron micrographs of a representative sample of the T. thermophilus HB27 wild type (A), the ΔpilF deletion mutant (B), and the pilF::kat insertion mutant (C). Electron microscopic investigations were conducted by shadowing the cells with platinum-carbon. A total of 250 to 300 cells of each strain were analyzed. Bars = 500 nm.

FIG 3

Natural competence of T. thermophilus HB27 and the complemented ΔpilF::bleo (pilF) mutant.

FIG 4

Twitching motility of cells of the T. thermophilus HB27 wild type (A), the pilF::kat insertion mutant (B), and the ΔpilF deletion mutant (C). The cells were inoculated on minimal medium containing 1% BSA and cultivated for 72 h at 64°C under a water-saturated atmosphere. Agar and cells were stained with Coomassie blue. The cells and the Coomassie dye were removed, and the twitching zone corresponding to the noncolored agar region was determined. The pictures were inverted. Experiments were done in triplicate and gave identical results. Bars = 0.5 cm.

FIG 5

Adhesion of cells of the T. thermophilus wild type (A), the pilF::kat insertion mutant (B), and the ΔpilF deletion mutant (C). Cells were stab inoculated through the solid medium down to the petri dish and cultivated on minimal medium containing 1% BSA for 72 h at 64°C. After removal of the agar, the petri dish was washed four times and adhering cells were stained with Coomassie blue. Experiments were done in triplicate and gave identical results. Bars = 0.5 cm.

FIG 6

Transcriptional organization of the pilF locus. To examine the transcriptional organization, RNA was isolated and subjected to RT-PCR analyses. (A) The primers and expected fragments are indicated and labeled 1 to 4. RNA was used as a negative control; chromosomal DNA was used as a positive control. (B) The resulting DNA fragments were separated on a 0.8% agarose gel. Lanes 1, chromosomal DNA from T. thermophilus; lanes 2, cDNA from T. thermophilus HB27; lanes 3, cDNA from T. thermophilus pilF::kat; lanes 4, RNA control.

FIG 7

Piliation of T. thermophilus and hyperpiliation of the pilT1 and pilT2 mutants. Electron microscopic investigations were conducted by shadowing the cells with platinum-carbon. A total of 250 to 300 cells were analyzed, and the results for one representative sample of cells of the wild type (A), the pilT1 mutant (B), and the pilT2 mutant (C) are shown. Bars = 500 nm.

FIG 8

Transformation frequencies of T. thermophilus HB27, the pilT1::kat (TTC1621) and ΔpilT2::hygro (TTC1415) mutants, and the pilT1 pilT2 double mutant.