Molecular analyses of the natural transformation machinery and identification of pilus structures in the extremely thermophilic bacterium Thermus thermophilus strain HB27

Abstract

Thermus thermophilus HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. To identify genes of the natural transformation machinery of T. thermophilus HB27, we performed homology searches in the partially completed T. thermophilus genomic sequence for conserved competence genes. These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria. Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency. One competence protein is similar to DprA of Haemophilus influenzae, seven are similar to type IV pilus proteins of Pseudomonas aeruginosa or Neisseria gonorrhoeae (PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in Deinococcus radiodurans R1. Analysis of the piliation phenotype of T. thermophilus HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent pil mutants of Thermus, with the exception of the pilF mutant, were devoid of pilus structures. These results suggest that pili and natural transformation in T. thermophilus HB27 are functionally linked.

FIG.1.

Structural organization and gene disruption strategy of conserved ORFs within potential competence loci in the genome of T. thermophilus HB27. Physical map of mutant loci that are involved (A) and not involved (B) in natural transformation. In the restriction maps of gene bank plasmids covering different conserved ORFs of the potential competence loci, only selected restriction sites are shown. The triangle indicating the kat gene denotes the insertion site of the Km^r marker gene. The arrows denote direction of transcription. +, wild-type transformation frequencies; ±, 100-fold-reduced transformation frequencies; −, not transformable. BI, BamHI; BII, BglII; EV, EcoRV; HII, HincII; HIII, HindIII; N, NcoI; P, PstI; XI, XbaI.

FIG. 2.

(A) Comparison of the pilD locus in different gram-negative proteobacteria and nonproteobacteria. orf2073 encodes a thermostable carboxypeptidase and orf2071 encodes a hypothetical protein in Thermus. ssr1853 encodes a hypothetical Synechocystis protein, and sll1059 encodes an adenylate cyclase. DR2065 is a pilD homologue in Deinococcus. The Deinococcus ORFs DR2063, DR2064, and DR2066 encode a polynucleotide phosphorylase, a hypothetical 17.3-kDa protein, and a conserved hypothetical protein, respectively. comC is a pilD homologue in B. subtilis. spoIIB and folC encode a sporulation factor and the enzyme folypoly-γ-glutamate synthetase-dihydrofolate synthetase, respectively. (B) Comparison of the genetic organization of the pilQ cluster in different gram-negative proteobacteria and nonproteobacteria. The Thermus ORFs orf559, orf566, and orf567 encode isopropylmalate dehydrogenase, chorismate synthase, and shikimate kinase, respectively. DR0775 is an aroC homologue and DR0776 is an aroK homologue in Deinococcus. slr1273 and sll1174 are predicted to encode hypothetical proteins in Synechocystis.

FIG. 3.

Representative sample showing the pilus structures on the surface of T. thermophilus HB27 wild-type cells. Electron microscopic investigations were conducted with uranylacetate-stained cells.

FIG. 4.

Alignment of the conserved central and C-terminal part of PilT-like proteins. Identical residues are indicated by grey shadows. The conserved cysteine residues found in PulE homologues are marked by a star (∗) and are boxed. B.s., B. subtilis; K.o., K. oxytoca; N.g. N. gonorrhoeae; T.t., T. thermophilus HB27.