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Journal of Bacteriology, August 2001, p. 4694-4701, Vol. 183, No. 16
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.16.4694-4701.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Requirement of Novel Competence Genes pilT and pilU of Pseudomonas stutzeri for Natural Transformation and Suppression of pilT Deficiency by a Hexahistidine Tag on the Type IV Pilus Protein PilAI

Stefan Graupner, Nicole Weger, Monika Sohni, and Wilfried Wackernagel*

Genetik, Fachbereich Biologie, Carl von Ossietzky Universität Oldenburg, D-26111 Oldenburg, Germany

Received 21 February 2001/Accepted 22 May 2001

The ubiquitous species Pseudomonas stutzeri has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termed pilT. The deduced amino acid sequence has identity with PilT of Pseudomonas aeruginosa (94%), Neisseria gonorrhoeae (67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [3H]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of pilT a gene, termed pilU, coding for a putative protein with 88% amino acid identity with PilU of P. aeruginosa was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable P. aeruginosa. When the pilAI gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in pilAI::Gmr cells but allowed transformability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the pilT mutation. Considering the hypothesized role of pilT+ in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the pilT-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.

* Corresponding author. Mailing address: Genetik, Fachbereich Biologie, Carl von Ossietzky Universität Oldenburg, Postfach 2503, D-26111 Oldenburg, Germany. Phone: 49-441-798 3298. Fax: 49-441-798 5606. E-mail: wilfried.wackernagel{at}uni-oldenburg.de.

Journal of Bacteriology, August 2001, p. 4694-4701, Vol. 183, No. 16
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.16.4694-4701.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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