Pseudomonas stutzeri Has Two Closely Related pilA Genes (Type IV Pilus Structural Protein) with Opposite Influences on Natural Genetic Transformation

Abstract

Pseudomonas stutzeri has type IV pili for which the pilA gene (here termed pilAI) provides the structural protein and which are required for DNA uptake and natural genetic transformation. Downstream of pilAI we identified a gene, termed pilAII, coding for a deduced protein with a size similar to that of PilAI with 55% amino acid sequence identity and with a typical leader peptide including a leader peptidase cleavage site. Fusions to lacZ revealed that pilAII is expressed only about 10% compared to pilAI, although the genes are cotranscribed as shown by reverse transcription-PCR. Surprisingly, insertional inactivation of pilAII produced a hypertransformation phenotype giving about 16-fold-increased transformation frequencies. Hypertransformation also occurred in pilAI pilAII double mutants expressing heterologous pilA genes of nontransformable bacteria, like Pseudomonas aeruginosa or Dichelobacter nodosus. The overexpression of pilAII decreased transformation up to 5,000-fold compared to that of the pilAII mutant. However, neither inactivation of pilAII nor its overexpression affected the amounts of [ 3 H]thymidine-labeled DNA that were competence-specifically bound and taken up by the cells. In the pilAII mutant, the transformation by purified single-stranded DNA (which depends on comA and exbB, as does transformation by duplex DNA) was also increased 17-fold. It is concluded that PilAII suppresses a step in transformation after the uptake of duplex DNA into the cell and perhaps before its translocation into the cytoplasm. The idea that the degree of the transformability of cells could be permanently adjusted by the expression level of an antagonistic protein is discussed.The gram-negative bacterium Pseudomonas stutzeri is ubiquitous and lives in terrestrial habitats and freshwater and marine environments. It is among the species capable of natural genetic transformation (7,21). This horizontal gene transfer mechanism involves the active uptake of free DNA by the cell from the environment and the heritable incorporation of its informational content into the genome. Transformation is thought to contribute to adaptation of populations to a changing environment and thereby to evolution and speciation (12,21). Recently it was shown by electron microscopy that cells of P. stutzeri strain JM300 have type IV pili (15), which are found in a large number of diverse gram-negative bacteria (18, 41). Type IV pili can mediate the interaction of bacteria with surfaces, including those of epithelial cells, and may allow the flagellum-independent translocation of bacteria on solid surfaces, such as on agar medium (41, 45). The phenomenon was termed twitching motility and has been observed in about 20 species, including P. stutzeri (5,18,41,45). Mutations preventing pilus formation abolished twitching motility in P. stutzeri (15). It was also found that the pili allow the infection of P. stutzeri by the pilus-specific phage PO4 (15), which was initially isolated with Pseudomonas aeruginosa as a host (4). Several genes involved ...

Type IV Pilin Proteins: Versatile Molecular Modules

“…Mutation of pilAI abolished piliation, while a pilAII mutant expressed surface pili and had increased transformability (159). Interestingly, the hypertransformation phenotype was maintained in a P. stutzeri pilAI pilAII double mutant expressing pilins from nonnaturally transformable species such as P. aeruginosa (159).…”

Biology of Pseudomonas stutzeri

“…They are probably located in the cytoplasm and in the inner membrane, respectively (125 (56,71,210,262,403). Surprisingly, the genetic inactivation of P. stutzeri pilAII produced a hypertransformation phenotype (127). It has been suggested that the role of PilAII is to interfere with DNA transport within the cell following DNA uptake.…”

“…Although much information has been obtained for Bacillus subtilis and Neisseria gonorrhoeae-see a review by Chen and Dubnau (67)-the transformation machinery of P. stutzeri has been studied only recently (125)(126)(127)(128)220). It has been demonstrated that P. stutzeri naturally transforms both duplex and single-stranded DNA using the same machinery.…”

“…PilAII therefore acts as a factor that is antagonistic to genetic transformation. Its controlled expression defines the level of transformability shown by naturally competent P. stutzeri cells (127). The second step of transformation consists of the translocation of DNA from the periplasm to the cytoplasm.…”

Regulation of Hypercompetence in Legionella pneumophila

“…The phenotype of enhanced competence, termed hypercompetence, has been described previously for bacterial mutants with defects in regulatory factors that increase or deregulate expression of the competence regulon (24,28,33,49,53). Hypercompetence can also result from mutations in components of the uptake machinery, as seen in P. stutzeri (18). Finally, hypercompetence can be induced indirectly: for example, an H. influenzae mutant with an altered peptidoglycan biosynthesis gene causes induction of the normal competence pathway (32).…”