Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of 3 H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the speciesspecific pilin.The soil bacterium Pseudomonas stutzeri is capable of natural genetic transformation (10). This phenomenon involves the binding of extracellular DNA to the bacterial cell, the active uptake of the bound DNA, and the heritable integration of its genetic information. Natural transformation has been observed in bacterial species from various taxonomic and trophic groups, including Proteobacteria, cyanobacteria, and Archaeobacteria, and is considered a major mechanism of horizontal gene transfer encompassing chromosomal and plasmid DNA (25,45,46).The physiological state in which cells are transformable is termed competence and is reached in the late log phase of broth-grown cultures of P. stutzeri (22). Competence is also achieved in media prepared from aqueous extracts of various soils (23,24). During these studies, it was found that P. stutzeri responds to limitations of single nutrients like C, N, or P by an up-to-290-fold stimulation of transformation (23,24). Other studies showed that P. stutzeri cells can take up DNA adsorbed on the surface of sand grains (22). Further, P. stutzeri is naturally transformable by broad-host-range plasmids like RSF1010 (3), even when these plasmids do not contain inserts of chromosomal P. stutzeri DNA (9). More recently, the transformation of P. stutzeri in...

Formation of sphingosine-1-phosphate (SPP) by sphingosine kinase serves as a signalling pathway for various membrane receptors. Here, we show that membrane depolarisation is another mechanism by which this pathway can be activated. Formation of [ 3 H]SPP as well as levels of endogenous SPP were rapidly and transiently increased in PC12 pheochromocytoma cells depolarised with high KCl. Time course and maximum were similar to those induced by bradykinin. Depolarisation-induced SPP production was also observed in RINm5F insulinoma cells, dependent on extracellular Ca 2+ and fully suppressed by verapamil, thus apparently caused by Ca