IDENTIFICATION OF GENES AFFECTING PRODUCTION OF THE ADHESION ORGANELLE OF CAULOBACTER-CRESCENTUS CB2

Transposon (Tn5) mutagenesis was used to identify regions in the genome involved with production, regulation, or attachment to the cell surface of the adhesive holdfast of the freshwater bacterium Caulobacter crescentus CB2. A total of 12,000 independently selected transposon insertion mutants were screened for defects in adhesion to cellulose acetate; 77 mutants were detected and examined by Southern blot hybridization mapping methods and pulsed-field gel electrophoresis. Ten unique sites of Tn5 insertion affecting holdfast function were identified that were clustered in four regions of the genome. Representative mutants of the 10 Tn5 insertion sites were examined by a variety of methods for differences in their phenotype leading to the loss of adhesiveness. Four phenotypes were identified: no holdfast production, production of a smaller or an altered holdfast, production of a holdfast that was unable to remain attached to the cell, and a fourth category in which a possible alteration of the stalk was related to impaired adhesion of the cell. With the possible exception of the last class, no pleiotropic mutants (those with multiple defects in the polar region of the cell) were detected among the adhesion-defective mutants. This was unexpected, since holdfast deficiency is often a characteristic of pleiotropic mutants obtained when selecting for loss of other polar structures. Overall, the evidence suggests that we have identified regions containing structural genes for the holdfast, genes involved with proper attachment or positioning on the caulobacter surface, and possibly regions that regulate the levels of holdfast production.