Identification and cloning of the genes required for production of the adhesive holdfast organelle of the marine Caulobacter MCS6

Caulobacters produce an organelle called a holdfast which allows the bacterium to firmly attach to surfaces. Tn5 insertion mutagenesis was used to identify genes affecting holdfast production or function in the marine strain MCS6. 12,000 Tn5- insertion mutants were produced and screened for adhesion defects by a newly developed assay involving the growth of cultures in polystyrene microtitre dish wells and detection of attached cells by staining with crystal violet. Among adhesion-defective mutants, those with multiple polar (pleiotropic) defects were excluded and the remainder were examined for the ability of Wheat Germ agglutinin to label the holdfast region, a feature of the wild-type holdfast. 41 mutants were isolated that produced no detectable or synthesized a reduced amount of holdfast. Southern blot and pulsed field gel electrophoresis (PFGE) analyses of these mutants indicated 11 unique sites of Tn5 insertion, clustered in three regions of the genome. In addition, 71 mutants were found that did not adhere to polystyrene or adhered poorly yet still produced a holdfast organelle as judged by Wheat Germ agglutinin binding. Southern blot and PFGE analyses of 15 of these mutants showed eight Tn5 insertion sites clustered in two regions of the genome. Glass slides treated with silane chemicals (producing surfaces with varying degrees of hydrophobicity and/or hydrophilicity) were used to attempt characterization of this phenotype; unexpectedly no generic pattern of differences in binding was found between the mutants and wild-type Caulobacters. In particular, no reduction in the ability of the mutants to bind to hydrophobic surfaces was noted; this might have been expected considering the inability to bind polystyrene. As a means of confirming their unique location and character of each genomic region, representatives of all 5 clusters were chosen and the Tn5-interrupted regions were cloned. The gene segments obtained were in turn used as probes to identify corresponding holdfast-related chromosome regions in a cosmid library of wild-type MCS6 DNA. Positive cosmid clones were transferred into the Tn5 -derived holdfast-defective mutants by conjugation. Among 6 groups tested, 4 of them showed almost full complementation, one showed poor complementation; for one group no complementation was obtained. In sum, several genetic regions responsible for holdfast mediated attachment in marine Caulobacter MCS6 were identified by Tn5 insertion mutagenesis. They were located at several discrete locations on the genome and, via the cloned segments obtained, are now accessible for further genetic analysis.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat