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Identification and cloning of DNA affecting production of the adhesive holdfast of Caulobacter crescentus CB2 Mitchell, David Walter


As part of a preliminary investigation into the biological and chemical properties of the freshwater Caulobacter crescentus strain CB2 holdfast (Hf), this research entailed the identification, phenotypic characterization, and cloning of genomic DNA that in some way contribute to the production or regulation of the Hf. Random transposon (Tn5) mutagenesis was used to identify genomic DNA encoding Hf-related functions. A library of approximately 16,000 independently selected mutants (P > 98%) was prepared from which approximately 12,000 colonies (P = 95%) were screened for 77 Hf-mutants that were aberrant in the ability to form large cellular masses adjoined at the Hf (termed rosettes) and/or the ability of colonies to adhere to cellulose-acetate. Ten unique sites of Tn5-insertion were identified from Southern blots of chromosomal DNA from all screened mutants using restriction enzymes that cut once or twice within the Tn5 element. Subsequently, using restriction enzymes that cut frequently or infrequently outside of the Tn5 element, the ten sites of Tn5-insertion were found to be clustered in four genomic regions. Restriction fragments of Tn5-mutated genomic DNA that were shown to contain all of the identified sites of Tn5-insertion within a given cluster were cloned from one representative mutant of each of the four regions. In attempting to ascribe functions to the regions of DNA that encode Hf-related functions, the phenotype of Hf-mutants was investigated on the basis of Hf-adhesiveness to glass and cellulose-acetate and on the basis of lectin-binding to Hf. Four phenotypes were identified: no Hf produced, a low amount of Hf produced, Hf produced and shed into the medium, and a fourth category where a possible alteration of stalk was related to impaired adhesion of the Hf material. The shedding phenotype is of particular interest as a possible means of isolating both wildtype and mutant Hf material for chemical analysis. To this end, a gene replacement strategy was used to transfer a Tn5-mutated DNA fragment cloned from one Hf-mutant into a previously prepared ultraviolet light-induced Hf-shedding mutant. Double mutants confirmed for both shedding and Tn5-induced Hf characteristics were isolated.

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