| Summary: | 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 of === Microbiology and Immunology, Department of === Graduate
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