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Characterization of Listeria innocua biofilm formation using TN917 transposon mutagenesis

by Shaw, William K.

Abstract (Summary)
Listeria sp. can grow on surfaces and be a member of multi-species biofilms within food processing plants, rendering them more resistant to sanitizers and other antimicrobial agents. Little is known of the cellular physiological processes involved in Listeria sp. growth on surfaces. The purpose of this study was to generate random transposon mutants in order to identify genetic determinants contributing to biofilm formation. Listeria innocua ATCC 51742 serotype 4b was selected for this study since it produces high levels of biofilm in a laboratory assay. Transposon mutagenesis was performed using plasmid pLTV3 (Tn917 ) with a temperature sensitive origin of replication. Mutants were selected by growth in the presence of erythromycin and lincomysin at 41�°C. A library, consisting of 2500 mutants, was further screened by use of a PVC microtiter plate assay at 32�°C at low nutrient levels. Reduced surface growth (RSG) mutants were defined as having a mean stained biofilm at 2 std dev below the mean of the wild-type strain. A total of 36 RSG mutants were isolated and further characterized by testing cellular motility and initial adhesion phenotypes. RSG mutants exhibited various phenotype profiles, suggesting a number of genes involved in biofilm formation of L. innocua . RSG 24 mutant was further characterized, showing an increased negative surface charge. RSG 24 mutant scanning electron microscopy and epifluorescence microscopy results showed reduced surface growth area coverage compared to wild-type. An arbitrary PCR, PCR product cloning and sequencing revealed that Tn917 transposition took place in lin2619, a gene homologous to mutarotase. Surface growth characteristics were restored to RSG 24 by complemetation of the lin2619 mutarotase gene in trans to levels comparable to the wild-type strain. Complementation in trans and results indicating altered cell surface characteristics, our study indicates that lin2619 is involved in L. innocua adhesion and surface growth.
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Advisor:

School:University of Massachusetts Amherst

School Location:USA - Massachusetts

Source Type:Master's Thesis

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Date of Publication:01/01/2005

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