Transposon Mutant Library Reveals Genes Involved in Biofilm Formation by Listeria monocytogenes 568 at a Simulated Food Plant Environmental Temperature of 15°C

Introduction: Listeria monocytogenes is a pathogenic foodborne microorganism noted for its ability to persist in the food processing environment. This phenomenon is attributed to the bacterium’s ability to form biofilms. However, much of the research on biofilm formation in this organism has been carried out at 37°C rather than temperatures more commonly associated with food processing.

Purpose: The purpose of this study was to identify the genes involved with biofilm formation of L. monocytogenes at 15°C.

Methods: A library of 11,000 Listeria monocytogenes 568 (serotype 1/2a) transposon mutants was created with plasmid pMC39. Biofilms were formed at 15°C and mutants with reduced or enhanced biofilm formation were selected after staining with crystal violet or safranin. The susceptibility of the mutants’ pre-formed biofilm to benzalkonium chloride was also determined.  Arbitrary PCR was used to generate sequence data and identify gene interruption sites.

Results: A total of 40 mutants were found to express altered phenotypes in two or three of the assays. Insertions were mapped into genes that encode for cell wall biosynthesis, motility, metabolism, and cell surface associated proteins. Benzalkonium chloride susceptible biofilm mutants contained interruptions in genes related to metabolism or cell surface proteins. In contrast, biofilm mutants selected in both the safranin and crystal violet assays were found to harbor the transposon in more varied targets, including three internalins and genes related to cell wall biosynthesis and motility.

Significance: This study carried out at a temperature common in food production environments revealed possible involvement of several genes not previously linked to biofilm formation as well as of genes also associated with biofilm formation at higher temperatures.