Purpose: The purpose of this study is to synthesize a deletion mutant of Lmo2555 and further characterize the functionality of LTA in L. monocytogenes biofilm formation.
Methods: Deletion mutant of Lmo2555 (Δlmo2555) was created by PCR amplification using two primer pairs, each with restriction sites upstream and downstream of the targeted gene. The two parts were joined together creating a deleted version of Lmo2555, which was cloned into a temperature sensitive shuttle vector pKSV7 in E. coli. The recombinant plasmids were transformed into L. monocytogenes wild type strain (LM21). Allelic exchange was performed in a two-step process. Initially, transformants were maintained under antibiotic selection at an elevated temperature (41 °C) which allows selection of cells which had undergone homologous recombination. Integration was confirmed by PCR amplification with primers specific for downstream region of pKSV7 plasmid and an upstream region of the chromosome. Selection for colonies that had undergone allelic exchange was performed by incubating isolates without antibiotic selection at a plasmid replication permissive temperature (32 °C).
Results: Approximately 360 colonies were screened for plasmid loss by antibiotic sensitivity and allelic exchange was confirmed using PCR amplification. Deletion mutant (Δlmo2555) showed a significantly less biofilm formation than wild type strain (LM21), indicating that LTA is involved in biofilm formation and in the structure of L. monocytogenes.
Significance: An understanding of the biological influences on biofilm formation in L. monocytogenes will ultimately result in unique strategies to prevent and remove biofilms in food processing environment.