Identification of genes necessary for growth of Listeria Monocytogenes at low temperatures

In this study transposon mutagenesis has been used to identify genes encoding for proteins essential for the growth of L. monocytogenes at 4°C. A library of transposon mutants of L. monocytogenes was generated using Tn917-LTV3 and screened for the ability to grow at 30 and 4°C. One mutant which exhibited visibly reduced growth in liquid medium and five mutants which had visibly reduced growth on solid medium, when incubated at 4°C but unaltered growth at 30°C, were selected for further investigation. Southern blots analysis revealed that there is single insertion of the transposon in the chromosome and that the insertion was at different sites in each mutant. Southern blotting also showed that none of the mutants were in genes of the cspL family. These observation suggested that different genes have been inactivated in these mutants. Sequencing analysis of the gene inactivated in one of the "solid mutants", revealed that the inactivated gene encodes a protein with homology to Bacillus subtilis transmembrane proteins belonging to a multicomponent transport system involved in the uptake of the osmoprotectants such as glycine betaine. Such an osmoprotectant uptake transport system has not been characterised before, at the molecular level, in Listeria monocytogenes. Growth of this L. monocytogenes mutant in defined medium at high osmolarity, either at 30 or 4°C, showed to be affected when compared to the growth of the wild type confirming that the inactivated gene is not only involved in cryotolerance but also in osmotolerance in this organism..