AN EFFECTIVE COUNTERSELECTION MARKER FOR LISTERIA MONOCYTOGENES

Construction of Listeria monocytogenes mutants by allelic exchange has been laborious and time consuming due to lack of proficient selection markers for the second recombination event. That is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a mutated counterselection marker based on the Bacillus subtilis Phenylalanyl-tRNA synthetase gene (pheS*)(A309G). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic p-chloro-phenylalanine analog (p-Cl-phe) as a substrate. When pheS* is introduced to L. monocytogenes and highly expressed under a constitutively active promoter, the bacteria become sensitive to p-Cl-phe supplemented to the media. This enabled us to utilize pheS* as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in L. monocytogenes. Furthermore, such an effective counterselection marker, that does not require any background genomic alterations, is a potent tool that can be modularly used to construct various selection systems such as in genetic screens. We expect this counter selection marker to be a valuable genetic tool in L. monocytogenes and other Gram-positive bacteria.^1–3

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