Genetic Characterization of Plasmid - Associated Triphenylmethane Reductase 1 in Listeria monocytogenes

27 The enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into 28 colorless, non-toxic derivatives, and TMR-producing microorganisms have been proposed as 29 bioremediation tools. Analysis of the genome of Listeria monocytogenes H7858 (1998-1999 hot 30 dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette 31 (bcrABC) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium 32 disinfectants also harbored a gene (tmr) highly homologous to TMR-encoding genes from 33 diverse Gram-negative bacteria. The pLM80-associated tmr was located two genes downstream 34 of bcrABC as part of a putative IS1216 composite transposon. To confirm the role of tmr in 35 triphenylmethane dye detoxification, we introduced various tmr-harboring fragments of pLM80 36 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed 37 resistance of the constructs to the triphenylmenhane dyes crystal violet (CV) and malachite 38 green. Transcriptional and subcloning data suggest that regulation of TMR is complex. 39 Constructs harboring fragments spanning bcrABC and tmr were CV-resistant and in such 40 constructs tmr transcription were induced by sublethal levels of either BC or CV. However, 41 constructs harboring only tmr and its upstream intergenic region could also confer resistance to 42 CV, albeit at lower levels. Screening a panel of BC-resistant L. monocytogenes strains revealed 43 that all those harboring bcrABC and adjacent pLM80 sequences, including tmr, were resistant to 44 CV and decolorized this dye. The findings suggest a potential role of TMR as a previously 45 unknown adaptive attribute for environmental persistence of L. monocytogenes. 46 47 48 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom