Photorhabdus luminescens is an enterobacterium establishing a mutualistic symbiosis with nematodes, that also kills insects after septicaemia and connective tissue colonization. The role of the bacterial mdtABC genes encoding a putative multidrug efflux system from the resistance/nodulation/cell division family was investigated. We showed that a mdtA mutant and the wild type had similar levels of resistance to antibiotics, antimicrobial peptides, metals, detergents and bile salts. The mdtA mutant was also as pathogenic as the wild-type following intrahaemocoel injection in Locusta migratoria , but had a slightly attenuated phenotype in Spodoptera littoralis . A transcriptional fusion of the mdtA promoter (P mdtA ) and the green fluorescent protein ( gfp ) encoding gene was induced by copper in bacteria cultured in vitro . The P mdtA -gfp fusion was strongly induced within bacterial aggregates in the haematopoietic organ during late stages of infection in L . migratoria , whereas it was only weakly expressed in insect plasma throughout infection. A medium supplemented with haematopoietic organ extracts induced the P mdtA -gfp fusion ex vivo , suggesting that site-specific mdtABC expression resulted from insect signals from the haematopoietic organ. Finally, we showed that protease inhibitors abolished ex vivo activity of the P mdtA -gfp fusion in the presence of haematopoietic organ extracts, suggesting that proteolysis by-products play a key role in upregulating the putative MdtABC efflux pump during insect infection with P . luminescens .

Summary The resistance‐nodulation‐division (RND)‐type efflux pumps AcrAB and MdtABC contribute to multidrug‐resistance (MDR) in Gram‐negative bacteria. Photorhabdus is a symbiotic bacterium of soil nematodes that also produces virulence factors killing insects by septicaemia. We previously showed that mdtA deletion in Photorhabdus laumondii TT01 resulted in no detrimental phenotypes. Here, we investigated the roles of the last two putative RND transporters in TT01 genome, AcrAB and AcrAB‐like (Plu0759‐Plu0758). Only ΔacrA and ΔmdtAΔacrA mutants were multidrug sensitive, even to triphenyltetrazolium chloride and bromothymol blue used for Photorhabdus isolation from nematodes on the nutrient bromothymol blue‐triphenyltetrazolium chloride agar (NBTA) medium. Both mutants also displayed slightly attenuated virulence after injection into Spodoptera littoralis. Transcriptional analysis revealed intermediate levels of acrAB expression in vitro, in vivo and post‐mortem, whereas its putative transcriptional repressor acrR was weakly expressed. Yet, plasmid‐mediated acrR overexpression did not decrease acrAB transcript levels neither MDR in TT01 WT. While no pertinent mutations were detected in acrR of the same P. laumondii strain grown either on NBTA or nutrient agar, we suggest that AcrR‐mediated repression of acrAB is not physiologically required under conditions tested. Finally, we propose that AcrAB is the primary RND‐efflux pump, which is essential for MDR in Photorhabdus and may confer adaptive advantages during insect infection.

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