Moiecutar Microbioiogy {1995) 16{5), 1001-1009 Importance of lipooligosaccharide structure in determining gonococcal resistance to hydrophobic antimicrobial agents resulting from the mfrefflux system Ctaressa E. Lucas.^'^ Kayla E. James C. Levin,^ Daniel C. Stein^ and William M. Shafer'*^* 'Department of Microbiology and Immunology, Emory University School of Medicine. Attanta, Georgia 30322, USA. ^Laboratories of Microbial Pathogenesis. VA Medical Research Sen/ice. VA Medical Center (Attanta). Decatur, Georgia 30033. USA. '^Department of Microbiology and Immunology, University of Maryland, College Park, Maryland 20742, USA. Summary Levels of gonococcal resistance to antimicrobial hydrophobic agents (HAs) are controlled by the mtr (multiple transferrable resistance) system, composed of the mtrRCDE genes. The mtrR gene encodes a tran- scriptional repressor that appears to regulate expres- sion of the upstream and divergent mtrCDE operon. The mfrCDE genes encode membrane proteins analo- gous to the MexABOprK proteins of Pseudomonas aerug/nosa that mediate export of structurally diverse antimicrobial agents. In this study we found that a single base pair deletion in a 13 bp inverted repeat sequence within the mtrR promoter resulted in increased resistance of gonococci to both crystal violet (CV) and erythromycin (ERY) as well as to the more lipophilic non-ionic detergent Triton X-100 (TX- 100). However, this cross-resistance was contingent on the production of a full-length lipooligosaccharide (LOS) by the recipient strain used in transformation experiments. Introduction of this mutation {mtrR-171) into three chemically distinct deep-rough LOS mutants by transformation resulted in a fourfold increase in resistance to TX-100 compared with a 160-fold increase in an isogenic strain producing a full-length LOS. How- ever, both wild-type and deep-rough LOS strains exhibited an eightfold increase in resistance to CV and ERY as a result of the mtrR-171 mutation. This sug- gests that gonococci have different LOS structural Received 6 December, 1994; revised 6 February, 1995; accepted 13 February, 1995. 'For correspondence. E-mail wshafer@unix. cc.emory.edu; Tei, (404) 728 7688; Fax (404) 329 2210. requirements for mfr-mediated resistance to HAs that differ in their lipophilic properties. Evidence is presented that gonococci exclude HAs by an energy- dependent efflux process mediated by the mfrsystem. Introduction The susceptibitity of Gram-negative bacteria to various antimicrobiai agents appears to be controiled by both the permeabiiity barrier associated with the outer membrane and by efflux systems that actively transport antibacterial compounds across the inner and outer membranes to the extraceiiuiar fluid {Nikaido. 1994). These systems working together or atone may account for the capacity of bacteria to express different levels ol resistance to structurally diverse antimicrobiai hydrophobic agents {HAs) including drugs, dyes and detergents. Lipopolysaccharide {LPS) structure of enteric pathogens is an important deter- minant in controlling permeability of the outer membrane to hydrophobic agents (Nikaido and Vaara, 1985). Unlike LPS produced by enteric pathogens, gonococci (Griffiss et ai. 1987) produce a glycolipid. termed iipooiigosac- charide {LOS), that tacks the 0-antigenic side-chains contained by LPS produced by enteric pathogens. LOS produced by Neisseria gonorrhoeae appears to be less important in controiting susceptibitity to HAs because trun- cation of the gonococca! LOS did not resuit in enhanced susceptibiiity to HAs {Guymon ef ai. 1982). Therefore, other ceti envetope components appear to controi perme- abiiity of the gonococcai ceii envelope to HAs. The mtr system {Maness and Sparling, 1973) is one membrane component that has been implicated (Guymon and Spar- ling. 1975; fVlorse et ai. 1982: Pan and Spratt, 1994) in determining levels of gonococcal susceptibitity to HAs. Recent genetic analysis of the mfrsystem revealed (Pan and Spratt, 1994; Hagman etai. 1995) its similarity to the acrRAB {Ma ef ai, 1993; 1994) and acrSEF {previously termed envRCD) {Kiein etai. 1991: Pan and Spratt, 1994) operons of Escherichia coii and the mexABoprK genes {Pooie et ai, 1993) of Pseudomonas aenjginosa. These genes encode membrane proteins that mediate energy- dependent efflux of structurally diverse antimicrobial agents {Ma etai, 1994; Nikaido. 1994). Hence, an efflux- based mechanism, as opposed to changes in celt enve- lope permeabiiity (Guymon and Sparling, 1975; Morse i£< 1995 Blackwell Science Lttl