The MtrR repressor binds the DNA sequence between the mtrR and mtrC genes of Neisseria gonorrhoeae

Abstract

Gonococcal resistance to antimicrobial hydrophobic agents (HAs) is due to energy-dependent removal ofHAs from the bacterial cell by the MtrCDE membrane-associated efflux pump. The mtrR (multiple transferrable resistance Regulator) gene encodes a putative transcriptional repressor protein (MtrR) believed to be responsible for regulation of mtrCDE gene expression. Gel mobility shift and DNase I footprint assays that used a maltose-binding protein (MBP)-MtrR fusion protein demonstrated that the MtrR repressor is capable of specifically binding the DNA sequence between the mtrR and mtrC genes. This binding site was localized to a 26-nucleotide stretch that includes the promoter utilized for mtrCDE transcription and, on the complementary strand, a 22-nucleotide stretch that contains the ؊35 region of the mtrR promoter. A single transition mutation (A3G) within the MtrR-binding site decreased the affinity of the target DNA for MtrR and enhanced gonococcal resistance to HAs when introduced into HA-susceptible strain FA19 by transformation. Since this mutation enhanced expression of the mtrCDE gene complex but decreased expression of the mtrR gene, the data are consistent with the notion that MtrR acts as a transcriptional repressor of the mtrCDE efflux pump protein genes.Resistance of Neisseria gonorrhoeae to structurally diverse hydrophobic agents (HAs) has been the subject of several recent investigations (5,6,10,20,23). Although earlier studies indicated that the mtr (multiple transferrable resistance) locus modifies the permeability barrier of the gonococcal cell envelope (3,4,13,16,22,24), more recent studies revealed that mtr encodes an energy-dependent efflux system (5,6,10,20,23). The membrane proteins (MtrC, MtrD, and MtrE) forming the efflux pump share considerable amino acid sequence similarity with other efflux proteins in Escherichia coli (12,17) and Pseudomonas aeruginosa (21). Active removal of toxic compounds by bacteria is an important mechanism of multiple antibiotic resistance (8, 17) and may confer a selective advantage on organisms, such as gonococci, when they colonize mucosal sites bathed in fluids containing antibacterial fatty acids and bile salts or encounter other environmental stresses (8,11,12,(16)(17)(18)23).Production of the MtrCDE efflux proteins is controlled at the level of transcription by both cis-and trans-acting factors involving the mtrR gene (5,6,20,23). The mtrR gene is located 250 bp upstream of the mtrCDE gene complex and transcribed divergently (5,6,20). It encodes a 210-amino-acid protein with a molecular mass of approximately 23 kDa that contains a putative helix-turn-helix motif near its N terminus (5,20,23). MtrR has considerable amino acid sequence similarities to several transcriptional repressors, most notably, the tetracycline repressor of pSC101 (1,7,20). Recent investigations have revealed that missense or deletion mutations within the mtrR coding region result in enhanced mtrCDE transcription and gonococcal resistance to HAs (6, 23). Thus, the available genetic dat...

Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family

“…Enhanced resistance of gonococci to HAs can be due to mutations in the mtrR gene, either as a result of a single basepair deletion in its promoter (28) or a missense mutation that causes a radical amino acid change in the helix-turn-helix motif of MtrR that abrogates binding of it to the mtrCDE promoter (29). Thus, isogenic transformants of strain FA19 or clinical isolates bearing mutations in their mtrR genes (30) were more resistant to PG-1 than strain FA19 (data not shown), displaying MGICs to PG-1 of 10-20 g͞ml.…”

Gonorrhea in Men Sex Men and Heterosexual Men

“…In clinical isolates were found mtrR mutations that alter the repressor protein and its binding to DNA, resulting in decreased repression of the mtrCDE operon (Lucas, 1997).…”

“…The complex of genes mtrCDE represents a transcriptional unit is located 250 bp upstream and divergently transcribed respect to mtrR gene, which is the gene that encodes a regulatory protein MtrR (repressor) of mtrCDE transcription (Lucas, 1997).…”

The Role of the Transcription Factors MtrR and MtrA in the Fitness of the Pathogen Neisseria gonorrhoeae

“…This deletion is frequently found in clinical isolates and confers greater resistance to hydrophobic agents and higher levels of mtrCDE transcription than that conferred by mutations in the mtrR-coding sequence (64,103,211 mtrA Mutants are Attenuated in vivo: We also compared the relative fitness of wild type gonococci and the mtrA mutant JF3, which is incapable of inducing expression of mtrCDE through the action of the MtrA activator protein (162). JF3 bacteria showed no fitness difference compared to wild type FA19Sm R gonococci in vitro (Fig.…”

“…There are numerous reports of clinical isolates of N. gonorrhoeae that harbor mutations in mtrR, the repressor gene of the mtrCDE operon, or its promoter region, which is located immediately upstream and divergently transcribed from the mtrCDE operon (65,103). These isolates exhibit increased resistance to penicillin (137,194), erythromycin, and azithromycin (184,205,210).…”