Cervimycin C resistance in Bacillus subtilis is due to a promoter up-mutation and increased mRNA stability of the constitutive ABC-transporter gene bmrA

Abstract

Two independent cervimycin C (CmC)-resistant clones of Bacillus subtilis were identified, each carrying two mutations in the intergenic region preceding the ABC transporter gene bmrA. In the double mutant, real-time PCR revealed an increased amount of bmrA mRNA with increased stability. Accordingly, isolation of membrane proteins yielded a strong band at 64 kDa corresponding to BmrA. Analyses showed that one mutation optimized the -35 box sequence conferring resistance to 3 μM CmC, while the +6 mutation alone had no effect, but increased the potential of the strain harboring the -35 mutation to grow at 5 μM CmC. Transcriptional fusions revealed an elevated bmrA promoter activity for the double mutant. Electrophoretic mobility shift assays (EMSAs) confirmed a 30-fold higher binding affinity of RNA polymerase for this mutant compared with the wild type, and the effect was due to the -35 box alteration of the bmrA promoter. In vitro transcription experiments substantiated the results of the EMSA. EMSAs in the presence of heparin indicated that the mutations did not influence the formation and/or the stability of open complexes. Half-life measurements demonstrated that the +6 mutation stabilized bmrA mRNA ≈ 2-fold. Overall, we found that an ABC transporter confers antibiotic resistance by the cumulative effects of two mutations in the promoter region.

Clinically relevant fluoroquinolone resistance due to constitutive overexpression of the PatAB ABC transporter in Streptococcus pneumoniae is conferred by disruption of a transcriptional attenuator

“… 27 Similarly, expression of another B. subtilis ABC transporter gene associated with antibiotic resistance, bmrA , has been shown to be increased by an upstream mutation that increases mRNA stability. 28 When combined with our results, these findings raise the intriguing possibility that transcriptional attenuation may be a common method of control of ABC transporter family efflux pumps in Gram-positive bacteria.…”

A comprehensive review of glycosylated bacterial natural products

“…There are 26 glycosylated tetracyclines reported to date including the cervimycins, 300–302 HKI10311129 303 dutomycin, 304,305 polyketomycin, 306–308 elloramycins, 176,309,310 tetracenomycins, 176,311,312 TAN-1518 A, B and X (also known as SF-2575), 313–315 and dactylocyclines, 316–319 the latter of which being the only member of non- Streptomyces origin ( Dactylosporangium ). Regiospecificity of tetracycline glycosylation is limited to C-4, C-8, C-11 and C-12a where all members with the exception of one subgroup (the C-8- C -glycosidic TAN-1518 analogs) are O -glycosides.…”

“…20). 300–302 The terminal rhodinose ( 44 ) of tetrasaccharide III is further modified with a 4′- O -dimethylmalonyl/or monomethylmalonyl ester. The structurally similar HKI10311129 303 displays an identical glycosylation pattern but lacks the terminal rhodinose ester modification while the related dutomycin and polyketomycin are C-4- O -glycosides bearing an a α-L-4′- epi -mycarosyl-(1′→4′)-β-D-amicetose (disaccharide II ) but lacking C-12a glycosylation.…”

Flexible-to-rigid transition is central for substrate transport in the ABC transporter BmrA from Bacillus subtilis

“…We here use the homodimeric multidrug ABC exporter BmrA from B. subtilis (130 kDa), which has been identified in the B. subtilis genome by homology with the human P-glycoprotein 18 . BmrA is able to transport multiple substrates, including Hoechst 33342 and doxorubicin 19 , and the antibiotic cervimycin C 20 . While high-resolution 3D structures of BmrA remain to be determined, different conformational states have been characterized for the apo and ADP:Mg:Vi-trapped forms using biochemical studies 21,22 , electron microscopy 23 , and EPR spectroscopy 24 .…”