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

Baylay AJ, Piddock LJ - J. Antimicrob. Chemother. (2014)

Bottom Line: To determine the effect of the detected terminator mutations, the WT and mutated patA leader sequences were cloned upstream of a GFP reporter.This study showed that a mutation in a Rho-independent transcriptional terminator structure confers overexpression of patAB and fluoroquinolone resistance.Understanding how levels of the PatAB efflux pump are regulated increases our knowledge of pneumococcal biology and how the pneumococcus can respond to various stresses, including antimicrobials.

View Article: PubMed Central - PubMed

Affiliation: Antimicrobials Research Group, School of Immunity and Infection, Institute of Microbiology and Infection and College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK.

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Fluorescence measured from pBAV1K-gfp82 reporter plasmid containing WT and mutant patA leader sequences during late-log (2.5 h), stationary (3.5 h) and late-stationary (5.5 h) phases of growth. Bars represent the fluorescence of pBAV1K-gfp82-WT and (a) v101 and v101c, (b) v168 and v168c and (c) v87 and v87c, adjusted to a standard OD600 of 0.5, relative to the fluorescence of empty pBAV1K-gfp82 vector. Error bars represent the standard deviation of three biological replicates. *Fluorescence significantly different from WT; P < 0.05, two-tailed Student's t-test.
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DKU449F4: Fluorescence measured from pBAV1K-gfp82 reporter plasmid containing WT and mutant patA leader sequences during late-log (2.5 h), stationary (3.5 h) and late-stationary (5.5 h) phases of growth. Bars represent the fluorescence of pBAV1K-gfp82-WT and (a) v101 and v101c, (b) v168 and v168c and (c) v87 and v87c, adjusted to a standard OD600 of 0.5, relative to the fluorescence of empty pBAV1K-gfp82 vector. Error bars represent the standard deviation of three biological replicates. *Fluorescence significantly different from WT; P < 0.05, two-tailed Student's t-test.

Mentions: It was predicted that reduced stability of the terminator loop would increase transcription of patAB, by increasing transcriptional read-through into the patA coding sequence.11 However, this had not been experimentally demonstrated. To investigate this, we synthesized four DNA fragments corresponding to the patA upstream region (−146 to −13 bp), flanked by restriction sites. One fragment corresponded to the WT (R6) sequence, and the remaining three introduced the mutations C(−33)T (v168), A(−47)C (v101) and G(−46)A (v87). These were cloned upstream of a promoter-less GFP allele in a plasmid named pBAV1K-gfp82, derived from pBAV1K-t5-gfp (construction described in the Materials and methods section). The resulting constructs were transformed into R6 cells and transformants selected using 100 mg/L kanamycin. Fluorescence from each construct was measured during growth of the strains in BHI broth. Compared with a strain carrying pBAV1K-gfp82 empty vector, the pBAV1K-gfp82-WT construct produced consistently higher fluorescence throughout the growth cycle (Figure 4). Introduction of the terminator mutations (constructs pBAV1K-gfp82-v168, pBAV1K-gfp82-v101 and pBAV1K-gfp82-v87) resulted in a significant 1.5- to 3-fold increase in fluorescence in all three cases after 2.5, 3.5 and 5.5 h of growth, respectively, representing late-log, stationary and late-stationary phases, respectively (Figure 4; P < 0.05, two-tailed Student's t-test).Figure 4.


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

Baylay AJ, Piddock LJ - J. Antimicrob. Chemother. (2014)

Fluorescence measured from pBAV1K-gfp82 reporter plasmid containing WT and mutant patA leader sequences during late-log (2.5 h), stationary (3.5 h) and late-stationary (5.5 h) phases of growth. Bars represent the fluorescence of pBAV1K-gfp82-WT and (a) v101 and v101c, (b) v168 and v168c and (c) v87 and v87c, adjusted to a standard OD600 of 0.5, relative to the fluorescence of empty pBAV1K-gfp82 vector. Error bars represent the standard deviation of three biological replicates. *Fluorescence significantly different from WT; P < 0.05, two-tailed Student's t-test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4319486&req=5

DKU449F4: Fluorescence measured from pBAV1K-gfp82 reporter plasmid containing WT and mutant patA leader sequences during late-log (2.5 h), stationary (3.5 h) and late-stationary (5.5 h) phases of growth. Bars represent the fluorescence of pBAV1K-gfp82-WT and (a) v101 and v101c, (b) v168 and v168c and (c) v87 and v87c, adjusted to a standard OD600 of 0.5, relative to the fluorescence of empty pBAV1K-gfp82 vector. Error bars represent the standard deviation of three biological replicates. *Fluorescence significantly different from WT; P < 0.05, two-tailed Student's t-test.
Mentions: It was predicted that reduced stability of the terminator loop would increase transcription of patAB, by increasing transcriptional read-through into the patA coding sequence.11 However, this had not been experimentally demonstrated. To investigate this, we synthesized four DNA fragments corresponding to the patA upstream region (−146 to −13 bp), flanked by restriction sites. One fragment corresponded to the WT (R6) sequence, and the remaining three introduced the mutations C(−33)T (v168), A(−47)C (v101) and G(−46)A (v87). These were cloned upstream of a promoter-less GFP allele in a plasmid named pBAV1K-gfp82, derived from pBAV1K-t5-gfp (construction described in the Materials and methods section). The resulting constructs were transformed into R6 cells and transformants selected using 100 mg/L kanamycin. Fluorescence from each construct was measured during growth of the strains in BHI broth. Compared with a strain carrying pBAV1K-gfp82 empty vector, the pBAV1K-gfp82-WT construct produced consistently higher fluorescence throughout the growth cycle (Figure 4). Introduction of the terminator mutations (constructs pBAV1K-gfp82-v168, pBAV1K-gfp82-v101 and pBAV1K-gfp82-v87) resulted in a significant 1.5- to 3-fold increase in fluorescence in all three cases after 2.5, 3.5 and 5.5 h of growth, respectively, representing late-log, stationary and late-stationary phases, respectively (Figure 4; P < 0.05, two-tailed Student's t-test).Figure 4.

Bottom Line: To determine the effect of the detected terminator mutations, the WT and mutated patA leader sequences were cloned upstream of a GFP reporter.This study showed that a mutation in a Rho-independent transcriptional terminator structure confers overexpression of patAB and fluoroquinolone resistance.Understanding how levels of the PatAB efflux pump are regulated increases our knowledge of pneumococcal biology and how the pneumococcus can respond to various stresses, including antimicrobials.

View Article: PubMed Central - PubMed

Affiliation: Antimicrobials Research Group, School of Immunity and Infection, Institute of Microbiology and Infection and College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK.

Show MeSH
Related in: MedlinePlus