Article Figures & Data
Figures
- Fig 1
Regions targeted by pre-rRNA probe pairs. The structures of the 16S and 23S pre-rRNA molecules are shown, including locations of mature rRNA termini (arrows) and regions targeted by electrochemical sensor probe pairs for the 16S 5′ tail (blue letters) and 16S and 23S splice sites (red letters).
- Fig 2
Comparison of pre-rRNA probe pairs. Probe pairs were tested for detection of E. coli in the log and stationary phases of growth. Log- and stationary-phase cells are expected to have high and low levels of pre-rRNA, respectively, yielding a high signal ratio for log-phase versus stationary-phase cells. Probe pairs for 16S pre-rRNA had good sensitivity for log-phase cells, but the signal ratio for log-phase versus stationary-phase cells was low. Some probe pairs targeting the splice sites at the termini of mature 16S and 23S rRNA had higher signal ratios for log-phase versus stationary-phase cells. The probe pair targeting the splice site at the 3′ terminus of 23S rRNA had the best combination of sensitivity and high signal ratio of log-phase versus stationary-phase cells.
- Fig 3
Variations in pre-rRNA and rRNA levels during E. coli growth. (A) Changes in mature rRNA, pre-rRNA, and their ratio were measured in overnight cultures that were subsequently inoculated into fresh MH growth medium and incubated for 7 h at 37°C. (B) Comparison of the mature/pre-RNA ratio and growth rate during different phases of growth. The growth rate curve is a weighted average determined from the change in CFU during each 30-min time period. Error bars estimated the standard deviations.
- Fig 4
E. coli cell volume versus rRNA copy number during different growth phases. (A) Correlation between the cell volume and rRNA copy number per cell at densities below an OD600 of ≤1.0. (B) Electron micrographs demonstrating progressively smaller cells over incubation time from log phase (2.5 h) to stationary phase (7 h). Error bars estimated the standard deviations.
- Fig 5
(A to D) Response of mature rRNA and pre-rRNA to antibiotics. Antibiotics have differential effects on rRNA and pre-rRNA. Rifampin and ciprofloxacin selectively inhibited transcription of new pre-rRNA, while the addition of chloramphenicol and gentamicin resulted in a selective decrease in mature rRNA. Error bars estimated the standard deviations.
- Fig 6
Comparison of ciprofloxacin-susceptible and -resistant E. coli strains. rRNA and pre-rRNA were measured in cultures of an E. coli clinical isolate susceptible to ciprofloxacin (EC103) and three ciprofloxacin-resistant isolates (EC135, EC139, and EC197). The amount of pre-rRNA in strain EC103 was significantly lower than that of the ciprofloxacin-resistant isolates within 15 min after addition of the antibiotic. Error bars estimated the standard deviations.
Tables
- Table 1
DNA oligonucleotides used in this study
Probe namea Sequenceb Pre16S 15 m 48D 5′-TTTTTCGTCTTGCGA-F Pre16S 15 m 63C 5′-B-GAGACTTGGTATTCA Pre16S 15 m R63D 5′-F-GAGACTTGGTATTCA Pre16S 15 m R48C 5′-TTTTTCGTCTTGCGA-B Pre16S 17 m R63D 5′-F-TTGAGACTTGGTATTCA Pre16S 17 m R46C 5′-TTTTTCGTCTTGCGACG-B Pre16S 19 m R63D 5′-F-TCTTGAGACTTGGTATTCA Pre16S 19 m R44C 5′-TTTTTCGTCTTGCGACGTT-B Pre16S 21 m R63D 5′-F-ACTCTTGAGACTTGGTATTCA Pre16S 21 m R42C 5′-TTTTTCGTCTTGCGACGTTAA-B Pre16S 17 m R60D 5′-F-AGACTTGGTATTCATTT Pre16S 17 m R43C 5′-TTCGTCTTGCGACGTTA-B Pre16S 19 m R60D 5′-F-TGAGACTTGGTATTCATTT Pre16S 19 m R41C 5′-TTCGTCTTGCGACGTTAAG-B Pre16S 21 m R60D 5′-F-CTTGAGACTTGGTATTCATTT Pre16S 21 m R39C 5′-TTCGTCTTGCGACGTTAAGAA-B Pre16S 17 m R66D 5′-F-CTCTTGAGACTTGGTAT Pre16S 17 m R49C 5′-TCATTTTTCGTCTTGCG-B Pre16S 19 m R66D 5′-F-CACTCTTGAGACTTGGTAT Pre16S 19 m R47C 5′-TCATTTTTCGTCTTGCGAC-B Pre16S 21 m R66D 5′-F-TTCACTCTTGAGACTTGGTAT Pre16S 21 m R45C 5′-TCATTTTTCGTCTTGCGACGT-B Pre16S 19 m 5′JxnD 5′-TTTGATGCTCAAAGAATTA-F Pre16S 21 m 5′JxnC 5-S-TCAAACTCTTCAATTTAAAAG Pre16S 21 m R5′JxnD 5-F-TCAAACTCTTCAATTTAAAAG Pre16S 19 m R5′JxnC 5′-TTTGATGCTCAAAGAATTA-S Pre16S 17 m 3′JxnD 5′-GAGGTGATCCAACCGCA-F Pre16S 20 m 3′JxnC 5-S-GAACGCTTCTTTAAGGTAAG Pre16S 20 m R3′JxnD 5-F-GAACGCTTCTTTAAGGTAAG Pre16S 17 m R3′JxnC 5′-GAGGTGATCCAACCGCA-S Pre23S 17 m 3′JxnDc 5′-AAGCCTCACGGTTCATT-F Pre23S 14 m 3′JxnCc 5′-S-GGCGTTGTAAGGTT Pre23S 14 m R3′JxnD 5′-F-GGCGTTGTAAGGTT Pre23S 17 m R3′JxnC 5′-AAGCCTCACGGTTCATT-S Mature rRNA 18 m 1484D 5′-GTTACGACTTCACCCCAG-F Mature rRNA 19 m 1502C 5′-S-GTTCCCCTACGGTTACCTT Synthetic target oligonucleotides Pre-rRNA 31 m 5′-AATGAACCGTGAGGCTTAACCTTACAACGCC Mature rRNA 37 m 5′-CTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAAC - Table 2
Pre-rRNA and mature rRNA quantitation during E. coli growth phases
Time point (min) OD600 CFU/ml Ratea Doublingb Generation time (min) rRNAc Pre-rRNAc Ratio Growth phase Overnight culture 2.307 6.67E + 09 6,009 2 2,720 Stationary phase 0 0 5.90E + 05 5,942 110 54 Lag phase 30 0 5.70E + 05 28,427 457 62 60 0.003 8.63E + 05 0.18 1.20 50.08 55,278 696 79 Log phase 90 0.008 2.30E + 06 0.43 2.83 21.19 78,741 970 81 120 0.024 6.20E + 06 0.43 2.86 21.00 91,049 1200 76 150 0.068 1.91E + 07 0.49 3.24 18.51 98,782 523 189 180 0.175 6.53E + 07 0.53 3.55 16.88 61,571 235 262 210 0.409 2.30E + 08 0.55 3.64 16.50 38,033 208 183 240 0.707 5.27E + 08 0.36 2.39 25.14 29,801 114 260 Early stationary phase 270 1.095 1.17E + 09 0.35 2.30 26.14 19,610 42 466 300 1.466 2.33E + 09 0.30 2.00 30.00 13,162 25 536 330 1.686 3.23E + 09 0.14 0.94 63.74 7,608 11 665 Stationary phase 360 1.834 4.43E + 09 0.14 0.91 65.87 5,734 8 723 390 1.957 5.03E + 09 0.06 0.37 163.81 4,899 7 706 420 2.051 5.73E + 09 0.06 0.38 159.68 6,230 6 1,049
