Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems
April 26th, 2019
In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.
Protein synthesis is error-prone. However, errors may be adaptive under physiological stress. We've previously shown that specific translation errors in mycobacterium are contributed to antibiotic tolerance.
Being able to measure specific error rates allows us to target this bacteria-adaptive mechanism. The main advantages of these techniques are the gain-of-function reporters can be exquisitely sensitive in measuring small error rates that are not easy to detect by mass spectrometry. The Nluc/GFP assay allows the medium-throughput screening since it avoids excessive handling and lysis of the bacteria.
Demonstrating the procedure will be PhD student Yue-Meng Chen. Today we will demonstrate the procedure of using these two reporters through this video. To begin, inoculate two milliliters of 7H9 medium with wild-type and mutated mycobacterial reporter strains.
Shake at 37 degrees Celsius for one to two days or until the OD at 600 nanometers reaches the stationary phase. Aliquot and dilute to obtain an OD at 600 nanometers around 0.5 to 1. Then, add ATC to a final concentration of 50 nanograms per milliliter.
Immediately add different doses of ksg to the culture according to the manuscript to measure its effects on mistranslation rates. Incubate at 37 degrees Celsius with shaking for four to six hours. Next, transfer the bacterial cultures to a two milliliter tube.
And centrifuge at 3, 220 times g at room temperature for five minutes to pellet down the bacteria. After the centrifugation step, discard the supernatant and disrupt the bacteria by adding 40 microliters of 1x passive lysis buffer. Transfer the resuspended bacterial lysate to a white 96-well plate, one well per sample, and shake at room temperature for 20 minutes.
Add 80 microliters of firefly substrate to each well. Shake for 15 seconds and measure the luminescence by a luminometer. Then, add 80 microliters of Renilla substrate to each well.
Shake for 15 seconds and measure the luminescence by the luminometer. Use the corrected values to calculate the mistranslation rates of each condition. To begin, inoculate two milliliters of 7H9 medium with the bacterial reporter strain.
Shake at 37 degrees Celsius for one to two days or until the OD at 600 nanometers reaches the stationary phase. Then, subculture in 50 milliliters of 7H9 medium and grow until the OD at 600 nanometers reaches the late stationary phase. Before aliquoting the bacteria to a 96-well plate, add ATC at a final concentration of 50 nanograms per milliliter and mix well.
Add 100 microliters per well to a clear, round-bottomed 96-well plate. Next, add different doses of ksg to selected wells to screen its effects on the mistranslation rates. Add 200 microliters of sterile water to edge wells of the plate to limit the evaporation from the sample wells.
Seal the plate, shake, and induce the samples at 37 degrees Celsius for 16 to 20 hours. Use a multi-channel pipette to take 80 microliters from each well. Transfer the samples to a black-bottomed 96-well plate and measure the GFP signal by the luminometer.
After measuring the GFP signal, centrifuge the plate at 3, 220 times g for 10 minutes. Then, transfer 50 microliters of the supernatant to a white-bottomed 96-well plate. Then, add 50 microliters of Nluc substrate to each well.
Mix well and measure the luminescence by the luminometer. Finally, determine the Nluc/GFP ratio by dividing the corrected Nluc luminescence value by GFP fluorescence. In this work, the Renilla firefly reporter system was used to measure the mistranslation rate in the presence of kasugamycin in wild-type and in a strain of S.Smegmatis which ksgA was deleted.
Results showed a clear dose-dependent manner of ksg decreasing mistranslation rate while in the ksgA-deletion strain, ksg was less potent and the baseline of mistranslation rate was higher due to the resistance to modulation by kasugamycin. Kasugamycin action on mistranslation was also measured using the Nluc/GFP reporter. Both Renilla firefly and Nluc/GFP reporter involve measuring luciferase activity.
Small pipetting errors could cause big fluctuations in readouts. For starters, we suggest increasing the number of each replicates to minimize the chance of getting unreliable readouts.
Overview
This article presents two complementary methods to measure translational error rates in Mycobacterium smegmatis using gain-of-function reporter systems. These techniques allow for accurate measurement of error rates in both low and high-throughput settings.
Key Study Components
Area of Science
- Neuroscience
- Microbiology
- Biotechnology
Background
- Protein synthesis is inherently error-prone.
- Translation errors can contribute to antibiotic tolerance in mycobacteria.
- Measuring specific error rates can help target adaptive mechanisms in bacteria.
- Gain-of-function reporters provide sensitive detection of small error rates.
Purpose of Study
- To measure specific rates of translational error and mistranslation.
- To utilize gain-of-function reporter systems for accurate error rate measurement.
- To demonstrate the procedures for using these reporter systems.
Methods Used
- Inoculation of mycobacterial strains in 7H9 medium.
- Use of ATC and ksg to measure effects on mistranslation rates.
- Measurement of luminescence using firefly and Renilla substrates.
- Calculation of Nluc/GFP ratios to assess mistranslation rates.
Main Results
- Kasugamycin decreased mistranslation rates in a dose-dependent manner.
- The ksgA-deletion strain showed higher baseline mistranslation rates.
- Both reporter systems effectively measured luciferase activity.
- Increased replicates are recommended to minimize readout fluctuations.
Conclusions
- The methods provide a reliable way to measure translational errors.
- Understanding mistranslation can inform antibiotic resistance mechanisms.
- These techniques can be applied in various experimental settings.
Frequently Asked Questions
Executive Industry Relevance
Measuring specific mistranslation rates in mycobacteria enables target validation of translational fidelity mechanisms linked to antibiotic tolerance. Gain-of-function reporter systems provide quantitative, reproducible readouts that support mechanistic de-risking in early discovery. These assays facilitate screening for compounds that modulate error rates, offering predictive confidence in lead identification for anti-infective programs.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates the role of translational errors in bacterial adaptation and antibiotic tolerance mechanisms.
- Operational Value: Enables functional validation of targets like GatCAB and ksgA through dose-response measurements of mistranslation modulation.
- Predictive Value: Supports hypothesis testing of mistranslation as a resistance mechanism, informing target confidence and portfolio triage.
Screening & Assay Development
- Scientific Value: Provides sensitive, gain-of-function detection of small error rates not easily captured by mass spectrometry.
- Operational Value: The Nluc/GFP system allows medium/high-throughput screening with minimal bacterial handling and lysis steps.
- Assay Readiness: Generates quantitative luminescence and fluorescence ratios enabling reliable compound evaluation across doses.
Translational & Preclinical Research
- Translational Continuity: Links mistranslation measurements to phenotypic outcomes like kasugamycin sensitivity, supporting preclinical validation.
- Mechanistic De-risking: Clarifies how genetic alterations (e.g., ksgA deletion) affect baseline error rates and compound response, reducing ambiguity in mechanism of action.
- Disease-Relevant System: Uses mycobacterial strains to model pathogen-specific translational fidelity, relevant for anti-infective target assessment.
Pipeline & Workflow Integration
The method fits within early discovery workflows, connecting target validation to screening and mechanistic follow-up in antimicrobial development.
- Discovery Biology: Supports hypothesis testing of translational errors as contributors to bacterial persistence and tolerance.
- Screening: Enables medium-throughput evaluation of small molecules like kasugamycin that affect mistranslation rates.
- Analytics: Delivers quantitative Nluc/GFP ratios and luminescence readouts for comparing error rates across conditions and genetic backgrounds.
- Translational Research: Connects in vitro error measurements to functional outcomes in mycobacterial strains, informing preclinical continuity.
- Enterprise Reuse: Establishes a reusable platform for assessing translational fidelity across model systems and compound libraries.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity by quantifying specific mistranslation events in pathogen-relevant contexts.
- Operational Value: Standardizes error rate measurement with sensitive, low-background reporters amenable to scaling.
- Strategic Value: Improves go/no-go decisions by identifying compounds that modulate translational fidelity, reducing late-stage biological risk.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on mechanistic links between mistranslation and antibiotic tolerance.
Implementation Considerations
- Requires expertise in microbiology, molecular cloning, and luciferase-based assays.
- Dependent on luminometer access for luminescence and fluorescence measurements.
- Necessitates standardization of inducer (ATC) and compound (kasugamycin) dosing across replicates.
- Involves adaptation considerations when transferring reporter systems to other bacterial models.
- Benefits from increased replicate numbers to minimize technical variability in readouts.
