Targeted metabolomics data for riboflavin, FMN and FAD in Mycobacteria tuberculosis and Mycolicibacterium smegmatis

Published Dec 19, 2025 on Dryad. https://doi.org/10.5061/dryad.pg4f4qs39

Data files

Dec 19, 2025 version files 41.54 GB

Abstract

Riboflavin biosynthesis is essential for most microorganisms, yet its production is tightly regulated due to the potential toxic effects of riboflavin and its derivatives. While regulatory mechanisms for this process have been described in other organisms, none have been identified in mycobacteria. Uncovering such a mechanism will be critical to fully exploit the riboflavin biosynthetic pathway as a pharmacological target. We previously observed that Mycobacterium tuberculosis (Mtb) and Mycolicibacterium smegmatis (Msm) can grow successfully under elevated riboflavin levels, prompting an investigation into how mycobacteria tolerate high intracellular riboflavin. Using a bioinformatic approach of proteomic data, we explored the global proteomic response of Mtb and Msm to elevated riboflavin. Our results revealed increased abundance of known flavin sequesters in response to high riboflavin levels, suggesting a role in flavin homeostasis. Using a transcription regulatory network analysis, we observed the employment of similar regulatory networks, most notably DosR, in both Mtb and Msm in response to elevated riboflavin. We also identified a potential link between the riboflavin-induced response and isoniazid resistance mechanisms, warranting further investigation. Overall, this study provides evidence for the involvement of flavin sequesters in maintaining flavin homeostasis and highlights the potential regulatory role of DosR in this process.

Dataset DOI: 10.5061/dryad.pg4f4qs39

Description of the data and file structure

This dataset contains raw targeted MS/MS data from wild-type Mycobacterium tuberculosis and Mycolicibacterium smegmatis treated with or without exogenous riboflavin, focusing on terminal metabolites of the riboflavin biosynthetic pathway. To assess how riboflavin availability influences the synthesis of FMN and FAD, a targeted high-resolution mass spectrometry (HRMS) assay was developed for riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The dataset includes qualitative and quantitative LC-MS/MS analyses and provides evidence for how mycobacteria regulate FMN and FAD synthesis from available riboflavin, contributing to key findings on flavin homeostasis in mycobacteria. All LC-MS/MS data are provided in original formats, enabling processing with free and accessible tools.

Growth and preparation of Mtb and Msm for metabolomics

Glycerol stocks of Mtb and Msm strains were used to start cultures on 7H11 agar plates supplemented with or without riboflavin. Plates were incubated for 2 weeks for Mtb and Msm for 4 to 5 days at 37ºC. Cells were scraped from plates into 7H9 media supplemented with OADC with or without riboflavin and grown at 37ºC until mid-log phase. Cells were collected via centrifugation, washed with mass spectrometry-grade water, and resuspended in Acetonitrile/Methanol/Water (2:2:1) and then transferred to screw-cap homogenization microtubes prefilled with 0.1 mm zircon beads. The cells were homogenized using the FastPrep-24 bead beater (MP Biomedical, SKU:116004500) at 6 m/s for 30 seconds (10 rounds), cooling on ice for 2 minutes between cycles. Homogenized cells were collected and spun down at 16,000 g for 20 minutes,e s the nd supernatant was collected for metabolomics. Supernatants were then stored at -20ºC until further processed.

Liquid Chromatography -MS/MS analysis

The supernatant collected for metabolomics was dried in a Savant and its weight determined. Dried samples were then resuspended in 3% Acetonitrile in Water (0.1% Formic Acid) to make a final concentration of 2 mg/ml for Mtb and 10 mg/ml for Msm. Samples were analyzed using the Agilent 1290 Infinity III Liquid Chromatography System attached to an Agilent 6546 quadrupole time-of-flight MS system. 2 µl of samples were loaded per injection. Reverse phase UHPLC was performed using InfinityLab Poroshell 120 EC-C18 Column (Agilent, Part Number: 693975-902) (2.1 x 100 mm, 2.7 µm). Mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in acetonitrile.LC separation was carried out at a flow rate of 0.32 µl/min using a linear gradient of 2% solvent B held for 3 minutes, and then to 50% solvent B over a duration of 15 minutes, then 50% solvent B to 99% solvent B over 7 minutes. 99% solvent B was held for 6.5 minutes and then returned to 2% solvent B for 1.5 minutes and held for 5 minutes for equilibration. The sample eluted within the first minute was diverted to waste to prevent clogging.

Mass spectra and tandem mass spectra were carried out using the Targeted MS/MS acquisition method in positive ion mode. Column temperature was set at 45 ºC. Source was operated with the following settings: 3500 capillary voltage, 10.0 l/min of dry gas at 40 psi, and 320°C. Post MS-inlet capillary, fragmentor was set to 120 V, nozzle voltage was set to 1000 V, and skimmer voltage was set to 40 V. The peak-to-peak voltage of the ion transfer octupole was set at 750 V. The mass analyzers for MS and MS/MS were set to scan between m/z 100 to 1700 at a scan rate of 4 spectra/sec and an isolation width of H1.3 amu. 3 precursors were selected per cycle with an absolute threshold of 200 and a relative threshold of 0.01%. Mass error tolerance was set to ± 20 ppm, and retention time exclusion tolerance was set to ± 0.2 minutes. Collision energy was alternated between 15 and 30 volts during the cycle.

Files and variables

File:250912_NO_FMN_FAD_Msm_Mtb.zip

Description: Contains .d raw HRMS data for the aforementioned metabolites in Mycobacterium tuberculosis and Mycolicibacterium smegmatis

File: HRMS_File_Annotation.xlsx

Description: Contains annotation information for all .d raw files. (Strain, Biological Replicate and Raw File Name, Treatment)

Naming Convention for files

Strain (Msm: Mycolicibacterium smegmatis, Mtb: Mycobacterium tuberculosis OR Blank)-Strain (Wild Type (WT))
Treated with Riboflavin (RF)
Biological Replicate (1, 2, 3)-NA: not applicable for blank
Type (test or blank)
File name (raw file names included)

e.g., For file name “250828_RibHexcl_038_Msm_RF_WT2.d"

Sample: Date of Collection: 08/28/25; Sample Table: Ri.bHexcl; Queue Number: 38; Organism: Mycolicibacterium smegmatis; Treated with riboflavin; Strain: Wild Type; Biological Replicate: 2

Code/software

Primary Software: Agilent Mass Hunter Qualitative Analysis Software V10.0 can be used to process and visualize .d files provided.

Alternative Software: Skyline (Metabolite interface) is a free and open software that can be used to process and visualize .d files provided.

Access information

Other publicly accessible locations of the data:

Data was derived from the following sources: