Cobalamin is present in cells of non-tuberculous mycobacteria, but not in Mycobacterium tuberculosis

Alina Minias,Filip Gąsior,Jarosław Dziadek,Tomasz Jagielski,Anna Brzostek

doi:10.1038/s41598-021-91430-w

Alina Minias, Filip Gąsior + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-91430-w

Copy DOI

Abstract

Cobalamin (vitamin B12) is a structurally complex molecule that acts as a cofactor for enzymes and regulates gene expression through so-called riboswitches. The existing literature on the vitamin B12 synthesis capacity in Mycobacterium tuberculosis is ambiguous, while in non-tuberculous mycobacteria (NTM) is rather marginal. Here we present the results of our investigation into the occurrence of vitamin B12 in mycobacteria. For detection purposes, immunoassay methods were applied to cell lysates of NTM and M. tuberculosis clinical and laboratory strains grown under different conditions. We show that whereas vitamin B12 is present in cells of various NTM species, it cannot be evidenced in strains of differently cultured M. tuberculosis, even though the genes responsible for vitamin B12 synthesis are actively expressed based on RNA-Seq data. In summary, we conclude that the production of vitamin B12 does occur in mycobacteria, with the likely exception of M. tuberculosis. Our results provide direct evidence of vitamin B12 synthesis in a clinically important group of bacteria.

Highlights

The melting curve analysis was performed at the end of each qPCR reaction to verify a single, specific product was generated
Raw RNA Seq reads were downloaded from European Nucleotide Archive Database (ENA)
We analyzed gene expression of M. abscessus subsp. abscessus grown in 7H9 medium supplemented with OADC40, M. smegmatis grown in 7H9 medium with glucose[41], and three experiments performed with M. tuberculosis H37Rv grown in 7H9 broth supplemented with OADC20, in a medium supplemented with cholesterol as a sole carbon source[21] and in human THP-1 derived macrophages three days post-infection

Summary

Introduction

Name Gene replacement bacA first flank bacA second flank cobIJ first flank cobIJ second flank Gene complementation metE promoter region Green fluorescence protein Southern blot bacA probe cobIJ probe qPCR cobG cobL cobO cobU cobD cobN sigA PCR confirmation of gene complementation pMV306 The melting curve analysis was performed at the end of each qPCR reaction to verify a single, specific product was generated. The threshold cycle (CT) value for each studied gene was normalized to the expression of msmeg_2758 (sigA) (ΔCT) and converted to linear form (2 − ΔCT).

Objectives

Results

Conclusion