Vitamin B ₁₂ uptake across the mycobacterial outer membrane is influenced by membrane permeability in Mycobacterium marinum

ABSTRACT Vitamin B 12 (B 12 ) serves as a critical cofactor within mycobacterial metabolism. While some pathogenic strains can synthesize B 12 de novo , others rely on host-acquired B 12 . In this investigation, we studied the transport of vitamin B 12 in Mycobacterium marinum using B 12 -auxotrophic and B 12 -sensitive strains by deleting metH or metE , respectively. These two enzymes rely on B 12 in different ways to function as methionine synthases. We used these strains to select mutants affecting B 12 scavenging and confirmed their phenotypes during growth experiments in vitro . Our analysis of B 12 uptake mechanisms revealed that membrane lipids and cell wall integrity play an essential role in cell envelope transport. Furthermore, we identified a potential transcription regulator that responds to B 12 . Our study demonstrates that M. marinum can take up exogenous B 12 and that altering mycobacterial membrane integrity affects B 12 uptake. Finally, during zebrafish infection using B 12 -auxotrophic and B 12 -sensitive strains, we found that B 12 is available for virulent mycobacteria in vivo . IMPORTANCE Our study investigates how mycobacteria acquire essential vitamin B 12 . These microbes, including those causing tuberculosis, face challenges in nutrient uptake due to their strong outer layer. We focused on Mycobacterium marinum , similar to TB bacteria, to uncover its vitamin B 12 absorption. We used modified strains unable to produce their own B 12 and discovered that M. marinum can indeed absorb it from the environment, even during infections. Changes in the outer layer composition affect this process, and genes related to membrane integrity play key roles. These findings illuminate the interaction between mycobacteria and their environment, offering insights into combatting diseases like tuberculosis through innovative strategies. Our concise research underscores the pivotal role of vitamin B 12 in microbial survival and its potential applications in disease control.