Significance: The mechanisms that the “dormant microbial majority” use to remain energized in nutrient-starved soil ecosystems have long remained elusive. In this work, we used an isolate of the highly abundant but poorly understood soil phylum Acidobacteria as a model for understanding microbial persistence mechanisms. When the bacterium entered a persistent state due to nutrient-exhaustion, we showed it could scavenge the trace concentrations of molecular hydrogen gas (H ₂ ) found in ambient air using a specialized high-affinity enzyme. These findings demonstrate that Acidobacteria can consume H ₂ and contribute to global hydrogen cycling. We propose that consumption of trace gases such as H ₂ provides a dependable general mechanism for dominant soil phyla to generate the maintenance energy required for long-term survival.

Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F₄₂₀H₂-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance. Bacterial pathogens often evolve resistance to antibiotics via mutations in the coding sequences of genes–frequently the target, an enzyme that metabolizes or transports the active drug, or an enzyme that activates a prodrug. In the case of tuberculosis, antibiotic resistance is a growing problem, with the rapid emergence of multi-drug resistant strains. New nitroimidazole-based antibiotic prodrugs, such as pretomanid and delamanid have the potential to help infected individuals, but we must guard against the evolution of resistance to these new compounds in Mycobacterium tuberculosis. In this report we use protein engineering to identify mutations that could potentially result in antibiotic resistance by knocking out the prodrug activating activity of the deazaflavin dependent nitroreductase (DDN), without completely abolishing its native menaquinone reductase activity. The retention of its native activity is important as DDN appears to be required for M. tuberculosis to emerge from hypoxia. Strikingly, when we analysed >15,000 M. tuberculosis genomes from clinical strains, we identified several that harboured mutations that we identified as abolishing prodrug activation in vitro. A hypervirulent Beijing strain N0008 from Vietnam (which had not been exposed to pretomanid in the clinic) was predicted, and confirmed, to be resistant to pretomanid, revealing that resistance to this drug has arisen through genetic drift and not selective pressure in this instance. These data show that by testing potential resistant mutations in the laboratory before large-scale use of antibiotics, we should be able to use them more judiciously in order to slow the spread of resistance.

New drugs are urgently needed to combat the global TB epidemic. Targeting simultaneously multiple respiratory enzyme complexes of Mycobacterium tuberculosis is regarded as one of the most effective treatment options to shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance. During infection and proliferation, the cytochrome bd oxidase plays a crucial role for mycobacterial pathophysiology by maintaining aerobic respiration at limited oxygen concentrations. Here, we present the cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 Å. In conjunction with atomistic molecular dynamics (MD) simulation studies we discovered a previously unknown MK-9-binding site, as well as a unique disulfide bond within the Q-loop domain that defines an inactive conformation of the canonical quinol oxidation site in Actinobacteria. Our detailed insights into the long-sought atomic framework of the cytochrome bd oxidase from M. tuberculosis will form the basis for the design of highly specific drugs to act on this enzyme.

The ability to persist in the absence of growth triggered by low-oxygen levels is a critical process for the survival of mycobacterial species in many environmental niches. MSMEG_5243 (fsq), a gene of unknown function in Mycobacterium smegmatis, is up-regulated in response to hypoxia and regulated by DosRDosS/DosT, an oxygen- and redox-sensing two-component system that is highly conserved in mycobacteria. In this communication, we demonstrate that MSMEG_5243 is a flavin-sequestering protein and henceforth refer to it as Fsq. Using an array of biochemical and structural analyses, we show that Fsq is a member of the diverse superfamily of flavin- and deazaflavin-dependent oxidoreductases (FDORs) and is widely distributed in mycobacterial species. We created a markerless deletion mutant of fsq and demonstrate that fsqis required for cell survival during hypoxia. Using fsq deletion and overexpression, we found that fsq enhances cellular resistance to hydrogen peroxide treatment. The X-ray crystal structure of Fsq, solved to 2.7 Å, revealed a homodimeric organization with FAD bound noncovalently. The Fsq structure also uncovered no potential substrate-binding cavities, as the FAD is fully enclosed, and electrochemical studies indicated that the Fsq:FAD complex is relatively inert and does not share common properties with electron-transfer proteins. Taken together, our results suggest that Fsq reduces the formation of reactive oxygen species (ROS) by sequestering free FAD during recovery from hypoxia, thereby protecting the cofactor from undergoing autoxidation to produce ROS. This finding represents a new paradigm in mycobacterial adaptation to hypoxia.

The discovery and application of bacteriocin-producing probiotics, such as Streptococcus salivarius K12 (BLIS K12), represent significant advances in the prevention and management of bacterial infections, particularly in the oral cavity and upper respiratory tract. Originally developed for its bacteriocin-mediated inhibition of the important bacterial pathogen Streptococcus pyogenes, BLIS K12 has more recently also demonstrated potential in the modulation and prevention of viral infections, including COVID-19. Emerging evidence also suggests a broader role for BLIS K12 in immune regulation, with implications for controlling hyperinflammatory responses and enhancing mucosal immunity. Of particular interest is recent work indicating that BLIS K12 can modulate antibody responses against viral antigens, such as the SARS-CoV-2 spike protein, positioning it as a unique adjunct in managing viral infections. This review chronicles the pathway of BLIS K12’s probiotic development, emphasizing its relevant bacteriocin mechanisms, oral health applications, emerging antiviral properties, and potential broader health benefits through immune modulation, all of which position it as a significant non-pharmacological adjunct in managing respiratory and immune health