MAGs-centric crack: how long will, spore-positive Frankia and most Protofrankia, microsymbionts remain recalcitrant to axenic growth?

Nearly 50 years after the ground-breaking isolation of the primary Comptonia peregrina microsymbiont under axenic conditions, efforts to isolate a substantial number of Protofrankia and Frankia strains continue with enduring challenges and complexities. This study aimed to streamline genomic insights through comparative and predictive tools to extract traits crucial for isolating specific Frankia in axenic conditions. Pangenome analysis unveiled significant genetic diversity, suggesting untapped potential for cultivation strategies. Shared metabolic strategies in cellular components, central metabolic pathways, and resource acquisition traits offered promising avenues for cultivation. Ecological trait extraction indicated that most uncultured strains exhibit no apparent barriers to axenic growth. Despite ongoing challenges, potential caveats, and errors that could bias predictive analyses, this study provides a nuanced perspective. It highlights potential breakthroughs and guides refined cultivation strategies for these yet-uncultured strains. We advocate for tailored media formulations enriched with simple carbon sources in aerobic environments, with atmospheric nitrogen optionally sufficient to minimize contamination risks. Temperature adjustments should align with strain preferences-28-29°C for Frankia and 32-35°C for Protofrankia-while maintaining an alkaline pH. Given potential extended incubation periods (predicted doubling times ranging from 3.26 to 9.60 days, possibly up to 21.98 days), patience and rigorous contamination monitoring are crucial for optimizing cultivation conditions.

Contrasted resistance of stone-dwelling Geodermatophilaceae species to stresses known to give rise to reactive oxygen species.

Stones in arid environments are inhabited by actinobacteria of the family Geodermatophilaceae like the genera Blastococcus and Modestobacter frequently isolated from altered calcarenites. Their habitat requires adaptation to light-induced and other stresses that generate reactive oxygen species. Here, we show that representative members of the species Blastococcus saxobsidens, Geodermatophilus obscurus, and Modestobacter multiseptatus are differentially adapted to stresses associated with arid environments. Whereas B. saxobsidens was found to be sensitive to gamma radiation (D(10)  = 900 Gy; 10% survival at 900 Gy), M. multiseptatus was moderately (D(10)  = 6000 Gy) and G. obscurus was highly tolerant (D(10)  = 9000 Gy). A difference in resistance to high-frequency (λ value = 254 nm) UV was shown by B. saxobsidens, M. multiseptatus, and G. obscurus, being sensitive, tolerant, and highly tolerant (D(10) of 6, 900, and > 3500 kJ m(-2) , respectively). Tolerance to desiccation, mitomycin C and hydrogen peroxide correlated with the ionizing radiation and UV resistance profiles of the three species and were correlated with the pigments synthesized. Resistance to heavy metals/metalloids did not follow the same pattern, with resistance to Ag(2+) and Pb(2+) being similar for B. saxobsidens, M. multiseptatus, and G. obscurus, whereas resistance to AsO4 3-, Cr(2+) , or Cu(2+) was greater for B. saxobsidens than for the other two species. The stress resistance profiles of M. multiseptatus and B. saxobsidens were reflected in different calcarenite colonization patterns. While M. multiseptatus was predominantly isolated from the first two millimeters of stone surface, B. saxobsidens was predominantly isolated from the deeper part of the stone where it is better protected from sun irradiation, suggesting that the response to light- and desiccation-induced oxidative stress is an important driver for niche colonization in the stone biotope.