Heme auxotrophy in abundant aquatic microbial lineages

Heme, a porphyrin ring complexed with iron, is a metalloprosthetic group of numerous proteins involved in diverse metabolic and respiratory processes across all domains of life, and is thus considered essential for respiring organisms 1,2 . Several microbial groups are known to lack the de novo heme biosynthetic pathway and therefore require exogenous heme from the environment 3 . These heme auxotroph groups are largely limited to pathogens 4,5 , symbionts 6,7 , or microorganisms living in nutrient-replete conditions 8 , whereas the complete absence of heme biosynthesis is extremely rare in free-living organisms 9 . Here, we show that the acI lineage, a predominant and ubiquitous free-living bacterial group in freshwater habitats, is auxotrophic for heme. We found that two recently cultivated acI isolates 10 require exogenous heme for their growth. According to whole-genome analyses, all ( n =20) isolated acI strains lacked essential enzymes necessary for heme biosynthesis, indicating that heme auxotrophy is a conserved trait in this lineage. Analyses of >24,000 representative genomes for species clusters of the Genome Taxonomy Database (GTDB) revealed that heme auxotrophy is widespread across abundant but not-yet-cultivated microbial groups, including Patescibacteria , Marinisomatota (SAR406), Actinomarinales (OM1), and marine group III Euryarchaeota . Our findings indicate that heme auxotrophy is a more common phenomenon than previously thought, and may lead to use of heme as a growth factor to increase the cultured microbial diversity.