bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426183; this version posted January 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 2 Heme auxotrophy in abundant aquatic microbial lineages 3 4 5 Suhyun Kim1, Ilnam Kang2*, Jin-Won Lee3, Che-Ok Jeon4, Stephen J. Giovannoni5, and 6 Jang-Cheon Cho1,6* 7 8 1Department of Biological Sciences, Inha University, Incheon 22212, Republic of Korea 9 2Center for Molecular and Cell Biology, Inha University, Incheon 22212, Republic of Korea 10 3Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea 11 4Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea 12 5Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA 13 6Department of Biological Sciences and Bioengineering, Inha University, Incheon 22212, Republic of Korea 14 15 *E-mail: [email protected] (Ilnam Kang); [email protected] (Jang-Cheon Cho) 16 17 Corresponding authors’ information: 18 *Ilnam Kang: Center for Molecular and Cell Biology, Inha University, Inharo 100, Incheon 22212, Republic of 19 Korea. Tel: +82-32-876-5541 20 *Jang-Cheon Cho: Department of Biological Sciences, Inha University, Inharo 100, Incheon 22212, Republic of 21 Korea. Tel: +82-32-860-7711 22 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426183; this version posted January 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 23 Heme, a porphyrin ring complexed with iron, is a metalloprosthetic group of numerous proteins involved 24 in diverse metabolic and respiratory processes across all domains of life, and is thus considered essential 25 for respiring organisms1,2. Several microbial groups are known to lack the de novo heme biosynthetic 26 pathway and therefore require exogenous heme from the environment3. These heme auxotroph groups are 27 largely limited to pathogens4,5, symbionts6,7, or microorganisms living in nutrient-replete conditions8, 28 whereas the complete absence of heme biosynthesis is extremely rare in free-living organisms9. Here, we 29 show that the acI lineage, a predominant and ubiquitous free-living bacterial group in freshwater habitats, 30 is auxotrophic for heme. We found that two recently cultivated acI isolates10 require exogenous heme for 31 their growth. According to whole-genome analyses, all (n=20) isolated acI strains lacked essential enzymes 32 necessary for heme biosynthesis, indicating that heme auxotrophy is a conserved trait in this lineage. 33 Analyses of >24,000 representative genomes for species clusters of the Genome Taxonomy Database 34 (GTDB) revealed that heme auxotrophy is widespread across abundant but not-yet-cultivated microbial 35 groups, including Patescibacteria, Marinisomatota (SAR406), Actinomarinales (OM1), and marine group 36 III Euryarchaeota. Our findings indicate that heme auxotrophy is a more common phenomenon than 37 previously thought, and may lead to use of heme as a growth factor to increase the cultured microbial 38 diversity. 39 40 We recently reported an approach for the robust cultivation of two acI strains10, thus establishing the first 41 stable cultures of the actinobacterial acI lineage, one of the most abundant bacterioplankton groups in freshwater 42 environments11-13. For long, efforts to cultivate the acI lineage using genome predictions and inferring 43 ecophysiological traits were unsuccessful. Stable axenic cultivation of two acI strains was first achieved by 44 supplying catalase as a critical growth supplement10, after which diverse sub-clades of the acI lineage14 were 45 isolated with this approach. In this previous work10, the two strains, “Candidatus Planktophila rubra” 46 IMCC25003 (hereinafter referred to as IMCC25003) and “Candidatus Planktophila aquatilis” IMCC26103 47 (hereinafter referred to as IMCC26103), grew robustly and reproducibly when bovine liver catalase was added to 48 growth media. Since the growth was concomitant with decreases in H2O2 concentrations caused by the addition 49 of catalase, it was suggested that the acI group might be sensitive to oxidative stress and the growth was due to 50 lowered H2O2 concentrations. However, two experimental observations could not be easily reconciled with this 51 interpretation. First, when H2O2 concentration was lowered by the addition of pyruvate, a chemical scavenger of 52 H2O2, the enhancement of acI growth was marginal. Second, the H2O2 concentration that allowed the growth of 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426183; this version posted January 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 53 the two acI strains in the catalase-supplemented laboratory experiment (~10 nM) was much lower than those 54 measured in the lake from which the strains were isolated (~60 nM). Therefore, we conjectured that "catalase is 10 55 essential for the growth of acI strains via mechanisms involving, but not restricted to, H2O2 decomposition" . 56 While further investigating this unsolved discrepancy, we discovered that all genomes of the acI lineage 57 were deficient in the heme biosynthetic pathway (Extended Data Fig. 1; discussed in more detail below), which 58 was also reported in a recent study15. This finding led us to speculate that acI bacteria require heme, an essential 59 cofactor of catalase, for their growth, rather than the H2O2 scavenging function of catalase. Here, based on 60 genome predictions and experimental evidence, we report that the acI lineage is auxotrophic for heme. To our 61 knowledge, these experiments with acI bacteria show for the first time that major and abundant lineages of free- 62 living microorganisms inhabiting natural aquatic environments can be heme auxotrophs. We further demonstrate 63 through comparative genomics that heme auxotrophy is found widely among many abundant prokaryotic groups. 64 All of twenty complete genome sequences from isolated acI strains were found to lack essential heme 65 biosynthesis genes. The heme biosynthetic pathway is complex and consists of three parts9 (Extended Data Fig. 66 1). In the upper part, 5-amino-levulinic acid (ALA) is synthesized via two different modules known as C4 and 67 C5. In the essential middle part, ALA is converted to uroporphyrinogen III (a common precursor of all heme 68 compounds), via a three-step process common to all organisms, designated as "COMMON." In the lower part, 69 heme synthesis from uroporphyrinogen III is mediated by three different modules, each designated PPH 70 (protoporphyrin-dependent), CPH (coproporphyrinogen-dependent), and SIRO (siroheme-dependent). Therefore, 71 six variant pathways, combinations of one of the two upper modules with one of the three lower modules, are 72 possible in theory for heme biosynthesis. All acI genomes lack any genes for the essential middle part of the 73 pathway and also lack most genes for the upper and lower parts, which strongly indicates that these strains 74 cannot synthesize heme and therefore require exogenous heme for survival. Hemes are iron-containing 75 porphyrins that are present as cofactors in a diverse range of bacterial proteins, including respiratory 76 cytochromes, non-respiratory-chain cytochromes, non-cytochrome hemoproteins, gas sensor proteins, 77 peroxidases, and catalases9. Given that heme-containing cytochromes are essential for respiration, it was 78 surprising to find that the acI lineage, which is comprised of free-living and obligately aerobic bacteria, does not 79 possess complete sets of heme biosynthesis coding sequences. 80 As the first step to test heme auxotrophy of acI bacteria, we investigated the effect of hemin (the oxidized 81 form of heme b) on the growth of the two acI strains, IMCC25003 and IMCC26103, belonging to the acI-A1 and 82 acI-A4 tribes, respectively. IMCC25003 growth was dependent on hemin in a catalase-free medium. Growth was 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426183; this version posted January 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 83 enhanced by hemin at concentrations as low as 10 pM, and enhancement was saturated at hemin concentrations 84 of 1 nM or higher, with maximum cell densities (1.8–2.5 × 107 cells mL-1) comparable to that obtained from 85 bovine liver catalase addition (10 U mL-1)(Fig. 1a). Given that the heme concentration in 10 U mL−1 catalase is 86 equivalent to ~50 nM, this amount of added catalase would be predicted to provide sufficient heme to support 87 full IMCC25003 culture growth. On the other hand, hemin showed no effect on the growth of IMCC26103 (Fig. 88 1b). 89 Since hemin enhanced the growth of IMCC25003, but not IMCC26103, we hypothesized that IMCC26103 90 requires additional growth factor(s) that are present in catalase preparations and we predicted that, similar to 91 IMCC25003, IMCC26103 would exhibit hemin-dependent growth if this additional factor was provided. A 92 reinvestigation of genome-inferred metabolic potential16 identified riboflavin, a precursor of FMN and FAD, as 93 the most likely additional growth factor required by IMCC26103 but not IMCC25003. The full gene set for de 94 novo biosynthesis of riboflavin was identified only in IMCC25003. IMCC26103 instead had a gene for the 95 riboflavin transporter PnuX, preceded by the FMN riboswitch (Extended Data Fig.