Globally distributed Myxococcota with photosynthesis gene clusters illuminate the origin and evolution of a potentially chimeric lifestyle

Domain: earth_science
Published: 2023-10-13
Editorial outcome: minor_revision

Abstract
Photosynthesis is a fundamental biogeochemical process, thought to be restricted to a few bacterial and eukaryotic phyla. However, understanding the origin and evolution of phototrophic organisms can be impeded and biased by the difficulties of cultivation. Here, we analyzed metagenomic datasets and found potential photosynthetic abilities encoded in the genomes of uncultivated bacteria within the phylum Myxococcota. A putative photosynthesis gene cluster encoding a type-II reaction center appears in at least six Myxococcota families from three classes, suggesting vertical inheritance of these genes from an early common ancestor, with multiple independent losses in other lineages. Analysis of metatranscriptomic datasets indicate that the putative myxococcotal photosynthesis genes are actively expressed in various natural environments. Furthermore, heterologous expression of myxococcotal pigment biosynthesis genes in a purple bacterium support that the genes can drive photosynthetic processes. Given that predatory abilities are thought to be widespread across Myxococcota, our results suggest the intriguing possibility of a chimeric lifestyle (combining predatory and photosynthetic abilities) in members of this phylum.

Introduction
Photosynthesis is an ancient biological process in which light energy is transformed into metabolic energy and reducing power for carbon dioxide fixation to produce biomass1. The light utilization of phototrophs expands their ecological niches, facilitates their cosmopolitan distribution, contributes significantly to global biogeochemical cycles, and completely altered the Earth’s surface and atmospheric environment during the Archaean and Proterozoic eons2. Bacterial chlorophototrophs include the oxygenic Cyanobacteria harboring both type I and type II photosynthetic reaction centers (RCI/II) like those found in eukaryotic plants and algae, as well as anoxygenic phototrophic prokaryotes that utilize a single RCI or RCII for light energy conversion3. It has been suggested that all RCs evolved from an anoxygenic common ancestor, living in an environment devoid of oxygen at ~3.5–3.2 Ga3. Multiple lines of evidence indicate that ancestral phototrophs evolved via a complex path to form diverse phototrophs and metabolisms4. Due to their ecological and evolutionary importance, a great deal of effort has been made to unravel the diversity of phototrophic microorganisms5. To date, prokaryotes using (bacterio)chlorophyll-based phototrophy have been found in eight bacterial phyla, namely Cyanobacteria (~200 years ago)6, Proteobacteria (~180 years ago)7, Bacteroidota (the class Chlorobia, 1906)8, Chloroflexota (1974)9, Firmicutes (1983)10, Acidobacteriota (2007)11, Gemmatimonadota (2014)12, and the recently discovered (2018) and cultured (2022) Eremiobacterota13,14. Each lineage contains a specific solar energy conversion device that varies in types of RC and antenna, as well as pigments biosynthetic pathways15, by which the energy is finally used for either autotrophic or heterotrophic growth, e.g., in photoautotrophic Cyanobacteria or in photoheterotrophic Firmicutes.

Myxococcota is an astonishing bacterial phylum because of their extraordinary social lifestyle (e.g., predation and fruiting body formation), which is unusual in the prokaryotes16,17. They were originally known as Myxobacteria affiliated within the class Deltaproteobacteria18, but were recently reclassified into a separate phylum according to the Genome Taxonomy Database (GTDB)19. Members of Myxococcota have distinct metabolic and structural characteristics. Most species exhibit the abilities of adventurous motility20,21 and social motility22,23. Predatory lifestyles have also been reported in many species of this phylum, and the “wolf pack” analogy has been widely used to describe their predatory behavior24. For instance, the model organism Myxococcus xanthus is able to feed on a broad range of microbes including bacteria25 and fungi26. Some members are saprophytic, and can produce antimicrobials to fend off competitors, e.g., the cellulose-degrading Sorangium cellulosum27. Therefore, the capacity to produce a plethora of secondary metabolites for predation23 or competition has made Myxococcota a rich source for bioactive secondary metabolites for decades besides Actinobacteria, Bacillus, and fungi28. Cultured Myxococcota have typically been isolated from (micro)-oxic soil habitats29. Recently, a novel group of bacterial predators30,31, Bradymonabacteria (representative of the order Bradymonadales of Myxococcota), was isolated in saline environments. Additionally, metagenomic studies have further expanded our knowledge of the wide distribution of Myxococcota in various habitats, including freshwater32, seawater33, and potentially anoxic habitats such as the human gut34. Therefore, Myxococcota are cosmopolitan and considered as keystone taxa that play significant roles in microbial interaction networks by their versatile capabilities.

In this study, we performed a global census for phototrophic prokaryotes, and found that the bacterial phylum Myxococcota contains potentially chlorophototrophic members that have a photosynthesis gene cluster (PGC) encoding all enzymes for uncultured and/or unclassified bacteriochlorophyll biosynthesis, reaction center and light-harvesting proteins, and other key enzymes. Results of the heterologous expression of their pigment biosynthesis genes strongly suggest that these genes can drive photosynthetic processes in their original myxobacterial background. Phylogenies of photosynthetic genes showed a monophyletic origin and vertical inheritance in the potentially phototrophic Myxococcota. Additionally, several members appear to have carbon fixation capacity with a complete Calvin-Benson-Bassham (CBB) cycle. Thus, our results indicate that the phylum Myxococcota has an ancient phototrophic history, and suggest that phototrophic Myxococcota may be playing overlooked roles in ecosystems.

Results
Discovery of potentially phototrophic Myxococcota
In order to provide a universal overview of potential phototrophs in prokaryotes, we first constructed a manually curated reference protein database with RCs, photosynthetic pigment biosynthesis enzymes, regulatory proteins, and antenna systems. We subsequently assembled genomes from public metagenomes and downloaded genomes from the National Center for Biotechnology Information (NCBI) prokaryotes database and the Earth’s Microbiome (GEM) catalog. Then genomes were used as queries for screening photosynthesis related homologs (identity >30%, e<1×10–20, coverage >75%) and the results were manually checked to identify potential phototrophs. A total of 8221 genomes containing a partial to complete (gene prevalence >50%) (bacterio)chlorophyll biosynthesis pathway were retained. Among these genomes, we found 32 MAGs that phylogenetically belong to the phylum Myxococcota and are thus placed outside the eight known phototrophic phyla, according to the taxonomy assignment by GTDB-Tk35 (Supplementary Data 1). All 32 MAGs contain genes coding for RCII (e.g., pufLM), photosynthetic pigment biosynthesis, regulatory proteins, and antenna systems, strongly suggesting chlorophototrophy in Myxococcota (Supplementary Data 2). The completeness of these 32 MAGs is >70% according to Gradient Boost Model in CheckM236, whereas the contamination of nearly all MAGs is below 5% (Supplementary Data 1).

We clustered these 32 MAGs on the basis of 95% whole-genome average nucleotide identities (ANIs) to uncover species-level diversity, revealing 18 candidate species-level taxa (Fig. S1 and Supplementary Data 1). To confirm the taxonomic relationship of these MAGs containing (near) complete sets of phototrophic genes, a species tree consisting of the 32 MAGs and reference bacterial genomes from the NCBI genome database was constructed based on a concatenated set of 37 marker genes (Fig. 1a and Supplementary Data 3). Reference genomes from the GTDB r207 (Supplementary Data 4 and 5) were also used to construct species trees (Figs. S1–S3) based on the concatenated alignment of 120 housekeeping genes35. These trees consistently determine that the Myxococcota MAGs with photosynthesis genes distribute in at least six discrete lineages classified into 14 genera and are interspersed with non-phototrophic members within this phylum. Specifically, three MAGs form a deep-branching cluster that shares ~45–49 whole-genome average amino acid identities (AAIs) and ~61–72 ANI with other myxococcotal classes (Fig. S1). We proposed the name Candidatus (Ca.) Kuafubacteria for this class-level lineage (placeholder name c__WYAZ01 in GTDB r207), from the sun-chasing giant Kua Fu in ancient Chinese mythology. Eight MAGs cluster within the family Myxococcaceae from the class Myxococcia (Fig. S2), and 21 MAGs form four clusters within the class Polyangia (Fig. S3). In Polyangia, six MAGs form a close cluster within the family Nannocystaceae, three MAGs belong to the family Polyangiaceae, and one MAG is clustered within the family Sandaracinaceae. We proposed the prefix Xihe (XiHe, sun goddess in Chinese mythology) combined with the environments where these MAGs were recovered to name these uncultured and/or unclassified lineages, e.g., Ca. Xihehalomonas phototrophica that was recovered from salt lagoon. Additionally, 11 MAGs form a cluster (f__SG8_38) within the Polyangia, and we tentatively named Ca. Houyibacteriaceae for this family-level lineage, after Hou Yi, a sun-related immortal in ancient Chinese mythology. A prologue for the proposed taxonomic names is provided in the Supplementary Note 1.

Phylogenic inference of photosynthesis genes
In the Proteobacteria, Gemmatimonadota, and Firmicutes, photosynthesis related genes are clustered into a continuous stretch with a characteristic ensemble of operons, named the PGC. A total of 1845 PGCs were retained from the 8221 potential phototrophic genomes. These PGCs were taxonomically affiliated within the phyla Myxococcota, Proteobacteria and Gemmatimonadota, while the PGCs of Firmicutes were excluded due to their short length and drastic difference with classic PGCs. Remarkably, for Myxococcota, all of the six families contain PGCs coding for all proteins of RCII, enzymes of bacteriochlorophyll and carotenoid biosynthesis, and regulatory proteins (Fig. 2). The phylogenies of PGC genes can provide robust evolutionary inference37, and here we constructed phylogenetic trees of the PGCs, as well as photosynthetic reaction center proteins (PufLM)38, magnesium chelatase encoded by bchHDI genes39, and light-independent protochlorophyllide reductase encoded by bchLNB genes40. Phylogenetic placements of the PGC, PufLM, BchHDI, and BchLNB trees all indicate that Myxococcota phototrophic genes are monophyletic but cluster within the proteobacterial clades (Figs. 1b and 2c, and Figs. S4 and S5).

The PGC gene organization patterns were identified for these genomes (Fig. 2c). Overall, the PGC arrangements of Myxococcota varies with different lineages but are still highly similar with those of Proteobacteria and Gemmatimonadota, such as the regular gene orders of bchIDO, crtCDEF-bchCXYZ, pufBALMC41. However, the operon containing bchP and bchG, separating by an ORF coding for a protein sharing a high identity with the light-harvesting complex assembly factor LhaA, which is commonly found in Proteobacteria and Gemmatimonadota, is found only in the family Ca. Kuafubacteriaceae. The other lineages of Myxococcota display a unique pattern in which the bchPG operon forms a compact arrangement by combining with the commonly occurring bchFNBHLM and pufH(puhA)-puhBC-bciE-acsF-puhE operons42.

The highly similar topologies of photosynthesis gene trees (Fig. 1b, and Figs. S4–S6) and the analogous PGC arrangement (Fig. 2), strongly indicate a vertical inheritance of chlorophototrophy in Myxococcota. Nevertheless, as all myxococcotal photosynthesis genes branch within the Proteobacteria clade on phylogenetic trees, we propose that one ancient Myxococcota lineage obtained an entire PGC from an early proteobacterium, leading to the origin of phototrophy of Myxococcota. The distribution of phototrophic lineages across its species trees is wide, with phototrophic clades scattered across 14 genera in six families from five orders in three classes of Myxococcota (Fig. 1a, and Fig. S1). It has been suggested that the compact arrangement of the PGC may facilitate horizontal transfer43, or be convenient for efficient regulation and gene expression44. It is unlikely that the 14 genera of the phylum Myxococcota independently laterally acquired the PGC multiple times from one lineage of phototrophic Proteobacteria. On the contrary, it may be best explained that the common ancestor of these Myxococcota lineages acquired phototrophic ability, with further multiple independent losses in other lineages (regressive evolution). In addition, it is widely accepted that the origin of the eukaryotic mitochondrion was an endosymbiotic process that involved the predation of an alphaproteobacterium. Therefore, it is likely that phototrophy in Myxococcota either (i) originated from an ancient acquisition by predation-based HGT from a proteobacterium, during which the entire PGC was integrated into the genome of a predatory ancestor, or (ii) originated from the ‘classical’ HGT via acquisition of a mobile genetic element, the scenario proposed for Gemmatimonadota12. Alternatively, as a recently reclassified phylum, Myxococcota were previously assigned to the class Deltaproteobacteria19. Thus, we also cannot rule out the possibility that photosynthetic ability was present in the last common ancestor of Proteobacteria and Myxococcota, resembling previous suggestions that photosynthesis was a common trait of all bacteria or a broad clade including all extant phototrophs45, followed by extensive losses in most lineages.

In all photosynthesis gene trees, the family Ca. Kuafubacteriaceae branched as an early diverging member within the phylum Myxococcota neighboring with the alphaproteobacterial lineage. The family Myxococcaceae contains two clades in the PufLM and BchHDI trees, with one clade neighboring with the Ca. Kuafubacteriaceae and the other clade located within the class Polyangia. The families Nannocystaceae, Polyangiaceae, Sandaracinaceae, and Ca. Houyibacteriaceae of the class Polyangia separate clearly, indicating an apparent vertical inheritance, with the exception that one clade of Polyangiaceae is located near the family Myxococcaceae in the BchHDI and BchLNB trees. Overall, the patchy distribution of phototrophs in diverse Myxococcota families is most likely caused by a combination of vertical inheritance of photosynthetic capacities, which is also supplemented by occasional HGT events between descendant lineages.

Estimating the date of origin of phototrophy in multiple lineages may provide key clues to determine the original inventors of phototrophy. For instance, based on our estimation by molecular dating via species tree, the last common ancestor of six phototrophic families of Myxococcota originated at ~3.09–2.67 Ga. Meanwhile, the last common ancestor of Proteobacteria and Myxococcota originated at ~3.56–3.17 Ga, close to the estimated origin of anoxygenic photosynthesis3. Thus, it is reasonable to assume that ancestral Myxococcota serve as an old phototrophic lineage that arose before the great oxygenation event (GOE) at ~2.33 Ga46,47. The early origin of phototrophy in Myxococcota, whose crown group is currently dominated by aerobic and/or facultative anoxygenic species, reflects the adaption of anaerobic ancestors to subsequent oxic environments. However, previous efforts to explain the early evolution of photosynthesis have been restricted to the limited phototrophic lineages known at the time48,49. The expansion in the number of phototrophic bacterial phyla will provide a crucial foundation for more accurate future reconstructions of the evolutionary history and relationships among phototrophs.

Light-driven metabolisms
We found a complete pathway for the biosynthesis of bacteriochlorophyll a in all of the six families belonging to the phylum Myxococcota (Fig. 3). To test the activity of a bacteriochlorophyll biosynthesis enzyme encoded in a myxococcotal PGC, the catalytic components of the enzyme chlorophyllide oxidoreductase (COR), encoded by genes bchYZ were chosen for heterologous expression. The overlapping bchYZ from a member of Ca. Kuafubacteriaceae were synthesized and cloned into a purple bacterial expression vector. A mutant of the model purple bacterium Rhodobacter (Rba.) sphaeroides lacking the native bchY and bchZ genes, along with the 8-vinyl reductase-encoding bciA50 was selected as the host, to determine if the myxococcotal source of the genes has the ability to synthesize bacteriochlorophyll a or bacteriochlorophyll b. Rba. sphaeroides, along with all purple bacteria, assembles light-harvesting complex 1 around its RCII, forming the RC–LH1 supercomplex, and this organism also uses light-harvesting complex 2 (LH2) as a peripheral antenna51. These complexes determine the absorption profile of the organism, resulting in characteristic features in the whole cell absorption spectrum at 800 nm and 850 nm, contributed by LH2, and a feature appearing as a shoulder at 875 nm, contributed by RC–LH1 (Fig. 4a). The mutant lacking bchYZ and bciA cannot assemble these complexes, thus lacks these absorption bands, and displays altered pigmentation (Fig. 4a). It has been demonstrated that synthesis of BChl b in this mutant background does not restore light harvesting complex assembly50. We found that expression of bchYZ from a member of the Ca. Kuafubacteriaceae restored the pigmentation of these cells to that of the wild-type strain, and also restored assembly of LH2 and RC–LH1, as determined by the appearance of their relevant absorption features in the whole cell spectrum, confirming their function as an enzyme of bacteriochlorophyll a biosynthesis (Fig. 3). The catalytic components of COR, BchYZ, require electrons from reduced ferredoxin that are shuttled through the BchX protein52. In our complementation experiment, the native Rba. sphaeroides BchX protein must interact with the myxococcotal BchYZ to form an active chimeric enzyme. Despite the time since the horizontal transfer of a purple bacterial PGC to the Myxococcota, our results demonstrate that an active enzyme can be formed by proteins encoded in PGCs from extant bacteria from different phyla, indicating their well conversed evolutionary history.

Myxococcota are known as social organisms with a predatory or saprophytic lifestyle, and many cultured strains can form fruiting bodies to resist extreme environments53, displaying flexible gene expression patterns that vary with environments54. Phototrophic capability may be an important feature for the survival of Myxococcota during intervals where prey and/or organic matter is limited. The metabolic model of aerobic anoxygenic phototrophs (AAPs) suggests that light can serve as an supplementary energy source to directly affect swimming speed of the marine proteobacterium Dinoroseobacter shibae55. In addition, it has been reported that bacteriochlorophyll synthesis of Roseobacter sp. was induced under organic substrate limitation56. Meanwhile, photoheterotrophic D. shibae was verified to use the additional energy from light to assimilate carbon dioxide for amino acid biosynthesis through the ethylmalonyl-CoA cycle (EMC, KEGG M00373)57, and light was shown to enhance survival of D. shibae during long-term starvation58. The genomes of Ca. Houyibacterium oceanica, recovered from surface seawater, contain near complete EMC pathways with the key enzyme crotonyl-CoA carboxylase/reductase (encoded by the gene ccr, K14446). Intriguingly, these potentially phototrophic Myxococcota from the same families show significantly larger genome sizes compared with other non-phototrophic families within Myxococcota (Wilcoxon rank sum tests, P<0.001; Fig. S7 and Supplementary Data 6). It might indicate that many additional genes are required for Myxococcota to perform phototrophic and other metabolic processes31. Therefore, incorporating phototrophic capability into genomes may supplement the energy requirement during motility, increase the production of secondary metabolites, and allow them to cope with survival challenges through flexibly regulated phototrophic lifestyle.

On the other hand, Myxococcota MAGs with phototrophic potential shared significantly higher GC content compared to either members with the same families or other members in this phylum (Wilcoxon rank sum tests, P<0.001; Fig. S7). It is suggested that higher GC content is associated with aerobic lifestyle59. The conversion of Mg-protoporphyrin IX monomethyl ester to 3,8-divinyl-protochlorophyllide a is a key step in the production of (bacterio)chlorophylls in all chlorophototrophs60 and is responsible for the characteristic green color associated with this family of pigments. This reaction is catalyzed by two distinct enzymes that use molecular oxygen (AcsF) or water (BchE) as oxygen donors, respectively. AcsF dominates in chlorophototrophs found in oxic environments (e.g., plants, Cyanobacteria, and AAPs), while BchE is essential for organisms performing photosynthesis in the absence of oxygen (e.g., Firmicutes and Chlorobia). MAGs from Myxococcota with PGC all encode an AcsF protein (Fig. S6), suggesting their phototrophy can operate with an aerobic lifestyle59. Intriguingly, all MAGs of Ca. Kuafubacteriaceae and some MAGs of Polyangiaceae, Myxococcaceae, and Ca. Houyibacteriaceae also contain bchE genes outside the PGCs, indicating that some Myxococcota members are potential facultative anaerobes that perform phototrophy in anoxic conditions. Phylogenetic evidence (Fig. S6) indicates that BchE sequences of Myxococcota form a monophyletic branch clustering near the clades of Gemmatimonadota and Proteobacteria (average identities ~60–80%), suggesting that they are functional enzymes. On the contrary, ten MAGs of Ca. Houyibacterium oceanica recovered from surface seawater do not encode bchE genes, suggesting that anaerobic phototrophy is not a practical metabolic option in the oxygen-replete surface ocean and therefore was lost because of selective pressure. This is consistent with previous studies indicating that Myxococcota species are usually cultured or recovered from oxic environments18.

Different genera from the phylum Myxococcota can be distinguished by their unique pigmentation, such as the orange color of the Chondromyces and the reddish to violet color of the Archangium18. Myxococcota with PGCs also contain diverse carotenoid biosynthesis pathways which may serve in light harvesting as accessory pigments and in photoprotection against harmful free radicals, or as antimicrobial agents and compounds with alternative functions. Besides the crtCDEF operon that is located in the PGC, multiple orthologs of carotenoids synthesis genes were dispersed throughout the chromosome. Some of them arranged occasionally as a carotenoid cluster, varying with different MAGs (Fig. S8). This complicated pattern suggests potential parallel acquisitions of carotenoid synthesis genes. Generally, all six families can synthesize spheroidene or spirilloxanthin (CrtF, K09846), but only Ca. Kuafubacteriaceae encode spheroidene monooxygenase (CrtA, K09847) to specifically recognize the C-2 position of spheroidene or the C-2 and C-2′ positions of spirilloxanthin to produce spheroidenone or di-ketospirilloxanthin61. Like many photosynthetic purple bacteria, the absence of CrtA is common in Myxococcota, while crtA genes are frequently located outside the PGC. Spheroidenone in Ca. Kuafubacteriaceae may serve as the main light harvesting carotenoid, as previously demonstrated in Roseobacter62. It was reported that spheroidenone is synthesized by anaerobic photoautotrophic organisms such as Rhodobacter sphaeroides and Rhodovulum marinum when grown under very low oxygen tension63. Besides, the potential to synthesize spirilloxanthin is consistent with the major carotenoids reported in Congregibacter litoralis KT7164. Except for Nannocystaceae, all families contain genes potentially coding for enzymes for synthesis of β-carotene (LcyB/CrtL1/CrtY, K06443; or CruA, K14605) and isorenieratene (CrtU/CruE, K09879) under suitable conditions. Additionally, Ca. Kuafubacteriaceae, Myxococcaceae, Polyangiaceae, and Nannocystaceae may further synthesize zeaxanthin (CrtR K02294; or CrtZ, K15746). These pigments may function in light harvesting, photoprotection, and sustaining membrane integrity65. Diverse carotenoids synthesized by Ca. Kuafubacteriaceae may play important roles in its physiology and ecology. Collectively, the substrate flexibility of many carotenoid biosynthesis enzymes makes it hard to predict the exact profile of pigments displayed by an organism based on its crt/cru gene complement. Therefore, further investigation will be necessary to determine the exact biosynthetic pathways employed by Myxococcota, and how they are regulated. Interestingly, proteorhodopsin (PR) was found in most MAGs of Ca. Houyibacterium oceanica belonging to the family Ca. Houyibacteriaceae, and a salt-lagoon-sourced MAG of Ca. Kuafuhalomonas phototrophica belonging to the family Ca. Kuafubacteriaceae (Supplementary Data 2). Meanwhile, the beta-carotene 15,15’-dioxygenase (Blh, K21817) can be identified in the genome of Ca. Kuafuhalomonas phototrophica but cannot be found in Ca. Houyibacterium oceanica. This phenomenon suggests that Ca. Kuafubacteriaceae and/or Ca. Houyibacteriaceae may have evolved the capability to complementarily perform both (BChl)-based and proton-pumping rhodopsin-based phototrophy in a single bacterium according to energy needs and environmental changes, as has recently been demonstrated for Tardiphaga sp.66 and Sphingomonas glacialis67.

Autotrophy and additional metabolic modes
Up until now, all cultured strains of Myxococcota are heterotrophic. The complete glycolysis (Embden-Meyerhof pathway, KEGG M00001) and citrate cycles (TCA cycle/Krebs cycle, KEGG M00009) are present in all genomes here, indicating heterotrophic lifestyle of members in these Myxococcota with PGC. We further checked carbon fixation pathways adopted by previously studied phototrophs, including the Calvin-Benson-Bassham (CBB) cycle (KEGG pathway M00165), reductive citrate cycle (rTCA cycle, KEGG M00173), 3-hydroxypropionate bi-cycle (3HP, KEGG M00376), for these Myxococcota containing photosynthesis genes. Astonishingly, we found some members of them may conduct autotrophic metabolism using the CBB cycle. Complete or near complete CBB cycles were identified in the families Nannocystaceae, Ca. Kuafubacteriaceae, and Myxococcaceae. Specifically, the genome of Ca. Xihebacterium glacialis from the family Nannocystaceae, recovered from glacier surface soil, contains a complete CBB cycle. Meanwhile, the genome of Ca. Xihepedomonas phototrophica of the family Myxococcaceae, recovered from Arctic peat soil, and genomes of Ca. Kuafubacterium phototrophica and Ca. Kuafucaenimonas phototrophica of the family Ca. Kuafubacteriaceae, also contain near complete CBB cycles with genes encoding the key enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK). Phylogenies place the RuBisCO sequences of aforementioned species into form I RuBisCO (Fig. S9), which is generally found in photosynthetic algae, plants, and bacteria70. The RuBisCO sequences of these families have average identities ~80–90% to the known protein sequences in the NCBI nr database, and they were clustered as three different subclades in the form I RuBisCO tree (Fig. S10), indicating that CBB cycle-based autotrophy may come from multiple historical HGT events, e.g., from Proteobacteria.

For other potential carbon, sulfur and nitrogen metabolic pathways, we found genomes of Ca. Kuafubacteriaceae and Myxococcaceae contain complete phosphate acetyltransferase-acetate kinase pathways (KEGG M00579). Furthermore, all genomes also encode complete assimilatory sulfate reduction (ASR, KEGG M00176), except for the genomes from the families Myxococcaceae and Nannocystaceae, which lack genes coding for phosphoadenosine phosphosulfate reductase (CysH). To our knowledge, sulfur respiration in Myxococcota has not been verified, but similar features have also been described in the MAGs assembled from sulfur and sulfide-rich Zodletone Spring71. Ca. Kuafubacteriaceae, Myxococcaceae, Polyangiaceae also have the potential for dissimilatory nitrate reduction (DNR, KEGG M00530). These additional features may complement the metabolic potentials of versatile Myxococcota, which have previously been reported including denitrification, dehalorespiration, and facultative anaerobic respiration72.

Together, multiple lineages of the phylum Myxococcota have the potential to perform phototrophy, which may be either photoautotrophic or photoheterotrophic lifestyles depending on their survival pressure, such as in extremely oligotrophic environments versus eutrophic environments. Consequently, Myxococcota with photosynthesis genes evolved diversified metabolic potentials. With the annotation predictions from their genomes, (i) Ca. Kuafubacteriaceae should be a versatile generalist with the ability to perform anoxygenic photosynthesis coupled to CBB cycle-based carbon fixation, possessing the potential to synthesize a broad range of carotenoids (e.g., astaxanthin, spheroidenone, isorenieratene and spirilloxanthin), as well as ASR and DNR capabilities, and display a facultative anaerobic lifestyle. (ii) Members of Myxococcaceae and Polyangiaceae share many metabolic features in common, such as synthesizing specific carotenoids (e.g., zeaxanthin, spheroidene, isorenieratene and spirilloxanthin), performing ASR or DNR, and the ability to survive either aerobically or as facultative anaerobes. (iii) Nannocystaceae is characterized by aerobic predation incorporating anoxygenic photosynthesis coupled to CBB cycle-based carbon fixation, carotenoid synthesis (e.g., spheroidene and spirilloxanthin), and partial ASR. The photosynthesis of aerobic Nannocystaceae expands our current understandings of AAPs, which were considered to be limited to photoheterotrophy. (iv) Sandaracinaceae may be aerobic photoheterotrophic predators with the ability to perform ASR and synthesize several carotenoids (e.g., spheroidene, isorenieratene and spirilloxanthin). (v) Phototrophic Ca. Houyibacteriaceae differentiated into two species, aerobic Ca. Houyibacterium oceanica that contain the EMC pathway and PR, and the facultative anaerobic Ca. Houyihalomonas phototrophica that lack the EMC pathway and PR. These two species share similar potential for chlorophototrophy, carotenoid synthesis and ASR with the family Sandaracinaceae.

Ecological and evolutionary implications
The discovery of potential photosynthetic capacity in Myxococcota which are known for their versatile lifestyle such as social and predatory behaviors, is intriguing. Despite studies finding that pigments play a role in mediating light-related functions, e.g., carotenoids function as photoreceptors in model organism M. xanthus73, photosynthetic genes (e.g., pufLM) have previously not been found in any species of Myxococcota. In this study, potential phototrophic members of Myxococcota were recovered from diverse habitats spanning large-scale geographical distance (Fig. 2a, b), including aquatic and terrestrial habitats, salt lagoon and freshwater lake, oligotrophic and eutrophic seawater, high- and low-latitude geographical zones, as well as anthropogenic ecosystems. Furthermore, we dug into global ocean metatranscriptomic analyses to investigate whether their phototrophic capacity is active in situ. We found that photosynthesis genes of Myxococcota were actively expressed on a global scale, e.g., substantial expression of PGC in both coastal and oceanic zones across tropical and subtropical oceans (Fig. 5). Predatory Myxococcota have already been shown to be keystone taxa in microbial food webs, shaping microbial community structure74. Consequently, the presence and expression of photosynthetic genes point to the possibility that Myxococcota are not only consumers shaping microbial communities by means of top-down control75, but also may be primary producers. Together with the widespread occurrence of Myxococcota in natural ecosystems (Supplementary Data 7), this taxon with multiple ecological roles should contribute substantially to global biogeochemical cycles. Moreover, the cosmopolitan distribution of myxococcotal descendants suggests that versatile metabolisms enable them survive across various habitats, and their multifunctionality expands our understanding of microbial evolution and ecology.

Considering that phototrophic potential occurs in at least four clades (i.e., the families Myxococcaceae, Polyangiaceae, Nannocystaceae, and Sandaracinaceae) that have many verified predatory members (Figs. S2 and S3), we suggest two assumptions to explain the origin of phototrophic capability of Myxococcota: (i) “Transitional State” assumption states that the ancestors of Myxococcota and/or Proteobacteria may have pioneered the invention of anoxygenic phototrophy in the late Archaean Earth when oxygen was accumulating, whereas the predatory capacity evolved later in Myxococcota; (ii) “Pillage and Plunder” (assimilating prey genes) assumption explain that if myxococcotal ancestors are predatory, frequent predatory behavior would provide more opportunities for them to horizontally gain foreign genes (e.g., PGC). It is uncertain whether the crown group of phototrophic members is simultaneously predatory (i.e., chimeric photosynthetic predators) or whether photosynthetic and predatory capacities represent two independent metabolic processes of different species (evolutionary adaptations to environmental pressure). In addition, current in silico analyses cannot directly prove that Myxococcota with PGCs actually perform photosynthesis in vivo. Consequently, cultivation of representatives of these Myxococcota will be required to validate the photosynthetic activity and hypotheses suggested in this study as well as other important capacities and bioactive resources in myxococcotal biology. Most importantly, although these implications are intriguing, there are still many mysteries to be explored within this fascinating lineage.

In conclusion, photosynthesis fuels the surface ecosystems on Earth. However, microbes employing bacteriochlorophyll-based reaction centers have been found in only eight out of the 148 currently-described bacterial phyla. According to a global-scale comparative genome analysis for phototrophic diversity, we obtained 32 genomes with chlorophototrophic genes belonging to a previously unrecognized predatory bacterial phylum, Myxococcota. Phototrophic members of this phylum possess and actively express bacteriochlorophyll biosynthesis and reaction center genes in distinct ecosystems across the globe. Heterologous expression of the pigment biosynthesis genes further suggest their ability to grow as phototrophs. Phototrophy in Myxococcota is not restricted to a single lineage, but occurs in at least six discrete families, interspersed with non-phototrophic members. Remarkably, Nannocystaceae, Ca. Kuafubacteriaceae, and Myxococcaceae encode (near) complete Calvin-Benson-Bassham (CBB) cycle with the form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The closely related phylogenies of photosynthesis genes from multiple lineages of Myxococcota suggest that phototrophy might be an ancient trait, with subsequent independent losses of phototrophic ability in many lineages. Overall, the discovery of anoxygenic phototrophy in Myxococcota extends the known distribution of phototrophic diversity in the Bacteria domain, and reveals overlooked ecological roles of Myxococcota in diverse ecosystems.

Methods
Data collection and treatments
Genomes used for mining potential phototrophs include two parts, MAGs binned from assembled metagenomic sequences and genomes downloaded from public databases. Metagenomic datasets were downloaded from the NCBI Sequence Read Archive public database (https://www.ncbi.nlm.nih.gov/sra). The Sickle algorithm version 1.33 (https://github.com/najoshi/sickle) was used to trim metagenomic sequencing reads. The trimmed reads were subsequently assembled by MEGAHIT76 version 1.2.9 with k-min 27 and k-max 127. The sequence coverage of contigs was calculated by mapping the trimmed reads to each contig using BBMap version 38.1877. The assembled metagenomic sequences were binned using MetaBAT78 version 2: 2.15 with 1.5 kb as contig length cut-offs. Additionally, a total of 474,091 genomes of prokaryotes were downloaded from the NCBI prokaryotes database (https://ftp.ncbi.nlm.nih.gov/genomes/genbank; 421,576; July 2022) as well as the GEM catalog79 (https://genome.jgi.doe.gov/portal/GEMs/GEMs.home.html; 52,515; February 2022), respectively. The metadata of Myxococcota genomes was determined manually by searching GEM metadata, NCBI Datasets (https://www.ncbi.nlm.nih.gov/data-hub/genome), NCBI Biosample (https://www.ncbi.nlm.nih.gov/biosample), and scientific literature.

Photosynthesis gene identification
All genomes were subjected to open reading frame (ORF) prediction with Prodigal80 version 2.6.3 with parameter -p single. A local database of representative sequences including bacteriochlorophyll and carotenoid biosynthesis, RC, electron transport, antenna systems, regulatory proteins, and cofactors, was collected from published literatures and KEGG database, and subsequently manually curated. The built local database was used as subject, and the predicted ORFs were queried against the database using DIAMOND81 version 2.0.14.152 with parameters coverage >75% and e<1×10–20. Genomes containing a partial to complete (bacterio)chlorophyll biosynthesis pathways were screened as potential chlorophototrophs. Taxonomic assignment was classified using GTDB-Tk version 2.0.035 and by placement in a concatenated ribosomal protein phylogeny82,83. A total of 8,221 genomes containing >50% of the genes of (bacterio)chlorophyll biosynthesis pathway were retained. These genomes were also screened for reaction center proteins, including the iron-sulfur containing RCI type utilized by green sulfur bacteria (the class Chlorobia of the phylum Bacteroidota), Heliobacteria (Firmicutes), and Chloracidobacteria (Acidobacteriota), the pheophytin-quinone RCII type employed by purple bacteria (Proteobacteria), green non-sulfur bacteria/filamentous anoxygenic phototrophs (FAP) (Chloroflexota), Gemmatimonadota, and Eremiobacterota, and photosystem I (PSI) and photosystem II (PSII) harbored by Cyanobacteria3,5–12,84.

To determine the phylogenies of concatenated PGCs, we first empirically set two type thresholds to locate the PGCs from all potential chlorophototrophs. A rigorous threshold is defined as at least 25 photosynthesis related genes should distribute on a fragment with no more than 50 continuous genes. Similarly, a moderate threshold represents that at least 12 photosynthesis genes should distribute on a fragment with no more than 24 continuous genes. The rigorous threshold is expected to determine all complete or nearly complete PGCs, while the moderate threshold should be able to ensure that fragmented PGCs are recovered even if the PGCs are incomplete or the supposed complete PGCs distribute in distinct genomic fragments. With the rigorous threshold, (near) complete PGCs and corresponding genomes were identified from the BLASTP results based on the software DIAMOND81. A total of 1,845 PGCs, with gene number ranging between 25 to 41, were retained from the 8,221 potential phototrophic genomes. Subsequently, all phototrophic genomes with PGCs were dereplicated using dRep85 version 3.4.0 with parameter -sa 0.95 to reduce redundancy and improve accuracy. PGC proteins occurring in at least 70% of non-redundant genomes were selected as PGC markers (Supplementary Data 8). The sequences of all PGC marker proteins and genes were retrieved from the non-redundant phototrophic genomes.

Evaluations and comparisons of genomes
Potential chlorophototrophic genomes were evaluated by CheckM86 version 1.1.3 using lineage-specific marker genes with parameter lineage_wf, and reference genomes with completeness >70% and contamination <10% were considered for further analyses. The approximate genome size was estimated by the following formula: approximate genome size equals assembly genome size/estimated completeness/(1+estimated contamination). The Wilcoxon rank-sum test was applied to compare the approximate genome size and GC content between phototrophic and non phototrophic genomes using the wilcox.test function in the “stats” package87. The completeness and contamination of Myxococcota MAG were also recertified by machine learning based CheckM236. To check the protein identities of Myxococcota to the most closely related sequences, the predicted ORFs were also queried against the NCBI nr protein database (August 2022) and eggnog88 database with the BLASTP algorithm (coverage >75% and e<1×10–20) using DIAMOND version 2.0.14.152 (Supplementary Data 9). Genomes were assessed for AAIs using CompareM version v0.1.2 with parameter aai_wf (https://github.com/dparks1134/CompareM). ANIs were estimated using OrthoANIu (v.1.2)89. CoverM (https://github.com/wwood/CoverM) was subsequently used to calculate the genome relative abundance based on coverage estimation. The web portal GhostKOALA on the KEGG website90 was employed for metabolic pathway analyses.

Phylogenetic analyses based on sequences of conserved proteins, PGCs, and PufLM/BchHDI/BchLNB/AcsF/BchE/RuBisCO
For phylogenomic analysis, 232 representative bacterial genomes were screened from the previously downloaded NCBI prokaryote genome database (https://ftp.ncbi.nlm.nih.gov/genomes/genbank). Based on a concatenated alignment of a set of 37 marker proteins, all MAGs of Myxococcota with photosynthesis genes and the reference genomes were used to construct a phylogenomic tree. The protein sequences of PGCs and single-protein markers (PufLM, BchHDI, BchLNB, AcsF, and BchE) were also retrieved from the genomes. The reference protein sequences of RuBisCO were retrieved from the published paper70. Protein sequences were aligned by the MAFFT91 algorithm version 7.490 with parameters --genafpair –ep 0 --maxiterate 1,000. The alignment was then filtered using trimAl92 version 1.4.1 with parameter -automated1. Subsequently, the trimmed 37 markers and PGCs were respectively concatenated into a single alignment, and were used to construct the phylogenetic trees by employing IQ-Tree93 and FastTree93, respectively. Divergence timing analyses were estimated based on the phylogenomic tree using the treePL94, and Reltime method95 as implemented in MEGA1196. Three different age constraints were used, including the potential fossil evidences of the cyanobacterial clades Nostocales and Stigonematales (~1.7 Ga), the predicted crown group of oxygenic Cyanobacteria (2.42-2.97 Ga), and the inferred bacterial root lineages (~3.8 Ga)97,98. Furthermore, the 32 MAGs and all high-quality Myxococcota genomes available from the GTDB r207 were used to construct phylogenetic trees based on the concatenated alignments of 120 conserved single-copy marker genes using GTDB-Tk35. Detailed descriptions of parameters of phylogenetic trees are provided in Supplementary Data 10.

Transcriptomic profiling of potential phototrophic Myxococcota
Metatranscriptomic datasets from Tara Oceans projects99–101 were downloaded from the NCBI Sequence Read Archive public database (https://www.ncbi.nlm.nih.gov/sra). The fastp algorithm version 0.12.4102 was used to trim raw metatranscriptomic sequencing reads. For the quantification of transcript expression, we used salmon version 1.9.0103 to map the trimmed metatranscriptomic reads to the six representative Myxococcota genomes. The abundance of each gene in metatranscriptomes was calculated as the metric-FPKB (fragments per kilobase per billion metatranscriptomic fragments) normalized based on the gene length and sequencing depth. For the profile of global expression of Myxococcota photosynthesis genes, we summed the FPKB of genes that belongs to the same function to calculate the functional abundance of each metatranscriptomic library.

Experimental validation for key enzymes involved in bacteriochlorophyll and carotenoid biosynthesis
Growth of described strains. Rba. sphaeroides strains were grown at 30°C under microoxic conditions in the dark in an orbital shaker at 180 rpm, or phototrophically in an anoxic sodium succinate medium27 (N27 medium)104 under illumination (20 μmol·photons·m−2·s−1) provided by 60 W halogen bulbs. All phototrophic culture transfers were performed in an anoxic chamber (Coy Laboratories). When carrying expression plasmids, medium was supplemented with kanamycin at 30 μg·ml−1. E. coli strains JM109105 and ST18 (DSM 22074)106 transformed with pBBRBB-Ppuf plasmids107 were grown in a rotary shaker at 37°C in lysogeny broth supplemented with 30 μg·ml−1 kanamycin. ST18 cells were supplemented with 50 μg·ml−1 5-aminolevulinic acid (ALA).

Complementation of Rba. sphaeroides mutants in trans. The exact sequences of Ca. Kuafubacteriaceae bchYZ and Nannocystaceae crtI could not be commercially-synthesized, nor could versions codon-optimized for expression in Rba. sphaeroides, due to their extreme GC-content. To overcome this, a codon optimization tool (www.idtdna.com/codon/optimization) was used to generate sequences optimized for expression in E. coli, codons used by Rba. sphaeroides at low frequency were manually changed, and restriction sites for subsequent cloning were also removed (sequences can be found in Supplementary Note 2). The resulting genes were synthesized flanked by BglII and SpeI restriction sites, these enzymes were used to excise these fragments, which were cloned in place of DsRed-Express2 in pBBRBB-Ppuf 107. The resulting plasmids isolated from JM109 were verified by DNA sequencing and conjugated into Rba. sphaeroides mutants from the ST18 strain of E. coli. Transconjugants harboring the plasmids were selected on N27 medium supplemented with kanamycin.

Absorption spectroscopy. Ultraviolet/visible/near-infrared absorption spectra were collected on a Cary 3500 spectrophotometer (Agilent Technologies) scanning between 300 and 1100 nm at 1 nm intervals with a 0.1 s integration time.

Pigment extraction. Rba. sphaeroides cultures were washed in 50 mM Tris-HCl pH 8.0 and pelleted by centrifugation. Total pigments were extracted, concentrated and reconstituted according to Namoon, et al.108.

Pigment analysis. Extracted carotenoids were separated on an Agilent 1100 HPLC system equipped with a Supelco Discovery HS C18 column (5 μm particle size, 120 Å pore size, 250 × 4.6 mm) maintained at 40˚C using a program modified from Magdaong, et al.109. Pigments were eluted on a 50 min isocratic gradient of 58:35:7 (v/v/v) acetonitrile/methanol/tetrahydrofuran. Elution of neurosporene and lycopene were monitored at 470 nm and 505 nm, respectively.

Automated nomenclature
The method Great Automatic Nomenclator (GAN)110 was employed to create linguistically correct taxonomic names for Myxococcota MAGs. Fourteen genus names were proposed for these uncultured Myxococcota (Supplementary Data 11) by combinatorial concatenation of three types of word roots (Chinese mythological figures correlated with the sun, the habitat terms, and universal Latin endings or diminutives, respectively) (Supplementary Data 12) and considering International Code of Nomenclature of Prokaryotes (ICNP) rules governing the use or elision of connecting vowels. The type materials for each proposed genus are designated as those with satisfied genome quality criterion111 (or the MAG with highest quality when MAGs do not meet the criteria) and environmental representativity. Higher-level taxonomic ranks were generated by concatenating the stem with an appropriate suffix according to the ICNP rules112.

Selection of representative MAGs for visualization
Representative genomes of photosynthesis gene-containing Myxococcota were selected for the data visualization of PGC arrangement, metabolic schemes, and expression pattern of photosynthesis genes. The MAGs were dereplicated using dRep85 version 3.4.0 with parameter -sa 0.95. Representative myxococcotal genomes were identified based on the genome quality, PGC quality, and completeness of metabolic pathways. In detail, for the visualization of PGC arrangement and expression pattern of photosynthesis genes, all PGCs were firstly compared, and representative MAGs with complete PGC and high genome quality were selected from each family. For the metabolic schemes, MAGs with high genome quality and complete metabolic pathways are labeled as representatives, and other MAGs that have additional genes to supplement the representatives are labeled as “Additional” genomes. We also selected the MAG LLY-WYZ-15_3 that was recovered from ocean as representative of Ca. Houyibacteriaceae to visualize PGC expressions for samples from Tara Oceans. The information of representative MAGs is listed in Supplementary Data 2.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this submission titled \"Globally distributed phototrophic prokaryotic predators illuminate the\norigin and evolution of a novel chimeric lifestyle\", Li and colleagues report their original finding\nthat a diverse group of myxobacteria (recently classified as Myxococcota) encodes whole gene\nclusters known to enable photosynthetic activity. Given that several myxobacteria are known\npredators, they set out to demonstrate that the parallel presence of homologous genes tied to\nphotosynthesis and heterotrophic predation mean that these species engage in chimeric life styles.\nAs such, this finding would be highly intriguing and of high interest to a larger scientific\ncommunity, especially for scientists in microbiology and botany. Yet, there are several major\ncaveats that I will highlight further down below, which prevents this contribution to substantiate\nthe claims it makes.\nIn more detail, the authors resorted to genome mining techniques to identify the presence of\nphotosynthesis-related genes in metagenomes. They de novo assembled metagenomic reads (that\nare dubbed Metagenome-Assembled Genomes, or MAGs) and queried assemblies against a\ndatabase containing an exhaustive list of relevant bacterial photosynthesis genes with parameters\nchosen to assure reasonable best matches (they also added a manual curation step of search\nresults). Within the set of identified MAGs the authors quite surprisingly identified conserved\nphotosynthesis-related gene clusters within the class of Myxococcota, including in several known\nfamilies, but also in two entirely novel groups (to which they assign the novel names,\nKuafubacteria, and Houyibacteriaceae). Given this observation, the authors would like to draw a\nstrong connection between phototrophy and heterotrophic predation, the latter off which is a\nfeature of many culturable myxobacteria, including Myxococcus xanthus.\nThe way they approach this, however, is shaky, and as such the connection between\nphotosynthesis and predation remains highly speculative. Below I substantiate my concerns and\ngive a verdict.\nThe good: identification of photosynthetis-related gene clusters in myxobacteria\nThere is no question in my mind that what the paper found is completely new: the identification of\nphotosynthetis-related gene clusters in MAGs, taxonomically classifiable as Myxococcota, of which\nseveral representatives are model systems for multicellular development, sociality, and predation.\nI really like their phylogenetic approaches, these meet accepted standards, and their graphical\nrepresentations are clear and aesthetically appealing. They also present phylogenetic evidence and\ngene expression data to further demonstrate that many or most of the complete sets of\nphotosynthesis genes are conserved and expressed in nature. This approach seems very robust to\nme, and demonstrates that photosynthesis may be important in several myxobacterial clades.\nThe bad (I): the interpretation of active photosynthesis from expression data\nNo myxobacterial research group to date (including the great Dale Kaiser) has previously found\nevidence for photosynthesis in myxobacteria. As such, the presence of these gene clusters and\neven the knowledge of gene expression is surprising and very exciting. But in my mind, in itself it\nis not direct proof that myxobacteria actually perform photosynthesis (however likely this appears\nbe). It has been shown in molecular detail that Myxoccocus xanthus, the most commonly used\nmodel system in the field, is able to respond to light (via photoreceptors) by producing pigments.\nThis line of research has been conducted mainly by the group of Montserrat Elías-Arnanz (see for\nexample: https://doi.org/10.1126/science.aay1436). I think it remains to be seen if the\nphotosynthesis-related genes have maintained their original purpose in myxobacteria (over all the\nbillions of years), or if they were repurposed for some other tasks. MAGs alone won't tell us.\nThe bad (II): the conceptualisation of and lines of evidence for predation\nThe way the authors want to tie potential photosynthesis to potential predation is rather weak.\nThis starts with the fact that it remains elusive what the authors think bacterial predation is, and\nwhat would be accepted hallmark genes involved in bacterial predation, especially for\nmyxobacteria. Myxobacteria form multicellular groups in order to overwhelm their prey, but can\nalso kill by contact-dependance. While predatory life-styles are wide-spread across the\nmyxobacteria, it is not yet clear if myxobacteria are generally predatory (see ref. 30 which the\nauthors cited). In fact, many are saprophytes and also produce antimicrobials to fend off\ncompetitors, e.g., the cellulose-degrader Sorangium cellulosum, which the authors erroneously list\nas facultative predator.\nWhile several killing mechanisms have been elucidated (eg, https://doi.org/10.7554/eLife.72409),\njust killing others is not predation. And, the general molecular basis of predation in myxobacteria\nis essentially still a mystery and the quest for elucidation of molecular elements has only recently\nbegun.\nTo counter this knowledge gap, the authors resort to a machine-learning approach to infer\ncommon “predatory” genes. It is not entirely clear how this works to me, and so I am sceptical of\nthe reported results., and this is mostly because of the list of genes it produces (see below).\nThese they report mainly in Tables S5 and S6, where they list what the authors define as\n“predators” (Table S5) and “predator-specific KEGG protein groups” (Table S6), respectively.\nBut not all members of Table S5 are predators, in fact Sorangium cellulosum is cellulolytic, as\nmentioned above, and can produces antimicrobial compounds meant for competitor control\n(killing) but not directly to use them as food source (they specialize on cellulose). Hence, any\nshared genes might not reflect predation per se, but simply antagonistic behaviors.\nIt is then not surprising, that Table S6 lists a lot of curious genes, which the authors call\npredation genes. Among them (on top) is listed lacI, the well characterized repressor of expression of genes\nencoding lactose degradation enzymes lacZ, lacY and lacA. LacI is a gene that is also present and\nperhaps better understood in E. coli, not known to be predatory. The list also includes dozens of\nseemingly unspecific genes including ammonium transporters. To me it remains unclear what\nthese genes represent, and how they can be labelled predation genes. And as such, I think this\nanalysis is not conclusive.\nThe ugly: ignoring well-established myxobacterial biology (which undermines their interpretation)\nThe most crucial issue arises from a lack of understanding of myxobacterial biology, which\nundermines the interpretation of the author's findings. Myxobacterial biology centers around two\nmain pillars: multicellular development and social motility, both of which are clearly much better\nunderstood in molecular detail than predation is. Instead of using flagella to swim, most well-\nstudied myxobacteria utilize two very different motility systems for movement across surfaces:\nsocial pili-driven motility (https://doi.org/10.1038/s41467-020-18803-z), and adventurous gliding\nmotility (https://doi.org/10.1073/pnas.1219982110). Yet, in Figure 3 the authors depict the\ntypical myxobacterium with a single flagellum not resembling anything that is usually studied in\nthe myxobacterial community. What is more, the standard myxobacterial response to starvation is\nfruiting body formation in which the cells engage in highly coordinated multicellular development\n(and sticking to a precise developmental program). This part of the life cycle is extremely well\nstudied, yet the authors only speculate that: “The phototrophic capability may be an important\nfeature for the survival of predatory Myxococcota during intervals where prey is limited.” (LL. 185\n– 186). In other words, they do not apply the more standard knowledge on development, but\nrather resort to a fictional myxobacterial model that fits their data best.\nThe verdict:\nIn essence, the authors find something quite amazing, and I think then overreach by setting out to\nestablish a link between the clear presence of photosynthesis-related genes in myxobacteria and\nthe possibility of a chimeric life-style involving heterotrophic predation. In that respect they fail, so\nI think it is best for them to thoroughly reconsider the scope of their study, such that they can\nembed their incredibly interesting finding in a bullet-proof context. For that they need to stick to\nwhat can be reasonably shown using omics techniques alone, and incorporate well-established\nfacts about myxobacterial biology that were gathered in the past around seventy years. The latter\nresearch has not found any evidence for photosynthesis in myxobacteria.\nSpecific comments:\n• The need to tone down any bold statement that remain unsubstantiated, (e.g., LL. 113-114: for\nthe phototrophic Myxococcota)\n• The very book chapter the authors cite as source that claims that all myxobacteria are predatory\n(ref. 30) states the following “It is not yet known whether all myxobacteria are predatory, however\nit seems likely that it is a common feature of the order; predators have been isolated from all\nthree myxobacterial sub-orders, including representatives from the Chondromyces, Corallococcus,\nEnhygromyxa, Myxococcus, Plesiocystis, Pyxidicoccus, Racemicystis, Sorangium and Stigmatella\ngenera (Amiri Moghaddam et al. 2016, 2018; Awal et al. 2016; Livingstone et al. 2017; Perez et\nal. 2016).” So, general predation is not verified truth but rather needs to be properly tested.\n• LL. 72-73 missing information what is meant by «near» completeness of (bacterio)chlorophyll\nbiosynthesis pathways.\n• L. 131 “The highly similar topologies of photosynthesis gene trees”. To make that statement, did\nthe authors perform a statistical test to test for (in)congruence of the tree topologies (eg, using a\nKishino–Hasegawa test)?\n• Figure 1:\no add color information to those taxa that are non-phototrophic (I think the color used is a shade\nof brown).\no better highlight all taxa that belong to the myxobacteria in the trees (ie, by a clearer line of\ndemarkation and circle sector enclosing all Myxococcota.", "reviewer_2": "The study by Li et al reports the first discovery of chlorophototrophic members of the predatory\nbacterial phylum Myxococcota and expands the number of phototrophic bacterial phyla to nine.\nThey show the photosynthetic genes are from a monophyletic origin and also show vertical\nevolution. The discovery represents the first time a chimeric lifestyle of photosynthesis and\npredation are integrated into a single prokaryotic cell.\nThey assembled genomes from the NCBI prokaryotic database and the Earth’s Microbiome catalog,\nwhich was used as queries for screening photosynthesis related homologs resulting in the\ndiscovery of the Myxococcota MAGs. They created gene trees from the photosynthetic genes to\ncompare them with genes from other phyla and inferred metabolic schemes based on the presence\nof genes. They also use metatranscriptional data to determine the expression of photosynthesis\ngenes for the different samples and propose the potential chimeric lifestyle of Myxococcota which\ninvolves photosynthesis, heterotrophy and predation.\nThe manuscript reads well and I think the work will be a significant addition to the field, especially\nin regards to the evolution of photosynthesis. The work supports the conclusions and claims. Below\nI have listed comments to address. The methods are sound and the work meets the expected\nstandards in the field.\nComments for the main text\nLine 39: Are all Myxococcota predatory or has it only been shown in the model organism\nMyxococcus xanthus?\nLine 51: Which microbes do they prey on?\nLine 78: I would remove the MAG that is only 38.32% as it’s not very complete plus the fact that\nyou’ve used a different model for completeness from all the other MAGs is questionable.\nLine 88: It can be difficult to use AAI percentages as an indicator for whether the MAGs should\nbe clustered at the class level as the range can be so varied. My suggestion would be to take\nrepresentatives from different classes and genera and identify what the normal AAI range is for\nthe genomes in the phylum Myxococcota. Otherwise you mention using GTDB-tk, you could use\nthe RED values. Were the MAGs assigned a new class within GTDB-tk? And this is what is shown in\nTable S1.\nLine 89: The correct etymology should be Kuafuibacteria, instead of Kuafubacteria. Also\nKuafuibacterium and Houyiibacterium, instead of Houyibacterium in Table S1. You should also\nprovide etymology for the genus and species names as well. The genus names that you currently\nhave in Table S1 doesn’t make any sense except for the one ending with -bacterium; aestuarium is\ntranslated as tidal flat or estuarium, lacus is a lake, caenum is mud. It would be great if you can\nsubstitute those endings, for instance with -microbium, -bacter, -lacustris (belonging to a lake).\nThis would give names such as Kuafuibacter, Kuafuimicrobium, Kuafuilacustris. You will need to\ndesignate (indicate) a type species of each new genus and type genome for each new species. You\nalso need to propose Youwenlacus (correct to Youwenilacustris or Youwenibacter) and provide its\netymology. It would be good to submit the names to the SeqCode.\nLine 97: What is the percentage of predation in the Myxococcota?\nLine 201: “…through an autonomously regulated phototrophic lifestyle.”\nLine 206 “lifestyle”.\nLine 220: How closely related are the genes?\nLine 371: “..not only be a consumer shaping…may be a primary producer.”\nLine 376: “..single prokaryotic cells can serve as a player at multiple..”\nLine 376: “rather than being confined to either decomposer, consumer or producer?”\nComments for methods\nLine 408: Did you trim the adaptors from the sequencing reads?\nLine 410: Remove the words “according to”, you can just say “The sequence coverage of contigs\nwas calculated by mapping the trimmed reads to each contig using BBMap version 38.18.\nLine 418: It says that the quantification of transcript expression was using salmon. But what did\nyou map the metatranscriptomic sequencing reads too? Were they the myxococcotal genomes or\nphotosynthetic genes from the whole metagenome?\nLine 452: Did you dRep your MAGs as well? Some of them are at 99-100% AAI (Fig S2).\nLine 489: Do you mean queried?\nLine 516: I’m not sure what is meant by rRNA genes. Did you find the rRNA genes and add them\nto the MSA?\nComments for figures and tables\nFig 1\nFig 1 is really difficult to see clearly. It would be better if you could zoom in on the Myxococcota.\nLine 802: 1,713.\nFig 2\nLine 824: They may also not have formed in clusters due to the fact that some of the MAGs have\nover 2,000 scaffolds.\nFig 3.\nWhich genomes are the representatives for each of the Myxococcota families? Are you using them\nall? What if the metabolic pathways are only found in half of the genomes? It would be good to\nhave the number of genomes that you’re using for the analysis in brackets next to each of the\nfamily names.\nFig 4\nWere the expression data mapped back to the genomes? In 4A, you may need to point out that\nthe green are photosynthesis genes and the grey are non-photosynthesis genes. Are there\nmultiple Myxococcota genomes from the same study? Are they expressed evenly? In 4B, what is\nthe representative genome of the family Ca. Houyibacteriaceeae?\nLine 860: Top highly expressed samples? Are the same levels of expression found in the other\ngenomes?\nFig 5\nLifestyle\nFig S1\nIn the legend, the completeness is 32.4% to 100% and the contamination is 0% to 8.4%, however\nin Supplementary Table 1, the contamination is up to 9.33%. It also looks like there are genomes\nin the tree that have even more contamination. It’s difficult to see the classes of interest in this\nfigure. I strongly suggest creating trees of the classes of interest and only including the type\nspecies for the other genera plus two or three extra genomes. The genome with the long branch is\na little worrying. I would remove that genome. Is it because it’s very incomplete? Instead of using\nunphototroph, I would use non-phototroph. How did you include the 16S rRNA genes? It’s very\ndifficult to determine how large the circles are. I would remove the different sizes from the legend.\nFig S2\nThe AAI between many of the Houyibacteriaceae is 99-100%. I would consider these MAGs to be\nthe same strain and would de-replicate them at least at the 95% ANI level. The same goes for\nsome of the MAGs in the Nannocystaceae. It would be good to also include a phylogenetic tree\nnext to the names so that we can also visually see how close they are. I now see in Table S1 that\nthe MAGs have been dereplicated but you don’t mention this in the main text. Are all the analyses\nperformed on all the MAGs or the dereplicated ones?\nFig S3\nIt’s difficult to know what the green squares were unless you zoom into the document at 300%.\nWhen I first looked at it I couldn’t see green in the legend. I’m not sure if there’s another way\nyou can show the numbers that will be easier to see. Is dark green 1 copy and light green 2 copies?\nFig S4\nYou may want to check that your supplementary files are in order. Fig S4 is first mentioned in line\n198 but Fig S5 and Fig S6 are mentioned on line 121.\nFig S5\nI would remove the legend for the bootstraps as it’s difficult to see how large the 0.9, 0.93,\n0.95, 0.98 and 1 circles are.\nFig S6\n1,201. Where are the Betaproteobacteria and why are they in brackets?\nFig S7\nIt took me a little while to understand what the different colours of the branches are. It maybe\nbest to remove the brackets.\nFig S8\npaper80\nTable S2\nMaybe to be consistent, just have the family name and the species name and remove the f__ for\nfamily. You should remove LLY-WYZ-19_1. Based on its completeness, this is probably why it’s\nmissing a lot of genes in the metabolism analysis."}, "author_response": "Dear Reviewers,\nThank you for your careful reading and professional suggestions. All comments here have\nbeen taken into full consideration during the preparation of our revised manuscript. Below are\nour point-by-point responses to the comments. Page and line numbers are consistent with\nthe revised manuscript. Responses of reviewers’ questions are in blue color, quotations of new\ntexts in the manuscript are in blown color.\nReviewer #1 (Remarks to the Author):\nIn this submission titled \"Globally distributed phototrophic prokaryotic predators illuminate the\norigin and evolution of a novel chimeric lifestyle\", Li and colleagues report their original finding\nthat a diverse group of myxobacteria (recently classified as Myxococcota) encodes whole gene\nclusters known to enable photosynthetic activity. Given that several myxobacteria are known\npredators, they set out to demonstrate that the parallel presence of homologous genes tied to\nphotosynthesis and heterotrophic predation mean that these species engage in chimeric life\nstyles. As such, this finding would be highly intriguing and of high interest to a larger scientific\ncommunity, especially for scientists in microbiology and botany. Yet, there are several major\ncaveats that I will highlight further down below, which prevents this contribution to substantiate\nthe claims it makes.\nIn more detail, the authors resorted to genome mining techniques to identify the presence of\nphotosynthesis-related genes in metagenomes. They de novo assembled metagenomic reads\n(that are dubbed Metagenome-Assembled Genomes, or MAGs) and queried assemblies\nagainst a database containing an exhaustive list of relevant bacterial photosynthesis genes\nwith parameters chosen to assure reasonable best matches (they also added a manual\ncuration step of search results). Within the set of identified MAGs the authors quite surprisingly\nidentified conserved photosynthesis-related gene clusters within the class of Myxococcota,\nincluding in several known families, but also in two entirely novel groups (to which they assign\nthe novel names, Kuafubacteria, and Houyibacteriaceae). Given this observation, the authors\nwould like to draw a strong connection between phototrophy and heterotrophic predation, the\nlatter off which is a feature of many culturable myxobacteria, including Myxococcus xanthus.\nThe way they approach this, however, is shaky, and as such the connection between\nphotosynthesis and predation remains highly speculative. Below I substantiate my concerns\nand give a verdict.\nResponse: We thank the reviewer for the constructive suggestion of our work. Based on the\nreviewer’s comments, we removed predation analysis, reduced and toned down the\ndescription of connection between photosynthesis and predation throughout the revised\nmanuscript. Instead, we focused more on photosynthesis analysis, and experimentally\nvalidated the functions of two important photosynthesis genes by heterologous expression.\nThe good: identification of photosynthetis-related gene clusters in myxobacteria\nThere is no question in my mind that what the paper found is completely new: the identification\nof photosynthetis-related gene clusters in MAGs, taxonomically classifiable as Myxococcota,\nof which several representatives are model systems for multicellular development, sociality,\nand predation. I really like their phylogenetic approaches, these meet accepted standards, and\ntheir graphical representations are clear and aesthetically appealing. They also present\nphylogenetic evidence and gene expression data to further demonstrate that many or most of\nthe complete sets of photosynthesis genes are conserved and expressed in nature. This\napproach seems very robust to me, and demonstrates that photosynthesis may be important\nin several myxobacterial clades.\nResponse: We thank the reviewer for the supportive comments.\nThe bad (I): the interpretation of active photosynthesis from expression data\nNo myxobacterial research group to date (including the great Dale Kaiser) has previously\nfound evidence for photosynthesis in myxobacteria. As such, the presence of these gene\nclusters and even the knowledge of gene expression is surprising and very exciting. But in my\nmind, in itself it is not direct proof that myxobacteria actually perform photosynthesis (however\nlikely this appears be). It has been shown in molecular detail that Myxoccocus xanthus, the\nmost commonly used model system in the field, is able to respond to light (via photoreceptors)\nby producing pigments. This line of research has been conducted mainly by the group of\nMontserrat Elías-Arnanz (see for example: https://doi.org/10.1126/science.aay1436). I think it\nremains to be seen if the photosynthesis-related genes have maintained their original purpose\nin myxobacteria (over all the billions of years), or if they were repurposed for some other tasks.\nMAGs alone won't tell us.\nResponse: Thanks for the comment. Although the photosynthesis gene clusters (PGCs) from\nMyxococcota are complete and phylogenetically cluster together with the functional\nphotosynthesis genes from Proteobacteria, we really agree with the reviewer that only\nanalyzing MAGs cannot conclude whether these myxococcotal photosynthesis-related genes\nmaintained their original purpose or were repurposed for other tasks. Thus, we performed\nheterologous expression of the chlorophyllide oxidoreductase genes bchYZ and phytoene\ndesaturase gene crtI in mutants of the model purple bacterial phototroph Rba. sphaeroides,\nand we demonstrate that these Myxococcota photosynthesis genes do function as enzymes\ninvolved in bacteriochlorophyll and carotenoid biosynthesis. Nevertheless, we toned down the\nstatements that are not experimentally tested by myxococcotal isolates. We here changed the\nphrase “phototrophic Myxococcota” to “potential phototrophic Myxococcota or Myxococcota\nwith photosynthesis genes”, and added the description for limitations of this study on Line 402-\n405: “Current in silico analyses cannot directly prove that Myxococcota with PGC actually\nperform photosynthesis in vivo. Consequently, future pure cultivation of representatives of\nthese Myxococcota will be required to validate the photosynthetic activity and hypotheses\nsuggested in this study as well as other important capacities in myxoccotal biology.”\nThe bad (II): the conceptualisation of and lines of evidence for predation\nThe way the authors want to tie potential photosynthesis to potential predation is rather weak.\nThis starts with the fact that it remains elusive what the authors think bacterial predation is,\nand what would be accepted hallmark genes involved in bacterial predation, especially for\nmyxobacteria. Myxobacteria form multicellular groups in order to overwhelm their prey, but can\nalso kill by contact-dependance. While predatory life-styles are wide-spread across the\nmyxobacteria, it is not yet clear if myxobacteria are generally predatory (see ref. 30 which the\nauthors cited). In fact, many are saprophytes and also produce antimicrobials to fend off\ncompetitors, e.g., the cellulose-degrader Sorangium cellulosum, which the authors\nerroneously list as facultative predator.\nWhile several killing mechanisms have been elucidated (eg,\nhttps://doi.org/10.7554/eLife.72409), just killing others is not predation. And, the general\nmolecular basis of predation in myxobacteria is essentially still a mystery and the quest for\nelucidation of molecular elements has only recently begun.\nTo counter this knowledge gap, the authors resort to a machine-learning approach to infer\ncommon “predatory” genes. It is not entirely clear how this works to me, and so I am sceptical\nof the reported results., and this is mostly because of the list of genes it produces (see below).\nThese they report mainly in Tables S5 and S6, where they list what the authors define as\n“predators” (Table S5) and “predator-specific KEGG protein groups” (Table S6), respectively.\nBut not all members of Table S5 are predators, in fact Sorangium cellulosum is cellulolytic, as\nmentioned above, and can produces antimicrobial compounds meant for competitor control\n(killing) but not directly to use them as food source (they specialize on cellulose). Hence, any\nshared genes might not reflect predation per se, but simply antagonistic behaviors.\nIt is then not surprising, that Table S6 lists a lot of curious genes, which the authors call\npredation genes. Among them (on top) is listed lacI, the well characterized repressor of\nexpression of genes encoding lactose degradation enzymes lacZ, lacY and lacA. LacI is a\ngene that is also present and perhaps better understood in E. coli, not known to be predatory.\nThe list also includes dozens of seemingly unspecific genes including ammonium transporters.\nTo me it remains unclear what these genes represent, and how they can be labelled predation\ngenes. And as such, I think this analysis is not conclusive.\nResponse: We thank the reviewer for the valuable comments. We agree with the comment\nthat the result of machine learning prediction is not conclusive, especially under the\nbackground that the general molecular basis of predation in myxobacteria is still not clear,\neven though the “predation-related genes”1-3 and the machine learning method for prediction\nof predatory behavior in Myxococcota have been reported by several groups4,5. So, we\ndecided to remove the analysis of “predation-prediction” in the revised manuscript as the\nreviewer suggested.\nOur previous effort was to do a machine learning prediction at the whole genome level, and\ncombined the “predator-specific KEGG protein groups” that was identified from another study3,\nto visualize the results. Therefore, it was not appropriate to indicate the names “predator-\nspecific KEGG protein groups” because these KEGG protein groups also contain proteins that\nare not specific to predators. Moreover, based on the current background of myxobacterial\nbiology, we cannot accurately list the “predator-specific KEGG protein groups” from the\nmachine learning approach, since many of the protein groups are unspecific and related with\nother functions or present in non-predatory microbes. We also thank the reviewer for pointing\nour error in labeling Sorangium cellulosum as predator (in fact they are cellulose-degrader).\nIn summary, we removed the analysis of “predation-prediction” in the revised manuscript. We\nare looking forward for future research that “predatory” genes and detailed mechanism are\nwell resolved and more species are verified as predator or not. Thereafter, machine learning\nor other methods may be more indicative and convincing.\nThe ugly: ignoring well-established myxobacterial biology (which undermines their\ninterpretation)\nThe most crucial issue arises from a lack of understanding of myxobacterial biology, which\nundermines the interpretation of the author's findings. Myxobacterial biology centers around\ntwo main pillars: multicellular development and social motility, both of which are clearly much\nbetter understood in molecular detail than predation is. Instead of using flagella to swim, most\nwell-studied myxobacteria utilize two very different motility systems for movement across\nsurfaces: social pili-driven motility (https://doi.org/10.1038/s41467-020-18803-z), and\nadventurous gliding motility (https://doi.org/10.1073/pnas.1219982110). Yet, in Figure 3 the\nauthors depict the typical myxobacterium with a single flagellum not resembling anything that\nis usually studied in the myxobacterial community. What is more, the standard myxobacterial\nresponse to starvation is fruiting body formation in which the cells engage in highly coordinated\nmulticellular development (and sticking to a precise developmental program). This part of the\nlife cycle is extremely well studied, yet the authors only speculate that: “The phototrophic\ncapability may be an important feature for the survival of predatory Myxococcota during\nintervals where prey is limited.” (LL. 185 – 186). In other words, they do not apply the more\nstandard knowledge on development, but rather resort to a fictional myxobacterial model that\nfits their data best.\nResponse: Thank you very much for these valuable suggestions.\ni) Myxobacteria have been paid much attention in the past decades because of their\nimportance. Identifying their common features, such as multicellular development,\nadventurous and social motility, is still challenging only to use the data of MAGs, just as the\nreviewer’s indication that “MAGs alone won't tell us”. Therefore, we deleted the single flagellum\nin the revised Figure 3 to avoid potential misleading after exploring the key genes for\nmovement as described below.\nFirst, to test the presence of flagella and pili related genes in the genomes of potential\nphototrophic Myxococcota, we used the web portal GhostKOALA on the KEGG website6. We\nfound that the present pattern of pili related genes of potential phototrophic Myxococcota\nresembles that of cultured species. This inference is mainly based on the cluster pattern that\nmany clades of potential phototrophic Myxococcota and cultured species clustered together\n(Response letter Fig. 1). For example, most of the genomes of potential phototrophic\nMyxococcota have PilY1 (essential for pilus extension in Myxococcus xanthus)7 and other\ngenes that may be important for Type IV pili assembly. In addition, it seems likely that potential\nphototrophic Myxococcota contain more flagellar related genes compared with cultured\nspecies (Response letter Fig. 2). Except for the LLY-WYZ-1_1, LLY-WYZ-3_1, and LLY-WYZ-\n15_3, other representatives of photosynthesis genes-containing Myxococcota clustered\ntogether, suggesting that gene groups of flagellar assembly of potential phototrophic\nMyxococcota are different compared with those of cultured myxobacterium. This is reasonable\nbecause many of the MAGs of potential phototrophic Myxococcota were collected from water-\nrelated environments, e.g., seawater, freshwater, and waste water, rather than soil\nenvironments. Under such environments, flagella might be very important for the movement\nof potential phototrophic Myxococcota.\nIn addition, we collected representative sequences of fruiting body formation (FruA, GenBank\naccession: ABF92619.1), social pili-driven motility (PilY1, GenBank accession: SDW47221.1),\nand adventurous gliding motility8 (AglR, flagella stator homologs of MotA, GenBank accession:\nAAO22892.1; MotA, GenBank accession: QDF08298.1) from type species Myxococcus\nxanthus. We used these sequences as database to search similar proteins from the MAGs of\npotential phototrophic Myxococcota using blastp (coverage >75% and e <1×10–20). We found\nthat all MAGs of potential phototrophic Myxococcota contain AglR and/or MotA, suggesting\nthe potential of adventurous motility in these members (Response letter Fig. 3). Interestingly,\nthe genes involved in fruiting body formation, adventurous and social motility were shared by\nthe LLY-WYZ-3_1 and cultured Myxococcus xanthus. We further used these sequences to\nconstruct phylogenetic tree by employing IQ-Tree9. We found that the sequences of FruA,\nPilY1, and AglR of LLY-WYZ-3_1 also shared close phylogenetic relationship with the\nsequences of Myxococcus xanthus (Response letter Fig. 5). These results suggest the\nmetabolic potentials of fruiting body formation, adventurous and social motility of LLY-WYZ-\n3_1. However, the above annotation analyses were only based on the KEGG database or\nprotein sequences from the cultured model organism Myxococcus xanthus6, hence, more\nexperimental analyses need further comprehensive and systematic investigation to identify\nthese important features in myxobacterial biology.\nii) To better interpret our findings, we added related knowledge in the introduction and refined\nour discussion with myxobacterial background knowledge throughout the revised manuscript.\nFor example, in the introduction on Line 40-52, we added the description: “Myxococcota is an\nastonishing bacterial phylum because of their extraordinary social lifestyle (e.g., predation and\nfruiting body formation), which is unusual in prokaryotes.” … “Most species exhibit the abilities\nof adventurous motility and social motility. Predatory lifestyles have also been reported in many\nspecies of this phylum, and the “wolf pack” analogy has been widely used to describe their\npredatory behavior”.…. “Some members of Myxococcota are saprophytic, and can produce\nantimicrobials to fend off competitors, e.g., the cellulose-degrading Sorangium cellulosum.\nTherefore, the capacity to produce a plethora of secondary metabolites for predation or\ncompetition makes Myxococcota a rich source for bioactive secondary metabolites for\ndecades besides Actinobacteria, Bacillus, and fungi.”\niii) We also added the description of fruiting body formation that is the standard myxobacterial\nresponse to starvation in the Discussion, e.g., on Line 216-218: “Myxococcota are known as\nsocial organisms with a predatory or saprophytic lifestyle, and many cultured strains can form\nfruiting bodies to resist extreme conditions, displaying flexible gene expression patterns that\nvary with environments.”\nResponse letter Fig. 1 The presence and abundance of pili related genes in the\nrepresentative genomes of potential phototrophic Myxococcota and cultured myxobacteria.\nResponse letter Fig. 2 The presence and abundance of flagella related genes in the\nrepresentative genomes of potential phototrophic Myxococcota and cultured myxobacteria.\nResponse letter Fig. 3 The heatmap depicts the blastp results using the predicted ORFs of\npotential phototrophic Myxococcota against the database of representative genes of fruiting\nbody formation (FruA, GenBank accession: ABF92619.1), social pili-driven motility (PilY1,\nGenBank accession: SDW47221.1), and adventurous gliding motility (AglR, GenBank\naccession: AAO22892.1; AglR, homologs of MotA, GenBank accession: QDF08298.1) from\ntype species Myxococcus xanthus.\nResponse letter Fig. 5 The phylogenies of potential FruA, PilY1, and AglR (MotA) sequences\nof potential phototrophic Myxococcota and cultured myxobacterium.\nThe verdict:\nIn essence, the authors find something quite amazing, and I think then overreach by setting\nout to establish a link between the clear presence of photosynthesis-related genes in\nmyxobacteria and the possibility of a chimeric life-style involving heterotrophic predation. In\nthat respect they fail, so I think it is best for them to thoroughly reconsider the scope of their\nstudy, such that they can embed their incredibly interesting finding in a bullet-proof context. For\nthat they need to stick to what can be reasonably shown using omics techniques alone,\nand incorporate well-established facts about myxobacterial biology that were gathered in the\npast around seventy years. The latter research has not found any evidence for photosynthesis\nin myxobacteria.\nResponse: Thank you for the inspiring verdict and constructive comments. We agree with the\nreviewer’s suggestions that only using a model of machine learning-based “predation\nprediction” without in vivo verification to link highly speculative predation to our findings of\nphotosynthesis-related genes in myxobacteria is not sufficiently convincing. We also note that\nthe accuracy and magnitude of training set as well as the performance of the resulting model\nof machine learning-based “predation prediction” in the previous version of our manuscript are\nlimited. Thus, we have removed the result of “predation prediction”, added the heterologous\nexpression validating the functions of photosynthesis genes, and re-discussed the findings in\nthis paper closely centered on omics data and followed the current myxobacterial biological\ncontext to the best of our knowledge.\nSpecific comments:\n• The need to tone down any bold statement that remain unsubstantiated, (e.g., LL. 113-114:\nfor the phototrophic Myxococcota)\nResponse: Thanks for the valuable suggestion. We toned down the statements that remain\nunsubstantiated, e.g., “phototrophic Myxococcota” to “potential phototrophic Myxococcota” or\n“MAGs of Myxococcota with photosynthesis genes”.\n• The very book chapter the authors cite as source that claims that all myxobacteria are\npredatory (ref. 30) states the following “It is not yet known whether all myxobacteria are\npredatory, however it seems likely that it is a common feature of the order; predators have\nbeen isolated from all three myxobacterial sub-orders, including representatives from the\nChondromyces, Corallococcus, Enhygromyxa, Myxococcus, Plesiocystis, Pyxidicoccus,\nRacemicystis, Sorangium and Stigmatella genera (Amiri Moghaddam et al. 2016, 2018; Awal\net al. 2016; Livingstone et al. 2017; Perez et al. 2016).” So, general predation is not verified\ntruth but rather needs to be properly tested.\nResponse: Thanks for the suggestion. We deleted the analysis of machine learning based\nprediction and toned-down the relevant description of “general predation” in the revised\nmanuscript.\n• LL. 72-73 missing information what is meant by «near» completeness of (bacterio)chlorophyll\nbiosynthesis pathways.\nResponse: Thanks for the suggestion. We modified the description in the Result on Line 81-\n82: “A total of 8,221 genomes containing a partial to complete (gene prevalence > 50%)\n(bacterio)chlorophyll biosynthesis pathway were retained.” We also added parameter to the\nMethods on Line 452-453: “Genomes containing a partial to complete (bacterio)chlorophyll\nbiosynthesis pathway were screened as potential chlorophototrophs.”\n• L. 131 “The highly similar topologies of photosynthesis gene trees”. To make that statement,\ndid the authors perform a statistical test to test for (in)congruence of the tree topologies (eg,\nusing a Kishino–Hasegawa test)?\nResponse: Thanks for the suggestion. We performed tree topology comparison to check the\ncongruence using Myxococcota MAGs with complete photosynthesis gene cluster (Response\nletter Table 1). The trees were all constructed by using IQ-tree with best-fitting models plus\nC60 model. The result of both Kishino-Hasegawa test10 and Shimodaira-Hasegawa test11\nusing the key photosynthesis marker type II reaction center PufLM and species trees using\ntwo different sets of marker genes showed that their topologies are not significantly different\n(p-value > 0.05). We also visualized these trees in the Response letter Fig. 5 to intuitively\ncompare tree topologies. We found that the tree topologies are consistent (both at the class\nlevel and at the family level).\nResponse letter Table 1. Tree topologies test for the congruence of photosynthesis gene\ntrees and species trees.\np-value p-value\nType II\nDifferent tested Kishino- Shimodaira-\nmarker sets Hasegawa Hasegawa\nreaction center\ntest test\n37 markers -4840.471955 0.0508 0.0935\nGTDB markers -4831.906993 0.0507 0.0935\nPufLM BchHDI -4831.907181 0.154 0.342\nBchLNB -4839.372352 0.0224 0.104\nPGC -4823.738882 0.154 0.342\nResponse letter Fig. 5 Phylogenies of photosynthesis gene trees and species trees using\nrepresentative genomes.\n• Figure 1:\no add color information to those taxa that are non-phototrophic (I think the color used is a\nshade of brown).\no better highlight all taxa that belong to the myxobacteria in the trees (ie, by a clearer line of\ndemarkation and circle sector enclosing all Myxococcota.\nResponse: Thanks for the comments. We re-visualized the Figure 1 as the reviewer suggested.\nIn the inner ring of the Figure 1, we used a shade of brown to indicate the lineages belonging\nto Myxococcota. We also used a green background to indicate phototrophic lineages, and\nused grey background to indicate non-phototrophic lineages. In addition, we added the\ndescription of caption on Line 925-929: “In the inner ring, lineages of Myxococcota are shaded\nwith brown background. In the middle ring, phototrophic lineages are colored with green\nbackground, and non-phototrophic lineages are colored with grey background. The outer ring\nof phylogeny is colored based on different types of reaction centers.”\nRevised Figure 1. Phylogenetic affiliations of the potential phototrophic Myxococcota MAGs,\nand corresponding PufLM and BchHDI protein sequences. (A) Phylogenomic affiliation of the\nMAGs based on 37 conserved protein sequences and using 232 representative reference\nbacterial genomes. Alignments were based on MAFFT and then filtered with trimAl. The tree\nwas built using IQ-Tree with 1,000 bootstrap replicates. The bootstrap supporting values above\n0.9 were indicated with solid circles. The phylogenetic tree was rooted at the midpoint. In the\ninner ring, lineages of Myxococcota are shaded with brown background. In the middle ring,\nphototrophic lineages are colored with green background, and non-phototrophic lineages are\ncolored with grey background. The outer ring of phylogeny is colored based on different types\nof reaction centers. All phyla are labeled near the phylogenies, with the exception of\nProteobacteria that are labeled based on different classes, e.g., Alphaproteobacteria and\nGammaproteobacteria (Betaproteobacteria shared same color with Gammaproteobacteria\nbased on the taxonomy in GTDB r207). Six potential phototrophic families of Myxococcota are\nalso labeled with different colors near their phylogenies. (B) The phylogenetic trees were\nconstructed based on the alignments of PufLM with 571 aligned positions and BchHDI with\n1,702 aligned positions. Alignments were based on MAFFT and then filtered with trimAl, and\nthe trees were built using IQ-Tree with 1,000 bootstrap replicates. The bootstrap supporting\nvalues above 0.9 are indicated with solid circles. Lineages of Myxococcota are colored with\nbrown for background. Members from Alphaproteobacteria, Gammaproteobacteria (including\nBetaproteobacteria), Gemmatimonadota, and six families of Myxococcota are assigned\ndifferent background colors. The outer ring of phylogeny is colored based on corresponding\nphylum.\nReviewer #2 (Remarks to the Author):\nThe study by Li et al reports the first discovery of chlorophototrophic members of the predatory\nbacterial phylum Myxococcota and expands the number of phototrophic bacterial phyla to nine.\nThey show the photosynthetic genes are from a monophyletic origin and also show vertical\nevolution. The discovery represents the first time a chimeric lifestyle of photosynthesis and\npredation are integrated into a single prokaryotic cell.\nThey assembled genomes from the NCBI prokaryotic database and the Earth’s Microbiome\ncatalog, which was used as queries for screening photosynthesis related homologs resulting\nin the discovery of the Myxococcota MAGs. They created gene trees from the photosynthetic\ngenes to compare them with genes from other phyla and inferred metabolic schemes based\non the presence of genes. They also use metatranscriptional data to determine the expression\nof photosynthesis genes for the different samples and propose the potential chimeric lifestyle\nof Myxococcota which involves photosynthesis, heterotrophy and predation.\nThe manuscript reads well and I think the work will be a significant addition to the field,\nespecially in regards to the evolution of photosynthesis. The work supports the conclusions\nand claims. Below I have listed comments to address. The methods are sound and the work\nmeets the expected standards in the field.\nResponse: We thank the reviewer for the supportive comments.\nComments for the main text\nLine 39: Are all Myxococcota predatory or has it only been shown in the model organism\nMyxococcus xanthus?\nResponse: Predation has been verified in many genera (Response letter Table 2), e.g.,\nCorallococcus, Myxococcus, and Stigmatella. Although predatory capacity seems likely to be\na common feature in cultured strains of Myxococcota, we cannot conclude that myxobacteria\nare generally predatory, just as the comment from Reviewer #1. Meanwhile, given that the\ngeneral molecular basis of predation in myxobacteria remains unclear, the result of predation-\nprediction based on machine learning (most phototrophic members are also predicted as\npredators) is not cogent enough. Therefore, even if the “predation-related genes”1-3,12 and the\nmachine learning method for prediction of predation behavior in Myxococcota have been\nreported by several groups4,5, we deleted the machine learning prediction and toned down the\ndiscussion of “the lifestyle depends on photosynthesis and predation of Myxococcota” as\nsuggested by Reviewer #1.\nResponse letter Table 2 The information of selected predatory Myxococcota genomes.\nLine 51: Which microbes do they prey on?\nResponse: Predatory species of Myxococcota are able to feed on a broad range of microbes\nlike soil bacteria13, pathogens14,15, Cyanobacteria16, and fungi, including yeast15,17,18.\nLine 78: I would remove the MAG that is only 38.32% as it’s not very complete plus the fact\nthat you’ve used a different model for completeness from all the other MAGs is questionable.\nResponse: Thanks for the suggestion. We have removed this MAG.\nLine 88: It can be difficult to use AAI percentages as an indicator for whether the MAGs should\nbe clustered at the class level as the range can be so varied. My suggestion would be to take\nrepresentatives from different classes and genera and identify what the normal AAI range is\nfor the genomes in the phylum Myxococcota. Otherwise you mention using GTDB-tk, you could\nuse the RED values. Were the MAGs assigned a new class within GTDB-tk? And this is what\nis shown in Table S1.\nResponse: Thanks for your valuable suggestions. i) The MAGs that we named Candidatus\n(Ca.) Kuafubacteria are assigned to the class with placeholder name c__WYAZ01 using\nGTDB-Tk. This class has six representative genomes in the GTDB r207 (the condition is\nconsistent in the recently updated GTDB r214) (Response letter Table 3). ii) We added\nrepresentative genomes from different classes and genera in Myxococcota to the AAI and ANI\nanalyses to indicate the taxonomic level in the revised Supplementary Figure S1. The results\nshowed that Ca. Kuafubacteria shared ~45–49 AAIs and ~61–72 ANIs with other classes.\nSuch a result is similar compared with other classes. Indeed, AAI and ANI values are so varied.\niii) Thus, we also placed the phylogenomic tree near the heatmap to indicate the taxonomic\nlevel according to your suggestion. Comprehensively, by combing the results of phylogenetic\ntrees, GTDB-Tk classification, AAI and AAI analyses, we determined that these MAGs should\nbe clustered at the class level. iv) We also checked the RED values using the software\nphylorank (https://github.com/dparks1134/PhyloRank). The RED analyses do not provide\nadditional supports because the six genomes of Ca. Kuafubacteria belong to a same family\nand currently other genomes from different families (and orders) of Ca. Kuafubacteria are\nunavailable. v) To facilitate comparisons for placeholder names and the names we proposed,\nwe added the taxonomy assignment of GTDB-Tk in the revised Table S1.\nResponse letter Table 3 NCBI and GTDB r207 taxonomy of the novel MAGs from Ca.\nKuafubacteria (c__WYAZ01). The screenshot was generated from the GTDB website\n(https://gtdb.ecogenomic.org/).\nLine 89: The correct etymology should be Kuafuibacteria, instead of Kuafubacteria. Also\nKuafuibacterium and Houyiibacterium, instead of Houyibacterium in Table S1. You should also\nprovide etymology for the genus and species names as well. The genus names that you\ncurrently have in Table S1 doesn’t make any sense except for the one ending with -bacterium;\naestuarium is translated as tidal flat or estuarium, lacus is a lake, caenum is mud. It would be\ngreat if you can substitute those endings, for instance with -microbium, -bacter, -lacustris\n(belonging to a lake). This would give names such as Kuafuibacter, Kuafuimicrobium,\nKuafuilacustris. You will need to designate (indicate) a type species of each new genus and\ntype genome for each new species. You also need to propose Youwenlacus (correct to\nYouwenilacustris or Youwenibacter) and provide its etymology. It would be good to submit the\nnames to the SeqCode.\nResponse: Thanks very much for this valuable comment. i) We have checked the etymology\nand changed all of the misassigned endings, e.g., Kuafucaenum to Kuafucaenimonas,\nDeshenbium to Xihebacterium. All changed names can be found in the Supplementary Table\nS1. ii) In addition, we found that in the case of “mythical people”, it seems likely that it is not\nrecommended to add ‘-i-’ to the end of the prefixes (Response letter Fig. 6). iii) We also added\nthe description of type species of each new genus and type genome for each new species and\nrelated etymology on Line 70-360 in the Supplementary Notes, according to your suggestion.\niv) It is very important to submit the names to the SeqCode, and we have uploaded the\ninformation to SeqCode. Nevertheless, the name with published work and genome accessions\nlinked to NCBI are required to complete the process of submitting names to SeqCode, so we\nwill finish the complete process once the genomes were published.\nRedacted\nEditorialNote:PartsofthisPeerReviewFilehavebeenredactedasindicatedtoremovethird-party\nmaterialwherenopermissiontopublishcouldbeobtained.\nLine 97: What is the percentage of predation in the Myxococcota?\nResponse: Predatory capacity seems likely to be a common feature in cultured strains of\nMyxococcota (Table S1), but we cannot conclude that myxobacteria are generally predatory.\nIn our previous version of the manuscript, we applied a machine learning method to predict\nthe predatory behavior, and the results showed that most of the potential phototrophic\nMyxococcota may be predators simultaneously (32/33). The training set of the machine\nlearning method was based on the genomes of predatory (20) or non-predatory species (5)\nthat have been experimentally verified according to published papers (Table S4). The number\nof predatory species may have been underestimated because many earlier studies did not\nsubmit sequences of predatory genomes to NCBI. Thus, the percentage of predatory behavior\nin cultured species of Myxococcota might exceed 80% (20/25). Whereas several other studies\ninferred that some uncultured species of Myxococcota are non-predatory or non-social,\nfor example, potential non-predatory species of the class c__UBA42485 and potential non-social\nspecies of the order o__JAFGXQ014. Nevertheless, as suggested by Reviewer #1, the\npredatory genes are still not clear, and detailed predatory mechanism is also unknown,\ntherefore, we deleted relevant analyses and toned down the description of this intriguing\nmetabolic type.\nLine 201: “…through an autonomously regulated phototrophic lifestyle.”\nResponse: Thanks for this suggestion. We changed the “autonomously” to “flexibly” on Line\n235-236: “..allow them to cope with survival challenges through flexibly regulated phototrophic\nlifestyle”.\nLine 206 “lifestyle”.\nResponse: Checked.\nLine 220: How closely related are the genes?\nResponse: The protein sequences of the genes showed ~60–80% identities to the sequences\nof Proteobacteria and Gemmatimonadota (Response letter Table 4).\nResponse letter Table 4 The blast results of myxococcotal BchE-like proteins from the NCBI\nwebsite (https://blast.ncbi.nlm.nih.gov/Blast.cgi).\nLine 371: “..not only be a consumer shaping…may be a primary producer.”\nResponse: Thanks for this suggestion. The sentence was changed on Line 383-384:\n“Myxococcota are not only consumers shaping microbial communities by means of top-down\ncontrol, but also may be primary producers.”\nLine 376: “..single prokaryotic cells can serve as a player at multiple..”\nResponse: This sentence was removed.\nLine 376: “rather than being confined to either decomposer, consumer or producer?”\nResponse: The sentence was deleted.\nComments for methods\nLine 408: Did you trim the adaptors from the sequencing reads?\nResponse: Thanks for this comment. The adaptors were trimmed with the software Scythe\n(https://github.com/vsbuffalo/scythe).\nLine 410: Remove the words “according to”, you can just say “The sequence coverage of\ncontigs was calculated by mapping the trimmed reads to each contig using BBMap version\n38.18.\nResponse: Thanks for this suggestion. We changed the sentence on Line 436-437: “The\nsequence coverage of contigs was calculated by mapping the trimmed reads to each contig\nusing BBMap version 38.18.”\nLine 418: It says that the quantification of transcript expression was using salmon. But what\ndid you map the metatranscriptomic sequencing reads too? Were they the myxococcotal\ngenomes or photosynthetic genes from the whole metagenome?\nResponse: Here we used the salmon software for quantification of transcript expression\n(https://doi.org/10.1038/nmeth.4197). We merged six representative myxococcotal genomes\ninto a single file, and mapped the metatranscriptomic sequencing reads to this file. The results\nare shown in the Fig. 5. In addition, to indicate the PGC expression pattern in a genome, we\nalso visualized the expression result of the genome LLY-WYZ-15_3 of Ca. Houyibacteriaceae\nso that we can identify the potential expression of an entire PGC from a single genome. This\nresult is shown in the Figure 5b. Relevant description was added in the Methods on Line 520-\n527: “For the quantification of transcript expression, we used salmon version 1.9.0 to map the\ntrimmed metatranscriptomic reads to the six representative Myxococcota genomes. The\nabundance of each gene in metatranscriptomes was calculated as the metric-FPKB\n(fragments per kilobase per billion metatranscriptomic fragments) normalized based on the\ngene length and sequencing depth. For the profile of global expression of Myxococcota\nphotosynthesis genes, we summed the FPKB of genes that belongs to the same function to\ncalculate the functional abundance of each metatranscriptomic library.”\nLine 452: Did you dRep your MAGs as well? Some of them are at 99-100% AAI (Fig S2).\nResponse: Thanks for this suggestion. We dRep analysis for the 32 MAGs. The dRep\nresult was used for the analyses of PGC (Fig. 2c, Fig. S4, S8), metabolisms (Fig. 3), gene\nexpressions (Fig. 4 and Fig.5), AAI and ANI (revised Fig. S1). In order to provide more detailed\ninformation, the sequences of all MAGs were used in the phylogenetic trees (Fig. 1, Fig. S2,\nS3, S5, S6, S9, S10) and genome comparisons (Fig. 2ab and Fig. S7).\nLine 489: Do you mean queried?\nResponse: Corrected. See Line 488.\nLine 516: I’m not sure what is meant by rRNA genes. Did you find the rRNA genes and add\nthem to the MSA?\nResponse: Thanks for this suggestion. We have deleted the unmatched description of “rRNA\ngenes”, as we originally intended to say the ribosome RNA protein sequences. The revised\nsentence is on Line 513-515: “…the 32 MAGs and all high-quality Myxococcota genomes\navailable from the GTDB r207 were used to construct phylogenetic trees based on the\nconcatenated alignments of 120 conserved single-copy marker genes using GTDB-Tk.”\nComments for figures and tables\nFig 1\nFig 1 is really difficult to see clearly. It would be better if you could zoom in on the Myxococcota.\nResponse: Thanks for this comment. We re-visualized the figure by simplifying dozens of\ncolors for different phyla. We hope this new version is clear now. In the inner ring, we used a\nshade of brown to indicate the lineages belonging to Myxococcota. We also used a green\nbackground to indicate phototrophic lineages, and used grey background to indicate non-\nphototrophic lineages. We also added the description of caption on Line 925-929: “In the inner\nring, lineages of Myxococcota are shaded with brown background. In the middle ring,\nphototrophic lineages are colored with green background, and non-phototrophic lineages are\ncolored with grey background. The outer ring of phylogeny is colored based on different types\nof reaction centers.”\nLine 802: 1,713.\nResponse: Revised.\nFig 2\nLine 824: They may also not have formed in clusters due to the fact that some of the MAGs\nhave over 2,000 scaffolds.\nResponse: Thanks for this comment. We added the description on Line 953-956:\n“Photosynthesis genes did not form apparent PGC (photosynthesis gene number <12 in any\nclusters) for the remaining 11 MAGs (average completeness ~84.6%), potentially attributed to\nincomplete assembly and/or binning of fragment reads in the next generation sequencing.”\nFig 3.\nWhich genomes are the representatives for each of the Myxococcota families? Are you using\nthem all? What if the metabolic pathways are only found in half of the genomes? It would be\ngood to have the number of genomes that you’re using for the analysis in brackets next to\neach of the family names.\nResponse: Thanks for this comment.\ni) Representatives of six Myxococcota families used for metabolic visualization are LLY-WYZ-\n15_3, LLY-WYZ-17_1, LLY-WYZ-3_1, LLY-WYZ-12_1, LLY-WYZ-9_1, and LLY-WYZ-13_1.\nThe accessions, taxonomy, and related information of representative genomes were also listed\nin the revised Supplementary Table S1. The selection of representative myxococcotal\ngenomes for metabolic visualization was mainly based on their genome quality and\ncompleteness of metabolic pathways (see Line 572-585).\nii) We note that all of these genomes have complete bacteriochlorophyll synthesis pathways,\nbut the completeness of other metabolic pathways varies with genomes. So, we used the half-\nfilled parts in snowflake to indicate the absence of genes in the representative genome but\npresent in other genomes of the same family. The genomes that have additional genes to\nsupplement the representatives are labels as “Additional” genome. For example,\nrepresentative LLY-WYZ-9_1 of the Polyangiaceae contains complete assimilatory sulfate\nreduction (ASR) pathway but lack the genes of dissimilatory nitrate reduction (DNR), while the\nLLY-WYZ-7_1 from the same family contains complete DNR pathway. In this case the DNR\npathway would be filled with half parts in snowflake to show the summary feature of this family,\nand LLY-WYZ-7_1 would be labeled as “Additional” genome.\n“Representative” genomes and “Additional” genomes were listed in the column named “Fig3-\nMetabolism” in the Supplementary Table S2. The list of additional genes could be found in the\ncolumn named “Fig3-Reasons” in the Supplementary Table S2.\niii) We have added the number of genomes in brackets next to each of the family names in the\nfigure. We also added the description on Line 973-974: “Half-filled parts in snowflake indicate\nthe absence of proteins in the representative genome but present in other genomes from the\nsame family.”\nFig 4\nWere the expression data mapped back to the genomes? In 4A, you may need to point out\nthat the green are photosynthesis genes and the grey are non-photosynthesis genes. Are there\nmultiple Myxococcota genomes from the same study? Are they expressed evenly? In 4B, what\nis the representative genome of the family Ca. Houyibacteriaceeae?\nResponse: Thanks for your suggestion. i) We mapped the trimmed metatranscriptomic reads\nto the coding sequences (CDS) of the six representative Myxococcota genomes (LLY-WYZ-\n15_3, LLY-WYZ-17_1, LLY-WYZ-5_1, LLY-WYZ-10_1, LLY-WYZ-9_1, and LLY-WYZ-13_1.). ii)\nIn the Fig. 5a, we used samples with highly expressed photosynthetic genes (considering both\nof the prevalence and abundance) to visualize the expression pattern (Response letter Fig. 7).\nWe refined the description of Fig. 5a in the caption on Line 1001-1004: “The expression level\n(FPKB, fragments per kilobase per billion metatranscriptomic fragments) of photosynthesis\ngenes (green) compared with non-photosynthesis genes (grey) of six representative\nMyxococcota genomes in typical samples with photosynthesis gene prevalence >50% and\nmean FPKB >3.” iii) Myxococcotal genomes were recovered from different samples. In\nmetatranscriptomic samples, the expression of the photosynthetic gene may be detected\nsimultaneously in multiple myxococcotal genomes with varied expression levels (Response\nletter Fig. 8), e.g., the expression of pufL gene from three genomes of the families Ca.\nHouyibacteriaceae, Nannocystaceae, and Polyangiaceae in the sample “080_DCM_0.8to5”\n(Response letter Table 5). The genome LLY-WYZ-15_3 of Ca. Houyibacteriaceae has the\nhighest prevalence and abundance of PGC genes compared with other genomes. This is\nreasonable because genomes of other families were all recovered from non-ocean\nenvironments, e.g., terrestrial or freshwater habitats. iv) Therefore, we select the genome LLY-\nWYZ-15_3 as representative of Ca. Houyibacteriaceae to visualize the expression data in the\nFig. 5b. v) The metatranscriptomic datasets were updated by replacing several published\nsamples without clear reference to samples with clear reference according to scientific\nliterature20-24.\nResponse letter Fig. 7 Typical samples (top 30 samples) for visualization were selected\nbased on the photosynthesis gene prevalence and mean abundance (prevalence >50% and\nmean FPKB >3).\nResponse letter Fig. 8 The expression of PGCs of representative genomes from the six\nfamilies in the samples with highly expressed photosynthetic genes (top 30 samples with\nphotosynthesis gene prevalence >50% and mean FPKB >3).\nResponse letter Table 5 The expression level of photosynthesis gene (pufL) from different\nfamilies of Myxococcota across samples.\nLine 860: Top highly expressed samples? Are the same levels of expression found in the other\ngenomes?\nResponse: Here we used samples with highly expressed photosynthetic genes (considering\nboth of the prevalence and abundance) to visualize the expression pattern (Response letter\nFig. 7). We refined the description of Fig. 5B in the caption on Line 1003-1007: “…in typical\nsamples with photosynthesis gene prevalence >50% and mean FPKB >3. b, The expression\nof PGC of representative genome (LLY-WYZ-15_3) from the family Ca. Houyibacteriaceae in\ntypical samples.” The expressions in other genomes are varied across samples (Response\nletter Table 6). For example, for the sample \"100_DCM_0.22to3\", the FPKB for bchL of three\ngenomes are about 267.58, 54.50, and 19.56, respectively.\nResponse letter Table 6 The expression level of photosynthesis gene (bchL) from different\nfamilies of Myxococcota across samples.\nFig 5\nLifestyle\nResponse: Revised.\nFig S1\nIn the legend, the completeness is 32.4% to 100% and the contamination is 0% to 8.4%,\nhowever in Supplementary Table 1, the contamination is up to 9.33%. It also looks like there\nare genomes in the tree that have even more contamination. It’s difficult to see the classes of\ninterest in this figure. I strongly suggest creating trees of the classes of interest and only\nincluding the type species for the other genera plus two or three extra genomes. The genome\nwith the long branch is a little worrying. I would remove that genome. Is it because it’s very\nincomplete? Instead of using unphototroph, I would use non-phototroph. How did you include\nthe 16S rRNA genes? It’s very difficult to determine how large the circles are. I would remove\nthe different sizes from the legend.\nResponse: Thanks for the suggestion. i) We re-visualized Fig. S1 by combining the\nphylogenetic tree and AAI and ANI analyses. The representative genomes used in this figure\nwere dereplicated and re-selected based on genome quality. In addition, reference genomes\nand their completeness and contamination which were evaluated by CheckM and CheckM2\nwere listed in the Supplementary Table S4. ii) We also created trees of the classes of interest\nin the Fig. S2 (Myxococcia) and Fig. S3 (Polyangia) following your suggestion to only include\nthe type species for the other genera plus two or three extra genomes. The genome numbers\nof Kuafubacteria are so limited that we did not construct a tree for this class, but the phylogeny\ncan be determined from the Fig. S1. Reference genomes were listed in the Supplementary\nTable S5. iii) The genome with the long branch was removed from the analysis because of the\nlow completeness. iv) We removed the typo “unphototroph”. v) We have deleted the wrong\ndescription of “rRNA genes”, which we originally meant ribosomal RNA protein sequences. vi)\nThe circles with different sizes from the legend were removed.\nFig S2\nThe AAI between many of the Houyibacteriaceae is 99-100%. I would consider these MAGs\nto be the same strain and would de-replicate them at least at the 95% ANI level. The same\ngoes for some of the MAGs in the Nannocystaceae. It would be good to also include a\nphylogenetic tree next to the names so that we can also visually see how close they are. I now\nsee in Table S1 that the MAGs have been dereplicated but you don’t mention this in the main\ntext. Are all the analyses performed on all the MAGs or the dereplicated ones?\nResponse: Thanks for the valuable comment. i) We dereplicated genomes, and use the dRep\ngenomes for AAI plot (Supplementary Figure S1). ii) We also added a phylogenetic tree next\nto the names of each MAG. iii) We added relevant description on Line 90-92: “We clustered\nthese 32 MAGs on the basis of 95% whole-genome average nucleotide identities (ANIs) to\nuncover species-level diversity, revealing 18 candidate species-level taxa”. iv) The dRep result\nwas used for the analyses of PGC (Fig. 2c, Fig. S4, S8), metabolisms (Fig. 3), gene\nexpressions (Fig. 4 and Fig. 5), AAI and ANI (revised Fig. S1). In order to provide more detailed\ninformation, the sequences of all MAGs were also used in the phylogenetic trees (Fig. 1, Fig.\nS2, S3, S5, S6, S9, S10) and genome comparisons (Fig. 2ab and Fig. S7).\nFig S3\nIt’s difficult to know what the green squares were unless you zoom into the document at 300%.\nWhen I first looked at it I couldn’t see green in the legend. I’m not sure if there’s another way\nyou can show the numbers that will be easier to see. Is dark green 1 copy and light green 2\ncopies?\nResponse: Thanks for this suggestion. In the revised manuscript, we have deleted this part.\nFig S4\nYou may want to check that your supplementary files are in order. Fig S4 is first mentioned in\nline 198 but Fig S5 and Fig S6 are mentioned on line 121.\nResponse: Checked.\nFig S5\nI would remove the legend for the bootstraps as it’s difficult to see how large the 0.9, 0.93,\n0.95, 0.98 and 1 circles are.\nResponse: Thanks for the suggestion. we have simplified the legend for the bootstraps.\nFig S6\n1,201. Where are the Betaproteobacteria and why are they in brackets?\nResponse: Checked. Betaproteobacteria has been classified as a clade of\nGammaproteobacteria as shown in the GTDB r207. We have clarified the description in the\ncaption in the revised Fig. S5: “Gammaproteobacteria (including Betaproteobacteria)”. We also\nclarified the description on Line 931-932: “Betaproteobacteria shared same color with\nGammaproteobacteria based on the taxonomy in GTDB r207”.\nFig S7\nIt took me a little while to understand what the different colours of the branches are. It maybe\nbest to remove the brackets.\nResponse: Removed.\nFig S8\npaper80\nResponse: Corrected.\nTable S2\nMaybe to be consistent, just have the family name and the species name and remove the f__\nfor family. You should remove LLY-WYZ-19_1. Based on its completeness, this is probably why\nit’s missing a lot of genes in the metabolism analysis.\nResponse: Thanks for the suggestion, we removed the “f__” for clarity. We also removed the\ngenome (LLY-WYZ-19_1) for its low completeness.\nReferences:\nIdentification of Myxobacterial Predation Genes and Demonstration of Formaldehyde Secretion as\na Potentially Predation-Resistant Trait of Pseudomonas aeruginosa. Frontiers in microbiology 10,\n2650, doi:10.3389/fmicb.2019.02650 (2019).\nThe ISME journal 7, 756-769, doi:10.1038/ismej.2012.149 (2013).\nstrategies in saline environments. Microbiome 8, 126, doi:10.1186/s40168-020-00902-0 (2020).\nPredation and Cellular Differentiation. Appl Environ Microbiol 87, e0170621,\ndoi:10.1128/AEM.01706-21 (2021).\nOligoflexia, and the phylum Thermodesulfobacteria into four phyla reflecting major functional\ncapabilities. Int J Syst Evol Microbiol 70, 5972-6016, doi:10.1099/ijsem.0.004213 (2020).\nCharacterization of Genome and Metagenome Sequences. J Mol Biol 428, 726-731,\ndoi:10.1016/j.jmb.2015.11.006 (2016).\nMyxococcus xanthus. Nat Commun 11, 5054, doi:10.1038/s41467-020-18803-z (2020).\nmoving in helical trajectories. Proc Natl Acad Sci U S A 110, E1508-1513,\ndoi:10.1073/pnas.1219982110 (2013).\nalgorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32, 268-274,\ndoi:10.1093/molbev/msu300 (2015).\ntree topologies from DNA sequence data, and the branching order in hominoidea. J Mol Evol 29,\n170-179, doi:10.1007/BF02100115 (1989).\nselection. Bioinformatics 17, 1246-1247, doi:10.1093/bioinformatics/17.12.1246 (2001).\ndoi:10.1038/s41586-022-04862-3 (2022).\nwith predatory performance by a social bacterium. Microb Ecol 65, 415-423, doi:10.1007/s00248-\n012-0135-6 (2013).\nability of the soil bacterium Myxococcus xanthus. Microbiology (Reading) 151, 1865-1874,\ndoi:10.1099/mic.0.27824-0 (2005).\nClinically-Relevant Pathogens, Exhibiting a Prey Range Which Cannot Be Explained by Phylogeny.\nFrontiers in microbiology 8, 1593, doi:10.3389/fmicb.2017.01593 (2017).\ndoi:10.1128/jb.104.1.453-461.1970 (1970).\nxanthus. J Bacteriol 188, 5888-5895, doi:10.1128/JB.00559-06 (2006).\nFungi, and Biocontrol Agents. Plant Dis 86, 889-896, doi:10.1094/PDIS.2002.86.8.889 (2002).\nMicrobiol 29, 289-298, doi:10.1016/j.tim.2020.10.009 (2021).\nvirome. Science 376, 156-162, doi:10.1126/science.abm5847 (2022).\n376, 1202-1208, doi:10.1126/science.abn6358 (2022).\nOcean Metatranscriptome. Cell 179, 1068-1083 e1021, doi:10.1016/j.cell.2019.10.014 (2019).\ndoi:10.1038/s41467-017-02342-1 (2018).\nsamples from the Tara Oceans Expedition (2009-2013). PANGAEA,\nhttps://doi.org/10.1594/PANGAEA.875582 (2017)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Here, I detail my second assessment of the submission titled “Globally distributed Myxococcota\nwith photosynthesis gene clusters illuminate the origin and evolution of a potentially chimeric\nlifestyle” by Li and colleagues.\nAs stated in my original review, the identification and characterisation of complete photosynthetic\ngene clusters in MAGs taxonomically classifiable as different groups of Myxococcota is\nextremely exciting, and highly original. This is because the myxobacteria are models for bacterial\ndevelopment and social evolution, and many aspects of their life cycles are known in molecular\ndetails. Yet, to date, none of its members have been implicated in photosynthesis. As such the\nwork will be highly interesting to a wider audience especially in the fields of microbiology, botany,\nand biochemistry.\nInitially, prior to revision, the draft did not effectively bolster its strong claims about a link\nbetween heterotrophic and prototrophic life-styles housed in the same myxobacterial cells. As such\nthe draft was shaky and highly speculative.\nYet, this has changed dramatically with the latest revision. And I am now very positive about\nthe presented revision.\nAs a brief recap, in my first assessment I expressed both clear praise but also strong reservation\nregarding the interpretation and framing of their results.\nIn brief, I warned that the following problems need considerable attention and revision:\n- the problematic interpretation of active photosynthesis from expression data alone\n- the shaky conceptualisation of and problematic lines of evidence for linking predation with\nphotosynthesis\n- the general ignorance toward well-established myxobacterial biology\nHowever, the authors now more than adequately addressed the problems and short-comings\nabove in their extensive and thorough revisions, by:\n- successfully demonstrating that key genes in both photosynthesis and sociality are functional in a\nheterologous expression model system using elegant, experimental tests,\n-providing more careful and adequate interpretation of their results in light of known\nmyxobacterial biology, especially by toning down on the speculative link between predation and\nphotosynthesis (they also scratched the problematic predation gene analysis),\n- addressing the important caveats provided by the reviewers, including the correct use of\ntaxonomic nomenclature with adequate tools, and otherwise problematic conclusions.\nIn conclusion, I can recommend the draft for publication with minor revisions, see below.\nMinor comments:\nl. 7:\nGrammar: “[…] Myxococcota, which were known for […]” - please replace with presence\nform (which ARE known …)\nll. 63-65:\nShaky phrasing: “Heterologous expression of their pigment biosynthesis genes indicate that they\ncan properly integrated into photosynthesis process.” – please rephrase adequately, e. g.,\n“Resulting heterologous expression of their pigment biosynthesis genes strongly suggests that\nthese genes can drive photosynthetic processes in their original myxobacterial background.” OR\nsimilar.\nL. 140:\nTechnical term: “vertical evolution” – that’s not a proper concept, did you mean *vertical\ninheritance* (see ll. 169 – 170, where you use that expression correctly)?\nFigure 1:\n- Thanks for taking into account my initial suggestions to improve the visibility of certain features\nin the circle tree (i.e., added color information on non-photrophic taxa, Fig. 1a, better highlight of\nall Myxococcota).\n- one remaining issue: Gamma- and Alphaproteobacteria cluster in a common group in Figure 1b.\nBut the labeling leads to mild confusion and should ideally be changed: currently a connector links\nthe reddish label “Gammaproteobacteria” with the differently colored circle sector (purple) which\nwidely defines “Alphaproteobacteria”. The latter label is also connected to the same sector on the\nbottom of the figures (Fig. 1b). This is not effective as is might lead to confusion, please\nadequately change the connector and or sector color.", "reviewer_2": "I have read the rebuttal and I feel that the authors have addressed my concerns in the revision."}, "author_response": "Dear Reviewers,\nThank you again for your careful reading and professional suggestions. All comments here\nhave been taken into full consideration during the preparation of the revised manuscript. Below\nare our point-by-point responses to the comments. Page and line numbers are consistent with\nthe revised manuscript. Responses of reviewers’ questions are in blue color, quotations of new\ntexts in the manuscript are in blown color.\nReviewer #1 (Remarks to the Author):\nHere, I detail my second assessment of the submission titled “Globally distributed\nMyxococcota with photosynthesis gene clusters illuminate the origin and evolution of a\npotentially chimeric lifestyle” by Li and colleagues.\nAs stated in my original review, the identification and characterisation of complete\nphotosynthetic gene clusters in MAGs taxonomically classifiable as different groups of\nMyxococcota is extremely exciting, and highly original. This is because the myxobacteria are\nmodels for bacterial development and social evolution, and many aspects of their life cycles\nare known in molecular details. Yet, to date, none of its members have been implicated in\nphotosynthesis. As such the work will be highly interesting to a wider audience especially in\nthe fields of microbiology, botany, and biochemistry.\nInitially, prior to revision, the draft did not effectively bolster its strong claims about a link\nbetween heterotrophic and prototrophic life-styles housed in the same myxobacterial cells. As\nsuch the draft was shaky and highly speculative.\nYet, this has changed dramatically with the latest revision. And I am now very positive about\nthe presented revision.\nAs a brief recap, in my first assessment I expressed both clear praise but also strong\nreservation regarding the interpretation and framing of their results.\nIn brief, I warned that the following problems need considerable attention and revision:\n- the problematic interpretation of active photosynthesis from expression data alone\n- the shaky conceptualisation of and problematic lines of evidence for linking predation with\nphotosynthesis\n- the general ignorance toward well-established myxobacterial biology\nHowever, the authors now more than adequately addressed the problems and short-comings\nabove in their extensive and thorough revisions, by:\n- successfully demonstrating that key genes in both photosynthesis and sociality are functional\nin a heterologous expression model system using elegant, experimental tests,\n-providing more careful and adequate interpretation of their results in light of known\nmyxobacterial biology, especially by toning down on the speculative link between predation\nand photosynthesis (they also scratched the problematic predation gene analysis),\n- addressing the important caveats provided by the reviewers, including the correct use of\ntaxonomic nomenclature with adequate tools, and otherwise problematic conclusions.\nIn conclusion, I can recommend the draft for publication with minor revisions, see below.\nResponse: We would like to thank the reviewer again for the positive remarks and the\nrecommendation for the manuscript to be published.\nMinor comments:\nl. 7:\nGrammar: “[…] Myxococcota, which were known for […]” - please replace with presence\nform (which ARE known …)\nResponse: Revised.\nll. 63-65:\nShaky phrasing: “Heterologous expression of their pigment biosynthesis genes indicate that\nthey can properly integrated into photosynthesis process.” – please rephrase adequately, e.\ng., “Resulting heterologous expression of their pigment biosynthesis genes strongly suggests\nthat these genes can drive photosynthetic processes in their original myxobacterial\nbackground.” OR similar.\nResponse: Thanks for the suggestion. We modified the description on Line 62-64: “Resulting\nheterologous expression of their pigment biosynthesis genes strongly suggests that these\ngenes can drive photosynthetic processes in their original myxobacterial background.”\nL. 140:\nTechnical term: “vertical evolution” – that’s not a proper concept, did you mean *vertical\ninheritance* (see ll. 169 – 170, where you use that expression correctly)?\nResponse: Thanks for the suggestion. We revised the “vertical evolution” and “vertical\nevolutionary history” to “vertical inheritance” on Line 8, Line 65, and Line 140.\nFigure 1:\n- Thanks for taking into account my initial suggestions to improve the visibility of certain\nfeatures in the circle tree (i.e., added color information on non-photrophic taxa, Fig. 1a, better\nhighlight of all Myxococcota).\n- one remaining issue: Gamma- and Alphaproteobacteria cluster in a common group in Figure\n1b. But the labeling leads to mild confusion and should ideally be changed: currently a\nconnector links the reddish label “Gammaproteobacteria” with the differently colored circle\nsector (purple) which widely defines “Alphaproteobacteria”. The latter label is also connected\nto the same sector on the bottom of the figures (Fig. 1b). This is not effective as is might lead\nto confusion, please adequately change the connector and or sector color.\nResponse: We thank the reviewer for the valuable comments. We modified the Figure 1 as the\nreviewer suggested by changing the connector to dashed lines to distinguish the\nAlphaproteobacteria and Gammaproteobacteria.\nReviewer #2 (Remarks to the Author):\nI have read the rebuttal and I feel that the authors have addressed my concerns in the revision.\nResponse: We would like to thank the reviewer again for the constructive comments on the\nsubmitted version and the positive remarks on the revisions we made."}]

Decision letter