**Description of the Class** *Chlorobia* **class nov. (Garrity and Holt 2001, 601EP)**

*Chlorobia* (Chlo.ro'bi.a. N.L. neut. n. *Chlorobium*, genus of the class, changing ending to denote a class; N.L. neut. pl. n. *Chlorobia*, class of the genus *Chlorobium*).

The class *Chlorobia* contains a single order (*Chlorobiales*) [8] and the description of this class is the same as that given below for the order *Chlorobiales.*

The type order is *Chlorobiales* Gibbons and Murray 1978 (Approved Lists 1980).

### **Emended Description of the order** *Chlorobiales* **Gibbons and Murray 1978 (Approved Lists 1980)**

The order is composed of two families: *Chlorobiaceae* and *Chloroherpetonaceae.* Members of this order form a monophyletic clade in phylogenetic trees based on 16S rRNA gene sequences and trees based on several individual proteins (viz., Fmo, GyrA, GyrB, PolA, RecA, and UvrD) and large datasets of concatenated protein sequences. The order comprises species that are anaerobic and strict photolithoautotrophs, which use reduced sulfur compounds as an electron source to fix CO2, as well as aerobic photoautotrophic organisms, which likely use photo-assimilated acetate and propionate as carbon sources. Species from this phylum generally contain genes encoding for photosynthetic reaction centers and bacteriochlorophyll biosynthesis. The members of this order can be reliably distinguished from all other bacteria based on the shared presence of 33 conserved signature indels (CSIs) in diverse proteins (Table 1) that are uniquely shared by the members

of this order. The CSIs specific for the order *Chlorobiales* are found in the following proteins: a major facilitator superfamily protein, a radical SAM-domain-containing protein, a Gfo/Idh/MocA family oxidoreductase, an acetyl-CoA carboxylase carboxyltransferase alpha subunit, a DNA mismatch repair protein, bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase, cytidylate kinase, deoxyribonucleotide triphosphate pyrophosphatase, a DNA polymerase III alpha subunit, DNA polymerase III subunits gamma and tau, fructose-1,6-bisphosphate aldolase (class II), glutamate-1-semialdehyde aminotransferase, glutamyltRNA reductase, glutamate-1-semialdehyde aminotransferase, glycyl-tRNA synthetase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, lactoylglutathione lyase, molecular chaperone DnaK, phosphoribosylformylglycinamidine synthase II, polynucleotide phosphorylase/polyadenylase, ribonuclease R, an RNA polymerase sigma-32 subunit, serine hydroxymethyltransferase, succinyl-CoA synthetase subunit alpha, thiazole synthase, tryptophanyl-tRNA synthetase, uroporphyrinogen decarboxylase, N-acetyl-alpha-Dglucosaminyl L-malate synthase BshA, and 7-carboxy-7-deazaguanine synthase QueE.

The type genus of this order is *Chlorobium* Nadson 1906 [81] (Approved Lists 1980).

#### **Emended Description of the family** *Chlorobiaceae* **Copeland 1956 (Approved Lists 1980)**

The family *Chlorobiaceae* contains the type genus *Chlorobium* [8,81] and the following validly published genera: *Ancalochloris* [8,14], *Chlorobaculum* [12], *Pelodictyon* Lauterborn [8], and *Prosthecochloris* [8,14]. The description of this family is partially based on that given by Imhoff [4]. Members of this family are non-motile photolithoautotrophs and they grow under anoxic conditions in the presence of limited light using reduced sulfur compounds as an electron source to fix CO2. Thiosulfate, hydrogen, and ferrous iron can also be used as reductants. Most of the members of this family have been isolated from anoxic aquatic settings in stratified lakes, where sunlight can penetrate. All members of this family except *Chlorobium ferroxidans* possess a dissimilatory sulfite reductase (DSR) system. Members of this family also form a monophyletic clade in phylogenetic trees based on 16S rRNA gene sequences and trees constructed based on large datasets of concatenated protein sequences. The members of this order can be clearly distinguished from members of the family *Chloroherpetonaceae* and all other bacteria based on the shared presence of eight conserved signature indels (CSIs) found in diverse proteins (listed in Table 2) that are uniquely shared by the members of this family. The proteins containing the CSIs specific for this family are as follows: biogenesis of lysosome-related organelles complex 1 subunit 2, DegT/DnrJ/EryC1/StrS aminotransferase, DNA gyrase subunit A, molecular chaperone HtpG, MiaB-like tRNA modifying protein, peptidase U32, peptide chain release factor 3, and polyphosphate kinase.

The type genus of this family is *Chlorobium* Nadson 1906 [81] (Approved Lists 1980).

#### **Description of the family** *Chloroherpetonaceae* **fam. nov.**

*Chloroherpetonaceae* (Chlo.ro.her'pe.to.na.ce'ae. N.L. neut. n. *Chloroherpeton*, type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. *Chloroherpetonaceae* the *Chloroherpeton* family).

This family contains the type genus *Chloroherpeton* and another *Candidatus* genus Thermochlorobacter. In addition, two uncultured organisms with assembled genomes known by the names *Chlorobium* sp. 445 and *Candidatus* Thermochlorobacteriaceae GBChlB are also members of this family. Like the *Chlorobiaceae* family, members of this family contain type-I reaction centers and chlorosomes. The sole cultured species from this family is *Chloroherpeton thalassium*, which like members of the *Chlorobiaceae* family is a photolithoautotroph that grows under anoxic conditions in the presence of light using reduced sulfur compounds as an electron source to fix CO2. However, unlike *Chlorobiaceae* species, which are nonmotile, cells of *Chloroherpeton thalassium* exhibit gliding mobility. In contrast to *Chloroherpeton*, the *Candidatus* species Thermochlorobacter aerophilum is an aerobic photoheterotroph that cannot oxidize sulfur compounds, cannot fix N2, and does not fix CO<sup>2</sup> autotrophically [27]. It is considered to have gliding and flexing motility and two copies of the type-1 NADH dehydrogenase complex similarly to *Chloroherpeton*

*thalassium*. Members of this family are characterized by their lack of a dissimilatory sulfite reductase (DSR) system, which is a protein present in all members of *Chlorobiaceae* except for *Chlorobium ferroxidans*. Members of this family form a monophyletic clade in a phylogenetic tree based on concatenated sequences of core proteins from the genomes of *Chlorobiales* species. The members of this family are also clearly differentiated from the *Chlorobiaceae* species in a pairwise matrix of the percentage of conserved proteins in the genomes. Furthermore, members of this family can be reliably distinguished from members of the family *Chlorobiaceae* and all other bacteria based on the shared presence of six conserved signature indels (CSIs) found in diverse proteins (Table 2) that are uniquely shared by members of this family. The proteins containing the CSIs specific for this family are: alkaline phosphatase family protein, dihydrolipoyl dehydrogenase, hypoxanthine phosphoribosyltransferase, SDR family oxidoreductase, RecQ family ATP-dependent DNA helicase, and UDP-glucose/GDP-mannose dehydrogenase family protein.

The type genus for this family is *Chloroherpeton* Gibson et al. 1985 [13,82].

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms10071312/s1, Figure S1. A bootstrapped maximum likelihood tree for different genome-sequenced *Chlorobia*/*Ignavibacteria* and related species/strains based on concatenated sequences of UvrD and PolA proteins. Figure S2. A bootstrapped maximumlikelihood tree for different genome-sequenced *Chlorobia*/*Ignavibacteria* and related species/strains based on concatenated sequences of GyrA and GyrB proteins. Figure S3. AAI matrix indicating the pairwise percentage average amino acid identities based on core proteins for the type species of different genera within the order *Chlorobiales*. Figure S4. Partial sequence alignment of the protein glycine-tRNA synthetase, showing a 1 aa insertion (boxed) that is specific for the order *Chlorobiales*. Figure S5. Partial sequence alignment of the protein RNA polymerase sigma factor RpoD/SigA, showing a 12 aa insertion (boxed) that is exclusive to all members belonging to the order *Chlorobiales*. Figure S6 Partial sequence alignment of the protein uroporphyrinogen decarboxylase, showing a 3 aa insertion (boxed) that is exclusive to all members belonging to the order *Chlorobiales*. Figure S7. Partial sequence alignment of the protein glutamyl-tRNA reductase, showing a 1 aa insertion (boxed) that is exclusive to all members belonging to the order *Chlorobiales*. Figure S8. Partial sequence alignment of the protein glutamate-1-semialdehyde 2,1-aminomutase, showing a 1 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales.* Figure S9. Partial sequence alignment of the protein class II fructose-1,6-bisphosphate aldolase, showing an 11 aa deletion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S10. Partial sequence alignment of the protein major facilitator superfamily transporter, showing a 1 aa insertion (boxed) that is specific for members of the order *Chlorobiales*. Figure S11. Partial sequence alignment of the protein DNA polymerase III subunit alpha, showing a 2–4 aa insert (boxed) that is specific for all members belonging to the order *Chlorobiales*. Figure S12. Partial sequence alignment of the protein acetyl-CoA carboxylase carboxyltransferase subunit alpha, showing a 6 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S13. Partial sequence alignment of the protein N-acetyl-alpha-D-glucosaminyl L-malate synthase BshA, showing a 3 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S14. Partial sequence alignment of the protein serine hydroxymethyltransferase, showing a 2 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S15. Partial sequence alignment of the protein deoxyribonucleotide triphosphate pyrophosphatase, showing a 3 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S16. Partial sequence alignment of the protein polynucleotide phosphorylase/polyadenylase, showing a 1 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S17. Partial sequence alignment of the protein polynucleotide phosphorylase/polyadenylase, showing a 17 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S18. Partial sequence alignment of the protein 7-carboxy-7-deazaguanine synthase QueE, showing a 3–7 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales*. While other *Chlorobia* species contain a 7 aa insertion, *Chlorobaculum* species have a 3 aa insertion in this position. Figure S19. Partial sequence alignment of the protein DNA polymerase III subunits gamma and tau, showing a 2 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S20. Partial sequence alignment of the protein DNA polymerase III subunits gamma and tau, showing a 4 aa insertion (boxed) that is

found in all members belonging to the order *Chlorobiales.* Figure S21. Partial sequence alignment of the protein cytidylate kinase, showing a 4 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S22. Partial sequence alignment of the protein 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, showing a 2–5 aa insertion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S23. Partial sequence alignment of the protein molecular chaperone DnaK, showing a 1 aa deletion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S24. Partial sequence alignment of the protein tryptophanyl-tRNA synthetase, showing an 8 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S25. Partial sequence alignment of the protein tryptophanyl-tRNA synthetase, showing a 5 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S26. Partial sequence alignment of the protein succinyl-CoA synthetase subunit alpha, showing a 7 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S27. Partial sequence alignment of the protein Gfo/Idh/MocA family oxidoreductase, showing a 4 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S28. Partial sequence alignment of the protein Gfo/Idh/MocA family oxidoreductase, showing a 2 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S29. Partial sequence alignment of the protein ribonuclease R, showing a 7–8 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S30. Partial sequence alignment of the protein ribonuclease R, showing a 4–5 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales.* Figure S31. Partial sequence alignment of the protein ribonuclease R, showing a 4 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S32. Partial sequence alignment of the protein bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase, showing a 2 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S33. Partial sequence alignment of the protein lactoylglutathione lyase, showing a 2 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S34. Partial sequence alignment of the protein thiazole synthase, showing a 1 aa insert (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S35. Partial sequence alignment of the protein DNA mismatch repair protein (MutS), showing a 1 aa deletion (boxed) that is found in all members belonging to the order *Chlorobiales*. Figure S36. Partial sequence alignment of the protein peptide chain release factor 3, showing a 1 aa deletion (boxed) that is specific for members of the family *Chlorobiaceae*. Figure S37. Partial sequence alignment of the protein U32 family peptidase, showing a 6 aa insertion (boxed) that is specific for members of the family *Chlorobiaceae*. Figure S38. Partial sequence alignment of the MiaBlike tRNA modifying protein, showing a 5 aa insertion (boxed) that is specific for members belonging to the family *Chlorobiaceae*. Figure S39. Partial sequence alignment of the molecular chaperone HtpG protein, showing a 3 aa insertion (boxed) that is specific for members of the family *Chlorobiaceae*. Figure S40. Partial sequence alignment of the DegT/DnrJ/EryC1/StrS family aminotransferase protein, showing a 1 aa deletion (boxed) that is specific for the family *Chlorobiaceae*. Figure S41. Partial sequence alignment of the biogenesis of lysosome-related organelles complex 1 subunit 2 protein, showing a 1 aa deletion (boxed) that is specific for members of the family *Chlorobiaceae*. Figure S42. Partial sequence alignment of the protein DNA gyrase subunit A, showing a 1 aa insertion (boxed) that is specific for members belonging to the family *Chlorobiaceae*. Figure S43. Partial sequence alignment of the protein hypoxanthine phosphoribosyltransferase, showing a 1 aa insertion (boxed) that is exclusively shared by all members of the family *Chloroherpetonaceae*. Figure S44. Partial sequence alignment of the protein dihydrolipoyl dehydrogenase containing a 1 aa insertion (boxed) that is specific for members of the family *Chloroherpetonaceae*. Figure S45. Partial sequence alignment of the protein SDR family oxidoreductase, showing a 4 aa insertion (boxed) that is specific for members of the family *Chloroherpetonaceae*. Figure S46. Partial sequence alignment of the protein RecQ family ATP-dependent DNA helicase, showing a 2 aa insertion (boxed) that is specific for members of the family *Chloroherpetonaceae*. Figure S47. Partial sequence alignment of the alkaline phosphatase family protein, showing a 5 aa insertion (boxed) that is specific for members of the family *Chloroherpetonaceae*. Figure S48. Partial sequence alignment of the protein tRNA dihydrouridine synthase DusB, showing a 1 aa insertion (boxed) that is specific for members of the order *Chlorobiales* and the "larger *Ignavibacteriae* clade".

**Author Contributions:** Conceptualization, R.S.G.; data curation, S.B.; formal analysis, S.B. and M.H.-A.; funding acquisition, R.S.G.; methodology, S.B., M.H.-A. and R.S.G.; project administration, R.S.G.; resources, R.S.G. and H.E.S.; software, R.S.G.; supervision, R.S.G.; validation, S.B. and R.S.G.; writing—original draft, R.S.G.; writing—review & editing, R.S.G. and H.E.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a Discovery Research Grant (RGPIN-2019-06397), an Alliance Research Grant from the Natural Science and Engineering Research Council of Canada (HES and RSG), and an ORF grant from the Ontario Ministry of Research, Innovation and Science.

**Data Availability Statement:** The data presented in this study are available in a publicly accessible repository (https://www.ncbi.nlm.nih.gov/genome/, accessed on 10 January 2022) and the Supplementary Material.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

