3.3.6. Cytochrome *b*<sup>5</sup>

Cytochrome *b*5, which is a well-known constituent of eukaryotic cells, has for the first time found in *Ect. vacuolata*, where its function is unknown [67]. It differs from its eukaryotic counterparts in having a cysteine disulfide and the presence of a signal peptide, which suggests that it is located in the periplasm. At least seven examples are now known from the genome sequences of *Ectothiorhodospiraceae*. These are *Ect. vacuolata* DSM 2111<sup>T</sup> *, Ect.* PHS-1*, Ect. shaposhnikovii* DSM 243<sup>T</sup> *, Ect. mobilis* DSM 237<sup>T</sup> *, Ect.* BSL-9*, Ect. haloalkaliphila* ATCC 51935<sup>T</sup> and *Ect. marina* DSM 241<sup>T</sup> .

The 10-heme cytochromes, MtrA and MtrF (or OmcA), as well as the outer membrane porin MtrB, are involved in the reduction and solubilization of insoluble iron and manganese minerals primarily in *Shewanella* species, as well as in other bacteria [68]. MtrAB are occasionally found in the purple sulfur bacteria, such as *Ect. vacuolata, Ect. shaposhnikovii, Ect. haloakaliphila, Ect.* BSL-9 and *Ect. marina*. In the species of *Ectothiorhodospiraceae*, these two genes are also associated with OmcA, as in *Shewanella*. Cytochrome *c*<sup>4</sup> or HiPIP are sometimes found associated with them, suggesting oxidation of FeII rather than reduction of FeIII, since they have high redox potentials. A HiPIP gene is adjacent to the MtrABF cluster in *Ect. vacuolata* DSM 2111<sup>T</sup> and *Hrs*. *halophila*, although not in other species considered herein.

#### 3.3.7. Arsenic Oxidation

The *Ectothiorhodospiraceae* and the *Halorhodospiraceae* are generally capable of oxidizing arsenic (III) to arsenic (V), but only a few species are capable of using As(III) as sole electron donor for growth, including *Ect*. species strain PHS-1 and *Ect*. species strain BSL-9 [40]. The enzymes involved are the molybdopterin proteins ArxA and ArrA, which are closely related. Most *Ectothiorhodospiraceae* have *arrABC* genes, but only *Ect*. PHS-1, *Ect*. BSL-9, *Ect. shaposhnikovii* DSM 243<sup>T</sup> , *Ect.* B14B, *Ect.* A-7Y, *Ect.* A-7R, and *Hrs. halophila* SL1<sup>T</sup> and BN9630, have five to eight *arx* genes. The authors of [40] examined four of these species plus *Ect. vacuolata* for oxidation of As(III) and checked for As(III) dependent growth and found that only PHS-1 and BSL-9 would grow with arsenic. Therefore, there has to be more to the story because *Ect. shaposhnikovii* and *Hrs. halophila* have the requisite genes but failed to grow.

#### 3.3.8. Photoactive Yellow Protein—PYP

Another interesting difference between the red- and green-colored *Halorhodospira* strains is that only the red strains have a pair of photoactive yellow proteins (PYP) [69]. It is completely absent in the green-colored strains but has been discovered in the *Thio-rhodospira sibirica* genome as well as *Halochromatium salexigens* (*Chromatiaceae*). The only proven role of PYP in purple bacteria is to reverse the effects of red light on the bacteriophytochrome in the hybrid protein PPR in *Rhodocista centenaria*, which is a PYP/bacteriophytochrome/histidine kinase [70]. The *Chromatiaceae* do not have PPR, but some species do have a hybrid PYP/bacteriophytochrome/diguanylate cyclase/phosphodiesterase, which is called PPD [71]. Interaction among the separate domains has not been demonstrated in PPD and the functional role of the PYP domain is likely to be different than it is in PPR.

#### 3.3.9. Photosynthesis Gene Clusters

As other phototrophic *Proteobacteria*, *Ectothiorhodospiraceae* and *Halorhodospira* species have photosynthesis gene clusters, including genes for carotenoid and bacteriochlorophyll biosynthesis, the photosynthetic reaction center and antenna proteins, as well as regulatory and sensory proteins. The structure of the gene clusters and the arrangement of genes show clear differences between *Ectothiorhodospira* and *Halorhodospira* species (Figure 3). *Microorganisms* **2022**, *10*, x FOR PEER REVIEW 15 of 24 of genes show clear differences between *Ectothiorhodospira* and *Halorhodospira* species (Figure 3).


**Figure 3.** Comparison of the *bch*B genomic region between representatives of *Halorhodospira* and *Ectothiorhodospira* species. Genes are colored based on their family membership. **Figure 3.** Comparison of the *bch*B genomic region between representatives of *Halorhodospira* and *Ectothiorhodospira* species. Genes are colored based on their family membership.

While in *Halorhodospira* species a cluster with *ppsR-bchFNBHLM* genes (the regulator gene *ppsR* is absent from *Hlr. abdelmalekii* and *Hlr. halochloris*) is present, in *Ectothiorhodospira* species an additional regulatory *ppaA* gene is combined with the *ppsR* gene (genes 12 and 13 in Figure 3). The presence of the two regulatory genes *ppsR* and *ppaA* in the photosynthetic gene While in *Halorhodospira* species a cluster with *ppsR-bchFNBHLM* genes (the regulator gene *ppsR* is absent from *Hlr. abdelmalekii* and *Hlr. halochloris*) is present, in *Ectothiorhodospira* species an additional regulatory *ppaA* gene is combined with the *ppsR* gene (genes 12 and 13 in Figure 3).

cluster is common to many phototrophic *Alpha*- and *Betaproteobacteria* as well as *Gemmatimonas*. The *ppaA* gene is absent from *Chromatiaceae* and *Halorhodospiraceae* and is found among phototrophic *Gammaproteobacteria* only in the genus *Ectothiorhodospira* (Figure 3). Both regulatory genes are absent from *Ets. Mongolicus.* Quite characteristic for *Ectothio-*The presence of the two regulatory genes *ppsR* and *ppaA* in the photosynthetic gene cluster is common to many phototrophic *Alpha*- and *Betaproteobacteria* as well as *Gemmatimonas*. The *ppaA* gene is absent from *Chromatiaceae* and *Halorhodospiraceae* and is found among phototrophic *Gammaproteobacteria* only in the genus *Ectothiorhodospira* (Figure 3). Both regulatory genes are absent from *Ets. Mongolicus.* Quite characteristic for *Ectothio-*

*rhodospiraceae,* and different to most other purple bacteria, including the *Halorhodospiraceae,* is a gene cluster *ChlG-ppsR-ppaA-bchFNB*, with the exclusion of *bchL* and *bchH* genes from the common *bchFNBHLM* gene cluster, as shown for representatives of the genus *Ectothiorhodospira* (Figure 3). Both *bchH* and *bchL* genes are at separate locations in these bacteria. All *Halorhodospira* species have an additional regulator gene *pufQ* which is located between *bchZ* and *pufB* (*bchCXYZ-pufQBALMC*). This regulator is found in many phototrophic *Alphaproteobacteria* but is absent from all *Ectothiorhodospiraceae* and from *Chromatiaceae* as well. All *Halorhodospira* species and *Ectothiorhodospiraceae* lack the aerobic Mg-protoporphyrin IX monomethylester oxidative cyclase (*acsF*) and therefore depend on anoxic conditions for bacteriochlorophyll biosynthesis using the anaerobic form of the enzyme (encoded by *bchlE*).

### *3.4. Habitats and Environmental Distribution*

Quite remarkably, species of phototrophic *Ectothiorhodospiraceae* and *Halorhodospiraceae* (*Ectothiorhodospira, Halorhodospira*, *Thiorhodospira* and *Ectothiorhodosinus* species), including their chemotrophic relatives, are characteristic inhabitants of marine and saline waters worldwide and preferably develop in alkaline and saline soda lakes. They are phylogenetically related to alkaliphilic chemotrophic sulfur-oxidizing bacteria of the genera *Thioalkalivibrio*, *Alkalilimnicola*, *Alkalispirillum* and are important sulfur-oxidizing chemotrophic bacteria in many alkaline soda lakes [72,73]. A recent review summarizes their occurrence in various types of salt and soda lakes in different geographic regions [74].

Although *Halorhodospira halophila* was first isolated from Summer Lake, Oregon [45,46] and *Hlr. neutriphila* originates from a marine saltern [49], the great majority of the studied strains originate from African soda lakes, most prominently those of the Wadi el-Natrun. This contains all strains of the green-colored species and a second strain of *Hlr. halophila* (9630). Several other strains assigned to this species (including, among others, BN9620, BN9622 and BN9624) are likely distinct on the species level from *Hlr. halophila*. In addition to strains from the Wadi el-Natrun, two isolates from Mongolian soda lakes (M38 and M39old) belong to this presumably new species.

The first intensively studied soda lakes with mass developments of red-colored and green-colored *Halorhodospira* species were those in the Wadi el-Natrun in Egypt [35,50,75]. While pH-optima of the type strain of *Hlr. halophila* SL1<sup>T</sup> (isolated from the highly alkaline and saline Summer Lake in Oregon) were found to be at 7.4–7.9 [45,46], isolates from the Wadi el-Natrun had pH optima at 8.5–9.0. Two more haloalkaliphilic species, the greencolored, bacteriochlorophyll *b*-producing *Hlr. halochloris* [47] and *Hlr. abdelmalekii* [48], originate from these soda lakes.

In addition, isolates of the moderate halophilic and alkaliphilic *Ectothiorhodospira* species *Ect. haloalkaliphila* [14,35] and *Ect. variabilis,* which is most closely related to *Ect. halo-alkaliphila* [36], were found in soda lakes of the Wadi el-Natrun and also in soda lakes from Siberia and Mongolia [36].

*Halorhodospira halophila* and two morphological distinct bacteria assigned to the genus *Ectothiorhodospira* were present in Mongolian soda lakes with high salinities (>15% salts), while other species of *Ectothiorhodospira* and *Thiorhodospira* were isolated from lakes with lower salinities of up to 5.5–6.0% salts. For the first time the moderately halophilic *Ectothiorhodosinus mongolicus* (salt optimum 1–7%) was isolated from one of these lakes [42]. Additionally, *Thiorhodospira sibirica* [43] and *Ect. magna* [44] were isolated from soda lakes of remote areas in Asia in Siberia, Mongolia and the Transbaikal region, but have so far not been found in other locations. Possibly the remote origin of these bacteria indicates their separate evolution and is a reason for their distant relationship to other strains of the *Ectothio-rhodospiraceae/Halorhodospiraceae* families.

Other *Ectothiorhodospira* species, especially *Ect. marina* and *Ect. mobilis*, prefer marine habitats, where they have been regularly observed and repeatedly been isolated from.

In recent years, proof of the presence of *Ectothiorhodospiraceae* in alkaline and saline lakes worldwide has been obtained by analysis of clone libraries. The analysis of 16S rRNA

gene clone libraries from several lakes of the Wadi el-Natrun (Lake Fazda, Lake Hamra and Lake UmRisha) revealed a great diversity of sequences related to *Ectothio-rhodospiraceae* species, including *Hlr. halochloris*, and *Ect. haloalkaliphila* [76]. In addition, communities of phototrophic purple bacteria in Chilean salt lakes of the Atacama Desert, Laguna Chaxa and Laguna Tebenquiche that were studied by clone libraries of *pufLM* gene sequences were found to contain bacteria related to *Ect. mobilis, Ect. variabilis* and *Hlr. halophila* as closest relatives [77,78].

#### **4. Systematic Conclusions**

The comparison of a large number of genome sequences of phototrophic purple sulfur bacteria in the present study clearly demonstrated the need to separate *Halorhodospira* species and relatives from the *Ectothiorhodospiraceae* and to place them in the new family *Halorhodospiraceae*. The comparison of the genomic phylogeny, including considerations of ANI, demonstrated that the phototrophic *Ectothiorhodospiraceae* as currently known represent two separate families, being almost equally distant from the *Chromatiaceae* family. Based on the significant differences of the extremely halophilic species from all other *Ectothiorhodospiraceae*, we propose to recognize these species as members of a new family, the *Halorhodospiraceae* fam. nov.

The herein proposed reclassification of the families of phototrophic purple sulfur bacteria with the separation of *Halorhodospira* species from the *Ectothiorhodospiraceae* family and the existence of three families of purple sulfur bacteria within the *Gammaproteobacteria* requires a careful reconsideration of species assignment to these families and sheds new light on the assignment of a number of chemotrophic species and genera to the *Ectothiorhodospiraceae* which have been made in recent years but do not warrant to be included within this family.

Among those bacteria that have been assigned to the *Ectothiorhodospiraceae* which without doubt cannot be considered as members of this family are *Acidiferrobacter thiooxydans* [79], now classified with *Acidiferribacteraceae* and *Acidiferribacterales* [80,81] and *Oceanococcus atlanticus* [82]. They do not fit into any of the three phototrophic *Chromatiales* families (Figure 1). In addition, *Halofilum ochraceum* [83], with ANI values to all considered *Ectothiorhodospiraceae* of only 65–68%, should not be included in *Ectothiorhodospiraceae* or *Halorhodospiraceae*. In addition, a few bacteria for which genome sequences at present are not available, should not be included in *Ectothiorhodospiraceae* or *Halorhodospiraceae* based on information available for 16S rRNA gene sequences and other properties. One of these species is *Natronocella acetinitrilica* [84]. Two other species are *Methylonatrum kenyense*, which is a gammaproteobacterium considered to be of unknown affiliation (genus incertae sedis), and *Methylohalomonas lacus* [85], which may be related to *Thioalkali-spira* species of the *Thioalkalispiraceae* [86].

Other species/genera that have been assigned to the *Ectothiorhodospiraceae* are distantly related to the phototrophic *Chromatiaceae, Ectothiorhodospiraceae* or *Halorhodospiraceae* by deep-branching lineages both in genome-based and 16S rRNA-based phylogenetic trees (Figures 1 and 2). These are *Thiohalospira halophila*, *Thiohalomonas denitrificans*, *Thiogranum longum*, *Thioalbus denitrificans*, *Inmirania thermothiophila* and the acidiphilic *Acidihalobacter prosperous* and related species, which therefore should not be considered as belonging to either *Ectothiorhodospiraceae* or *Halorhodospiraceae*. Their placement in one or more new separate families needs to be evaluated and these bacteria will not be further considered in the present discussion.

Halophilic and alkaliphilic chemotrophic bacteria that are most closely related to phototrophic *Ectothiorhodospiraceae* are species of the genus *Thioalkalivibrio*, particularly those belonging to the *Thioalkalivibrio sulfidophilus* cluster (Figures 1 and 2). Species of both clusters of *Thioalkalivibrio* are the only known chemotrophic bacteria that can be con-sidered to be included in the *Ectothiorhodospiraceae*.

The new family *Halorhodospiraceae* is represented by two major groups of phototrophic bacteria, the red-colored *Hlr. halophila* and relatives and the green-colored *Hlr. halochloris* and relatives. Phenotypic and phylogenetic differences suggest a separation of the greencolored species in a separate genus, with ANI values of the type species of 70.4–71.3 to strains of *Hlr. halophila*. Although *Hlr. neutriphila* appears phylogenetically and phenotypically (characteristically by the preference for neutral pH and high G + C content of 72 mol%) distinct from *Hlr. halophila*, an ANI >77% to *Hlr. halophila* (Table 3) and 16S rRNA identity of 96.5% to the type strain of *Hlr. halophila* SL1 support recognizing this bacterium as a distinct species of the genus *Halorhodospira*. This is in line with proposals made by others suggesting genus delineations at ANI values close to 74% [51,87].

According to both the phylogenomic and the 16S rRNA tree, a phylogenetic cluster distinct to these phototrophic bacteria is represented by a group of chemotrophic bacteria, including chemotrophic alkaliphilic and halotolerant species, the nitrifying *Nitrococcus mobilis*, *Aquisalimonas asiatica*, *Spiribacter salinus*, *Spiribacter (Halopeptonella) vilamensis* and *Arhodomonas aquaeolei* (Figures 1 and 2). According to 16S rRNA phylogeny, this cluster includes *Alkalispirillum mobilis* and *Alkalilimnicola ehrlichii* and relatives as well, which, according to the genomic tree, form a separate lineage closer to *Halorhodospira*. The phylogenetic distance of the whole group of chemotrophic relatives suggests the exclusion of these bacteria from the *Halorhodospiraceae* and placing them within one or more separate new families. Accordingly, the *Halorhodospiraceae* are represented exclusively by phototrophic bacteria.

Based on the present data, *Ectothiorhodosinus mongolicus* [42] and *Thiorhodospira sibirica* [43] represent genera distinct from *Ectothiorhodospira*. *Ect. magna* [44], though distantly related to other species of the genus, is placed inside a group of species together with *Ect. shaposhnikovii* and *Ect. vacuolata*. ANI values among all studied strains/species of the genus *Ectothiorhodospira* are >74.9% which is in line with a proposed genus demarcation of approximately 74% [88]. Other strains (strains A-7Y, A-7R and B14B) with ANI values to related *Ectothiorhodospira* species below 90% (87.8–89.4) presumably represent a new species of this genus. Although little information is known, and genome sequences are not available for *Ect. salini* and "*Ect. imhoffii*", both species appear most closely related to *Ect. mobilis* and *Ect. marismotui* according to 16S rRNA gene sequences (Figure 2; [88,89]).

Characteristics of the three families.

There are three families of the *Chromatiales* that are characterized by the presence of phototrophic purple sulfur bacteria forming coherent clusters that are, with very few exceptions, clearly distinct from their chemotrophic relatives. These families can be distinguished by a number of phenotypic properties as well as on the basis of genomic properties.

The *Chromatiaceae* primarily live in fresh water or marine habitats; they are phototrophic and primarily autotrophic. When oxidizing sulfide and thiosulfate, intermediate sulfur is stored in the periplasmic space, enclosed within a proteinaceous membrane containing one to five small, related sulfur globule proteins. The thiosulfate dehydrogenase, SoxA, and its electron-acceptor, SoxX, exist as separate subunits. The small protective thiol, glutathione, is present as the amide.

*Ectothiorhodospiraceae* primarily live in shallow alkaline desert soda lakes and also marine shallow waters and tolerate up to about 5–7% salt. They are primarily phototrophic and autotrophic. When oxidizing sulfide and thiosulfate, the intermediate sulfur is excreted into the growth medium without a membrane. SoxA and SoxX exist as separate subunits. Glutathione is present as the amide.

The *Halorhodospiraceae* are found in soda lakes, require elevated salt concentrations for growth and some species can even live in saturated brines. Intermediate elemental sulfur is excreted into the growth medium. SoxX is fused to the N-terminus of SoxA. Glutathione is not amidated. They are phototrophic bacteria forming two distinct groups of species, red-colored species producing bacteriochlorophyll *a* and green-colored species producing bacteriochlorophyll *b*. The red-colored species likely use the small iron–sulfur protein HiPIP as mediator between the *bc*<sup>1</sup> complex and the photosynthetic reaction center PufLM. On the other hand, the green species are likely to use the small soluble cytochrome *c*<sup>5</sup> as mediator.
