*3.7. Endosporulation*

A likely universal trait of heliobacteria is the ability to form endospores [11], differentiated and largely dormant cells that are highly resistant to environmental extremes, such as heat and desiccation. Genomic comparisons of *Hrs. convoluta* and *Hbt. modesticaldum* revealed high similarity between endosporulation genes in each species. For example, genes encoding key sporulation sigma factors (σH, σE, σF, σG, σK) in *Hbt. modesticaldum* were also identified in the *Hrs. convoluta* genome. Like *Hbt. modesticaldum*, *Hrs. convoluta* lacked the *spo0M* gene functioning to regulate stage 0 development of endosporulation [80] and the *spoIIB* gene necessary for robust sporulation in *B. subtilis* [81]. This may help explain the sporadic (as opposed to consistent) production of endospores in serially subcultured

cells of *Hrs. convoluta* strain HH<sup>T</sup> [21], as the deletion of either *spo0M* or *spoIIB* in *B. subtilis* results in impairment of endosporulation [80,81]. Additionally, the 20 *cot* genes encoding proteins that comprise the protective spore coat for *B. subtilis*, including *cotH* required for spore coat assembly [82], did not show significant similarity to genes in *Hbt. modesticaldum* [6] or *Hrs. convoluta*. Likewise, key proteins that coordinate spore coat assembly and composition in *Clostridioides* (*Clostridium*) *di*ffi*cile*, including CotA and CotB [83], showed no sequence similarity to genes in *Hrs. convoluta*. Despite these deficiencies, cells of *Hrs. convoluta* strain HH<sup>T</sup> were still capable of forming heat-resistant endospores, even if sporadically [21]. These findings sugges<sup>t</sup> shared biosynthetic and regulatory mechanisms governing endosporulation in *Hbt. modesticaldum* and *Hrs. convoluta* that di ffer in some respects from those that govern endosporulation in species of *Bacillus* and *Clostridium*.

#### *3.8. Molecular Adaptations to Alkaliphily in* Heliorestis convoluta

Alkaliphilic bacteria employ several mechanisms to maintain intracellular pH homeostasis in their highly alkaline environments. Experimental work conducted with alkaliphiles revealed that these organisms maintain a lower cytoplasmic pH than their external environment—up to a 2.3 pH unit di fference—for optimal enzyme activity and cellular functioning [84,85]. Despite its optimal growth pH of 8.5–9 and ability to grow slowly at pH 10 [21], it is likely that *Hrs. convoluta* maintains a cytoplasmic pH at or below pH 8, as is true from studies of several alkaliphilic strains of *Bacillus* [84,86].

Cytoplasmic pH homeostasis in *Hrs. convoluta* is likely supported by the presence of a Na+/H<sup>+</sup> antiporter encoded by *nhaA* (FTV88\_0116). To maintain cytoplasmic pH at homeostatic levels, the Na+/H<sup>+</sup> antiporter operates in an electrogenic manner, facilitating the import of twice as many H<sup>+</sup> as Na<sup>+</sup> exported [87,88]. The inward movement of protons through the antiporter acidifies the cytoplasm to maintain a pH closer to neutral [87–89]. The NhaA protein from *Hrs. convoluta* was found to be 87% identical in amino acid sequence to NhaA from *Heliorestis acidaminivorans*—also an alkaliphile—but only 50% identical to NhaA from the neutrophile *Hbt. modesticaldum*. The NhaA enzyme may, therefore, be a good candidate to study which amino acid residues facilitate antiporter activity in alkaline versus neutral environments.

In addition to its role in cytoplasmic pH maintenance, the Na+/H<sup>+</sup> antiporter generates a sodium motive force (SMF) that has been shown to be important for secondary active transport of various substrates [89–91] (see Figure 3 for examples in *Hrs. convoluta*). The use of Na<sup>+</sup>-coupling for transport is potentially more important in *Hrs. convoluta* than in its neutrophilic relative, *Hbt. modesticaldum*, as genes encoding multiple Na<sup>+</sup>-dependent transporters (FTV88\_2418 and FTV88\_1400) and a Na+/Ca<sup>+</sup> antiporter (FTV88\_2739) in *Hrs. convoluta* showed little to no significant similarity with genes in *Hbt. modesticaldum*.

The more neutral cytoplasm compared to the alkaline extracellular milieu would seemingly create an outward-directed bulk PMF rather than the inward-directed PMF needed to drive ATP synthesis [89–91]. Despite this, most alkaliphiles, including *Hrs. convoluta*, still employ a PMF rather than a SMF to power ATP synthase [89,92]. Alkaliphilic bacteria must therefore have mechanisms in place to prevent H<sup>+</sup> equilibration with the external environment so that an e ffective local PMF can be established. To this end, carotenoids, which are produced in large quantities by alkaliphilic heliobacteria [93], have been proposed to play a role in organizing proton pumps close to ATP synthases in the membrane, thus facilitating more e fficient ATP generation [91,94,95]. In addition, cardiolipin, a glycerophospholipid that assists in membrane domain organization, may also help prevent H<sup>+</sup> equilibration by functioning as a proton sink for the <sup>H</sup><sup>+</sup>-coupled ATP synthase [96–98]. By functioning in this capacity, cardiolipin allows for the retention of H<sup>+</sup> near the surface of the cell membrane so that they are unable to spontaneously di ffuse into the alkaline environment. Notably, a gene encoding cardiolipin synthase was present in *Hrs. convoluta* (FTV88\_2523), but no corresponding homolog was identified in *Hbt. modesticaldum*.

In addition to producing proteins and other molecules that counteract the pH di fference between the cytoplasm and environment and the consequences thereof, homologous proteins also have amino

acid substitutions that optimize the functioning of normal processes for the alkaline environment. In alkaliphiles, the portions of extracellular enzymes that are exposed to the external environment tend to have decreased numbers of basic residues (arginine, histidine, or lysine), with acidic amino acids (aspartate or glutamate) or neutral residues in their place [99–101]. In a noteworthy example, the amino acid sequence for cytochrome *c*553 (PetJ) of *Hrs. convoluta* contained 13 more acidic amino acid residues and 11 fewer basic residues than PetJ of *Hbt. modesticaldum* (Figure 8A). In line with previous discussion, the elevated number of acidic residues and corresponding decrease in basic residues in the externally-functioning *Hrs. convoluta* PetJ should contribute to OH− repulsion and H<sup>+</sup> attraction near the membrane surface and help maintain the PMF [89]. Although additional investigation of the cell surface of *Hrs. convoluta* is required to confirm its electrochemical nature, genomic data sugges<sup>t</sup> that this phototroph can sequester H<sup>+</sup> near the cell surface to create an effective PMF for ATP synthesis and flagellar motility.




**Figure 8.** Amino acid sequence alignments for cytochrome *c*553 and ATP synthase Fo alpha subunit of *Heliorestis convoluta* with related species. (**A**) The sequence alignment for cytochrome *c*553 of *Hrs. convoluta* and *Heliobacterium modesticaldum*. Acidic amino acid residues (red), aspartate (D) and glutamate (E), and basic amino acid residues (blue), arginine (R) and lysine (K), that differed between each species were indicated by a colored dash directly above or below the residue. Acidic or basic amino acids in the gap (–) regions were not marked. (**B**) Sequence alignment for ATP synthase Fo alpha subunit of *Hrs. convoluta* and *Bacillus pseudofirmus* OF4. The lysine residue of interest at position 180 in *B. pseudofirmus* aligns with Lys<sup>182</sup> in *Hrs. convoluta* (red box). (**C**) Sequence alignment for ATP synthase Fo alpha subunit of *Hrs. convoluta* and *Hbt. modesticaldum*. The lysine residue of interest at position 182 in *Hrs. convoluta* aligns with Gly<sup>179</sup> in *Hbt. modesticaldum* (red box). All sequence alignments were generated using the BLAST algorithm.

In a similar way, several key amino acid residues and motifs in ATP synthase have been found to contribute to optimal functioning of the enzyme at different pH levels [89,91,102,103]. For example, a lysine residue found at position 180 in the Fo alpha subunit of ATP synthase in *Bacillus pseudofirmus* OF4 was determined to favor <sup>H</sup><sup>+</sup>-powered ATP synthesis at an alkaliphilic pH due to its basic properties [102,104–106]. As expected, a corresponding Lys<sup>182</sup> in the *Hrs. convoluta* Fo alpha subunit (Figure 8B) can presumably capture protons optimally from the alkaline environment and release them into the rotor subunit of ATP synthase at an external basic pH near the high pKa of the side chain [102]. The lysine residue would be detrimental to ATP synthesis in a neutral pH range, as H<sup>+</sup> would be retained on the residue side chain at ~pH 7 (below the side chain pKa). This highlights the significance of a glycine residue at the corresponding position in the Fo alpha subunit in neutrophilic bacteria, including *Hbt. modesticaldum* [102] (Figure 8C).

Several alkaliphilic bacteria use a SMF to power flagellar motor proteins, thus reserving the valuable and limited PMF for ATP production [107,108]. Research conducted with alkaliphilic *Bacillus* spp. concluded that a highly conserved valine residue is present in H<sup>+</sup>-driven (MotB) flagellar motor protein sequences, whereas a leucine residue takes the place of this valine in Na<sup>+</sup>-driven (MotS) motor protein sequences [107,108]. The alkaliphilic *Bacillus* spp. contained MotS with the conserved leucine amino acid, allowing these bacteria to use a SMF to power Na<sup>+</sup>-coupled flagellar motility [107,108]. Interestingly, MotB—with its conserved valine—was identified in both *Hrs. convoluta* and *Hbt. modesticaldum*, suggesting that a PMF is used to power motility in both alkaliphilic and neutrophilic heliobacteria. Genomic analyses confirmed the presence of a core set of 24 genes (*fliCDEFGHIMNPQR*, *flgBCDEFGKL*, *motAB*, *flhAB*) in *Hrs. convoluta* that are essential for PMF-driven swimming motility in numerous flagellated bacteria [109].
