Cyt<sup>c</sup> BV509\_15885

The transcript encoding the gene BV509\_15885 is annotated as a cyt*c*. RNA-Seq data did not identify this cyt*<sup>c</sup>* as significantly upregulated during pEEU (log2fold change −0.48), but it was identified through heme staining (membrane fraction) followed by mass spectroscopy compared to hydrogen grown cultures [12]. This cyt*<sup>c</sup>* contains 5 heme-binding motifs and a predicted Sec signal peptide. BLASTp analysis identified homologs of this cyt*<sup>c</sup>* in strain AB19 and *R. visakhapatnamense* strain JA181. Homologs in the *R. sulfidophilum* isolates show 71–75% identity.

#### 3.3.2. Transcriptional Regulators

To understand expression patterns and potential regulators of pEEU genes, the pEEU vs. hydrogen transcript data was analyzed for any upregulated transcriptional regulators.

#### Mar Family Transcription Regulator BV509\_01095

BV509\_01095 encodes a hypothetical protein identified as a member of the multi antibiotic resistance family transcriptional regulator (log2fold change ~6, *p* < 0.001). InterProScan predicts phosphorelay signal transduction function. BV509\_01095 is present only in strain AB19 and strain AB26 via BLASTp analysis, but not *R. visakhapatnamense* strain JA181 or any of the *R. sulfidophilum* isolates.

#### Transcriptional Regulator BV509\_15825

This transcript is another transcriptional regulator upregulated during pEEU in strain AB26 (log2fold change ~4, *p* < 0.0001). This regulator is also predicted to be involved in signal transduction response regulation via InterProScan, with DNA-binding wing-helix domain and tetratricopeptide repeat domain. BLASTp reveals this transcription regulator is unique to strain AB19, strain AB26, and *R. visakhapatnamense* strain JA181 but not the *R. sulfidophilum* isolates.

#### **4. Conclusions**

With the current accessibility of genome sequencing and ability to isolate bacterial species from diverse environments, this paper shows that taxonomy requires continuous addressing. Here, we provide a current taxonomic classification of 15 *Rhodovulum* spp. isolated from a marine estuary, specifically the re-classification of two of the original 15 isolates previously identified as *R. sulfidophilum* now as *R. visakhapatnamense*. These isolates are capable of many modes of metabolism, including photoferrotrophy and pEEU. To expand the characterization of *R. visakhapatnamense*, which at present is represented by 3 genomes assembled at the scaffold level, we describe the genome contents of *R. visakhapatnamense* strain AB26, and pair the genome data with expression data under anaerobic photoheterotrophy (acetate), photoautotrophy (H<sup>2</sup> or thiosulfate as electron donor), and photosynthetic extracellular electron uptake (poised electrode as electron donor). Upregulated transcripts under pEEU of interest to the analysis include *c*-type cytochromes and transcriptional regulators. BLASTp shows some transcripts of interest are unique to *R. visakhapatnamense* genomes and could be indicative of adaptation of molecular mechanisms for pEEU explained by speciation.

What will be interesting to observe in the future is continued comparison of expression profiles and proteomes under pEEU and photoferrotrophy between *R. sulfidophilum* and *R. visakhapatnamense* strains from Woods Hole, as this data points to differences between species. Mutational analysis of genes of interest in each species will be necessary to determine their role under these growth conditions. With the data presented here, we can begin to characterize the metabolic pathway of pEEU in *Rhodovolum* species.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms10061235/s1, File S1: database files for phylogenetic trees (Figure S1: Average Nucleotide Identity (ANI) heatmap comparing AB26, AB19, and AB28 to other closely related *Rhodovulum* strains. The heatmap shows AB19 and AB26 share >99% ANI to each other, and 98% ANI to *Rhodovulum visakhapatnamense*. AB28 does not show increased ANI values to any related species, despite the relatively low (~94%) ANI to the other Woods Hole isolates (Figure 2). ANI values were calculated using JSpeciesWSd and visualized as a heatmap using Morpheus. Table S1: General Genome Features of Woods Hole isolates and publicly available *Rhodovulum visakhapatnamense* and *Rhodovulum sulfidophilum* genomes from NCBI and IMG/JGI databases); File S2: RNASeq Heatmaps base files.

**Author Contributions:** E.J.D. carried out all analysis and writing. A.B. contributed to writing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the following grants to A.B.: The David and Lucile Packard Foundation Fellowship (201563111), the U.S. Department of Energy (grant number DESC0014613), and the U.S. Department of Defense, Army Research Office (grant number W911NF-18-1-0037), Gordon and Betty Moore Foundation, National Science Foundation (Grant Number 2021822, Grant Number 2124088, and Grant Number 2117198), the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DEAC5207NA27344 (LLNL-JRNL-812309), and a DEPSCoR grant (FA9550-21-1-0211). A.B. was also funded by a Collaboration Initiation Grant, an Office of the Vice-Chancellor of Research Grant, and an International Center for Energy, Environment, and Sustainability Grant from Washington University in St. Louis. E.J.D. is supported by an Institutional Training Grant in Genomic Science from the NIH (T32 HG000045-18).

**Data Availability Statement:** Sequencing reads were deposited in the NCBI database under Bio-Project PRJNA546270, BioProject PRJNA692994 and BioProject PRJNA693004. Files for trees and heatmaps in supplement.

**Acknowledgments:** We would like to thank Eric Conners for his assistance with manuscript editing.

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

#### **References**

