Comparative Genome Analysis Provides Molecular Evidence for Reclassification of the Photosynthetic Bacterium Rhodobacter sphaeroides EBL0706 as a Strain of Luteovulum azotoformans

In this study, we conducted a genome-wide comparative analysis of a former Rhodobacter sphaeroides strain EBL0706, which is now recorded as Luteovulum sphaeroides EBL0706. The genome of EBL0706 was compared with that of Luteovulum azotoformans ATCC 17025, Luteovulum azotoformans KA25, and Luteovulum sphaeroides 2.4.1. The average nucleotide identity (ANI), tetra nucleotide signatures (Tetra), digital DNA–DNA hybridization (dDDH) values, comparative genome, and phylogenetic analysis proposed that EBL0706 is a strain of Luteovulum azotoformans. Functional annotations identified a total of 4034 protein-coding genes in the genome of EBL0706, including a complete photosynthetic gene cluster. This study provides genomic molecular verification for the strain EBL0706 to be reclassified to Luteovulum azotoformans.


Introduction
The Rhodobacter genus is comprised of heterogenous members showing flexibility in ecophysiology and metabolic capability [1][2][3]. Members of the genus can fix atmospheric nitrogen and carry out anoxygenic photosynthesis, thereby allowing them to adapt to various environments and play key roles in global biogeochemical cycles [4][5][6]. Furthermore, Rhodobacter species have been model organisms for studying bacterial photosynthesis [7]. Their single photosynthetic system consists of the light-harvesting complex I (LH1), the light-harvesting complex II (LH2), and the reaction center (RC) [8,9], showing structural and functional similarities to the light system II of higher plants [10].
The cells of the anaerobic culture of strain EBL0706 are ovoid and brown, with a diameter range from 0.8 µm to 1.2 µm. The cell can carry out binary fission reproduction. The cell has a single polar flagellum and a vesicular intima structure. The reddish aerobic culture of strain EBL0706 uses biotin as a growth factor. Small molecular organic matters, such as sodium acetate, sodium succinate, and glycerol, can support the growth of EBL0706. 2

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This is not the case, however, for sodium benzoate and sodium tartrate (Table S1) [11]. Previous studies have revealed the uniquity of "Rhodobacter sphaeroides" strain EBL0706 for its outstanding antioxidant capacity [12] and the ability to synthesize a variety of bioactive substances, such as carotenoids [13,14], chlorophylls [15], superoxide dismutase [16,17], and vitamin B12 [18]. In addition, studies have demonstrated the potential of this strain for environmental remediation on pollutants, such as dichlorvos [19] and oil [20].
Recently, the Rhodobacter genus was further reclassified as a new genus Luteovulum gen. nov. [6,21], and also had another genus name, Cereibacter, in the NCBI database. The genus Luteovulum awaits appropriate action by the research community to be transferred to another genus; we propose Luteovulum here as a temporary name noted in our study. In the NCBI database, the Luteovulum genus currently contains six species: Luteovulum sphaeroides, Luteovulum johrii, Luteovulum ovatum, Luteovulum azotoformans, Luteovulum alkalitolerans, and Luteovulum changlensis. Within the six species, 28 strains were identified (https://www. ncbi.nlm.nih.gov/taxonomy/?term=Luteovulum) (accessed on 10 January 2021). So far, the complete genome sequences of eight strains among them can be obtained from public databases. Rhodobacter sphaeroides strain EBL0706 is currently classified and deposited in the NCBI database as Luteovulum sphaeroides EBL0706.
Here, a combination of phylogenetic analyses, including comparative genomics, average nucleotide identity (ANI), tetra nucleotide signatures (Tetra), and digital DNA-DNA hybridization were used to reclarify the taxonomic position of "Rhodobacter sphaeroides" strain EBL0706 to Luteovulum azotoformans.

Medium and Growth Conditions
The EBL0706 culture was obtained from China General Microbiological Culture Collection Center (CGMCC) under the identity number of CGMCC No. 0645. The strain was inoculated and resuscitated in sterile Luria-Bertani (LB) medium (Solarbio, Beijing, China) (10.0 g/L tryptone, 5.0 g/L yeast extract, and 10.0 g/L NaCl; pH 7.0) at 32 • C for 24 h at 3000 lx light intensity.

Sequencing and Genome Assembly
The whole genome DNA was extracted by Bacterial Genomic DNA Extraction Kit (Solarbio, Beijing, China) according to the user manual. Then, the genome of strain EBL0706 was sequenced using Illumina HiSeq2000 (Illumina, San Francisco, CA, USA) and Pacific Biosciences II (Pacific Biosciences, San Francisco, CA, USA) sequencing platforms. Highquality reads were assembled by SOAPdenovo v2.04 [22]. Inner gaps that exist in the scaffolding were filled with GapCloser [23]. Pacific Biosciences SMART analysis software 1.2 was used to generate long "filtered sub-reads" from the instrument. The quality of the genome obtained was assessed through CheckM [24].
Data were analyzed on the Major BioCloud Platform (www.majorbio.com) (accessed on 11 December 2020). The complete genome project has been deposited in the National Center for Biotechnology Information (NCBI) as CP031750-CP031755.

Phylogenetic Tree
The target and reference 16S rRNA gene sequences were obtained from EzTaxon (https://www.ezbiocloud.net/) (accessed on 20 January 2021). MEGA 7.0 was used to construct 16S rRNA gene phylogenetic trees based on the neighbor-joining method. A rooted phylogenetic tree was constructed using RAxML (v. 8.2.8) software based on 20 single-copy core gene sequences, showing relationships between 10 whole genomes (Table S2) from the NCBI database. The RAxML analyses were run with rapid bootstrap analysis and 1000 replicates.

Comparative Genomics
Comparative genomic analysis to verify homology was carried out using Sibelia Software [31]. Strains that phylogenetically close to EBL0706 in the 16S rRNA tree were selected for the comparative analysis. These strains were L. azotoformans ATCC 17025, L. azotoformans KA25, and L. sphaeroides 2.4.1 [32,33]. L. azotoformans ATCC 17025 is formerly L. sphaeroides ATCC 17025 in the NCBI database and Rhodobacter azotoformans ATCC 17025 in the American Type Culture Collection. Genomic sequences of these strains were obtained from the NCBI database.
Genome-wide analysis identified 19 genomic islands (GIs) in strain EBL0706. Genes related to these GIs are listed in Table S4. GIs of strain EBL0706 carry functional genes, such as ABC transporter protein family, heme biosynthesis protein HemY, integrases, and transposases [37]. Microorganisms 2021, 9, x FOR PEER REVIEW 4 of 10 Genome-wide analysis identified 19 genomic islands (GIs) in strain EBL0706. Genes related to these GIs are listed in Table S4. GIs of strain EBL0706 carry functional genes, coding sequences (CDSs), colored on clusters of orthologous groups (COG) functional categories, on the forward strand; tRNA and rRNA on the forward and reverse strand; CDSs on the reverse strand; GC content (plotted as the deviation from the average GC content of the entire sequence; outward plots as positive values and inward plots as negative values) and GC skew (G -/G + C, the leading chain and the lagging chain can be judged by the change of GC skew, generally the leading chain GC skew > 0, the lagging chain GC skew < 0).
Phagic genes, such as antibiotic resistance genes and virulence genes, can facilitate bacteria to adapt to hostile environments [38,39]. In the genome of strain EBL0706, five prophage elements and a total of 162 protein-coding genes were identified. Among the 162 proteins, 67 were phage proteins (Table S5).

Photosynthetic Genes
Strain EBL0706 harbors photosynthesis-relating operons, such as puc, puf, and puh, composed of the light-harvesting I (LH1), the light-harvesting II (LH2), and the reaction center (RC). The gene clusters encoding these photosynthetic apparatuses are shown in Figure 2. Operon puc encodes LH2. Operon puf encodes LH1, RC-L subunit, and RC-M subunit. Operon puh encodes RC-H subunit. In addition, bch and crt are involved in bacterial chlorophyll and carotenoid synthesis, respectively. The main pigments in the photosynthetic apparatus are bacterial chlorophyll and carotenoids. These pigments are bound to membrane proteins such as LH1, LH2, and RC to form a complete photosynthetic machinery (Figure 2). In strain EBL0706, the main function of bchI is photon absorption, while crt responds to damage from photo-oxidation, dissipates excess radiation energy, and maintains the photosynthetic apparatus. The light absorption of LH1 is affected by pufX, which can change the binding state of LH1 and RC to influence the electron transfer between the two functional assemblages. During photosynthesis, LH2 absorbs and transfers photons to RC through LH1, followed by charge separation. ATPs can be produced through the series of electron transfer [8].
Microorganisms 2021, 9, x FOR PEER REVIEW 5 of 10 such as ABC transporter protein family, heme biosynthesis protein HemY, integrases, and transposases [37]. Phagic genes, such as antibiotic resistance genes and virulence genes, can facilitate bacteria to adapt to hostile environments [38,39]. In the genome of strain EBL0706, five prophage elements and a total of 162 protein-coding genes were identified. Among the 162 proteins, 67 were phage proteins (Table S5).

Photosynthetic Genes
Strain EBL0706 harbors photosynthesis-relating operons, such as puc, puf, and puh, composed of the light-harvesting I (LH1), the light-harvesting II (LH2), and the reaction center (RC). The gene clusters encoding these photosynthetic apparatuses are shown in Figure 2. Operon puc encodes LH2. Operon puf encodes LH1, RC-L subunit, and RC-M subunit. Operon puh encodes RC-H subunit. In addition, bch and crt are involved in bacterial chlorophyll and carotenoid synthesis, respectively. The main pigments in the photosynthetic apparatus are bacterial chlorophyll and carotenoids. These pigments are bound to membrane proteins such as LH1, LH2, and RC to form a complete photosynthetic machinery (Figure 2). In strain EBL0706, the main function of bchI is photon absorption, while crt responds to damage from photo-oxidation, dissipates excess radiation energy, and maintains the photosynthetic apparatus. The light absorption of LH1 is affected by pufX, which can change the binding state of LH1 and RC to influence the electron transfer between the two functional assemblages. During photosynthesis, LH2 absorbs and transfers photons to RC through LH1, followed by charge separation. ATPs can be produced through the series of electron transfer [8].  Table S6.

Phylogenetic Analysis
We found that the 16S rRNA gene sequence of strain EBL0706 shared 100% similarity with its closest type strain L. azotoformans ATCC 17025 (Table S7). Two phylogenetic trees were constructed to show the same results; the strain EBL0706 was found to be a sister to L. azotoformans. One was based on 16S rRNA genes ( Figure 3a) and another was constructed according to 20 single-copy core gene sequences of reference strains in the genera of Luteovulum and Rhodobacter (Figure 3b).  Table S6.

Phylogenetic Analysis
We found that the 16S rRNA gene sequence of strain EBL0706 shared 100% similarity with its closest type strain L. azotoformans ATCC 17025 (Table S7). Two phylogenetic trees were constructed to show the same results; the strain EBL0706 was found to be a sister to L. azotoformans. One was based on 16S rRNA genes ( Figure 3a) and another was constructed according to 20 single-copy core gene sequences of reference strains in the genera of Luteovulum and Rhodobacter (Figure 3b).

ANI, TETRA, and dDDH Analyses
ANI, TETRA, and dDDH values between strain EBL0706 and different Luteovulum strains were calculated ( Table 2). The ANI value of strain EBL0706 against L. azotoformans ATCC 17025 and L. azotoformans KA25 T were 98.13% and 99.56%, respectively. Both were higher than the defined threshold (95%). In contrast, the ANI value of strain EBL0706 against that of L. sphaeroides was down to 84.7-85%, indicating strain EBL0706 was phylogenetically close to L. azotoformans. Therefore, strain EBL0706 should be reclassified as L. azotoformans rather than L. sphaeroides. The results of TETRA and dDDH also supported the conclusion (Table 2). Microorganisms 2021, 9, x FOR PEER REVIEW 6 of 10

ANI, TETRA, and dDDH Analyses
ANI, TETRA, and dDDH values between strain EBL0706 and different Luteovulum strains were calculated ( Table 2). The ANI value of strain EBL0706 against L. azotoformans ATCC 17025 and L. azotoformans KA25 T were 98.13% and 99.56%, respectively. Both were higher than the defined threshold (95%). In contrast, the ANI value of strain EBL0706 against that of L. sphaeroides was down to 84.7-85%, indicating strain EBL0706 was phylogenetically close to L. azotoformans. Therefore, strain EBL0706 should be reclassified as L. azotoformans rather than L. sphaeroides. The results of TETRA and dDDH also supported the conclusion (Table 2).

Comparative Genome Analyses
The synteny analysis of the whole genome of strain EBL0706 and Luteovulum strains (L. azotoformans ATCC 17025, L. azotoformans KA25, and L. sphaeroides 2.4.1) were carried out. In total, 193 syntenic blocks existed among the four strains (Table S8). Strain EBL0706 has the maximum synteny with L. azotoformans ATCC 17025, which was 92.2% in terms of the shared region [40]. However, only 43.5% syntenic regions of EBL0706 were shared with L. sphaeroides 2.4.1 (Figure 4). The synteny analysis of the whole genome of strain EBL0706 and Luteovulum strains (L. azotoformans ATCC 17025, L. azotoformans KA25, and L. sphaeroides 2.4.1) were carried out. In total, 193 syntenic blocks existed among the four strains (Table S8). Strain EBL0706 has the maximum synteny with L. azotoformans ATCC 17025, which was 92.2% in terms of the shared region [40]. However, only 43.5% syntenic regions of EBL0706 were shared with L. sphaeroides 2.4.1 (Figure 4).

Conclusions
In this study, the complete genome of strain EBL0706 was analyzed. Phylogenetic investigation based on 16S rRNA genes and complete genomes revealed that strain EBL0706 was phylogenetically close to L. azotoformans. The ANI, TETRA, and dDDH analyses further verified the taxonomic relationship between strain EBL0706 and the species of L. azotoformans. The functional analysis of the whole genome sequence of strain EBL0706 indicated that this strain encoded a complete photosynthetic apparatus and shared a major part of the genomic synteny with L. azotoformans. Therefore, we argue that former Rhodobacter sphaeroides strain EBL0706 should be reclassified as a strain of Luteovulum azotoformans.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: Differential characteristics of strain EBL0706 and related species.

Conclusions
In this study, the complete genome of strain EBL0706 was analyzed. Phylogenetic investigation based on 16S rRNA genes and complete genomes revealed that strain EBL0706 was phylogenetically close to L. azotoformans. The ANI, TETRA, and dDDH analyses further verified the taxonomic relationship between strain EBL0706 and the species of L. azotoformans. The functional analysis of the whole genome sequence of strain EBL0706 indicated that this strain encoded a complete photosynthetic apparatus and shared a major part of the genomic synteny with L. azotoformans. Therefore, we argue that former Rhodobacter sphaeroides strain EBL0706 should be reclassified as a strain of Luteovulum azotoformans.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/microorganisms9081754/s1, Table S1: Differential characteristics of strain EBL0706 and related species. Table S2: The list of genomes used in the study. Table S3: General feature of the strain EBL0706 genome, Table S4: List of genes associated with these GIs in the strain EBL0706, Table  S5: The intact prophage identified in the strain EBL0706, Table S6: Photosynthetic gene cluster in the strain EBL0706, Table S7: Blastx analysis of 16S rRNA gene against NCBI collection, Table S8: Comparison of multiple alignment blocks between the four strains.

Data Availability Statement:
The data presented in this study are openly available in the NCBI; the accession number has been listed in the article.