microorganisms

Article Genomic and Metabolic Insights into Denitrification, Sulfur Oxidation, and Multidrug Efflux Pump Mechanisms in the Bacterium sediminis sp. nov.

1,2, 1, 1 3 1 1 Chun-Zhi Jin †, Ye Zhuo †, Xuewen Wu , So-Ra Ko , Taihua Li , Feng-Jie Jin , Chi-Yong Ahn 3 , Hee-Mock Oh 3 , Hyung-Gwan Lee 3 and Long Jin 1,*

1 Co-Innovation Centre for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing 210-037, China; [email protected] (C.-Z.J.); [email protected] (Y.Z.); [email protected] (X.W.); [email protected] (T.L.); [email protected] (F.-J.J.) 2 Industrial Biomaterial Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Korea 3 Cell Factory Research Centre, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Korea; [email protected] (S.-R.K.); [email protected] (C.-Y.A.); [email protected] (H.-M.O.); [email protected] (H.-G.L.) * Correspondence: [email protected]; Tel./Fax: +86-25-8542-7210 These authors contributed equally to this work. †  Received: 27 December 2019; Accepted: 13 February 2020; Published: 15 February 2020 

Abstract: This contains both phototrophs and nonphototrophic members. Here, we present a high-quality complete genome of the strain CHu59-6-5T, isolated from a freshwater sediment. The circular chromosome (4.39 Mbp) of the strain CHu59-6-5T has 64.4% G+C content and contains 4240 genes, of which a total of 3918 genes (92.4%) were functionally assigned to the COG (clusters of orthologous groups) database. Functional genes for denitrification (narGHJI, nirK and qnor) T were identified on the genomes of the strain CHu59-6-5 , except for N2O reductase (nos) genes for the final step of denitrification. Genes (soxBXAZY) for encoding sulfur oxidation proteins were identified, and the FSD and soxF genes encoding the monomeric flavoproteins which have sulfide dehydrogenase activities were also detected. Lastly, genes for the assembly of two different RND (resistance-nodulation division) type efflux systems and one ABC (ATP-binding cassette) type efflux system were identified in the Rhodoferax sediminis CHu59-6-5T. Phylogenetic analysis based on 16S rRNA sequences and Average Nucleotide Identities (ANI) support the idea that the strain CHu59-6-5T has a close relationship to the genus Rhodoferax. A polyphasic study was done to establish the taxonomic status of the strain CHu59-6-5T. Based on these data, we proposed that the isolate be classified to the genus Rhodoferax as Rhodoferax sediminis sp. nov. with isolate CHu59-6-5T.

Keywords: Rhodoferax; Rhodoferax sediminis; denitrification; sulfur oxidation; RND efflux systems

1. Introduction Rhodoferax are frequently found in stagnant aquatic environments exposed to light [1], and some have been isolated from fresh water, sewage, sludge and sediments [2–7]. In the case of Rhodoferax antarcticus, strains were first isolated from microbial mats collected from saline ponds [2]. Rhodoferax growth occurs in a temperature range from 2 to 35 ◦C[1–7]. Rhodoferax fermentans, , Rhodoferax koreense, Rhodoferax lacus and Rhodoferax bucti are mesophilic species with an optimal growth temperature between 25 and 30 ◦C[1,3,5–7]. The other two species, R. antarcticus and

Microorganisms 2020, 8, 262; doi:10.3390/microorganisms8020262 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 262 2 of 17

Rhodoferax saidenbachensis, are psychrotolerant with optimal growth temperatures ranging from 15 to 20 ◦C, but they are capable of growth at temperatures near 0 ◦C[2,4]. Rhodoferax is also well known for its diverse metabolic pathways. Members of the Rhodoferax species are able to grow phototrophically, aerobically and anaerobically. Two species in this genus, R. fermentans and R. antarcticus, grow photoheterotrophically using carbon sources such as acetate, pyruvate, lactate and succinate. Genomic features that provide photosynthesis gene clusters of the R. antarcticus have already been reported [8]. In contrast, R. ferrireducens is able to grow anaerobically using organic electron donors to reduce ferric iron (Fe+3) to ferrous (Fe+2)[1–7]. As of 2019, seven genomes have been sequenced from the genus Rhodoferax. Some of them contain the genes for the RuBisCo (ribulose 1, 5-bisphosphate carboxylase/oxygenase), several heavy metal resisting genes, and putative arsenite efflux pump genes [9]. Recently, the species R. ferrireducens was applied to the area of sustainable energy microbial fuel cells (MFC), where a bacterial suspension was used as a source of electrons for the [10]. Additionally, Rhodoferax is one of most abundant groups in the lakes of northeastern Germany [11], understanding the ecology of the genus Rhodoferax and their roles in geochemical cycles, as well as their physiology, would contribute to the understanding of its functions in environments. In the present study, a non-phototrophic member in this genus Rhodoferax obtained from freshwater was investigated at the genome and metabolic level, and a polyphasic approach was applied to establish its taxonomic status.

2. Material and Methods

2.1. Sampling, Isolation and Cultivation of the Strain

T Strain CHu59-6-5 was recovered from a 67 cm-long sediment core (36◦22’30” N, 127◦33’58” E) collected at a water depth of 17 m from the Daechung Reservoir using a modified gravity corer (Wildco, FL, USA) (Figure1). The Daechung Reservoir is located in the central region of South Korea, which is a large branch-type lake, and the reservoir has a 72 m-high dam with a gross storage capacity of 1490 Mm3, where cyanobacterial blooms occur every year because of eutrophication and global warming [12–15]. Approximately one gram of sediment sample was applied to the serial dilution 6 7 method in a 0.85% saline solution. A total of 100 µl aliquot of each serial dilution (10− or 10− ) was spread on onto modified 1/10 R2A medium (Difco, NJ, USA), which is specifically used for isolating aquatic bacteria [16] and incubated at 25 ◦C under aerobic heterotrophic conditions. A colorless colony T CHu59-6-5 was isolated after 6 days and routinely subcultured on an R2A agar at 30 ◦C for 48 h. For long term preservation of the culture, glycerol stocks (20% v/v) were prepared and stored at 70 C. − ◦ Strains were cultured on R2A agar for most physiological experiments.

2.2. Morphological, Physiological and Chemotaxonomic Characteristics The cell morphology was examined using transmission electron microscopy (Philips CM-20) after negative staining with 1 % (w/v) phosphotungstic acid, and the motility was checked in a phase-contrast microscope (Nikon Optiphot, 1000 magnification) after 2 days of incubation in R2A at × 30 ◦C. Gram staining was performed by using a Gram stain kit (Becton Dickinson) according to the manufacturer’s instructions. The activity of oxidase was tested using the oxidase reagent (bioMérieux), and catalase activity was determined by observing the production of O2 bubbles after dropping 3% (w/v)H2O2 on a fresh culture grown for 48 h on R2A medium [17]. Cell growth was investigated in different bacteriological media: R2A agar, tryptic soy agar (TSA; Difco, NJ, USA), Luria-Bertani (LB; Difco, NJ, USA) and nutrient agar (NA; Difco, NJ, USA). The growth temperature range was checked at 4, 8, 15, 20, 30, 37 and 42 ◦C. The pH range for growth was determined by measuring the OD values of R2A broth cultures after 3 days. Then, pH was adjusted to 5–10 at intervals of 1 pH unit with appropriate biological buffers [18,19]. Tolerance of NaCl was checked on R2A agar with different NaCl concentrations (0%–5%, w/v). Duplicated antibiotic-susceptibility was conducted using filter-paper disks containing the following: amikacin (30 µg mL 1), ampicillin/sulbactam · − Microorganisms 2019, 7, x FOR PEER REVIEW 2 of 17 species with an optimal growth temperature between 25 and 30 °C [1,3,5–7]. The other two species, R. antarcticus and Rhodoferax saidenbachensis, are psychrotolerant with optimal growth temperatures ranging from 15 to 20 °C, but they are capable of growth at temperatures near 0 °C [2,4]. Rhodoferax is also well known for its diverse metabolic pathways. Members of the Rhodoferax species are able to grow phototrophically, aerobically and anaerobically. Two species in this genus, R. fermentans and R. antarcticus, grow photoheterotrophically using carbon sources such as acetate, pyruvate, lactate and succinate. Genomic features that provide photosynthesis gene clusters of the R. antarcticus have already been reported [8]. In contrast, R. ferrireducens is able to grow anaerobically using organic electron donors to reduce ferric iron (Fe+3) to ferrous (Fe+2) [1–7]. As of 2019, seven genomes have been sequenced from the genus Rhodoferax. Some of them contain the genes for the RuBisCo (ribulose 1, 5-bisphosphate carboxylase/oxygenase), several heavy metal resisting genes, and putative arsenite efflux pump genes [9]. Recently, the species R. ferrireducens was applied to the area of sustainable energy microbial fuel cells (MFC), where a bacterial suspension was used as a source of electrons for the bacteria [10]. Additionally, Rhodoferax is one of most abundant groups in the lakes of northeastern Germany [11], understanding the ecology of the genus Rhodoferax and their roles in geochemical cycles, as well as their physiology, would contribute to the understanding of its functions in environments. In the present study, a non-phototrophic member in this genus Rhodoferax obtained from freshwater was investigated at the genome and metabolic level, and a polyphasic approach was applied to establish its taxonomic status.

2. Material and Methods

2.1. Sampling, Isolation and Cultivation of the Strain

Strain CHu59-6-5T was recovered from a 67 cm-long sediment core (36°22’30” N, 127°33’58” E) collectedMicroorganisms at a2020 water, 8, 262 depth of 17 m from the Daechung Reservoir using a modified gravity corer3 of 17 (Wildco, FL, USA) (Figure 1). The Daechung Reservoir is located in the central region of South Korea, which is a large branch-type lake, and the reservoir has a 72 m-high dam with a gross storage capacity 1 1 1 1 of(20 1490µg mLMm− 3,, 1:1),where chloramphenicol cyanobacterial (30 bloomsµg mL occur− ), erythromycin every year because (30 µg mL of− eutrophication), gentamicin (30 andµg globalmL− ), · 1 · 1 · 1 · 1 warmingkanamycin [12 (30–15].µg Approximatelyml− ), lincomycin one (15 gramµg mL of− s),ediment nalidixic sample acid (30 wasµg mLapplied− ), rifampicin to the serial (30 µ dilutiong mL− ), · 1 · 1 · 1 · methodspectinomycin in a 0.85% (25 salineµg mL solution.− ), streptomycin A total of 100 (25 µlµg aliquotmL− ), of teicoplanin each serial (30dilutionµg mL (10− −),6 or tetracycline 10−7) was 1 · 1 · · spread(30 µg onmL onto− , and modified vancomycin 1/10 R2A (30 µmediumg mL− ). (Difco Susceptibility, NJ, USA results), which were is specifically recorded as used positive for isolating at zones · · aquaticwith diameters bacteria greater[16] and than incubated 10 mm afterat 25 incubation °C under aerobic at 30 ◦C heterotrophic for 2 days. Carbon conditions. source A utilization, colorless colonyenzyme CHu59 activities-6-5T andwas additionalisolated after physiological 6 days and androutinely biochemical subcultured characterization on an R2A agar were at performed 30 °C for 48using h. For API long 20NE, term API preservation ID 32GN andof the API culture, ZYM kitsglycerol (bioM stocksérieux, (20% l’Etoile, v/v) were France) prepared and the and Biolog stored GN2 at −MicroPlate70 °C. Strains following were cultured the manufacturer’s on R2A agar instructions.for most physiological experiments.

Figure 1. Sampling site and location of the Daechung Reservoir. Map shows the location of the

sampling site on the shore in the vicinity of the Daechung Reservoir dam in the central region of South Korea. A 67 cm-long sediment core was collected at a 17 m water depth. IS, isolation section.

For the comparative whole-cell fatty acid profile, strains CHu59-6-5T, R. saidenbachensis DSM 22694T, R. lacus KACC 18983T, R. bucti KCTC 62564T and R. koreense KCTC 52288T were cultured on R2A agar for 3 days at 30 ◦C. Cell harvesting standardization was done following the method described by Jin et al. [20], and the extracted fatty acids were analyzed by gas chromatography (GC) (Hewlett Packard 6890, Kyoto, Japan) and identified in the TSBA 6 (Trypticase Soy Broth Agar) database using the Sherlock software 6.1. Respiratory isoprenoid quinones were extracted and analyzed by high performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) with the YMC-Pack ODS-A column, following the method described by Komagata and Suzuki [21]. Polar lipids were extracted according to the method of Tindall [22]. The biomass used for lipid extraction was obtained from cultures growing on R2A agar plates at 30 ◦C for 3 days. Several spraying reagents were applied for visualizing the spots on the two-dimensional thin layer chromatography (TLC) on silica gel 60 F254 plates (Merck): molybdatophosphoric acid for total spots, ninhydrin for aminolipids, molybdenum blue for phospholipids and alphanaphthol solution for glycolipids.

2.3. Genomic and Phylogenetic Analyses Whole genomic DNA was extracted using a FastDNATM SPIN kit according to the manufacturer’s instructions. The 16S rRNA gene of strain Chu59-6-5T was amplified by PCR (polymerase chain reaction) with the following universal primer sets: 27F (5’-AGA GTT TGA TCM TGG CTC AG-3’; Escherichia coli position 8-27) and 1492R (5’-TAC GGY TAC CTT GTT ACG ACT T-3’; E. coli position 1492-1510) [23], and identified in the server of EzBioCloud [24]. For the phylogenetic analysis, 16S rRNA gene sequences of strain CHu59-6-5T and closely related species were aligned with clustal x [25] and edited with bioedit [26]. Based on the aligned sequences, the phylogenetic tree was reconstructed using Microorganisms 2020, 8, 262 4 of 17 the following algorithms in the mega7 software [27]: neighbor-joining [28], maximum-parsimony [29] and maximum-likelihood methods [30]. In each case, bootstrap values were calculated based on 1000 resamplings of the sequences [31]. The whole genome sequencing was carried out using the SMRT (Single Molecule, Real-Time) platform at Novogene Biotechnology (Beijing, China) together with Illumina next-generation sequencing technology. The low-quality read filtration and assemblage of the draft genome were conducted using SMRT version 5.0.1. The genome annotation of strain CHu59-6-5T was annotated in the RAST pipeline, and a sequence-based comparison was made using the SEED Viewer [32,33]. The predicted protein coding sequences (CDSs) were submitted to the COG (Clusters of Orthologous Groups) database (http://www.ncbi.nlm.nih.gov/COG/) to generate the functional category and summary statistics [34,35]. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values were calculated using the OrthoANI tool in EZBioCloud web server [36].

3. Results and Discussion

3.1. Physiological Tests

T Strain CHu59-6-5 formed visible colonies at 48 h on R2A agar when incubated at 30 ◦C. Cell growth was found to occur at temperatures ranging from 4 to 37 ◦C, and quite good growth was observed at 4 ◦C after 10 days; however, no growth was observed at 0 or 42 ◦C. Growth was found to occur at pH 6–9; however, no growth was observed at pH 5 or 10. The cells were found to be Gram-stain-negative, catalase and oxidase-positive, motile by gliding, and short rod-shaped (Supplementary Figure S1). The cells were found to assimilate d-alanine, 2,3-butanediol, citrate, d-galactose (weakly), glycogen, β-hydroxybutyric acid, α-ketoglutaric acid, d,l-lactic acid, l-proline (weakly), succinamic acid, succinic acid monomethyl ester and turanose (weakly) but not the rest (API 20NE, API ID 32GN test strips and Biolog GN2). The cells were found to be positive for the following enzyme activities (API ZYM test strip): acid phosphatase (weakly), esterase (C4), esterase lipase (C8) and leucine arylamidase but not the rest (Table1). The cells were found to be susceptible to amikacin (30 µg mL 1), ampicillin/sulbactam (1:1; · − µg mL 1), chloramphenicol (30 µg mL 1), erythromycin (30 µg mL 1), gentamicin (30 µg mL 1), · − · − · − · − kanamycin (30 µg mL 1), rifampicin (30 µg mL 1), spectinomycin (25 µg mL 1), streptomycin · − · − · − (25 µg mL 1), teicoplanin (30 µg mL 1), tetracycline (30 µg mL 1) and but resistant to lincomycin · − · − · − (15 µg mL 1), nalidixic acid (30 µg mL 1) and vancomycin (30 µg mL 1). · − · − · − Table 1. Features that differentiate strain CHu59-6-5T from the most closely related species in the genus Rhodoferax.

R. saidenbachensis R. lacus KACC R. bucti KCTC Characteristic CHu59-6-5T R. koreense KCTC 52288T DSM 22694T 18983T 62564T § Isolation source Sediment Sediment* Freshwater† Freshwater‡ Sludge § Morphology Short rods Short rods* Rods† Curved rods‡ Rods Colony colour Colourless Colourless Colourless Peach brown Colourless Motility + + + - + § Growth temperature 4–37 4–30* 4–30† 15–35‡ 4–30 Oxidase/catalase +/+ +/ +/+ /+ /+ − − − Urease + + + − − Aesculin hydrolysis + + − − − Enzyme activity: alkaline phosphatase + + + + − esterase (C4) + + + + − esterase lipase (C8) + + + + − α-glucosidase + − − − − α-galactosidase + − − − − β-galactosidase + + − − − leucine arylamidase + + + + − naphthol-AS-BI-phosphohydrolase + + + − − Carbon utilization: l-alanine + + − − − l-arabinose + − − − − citrate + − − − − l-fucose + − − − − gluconate + − − − − d-glucose + + + + − histidine + − − − − 3-hydroxy-benzoate + − − − − Microorganisms 2020, 8, 262 5 of 17

Table 1. Cont.

R. saidenbachensis R. lacus KACC R. bucti KCTC Characteristic CHu59-6-5T R. koreense KCTC 52288T DSM 22694T 18983T 62564T 4-hydroxy-benzoate + − − − − 3-hydroxy-butyrate + + − − − d,l-lactate + + + − − 2-ketogluconate + + − − − 5-ketogluconate + + − − − maltose + − − − − d-mannitol + + + − − d-mannose + - − − − d-melibiose + - − − − l-proline + − − − − d-ribose + + − − − d-sorbitol + − − − − d-sucrose + − − − − § DNA G+C content (mol%) 64.4 60.3–61* 62.3† 61.2‡ 60.3 All data are from this study unless indicated. +, positive; , negative. Note: * Data from Kaden et al. [4]; Data from Park et al. [6]; Data from Zhou et al. [7]; § Data from− Farh et al. [5]. † ‡

3.2. Chemotaxonomy

The major fatty acids (>10%) were sorted into three groups C16:1 ω7c and/or C16:1 ω6c (29.6%), T C16:0 (21.6%) and C17:0 cyclo (15.0%) (Table2). The profile of major fatty acids in the strain CHu59-6-5 was consistent with the components in species from the genus Rhodoferax, although some qualitative and quantitative differences were found. The strain CHu59-6-5T was observed to have quite large amounts of C10:0 3-OH (6.4 %), C17:0 (6.9 %) and C15:1 ω6c (6.4 %), which were not found, or barely found, in the reference strains (Table2). The major predominant respiratory ubiquinone was Q-8. The polar lipids consisted of phosphatidylethanolamine (PE), three unidentified phospholipids (PL1-3), two unidentified aminophospholipids (APL1-2) and four unidentified lipids (L1-4) (Supplementary Figure S2). The polar lipid profile of strain CHu59-6-5T was similar to those of R. saidenbachensis DSM 22694T, R. lacus KACC 18983T, R. bucti KCTC 62564T and R. koreense KCTC 52288T in that phosphatidylethanolamine and the unidentified polar lipid were the major polar lipids, but could be differentiated from those of the reference strains in the presence or absence of several other polar lipids.

Table 2. Cellular fatty acid compositions (%) of strain CHu59-6-5T and the type strains of related species of the genus Rhodoferax.

R. saidenbachensis R. lacus KACC R. bucti KCTC R. koreense Fatty Acids CHu59-6-5T DSM 22694T 18983T 62564T KCTC 52288T Saturated C 0.9 11:0 − − − − C12:0 3.0 1.3 1.1 0.5 12.0 C 1.3 13:0 − − − − C14:0 2.9 0.2 0.7 0.3 1.7 C 0.5 1.3 1.1 15:0 − − C16:0 21.6 33.6 28.2 29.3 24.7 C 6.9 0.4 1.0 4.6 17:0 − C 1.2 1.3 2.0 0.3 18:0 − Unsaturated − C ω5c 1.3 0.5 0.9 3.0 14:1 − C ω6c 7.5 15:1 − − − − C ω6c 0.2 0.5 15:1 − − − C ω5c 1.8 16:1 − − − − Hydroxy − C 3-OH 1.5 0.8 0.9 1.8 8:0 − C 3-OH 0.9 9:0 − − − − C 3-OH 6.4 10.7 10:0 − − − C 2-OH 1.2 16:1 − − − − C 3-OH 1.2 17:0 − − − − Cyclo − C cyclo 15.0 9.9 17:0 − − − Summed features ‡ − 3 29.6 53.6 59.3 60.2 26.5 8 0.8 6.1 4.2 2.8 2.3 Data are from the present study. , not detected. Note: Summed Feature 3 contains C ω7c and/or C ω6c; − ‡ 16:1 16:1 summed feature 8 contains C18:1 ω7c and/or C18:1 ω6c. Microorganisms 2019, 7, x FOR PEER REVIEW 6 of 17

Data are from the present study. −, not detected. Note: ‡ Summed Feature 3 contains C16:1 ω7c and/or Microorganisms 2020, 8, 262 6 of 17 C16:1 ω6c; summed feature 8 contains C18:1 ω7c and/or C18:1 ω6c.

3.3.3.3. Genomic Analysis: The Taxonomic StatusStatus TheThe 16S16S rRNArRNA genegene sequencesequence (1478(1478 nt)nt) ofof strainstrain CHu59-6-5CHu59-6-5TT was compared against thethe 16S rRNA genegene sequencessequences ofof representative representative species species within within the the genus genusRhodoferax Rhodoferaxand and related related genera. genera. We We used used the EzTaxon-ethe EzTaxon server-e serv (www.ezbiocloud.neter (www.ezbiocloud.net)) to search to search for their for their closest closest members. members. The resultsThe results show show that strainthat strain CHu59-6-5 CHu59T-6shares-5T shares a 98.8% a 98.8% pairwise pairwise similarity similarity with withRhodoferax Rhodoferax saidenbachensis saidenbachensisED16 ED16TT,, 98.4%98.4% withwith RhodoferaxRhodoferax lacuslacus IMCC26218IMCC26218TT,, 98.2%98.2% withwith RhodoferaxRhodoferax buctibucti GSA243-2GSA243-2TT and lessless thanthan 98.0%98.0% withwith otherother speciesspecies inin thethe genusgenus RhodoferaxRhodoferax.. TheThe strainstrain CHu59-6-5CHu59-6-5TT also shares highhigh similaritiessimilarities withwith otherother speciesspecies thanthan justjust membersmembers ofof RhodoferaxRhodoferax:: 98.1%98.1% withwith CurvibacterCurvibacter delicatesdelicates NBRCNBRC 1491914919TT CurvibacterCurvibacter fontanusfontanus AQ9AQ9TT,, 97.8%97.8% withwith CurvibacterCurvibacter fontanusfontanus AQ9AQ9TT,, 97.5%97.5% withwith VariovoraxVariovorax boronicumulansboronicumulansBAM-48 BAM-48TT andand 97.3% 97.3% with withVariovorax paradoxus paradoxusNBRC NBRC 15149 15149T. However,T. However, strain strain CHu59-6-5 CHu59T-6clustered-5T clustered clearly clearly with thewith species the species of Rhodoferax of Rhodoferaxfrom the from topology the topology of the phylogenetic of the phylogenetic tree (Figure tree2 ).(Figure To determine 2). To determine genomic relatednessgenomic relatedness with other withRhodoferax other Rhodoferaxspecies and species its genomic and its characteristics, genomic characteristics, whole genome whole sequence genome of strainsequence CHu59-6-5 of strainT CHu59was obtained-6-5T was by obtained the PacBio by platformthe PacBio together platform with together the Illumina with the MiSeq Illumina platform. MiSeq Theplatform. draft genomeThe draft sequence genome ofsequence strain CHu59-6-5 of strain CHu59T was- deposited6-5T was deposited at DDBJ/EMBL at DDBJ/EMBL/GenBank/GenBank with the accessionwith the accession number CP036282. number CP036282. The genomic The DNAgenomic G+ CDNA content G+C of content the strain of the was strain 64.4 mol%,was 64.4 which mol%, is withinwhich theis within range reportedthe range for reported the genus for Rhodoferaxthe genus (59.52–66.2Rhodoferax mol%).(59.52–66.2 The mol%). observed The ANI observed and dDDH ANI valuesand dDDH between values strain between CHu59-6-5 strainT andCHu5 all9 species-6-5T and of allRhodoferax species wereof Rhodoferax 74.3%–76.9 were % 74.3% and 21.3%–23.3–76.9 % and % (Table21.3%3–),23.3 respectively, % (Table 3), which respectively, were much which lower were than much the species lower separationthan the species threshold separation of 95%–96% threshold and inof fact95% fall–96% in and the intergenericin fact fall in range the intergeneric [37–39]. range [37–39].

85 Rhodoferax antarcticus ANT.BRT (AF084947) 94 Rhodoferax fermentans FR2T (D16211) 71 Rhodoferax ferrireducens T118T (CP000267) Rhodoferax lacus IMCC26218T (KX505858) Rhodoferax saidenbachensis ED16T (FJ755906) Rhodoferax sediminis CHu59-6-5T (MF770245) 82 Rhodoferax bucti GSA243-2T (MF352137) Rhodoferax koreense DCY110T (KU519435) Curvibacter delicatus NBRC 14919T (BCWP01000019) 56 54 Curvibacter fontanus AQ9T (AB120963) Curvibacter lanceolatus ATCC 14669T (AB021390) 100 Curvibacter gracilis 7-1T (AB109889)

100 Limnohabitans parvus II-B4T (FM165536) Limnohabitans planktonicus II-D5T (LFYT01000006) 97 Limnohabitans australis MWH-BRAZ-DAM2DT (FM178226) Caenimonas koreensis EMB320T (DQ349098) 57 Caenimonas terrae SGM1-15T (GU181268) Ramlibacter solisilvae 5-10T (CP010951)

57 Variovorax humicola UC38T (KT301941) 60 Variovorax soli DSM 18216T (DQ432053)

97 Gsoil 3165T (AB245358) Variovorax boronicumulans BAM-48T (AB300597) 63 T 57 ATCC 17713 (AJ420329) T 54 Variovorax gossypii JM-310 (KR349468) 50 Variovorax guangxiensis GXGD002T (JF495126) Hydrogenophaga taeniospiralis ATCC 49743T (AF078768)

0.005

FigureFigure 2.2. PhylogeneticPhylogenetic treetree constructedconstructed usingusing thethe neighbor-joiningneighbor-joining method in megaMEGA 77 whichwhich depictsdepicts T thethe phylogeneticphylogenetic relationshiprelationship ofof strainstrain CHu59-6-5CHu59-6-5T amongamong relatedrelated taxa.taxa. TheThe treetree waswas reconstructedreconstructed basedbased onon 13991399 nucleotides.nucleotides. Numbers at branching points refer to bootstrapbootstrap values (1000 resamplings,resamplings, valuesvalues aboveabove 50%50% shown).shown). Bar, 0.50.5 substitutionsubstitution perper 100100 ntnt positions.positions. FilledFilled circlescircles indicateindicate thatthat thethe correspondingcorresponding nodes nodes were were also also calculated calculated in trees in generated trees generated with the with algorithms the algorithms of maximum-likelihood of maximum- andlikelihood maximum-parsimony. and maximum-parsimony.

Microorganisms 2020, 8, 262 7 of 17

Table 3. General features, and relationship of the genomes between strain CHu59-6-5T and strains of its closely related species of the genus Rhodoferax.

R. saidenbachensis R. lacus R. bucti R. koreense R. ferrireducens R. fermentans Attribute CHu59-6-5T DSM 22694T KACC 18983T KCTC 62564T KCTC 52288T T118T KACC 15304T Genome size (bp) 4,387,497 4,264,855 4,900,405 3,673,501 5,895,641 4,969,784 4,467,741 G + C content (%) 64.4 60.9 62.3 61.2 66.2 59.9 56.9 N50 4,387,497 4,264,855 234,657 937,707 5,800,473 4,712,337 4,447,702 L50 1 1 6 2 1 1 1 Number of contigs 1 1 72 8 3 2 2 Number of coding sequences 4191 4030 4420 3436 5346 4339 4176 Number of tRNA 43 46 44 44 47 45 53 Number of rRNA 3 6 7 4 9 6 12 ANI (%) 100 75.4 74.9 74.4 76.9 75.2 74.3 dDDH (%) 100 22.1 21.3 23.3 20.9 22.3 22.4 GenBank Accession number CP035503 CP019239 QFZK00000000 VAHD00000000 CP019236 CP000267 MTJN00000000 Data are from the present study unless indicated. MicroorganismsMicroorganisms 2020, 8, 2020x FOR, 8 ,PEER 262 REVIEW 8 of 17 8 of 17

3.4. Genome Properties 3.4. Genome Properties The genome of strain CHu59-6-5T consists of a single circular chromosome of 4,387,497 base pairs The genome of strain CHu59-6-5T consists of a single circular chromosome of 4,387,497 base pairs with awith G+C acontent G+C content of 64.4 of% 64.4%(Figure (Figure 3). Of 3the). Of 4240 the genes 4240 genesidentified identified in the intotal the genome, total genome, 4191 were 4191 were proteinprotein-encoding-encoding genes, genes,49 were 49 ribosomal were ribosomal or transfer or transfer RNAs, RNAs, and 133 and were 133 putative were putative pseudogenes pseudogenes (Table(Table 3 and3 Supplementary and Supplementary Table TableS1). A S1). total A of total 3918 of genes 3918 genes (92.4%) (92.4%) were werefunctionally functionally assigned assigned to to the COGthe database. COG database. The distribution The distribution of the ofgenes the genesinto the into COG the functional COG functional categories categories is presented is presented in in Rhodoferax Table Table 3 and3 and Supplementary Supplementary Figure Figure S3. S3. Similar Similar to to the the closely closely related related Rhodoferax strains,strains, abundant abundant genes genes related to to amino amino acid acid transport transport and and metabolism metabolism (COG (COG category category E), E), transcription transcription (COG (COG category category K), K), andand energy energy production production and and conversion conversion (COG (COG category category C) were C) wereobserved. observed. Unexpectedly, Unexpectedly, 27.7% 27.7%of of genes were assigned to the unknown function COG category in the genome of strain CHu59-6-5T genes were assigned to the unknown function COG category in the genome of strain CHu59-6-5T (Supplementary(Supplementary Table TableS2). S2).

Figure 3. Graphic representation of circular genome plot of CHu59-6-5T. From the inner to the outer Figure 3. Graphic representation of circular genome plot of CHu59-6-5T. From the inner to the outer circle: The circles represent from outside to inside with the first and last circle showing the genomic circle: The circles represent from outside to inside with the first and last circle showing the genomic position. The second and fifth circle shows the predicted protein coding sequences according to the position. The second and fifth circle shows the predicted protein coding sequences according to the COG (Clusters of Orthologous Group) database. The third and fourth circle shows protein-coding COG (Clusters of Orthologous Group) database. The third and fourth circle shows protein-coding regions. The sixth circle represents variation in G + C content. The seventh circle shows a GC skew ([G regions. The sixth circle represents variation in G + C content. The seventh circle shows a GC skew C]/[G + C]) plot of the genome. ([G − C]/[G− + C]) plot of the genome.

3.5. Carbon Metabolism Two anoxygenic phototrophic species in the genus Rhodoferax, R. fermentans and R. antarcticus have been reported. Both members are confirmed to have gene clusters including genes encoding

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3.5. Carbon Metabolism Two anoxygenic phototrophic species in the genus Rhodoferax, R. fermentans and R. antarcticus haveMicroorganisms been reported.2020, 8, x FOR PEER Both REVIEW members are confirmed to have gene clusters including9 of 17 genes encoding light-harvesting complex and RuBisCo (ribulose 1, 5-bisphosphate carboxylase/oxygenase) geneslight-harve[1,2,8sting,40,41 complex]. Genes and of RuBisCo complete (ribulose sets of 1, glycolysis 5-bisphosphate and the carboxylase/oxygenase) citric acid cycle were genes identified in[1,2,8,40,41 the genome]. Genes of strain of complete CHu59-6-5 sets T of. glycolysis Furthermore, and the genes citric for acid the cycle Entner–Doudoro were identifiedff inpathway the and genome of strain CHu59-6-5T. Furthermore, genes for the Entner–Doudoroff pathway and pentose pentose phosphate pathway were also detected (Figure4). No genes for encoding RuBisCO (ribulose phosphate pathway were also detected (Figure 4). No genes for encoding RuBisCO (ribulose 1,5- 1,5-bisphosphate carboxylase/oxygenase) or light-harvesting complex were found in the genome bisphosphate carboxylase/oxygenase) or light-harvesting complex were found in the genome of of strain CHu59-6-5T, which means that strain CHu59-6-5T is not able to fix CO [41,42]. Strain strain CHu59-6-5T, which means that strain CHu59-6-5T is not able to fix CO2 [41,42]. Strain2 CHu59- T CHu59-6-56-5T containscontains genes encoding genes encoding phosphoenolpyruvate phosphoenolpyruvate (PEP) carboxylase (PEP) carboxylase(EC 4.1.1.31), (ECbut lacks 4.1.1.31), genes but lacks genesencoding encoding pyruvate pyruvate–phosphate–phosphate dikinase. dikinase. This means This thatmeans some that other some enzyme other enzyme together together with PEP with PEP carboxylasecarboxylase assistsassists in adding adding CO CO2 to2 toPEP PEP [43]. [43 ].

Fructose Ribose Glycerol-3-phosphate Cu2+ Mg2+ Phosphate Dipeptide

ATP ADP ATP ADP ATP ADP ATP ADP ATP ADP ATP ATP ADP ATP ADP Branched- chain amino acid Glycolysis Denitrification ADP ADP Enter-Doudoroff Pentose Phosphate - Ferric siderophore Pathway Pathway PEP NO2

ATP D-Gluconate D-Glucose Nar ATP - - NO3 NO2 NH3 Glutamine Gluconate-6P Glucose-6P Pyruvate Nir Nitrate CO2 NO GS/GOGAT ADP qNor 2-Dehydro-3-deoxy-gluconate-6P Fructose-6P

C4-dicarboxylate Acetyl-CoA N2O Glutamate Fructose-1,6 P2 Citrate ATP Pyruvate Glyceraldehyde-3P Alanine Oxaloacetate Urea Sulfur Aspartate Citric Acid oxidation Malate Cycle ADP 24 pilus gens HS- L-Arginine Isocitrate pilABCDMNOPSeTXY1 FSD/SoxH L-Proline Glyoxylate ATP 0 2- Fumarate S SO4 Tyrosine Methionine Succinate 2-Oxoglutarate 2- S2O3 NAD+ Succinyl-CoA ADP NADH NADH O2 K+

+ H2O NAD ATP ADP ATP ADP ATP ADP ATP ADP Succinate Fumarate

e- e-

Q pool Q pool Q pool Cyt c2 Q pool

2H+/2e- Succinate dehydrogenase NADH ubiquinone 2H+ NADH dehydrogenase (Complex II) oxidoreductase Cytochrome C oxidase Alkanesulfonate Oligopeptide Bicarbonate Tungstate (Complex I) (Complex I) (cbb3)

Figure 4. Model predictions for metabolism and some transporters of strain CHu59-6-5T. Nitrogen T metabolicFigure 4. Model and sulfur predictions oxidation for metabolism pathways and are indicated.some transporters Several of transporters strain CHu59 and-6-5 electron. Nitrogen transport metabolic and sulfur oxidation pathways are indicated. Several transporters and electron transport systems are shown. Two electron chains, complex I (NADH dehydrogenase) and complex II (succinate systems are shown. Two electron chains, complex I (NADH dehydrogenase) and complex II dehydrogenase), were observed. Complex II usually parallel electron transport to complex I. A C-family (succinate dehydrogenase), were observed. Complex II usually parallel electron transport to complex heme-copper cbb3 oxidase, which presumably serves as the terminal electron acceptor during aerobic I. A C-family heme-copper cbb3 oxidase, which presumably serves as the terminal electron acceptor respiration,during aerobic was respiration, identified. was identified. The genome The genome also encodes also encodes genes genes related related to to the the type IV IV pilus. pilus. PEP, phosphoenolpyruvate;PEP, phosphoenolpyruvate; GS/GOGAT: GS/GOGAT glutamine: glutamine synthetase /glutamate synthetase/glutamate oxoglutarate oxoglutarate aminotransferase. aminotransferase. 3.6. Denitrification 3.6. DenitrificationDenitrification can take place in both terrestrial and marine ecosystems and is one of the main branchesDenitri of thefication global can nitrogen take place cycle in both supported terrestrial by and bacteria. marine Typically, ecosystems denitrification and is one of the occurs main in anoxic environments,branches of the whereglobal nitrogen the concentration cycle supported of dissolvedby bacteria. and Typically, available denitrification oxygen is occurs depleted in anoxic and nitrate environments, where the concentration of dissolved and available oxygen is depleted and nitrate (NO3−) or nitrite (NO2−) can be used as a substitute terminal electron acceptor instead oxygen [44–46]. − − (NOKey3 ) or functional nitrite (NO genes2 ) can (benarGHJI used as, nirKa substituteand q norterminal) for denitrification electron acceptor were instead identified oxygen on[44– the46]. genomes Key functional genes (narGHJI, nirK and qnor) for denitrification were identified on the genomes of the strain CHu59-6-5T except for Nos genes. Currently, denitrification starts with a membrane bound of the strain CHu59-6-5T except for Nos genes. Currently, denitrification starts with a membrane (narGHI) in the mesophilic models of bacteria [47,48]. Genes encoding nitrate reductase (narGHJI) and bound (narGHI) in the mesophilic models of bacteria [47,48]. Genes encoding nitrate reductase nitrate(narGHJI)/nitrite and transporters nitrate/nitrite (transportersnaNiT) were (naNiT identified) were (Figureidentified5A). (Figure Nitrite 5A). produced Nitrite produced in the cytoplasmin the by NarGHJIcytoplasm will by then NarGHJI be secreted will then to the be periplasm secreted to through the periplasm a membrane through transporter a membrane (NaNiT) transporter and reduced (NaNiT) and reduced to nitric oxide (NO) by copper-containing nitrite reductase (NirK). Strain

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Microorganisms 2020, 8, x FOR PEER REVIEW 10 of 17 to nitric oxide (NO) by copper-containing nitrite reductase (NirK). Strain CHu59-6-5T possesses two quinone-dependentCHu59-6-5T possesses nitric two oxide quinone reductases-dependent (qNor). nitric As theoxide NO reductases is highly toxic,(qNor). it needsAs the toNO be is reduced highly to N O immediately by a membrane-bound nitric oxide reductase (Nor) (Figure5B). Depending on the 2toxic, it needs to be reduced to N2O immediately by a membrane-bound nitric oxide reductase (Nor) type(Figure of Nor 5B). enzyme Depending used, on electrons the type forof Nor this enzyme reaction used, can be electrons transferred for this by reaction a periplasmic can be cytochrometransferred c T (cNorby )a orperiplasmic by quinones cytochrome (qNor) [c47 (cNor,49–51) or]. by Strain quinones CHu59-6-5 (qNor) [47,49does– not51].contain Strain CHu59 N2O reductase-6-5T does not (Nos) genescontain for theN2 finalO reductase step of denitrification. (Nos) genes for In themany final denitrifying step of denitrification. microorganisms, In N many2O is finally denitrifying reduced to Nmicroorganisms,2 by a periplasmic N2O reductase is finally (NosZ), reduced in whichto N2 aby cytochrome a periplasmic c or areductase type I copper (NosZ), protein in which has to a act ascytochrome an electron donorc or a [type52]. I However, copper protein the cases has into whichact as an organisms electron donor without [52].nos However,genes still the can cases produce in N2whichgas have organisms been studied,without nos supporting genes still the can idea produce that aN di2 gasfferent have type been ofstudied, N2O reductase supporting needs the idea to be discoveredthat a different [53,54 type]. of N2O reductase needs to be discovered [53,54].

(A)

(B)

T FigureFigure 5. 5.(A ()A Denitrification) Denitrification gene gene clusters clusters locatedlocated on draft genome of of strain strain CHu59 CHu59-6-5-6-5 . TColor. Color key: key: pink, nor genes; light orange, nir genes; blue-grey, cco genes (cytochrome c oxidase subunit); green, pink, nor genes; light orange, nir genes; blue-grey, cco genes (cytochrome c oxidase subunit); green, nnr genes (NO sensing protein (NnrS); denitrification regulatory protein (NnrU); white, dnr gene (NO nnr genes (NO sensing protein (NnrS); denitrification regulatory protein (NnrU); white, dnr gene (NO responding transcriptional regulator); blue, naNiT genes (nitrate/nitrite transporter); yellow, naRas responding transcriptional regulator); blue, naNiT genes (nitrate/nitrite transporter); yellow, naRas gene (nitrite reductase [NAD(P)H] large subunit (NaRas1), Nitrite reductase [NAD(P)H] small gene (nitrite reductase [NAD(P)H] large subunit (NaRas1), Nitrite reductase [NAD(P)H] small subunit subunit (NaRas2); purple, reg genes; gold, nar genes; grey, naT gene (nitrate ABC transporter). (B) The (NaRas2); purple, reg genes; gold, nar genes; grey, naT gene (nitrate ABC transporter). (B) The substance substance transformation in the denitrification process was identified from the genome. transformation in the denitrification process was identified from the genome.

3.7.3.7. Sulfur Sulfur Oxidation Oxidation Most of the sulfur-oxidizing bacteria are autotrophic and use reduced sulfur as electron donors Most of the sulfur-oxidizing bacteria are autotrophic and use reduced sulfur as electron donors for for carbon dioxide fixation [55]; chemolithotrophic microorganisms obtain energy by oxidizing carbon dioxide fixation [55]; chemolithotrophic microorganisms obtain energy by oxidizing inorganic inorganic compounds for their structural components to survive, grow and reproduce [56,57]. Some compounds for their structural components− to0 survive, grow and reproduce [56,57]. Some inorganic inorganic forms of reduced sulfur (H0 2S/HS , S ) can be oxidized by chemolithotrophic sulfur-oxidizing forms of reduced sulfur (H2S/HS−,S ) can be oxidized by chemolithotrophic sulfur-oxidizing bacteria bacteria coupled to the reduction of oxygen (O2) or nitrate (NO3−) [56,58]. coupledThe to the functional reduction genes of oxygen for sulfur (O2) or oxidation nitrate (NO (sox3−) )[ participating56,58]. in a thiosulfate-oxidizing multienzymeThe functional system genes include for sulfur SoxXYZABDEFGH, oxidation (sox) participating which is able in ato thiosulfate-oxidizing oxidize various reduced multienzyme sulfur systemcompounds include to SoxXYZABDEFGH, sulfates [57]. The strain which CHu59 is able-6-5T topossesses oxidize g variousenes (soxBXAZY reduced) sulfurencoding compounds for sulfur to T sulfatesoxidation [57]. proteins The strain (Figure CHu59-6-5 6), soxB, possessessoxXA and genes soxZY (soxBXAZY genes are suggested) encoding to for be sulfuressential oxidation to thiosul proteinsfate (Figureoxidation6), soxB [59,60]., soxXA Theand SoxYZsoxZY complexgenes appears are suggested as hetero to- be and essential homo-dimers to thiosulfate with protein oxidation disulfide [59,60 ]. Thelinked SoxYZ subunits, complex SoxB appears contains as a dinuclear hetero- and manganese homo-dimers cluster withwhich protein is proposed disulfide to function linked as subunits,sulfate SoxBthiohydrolase contains a dinuclearand interacts manganese with the clusterSoxYZ complex which is [61 proposed,62]. The to FSD function and soxF as sulfate gene encoding thiohydrolase the andmonomeric interacts flavoproteins with the SoxYZ which complex have sulfide [61,62 dehydrogenase]. The FSD and activitiessoxF gene has also encoding been detected. the monomeric Those

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Microorganisms 2020, 8, x FOR PEER REVIEW 11 of 17 flavoproteins which have sulfide dehydrogenase activities has also been detected. Those sulfur oxidationsulfur oxidationSox systems Sox systems indicate indicate that the strainthat the CHu59-6-5 strain CHu59T appears-6-5T appears to be capable to be ofcapable thiosulfate of thiosulfate oxidation tooxidation sulfate. to sulfate.

T FigureFigure 6. 6.Sulfur Sulfur oxidationoxidation genegene clustercluster ofof strain CHu59-6-5CHu59-6-5T.. Color Color key: key: golden, golden, FSDFSD genegene (cytochrome (cytochrome ccsubunit subunit of of flavocytochrome flavocytochromec csulfide sulfide dehydrogenase);dehydrogenase); blue;blue; soxsox genes.

3.8.3.8. RNDRND andand ABCABC EEffluxfflux SystemsSystems BacterialBacterial e effluxfflux pumps pumps are are categorized categorized into into five five families: families: ABC, ABC, the the ATP ATP-binding-binding cassette cassette superfamily;superfamily; MFS,MFS, thethe majormajor facilitatorfacilitator superfamily; MDR, MDR, the the small small multidrug multidrug resistance resistance family; family; RND,RND, the the resistance-nodulation resistance-nodulation divisiondivision superfamily;superfamily; and MATE, the multidrug multidrug and and toxic toxic compound compound extrusionextrusion family family [63 [63,64,64].]. RND RND e ffleffluxux systems, systems, a categorya category of of bacterial bacterial effl effluxux pumps, pumps, play play a prominent a prominent role inrole both in intrinsicboth intrinsic and acquired and acquired multidrug multidrug resistance resistance identified identified in Gram-negative in Gram-negative bacteria bacteria and catalyze and thecataly activeze e thefflux active of a wide efflux variety of a of wide antibacterial variety of substrates antibacterial including substrates antibiotics including and chemotherapeutic antibiotics and agentschemotherapeutic [65,66]. RND agents efflux [65,66 systems]. RND are efflux normally systems composed are normally of three composed proteins: of an three inner proteins: membrane an transporter,inner membrane an outer transporter, membrane an protein outer membrane that functions protein as a pore, that functions and a periplasmic as a pore, protein and a thatperiplasmic interacts withprotein both that the interacts inner and with outer both membrane the inner and proteins outer membrane to provide proteins a conduit to forprovide the extrusion a conduit offor small the moleculesextrusion [of67 small]. molecules [67]. TwoTwo di differentfferent RND RND typetype eeffluxfflux systemssystems andand one ABC type efflux efflux system have have been been detected detected in in T T thethe strain strain CHu59-6-5 CHu59-6-5T.. The genome of the strainstrain CHu59-6-5CHu59-6-5T contaicontainsns three three-gene-gene operon operon cmeABCcmeABC (Figure(Figure7 A).7A). CmeABC isis also also a atripartite tripartite efflux efflux system system composed composed of three of three proteins proteins CmeACmeA, CmeB, CmeB and andCmeCCmeC: the: the inner inner membrane membrane protein protein CmeBCmeB, the, the periplasmic periplasmic fusion fusion protein protein CmeACmeA, and, and the the outer outer membranemembrane proteinprotein CmeCCmeC [[68].68]. A TetR familyfamily regulator gene AcrRAcrR waswas present present just just upstream upstream of of the the operon.operon. TheThe AcrR regulatorregulator is is known known to to act act to to repress repress the the AcrABAcrAB operonoperon in E. in coliE. coli[69].[ 69Except]. Except for the for thecomplete complete CmeABCCmeABC genegene cluster, cluster, three three more more CmeCCmeC genesgenes encoding encoding outer outermembrane membrane lipoproteins lipoproteins were found throughout the genome. The genome also has been found to have complete genes for the were found throughout the genome. The genome also has been found to have complete genes for AcrAB-TolC efflux pump, which has shown to be resistant to chloramphenicol, fluoroquinolone, the AcrAB-TolC efflux pump, which has shown to be resistant to chloramphenicol, fluoroquinolone, tetracycline, novobiocin, rifampin, fusidic acid, nalidixic acid and β-lactam antibiotics [70]. Except for tetracycline, novobiocin, rifampin, fusidic acid, nalidixic acid and β-lactam antibiotics [70]. Except for the complete AcrAB-TolC gene cluster, two more AcrB genes encoding the inner membrane proteins the complete AcrAB-TolC gene cluster, two more AcrB genes encoding the inner membrane proteins were detected through the genome. Interestingly, the genome of the strain CHu59-6-5T possesses an were detected through the genome. Interestingly, the genome of the strain CHu59-6-5T possesses ABC-type efflux pump MacAB-TolC (Figure 7B), which has resistance to a variety of macrolides, an ABC-type efflux pump MacAB-TolC (Figure7B), which has resistance to a variety of macrolides, aminoglycosides and polymyxins [71]. TolC is an outer membrane protein which interacts with aminoglycosides and polymyxins [71]. TolC is an outer membrane protein which interacts with several several inner membrane efflux pumps to expel antibiotics and export virulence factors from bacteria inner membrane efflux pumps to expel antibiotics and export virulence factors from bacteria [72], and [72], and works in combination with other RND, ABC, and MFS efflux pumps [73,74]. The genome works in combination with other RND, ABC, and MFS efflux pumps [73,74]. The genome of strain of strain CHu59-6-5T possesses only one TolC gene, presumably working cooperatively as outer CHu59-6-5T possesses only one TolC gene, presumably working cooperatively as outer membrane membrane proteins of AcrAB-TolC and MacAB-TolC. Those efflux systems may function proteins of AcrAB-TolC and MacAB-TolC. Those efflux systems may function complementarily with complementarily with them and can work sequentially when one fails. them and can work sequentially when one fails.

3.9. Motility All the members of genus Rhodoferax are motile by polar flagella except for Rhodoferax bucti [1–7]. This genetic evidence of motility by flagella and type IV pili in Rhodoferax antarcticus was found in previous observations by Baker et al. [8], in which the genes coding proteins synthesizing polar flagella, fliEFGHIJKLMNPQR, were well studied. The genome of strain CHu59-6-5T contains genes for motility via IV pili but not flagella. Type IV pili, important virulence factors in many bacterial pathogens, generate motile forces called twitching or gliding motility in Bacteria and Archaea. The biogenesis of Type IV pili relies on macromolecular (A)

Microorganisms 2020, 8, x FOR PEER REVIEW 11 of 17

sulfur oxidation Sox systems indicate that the strain CHu59-6-5T appears to be capable of thiosulfate oxidation to sulfate.

Figure 6. Sulfur oxidation gene cluster of strain CHu59-6-5T. Color key: golden, FSD gene (cytochrome c subunit of flavocytochrome c sulfide dehydrogenase); blue; sox genes.

3.8. RND and ABC Efflux Systems Bacterial efflux pumps are categorized into five families: ABC, the ATP-binding cassette superfamily; MFS, the major facilitator superfamily; MDR, the small multidrug resistance family; RND, the resistance-nodulation division superfamily; and MATE, the multidrug and toxic compound extrusion family [63,64]. RND efflux systems, a category of bacterial efflux pumps, play a prominent role in both intrinsic and acquired multidrug resistance identified in Gram-negative bacteria and catalyze the active efflux of a wide variety of antibacterial substrates including antibiotics and chemotherapeutic agents [65,66]. RND efflux systems are normally composed of three proteins: an inner membrane transporter, an outer membrane protein that functions as a pore, and a periplasmic protein that interacts with both the inner and outer membrane proteins to provide a conduit for the extrusion of small molecules [67]. Two different RND type efflux systems and one ABC type efflux system have been detected in the strain CHu59-6-5T. The genome of the strain CHu59-6-5T contains three-gene operon cmeABC (Figure 7A). CmeABC is also a tripartite efflux system composed of three proteins CmeA, CmeB and CmeC: the inner membrane protein CmeB, the periplasmic fusion protein CmeA, and the outer membrane protein CmeC [68]. A TetR family regulator gene AcrR was present just upstream of the operon. The AcrR regulator is known to act to repress the AcrAB operon in E. coli [69]. Except for the complete CmeABC gene cluster, three more CmeC genes encoding outer membrane lipoproteins were found throughout the genome. The genome also has been found to have complete genes for the AcrAB-TolC efflux pump, which has shown to be resistant to chloramphenicol, fluoroquinolone, tetracycline, novobiocin, rifampin, fusidic acid, nalidixic acid and β-lactam antibiotics [70]. Except for

Microorganismsthe complete2020 AcrAB, 8, 262-TolC gene cluster, two more AcrB genes encoding the inner membrane proteins12 of 17 were detected through the genome. Interestingly, the genome of the strain CHu59-6-5T possesses an ABC-type efflux pump MacAB-TolC (Figure 7B), which has resistance to a variety of macrolides, assembliesaminoglycosides composed and of polymyxins 15 conserved [71]. proteins TolC (PilC, is an PilD, outer PilE, membrane PilF, PilG, PilH, protein PilI, which PilJ, PilK, interacts PilM, PilN, with PilO,several PilP, inner PilQ membrane and PilW) efflux in model pumps Gram-negative to expel antibiotics bacteria [and75,76 export]. The virulence strain CHu59-6-5 factors fromT contains bacteria all 15[72 necessary], and works genes in forcombination the proteins, with and other thetype RND, IV ABC, pilus and genes MFS in the efflux strain pumps CHu59-6-5 [73,74T].are The located genome in aof more strain typical CHu59 pattern-6-5T throughoutpossesses only the chromosome one TolC gene, (Supplementary presumably Figure working S4). cooperatively Unfortunately, as no outer pili ofmembrane any type have proteins been of found AcrAB in the-TolC cultured and MacABCHu59-6-5-TolC.T cells Those (Supplementary efflux systems Figure may S1), function but the glidingcomplementarily motility was with observed them and by can a phase-contrast work sequentially microscope. when one fails.

Microorganisms 2020, 8, x FOR PEER REVIEW 12 of 17 (A)

(B)

FigureFigure 7. 7. ThThee gene gene clusters clusters of of RND RND and and ABC ABC type type efflux efflux pump pump in in the the genome genome of of strain strain CHu59 CHu59-6-5-6-5TT (A(A).). Three Three types types of of tripartite tripartite efflux efflux pump. pump. From From left left to to right, right, CmeA CmeABCBC pump, pump, AcrAB AcrAB-TolC-TolC pump pump and and MacABMacAB-TolC-TolC pump pump ( (BB).).

3.9.4. ConclusionsMotility T AllTo the summarize members our of investigations,genus Rhodoferax the are strain motile CHu59-6-5 by polar flagellafrom a freshwaterexcept for Rhodoferax sediment belonging bucti [1– 7].to This the genusgeneticRhodoferax evidence wasof motility studied by through flagella genome and type analysis, IV pili in together Rhodoferax with antarcticus a polyphasic was approach.found in T previousThe phylogenetic observations analysis by Baker indicated et al. that [8], the in strain which CHu59-6-5 the genes was coding affi liated proteins closely synthesizing with the species polar flagella,of Rhodoferax fliEFGHIJKLMNPQR,, based on the phylogenetic, were well studied. genomic and physiological differences; thus, we propose the T strainThe CHu59-6-5 genome of asstrain a novel CHu59 species,-6-5T Rhodoferaxcontains genes sediminis for motilitysp. nov., via in IV the pili family but notComamonadaceae flagella. Type. IV pili, importantUnlike two virulence phototrophs factors in many the genus bacterialRhodoferax pathogens,, phototrophic generate motile systems forces were called not twitching detected. T orRhodoferax gliding motility sediminis inCHu59-6-5 Bacteria andpossesses Archaea. genes The encodingbiogenesis for of denitrification Type IV pili andrelies nitrate on macromolecular ammonification assembliesrelated genes composed (GS/GOGAT of 15 conserved pathway: glutamineproteins (PilC, synthetase PilD, PilE,/glutamate PilF, PilG, oxoglutarate PilH, PilI, aminotransferase) PilJ, PilK, PilM, T PilN,(data PilO, not shown). PilP, PilQRhodoferax and PilW) sediminis in modelCHu59-6-5 Gram-negativealso contains bacteria genes [75,76 for]. sulfurThe strain oxidation CHu59 and-6-5 allT containsthe essential all 15 genes necessary encoding genes for for proteins the proteins, of CmeABC, and the AcrAB-TolC type IV pilus and genes MacAB-TolC in the strain multidrug CHu59 e-6ffl-5uxT T aresystems. located Besides in a more this, the typical strain pattern CHu59-6-5 throughoutcontains the all necessarychromosome genes (Supplementary for type IV pilus Figure proteins. S4). T Unfortunately,The genome no pili sequence of any andtype comparativehave been found genome in the analyses cultured of CHu59Rhodoferax-6-5T cells sediminis (SupplementaryCHu59-6-5 Figureprovide S1) a, geneticbut theblueprint gliding motility and physiological was observed characteristics by a phase- whichcontrast help mic usroscope to understand. the different metabolism and evolutionary life of the genus Rhodoferax. 4. ConclusionsTaxonomic Description of proposed Rhodoferax sediminis sp. nov. Etymology: Rhodoferax sediminis (se.di0mi.nis. L. gen. n. sediminis of sediment). To summarize our investigations, the strain CHu59-6-5T from a freshwater sediment belonging Cells are Gram-stain-negative, motile by gliding and rods (0.8–1.3 µm long; 0.5–0.7 µm wide). to the genus Rhodoferax was studied through genome analysis, together with a polyphasic approach. Colonies grown on R2A agar are colorless. Cells are catalase and oxidase positive. Growth occurs on R2A The phylogenetic analysis indicated that the strain CHu59-6-5T was affiliated closely with the species at temperatures from 4 to 37 ◦C (optimum temperature 25–30 ◦C) but not 42 ◦C. The pH range for growth of Rhodoferax, based on the phylogenetic, genomic and physiological differences; thus, we propose the strain CHu59-6-5T as a novel species, Rhodoferax sediminis sp. nov., in the family . Unlike two phototrophs in the genus Rhodoferax, phototrophic systems were not detected. Rhodoferax sediminis CHu59-6-5T possesses genes encoding for denitrification and nitrate ammonification related genes (GS/GOGAT pathway: glutamine synthetase/glutamate oxoglutarate aminotransferase) (data not shown). Rhodoferax sediminis CHu59-6-5T also contains genes for sulfur oxidation and all the essential genes encoding for proteins of CmeABC, AcrAB-TolC and MacAB- TolC multidrug efflux systems. Besides this, the strain CHu59-6-5T contains all necessary genes for type IV pilus proteins. The genome sequence and comparative genome analyses of Rhodoferax sediminis CHu59-6-5T provide a genetic blueprint and physiological characteristics which help us to understand the different metabolism and evolutionary life of the genus Rhodoferax. Taxonomic Description of proposed Rhodoferax sediminis sp. nov. Etymology: Rhodoferax sediminis (se.di′mi.nis. L. gen. n. sediminis of sediment). Cells are Gram-stain-negative, motile by gliding and rods (0.8–1.3 µm long; 0.5–0.7 µm wide). Colonies grown on R2A agar are colorless. Cells are catalase and oxidase positive. Growth occurs on R2A at temperatures from 4 to 37 °C (optimum temperature 25–30 °C ) but not 42 °C . The pH range for growth is from pH 6.0 to 9.0 (optimum pH 6-7) but not at pH 5.0 and 10.0. Growth occurs in the

Microorganisms 2020, 8, 262 13 of 17 is from pH 6.0 to 9.0 (optimum pH 6-7) but not at pH 5.0 and 10.0. Growth occurs in the presence of 1% NaCl, but not with 2% or above. Cells are found to be positive for nitrate reduction but negative for indole production, glucose acidification, urease, aesculin hydrolysis, gelatin hydrolysis and β-galactosidase. Cells utilize d-alanine, 2,3-butanediol, citrate, d-galactose (weakly), d-glucose, β-hydroxybutyric acid, α-ketoglutaric acid, d,l-lactic acid, l-proline (weakly), succinamic acid, succinic acid monomethyl ester and turanose (weakly), but not acetic acid, N-acetyl-d-galactosamine, N-acetyl-glucosamine, cis-aconitic acid, adipate, adonitol, l-alaninamide, l-alanine, l-alanylglycine, l-asparagine, l-aspartic acid, γ-aminobutyric acid, 2-aminoethanol, l-arabinose, l-arabitol, bromosuccinic acid, caprate, dl-carnitine, d-cellobiose,α-cyclodextrin, dextrin, i-erythritol, formic acid, d-fructose, l-fructose, l-fucose, d-galactonic acid lactone, d-galacturonic acid, gentiobiose, d-gluconate, α-d-glucose-1-phosphate, d-glucose-6-phosphate, d-glucosaminic acid, α-d-glucose, glucuronamide, d-glucuronic acid, l-glutamic acid, glycerol, dl-α-glycerol phosphate, glycogen, glycyl l-aspartic acid, glycyl l-glutamic acid, l-histidine, hydroxy-l-proline, 3-hydroxybenzoate, 4-hydroxybenzoate, α-hydroxybutyric acid, γ-hydroxybutyric acid, p-hydroxyphenylacetic acid, inosine, inositol, myo-inositol, itaconate, α-ketobutyric acid, 2-ketogluconate, 5-ketogluconate, α-ketovaleric acid, α-d-lactose, lactulose, l-leucine, malate, malonate, maltose, d-mannitol, d-mannose, d-melibiose, methyl β-d-glucoside, l-ornithine, phenylacetate, l-phenylalanine, phenylethylamine, propionic acid, d-psicose, putrescine, l-pyroglutamic acid, pyruvic acid methyl ester, quinic acid, d-raffinose, l-rhamnose, d-ribose, d-saccharic acid, salicin, sebacic acid, d-serine, l-serine, d-sorbitol, suberate, succinic acid, sucrose, l-threonine, thymidine, d-trehalose, Tween 40, Tween 80, uridine, urocanic acid, valerate or xylitol. Cells are found to be positive for the following enzyme activities: acid phosphatase (weakly), esterase (C4), esterase lipase (C8) and leucine arylamidase. Cells are found to be negative for the following enzyme activities: N-acetyl-β-glucosaminidase, alkaline phosphatase, α-chymotrypsin, cystine arylamidase, α-fucosidase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-glucuronidase, lipase (C14), α-mannosidase, naphthol-AS-BI-phosphohydrolase, trypsin and valine arylamidase. The only respiratory quinone is ubiquinone Q-8. The major polar lipids are phosphatidylethanolamine, two unidentified phospholipids and one unidentified aminophospholipid. The major fatty acids are grouped into three categories C16:1 ω7c and/or C16:1 ω6c,C16:0 and C17:0 cyclo. The G+C content of the DNA is 64.4 mol%. The strain type CHu59-6-5T was isolated from a sediment sample of Dachung reservoir, Daejeon, Republic of Korea, and deposited at two different culture collections within the assigned numbers of KCTC 62554T and JCM 32677T, respectively. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence the whole genome sequence of strain CHu59-6-5T are MF770245 and CP035503, respectively.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/2076-2607/8/2/262/s1. Author Contributions: C.-Z.J., Y.Z. and L.J. conceived and designed the experiments; C.-Z.J., Y.Z., X.W., S.-R.K., and L.J. performed the experiments; T.L., F.-J.J., C.-Y.A. and H.-M.O. analysed the data; C.-Z.J., H.-G.L. and L.J. wrote the manuscript; and all co-authors edited numerous drafts. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Natural Science Foundation of Jiangsu Province (BK20191387), National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP; Ministry of Science, ICT & Future Planning) (NRF-2018R1C1B3009513) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Conflicts of Interest: The authors declare no conflict of interest.

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