Aquibium pacificus sp. nov., a Novel Mixotrophic Bacterium from Bathypelagic Seawater in the Western Pacific Ocean

A novel Gram-stain-negative, facultatively anaerobic, and mixotrophic bacterium, designated as strain LZ166T, was isolated from the bathypelagic seawater in the western Pacific Ocean. The cells were short rod-shaped, oxidase- and catalase-positive, and motile by means of lateral flagella. The growth of strain LZ166T was observed at 10–45 °C (optimum 34–37 °C), at pH 5–10 (optimum 6–8), and in the presence of 0–5% NaCl (optimum 1–3%). A phylogenetic analysis based on the 16S rRNA gene showed that strain LZ166T shared the highest similarity (98.58%) with Aquibium oceanicum B7T and formed a distinct branch within the Aquibium genus. The genomic characterization, including average nucleotide identity (ANI, 90.73–76.79%), average amino identity (AAI, 88.50–79.03%), and digital DNA–DNA hybridization (dDDH, 36.1–22.2%) values between LZ166T and other species within the Aquibium genus, further substantiated its novelty. The genome of strain LZ166T was 6,119,659 bp in size with a 64.7 mol% DNA G+C content. The predominant fatty acid was summed feature 8 (C18:1ω7c and/or C18:1ω6c). The major polar lipids identified were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), glycolipid (GL), and phosphatidylglycerol (PG), with ubiquinone-10 (Q-10) as the predominant respiratory quinone. The genomic annotation indicated the presence of genes for a diverse metabolic profile, including pathways for carbon fixation via the Calvin–Benson–Bassham cycle and inorganic sulfur oxidation. Based on the polyphasic taxonomic results, strain LZ166T represented a novel species of the genus Aquibium, for which the name Aquibium pacificus sp. nov. is proposed, with the type strain LZ166T (=MCCC M28807T = KACC 23148T = KCTC 82889T).


Introduction
The genus Aquibium, derived from its isolation from aquatic environments, was first proposed by Kim et al. and classified within the family Phyllobacteriaceae [1].At the time of writing, only three species within the genus Aquibium have been validly published according to the List of Prokaryotic names with Standing in Nomenclature (LPSN, http://lpsn.dsmz.de/search?word=Aquibium accessed on 20 June 2024) [2].The type species is Aquibium microcysteis, with the other two species, Aquibium carbonis and Aquibium oceanicum, having been reclassified from the genus Mesorhizobium [3].While most of the species of Mesorhizobium were isolated from the rhizospheres of leguminous plants, the Aquibium species originated from aquatic environments such as coal bed water [4], deep-sea water [5], and cultures of Microcystis aeruginos [1].The genomic analysis suggested that Aquibium species might be ancestral to those in Mesorhizobium, with some Mesorhizobium members acquiring nitrogen-fixing genes over the course of evolution [6].Species within Aquibium consistently exhibit characteristics such as being Gram-straining-negative, aerobic, and catalase-and oxidase-positive.The genomic DNA G+C content, predominant

Isolation and Culture
Strain LZ166 T was isolated from the bathypelagic seawater of the western Pacific Ocean at station CTD-11 (1000 m depth, 16 • 13 ′ 12 ′′ N, 130 • 22 ′ 12 ′′ E) during the DY60 cruise in January 2021.Seawater samples were collected and filtered through a 0.2 µm polycarbonate (PC) membrane (Merck, Rahway, NJ, USA) on board.The membrane was then immediately transferred to Axygen screw cap tubes containing 30% (v/v) glycerol, and subsequently stored at −80 • C for preservation prior to laboratory analysis.Once in the laboratory, microbes attached to the membrane were washed off and serially diluted with sterile artificial seawater and spread onto Marine Agar (MA, BD Difco, Sparks, MD, USA) plates.Individual colonies were isolated and transferred to fresh MA plates to obtain pure cultures.Strain LZ166 T was successfully isolated and cultivated following these procedures.For long-term preservation, cultures were mixed with 30% glycerol and stored at −80 • C.
Closely related type strains, including A. microcysteis NIBR3 T and A. oceanicum B7 T , were purchased from the Korean Agricultural Culture Collection (KACC) and the Marine Culture Collection of China (MCCC), respectively.These type strains were used for comparison of phenotypic, physiological, and chemotaxonomic characteristics with the isolated strain LZ166 T in this study.
Additionally, strain LZ166 T has been deposited in multiple culture collections, including the MCCC (accession number = MCCC M28807 T ), the KACC (accession number = KACC 23148 T ), and the Korean Culture Type Collection (KCTC, accession number = KCTC 82889 T ).
Catalase activity was assessed by the formation of oxygen bubbles when the cells were exposed to a 3% (w/v) H 2 O 2 solution [1].Oxidase activity was determined by the oxidation of 1% (w/v) N,N,N,N-tetramethyl-1,4-phenylenediamine (BioMérieux, Lyon, France).The hydrolysis of skimmed milk, starch, cellulose, Tween 20, 40, 60, and 80 was examined on MA plates supplemented with the corresponding substrates.Additional biochemical properties were identified using API ZYM [13] and 20NE strips (BioMérieux, Lyon, France) following the manufacturer's protocol.Utilization of various carbon sources by strain LZ166 T was assessed with the Biolog GEN III Microplates (Biolog, Hayward, CA, USA) following the manufacturer's instructions.

Morphology and Physiology
Strain LZ166 T is Gram-stain-negative, facultatively anaerobic, and catalase-and oxidase-positive.The cells are short rod-shaped (1.3-1.7 µm long; 1.0-1.1 µm wide), non-spore-forming, and motile by a lateral flagella (Figure S1).After incubation for 3 days on an MA plate at 28 • C, circular (1-2 mm in diameter), convex, smooth, creamy-white, and non-transparent colonies were observed.Growth was observed at temperatures ranging from 10 to 45 • C (optimum 34-37 • C), in NaCl concentrations of 0-5% (w/v) (optimum 1-3%), and at a pH range of 5-10 (optimum pH 6-8).The physiological and biochemical differences among strain LZ166 T , A. microcysteis NIBR3 T , and A. oceanicum B7 T are detailed in Table 1.The strain was positive for skimmed milk hydrolysis but negative for cellulose, starch, Tween 20, 40, 60, and 80 hydrolysis.Additionally, the R2A and LB plates supported the heterotrophic growth of LZ166 T but the NA plate did not.Anaerobic growth was observed in the MB medium.While most of the API ZYM results were consistent between LZ166 T and the reference species, differences were noted in the activity of alkaline phosphatase and the utilization of D-mannitol, potassium gluconate, and malic acid.Moreover, the Biolog test suggested that LZ166 T was capable of utilizing a range of organic carbon sources, including dextrin, D-cellobiose, gentiobiose, D-turanose, α-D-glucose, and D-fucose, for heterotrophic growth (Table S1).
Table 1.Comparison of the phenotypic characteristics of strain LZ166 T with reference strains of the genus Aquibium.

16S rRNA Gene Phylogeny
The 16S rRNA gene sequence analysis indicated that LZ166 T exhibited the highest sequence similarity of 98.58% with A. oceanicum B7 T , followed by A. carbonis B2.7 T (97.94%) and A. mycrocysteis NIBR3 T (96.71%), which were lower than the cut-off value of 98.65% [33].A detailed ML tree (Figure S3) and a concise ML tree (Figure 1) based on the 16S rRNA gene sequences were constructed and both of them clearly revealed that strain LZ166 T formed a separate lineage within the genus Aquibium.This topology was further confirmed by the ME (Figure S4) and NJ methods (Figure S5).The collective findings from the 16S rRNA gene phylogeny suggest that strain LZ166 T could represent a potential novel species within Aquibium.
sequence similarity of 98.58% with A. oceanicum B7 , followed by A. carbonis B2.7 (97.94%) and A. mycrocysteis NIBR3 T (96.71%), which were lower than the cut-off value of 98.65% [33].A detailed ML tree (Figure S3) and a concise ML tree (Figure 1) based on the 16S rRNA gene sequences were constructed and both of them clearly revealed that strain LZ166 T formed a separate lineage within the genus Aquibium.This topology was further confirmed by the ME (Figure S4) and NJ methods (Figure S5).The collective findings from the 16S rRNA gene phylogeny suggest that strain LZ166 T could represent a potential novel species within Aquibium.

Genomic Features
A total of 10,595,752 raw reads were produced with 258X sequencing depth.The assembled genome size of strain LZ166 T is 6,119,659 bp with a chromosomal DNA G+C content of 64.7 mol%, which is similar to the other species within the genus Aquibium, which ranges from 65.1-67.9mol% [1].Moreover, fifty tRNA genes for twenty amino acids as well as one gene each for 5S rRNA, 16S rRNA, and 23S rRNA were also identified in the genome of LZ166 T .A total of 5513 protein-coding sequences (CDSs) were predicted, with the majority (5220/5513, 94.7%) assigned to a putative function based on the COG categories, while the rest were annotated as hypothetical proteins.Detailed information on the gene classification according to the RAST, COG, and CAZy databases is provided in Fig- ure S6.
The ANI and AAI values between LZ166 T and the other closely related species in the genus Aquibium are in the ranges of 76.79-90.73%and 79.03-88.50%,respectively (Table S3).These values are below the 95-96% cut-off thresholds previously proposed for species delineation [34][35][36].The dDDH estimated values between LZ166 T and the other three species, A. microcysteis NIBR3 T , A. oceanicum B7 T , and A. carbonis B2.3 T , were 22.8%, 36.1%, and 22.2%, respectively (Table S3), all of which are below the dDDH standard cut-off value of 70% [36,37].Furthermore, a whole-genome phylogenomic tree (Figure 2) was also constructed, which corroborated the phylogenetic relationships derived from the 16S rRNA genesʹ phylogeny and genetic relatedness, as depicted in Figure 1.Altogether, these results suggest that strain LZ166 T represents a novel species within the genus Aquibium.

Genomic Features
A total of 10,595,752 raw reads were produced with 258X sequencing depth.The assembled genome size of strain LZ166 T is 6,119,659 bp with a chromosomal DNA G+C content of 64.7 mol%, which is similar to the other species within the genus Aquibium, which ranges from 65.1-67.9mol% [1].Moreover, fifty tRNA genes for twenty amino acids as well as one gene each for 5S rRNA, 16S rRNA, and 23S rRNA were also identified in the genome of LZ166 T .A total of 5513 protein-coding sequences (CDSs) were predicted, with the majority (5220/5513, 94.7%) assigned to a putative function based on the COG categories, while the rest were annotated as hypothetical proteins.Detailed information on the gene classification according to the RAST, COG, and CAZy databases is provided in Figure S6.
The ANI and AAI values between LZ166 T and the other closely related species in the genus Aquibium are in the ranges of 76.79-90.73%and 79.03-88.50%,respectively (Table S3).These values are below the 95-96% cut-off thresholds previously proposed for species delineation [34][35][36].The dDDH estimated values between LZ166 T and the other three species, A. microcysteis NIBR3 T , A. oceanicum B7 T , and A. carbonis B2.3 T , were 22.8%, 36.1%, and 22.2%, respectively (Table S3), all of which are below the dDDH standard cut-off value of 70% [36,37].Furthermore, a whole-genome phylogenomic tree (Figure 2) was also constructed, which corroborated the phylogenetic relationships derived from the 16S rRNA genes' phylogeny and genetic relatedness, as depicted in Figure 1.Altogether, these results suggest that strain LZ166 T represents a novel species within the genus Aquibium.
According to the RAST annotation, 1398 genes were detected in the genome of strain LZ166 T and could be assigned to 279 subsystems belonging to 25 categories.Among the 25 categories, amino acids and derivatives (241) was the most common, followed by carbohydrates (193), protein metabolism (172), and cofactors, vitamins, prosthetic groups, and pigments (107).Most of the genes in the amino acids and derivatives category are connected to branched-chain amino acids.Most of the genes in the carbohydrates category were relevant to the TCA cycle belonging to the central carbohydrate metabolism subcategory (Figure S6A).The COG analysis suggested that, except those with unknown functions (S, 22.45%), amino acid transport and metabolism (E, 12.53%) was the most abundant category, followed by carbohydrate transport and metabolism (G, 7.91%), transcription (K, 7.78%), energy production and conversion (C, 7.45%), and lipid transport and metabolism (I, 6.84%) (Figure S6B).Additionally, a total of 71 genes in the genome of strain LZ166 T matched the CAZy families.Glycosyl transferases (GTs) was the largest CAZy family with thirty-eight genes, followed by glycoside hydrolases (GHs, twenty-nine genes), carbohydrate-binding modules (CBMs, six genes), carbohydrate esterases (CEs, three genes), and polysaccharide lyases (PLs, one gene).However, auxiliary activities (AAs) were not detected (Figure S6C).According to the RAST annotation, 1398 genes were detected in the genome of strain LZ166 T and could be assigned to 279 subsystems belonging to 25 categories.Among the 25 categories, amino acids and derivatives (241) was the most common, followed by carbohydrates (193), protein metabolism (172), and cofactors, vitamins, prosthetic groups and pigments (107).Most of the genes in the amino acids and derivatives category are connected to branched-chain amino acids.Most of the genes in the carbohydrates category were relevant to the TCA cycle belonging to the central carbohydrate metabolism subcategory (Figure S6A).The COG analysis suggested that, except those with unknown functions (S, 22.45%), amino acid transport and metabolism (E, 12.53%) was the most abundant category, followed by carbohydrate transport and metabolism (G, 7.91%), transcription Phylogenomic tree of LZ166 T and its functional genes involved in carbon, sulfur, and nitrogen metabolism in comparison with closely related species.The bold font represents the novel species identified in this study.Bradyrhizobium japonicum USDA 6 T (GFC_000284375.1)was used as the out group.Bar, 0.05 substitutions per nucleotide position.Pink, green, and yellow blocks, presence of corresponding carbon, sulfur, and nitrogen functional genes, respectively.White blocks, absence or partial presence of corresponding functional genes.rcbL, ribulose-1,5-bisphosphate carboxylase/oxygenase gene large chain.prk, phosphoribulokinase gene.CA, carbonic anhydrase gene.coxMSL, carbon monoxide dehydrogenase (form I) gene.coxSLM, carbon monoxide dehydrogenase (form II) gene.sox, thiosulfate oxidation genes cluster (soxABCDXYZ).fcc, flavocytochrome c-sulfide dehydrogenase gene.sqr, sulfide:quinone oxidoreductases gene.soe, sulfite dehydrogenase (quinone) gene.cys, assimilatory sulfate reduction genes cluster (cysNDCHJIK).sat, sulfate adenylyltransferase gene.aprAB, adenylylsulfate reductase gene.dsr, dissimilatory sulfite reductase gene.nifH, nitrogenase gene.nirK, copper-containing nitrite reductase (denitrification) gene.nosZ, nitrous oxide reductase gene.norB nitric oxide reductase gene.napA, periplasmic dissimilatory nitrate reductase gene.narG, membrane-bound dissimilatory nitrate reductase gene.nirBD, dissimilatory nitrite reductase (NADH) gene.nrfAH, dissimilatory nitrite reductase (cytochrome c-552).nasD, assimilatory nitrite reductase gene.nasA, assimilatory nitrate reductase.glnA, glutamine synthase gene.gltB, glutamate synthase gene.ure, urease gene.gdh, glutamate dehydrogenase gene.

Carbon Metabolism
The draft genome sequence of strain LZ166 T was utilized to infer its metabolic profiles, which was subsequently compared with those of three other Aquibium species (Figure 2).LZ166 T is capable of obtaining carbon from both inorganic and organic sources.A distinctive feature of the LZ166 T genome is its potential for carbon dioxide fixation via the Calvin-Benson-Bassham (CBB) cycle.The genome contains a complete set of genes nec-essary for the CBB cycle, including those encoding key enzymes such as ribulose-1,5bisphosphate carboxylase (Rubisco) and phosphoribulokinase (PRK) [38].The Rubisco enzyme catalyzes the carboxylation of ribulose-1,5-bisphosphate, resulting in the formation of 3-phosphoglycerate [39].To date, four forms of Rubisco have been identified, with form I being the most prevalent [40].The genome of LZ166 T encodes form I RubisCO (RcbL and RcbS), which shows the highest similarity (88.8% and 84.7%, respectively) to the enzymes from Mesorhizobium mediterraneum.Although the rcbL and rcbS genes were predicted in the genome of LZ166 T , no complete carbon fixation pathways have been identified in the genomes of the other Aquibium species.This indicates that the rcbL, rcbS, and prk genes may serve as genetic markers to distinguish strain LZ166 T from the other members of the genus.The presence of the CBB pathway hints that LZ166 T may be capable of autotrophic metabolism.Additionally, the genome of strain LZ166 T also encodes the genes involved in the Embden-Meyerhof pathway (EMP), hexose monophosphate pathway (HMP), Entner-Doudoroff pathway (ED), and tricarboxylic acid cycle (TCA), indicating a heterotrophic lifestyle that relies on organic matter for carbon and energy [41].Overall, the presence of both carbon fixation and organic matter breakdown genes in the genome of LZ166 T suggests a mixotrophic strategy, potentially allowing it to adapt to the dynamic and variable conditions of the marine environment [11].
The genome of strain LZ166 T , in common with those of the other Aquibium species, includes all the key genes that encode the aerobic carbon monoxide dehydrogenase (CoxSLM).While the other Aquibium species only contain the form II cox gene, LZ166 T distinctively harbors both the form I and form II cox genes (Figure 2 and Figure S7).Moreover, at least three copies of cox gene clusters could be found in the genome of LZ166 T .This dual presence of cox genes indicates that LZ166 T might have the capacity to oxidize carbon monoxide (CO).The co-existence of the CBB pathway for carbon fixation and the CO oxidation pathway in the genome of LZ166 T suggests that this strain could utilize the energy derived from CO oxidation.This capability might, in turn, facilitate its autotrophic growth [42].It seems not surprising as some CO oxidizers have been confirmed to fix CO 2 through the CBB cycle by using the energy obtained from CO oxidation [42].

Sulfur Metabolism
Sulfur metabolism is crucial for bacteria, providing not only the essential sulfur element but also a source of energy.The genome of strain LZ166 T encodes the complete set of genes for the SOX pathway (soxABCDXYZ), which is responsible for oxidizing thiosulfate to sulfate [43].In addition to the SOX system, the LZ166 T genome also includes genes encoding flavocytochrome c-sulfide dehydrogenase (fcc) and sulfide:quinone oxidoreductase (sqr) genes, indicating that this strain potentially possesses the capacity to oxidize sulfide to elemental sulfur [43].The presence of the SOX system, fcc, and sqr in the other Aquibium species suggests that sulfur oxidation is probably a common feature of this genus.However, an exception is A. oceanicum B7 T , which lacks the fcc gene despite its phylogenetic proximity to strain LZ166 T .Furthermore, the Aquibium species, including LZ166 T , encodes the sulfite dehydrogenases (soe) gene, which plays a role in sulfite oxidation [43,44].Strain LZ166 T and the other Aquibium species possessed a complete assimilatory sulfate reduction pathway (cysND, cysC, cysH, and cysJI), indicating that they can reduce sulfate into sulfide, even to L-cysteine (cysK) [45].The presence of multiple sulfur oxidation pathways in the genome of LZ166 T underscores its capacity to oxidize sulfide, sulfite, or thiosulfate, thereby conserving energy and potentially supporting chemoautotrophic growth.

Nitrogen Metabolism
The analysis of nitrogen metabolism using KEGG annotation revealed the absence of nitrogenase (nifH) genes in strain LZ166 T as well as in the other Aquibium species, a distinguishing feature between the genera Aquibium and Mesorhizobium [1].Strain LZ166 T was found to harbor an incomplete denitrification pathway, characterized by the presence of key genes of nirK and nosZ, which encode nitrite reductase [46] and nitrous oxide reductase [47], respectively.This genetic profile implies the potential for LZ166 T to utilize nitrite or nitrous oxide as electron acceptors.However, neither the nitric oxide reductase genes (norB) [48] nor the dissimilatory nitrate reductase genes (napA and narG) [46] and the nitrite reductase genes (nrfAH and nirBD) [49] could be identified in the genome.Additionally, the assimilatory nitrate reduction pathway was annotated in the genome of LZ166 T , which contained the nasDE genes, responsible for the conversion of nitrite to ammonia [50].However, the assimilatory nitrate reductase gene, nasA [51], is absent in the LZ166 T genome.The genome of strain LZ166 T encodes the glutamine synthase (glnA) and glutamate synthase (gltB) genes, enabling the assimilation of ammonium [51].Furthermore, the presence of the urease (ure) and glutamate dehydrogenase (gdh) genes provided genomic evidence for strain LZ166 T to transform nitrogen from organic to inorganic forms [46,51].
A comprehensive overview of the metabolic pathways involved in the carbon, nitrogen, and sulfur cycles for strain LZ166 T (Figure 3) and the reference strains of the genus Aquibium is presented in Figure 2. Consequently, the genomic functional analysis underscores the metabolic versatility of strain LZ166 T .
guishing feature between the genera Aquibium and Mesorhizobium [1].Strain LZ166 T wa found to harbor an incomplete denitrification pathway, characterized by the presence o key genes of nirK and nosZ, which encode nitrite reductase [46] and nitrous oxide reduc tase [47], respectively.This genetic profile implies the potential for LZ166 T to utilize nitrit or nitrous oxide as electron acceptors.However, neither the nitric oxide reductase gene (norB) [48] nor the dissimilatory nitrate reductase genes (napA and narG) [46] and the n trite reductase genes (nrfAH and nirBD) [49] could be identified in the genome.Addition ally, the assimilatory nitrate reduction pathway was annotated in the genome of LZ166 which contained the nasDE genes, responsible for the conversion of nitrite to ammoni [50].However, the assimilatory nitrate reductase gene, nasA [51], is absent in the LZ166 genome.The genome of strain LZ166 T encodes the glutamine synthase (glnA) and gluta mate synthase (gltB) genes, enabling the assimilation of ammonium [51].Furthermore, th presence of the urease (ure) and glutamate dehydrogenase (gdh) genes provided genomi evidence for strain LZ166 T to transform nitrogen from organic to inorganic forms [46,51] A comprehensive overview of the metabolic pathways involved in the carbon, nitro gen, and sulfur cycles for strain LZ166 T (Figure 3) and the reference strains of the genu Aquibium is presented in Figure 2. Consequently, the genomic functional analysis under scores the metabolic versatility of strain LZ166 T .S1: The Biolog GNIII test of strain LZ166 T .Table S2: Cellular fatty acid compositions of strain LZ166 T and its reference strains.Table S3: The average nucleotide identity(ANI), average amino identity(AAI) and digital DNA-DNA hybridization (dDDH) value (%) between strain LZ166 T and its close-related strains in Aquibium.

Figure 1 .
Figure 1.Maximum likelihood phylogenetic tree based on 13 16S rRNA gene sequences showing the positions between strain LZ166 T and other closely related phylogenetic neighbors.Bootstrap numbers (>70%) were shown with 1000 calculations.The bold font represents the novel species identified in this study.Bradyrhizobium japonicum DSMZ_30131 T (X87272) was used as the out group.Bar, 0.01 substitutions per nucleotide position.

Figure 1 .
Figure 1.Maximum likelihood phylogenetic tree based on 13 16S rRNA gene sequences showing the positions between strain LZ166 T and other closely related phylogenetic neighbors.Bootstrap numbers (>70%) were shown with 1000 calculations.The bold font represents the novel species identified in this study.Bradyrhizobium japonicum DSMZ_30131 T (X87272) was used as the out group.Bar, 0.01 substitutions per nucleotide position.

Table 2 .
Comparison of major fatty acids (>5%) between strain LZ166 T and closely related Aquibium species.