Zwartia vadi sp. nov., a Novel Species of the GKS98 Cluster Isolated from a Stream, and the Reclassification of ‘Achromobacter Panacis’ as Zwartia panacis comb. nov

A Gram-stain-negative, aerobic, motile by gliding, and rod-shaped bacterium, designated IMCC34845T, was isolated from a freshwater stream in the Republic of Korea. The results of 16S rRNA gene-based phylogenetic analyses showed that strain IMCC34845T was affiliated with the genus Zwartia and was most closely related to ‘Achromobacter panacis’ DCY105T (100%) and Zwartia hollandica LF4-65T (98.9%). The whole-genome sequence of strain IMCC34845T was 3.2 Mbp in size with a 51.5% DNA G+C content. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain IMCC34845T and ‘Achromobacter panacis’ KCTC 42751T were 77.7% and 20.7%, respectively, revealing that they are independent species. Moreover, the strains IMCC34845T and KCTC 42751T exhibited ≤ 72.5% ANI and ≤18.5% dDDH values with closely related species Zwartia hollandica LF4-65T, further supporting that the two strains represent each novel species of the genus. The major respiratory quinone of strain IMCC34845T was ubiquinone-8 (Q-8), and the predominant cellular fatty acids were C16:0 (41.3%) and C17:0 cyclo (34.5%). The major polar lipids of the strain were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified phospholipids, and unidentified aminolipids. Based on the phylogenetic, genomic, physiological, and chemotaxonomic characteristics, strain IMCC34845T was considered to represent a novel species within the genus Zwartia, for which the name Zwartia vadi sp. nov. is proposed. The type of strain is IMCC34845T (=KCTC 92920T = NBRC 114902T). Furthermore, based on the taxonomic data, ‘Achromobacter panacis’ is proposed to be reclassified as Zwartia panacis comb. nov.


Introduction
The GKS98 [1] or betIII [2] cluster, belonging to the bacterial family Alcaligenaceae [3], has been recognized as one of the typical bacterial assemblages in freshwater ecosystems [2].The GKS98 cluster was originally designated based on 16S rRNA sequences retrieved from Lake Gossenköllesee [4].Subsequently, its presence has been identified in diverse freshwater systems through numerous cultivation-independent investigations [1,2,[5][6][7][8].The first proposal of the taxa affiliated with the GKS98 cluster was suggested by Hahn et al. in 2022 [9], revealing that the cluster represents three genera: Sheuella, Jezberella, and Zwartia.The classification was based on the proposal of two novel species, Jezberella montanilacus and Zwartia hollandica, and the reclassification of Orrella amnicola [10] as Sheuella amnicola.
Although some other strains of the cluster have previously been isolated [11,12], there are currently only the three validly described species within the cluster.
As of July 2023, the genus Zwartia is represented by a sole species, Zwartia hollandica, exhibiting characteristics of an aerobic, unpigmented, rod-shaped, and chemoorganotrophic bacterium [9].This species is characterized by the presence of C 16:1 ω7c and C 18:1 ω7c as major fatty acids, ubiquinone-7 (Q-7) and ubiquinone-8 (Q-8) as major respiratory quinones, and phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and aminophospholipid as major polar lipids [9].In the present study, we report the isolation of a bacterium, the designated strain IMCC34845 T , from a freshwater stream.Based on the phylogenetic, genomic, physiological, and chemotaxonomic characteristics of the strain, we propose the inclusion of strain IMCC34845 T in the genus Zwartia as a novel species.The taxonomic evidence collected from this study also confirms that 'Achromobacter panacis' KCTC 42751 T [13] is clearly distinct from the genus Achromobacter and should, therefore, be transferred to the genus Zwartia.

Isolation and Maintenance
Strain IMCC34845 T was isolated from a freshwater sample collected at Wangsuk stream, a tributary of the Han River, Republic of Korea.The sampling station is situated downstream of a wetland area, which serves as both the site for effluent discharge from sewage treatment and retention of water within the wetland for 24 h prior to its introduction into the stream.This attribute emphasizes the ecological value of the site, thereby reinforcing its essential role as a central urban habitat characterized by an elevated standard of water quality.A water sample (37 • 36 41.4 N, 127 • 8 52.4 E) was collected in June 2018, and an aliquot of the sample was inoculated onto Reasoner's 2A (R2A) agar (BD Diagnostics, Le Pont de Claix, France).After incubation at 20 • C for 2 weeks, strain IMCC34845 T was isolated as a single colony, routinely cultured on R2A at 30 • C, and stored at −80 • C in 10% (v/v) glycerol suspension.For phenotypic and genomic comparisons, two closely related species, 'Achromobacter panacis' KCTC 42751 T and Z. hollandica LF4-65 T , were obtained from the Korean Collection for Type Cultures (KCTC) and the original isolator, respectively.These species were used as experimental controls for further characterization.

16S rRNA Gene Sequence Analysis
The 16S rRNA gene of strain IMCC34845 T was amplified via PCR using the universal bacterial primers 27F and 1492R [14] and sequenced using the Sanger sequencing method (Biofact Co., Daejeon, Korea).The resultant 16S rRNA gene sequence of strain IMCC34845 T (1448 bp) was identified using BLASTn searches in the GenBank database and the 16Sbased ID in EzBioCloud [15].For phylogenetic analysis, 16S rRNA gene sequences of strain IMCC34845 T and closely related type strains were aligned using the Silva Incremental Aligner and imported into the ARB software v.6.06 [16].Based on the aligned sequences of strain IMCC34845 T and the phylogenetically related species, phylogenetic trees were generated using the following tree-inferring methods implemented in the MEGA X program [17]: the maximum likelihood method [18] with the Tamura-Nei model, the neighbor-joining method [19] with Jukes-Cantor correction, and the minimum evolution method [20] with Jukes-Cantor correction.The topology of the phylogenetic trees was evaluated by using bootstrap analyses based on 1000 random replicates [21].
To determine genomic relatedness and perform comparative genomic analysis, the genome sequences of Z. hollandica LF4-65 T (GenBank accession, JAHXRI000000000), S. amnicola NBD-18 T (JAAGRN000000000), and J. montanilacus MWH-P2sevCIIIb T (PVTV00000000) were downloaded from GenBank.The genome relatedness value was determined using the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values through the OrthoANI algorithm [24] and genome-to-genome distance calculator (GGDC 2.1) [25], respectively.To infer a genome-based phylogenetic tree, 81 universal bacterial core genes were extracted using the up-to-date bacterial core gene set and pipeline (UBCG2) [26] and employed to reconstruct the phylogenetic tree using RAxML [27].Pan-and coregenome analyses were performed using the GET_HOMOLOGUES software v.3.3.2 [28] with the orthoMCL algorithm, utilizing protein sequences predicted by Prokka [29].Prior to analyzing the distribution of Clusters of Orthologous Groups (COGs) categories and metabolic pathways, protein-coding genes of each genome were obtained using Prokka [29].Assignment of the proteins into COG functional categories [30] was performed using RPS-blast search (e-value cutoff, 0.01) [31] against the COG profile database.Metabolic pathways were predicted using BlastKOALA [32], and the results were confirmed with KofamKOALA [33].
For the analysis of cellular fatty acid methyl esters (FAME), cells were harvested from colonies grown on the third quadrant sectors of the plates after incubation on R2A at 30 • C for 5 days.The FAME profile was analyzed using gas chromatography (Agilent 7890 GC, Santa Clara, CA, USA) with the Sherlock Microbial Identification System version 6.1 (MIDI) and a TSBA6 database [36].Respiratory isoprenoid quinone was extracted following the procedures described by Minnikin et al. [37] and examined via reverse-phase partition chromatography [38] on Merck HPTLC RP-18F254 (Sigma-Aldrich, St. Louis, MO, USA) reverse-phase thin-layer plates.For the extraction and separation of polar lipids, previously described methods [37] were employed, and two-dimensional thinlayer chromatography (TLC) was performed on silica gel 60 F254 plates (Merck, Rahway, NJ, USA).Visualization of all polar lipids on the TLC plates was achieved by spraying with 10% (w/v) molybdatophosphoric acid (Sigma-Aldrich).Specific lipids containing functional groups were identified by spraying 0.2% (w/v) ninhydrin (Sigma-Aldrich) for aminolipids, 1.3% (w/v) molybdenum blue (Sigma-Aldrich) for phospholipids, 2.4% (w/v) alpha-naphthol solution for glycolipids, and Dragendorff solution for choline.

Whole Genome Analysis and Genomic Relatedness
The draft genome sequence of strain IMCC34845 T was 3,232,556 bp long with 443× genome coverage, comprising 72 contigs with 51.5% of DNA G+C (Table 1).The genome completeness and contamination values estimated by CheckM were 100% and 0.12%, respectively.The annotated genome contained a single copy of the 16S rRNA gene, which exhibited 100% similarity to the amplified 16S rRNA gene sequence.The general genomic features of IMCC34845 T , KCTC 42751 T , and LF4-65 T , annotated using Prokka, are summarized in Table 1.The OrthoANI and dDDH values between strains IMCC34845 T and KCTC 42751 T were 77.7% and 20.7%, respectively.Analyses based solely on 16S rRNA gene sequences fall short in effectively distinguishing bacterial species exhibiting substantial sequence resemblance.Consequently, ANI values of 95-96% have gained wide recognition as the threshold equivalent to a DDH value of 70% for species demarcation [39][40][41].Remarkably, despite there being an evident 100% similarity in the 16S rRNA gene sequence between the two strains, these values remain below the recommended ANI and dDDH thresholds, unambiguously indicating that the two strains represent two distinct bacterial species.Similarly, even with a >98.7% 16S rRNA gene sequence similarity between the two strains and Z. hollandica LF4-65T, the genomic relatedness values ranged from 72.2% to 72.5% (for ANI) and from 18.3% to 18.5% (for dDDH), confirming that the two strains represent novel species apart from Z. hollandica.The low ANI values, despite the very high 16S rRNA gene sequence similarity within the genus Zwartia, highlights the significance of genomebased comparison for precise bacterial species classification.This aspect bears particular importance as microbiologists adhering to the 98.7% similarity criterion might dismiss new strains due to an excessive similarity with the described species, thereby potentially overlooking emergent species.The ANI and dDDH values among strain IMCC34845 T and the phylogenetically related species are summarized in Table 2.
In the phylogenomic tree, strains IMCC34845 T and KCTC 42751 T formed a robust clade with Z. hollandica LF4-65 T and was distinct from other genera belonging to the GKS98 cluster, such as J. montanilacus MWH-P2sevCIIIb T and S. amnicola NBD-18 T (Figure 2).These genomic DNA relatedness values and phylogenomic positions clearly indicated that strains IMCC34845 T and KCTC 42751 T represent each novel species of the genus Zwartia.

Comparative Genomic Analyses
In the core-and pan-genome analyses, strains IMCC34845 T , KCTC 42751 T , and LF4-65 T shared 2108 protein clusters, accounting for only 68.8-71.4% of the protein-coding genes predicted in each genome (Figure 3).The genomes of the three strains encoded various central carbohydrate pathways, including the Embden-Meyerhof-Parnas pathway, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the non-oxidative pentose phosphate pathway, and 5-phospho-α-D-ribose-1-diphosphate biosynthesis (PRPP biosynthesis).The three genomes also featured the SOX complex and the nitrogen regulation two-component system (NtrB/NtrC family).Additionally, the three strains carried the genes for carbonic anhydrase, which facilitates the hydration of dissolved CO 2 into carbonic acid and dissociates to produce hydrogen carbonate at neutral pH levels.This suggests that the genus Zwartia may play a crucial role in sulfur, nitrogen, and carbon cycling in their habitats.However, there were differences in some nitrogen-and sulfur-related metabolic genes among the three genomes.Specifically, these differences included the presence of genes for nitrate reductase (NarGHI), glutamate dehydrogenase, cyanate lyase, sulfite reductase, and dimethyl-sulfone monooxygenase, which were encoded in the genomes of strains IMCC34845 T and KCTC 42751 T but were absent in Z. hollandica LF4-65 T (Table 1).The identification of orthologous genes based on the COG functional categories showed that translation, ribosomal structure, and biogenesis (J); cell wall/membrane/envelope biogenesis (M); energy production and conversion (C); amino acid transport and metabolism (E); coenzyme transport and metabolism (H); lipid transport and metabolism (I); and inorganic ion transport and metabolism (P) are enriched (≥5.0%) in the genome of IMCC34845 T (Table S1).

Taxonomic Conclusions
Phylogenetic inference, genome analyses, and chemotaxonomic characteristics supported the assignment of strain IMCC34845 T to the genus Zwartia.However, due to the low level of DNA-DNA relatedness and several different phenotypic characteristics compared to its close phylogenetic neighbors, it is evident that strain IMCC34845 T represents a novel species of the genus Zwartia.Therefore, we propose the name Zwartia vadi sp.nov.for strain IMCC34845 T .Additionally, 'Achromobacter panacis' KCTC 42751 T , which is a species with an invalidly published name, exhibited significant differences from all recognized Achromobacter species based on both 16S rRNA and genome sequence comparisons.Thus, we propose its reclassification as a novel species within the genus Zwartia, and the name Zwartia panacis comb.nov. is proposed.

Description of Zwartia panacis comb. nov.
Basonym: 'Achromobacter panacis ' Singh et al., 2017.The characteristics of the species are as given by Singh and colleagues for the type strain of 'Achromobacter panacis'.The type strain is DCY105 T (=KCTC 42751 T = CCTCCAB 2015193 T ).The length of the draft whole-genome sequence of the type strain is 3.3 Mbp.The DNA G+C content of the type strain is 52.0%.The GenBank accession number of the draft whole-genome sequence of the type strain is JAUHHF000000000.

Figure 1 .
Figure 1.Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences showing the relationships among strain IMCC34845 T , related type strains of the GKS98 cluster, and other species of the family Alcaligenaceae.Bootstrap values (expressed as percentages of 1000 replications) over 70% are shown at nodes for maximum likelihood, neighbor-joining, and minimum evolution methods, respectively.Filled circles indicate that the corresponding nodes were recovered by all treeing methods.Open circles indicate that the corresponding nodes were recovered by any two out of three methods.Burkholderia cepacia ATCC 25416 T (AF097530) was used as an outgroup.Bar, 0.02 substitutions per nucleotide position.

Figure 2 .
Figure 2. Phylogenomic tree based on concatenated multiple alignment of 81 genes showing the relationship between IMCC34845 T and closely related species of the family Alcaligeneceae.The tree was generated using UBCG2 pipeline with the concatenation of 81 gene sequences.GenBank accession numbers are shown in parentheses.Percentage bootstrap values are given at branching points.Bar, 0.2 substitutions per nucleotide position.

Figure 3 .
Figure 3. Venn diagram illustrating the number of unique and shared protein-coding genes in the genomes of three Zwartia species.