Craterilacuimicrobium aquaticum gen. nov. sp. nov., a novel member of the order Neisseriales isolated from crater lake

doi:10.21203/rs.3.rs-7595832/v1

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Craterilacuimicrobium aquaticum gen. nov. sp. nov., a novel member of the order Neisseriales isolated from crater lake

https://doi.org/10.21203/rs.3.rs-7595832/v1

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Strain TC5R-5^T, a white rod-shaped bacterium, was isolated from Aershan Tianchi crater lake in northern China. It is Gram-stain-negative, catalase- and oxidase- positive. Optimal growth occurs at 20–30°C, pH of 6.0–7.0 and in the presence of 0-0.5% (w/v) NaCl. Based on 16S rRNA gene sequences analysis, strain TC5R-5^T exhibits the highest similarity (94.3%) to Chromobacterium alkanivorans IITR71^T. The genomic DNA G + C content of strain TC5R-5^T is 51.4% according to the genome sequence. The predominant cellular fatty acids are summed feature 3 (comprising C_16:1 ω7c /C_16:1 ω6c) and C_16:0. The predominant quinone is ubiquinone Q-8, with Q-7 present in moderate amounts. The polar lipid profile comprises phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), an unidentified aminophospholipid (APL), and an unidentified phospholipid (PL). Through polyphasic taxonomy and molecular phylogenetic analysis, strain TC5R-5^T is proposed to represent a novel species of a new genus of the family Chromobacteriaceae within the order Neisseriales, for which the name Craterilacuimicrobium aquaticum gen. nov. sp. nov. is proposed. The type strain is TC5R-5^T (= CGMCC 1.17058^T = KCTC 72736^T).

crater lake

Craterilacuimicrobium

Da Hinggan mountain

Neisseriales

The order Neisseriales was reorganized by Adeolu et al. in 2013, dividing it into the families Neisseriaceae and Chromobacteriaceae based on phylogenomic and molecular signatures [Adeolu and Gupta, 2013]. Chen et al. classified 37 genera within the order Neisseriales, encompassing 122 species, into two revised families Neisseriaceae and Chromobacteriaceae, along with three new families namely Aquaspirillaceae fam. nov., Chitinibacteraceae fam. nov., and Leeiaceae fam. nov. [Chen et al., 2021]. The family Chromobacteriaceae contained the type genus Chromobacterium [De Ley et al., 1978] and other genera like Aquitalea [Lau et al., 2006], Craterilacuibacter [Liu et al., 2020], Crenobacter [Dong et al., 2015], Gulbenkiania [Vaz-Moreira et al., 2007], Paludibacterium [Kwon et al., 2008], Pseudogulbenkiania [Lin et al., 2008] and Vogesella [Grimes et al., 1997]. Strains of these genera were predominantly isolated from aqueous and soil habitats, such as soil, rice field and water samples. Aquitalea magnusonii [Lau et al., 2006], Gulbenkiania indica [Jyoti et al., 2010], Gulbenkiania mobilis [Vaz-Moreira et al., 2007], Vogesella lacus [Chou et al., 2009], and Pseudogulbenkiania subflava [Lin et al., 2008] were isolated from various aquatic environments, such as humic lake, sulfur spring, cold spring and pond.

During the exploration of bacterial diversity of the Aershan Tianchi crater lake, strain TC5R-5^T was isolated from a water sample. Due to its low 16S rRNA similarity (94.3%) with the currently valid published species, a series of experiments were conducted to determine the taxonomic status of strain TC5R-5^T. Using polyphasic taxonomy, strain TC5R-5^T was classified as a novel species of a new genus within the order Neisseriales. Despite minor topological discrepancies among phylogenomic trees generated through different analytical methods, the collective evidence strongly supports its provisional assignment to the family Chromobacteriaceae as the most coherent and biologically plausible placement.

Strain Isolation

Strain TC5R-5^T was isolated from water samples collected from the surface water at a depth of 0.5 m in Aershan Tianchi crater lake, September 2015, located on Da Hinggan Ling Mountain in northern China (120.41°E, 47.32°N). The water temperature was 12°C. The samples were diluted from 10^− 1 to 10^− 5 gradient with sterile 0.85% NaCl (w/v) and cultured on R2A agar medium (Difco) at 25°C for 7 days. Single colonies were picked up and purified by streaking on R2A plate once again. The strains were preserved by lyophilization and in liquid nitrogen, respectively.

16S rRNA Gene Sequencing and Phylogenetic Analysis

After being cultivated on R2A agar at 25℃, the genomic DNA of strain TC5R-5^T was extracted using the Genomic DNA Rapid Isolation Kit for bacterial cells (BioDev-Tech. Beijing, China) and the 16S rRNA gene of strain TC5R-5^T was amplified with the universal primers 27F and 1492R [Lane and Goodfellow, 1991]. An almost complete 16S rRNA gene sequence were obtained and analysed using the GenBank databases (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and EzBioCloud (https://www.ezbiocloud.net/) [Yoon et al., 2017]. Sequences of related taxa were downloaded and aligned using Clustal W [Larkin et al., 2007]. Phylogenetic trees were constructed using neighbor-joining, maximum-likelihood and maximum-parsimony methods in MEGA7.0 [Felsenstein, 1981; Fitch, 1971; Saitou and Nei, 1987; Tamura et al., 2011]. The evolutionary distance matrices were calculated using Kimura’s two parameter model [Kimura, 1980], and bootstrap values were determined based on 1000 replications [Felsenstein, 1985].

Genome Features and Phylogenomic Analysis

The genome of strain TC5R-5^T was sequenced using the Illumina HiSeq X platform at Majorbio Co., Ltd. The qualified samples were pair-end sequenced, and raw sequencing data were acquired. Quality control used SeqPrep v0.20.0, the genome sequence was de novo assembled using SOAP v2.04. Contigs exceeding 400 bp in length were retained for gene prediction. CDS prediction was carried out using Glimmer v3.02 (http://ccb.jhu.edu/software/glimmer/index.shtml) and GeneMarkS v4.3 (http://topaz.gatech.edu/GeneMark). The rRNA and tRNA were predicted using Barrnap v0.8; (https://github.com/tseemann/barrnap/) and tRNA-scan-SE v2.0 (http://trna.ucsc.edu/software/), respectively. Average Nucleotide Identity (ANI) was determined through the OrthoANI algorithm on Ezbiocloud [Yoon et al., 2017]. The dDDH values were calculated using the TYGS with the GBDP method [Meier-Kolthoff and Göker, 2019]. Amino acid identity (AAI) was calculated using an AAI calculator (http://enve-omics.ce. gatech.edu/aai/index) [Luo et al., 2014]. The ANI, dDDH and AAI values were compared with the thresholds established in literature [Chun et al., 2018; Richter and Rosselló-Móra, 2009]. The genes were classified into subsystems and clusters of orthologous groups (COGs) using the Rapid Annotation using Subsystem Technology (RAST) server [Aziz et al., 2008]. Genomic annotation was carried out using the NR (ftp://ftp.ncbi.nlm.nih.gov/blast/db/), KEGG (http://www.genome.jp/kegg/) and eggNOG (http://eggnogdb.embl.de/#/app/home) algorithms, respectively. The software antiSMASH 6.0 was utilized to predict the genes responsible for the secondary metabolites [Kai et al., 2021].

To confirmed the phylogenetic position of strain TC5R-5^T, three genome-based phylogenetic trees were reconstructed using different methods: one used the TYGS platform [Meier-Kolthoff and Göker, 2019], another was reconstructed with IQ-TREE v2.1.2 and based on single copy genes using MFP as the model and 1000 ultrafast bootstrap repeats [Minh et al., 2020], and the third phylogenomic tree was generated based on core genes using UBGC2.0 on EzBiocloud.

Morphological and Physiological Analyses

The morphological and physiological characteristics were determined through the following experiments. Cell morphology was observed using phase-contrast microscope and transmission electron microscope (JEM1400; JEOL) after 2 days of growth on R2A agar at 25℃ [Winey et al., 2014]. Cells were stained with uranium acetate before observation. Motility was assessed by inoculating strain TC5R-5^T into semi-solid R2A agar medium (0.5% agar) and observing its growth trace in the medium. A single colony of strain TC5R-5^T was circular, smooth, and white-colored. Cells were 0.8–0.9 µm in width and 3.0–4.0 µm in length. The optimal growth conditions of strain TC5R-5^T, including temperature range, pH and NaCl concentrations were confirmed as described below. Strain TC5R-5^T was cultured in R2A broth with NaCl concentrations ranging from 0 to 5.0% (w/v, at intervals of 0.5%). The pH variations (4.0, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, 10.0) was achieved using specific buffers: 100mM acetate buffer (for pH 4.0–5.0), 100mM phosphate buffer (pH 6.0–8.0) or 100mM NaHCO₃/Na₂CO₃ buffer (pH 9.0–10.0). Growth at different temperatures (4, 15, 20, 25, 30, 35, 37, 40 and 45°C) was evaluated in R2A broth under the optimal NaCl concentration. Gram-staining, anaerobic growth and DNase activity were measured following previously described methods [Gerhardt et al., 1994]. The catalase and oxidase activities were assessed using 3% H₂O₂ solution and 1% tetra-methyl-p-phenylenediamine (bioMérieux), respectively. Strain TC5R-5^T and its related reference strains, such as Chromobacterium alkanivorans IITR-71^T, Chromobacterium violaceum ATCC 12472^T, Aquitalea magnusonii TRO-001DR8^T, Paludibacterium yongneupense 5YN8-15^T, Pseudogulbenkiania subflava DSM 22618^T and Gulbenkiania mobilis E4FC31^T, were cultured on R2A agar at 25°C for 2 days for comparison. Craterilacusibacter sinensis B2N2-7^T was cultivated in PYG medium. Enzymatic and physiological activities were determined using a set of enzymatic tests API ZYM and 20NE (BioMérieux), and acids production was tested using API 50CH (BioMérieux) strips. Carbon sources utilization was measured using the GEN III MicroPlate system (Biolog) following the manufacturer’s protocol.

Chemotaxonomic Analyses

Strain TC5R-5^T was cultured in R2A broth at 25°C with 180 rpm for 48h and harvested during the late exponential phase. Cells were collected for further analysis of fatty acids, respiratory quinones, and polar lipids. Fatty acids were extracted following the standard protocol by Miller and Kuykendall and analysed using the Sherlock Microbial Identification System (MIDI) [Miller, 1982; Sasser, 1990]. The collected cells were freeze-dried for chemotaxonomic analysis. Polar lipids were extracted as per the protocol outlined by Komagata and Suzuki (1987) [Komagata and Suzuki, 1987] and separated using two-dimensional silica gel thin layer chromatography. The first direction involved a mixture of chloroform: methanol:water (65:25:4, v/v/v), while the second direction used a mixture of chloroform : acetic acid : methanol : water (80:15:12:4, v/v/v/v). Finally, molybdatophosphoric acid, molybdenum blue, ninhydrin and anisaldehyde reagent were utilized to detect total polar lipid, phospholipids, aminolipids, and glycolipids [Minnikin et al., 1993]. The specific components of respiratory quinones were extracted, separated, purified and analysed by HPLC following the previously described approach [ Collins, 1985; Wu et al., 1989].

Phylogenetic and phylogenomic analysis

Based on 16S rRNA gene sequence comparisons, strain TC5R-5^T exhibited the highest similarity (94.3%) to Chromobacterium alkanivorans IITR-71^T [Bajaj et al., 2016]. Phylogenetic analysis using the maximum-likelihood method with the GTR + G + I nucleotide substitution model placed strain TC5R-5^T within a clade containing the genera Chromobacterium, Aquitalea, Gulbenkiania, and Pseudogulbenkiania (Fig. 1). Consistent with this, phylogenetic trees reconstructed using neighbor-joining and maximum-parsimony methods also supported the close affiliation of strain TC5R-5^T with members of the family Chromobacteriaceae (order Neisseriales). The topological structures of the neighbor-joining and maximum-parsimony trees were congruent with that of the maximum-likelihood tree (see Supplementary Material Figure S1).

To further clarify the phylogenetic position of strain TC5R-5T, phylogenomic trees were reconstructed using TYGS, IQ-TREE, and UBGC. The maximum-likelihood tree generated with IQ-TREE v2.1.2 under the MFP model and 1000 ultrafast bootstrap replicates, with selected species from Neisseriales and Betaproteobacteria as outgroups, placed strain TC5R-5^T within a cluster comprising Chromobacterium species. This branch was neighbored by the genera Aquitalea, Gulbenkiania, Paludibacterium, and Pseudogulbenkiania, all belonging to the family Chromobacteriaceae (Fig. 2a). A similar phylogenetic placement was supported by the bacterial core gene-based tree (Fig. 2b), in which strain TC5R-5^T reliably grouped with members of the Chromobacteriaceae, including species of Paludibacterium, Aquitalea, Chromobacterium, Vogesella, and Gulbenkiania, among others. These results suggest that strain TC5R-5^T may represent a novel species within this family. In contrast, the TYGS-based phylogenomic tree revealed a divergent topology (Fig. 2c). Here, strain TC5R-5^T clustered with ‘Halomonas humidisoli’, Vreelandella glaciei, and Andreprevotia sp. However, based on physiological and ecological characteristics, the strain shows no clear affiliation with Halomonas species. Notably, it shares certain similarities with Andreprevotia chitinilytica JS11-7^T, a member of the family Chitinibacteraceae.

The ANI and dDDH values between strain TC5R-5^T and its closest relative, Chromobacterium alkanivorans IITR-71^T, were 73.5% and 13.6%, respectively—both well below the established species demarcation thresholds of 95–96% for ANI and 70% for dDDH. Additionally, the AAI value between the two strains was 52.8%, which fell beneath the proposed genus boundary range of 60–80% [Luo et al., 2014].

To further evaluate its taxonomic position, ANI values were compared between strain TC5R-5^T and several reference strains: the phylogenetically closest relatives within the Chromobacteriaceae (based on 16S rRNA and IQ-TREE/UBGC phylogenomic trees), Chromobacterium alkanivorans IITR-71^T and Aquitalea palustris CCM7557^T, as well as the top hits from the TYGS-based tree, including Andreprevotia chitinilytica DSM 18519^T, Halomonas humidisoli WN018^T, and Vreelandella glaciei DD39^T (Table S1). The ANI values between strain TC5R-5^T and the Chromobacteriaceae members C. alkanivorans IITR-71^T and A. palustris CCM7557^T (73.48%, 72.49%) were notably higher than those with A. chitinilytica DSM 18519^T, H. humidisoli WN018^T, and V. glaciei DD39^T (68.49%, 64.26%, 64.73%). These comparisons were also supported by substantially greater genome coverage (26.78% and 25.03% vs. 9.92%, 5.01%, and 4.91%, respectively). Together, these results corroborate that strain TC5R-5^T is phylogenetically more closely affiliated with the Chromobacteriaceae.

In summary, integrated phylogenetic analyses based on both 16S rRNA gene and whole-genome sequences support the classification of strain TC5R-5^T as a novel species of a new genus within the order Neisseriales. Although some discrepancies were observed among phylogenomic trees constructed using different methods, current evidence indicates that its placement within the family Chromobacteriaceae is the most appropriate and rational provisional assignment.

Genome Features and Biosynthetic Gene Cluster

The complete genome of strain TC5R-5^T comprises 3.44 Mb with a total of 3408 genes, including 3274 protein-coding sequences (CDS). The genomic DNA G + C content, derived from sequencing data, was determined to be 51.4%. A comparative summary of genomic features between strain TC5R-5^T and its phylogenetic relatives is provided in Table 1. Annotation via RAST revealed that 29% of the chromosomal genes could be assigned to functional categories, distributed across 291 subsystems within 27 broader functional groups. The most abundant subsystems were ‘Amino Acids and Derivatives’, ‘Protein Metabolism’, and ‘Cofactors, Vitamins, Prosthetic Groups, Pigments’ (Figure S2). AntiSMASH analysis identified a biosynthetic gene cluster (BGC) located on scaffold 7, spanning 24,974 bp, which showed high similarity to the rhizomide BGC from Paraburkholderia rhizoxinica HKI 454 (NC_014718.1; Figure S3). According to MIBiG annotation, this cluster is predicted to encode a nonribosomal peptide synthetase (NRPS) consisting of seven modules and containing a starter condensation domain, suggesting the potential production of a lipopeptide-class compound.

Table 1
Comparison of genome features between strain TC5R-5^T and the type species of genus in the family Strains: 1. TC5R-5^T (JACCFC000000000); 2. *Chromobacterium alkanivorans* IITR-71^T (JAFHKL000000000); 3. *Chromobacterium violaceum* ATCC 12472^T (AE016825); 4. *Aquitalea magnusonii* DSM 25134^T (QJKC00000000); 5. *Paludibacterium yongneupense* 5YN8-15^T (AUGZ00000000); 6. *Pseudogulbenkiania subflava* DSM 22618^T (FXAG00000000); 7. *Gulbenkiania mobilis* E4FC31^T (SMDA00000000); 8. *Craterilacuibacter sinensis* B2N2-7^T (WSSB00000000); 9. *Crenobacter luteus* YIM 78141^T (SLXG00000000); 10. *Vogesella indigofera* DSM 3303^T (RBID00000000).
	1	2	3	4	5	6	7	8	9	10
Genome size (Mb)	3.5	5.2	4.8	4.4	4.3	4.4	2.9	3.2	2.9	3.6
GC content (%)	51.4	64.5	64.5	60.5	62.2	63.4	63.5	59.5	68.3	64.5
Number of genes	3408	4839	4529	4198	4125	4064	2805	3153	2845	3454
Number of CDS	3274	4733	4407	4099	4010	3979	2717	3041	2760	3333

Phenotypic and Physiological Characteristics

Colonies of strain TC5R-5^T were white and non-transparent. The cells were rod-shaped, measuring approximately 0.8–0.9 µm in width and 3.0–4.0 µm in length, and exhibited a single polar flagellum (Fig. 3). Cells were Gram-stain-negative, catalase- and oxidase-positive, but did not display DNase activity. Growth occurred only under aerobic conditions. The strain tolerated NaCl concentrations ranging from 0 to 0.5% (w/v), and was capable of growth at temperatures between 15–30°C and pH 6.0–8.0, with optimal growth observed at 20–30°C and pH 6.0–7.0.

Substrate degradation assays were performed on R2A agar supplemented with various compounds (5.0% casein, 1.0% aesculin, 1.0% Tween 20, 1.0% Tween 80, 1.0% starch; w/v). Strain TC5R-5^T demonstrated limited carbon source utilization and acid production capabilities. It also tested negative for numerous enzymatic activities and biochemical reactions when compared to type strains of related genera within the family Chromobacteriaceae. A summary of its morphological and physiological characteristics is provided in Table 2.

Table 2
Differential characteristics between strain TC5R-5^T and most related phylogenetic neighbors Strains: 1. TC5R-5^T; 2. *Chromobacterium alkanivorans* IITR-71^T; 3. *Chromobacterium violaceum* ATCC 12472^T; 4. *Aquitalea magnusonii* TRO-001DR8^T; 5. *Paludibacterium yongneupense* 5YN8-15^T; 6. *Pseudogulbenkiania subflava* DSM 22618^T; 7. *Gulbenkiania mobilis* E4FC31^T; 8. *Craterilacusibacter sinensis* B2N2-7^T; 9. *Crenobacter luteus* YIM 78141^T; 10. *Vogesella indigofera* DSM 3303^T. +, Positive; -, negative; w, weakly positive. All of the data are acquired from this experiment except where indicated otherwise. ^a,b,c,d,e,f Data from corresponding literatures [Liu et al., 2020; Dong et al., 2015; Subhash et al., 2013; Sedláček et al., 2016; Sheu et al., 2014; Vaz-Moreira et al., 2007].
	1	2	3	4	5	6	7	8^a,	9^b	10^c
Reactions (API 20E&20NE)
Fermentation of glucose	-	+	+	+	+	-	-	-	+	-
Arginine dihydrolase	-	+	+	+	-	-	+	+	-	+
Hydrolysis of gelatin	-	+	+	-	-	-	-	-	-	-
Enzyme activity (API ZYM )
α-Chymotrypsin	-	-	+	-	+	+	-	-	-	-
N-Acetyl-β- glucosaminidase	-	+	+	-	w	-	-	-	-	-
Utilization of substrates (Biolog GENIII)
D-Trehalose	-	w	w	-	+	+	-	-	+	-
Inosine	-	+	w	-	-	-	-	-	-	-
Glycerol	-	+	w	+	-	-	-	-	-	+
Gelatin	-	+	+	-	-	-	-	-	-	-
L-Arginine	-	w	w	+	-	+	+	w	+	+
Pectin	w	-	-	-	+	+	-	w	+	-
Mucic acid	w	-	-	-	w	-	-	-	-	-
α –Keto-glutaric acid	+	w	-	+	w	-	-	w	-	-
Formic acid	+	-	w	w	-	+	-	w	-	+
Acids production (API 50CH)
D-Glucose	+	-	+	+	+	+	w	-	+	w
D- Mannose	-	-	+	-	-	-	-	-	-	-
Trehalose	-	+	+	-	+	+	-	-	-	-
Respiratory quinone	Q-8, Q-7	Q-8, Q-7	ND	Q-8^d	Q-8^e	Q-8^e	Q-8, Q-9^f	Q-8	Q-8	ND
Polar Lipid
PE	+	+	ND	+^d	+^[e]	ND	ND	+	+	+^c
PG	+	+	ND	+	+	ND	ND	+	-	+
DPG	+	+	ND	+	+	ND	ND	+	+	+
APL	1	1	ND	1	1	ND	ND	2	-	3
AL	-	4	ND	-	-	ND	ND	-	-	-
PL	1	-	ND	2	5	ND	ND	-	3	1
L	-	-	ND	2	-	ND	ND	3	-	-
AGP	-	-	ND	-	-	ND	ND	-	1	-

Chemotaxonomic Characteristics

The cellular fatty acid profile of strain TC5R-5^T was presented in Table 3. The predominant component was summed feature 3 (C_16:1 ω7c/C_16:1 ω6c; 51.6%), which was notably higher than those observed in related reference strains. In contrast, the content of summed feature 8 (C_18:1 ω7c/C_18:1 ω6c) in strain TC5R-5^T was less than half of that detected in Chromobacterium alkanivorans IITR-71^T. Overall, the principal fatty acids in strain TC5R-5^T and all type strains of the family Chromobacteriaceae were summed were summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) and C_16:0. The major polar lipids of strain TC5R-5^T were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), one unidentified aminophospholipid (APL1), and one unidentified phospholipid (PL1) (Figure S4). For comparative classification, the polar lipid profile of Chromobacterium alkanivorans IITR-71^T was also examined. The results indicated the presence of four aminolipids and one aminophospholipid in Chromobacterium alkanivorans IITR-71^T, which were absent in TC5R-5^T. Moreover, the comparison between strain TC5R-5^T and the type species of related genera in the family Chromobacteriaceae were also conducted. As shown in Table 2, polar lipids of most of type strains, except Crenobacter luteus YIM 78141^T, consisted of PE, PG and DPG. The predominant respiratory quinone of strain TC5R-5^T was ubiquinone 8 (Q-8), being in line with the quinone profile of members of the family Chromobacteriaceae.

Table 3
Whole-cell fatty acid profiles of the novel isolates and the type strains of related species of genera 1.strain TC5R-5^T; 2. *Chromobacterium alkanivorans* IITR-71^T; 3. *Chromobacterium violaceum* ATCC 12472^T; 4. *Aquitalea magnusonii* TRO-001DR8^T; 5. *Paludibacterium yongneupense* 5YN8-15^T; 6. *Pseudogulbenkiania subflava* DSM 22618^T; 7. *Gulbenkiania mobilis* E4FC31^T; 8. *Craterilacusibacter sinensis* B2N2-7^T; 9. *Crenobacter luteus* YIM 78141^T; 10. *Vogesella indigofera* DSM 3303^T. All data were from this study unless otherwise indicated. *Summed features are groups of two acids that cannot be separated by GLC using the MIDI system. ^a Data from Lau et al., ^b Data from Liu et al., ^c Data from Dong et al.
	1	2	3^a	4	5	6	7	8^b	9^c	10^b
C_12:0	7.1	10.5	5.6	7.2	2.9	6.5	3.6	2.0	5.1	3.8
C_14:0	2.2	3.1	1.7	1.3	3.8	0.8	0.8	3.6	-	0.5
C_16:0	26.7	20.5	23.9	30.5	29.8	28.7	24.9	33.8	27.2	24.9
C_10:0 3-OH	3.6	4.6	5.2	3.6	-	3.3	3.2	2.9	2.6	7.2
C_12:0 3-OH	2.7	2.2	5.0	2.2	3.5	2.6	3.2	3.3	2.3	-
Summed feature 3*	51.6	44.8	35.8	40.8	34.4	38.2	43.5	31.1	44.0	45.6
Summed feature 8*	5.1	12.1	15.0	10.7	13.2	11.8	9.1	1.0	-	7.9
* Summed Features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed feature 3, C_16:1 ω7c/C_16:1 ω6c; Summed feature 8, C_18:1 ω7c/C_18:1 ω6c. Data from corresponding literatures.

Taxonomic Conclusion

We conducted comprehensive phylogenetic analysis based on the 16S rRNA gene and whole genome alignments to determine the phylogenetic position of strain TC5R-5^T.

Phylogenetic trees reconstructed from the 16S rRNA gene consistently placed the strain within the family Chromobacteriaceae. This assignment was further supported by phylogenomic analyses performed with IQ-TREE using single-copy genes and with UBGC based on core genes. In contrast, the phylogenomic tree generated with the TYGS online tool showed that strain TC5R-5ᵀ formed a cluster with Halomonas humidisoli WN018ᵀ, Vreelandella glaciei DD39ᵀ, and Andreprevotia chitinilytica JS11-7ᵀ.

Subsequently, we calculated the Average Nucleotide Identity (ANI) and genome coverage between strain TC5R-5^T with its phylogenetic neighbors identified in the phylogenomic trees (Table S1). The results showed that strain TC5R-5^T shared higher ANI values with Chromobacterium alkanivorans IITR-71^T and Aquitalea palustris CCM7557^T (73.48%, 72.49%) than with Andreprevotia chitinilytica DSM 18519^T, Halomonas humidisoli WN018^T, and Vreelandella glaciei DD39^T (68.49%, 64.26%, 64.73%). Genome coverage was also notably greater with the former group (26.78% and 25.03%, respectively) compared to the latter (9.92%, 5.01%, and 4.91%, respectively). These findings further corrobrorated that strain TC5R-5ᵀ is phylogenetically affiliated with the family Chromobacteriaceae.

In summary, comprehensive phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences consistently support the classification of strain TC5R-5^T as representing a novel genus and species within the order Neisseriales. Although minor topological variations were observed among phylogenomic trees constructed using different analytical methods, the overall evidence strongly supported its provisional classification within the family Chromobacteriaceae as the most coherent and biologically plausible placement.

Description of Craterilacuimicrobium gen. nov.

Craterilacuimicrobium gen. nov. (Cra.te.ri.la.cu.i.mi.cro'bium. L. masc. n. crater, a crater; L. masc. n. lacus, a lake; N.L. neut. n. microbium, a microbe; N.L. neut. n. Craterilacuimicrobium, a microbe from a crater lake)

Cells rod-shaped, motile, Gram-stain-negative and strictly aerobic. Catalase- and oxidase-positive. The major fatty acids consist of summed feature 3 (C_16:1 ω7c/C_16:1 ω6c). The predominant ubiquinone is Q-8, with a small amount of Q-7. The main polar lipids include phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), one unidentified aminophospholipid (APL), and one unidentified phospholipid (PL). The type species is Craterilacuimicrobium aquaticum.

Description of Craterilacuimicrobium aquaticum sp. nov.

Craterilacuimicrobium aquaticum sp. nov. (a.qua'ti.cum. L. neut. adj. aquaticum, living, growing or found in or by water, aquatic)

Cells are Gram-stain-negative, strictly aerobic, motile, and short rod-shaped. Colonies are circular, smooth, white-colored after incubating for 48 h at 25°C on R2A agar. Growth occurs at 15–30°C (optimum, 20–30°C), at pH 6.0–8.0 (optimum, 6.0–7.0) and with 0-0.5% NaCl (optimum, 0-0.5%, w/v). Catalase- and oxidase-activity present. DNase activity is not present. Positive for hydrolyzation of Tween 20, but negative for degradation of casein, cellulose, starch, aesculin and Tween 80. In the API 20 NE and 20E tests, strain TC5R-5^T could reduce nitrate to nitrite. Voges-Proskauer-test and fermentation of glucose are positive. Citrate not utilized, indole not produced, no arginine dihydrolase activity, no hydrolysis of gelatin and esculin. Does not produce H₂S. The activities of ornithine decarboxylase, lysine decarboxylase, tryptophane deaminase, urease, ortho-nitrophenyl-β-D-galactopyranosidase and para-nitrophenyl-β-D-galactopyranosidase are negative. In the API ZYM test, positive for alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, but negative for esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Acids could be produced from glucose in API 50CH, but not other carbon sources. In the BIOLOG GEN III MicroPlate system, α-D-glucose, D-glucose-6-PO₄, L-alanin, L-glutamic acid, L-histidine, L-serine, methyl pyruvate, L-lactic acid, α-keto-glutaric acid, γ-amino-butrytic acid, β–hydroxyl-D, L-butyric acid, acetoacetic acid, formic acid are utilized as sole carbon resource, and dextrin, D-frucose, D-fucose, L-fucose, D-fructose-6-PO₄, D-serine, glycyl-L-proline, L-aspartic acid, L-pyroglutamic acid, pectin, D-galacturonic acid, D-glucuronic acid, glucuronamide, mucic acid, D-lactic acid metyl ester, citric acid, D-malic acid, L-malic acid, Tween 40, α-hydroxy-butyric acid, propionic acid, acetic acid are weakly utilized. The genomic DNA G + C content is 51.4% according to the genomic sequencing data. The major fatty acids (> 10%) are summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) and C_16:0. The predominant respiratory quinone is Q-8 with a trace amount of Q-7. The main polar lipids are phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), one unidentified aminophospholipid (APL) and one unidentified phospholipid (PL).

The type strain TC5R-5^T (= CGMCC 1.17058^T = KCTC 72736^T ) was isolated from water of Aershan Tianchi crater lake in the Inner Mongolia Autonomous Region of China. The GenBank/EMBL/DDBJ accession number for 16S rRNA gene of strain TC5R-5^T is OK487579 and the DDBJ/ENA/GenBank accession number of the Whole Genome Shotgun project of TC5R-5^T is JACCFC000000000.

ANI, average nucleotide identity; APL, unidentified aminophospholipid; CDS, coding sequences; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL, unidentified phospholipid; AGP, aminoglycophospholipid; L, unidentified polar lipid; NRPS, nonribosomal peptide synthetase.

Conflicts of interest:

The authors declare that there are no conflicts of interest.

Ethical approval:

The data reported in this article is not derived from any studies or tests on human volunteers or animals.

Informed consent:

All the authors have read and approved the final manuscript for submission.

Funding information

This work was financially supported by the National Natural Science Foundation of China (31970004), the Strategic Biological Resources Capacity Building Project of Chinese Academy of Sciences, China (KFJ-BRP-017-73) and the Key Research and Development Project of Xizang Autonomous Region, China (XZ202401ZY0061).

Author Contribution

You-Jun Liao: Conceptualization, Resources, Data curation, Methodology; Xuan Zhang : Investigation, Formal analysis; Zi-Xuan Liu: Visualization, Writing—Original Draft and Editing; Ai-Hua Li: Funding acquisition, Project administration, Writing—Reviewing.

Acknowledgement

The authors gratefully acknowledge the help from Prof. Teun Boekhout (College of Sciences, King Saud University, Saudi Arabia) to improve the English text, the help from Dr. Jing-Nan Liang for the TEM observation, and the assistance from Prof. Stefano Ventura (Research institute on Terrestrial Ecosystems, Firenze Unit, Italy) for his advice on bacterial nomenculature.

Data Availability

The GenBank/EMBL/DDBJ accession number for 16S rRNA gene of strain TC5R-5T is OK487579. The Whole Genome Shotgun project of strain TC5R-5T has been deposited at DDBJ/ENA/GenBank under the accession number JACCFC000000000. This strain is available at China General Microbiology Culture Collection Center (CGMCC) under the accession no. CGMCC 1.17058T.

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Craterilacuimicrobium aquaticum gen. nov. sp. nov., a novel member of the order Neisseriales isolated from crater lake

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Abstract

Figures

Introduction

Material and Methods

Strain Isolation

Genome Features and Phylogenomic Analysis

Morphological and Physiological Analyses

Chemotaxonomic Analyses

Results and Discussion

Phylogenetic and phylogenomic analysis

Genome Features and Biosynthetic Gene Cluster

Phenotypic and Physiological Characteristics

Chemotaxonomic Characteristics

Taxonomic Conclusion

Abbreviations

Declarations

Funding information

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

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