Acerihabitans arcticus sp. nov., a first psychrophilic member of the family Pectobacteriaceae and able to reduce Fe(III)

doi:10.21203/rs.3.rs-7027023/v1

A novel psychrophilic strain TG2^T was isolated from an anaerobic enrichment obtained from a tundra soil sample selected on the Bykovsky Peninsula (Russia). Cells were facultatively anaerobic, Gram-stain-negative, non-spore-forming motile short rods. The novel isolate grows at 0–25°C (optimum 8°C), pH 6.0–8.0 (optimum 7.0–7.5), up to 1.0% (w/v) NaCl (optimum 0.1–0.5%). Strain TG2^T was a chemoorganoheterotroph. Lactate, acetate and formate were the major products of glucose and pectin fermentation. The major cellular fatty acids were С_16:1ω8c, С_17:0 cyclo and С_18:0 cyclo. The polar lipids were phosphatidylcholine, phosphatidylethanolamine phosphatidylglycerol, and four unidentified polar lipids. Phylogenomic analysis based on 81 core gene sequences showed that strain TG2^T belonged to the family Pectobacteriaceae. The closest phylogenetic neighbor was Acerihabitans arboris SAP-6^T (98.2% 16S rRNA gene sequence similarity). The genome of strain TG2^T was 5.3 Mbp in size with 51.1 mol% of G + C content. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain TG2^T and its closest relative were 78.6 and 22.6%, respectively. Based on the phylogenetic, phenotypic, chemotaxonomic and genomic data, new bacterium represents a novel species of the genus Acerihabitans in the family Pectobacteriaceae, for which the name Acerihabitans arcticus sp. nov. is proposed. The type strain is TG2^T (= VKM B-3773^T = JCM 39549^T)

The family Pectobacteriaceae currently contains the type genus Pectobacterium (Waldee 1945), as well as validly published genera Acerihabitans (Lee et al. 2021), Biostraticola (Verbarg et al. 2008), Brenneria (Hauben et al. 1998), Dickeya (Samson et al. 2005), Lonsdalea (Brady et al. 2012), Musicola (Hugouvieux-Cotte-Pattat et al. 2021), Prodigiosinella (Hugouvieux-Cotte-Pattat et al. 2024), Sodalis (Dale and Maudlin 1999) and genus Symbiopectobacterium was recently described as being closely related to Pectobacterium (Nadal-Jimenez et al. 2022). Presently, the genera Biostraticola and Sodalis are proposed for reclassification into the novel family "Bruguierivoracaceae" based on phylogenomic and phenotypic evidence (Li et al. 2021). The whole-genome analyses demonstrated that these genera form a distinct monophyletic clade, evolutionarily divergent from Pectobacteriaceae. Notably, the genera Biostraticola and Sodalis lack pectinase activity – a defining characteristic of the family Pectobacteriaceae.

Members of the family Pectobacteriaceae produce acid from N-acetylglucosamine and are negative for arginine dihydrolase, orthinine decarboxylase and lysine decarboxylase. These bacteria are mesophilic non-spore-forming, catalase-positive, oxidase-negative, and do not produce hydrogen disulfide (Adeolu et al. 2016). Members of the family Pectobacteriaceae inhabit a number of different ecological niches and have been found in soil, water and in association with living organisms including plants and insects (Brenner and Farmer 2005). Some of them such as are major plant pathogens. Thus, members of Brenneria and Lonsdalea genera are responsible for tree diseases (Brady et al. 2012), while bacteria of Dickeya, Musicola, and Pectobacterium genera affect a wide range of hosts, provoking soft-rot diseases due to the action of extracellular pectinases that degrade the plant cell wall (Hugouvieux-Cotte-Pattat et al. 2020; Van Gijsegem et al. 2021). Most of the characterized strains of these three soft-rot genera were isolated from diseased crops or ornamental plants (Parkinson et al. 2014; Hugouvieux-Cotte-Pattat et al. 2019; Oulghazi et al. 2019a; Oulghazi et al. 2019b; Waleron et al. 2019; Pédron et al. 2019; Ben Moussa et al. 2021). Recently Prodigiosinella aquatilis as the type species of the new bacterial genus Prodigiosinella was isolated from the water samples collected in coastal saline wetland lakes near the Mediterranean Sea. This environment is distinct due to its saline conditions, influencing the phenotypic and genomic characteristics of the isolated free-living strains of the genus. In this study, we describe a novel psychrophilic strain of Acerihabitans sp. isolated from an Arctic tundra soil sample (Bykovsky Peninsula, Russia) able to grow at 0 ^oC and reduce Fe(III) in anaerobic conditions.

Isolation and cultivation

Strain TG2^T was isolated from an enrichment culture of the iron-reducing bacteria IRB obtained from the upper horizon (depth 8–17 cm) of tundra soil sample collected in the western part of the Ivashkina lagoon on the Bykovsky Peninsula, Russia in 2021 (71.72N 129.28E). The horizon was represented by wet, dense, blue-gray peat with a high content of evenly distributed loamy material. The soil samples were transported and stored frozen at − 20°C until the start of the microbiological studies. To obtain the IRB enrichments, the sample of soil (1.0 g) was added to 100 mL of the Medium I containing (l^–1): 2.5 g NaHCO₃, 1.0 g NaCl, 0.7 g NaH₂PO₄, 0.2 g MgCl₂ .6H₂O, 1.0 g NH₄Cl, 0.1 g CaCl₂.2H₂O, 0.002 g yeast extract (Difco), 1.0 ml trace element solution SL-10 (medium 320; DSMZ), 10.0 ml vitamin solution (Wolin et al. 1963). Various monosaccharides (4.0 g L^− 1) served as the carbon source and as the electron donors, while Fe(III)-citrate (30 mM) served as the electron acceptor. The final pH was adjusted to 7.0. High-purity nitrogen was used as the gas phase. The enrichments were incubated at 8°C for 30 days. A pure culture, designated strain TG2^T, was performed according to the Hungate anaerobic method (Hungate 1969) by the serial tenfold dilution method on the Medium I with xylose as the substrate. Strain TG2^T was deposited in the All-Russian Collection of Microorganisms (VKM) and Japan Collection of Microorganisms (JCM), as lyophilized cultures with accession numbers of VKM B-3773^T and JCM 39549^T, respectively.

Genome sequencing analyses and phylogenetic analysis

To determine the phylogenetic assignment of strain TG2^T, its genome was sequenced. Genomic DNA preparation and sequencing were performed by the BioSpark Company (Troitsk, Russia). Genomic DNA was isolated with the FastDNA spin kit (MP Biomedicals, USA) by the column method with the deposition on silica gel. The libraries were synthesized using KAPA HyperPlus kits (Kapa Biosystems, USA) in accordance with the manufacturer’s recommendations. Sequencing was performed on the Illumina NovaSeq 6000 platform, and a paired-end library with a total of 2,786,968 read pairs and a read length of 2 x 150 bp was obtained. The Kbase (Arkin et al. 2018) online platform with associated tools was used for genome assembly and analysis. The quality of the reads was assessed using the FastQC v.0.12.1 program (Andrews 2010). The reads were edited and filtered using Trimmomatic v. 0.36 (Bolger et al. 2014) with adapter clipping (adapter library TruSeq3-PE-2). De novo genome assembly was performed using the metaSPAdes genome assembler (version 3.13.0) (Bankevich et al. 2012). Genome completeness and contamination were evaluated using CheckMv.1.0.18 (Parks et al. 2015). A maximum-likelihood phylogenetic tree was reconstructed from a concatenated alignment of 81 core genes using the UBCG v.2 pipeline (Na et al. 2018). Nucleotide sequences were retrieved from the RefSeq database. Core gene identification was performed with Prodigal v.2.6.3 (Hyatt et al. 2010) for gene prediction and HMMER v.3.3.2 (Eddy 2011) for profile searches. Multiple sequence alignment was generated using MAFFT v.7.490 (Katoh and Standley 2013) with the L-INS-i algorithm (iterative refinement method for < 200 sequences). Tree topology was inferred with 1,000 bootstrap replicates to assess branch support (Felsenstein 1985). The final dendrogram was visualized and annotated in TreeViewer v.2.2.0. dDDH values and confidence intervals were calculated using the Genome-to-Genome Distance Calculator available at http://ggdc.dsmz.de. Average amino acid (AAI) values were calculated using the open-source software package GET_HOMOLOGUES v. 07112023. The search for homologues of protein sequences was carried out using the COG algorithm (Contreras-Moreira and Vinuesa 2013). ANI values between pairwise-genome sequences of strain TG2^T and its phylogenetic neighbors were calculated using the online service https://www.ezbiocloud.net/tools/ani. Alignment of reads to genomes, determination of coverage, and the number of aligned reads were carried out using the bowtie2 v. 2.3.2 program (Langmead and Salzberg 2012).

Morphological, physiological and biochemical characterization

Cell morphology and motility were studied using phase-contrast microscopy (Olympus BX41) at ×1300 magnification equipped with a ProgRes SpeedXT core5 camera (Jenoptik, Germany) with cells grown under anaerobic conditions on the Medium I with xylose at 8°C. Electron microscopic analysis was performed as described earlier (Suzina et al. 2022). Colonies were obtained by the “roll-tube” method using Medium I with Bacto agar (0.2%, w/v) with xylose (4.0 g L^− 1) as substrate at 8°C. Gram-staining was performed following a standard protocol (Smibert and Krieg 1994). The temperature range for growth was determined by incubating at 0, 3, 8, 12, 18, 25, 30, 37°C. The effect of pH was examined at pH 6.0–10.0. The NaCl concentrations were tested from 0 to 0.5% (w/v) with 0.05% intervals and from 1.0 to 5.0% (w/v) at intervals of 0.5% NaCl. The ability to reduce Fe(III)-citrate (10 mM), nitrate (10mM), ferrihydrite (10 mM) was added as amorphous iron (III) oxide, prepared by titration of acidic FeCl₃ solution with 10% (w/v) NaOH to pH 7.0 (Lovley et al. 1993) was assessed in Hungate tubes containing anaerobic Medium I supplemented with xylose as the electron donor and carbon source. Potentially fermentable substrates were examined in Medium I in the absence of an electron acceptor. The following substrates were tested: organic acids (20 mM), monosaccharides and polysaccharides (4.0 g L^− 1), and alcohols (0.1%, v/v). Bacterial biomass (measured as OD₆₀₀ on a spectrophotometer Spekol 221 (Germany)) was determined at the beginning and end of growth and all results were recorded after 7 days of incubation at 8°C. The effect of antibiotics on strain TG2^T was identified by transferring the culture into fresh Medium I containing (mg L^− 1) bacitracin (100), kanamycin (200), streptomycin (200), tetracycline (100), methycillin (1000), gentamycin (10), cephalosprorin C (100), and amikacin (100). Tests were performed in duplicate with a non-antibiotic control for 1 week at the optimum temperature and pH. All tests were carried out in triplicate and confirmed by two additional transfers. Oxidase and catalase activities were detected with N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride and 3 % H₂O₂ solutions, respectively. Enzymatic activities, carbon metabolic profiles were analyzed using the API ZYM, API 50CH system, respectively, according to the manufacturer′s protocol (BioMérieux, France). The results were recorded after 24 h incubation at 8 °C for the API ZYM strip and after 7 days of incubation at 8 °C for the API 50CH strip.

Analytical techniques

Nitrite was analysed according to Gries-Romijn van Eck (1966). Fe(III) reduction was determined calorimetrically by formation of a stable colored complex of Fe(II) with ferrozine (Viollier et al. 2000). Products of carbohydrate fermentation in the culture medium were assayed analyzed by HPLC (Shimadzu, Japan). Separation was performed on a Repro-Gel H + column (9 µm, 250×8 mm) (Dr. Maisch, Germany) using 1 mM H₂SO₄, 0.5 ml/min at 50°C for elution. Registration was performed spectrophotometrically at a wavelength of 210 nm.

Chemotaxonomic characterization

For the analysis of the cellular fatty acids, the cells of strain TG2^T were grown in Medium I with xylose and harvested in the late exponential growth phase. Cellular fatty acids (CFA) profiles were determined by GC-MSas described earlier by Karnachuk et al. (2024). Polar lipids were extracted from the freeze-dried biomass with water–methanol–chloroform mixture (0.8:2:1) and separated with 2D thin-layer chromatography high-efficient 100×100 mm plates (Sorbfil, Krasnodar, Russia) using eluent systems (1stD CHCl₃–MeOH–H₂O, 75:25:2.5; 2ndD CHCl₃–MeOH–CH₃COOH–H₂O, 80:9:12:2) recommended by Christie (Christie and Han 2010). Various lipid classes were revealed by the spray reagents. Reference samples were lecithin and E. coli biomass.

Strain identification and phylogenomic analysis

The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) and RAST. The assembled genome of strain TG2^T was 5.3 Mbp in size and contained 108 contigs with a maximum length of 420,954 bp. The scaffold N50 value was 150.9 kb, and the L50 value was 12. Reads alignment demonstrated that the genome accounted for 68.33% of the reads, with coverage of 106.34 ± 40.32. Genome completeness was 100%, contamination was 0.96%. In the phylogenomic tree based on 81 core gene sequences, strain TG2^T clustered with members of the family “Bruguierivoracaceae” and one representative of the family Pectobacteriaceae, forming a distinct monophyletic clade. The closest related type strain was A. arboris SAP-6^T the only member of the genera Acerihabitans (assembly GCA_010131535.1, WGS WUBS01 project), isolated from tree sap (Jeju Island, South Korea) (Fig. 1). The dDDH value between the strains was 22.6%, while the ANI was 78.2% and AAI was 80.3% (Table 1). The 16S rRNA gene was extracted from the genome. Comparison of its nucleotide sequence between strains TG2^T (1,551 bp) and SAP-6^T (MN737198.1) revealed 98.2% similarity. Given the low dDDH and ANI values, along with differences in 16S rRNA sequences, it can be concluded that strain TG2^T represents a novel species within the genus Acerihabitans.

Table 1

dDDH, ANI and AAI values (%) between strain TG2^T and closely relative species
Strain TG2^T in comparison with:	GenBank ID	dDDH	ANI	AAI
Acerihabitans arboris SAP-6^T	GCA_010131535.1	22.6	78.2	80.3
Sodalis ligni dw23^T	GCA_004346745.1	21.6	75.6	77.3
Biostraticola tofi DSM 19580^T	GCA_004343195.1	19.9	73.3	74.7

The proposed and generally accepted species boundary for ANI and dDDH values are 95⁓96 and 70%, respectively (Chun et al. 2018), for AAI values are 95% (Konstantinidis and Tiedje (2005)

Genome information

The genome of strain TG2^T contained 4,950 genes, including 4,734 protein-coding genes, 61 tRNA genes, and 2 rRNA genes (one 16S and one 23S rRNA). The strain was characterized by a low genomic G + C content (51.1 mol%), showing a 5.9% difference from A. arboris SAP-6^T and 2.9–3.9% differences from other closely related type strains (Table S1).

The genome of the strain contained genes encoding key proteins involved in extracellular cellulose biosynthesis, including: UDP-forming cellulose synthase catalytic subunit BcsA (EC 2.4.1.12), cellulose biosynthesis cyclic di-GMP-binding regulatory protein BcsB, cellulose synthase subunit BcsC-related outer membrane protein, and cellulose biosynthesis protein BcsD.

Additionally, were identified two endoglucanases of the GH8 family, several beta-glucosidases, including phospho-beta-glucosidases (GH1 and GH4 families) and BglX (GH3 family). Other annotated enzymes included: cellobiose transporters (various PTS types, 6 genes), pectinases (putatively GH43 family), amylases (GH13 family), lysozyme (GH24), alpha-xylosidase (GH31), murein transglycosylases A and B. The genomic presence of a complete cellulose synthase operon (bcsAB-bcsD) along with accessory proteins (BcsC) demonstrates capacity of strain TG2^T for bacterial cellulose biosynthesis, which serves as a critical structural element in biofilms, conferring mechanical stability and stress resistance. Endoglucanases (GH8) and β-glucosidases (GH1, GH3, GH4) indicate the ability of the new bacterium to hydrolyze cellulose and cellobiose. The presence of these enzymes suggests that the strain can utilize plant-derived substrates. Specifically, amylases (GH13 family) indicate potential starch degradation capability. Although the A. arboris SAP-6^T genome encoded an alpha-amylase genes (amyA), no starch hydrolysis was detected under laboratory growth conditions.

The genome of strain TG2^T contained the genes encoding undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase (EC 2.4.2.43) and lipid IV(A) 4-amino-4-deoxy-L-arabinosyltransferase (EC: 2.4.2.44) which play a key role in lipopolysaccharide (LPS) modification. Strain TG2^T genome encoded a high number of cold-shock domains (CSD), genes related with a psychrophilic phenotype. Genomic analysis revealed iron metabolism genes in strain TG2^T, comprising: 22 cytochrome c, d, O biogenesis genes, specific cytochrome genes (c-553, b-562, b-561), and three additional cytochrome-containing protein subunits. We found genes encoding respiratory NarK family nitrate/nitrite MFS transporter, two subunits nitrate reductase (alpha, beta) and respiratory nitrate reductase subunit gamma. These genes play an important role in nitrogen metabolism, particularly in denitrification, nitrogen assimilation, and anaerobic respiration processes.

Morphological, physiological, and biochemical characteristics

Cells of strain TG2^T were Gram-negative, motile, non-spore-forming short rods (0.6–0.7 x 1.0–2.0 µm). At the periphery of the cell wall surface, outer membrane vesicles (OMVs) which are known to be involved in biofilm formation were observed. The cells were surrounded by an outer restrictive layer of a capsule (ORLC) or a sheath (Fig. 2). The strain was facultative anaerobic with a slight preference for the anaerobic life style. Cells were catalase-positive and oxidase-negative. Colonies appeared after 4 weeks of incubation at 8°C and pH 7.5 were 1–3 mm in diameter, circular with smooth, entire margins, convex or slightly raised, and white in colour. Strain TG2^T was capable of forming biofilm. The biofilm presumably contained lipopolysaccharides and cellulose. The possibility of polymeric substances biosynthesis was confirmed through identification of the key genes associated with their synthetic pathways. Strain TG2^T could grow between 0 and 25°C, and the optimum growth was at 8°C. The isolate demonstrated growth within pH 6.0– 8.0 (optimum pH 7.0–7.5) and at NaCl concentrations from 0 to 1.0% with an optimum at 0.1–0.5% (w/v).

Strain TG2^T was capable of growth on sugars (fructose, galactose, glucose, mannose, rhamnose, D-sorbitol), polysaccharides (cellulose, dextrin, maltodextrin, pectin, starch). No growth was observed with citrate, lactose, malate, alginate, xylan, methanol, and ethanol. Weak growth occurred with glycerol. The strain did not require yeast extract for growth and did not grow autotrophically on H₂/CO₂ (80/20 (v/v), 1.5 atm). Growth on the starch- and pectin-containing media was corroborated by the evidence of genes encoding starch- and pectin-degrading enzymes. Strain TG2^T degraded esculin and reduced nitrates to nitrites. Since the strain was isolated from an Fe(III)-reducing enrichment, its ability to reduce iron was tested. Strain TG2^T was capable of reducing Fe(III)-citrate and ferrihydrite under anaerobic conditions, but only with a fermentable substrate. In the presence of xylose, nearly 3.4 mM Fe(II) was produced after 1 month of the incubation at 8 ^oC. Strain TG2^T demonstrated a high activity of alkaline phosphatase, acid phosphatase, and β-galactosidase. It showed positive reactions for esterase (C4), naphthol-AS-BI-phosphohydrolase, and β-glucuronidase. Negative results were obtained for lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. The isolate was capable of fermenting glucose and pectin, producing lactate, formate, and acetate as the major fermentation products. The strain was sensitive to tetracycline, kanamycin, gentamicin, cephalosporin C, amikacin, and streptomycin, while demonstrating resistance to bacitracin and methicillin.

Differential physiological and biochemical properties of strain TG2^T and closely related species are given in Table 2. Strain TG2^T was phenotypically distinguished from its closest relative A. arboris SAP-6^T by its inability to utilize citrate and malate. While A. arboris SAP-6^T and S. ligni dw23^T failed to grow with starch, strain TG2^T shared this capability with B. tofi DSM 19580^T when starch was tested as a sole carbon source. Notably, strain TG2^T showed a restricted carbohydrate metabolism compared to related strains, lacking utilization of maltose, melibiose and sucrose - traits typically associated with plant-associated microorganisms.

Chemotaxonomic characteristics

The dominant fatty acids of strain TG2^T were С_16:1ω8c (20.8%), С_17:0 cyclo (24.3%), and С_18:0 cyclo (26.2%). (Table 3). The membrane lipid profile of strain TG2^T was distinct from that of A. arboris SAP-6^T, characterized by a more diverse CFA composition. The prominence of C_17:0 cyclo and C_18:0 cyclo isoforms implies evolutionary adaptations to low-temperature environments. The polar lipid of strain TG2^T consisted of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and four unidentified compounds (UL1–UL4). The presence of PC, PE, and PG is typical for bacterial membranes. Diphosphatidylglycerol (DPG) was detected in A. arboris SAP-6^T and S. ligni dw23^T which may indicate differences in membrane stability and adaptation strategies between the closely related species and strain TG2^T (Table 2). The absence of aminophospholipid (APL) suggests that strain TG2^T may employ alternative membrane stabilization mechanisms, such as an increased proportion of unsaturated fatty acids or cyclopropane fatty acids to maintain membrane integrity under physiological stress.

Table 2

**Differential characteristics of strain TG2**^T **and closely related species** Taxa: 1, Strain TG2^T (data from this study); 2, *Acerihabitans arboris* SAP-6^T (Lee et al. 2021); 3, *Sodalis ligni* dw23^T (Chaput et al. 2022); 4, *Biostraticola tofi* DSM 19580^T (Verbard et al. 2008). +, Positive; -, negative; w, weak; A, aerobic; FA, facultatively anaerobic; ND, not determined; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PC, phosphatidylcholine; APL, unidentified aminophospholipid; PL, unidentified phospholipids, AL, unidentified aminolipid; UL, unidentified polar lipids; PI, phosphatidylinositol; L, unidentified lipid, GL, unidentified glycolipid.
Characteristic	1	2	3	4
Growth under anaerobic conditions	FA	FA	A	FA
Temperature range, ^oC (optimum)	0–25 (8)	4–37 (30)	25–37 (30)	5–30 (25–30)
pH range (optimum)	6–8 (7.0-7.5)	6–8 (7–8)	5.5–8.5 (7.0)	5.0–9.0 (8.0–9.0)
NaCl range, % (optimum)	0–1 (0.1–0.5)	0–4 (1–2)	0–3	0–6 (0–1)
Nitrate reduction	+	+	-	+
Aesculin degradation	+	+	-	+
Citrate	-	w	ND	-
Malate	-	+	-	+
Starch	+	-	-	+
Carbohydrate fermentation	glucose, pectin	glucose, mannose	glucose, fructose	glucose, galactose
Fermentation products	lactate, acetate, formate	lactate, acetate	succinate, acetate	acetate, ethanol
Acid production from:
Amygdalin	-	+	ND	-
Arbutin	+	+	ND	w
D-Adonitol	-	+	-	-
L-Arabinose	-	+	+	+
D-Arabitol	+	+	-	+
Cellobiose	+	+	-	+
D-Dulcitol	-	+	-	-
Glycerol	w	w	+	+
Lactose	-	-	+	w
Maltose	-	+	+	+
Melibiose	-	-	+	-
Raffinose	-	-	+	-
D-Ribose	-	+	+	+
D-Sorbitol	+	+	+	-
Gentibiose	-	+	ND	+
Salicin	+	+	+	-
Sucrose	-	+	-	-
Polar lipids	PC,4UL	DPG, PC, APL, 2PL, 2L	DPG, AL, PL, L	PI, APL
DNA G + C, mol%	51.1	57.0	55.0	54.0
Source of the isolation	Tundra soil (Russia)	Sap drawn from Acer pictum (South Korea)	Temperate forest soil (USA)	Biofilm of a tufa deposit (Germany)

Table 3

Cellular fatty acid composition of strain TG2^T in comparison with *Acerihabitans arboris* SAP-6^T
Fatty acids, %	1	2
C_11:0	0.2	ND
C_12:0	1.8	6.1
C_14:0	ND	1.4
C_15:0	5.0	ND
C_16:0	8.3	31.8
C_16:1 ω8c	20.8	ND
C_16:1 ω6c	0.3	17.5
C_17:0	4.2	0.5
C_17:0 cyclo	24.3	20.1
C_17:0 cyclo9,10	4.5	ND
C_18:0	ND	0.7
C_18:0 cyclo	26.2	ND
C_18:1 ω7c and/orC_18:1 ω6c	ND	8.1
3OH-C_12:0	ND	ND
3OH-C_13:0 branched	2.1	ND
3OH-C_13:0	0.2	ND
C_19:0 cyclo	0.6	4.6
3OH-C_14:0	0.3	8.7
OH-C_n:0	1.1	ND

Strains: 1, strain TG2^T; 2, A. arboris SAP-6^T (Lee et al. 2021). ND, not detected.

Taxonomic conclusion and ecological role

Phylogenetic and phenotypic analyses placed strain TG2^T within the genus Acerihabitans, while revealing distinctive traits that differentiate it from its closest relatives. The novel isolate was capable of growth at 0°C, with an optimum temperature of 8°C, distinguishing it from its mesophilic closely species. The strain exhibited restricted salt tolerance (0–1%) and a growth-limiting pH range (6.0–8.0). The limited disaccharide fermentation profile, combined with psychrophilic traits, suggests that strain TG2^T is adapted to low-temperature environments with restricted availability of carbohydrate substrates. Increased cyclopropane fatty acid content in the cell wall of strain TG2^T maintains membrane fluidity homeostasis under stress conditions. Distinct lipid profile and a lower DNA G + C content (51.1 mol%) further support the genetic and metabolic divergence of strain TG2^T from its closest relatives. Strain TG2^T demonstrated the ability to reduce iron and nitrate. Genomic analysis revealed the presence of genes encoding cytochrome c, d, and O biogenesis, as well as specific cytochrome genes. Additionally, we identified genes for a NarK family nitrate/nitrite MFS transporter, two subunits of nitrate reductase (alpha and beta), and the gamma subunit of respiratory nitrate reductase. The iron- and nitrate-reducing capabilities of this strain may play a significant role in iron, nitrogen, and carbon cycling in Arctic ecosystems.

Based on the chemotaxonomic difference and genome- and 16S rRNA gene-based phylogenetic distinctness, strain TG2^T represents a novel species within the genus Acerihabitans, for which the name Acerihabitans arcticus, sp. nov. is proposed.

Description of Acerihabitans arcticus sp. nov.

Acerihabitans arcticus (arc′tic.us. L. masc. adj. arcticus northern, Arctic).

Cells are Gram-negative, facultatively anaerobic, motile, non-spore-forming short rods (0.6–0.7 x 1.0–2.0 µm). Catalase-positive and oxidase-negative. Colonies are 1–3 mm in diameter, circular with smooth, entire margins, convex or slightly raised, and white after 4 weeks of incubation at 8°C. Growth is observed at 0–25°C (optimum 8°C), at pH 6.0– 8.0 (optimum pH 7.0–7.5) and in presence of up to 1.0% (w/v) NaCl, (optimum 0.1–0.5%). Utilized a broad range of carbon source for growth, such us fructose, galactose, glucose, mannose, rhamnose, D-sorbitol, cellulose, dextrin, maltodextrin, pectin, starch. Does not utilize citrate, lactose, malate, alginate, xylan, methanol, and ethanol. Lactate, acetate, formate were the major products of glucose and pectin fermentation. Does not grow autotrophically. Aesculin degradation is observed and nitrate is reduced to nitrite. Capable of using Fe(III) as an electron sink in the presence of xylose. Alkaline phosphatase, acid phosphatase, β-galactosidase, esterase (C4), naphthol-AS-BI-phosphohydrolase, and β-glucuronidase are present but lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase are absent. Acid is produced from N-acetylglucosamine, aesculin, l-arabinose, d-arabitol, arbutin, cellobiose, d-fructose, d-galactose, gentibiose, gluconate, d-glucose, glycerol (weak), inositol, maltose, d-mannitol, d-mannose, l-rhamnose, salicin, d-sorbitol, trehalose, starch and d-xylose but not from l-arabitol, d-adonitol, amygdalin, d-arabinose, dulcitol, erythritol, d-fucose, l-fucose, glycogen, inulin, 2-ketogluconate, 5-ketogluconate, lactose, d-lyxose, melezitose, melibiose, methyl-α-d-glucoside, methyl-α-d-mannoside, methyl-β-d-xyloside, d-ribose, raffinose, sucrose, l-sorbose, d-tagatose, turanose, d-xylitol and l-xylose. Cells are resistant to bacitracin and methicillin. The polar lipids are phosphatidylcholine, phosphatidylethanolamine phosphatidylglycerol, and four unidentified lipids. The major cellular fatty acids are С_16:1ω8c, С_17:0 cyclo and С_18:0 cyclo.

The type strain TG2^T (= VKM B-3773^T = JCM 39549^T) was isolated from tundra soil, Bykovsky Peninsula, Russia. The DNA G + C content of the type stain is 51.1 mol%. The accession number of the genome sequence is JAYGJO000000000.1 and PP024248 at the 16S rRNA sequence.

ANI: Average nucleotide identity; AAI: Average amino acid identity; dDDH: digital DNA–DNA hybridization; IRB: iron-reducing bacteria; CFA: cellular fatty acids; PGAP: Prokaryotic Genome Annotation Pipeline.

Funding information

This work was supported by the Russian Science Foundation, (project № 25-24-00337).

Author Contribution

A. G. Z., investigation, writing and approving the manuscript; V. E. T., bioinformatic analysis, methodology; N. E. S., microscopic investigations; D. S. K., investigation (fatty acid analysis of cell walls); A. A. K., investigation (polar lipid identification); O. I. M., investigation (products of carbohydrate fermentation identification); V. A. S., writing and approving the manuscript. All authors reviewed the manuscript.

Data Availability

The genome sequence of Acerihabitans sp. strain TG2T is available in GenBank under accession number JAYGJO000000000.1 (BioProject accession number PRJNA1041642 and BioSample accession number SAMN38289666), and the 16S rRNA nucleotide sequence was deposited under accession number PP024248.

Authors and Affiliations

Federal Research Center «Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences», G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki, 5, Pushchino, Moscow Region, Russia, 142290

Anastasiya G. Zakharyuk, Vladimir E. Trubitsyn, Natalia E. Suzina, Oleg I. Melnikov & Viktoria A. Shcherbakova

Gubkin University, Leninsky Prospect 65-1, 119991, Moscow, Russia.

Dmitry S. Kopitsyn, Aleksandra A. Kuchierskaya

Contributions

A. G. Zakharyuk, investigation, writing and approving the manuscript; V. E. Trubitsyn, bioinformatic analysis, methodology; N. E. Suzina, microscopic investigations; D. S. Kopitsyn, investigation (fatty acid analysis of cell walls); A. A. Kuchierskaya, investigation (polar lipid identification); O. I. Melnikov, investigation (products of carbohydrate fermentation identification); V. A. Shcherbakova, writing and approving the manuscript. All authors reviewed the manuscript.

Conflict of interest

The authors declare no competing interests.

Ethical approval

This article does not contain any studies with human participants and/or animals performed by any of the authors.

Consent to participate

All authors gave their consent to participate in this study.

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No competing interests reported.

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Acerihabitans arcticus sp. nov., a first psychrophilic member of the family Pectobacteriaceae and able to reduce Fe(III)

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