Pseudomonas aquigelida sp. nov., an Antarctic bacterium isolated from seawater of Fildes Bay, King George Island

doi:10.21203/rs.3.rs-3342923/v1

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Pseudomonas aquigelida sp. nov., an Antarctic bacterium isolated from seawater of Fildes Bay, King George Island

https://doi.org/10.21203/rs.3.rs-3342923/v1

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The isolate Pseudomonas sp. IB20^T was obtained from Fildes Bay seawater, King George Island, Antarctica. To characterize this novel species, a polyphasic approach was addressed, and phenotypic, phylogenetic, phylogenomic, and chemotaxonomic analyses were performed. IB20^T is a rod-shaped and motile bacterium because of multiple polar flagella and shows an optimal growth temperature of 25°C. Phylogenetic analyses based on 16S rRNA gene sequence and multilocus sequence analysis (MLSA) demonstrated that this strain belongs to the Pseudomonas fluorescens group presenting the highest similarity of multilocus sequence with Pseudomonas antarctica LMG22709^T. To further characterize this strain, genomic analyses were made. The average nucleotide identity (ANIb) and average amino acid identity (AAI) scores of all species phylogenetically closest to IB20^T were less than 95–96%, values currently accepted to describe a novel species. P. antarctica CMS 35^T shows the highest ANIb and AAI scores (92,67%, and 95,98%, respectively). A remarkable feature in the IB20^T genome is the presence of the type III secretion system (T3SS), one of the most important virulence elements in pathogenic members of the Pseudomonas genus. This T3SS was not found in any of the genomes of the strains phylogenetically closest to IB20^T. Finally, chemotaxonomic analyses demonstrated that the primary fatty acids making up their cells include summed feature 3 (C_16:1ω7c/C_{15:0iso 2−OH}), C_16:0, and C_18:1ω7c. After conducting comprehensive phenotypic and genotypic analyses, we can propose the IB20^T strain as a novel species within the Pseudomonas genus. Consequently, we designate it as Pseudomonas aquigelida, with the type strain being IB20.

The Pseudomonas genus belongs to the Pseudomonadaceae family and the γ-proteobacteria class (Liu et al. 2021). To date, this genus comprises more than 250 formal species, mainly including gram-negative, aerobic, non-spore-forming, and motile bacilli (Sawada et al. 2020). Pseudomonas genus are well known for their great metabolic and genetic diversity (Koehorst et al. 2016), which results in a wide arrays of metabolic and physiological features such as: i) use a diverse range of compounds as a carbon and energy source; ii) degrade a variety of recalcitrant and toxic chemicals; iii) synthesize low molecular weight compounds and other products used for ecological competition with other microorganisms; and iv) efficient response to abiotic conditions (desiccation, temperature changes, etc). These characteristics provide these organisms with a high adaptability to various environments, allowing them to colonize virtually any environment, no matter how inhospitable it may be on Earth (Arnau et al. 2015). Indeed, members of the Pseudomonas genus have been isolated from desert areas, marine and freshwater (Hahn et al. 2000), soil and sediments or even as commensal and/or pathogens from plants (Zhang et al. 2018; Xin et al. 2018), fungi (Munsch et al. 2000) and different animals, including humans (Oliver et al. 2015; Zhao et al. 2018).

From a taxonomic point of view, the characterization of a novel bacterial species has evolved in parallel with the availability of new methodologies. The first formal description of the Pseudomonas genus was performed by Migula (1895) only based on a morphological and biochemical characterization. At present, the definition of new bacterial species is carried out using a polyphasic approach including phenotypic, chemotaxonomic, and genomic analyses, like Multilocus Sequence Analysis (MLSA); calculation of the average nucleotide identity (ANI) or average amino acid identity (AAI) scores; and digital DNA-DNA hybridization (dDDH) (Arnau et al. 2015; Liu et al. 2021).

The first report of isolation of members of the genus Pseudomonas in Antarctica was made in 1976 (Kriss et al. 1976), but just in 1989, the first novel species was characterized (Shivaji et al. 1989). Subsequently, Reddy et al. (Reddy et al. 2004) described three novel species: Pseudomonas antarctica, Pseudomonas meridiana, and Pseudomonas proteolytica, all associated with cyanobacteria. Subsequently, eleven new formal species isolated in different places of this continent have been described: Pseudomonas guineae (Bozal et al. 2007) and Pseudomonas deceptionensis (Carrión et al. 2011), both isolated in Deception Islands from soil and marine sediments respectively; Pseudomonas pelagia (Koh et al. 2013) isolated from Antarctic Ocean in association with green algae; Pseudomonas extremaustralis (López et al. 2009) isolated from water samples in Cierva Point; Pseudomonas prosekii (Kosina et al. 2013) (unknown source); Pseudomonas gregormendelii (Kosina et al. 2013) from marine sediments and Pseudomonas leptonychotis (Nováková et al. 2020) from seals microbiota, all of them from James Ross Island; Pseudomonas yamanorum (Arnau et al. 2015) isolated from soils in “Isla de los Estados”, (Ushuaia); Pseudomonas fildesensis (Pavlov et al. 2020) from King George Island soils; Pseudomonas neustonica (Jang et al. 2020) in The Ross Sea waters; and recently Pseudomonas versuta (See-Too et al. 2017) from soils located in the Ryder Bay, on Adelaide Island.

In addition to the formal description and characterization of new species, many isolates identified as Pseudomonas sp. have been reported in Antarctica; all of them seem to be adapted to fluctuating conditions of temperature, humidity, availability of carbon sources, and to the presence of antimicrobial compounds (Carrión et al. 2011; Arnau et al. 2015; Vásquez-Ponce et al. 2018; Higuera-Llantén et al. 2018). In this study, we describe a novel species of Pseudomonas isolated from seawater from Fildes Bay on King George Island (Antarctica) using a polyphasic approach. Our results suggest that the strain IB20^T has been classified as a member of the Pseudomonas fluorescens subgroup and can be formally proposed as a new species. Hence, we propose the name Pseudomonas aquigelida for the classification of this microorganism.

Habitat and isolation

The Pseudomonas strain IB20^T, as described in the current study, was isolated from a seawater sample collected at Fildes Bay, King George Island, Antarctica (coordinates: 62°11´50.3´´ S, 58°54´50.3´´ W) in February 2012. The sampling took place during an Antarctic summer research expedition aimed at identifying Pseudomonas spp. with significant biotechnological potential. In-situ filtration of 10 liters of seawater was conducted using a 0.1 mm filter, followed by concentration through a 0.2 µm filter. The resulting filtrates were stored at 4°C until reaching the laboratory facilities of the "Instituto Antártico Chileno, INACH," situated on King George Island, Antarctica. Under aseptic conditions, the filter-retained material was re-suspended in 3 ml of 150 mM NaCl. Subsequently, 100 µL of the supernatant was directly plated onto Nutrient Agar (BD™ DIFCO™, New Jersey, NJ, USA) in triplicate. The plates were then incubated at temperatures of 4, 10, 15, and 25°C for 5 days under aerobic conditions. To isolate and purify bacteria belonging to the genus Pseudomonas, individual colonies were streaked onto Pseudomonas Isolation Agar (PIA) plates (SIGMA-Aldrich, Los Angeles, CA, USA) and incubated for 72 hours, following the temperature regimen. Isolated colonies from the PIA plates were individually selected and preserved at both 4 and − 80°C in a 15% glycerol (v/v). Strain IB20^T was obtained from a 15°C master plate and subsequently chosen for comprehensive characterization. For routine cultivation purposes, IB20^T was cultured in Lysogeny Broth (LB) (BD™ DIFCO™, New Jersey, NJ, USA) at 25°C under aerobic conditions.

Morphological characterization

The purity of the IB20^T strain and the characteristics of its colony morphology were examined using phase-contrast microscopy (Eclipse 80i, Nikon, Japan) following 48 hours of growth at 25°C on LB agar plates. The Gram nature of the strain was determined based on the methodology outlined by Coico et al. (2006). For electron microscopy visualization (Philips Tecnai 12, Biotwin), individual and isolated colonies were employed. These colonies were first fixed with glutaraldehyde (2.0% v/v) and then stained with uranyl acetate (1% v/v, pH 7.0). The cell size was quantified as the average width and length of at least ten cells.

Physiological and biochemical characterization

For all physiological and biochemical characterizations, an individual colony was picked and grown in LB broth, and then incubated at 25 ºC for 24 hours. The optimal growth temperature (25°C) was previously determined by our research group in LB medium, resulting in a doubling time of 43 ± 8 minutes (Vásquez-Ponce et al. 2018). To assess the presence of relevant enzymatic activities and the assimilation of different substrates as the sole energy and carbon source, phenotypic tests were conducted using API 20NE and GN2 Biolog systems, following the methodologies proposed by the manufacturers. The results were evaluated after incubation at 25°C for 24 hours and were previously published by our group in 2018 (Vásquez-Ponce et al. 2018). The phenotypic and biochemical characteristics of IB20^T were compared with those of the type strains of the seven closely related species, as identified through phylogenetic analyses (Table 1).

Table 1

Overall genome-related indexes for the strain *P. aquigelida* sp. nov. IB20^T and the closely related species of the *P. fluorescens* lineage (*16S rRNA* sequence similarity > 98.7%). ANIb (average nucleotide identity by BLAST) values were calculated using the Pairwise Genome Comparison Tool of the web platform JSpecies WS. dDDH (digital DNA-DNA hybridization) values were obtained using the Genome-to-Genome Distance Calculator 3.0 (Richter et al. 2016). IB20^T was used as query (accession number NMRE00000000.1).
Type strain	Accession number	ANIb (%)	AAI (%)	dDDH (%)
Pseudomonas antarctica CMS 35 ^T	JXDI00000000.1	92.67	95.98	49.70
Pseudomonas fluorescens NCTC 10038 ^T	LS483372.1	89.24	93.99	38.10
Pseudomonas edaphica RD25 ^T	VBVZ00000000.1	89.05	93.70	37.80
Pseudomonas salomonii ICMP 14252 ^T	FNOX00000000.1	88.91	93.50	37.20
Pseudomonas costantinii LMG 22119 ^T	MDDR00000000.1	88.71	93.51	37.50
Pseudomonas fildesensis KG01 ^T	LFMW00000000.1	88.15	92.55	55.50
Pseudomonas kitaguniensis MAFF 212408 ^T	VUAZ00000000.1	87.95	91.59	51.90
Pseudomonas grimontii DSM 17515 ^T	FNKM01000002.1	87.83	91.89	35.50
Pseudomonas marginalis ICMP 3553 ^T	LKEG01000000.1	87.71	92.26	34.70
Pseudomonas allii MAFF 301514^T	JABUHT000000000.1	87.66	91.80	52.40
Pseudomonas extremorientalis LMG 19695 ^T	MDGK00000000.1	87.61	92.18	34.40
Pseudomonas haemolytica DSM 108987 ^T	VOIW00000000.1	87.57	92.27	34.70
Pseudomonas veronii DSM 11331 ^T	JYLL01000000.1	87.56	91.28	35.20
Pseudomonas lurida LMG 21995 ^T	PDJB00000000.1	87.52	92.44	34.10
Pseudomonas canadensis 2–92 ^T	AYTD00000000.1	87.50	92.40	34.10
Pseudomonas cyclaminis MAFF 301449 ^T	JADDUM000000000.1	87.49	91.29	48.90
Pseudomonas simiae CCUG 50988 ^T	MDFH00000000.1	87.33	92.36	34.10
Pseudomonas extremaustralis 14 − 3 ^T	AHIP01000000.1	87.09	90.63	34.40
Pseudomonas nabeulensis E10B ^T	QUZT00000000.1	86.99	91.64	48.30
Pseudomonas sivasensis P7 ^T	JAAOWU000000000.1	86.88	91.53	51.50
Pseudomonas pisciculturae P115 ^T	JACVZE000000000.1	86.76	91.56	50.40
Pseudomonas orientalis DSM 17489 ^T	JYLM00000000.1	86.73	91.18	33.20
Pseudomonas kairouanensis KC12 ^T	QUZU00000000.1	86.70	91.15	47.30
Pseudomonas trivialis DSM 14937 ^T	JYLK00000000.1	86.40	90.50	32.80
Pseudomonas poae DSM 14936 ^T	JYLI00000000.1	86.19	90.60	32.40
Pseudomonas rhodesiae LMG 17764 ^T	LT629801.1	85.98	90.69	31.80
Pseudomonas spelaei CCM 7893 ^T	WNNK00000000.1	84.75	89.65	44.70
Pseudomonas brenneri DSM 15294 ^T	LT629800.1	84.60	88.66	29.90
Pseudomonas karstica CCM 7891 ^T	WLYI00000000.1	84.22	89.64	39.90
Pseudomonas kielensis MBT-1 ^T	JACMYG000000000.1	80.96	82.41	28.80
Pseudomonas prosekii LMG 26867 ^T	LT629762.1	80.67	82.94	25.10
Pseudomonas arsenicoxydans CECT 7543 ^T	LT629705.1	80.57	83.10	25.00

Molecular phylogenetic analysis

To begin the comparative analysis of IB20^T with other closely related Pseudomonas species, we initially sequenced the 16S gene and developed phylogenetic analyses. Genomic DNA extraction was carried out using the AxyPrep™ Bacterial Genomic DNA Miniprep Kit (Axygen® Biosciences, Corning). Subsequently, PCR amplification for the 16S gene was conducted using the universal primer pairs: 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). The resulting PCR product was cloned into the pJET 1.2 vector (Thermo Scientific, USA) and subsequently sequenced at Macrogen Inc (Seoul, Korea). The obtained sequence was subjected to analysis using the 16S-based ID tool available on the EZBioCloud Server (https://www.ezbiocloud.net) (Pavlov et al. 2020). To validate these findings, a multi-locus sequence analysis (MLSA) was conducted utilizing the housekeeping genes 16S rRNA, rpoB, rpoD, and gyrB, following the methodology outlined by Bennasar et al. (2010). The analysis included the 32 most closely related Pseudomonas species, selected based on 16S rRNA sequence similarity (> 98.8%), as presented in Table 1. The gene sequences of IB20^T were obtained through PCR amplification and subsequent sequencing at Macrogen Inc, Korea. Comparative gene sequences of the target species were retrieved from the GenBank/EMBL databases as of February 2023. To construct concatenated sequences, we employed the concatenation tool within the Mesquite System for Phylogenetic Computing v.3.40 (Chiba et al. 2007). Multiple sequence alignments for the multilocus sequences were conducted using the Multiple Sequence Comparison by Log-Expectation (MUSCLE) tool integrated (Edgar 2004). Subsequently, pairwise similarity comparisons and maximum-likelihood phylogenetic trees were generated using MEGA-X v.10.2.6 software (Kumar et al. 2018). Sites with less than 100% coverage were excluded, and bootstrap analysis was performed with 1000 iterations.

Genomic characterization

Whole-genome sequencing (WGS) was conducted using the Illumina HiSeq XTM TEN platform with a PE150-plus index read at Novogene (Sacramento, CA, USA). Assembly and gene annotation were executed using Velvet V1.2.03 (Zerbino and Birney 2008) and RAST (Aziz et al. 2008), respectively. Three overall genome-relatedness indexes were calculated using the WGS data: i) average nucleotide identity by BlastN (ANIb); ii) digital DNA–DNA hybridization (dDDH); and iii) average amino acid identity (AAI). Genome sequences of closely related species in the Pseudomonas genus were retrieved from GenBank (Table 1). ANIb and dDDH values between the IB20^T genome and the selected type strains were calculated using the Pairwise Genome Comparisons tool from the JSpeciesWS online platform (Richter et al. 2016) and the GGDC 3.0 web server, respectively.

Finally, characterization of the virulence factors present in the bacterial genome was performed. For this purpose, a predictive analysis was carried out by identifying virulence factors using ABRicate (https://github.com/tseemann/abricate) (Seemann 2018) and the Virulence Factor Database (VFDB) (Liu et al. 2019).

Chemotaxonomic analysis

Chemotaxonomic analysis was conducted to ascertain the complete fatty acid composition of IB20^T. These analyses were carried out by the Identification Service at the German Collection of Microorganisms and Cell Cultures (DSMZ). In brief, IB20^T bacterial cells were cultivated for 24 hours at 25°C under aerobic conditions on LB medium. Cellular fatty acids within the whole cells were analyzed following their conversion into fatty acid methyl esters (FAMEs) through saponification, methylation, and extraction. This process involved minor adjustments to the methodology outlined by Miller (1982) and Kuykendall et al. (1988). The mixtures of fatty acid methyl esters were separated using gas chromatography and subsequently detected using a flame ionization detector, employing the Sherlock Microbial Identification System (MIS) developed by MIDI (Microbial ID, Newark, DE 19711, U.S.A.).

Antimicrobial susceptibility

Antimicrobial susceptibility tests were conducted in Mueller-Hinton medium using the broth microdilution methodology in accordance with CLSI standards (CLSI, 2018). Furthermore, epsilometry tests were carried out using the E-test (Biomerieux®), following the methodology recommended by the manufacturer. Both techniques were used to determine the minimum inhibitory concentration (MIC).

Morphological characterization

The IB20^T colonies displayed a highly convex circular shape with small dimensions, approximately 1.5 mm in diameter. Although they initially appeared to have regular edges, closer inspection with a 10x binocular loupe revealed slight irregularities. These colonies exhibited a vivid greenish-yellow color and a somewhat mucous texture. The cells of IB20^T were identified as gram-negative bacilli through Gram staining. Transmission electron microscopy revealed that IB20^T exhibited a rod-shaped morphology, measuring 1.4–1.6 µm in length and 0.3–0.4 µm in width. Additionally, the presence of one or multiple polar flagella was confirmed (Fig. 1).

Molecular phylogenetic analysis

The 16S identification analysis carried out using the 16S-based ID tool from the EZBioCloud server confirmed that IB20^T is categorized within the genus Pseudomonas, exhibiting a similarity of > 98.8% with 32 validly published Pseudomonas species (Table 1). The most significant similarities were noted with P. antarctica LMG 22709^T (99.79%), P. meridiana DSM 15319^T (99.73%), and Pseudomonas kitaguniensis MAFF 212408^T (99.72%). The 16S rRNA sequence was deposited in the NCBI GenBank database under accession number KT377042.2. Subsequently, a phylogenetic reconstruction based on the 16S rRNA gene sequences was performed, incorporating P. aeruginosa DSM 50071^T as an outgroup. The outcomes of this analysis indicated that IB20^T is part of an extensive cluster alongside P. antarctica LMG 22709^T and P. meridiana DSM 15319, both of which belong to the P. fluorescens subgroup (Fig. 2).

To validate the results obtained from the 16S phylogenetic analysis, a parallel evaluation was conducted utilizing a concatenated sequence of the selected MLSA genes. According to these analyses, IB20^T was positioned within a monophyletic and robust clade with P. antarctica LMG 22709^T, sharing a common closest ancestor with P. fluorescens ATCC 13525^T and Pseudomonas haemolytica DSM 108987^T. Furthermore, there was close relatedness with Pseudomonas costantinii CFBP 5705^T and P. kitaguniensis MAFF 212408^T, underscoring IB20^T's affiliation with the P. fluorescens subgroup (Fig. 3). Despite the notable similarity observed with the 16S rRNA of P. meridiana DSM 15319^T, it occupied a separate clade from IB20^T. Apart from P. antarctica LMG 22709^T (97.16%), none of the phylogenetically nearest species identified through MLSA displayed pairwise evolutionary similarities of the multilocus sequences greater than 97.0% (species cut-off) when compared to IB20^T.

Genomic characterization

The resulting genome size of IB20^T was 6,484,439 base pairs (bp), with a sequencing coverage of 25-fold. The genome encoded a total of 5,973 open reading frames (ORFs) and 54 tRNAs. The G + C content was 59.84 mol%, falling within the expected range described for the Pseudomonas genus (Palleroni 2010). The whole-genome sequencing (WGS) data for IB20^T were deposited in GenBank under accession number GCA_002263605.1. The percentage values of ANIb, AAI and dDDH between IB20^T, and closely related strains were lower than the established cut-off values for defining new species (ANIb: <95%; AAI: 95–96%; and < 70% for dDDH). The strain most closely related to IB20^T at the phylogenomic level was P. antarctica CMS 35^T (ANIb: 92.67%; AAI: 95.98%; dDDH: 49.70%). AAI scores were calculated using the online resource from the Konstantinidis group (http://enve-omics.ce.gatech.edu/aai/) (Konstantinidis and Tiedje 2005). The highest AAI value between the predicted proteomes of IB20^T and the most closely related species was 95.98%, which is above the threshold used for delineating prokaryotic species (Sangal et al. 2018) (Table 1).

However, the most interesting genomic feature of IB20^T was the presence of genes related to the production of the type III secretion system (T3SS) (Fig. 4). T3SS is a macromolecular complex strongly associated with bacterial virulence and is a distinctive feature of pathogenic bacteria like P. aeruginosa and Pseudomonas syringae. The presence of T3SS is uncommon in non-pathogenic Pseudomonas species, and its role in such strains remains poorly understood. This analysis revealed that IB20^T possessed at least 25 genes potentially involved in T3SS production. In contrast, the other phylogenetically related strains lacked genes associated with the production of this macromolecular complex. Similarly, IB20^T was the only strain in this analysis that possessed the complete set of genes related to pyochelin biosynthesis. Pyochelin, along with pyoverdine, constitutes one of the major siderophores produced by P. aeruginosa. It plays a crucial role in the arsenal of virulence factors that enable the survival of this microorganism under conditions of iron scarcity (Braud et al. 2009). Detailed information regarding this analysis is presented in the supplementary table 1.

Phenotypic characterization

For the initial phenotypic characterization, IB20^T exhibited a positive phenotype for catalase and oxidase activities. It displayed the ability to grow at temperatures ranging from 4 to 30°C, with an observed optimum temperature of 25°C. Moreover, it demonstrated resilience under NaCl concentrations of 1–5% (with an optimum of 3.5% w/v) and within a pH range of 4–9 (optimum pH of 7) when cultivated in LB medium. These phenotypic characteristics are common features among environmental Pseudomonas isolates (Garrido-Sanz et al. 2016). Additionally, a comparison of the results obtained from the GN2 Biolog assay with those of the seven closest strains identified in the phylogenetic analysis revealed several phenotypic differences between IB20^T and P. antarctica, its most closely related species. For instance, unlike P. antarctica LMG 22709^T, IB20^T was able to reduce nitrate. However, this trait seems to be uncommon among strains closely related to IB20^T. Furthermore, IB20^T exhibited gelatinase and arginine dihydrolase activities-characteristics absent in P. antarctica LMG 22709^T. In contrast to P. antarctica LMG 22709^T, IB20^T could degrade arginine and utilize L-proline and L-arabinose as sole carbon and energy sources. Notably, all other strains considered in this analysis demonstrated the ability to degrade and assimilate these molecules (Table 2). This suggests that P. antarctica LMG 22709^T might have lost these capabilities during its evolutionary process. Comprehensive details of all phenotypic characteristics evaluated in IB20^T are provided in Tables S2 and S3.

Finally, an antibiotic susceptibility test was conducted, revealing that IB20^T exhibited high levels of resistance to erythromycin, polymyxin, and certain beta-lactam antibiotics (Table 3). Although this isolate demonstrated susceptibility to carbapenems and third- and fourth-generation cephalosporins, it displayed elevated MIC values for penicillins (including carbenicillin, a modified penicillin), as well as first- and second-generation cephalosporins. These MIC values were notably higher than those observed in the human pathogen and multidrug-resistant reference strain P. aeruginosa PAO1 (Table 3).

Table 3

**Differential phenotypic characteristics of IB20**^Tand the closest phylogenetically related species of the genusPseudomonas. Phenotypic characterization was performed using the API 20NE and Biolog GN2 systems. (+) positive; (-) negative; NA, data not available. 1, *P. aquigelida* IB20^T; 2, *P antarctica* LMG 22709^T [data from Reddy et al. (2004)]; 3, *P. haemolytica* DSM 108987^T [data from Hofmann et al. (2020)]; 4, *P. fluorescens* ATCC 13525^T [data from Hofmann et al. (2020) and Sawada et al. (2020)]; 5, *P. edaphica* RD25^T [data from Ramírez-Bahena et al. (2019)]; 6, *P. salomonii* CFBP 2022^T [data from Gardan et al. (2002) and Sawada et al. (2020)]; 7, *P. costantinii* CFBP 5705^T [data from Munsch et al. (2002)]; 8, *P. kitaguniensis* MAFF 212408^T [data from Sawada et al. (2020)). All the strains are positive for oxidase and catalase reaction and effectively assimilate D-glucose, D-mannose, trisodium citrate, and myo-Inositol. None of them assimilate D-raffinose, D-maltose, adipic acid.
Characteristic		1	2	3	4	5	6	7	8
	DNA G + C (mol%)	59.9	60.7	60.0	60.2	59.9	60.2	57.5	59.5
	Growth 37 ºC	-	-	-	-	-	+	-	-
	Fluorescent Pigments	+	-	+	+	NA	+	+	+
	Motility	Single or multiple polar flagella	Motile	Motile	Motile	Multiple polar or subpolar flagella	Motile	Single polar flagellum	Single or multiple polar flagella
Activity
	Nitrate reduction	-	+	-	-	-	-	-	-
	Arginine dihydrolase	+	-	+	+	+	+	+	+
	Gelatin hydrolysis	+	-	+	-	-	+	+	+
Assimilation
	L-arabinose	+	-	+	+	+	+	+	+
	D-mannitol	-	+	+	+	+	NA	+	+
	N-acetyl-glucosamine	+	+	-	+	+	-	+	+
	Potassium gluconate	+	NA	-	+	+	NA	+	+
	Malate	+	NA	NA	+	-	+	NA	+
	Tween 40	+	+	NA	+	w	+	-	+
	D-arabitol	-	NA	+	+	+	NA	+	+
	D-cellobiose	-	-	+	NA	-	NA	-	NA
	meso-Erythritol	+	+	+	+	+	+	+	NA
	Gentibiose	-	NA	+	NA	-	NA	-	-
	α-D-lactose	-	-	+	-	-	NA	-	-
	D-melibiose	-	-	+	-	-	-	-	NA
	L-rhamnose	-	-	+	-	-	-	-	-
	D-sorbitol	-	+	+	+	+	+	+	+
	Sucrose	-	-	-	-	-	+	+	-
	L-proline	+	-	NA	+	+	NA	+	NA
	L-pyroglutamic acid	+	+	NA	+	+	-	+	+

Chemotaxonomic analysis

In the chemotaxonomic analysis, three major components were detected in IB20^T: summed feature 3 (C16:1ω7c/C15:0iso 2-OH), C16:0, and C18:1ω7c. Additionally, a small proportion of hydroxylated fatty acids (C12:0 2-OH, C12:0 3-OH, C10:03-OH, C12:0) as well as C14:0, C18:0, and C17:0 cyclo were identified. While the general fatty acid profile of IB20^T closely resembles that of related strains, there are some slight differences worth noting. For instance, a composition of 15% of the C18:1 ω7c is present, a feature seemingly uncommon among most related strains except for P. haemolytica DSM 108987^T, which appears to be the only other strain sharing this characteristic. Likewise, the summed feature (C16:1ω7c / C15:0 iso 2-OH) and summed feature 8 (C18:1ω7c / C18:1ω6c) could not be detected in IB20^T. Interestingly, the summed feature (C16:1ω7c / C15:0 iso 2-OH) is absent in these strains (with the exception of P. haemolytica DSM 108987^T), whereas most strains exhibit a composition of 12–20% for summed feature 8 (C18:1ω7c / C18:1ω6c) a characteristic that IB20^T lacks. For a comprehensive breakdown of this analysis, please refer to Table 4.

Table 4

**Cellular fatty acids composition (percentages) for IB20**^Tand the most closely relatedPseudomonasspecies. The results are shown as percentages of the total fatty acids detected on the evaluated sample. ND, fatty acid not detected; TR, trace amount (< 1%). 1.P. aquigelida IB20^T; 2.P. antarctica LMG 22709^T; 3.P. haemolytica DSM 108987^T; 4.P. fluorescens ATCC 13525^T;5.P. edaphica RD25^T; 6.P. salomonii CFBP 2022^T; 7.P. costantinii CFBP 5705^T; 8.P. kitaguniensis MAFF 212408^T. Data of P. salomonii and P. edaphica were retrieved from Ramirez-Bahena et al. (2019). Data of *P. antarctica*, *P. fluorescens*, *P. costantinii* y *P. kitaguniensis* were obtained from Sawada et al. (2020). Data of *P. haemolytica* were retrieved from Hofmann et al. (2020).
Fatty Acid	1	2	3	4	5	6	7	8
C _12:0	2.66	1.8	1.9	2.5	2.7	1.7	2.0	1.5
C _14:0	TR	TR	TR	TR	TR	TR	TR	TR
C _16:0	29.04	26.7	31.1	27.8	31.4	33.2	33.5	32.4
C _17:0 cyclo	0.59	1.2	9.0	1.5	1.0	2.6	1.7	2.8
C _18:0	TR	TR	TR	TR	1.3	1.5	TR	TR
C _18:1 ω7c	14.84	ND	15.9	ND	ND	ND	ND	ND
C_10:0 3-OH	4.14	4.1	2.4	3.9	3.2	2.4	3.2	2.8
C _12:0 2-OH	5.49	5.4	4.7	5.9	3.8	4.2	5.1	5.5
C _12:0 3-OH	5.39	4.6	4.1	4.9	4.1	3.9	4.3	4.0
Summed feature 3	36.6	34.1	ND	36.2	31.6	32.0	34.8	36.5
Summed feature	ND	ND	28.3	ND	ND	ND	ND	ND
Summed feature 8	ND	20.0	ND	15.5	20.0	17.4	13.6	12.7
Summed feature 3: C _{16: 1} ω7c / C _16:1 ω6c
Summed feature: C_{16 : 1ω7c} / C_{15 : 0 iso 2−OH}
Summed feature 8: C _18:1 ω7c / C _18:1 ω6c

Description of Pseudomonas aquigelida sp. nov.

Pseudomonas aquigelida sp. nov. [a.qui.ge´li.da. L. fem. n. aqua, water; L. fem. adj. gelida, gelid; N.L. fem. n. aquigelida, of/from gelid water] is a bacterium isolated from seawater collected in Fildes Bay, King George Island (South Shetlands Islands, Antarctica).

IB20^T cells are gram-negative, rod-shaped (1.4–1.6 µm in length x 0.3–0.4 µm in width). At the phenotypic level, it exhibits positive oxidase activity and can grow at temperatures between 4 and 30°C. No growth was observed at 37°C. IB20 cells are motile and possess one or more polar flagella when viewed by transmission electron microscopy. According to API 20 NE and BIOLOG GN2 test results, IB20^T is positive for arginine dihydrolase and gelatin hydrolysis and negative for glucose fermentation, reduction of nitrates, indole production, urease, aesculin hydrolysis, and β-galactosidase. In the same line, IB20^T can assimilate L-proline, L-serine, L-ornithine, inosine, D-trehalose, thymidine, L-leucine, and L-threonine. The main fatty acids contained in the cell of IB20^T included summed feature 3 (C_16:1 ω7c/C_15:0 iso 2-OH), C _16:0, and C _18:1 ω7c).

In brief, genotypic, and phenotypic results shown in this study support the establishment of the Antarctic strain IB20^T as a novel species in the genus Pseudomonas. For this reason, we formally propose the name Pseudomonas aquigelida for this novel specie and IB20^T as the type strain.

Funding information

This study was supported by Instituto Antártico Chileno (INACH) project DT 02-13, the ANID Millennium Science Initiative/Millennium Initiative for Collaborative Research on Bacterial Resistance, MICROB-R, NCN17-08, and by a FONDECYT 11150858 grant. M.S. Pavlov and S. Higuera-Llantén were recipients of a doctoral fellowship from ANID.

Acknowledgments:

We thank Dr. Paris Lavin (Universidad de Antofagasta) for sampling and bacteria isolation support.

Conflicts of interest

The authors declare that there are no conflicts of interest.

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Pseudomonas aquigelida sp. nov., an Antarctic bacterium isolated from seawater of Fildes Bay, King George Island

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