DOI: 10.15468/nc6jhg
title: Bosea psychrotolerans sp. nov., a psychrotrophic alphaproteobacterium isolated from Lake Michigan water

description: This dataset contains the digitized treatments in Plazi based on the original journal article Albert, Richard A., McGuine, Molly, Pavlons, Shawn C., Roecker, Jon, Bruess, Jennifer, Mossman, Shane, Sun, Sona, King, Mike, Hong, Sunhee, Farrance, Christine E., Danner, Joseph, Joung, Yochan, Shapiro, Nicole, Whitman, William B., Busse, Hans-Jürgen (2019): Bosea psychrotolerans sp. nov., a psychrotrophic alphaproteobacterium isolated from Lake Michigan water. International Journal of Systematic and Evolutionary Microbiology 69 (5): 1376-1383, DOI: 10.1099/ijsem.0.003319, URL: http://dx.doi.org/10.1099/ijsem.0.003319AbstractThree strains of a Gram-stain negative bacterium were isolated from Lake Michigan water. 16S rRNA gene sequence analysis revealed that strain 1131 had sequence similarities to Bosea vaviloviae LMG 28367 T, Bosea lathyri LMG 26379 T, Bosea lupini LMG 26383 T, Bosea eneae CCUG 43111 T, Bosea vestrisii CCUG 43114 T and Bosea massiliensis CCUG 43117 T of 99.8, 99.1, 98.4, 98.4, 98.4 and 98.2 %, respectively. The average nucleotide identity value between strain 1131 T and Bosea vaviloviae Vaf-18 T was 93.4 % and the DNA relatedness was 38 %. The primary cellular fatty acids of strain 1131 T were C 16: 1 Ɯ 7 c and C 18: 1 Ɯ 7 c. The primary polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major compound in the quinone system was ubiquinone Q-10 and in the polyamine pattern sym - homospermidine was predominant. Additional phenotypic characteristics included growth at 5 – 35 Ǫ C, pH values of pH 5.5 – 8.0, a salt tolerance range of 0.0 – 1.2 % (w/v), and production of an unknown water soluble brown pigment. After phenotypic, chemotaxonomic and genomic analyses, this isolate was identified as a novel species for which the name Bosea psychrotolerans is proposed. The type strain is 1131 T (NRRL B-65405= LMG 30034).Microbial profiling of fresh water using both culture-dependent and culture-independent methods has revealed that fresh water is composed of both diverse and unique microbial communities. Specially, representatives from multiple phyla and classes including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Verrucomicrobia, Armatimonadetes and Planctomycetes have been isolated from fresh water. Betaproteobacteria are the most common, although Actinobacteria and Bacteroidetes can be nearly as common in some samples. The same phyla and classes have been reported from potable fresh water [1 – 8].A group of five lakes, collectively called the Great Lakes, found in the upper Midwest of the USA, contain the greatest amount of fresh water in the world, comprising approximately 5500 cubic miles of fresh water. Lake Michigan, which is the second largest, contains about 1180 cubic miles of fresh water and serves as a source of potable water for over 7 million people. Using culture-independent techniques, the microbial community found in Lake Michigan water near the city of Milwaukee, WI (USA) has been reported to be primarily composed of Betaproteobacteria followed by Bacteroidetes and Alphaproteobacteria [9 – 11]. However, there are no published reports that characterized the microbial community in Lake Michigan potable water, although Albert and co-workers [12 – 15] have isolated several novel species and a novel genus from Lake Michigan potable water.The genus Bosea was initially described by Das et al. [16] and subsequently emended by La Scola et al. [17]. It presently comprises ten species, including Bosea thiooxidans [16], Bosea eneae [17], Bosea lathyri [18], Bosea lupini [18], Bosea massiliensis [17], Bosea minatitlanensis [19], Bosea robiniae [18] and Bosea vaviloviae [20]. Members of the genus Bosea have been isolated from a variety of sources including hospital water supplies, anaerobic digesters and legume root nodules.Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences showing relationships among strains 1131 T, 13 and 1175 and members of the genus Bosea. Numbers at nodes represent the percentages of bootstrap support based on neighbour-joining analyses of 1000 resampled datasets. Values less than 70 % were not reported. Filled circles indicate that the corresponding nodes (groupings) were also recovered in the maximum-likelihood tree. Bradyrhizobium japonicum USDA 6 T (AP012206) was used as the outgroup. Bar, 0.002 nucleotide substitutions per position.As part of a taxonomic investigation of the bacterial population in Lake Michigan potable water, samples were collected from the Port Washington, Wisconsin community water system. Water samples were analysed using plate count agar (PCA; Difco) and incubated at 32 Ǫ C for 5 days. Multiple colonies were isolated and characterized. From a water sample collected on 12 November 2013 one strain, designated 1131 T, was determined to be a novel species of the genus Bosea. Additional strains similar to strain 1131 T were subsequently isolated from water samples collected on 29 December 2013 (strain 1175) and 14 January 2015 (strain 13). In this paper we report on the characterization of these strains. Based upon phenotypic (morphological, physiological and chemotaxonomic traits) and genotypic studies, strain 1131 T represents previously undescribed species of the genus Bosea for which we propose the name, Bosea psychrotolerans. For comparison to other Bosea species the type strains B. vaviloviae LMG 28367 T (=Vaf-18 T), B. lathyri LMG 26379 T (=DSM 26656 T) and B. lupini LMG 26383 T were obtained from the BCCM / LMG (Gent, Belgium) while Bosea eneae CCUG 43111 T, Bosea vestrisii CCUG 43114 T and Bosea massiliensis CCUG 43117 T were obtained from the CCUG (Göteborg, Sweden).The isolation of DNA and generation of PCR products for 16S rRNA sequence analysis and assembly were performed using the procedure of Hong et al. [21]. The identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarity were achieved using the EzBioCloud server (www.ezbiocloud.net) [22]. When compared to the 16S rRNA sequence generated by genomic sequencing (Illumina), the sequence generated by Sanger sequencing was identical to the one generated by genomic sequencing.Phylogenetic and molecular evolutionary analyses were conducted by using MEGA 6.06 software [23]. Evolutionary distance was calculated on the basis of the Jukes and Cantor model [24] and phylogenetic trees were inferred on the basis of the neighbour-joining [25] and maximum-likelihood [26] models. The tree topologies were evaluated by bootstrap analysis based on 1000 resamplings of the sequences [27]. All of the phylogenetic trees generated in this study indicated that strain 1131 T belonged to genus Bosea and the closest neighbour was B. vaviloviae Vaf-18 T (Fig. 1). Sequence similarities of the 16S rRNA genes between strain 1131 T and strains B. vaviloviae LMG 28367 T, B. lathyri LMG 26379 T, B. lupini LMG 26383 T, B. eneae CCUG 43111 T, B. vestrisii CCUG 43114 T and B. massiliensis CCUG 43117 T were 99.8, 99.1, 98.4, 98.4, 98.4 and 98.4 %, respectively. Strain 1131 T and strains 13 and 1175 shared a 16S rRNA sequence similarity of 100 %.Fig. 2. Phylogenetic tree based on the concatenated sequences (1933 bp) of five housekeeping gene fragments (rpoB, gyrB, atpD, dnaK and recA) of Bosea species with validly published names and strains 1131 T, 1175 and 13. Numbers at nodes are given in percentages and represent the levels of bootstrap support based on neighbour-joining analyses of 1000 resampled datasets. Values &lt;70 % are not reported. Filled circles indicate that the corresponding nodes (groupings) were also recovered in the maximum-likelihood tree. Bradyrhizobium japonicum USDA 6 T (AP012206) was used as the outgroup. Bar, 0.01 nucleotide substitutions per position. Sequence accession numbers for all strains are reported in Table S2.Similar to what has been previously reported for other members of the genus Bosea, the 16S rRNA gene sequence of strain 1131 T did not clearly discriminate between strain 1131 T and other members of the genus. Thus multiple approaches were used to determine the relationship between strain 1131 T and other members of the genus Bosea. The genetic similarity between strains 1131 T, B. vaviloviae LMG 28367 T and B. lathyri LMG 26379 T were compared by DNA–DNA hybridization using the method of [28] with photobiotin-labeled DNA probes and microdilution wells. Five replications were performed for each sample. The highest and lowest values were excluded and the remaining three values were used to determine the mean percent similarity. The experimental controls had values consistent with demonstrating that the experiment was correctly performed. By this method strain 1131 T and B. vaviloviae LMG 28367 T and B. lathyri LMG 26379 T had DNA:DNA relatedness values of 38 and 12 %, respectively. These values are consistent with the placement of strain 1131 T in a separate species distinct from B. vaviloviae and B. lathyri [29].The draft genome of strain 1131 T was sequenced at the DOE Joint Genome Institute (JGI) using Illumina technology [30] as part of the GEBA Phase III study [31]. An Illumina standard shotgun library was reconstructed and sequenced using the Illumina HiSeq-25001 TB platform which generated 7 132 838 reads totaling 1 077 058 538 bp. Raw Illumina sequence was quality filtered using BB Tools [32] per SOP 1061. The following steps were then performed for assembly: (1) artifact filtered and normalized Illumina reads were assembled using SPAdes (version 3.10.1; -phred-offset 33 -cov-cutoff auto -t 16 –m 115 – careful –k 25,55,95–12) [33]; (2) 200 bp was trimmed from all contig ends and contigs discarded if the length was &lt;1 kbp or read coverage was less than two (BB Tools reformat.sh: minilength). The final assembly was based on 1 053 118 370 bp of Illumina data with a mapped coverage of 164.3×. Genome annotation was performed using the DOE-JGI annotation pipeline [34]. Additional analysis and functional annotation were performed within the Integrated Microbial Genomes (IMG) platform [35] developed by JGI.The average nucleotide identity (ANI) values [36 – 38] between the genome of strain 1131 T and those of B. vaviloviae Vaf 18 T and B. lathyri DSM 26656 T were determined by using JSpeciesWS (http://species.ribohost.com/jspeciesws/) [39]. The ANI value between the strain 1131 T and B. vaviloviae Vaf-18 T was 93.4 %. When compared to B. lathyri LMG 26379 T the ANI value was 81.4 %. Both values are less than the value of 95 %, which is the presently accepted cut-off value to determine if two genomes are from the same species or not, indicating that strain 1131 T is a distinct species from B. vaviloviae and B. lathyri.Table 1. Morphological and physiological characteristics of strains 1131 T, 1175, 13, B. vaviloviae LMG 28367 T, B. lathyri LMG 26379 T, B. lupini LMG 26383 T, B. eneae CCUG 43111 T, B. vestrisii 43114 T, B. massiliensis 43117 T. All data from the present studyStrains: 1, 1131 T; 2, 1175; 3, 13; 4, B. vaviloviae LMG 28367 T; 5, B. lathyri LMG 26379 T; 6, B. lupini LMG 26383 T; 7, B. eneae CCUG 43111 T 8, B. vestrisii 43114 T; 9, B. massiliensis 43117 T. All species share the characteristics of being Gram-stain-negative rods, motile; negative for anaerobic growth, cell bound orange pigments, use of sucrose, D- lactose, D- maltose, D- rhamnose, raffinose, D- inositol, L-threonine and hydrolysis of casein, gelatin and starch.1 2 3 4 5 6 7 8 9 Catalase W+ W+ W+- W+ + W+ W+ W+ W+ Oxidase ‒ ‒ ‒ ‒ + ‒ ‒ ‒ ‒ Temperature growth range (Ǫ C) 5–35 5–35 5–35 10–40 5–35 10–35 10–40 10–40 10–45 NaCl, % (w/w) tolerance 0–1.2 0–1.4 0–1.2 0–1.4 0–1.2 0–1.6 0–1.8 0–1.8 0–1.2 pH growth range 5.5–8.0 5.5–7.5 5.5–7.5 6.0–7.5 6.0–8.0 6.0–9.0 6.0–8.5 6.0–8.5 6.5–8.0 Nitrate reduction + + + ‒ ‒ ‒ ‒ ‒ + Water soluble brown pigment + + + ‒ ‒ ‒ ‒ ‒ ‒ Substrate assimilation: D- Glucose + + + ‒ + + + + ‒ Sucrose ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ D- Fructose W+ W+ W+ ‒ + + ‒ + ‒ Cellobiose + + + + + ‒ ‒ + ‒ D- Mannose ‒ ‒ ‒ ‒ + + + + ‒ D- Mannitol ‒ ‒ ‒ ‒ ‒ ‒ ‒ + ‒ D- Glycerol ‒ ‒ ‒ ‒ + + + + ‒ D- Xylose W+ W+ W+ + + W+ W+ + ‒ D- Galactose + + + + + + + + ‒ D- Arabinose + + + + W+ W+ + + ‒ Trehalose ‒ ‒ ‒ + ‒ ‒ ‒ ‒ ‒ D- Ribose + + + + + W+ + + ‒ Sodium gluconate + + + + + ‒ + + ‒ Tween 20 + + W+ + + ‒ ‒ + ‒ Tween 80 + + + + ‒ W+ + + ‒ Sodium citrate + + + ‒ + ‒ ‒ + ‒ Sodium acetate ‒ ‒ ‒ ‒ + ‒ + ‒ ‒ Sodium propionate ‒ ‒ ‒ ‒ ‒ ‒ ‒ W+ ‒ L- Lysine ‒ ‒ ‒ ‒ ‒ ‒ + + ‒ L- Histidine ‒ ‒ ‒ + + + ‒ + ‒ L- Leucine + + + + + + + + ‒ L- Valine W+ W+ + + W+ W+ W+ ‒ ‒Multilocus sequence analysis (MLSA) using housekeeping gene sequences has been previously used to resolve different species in the genus Bosea [18, 20]. In the present study, MLSA, based on partial sequences of five protein coding genes, rpoB, gyrB, atpD, dnaK and recA were used to estimate the phylogenetic relationship among strains 1131 T, 1175 and 13 and other member of the genus Bosea, including, B. vaviloviae Vaf-18 T, B. lathyri LMG 26379 T, B. lupini LMG 26383 T, B. eneae CCUG 43111 T, B. vestrisii CCUG 43114 T, B. minatitlanensis LMG 26207 T, B. robiniae LMG 26381 T, B. thiooxidans LMG 26210 T and B. massiliensis CCUG 43117 T. The source of each sequence is summarized in Table S1 (available in the online version of this article), and the primer sequences used in this study for all five housekeeping genes are listed in Table S2. Prior to concatenating the gene sequences, the gene sequences were trimmed to the same length. The concatenated gene sequences were aligned using the CLUSTAL_W program contained in the MEGA 6.06 software package [23] and phylogenetic trees were reconstructed using the neighbour-joining method. Evolutionary distances were calculated using the Jukes and Cantor model [24] and the bootstrap analysis with 1000 replicates was performed to assess support for the clusters. The three novel strains formed a monophyletic cluster in the genus Bosea with a high bootstrap value (100 %). This cluster was genetically distinct from Bosea vaviloviae Vaf- 18 T and other species in the genus Bosea, clearly demonstrating that strain 1131 T, 1175 and 13 represent a novel species within the genus Bosea (Fig. 2). The sequence similarity between the three strains and different Bosea type strains for gyrB ranged from 98.0 to 89.9 %, for rpoB from 98.6 to 92.3 %, for atpD from 89.7 to 86.2 %, for dnaK from 98.0 to 87.4 % and for recA from 97.1 to 78.8 %.Table 2. Cellular fatty acid composition of strains 1131 T, 1175, 13, B. vaviloviae LMG 28367 T, B. lathyri LMG 26379 T, B. lupini LMG 26383 T, B. eneae CCUG 43111 T, B. vestrisii CCUG 43114 T, B. massiliensis CCUG 43117 T. All data are from the present studyNot reported are cellular fatty acids at &lt;0.50 %.Strains: 1, 1131 T; 2, 1175; 3, 13; 4, B. vaviloviae LMG 28367 T; 5, B. lathyri LMG 26379 T; 6, B. lupini LMG 26383 T; 7, B. eneae CCUG 43111 T 8, B. vestrisii CCUG 43114 T; 9, B. massiliensis CCUG 43117 T1 2 3 4 5 6 7 8 9 14: 0 0.5 0.5 0.4 0.5 0.2 0.3 0.1 0.1 0.0 15: 0 1.6 1.6 1.5 1.3 2.2 0.4 0.5 0.2 0.2 16: 0 3OH 4.8 5.2 4.7 5.2 3.5 4.7 4.6 4.9 4.0 16: 1 Ɯ 7 c 17.1 16.9 16.4 13.4 12.4 11.3 6.9 10.2 6.6 16: 1 Ɯ 5 c 6.6 6.4 5.8 2.7 7.3 3.8 6.4 5.4 5.3 16: 0 5.0 5.0 5.1 7.2 3.7 5.4 4.9 5.3 2.3 17: 1 Ɯ 8 c 1.6 1.4 1.4 0.9 2.3 0.5 0.8 0.2 0.8 17: 0 cyclo 2.7 3.6 3.1 6.0 4.0 2.2 1.8 1.6 0.0 17: 0 0.9 0.8 0.9 0.7 1.0 0.4 1.2 0.2 0.6 18: 1 Ɯ 7 c 53.1 52.4 53.9 52.2 55.4 65.4 65.1 66.0 75.1 18: 0 0.5 0.4 0.5 0.4 0.4 0.3 0.8 0.3 0.8 18: 1 Ɯ 7 c 11-methyl 0.4 0.4 0.4 0.4 0.3 0.3 0.4 0.3 1.1 17: 0 3OH 0.4 0.4 0.4 0.4 1.0 ND 0.3 ND 0.2 19: 0 cyclo Ɯ 10 c 0.4 0.3 0.3 0.4 0.3 0.2 0.4 0.2 0.0 19: 0 cyclo Ɯ 8c 3.8 4.1 4.4 7.3 5.2 4.3 5.3 4.3 2.0 20: 4 Ɯ 6,9,12,15 c 0.4 0.4 0.4 0.4 0.4 0.3 0.2 0.3 0.0The clonal relationship between the three strains was examined by genomic fingerprinting using six RAPD-PCR primers (Fig. S1). One primer showed that strain 13 was not clonally related to strains 1131 T and 1175, while a different primer showed that strain 1131 T was not clonally related to strains 13 and 1175. However, the finding that the three strains had several similar fingerprints supported the MLSA results indicating that strains 1131 T, 13 and 1175 are members of the same species.When grown on PCA (Difco) at 25 Ǫ C for 5 days, colonies of strain 1131 T produced copious amounts of extracellular material while isolated colonies were circular, flat, 1–3 mm in diameter, and with an entire edge. Cells were rod-shaped, 2–3 µm long. When grown on plate count agar (Difco) at 25 Ǫ C for 2 days growth occurred as a rosette (Fig. S2). Cells were prepared for SEM imaging by fixing the cells in a glutaraldehyde buffer, multiple risings, then depositing onto Nucelopore membranes. After dehydration and drying followed by coating samples, were examined using a Hitachi S- 4800 field emission scanning electron microscope (personal communication, Heather Owen, Microscopy Centre, UW- Milwaukee).Strain 1131 T grew well on R2A (Difco), PCA (Difco), tryptic soy agar (Difco), eosine methylene blue (Difco), MacConkey agar (Difco), and sabouraud dextrose agar (Oxoid) with reduced growth on violet red bile agar (Difco). Growth did not occur on cetrimide agar (Difco) and Pseudomonas isolation agar (Difco). When grown on PCA and incubated at 25 Ǫ C for at least 96 h strains 1131 T, 13 and 1175 produced a brown pigment that diffuses into the agar. The brown pigment was not produced when grown on TSA, TSBA and in plate count broth (Difco). This attribute was not observed with the reference stains B. vaviloviae LMG 28367 T, B. lathyri LMG 26379 T, B. lupini LMG 26383 T, B. eneae CCUG 43111 T, B. vestrisii CCUG 43114 T and B. massiliensis CCUG 43117 T.The physiological and biochemical characterizations reported in Table 1 were performed as follows. All tests were performed at 25 Ǫ C, except while examining growth temperature range, using 24–48 h old cultures grown on PCA. Catalase activity, oxidase test, Gram staining were performed using standard procedures [40]. The temperature growth range was determined by visual examination for growth on PCA plates. Prior to inoculation, the plates were incubated at the test temperature for 24 h. After inoculation, plates were incubated from 2 to 10 days. Growth at various concentrations (w/v) of NaCl was determined using plate count broth as the base supplemented with a range of concentrations (0.5 % w/v) of NaCl. The pH growth range was determined using phosphate buffered plate count broth at 25 Ǫ C for 7 days with shaking aeration [41]. After autoclaving, the pH value of the broth was checked prior to inoculation. The ability to utilize various substrates including sugars, fatty acids and amino acids was determined as described by Albert et al. [15] except that the base growth medium was supplemented with 0.0025 % yeast extract (Difco). After inoculation, tubes were incubated at 25 Ǫ C with shaking aeration. The presence of turbidity was used to indicate if the substrate was supporting growth. Anaerobic growth was determined by visually monitoring for growth on plates containing PCA supplemented with 0.1 % sodium nitrate. Prior to inoculation, the PCA plates were incubated under anaerobic conditions for 24 h at 25 Ǫ C. Anaerobic conditions were generated by using H 2 and CO 2 GasPaks (BBL) in anaerobic jars. After inoculation, the plates were placed in the anaerobe jars and anaerobic conditions were regenerated using the H 2 and CO 2 GasPaks. Ten days after inoculation, the plates were visually examined for growth. Production of cell-bound orange pigments was determined by the technique of Yabuuchi et al. [42].Results of physiological and biochemical characterization of strains 1131 T, 13 and 1175 are summarized in Table 1. The temperature and pH growth range for strain 1131 T was 5–37 Ǫ C and 5.5 to 8.0, respectively, while the [NaCl] for growth was 0.0 to 1.2 % (w/v). Similar results were seen with strains 13 and 1175 although the upper pH limit for growth for strains 13 and 1175 was 7.5 and the upper [NaCl] for growth was 1.4 % (w/v) for strain 1175. Minor differences between the three strains were also seen in their respective ability to use different compounds as carbon and energy sources. Cell-bound orange pigments were not produced by any of these strains.The fatty acid profile of strain 1131 T and strains 13 and 1175 was determined by growing all strains on PCA at 28 Ǫ C for 24 h. After growth, bacteria were saponified, and the liberated fatty acids were methylated and analysed by capillary gas-liquid chromatography (Agilent HP 6890) by the Sherlock system (MIDI 6.2) according to the manufacturer’ s instructions. The fatty acid profile of strain 1131 T, 13 and 1175 are reported in Table 2. For strain 1131 T, the primary fatty acids, which comprised approximately 70 % of the total, were C 16:1 Ɯ 7c and C 18:1 Ɯ 7c. These two fatty acids were also the primary fatty acids for strains 13 and 1175.Comparing the fatty acid profile of strain 1131 T to that of other members of the genus Bosea (Table 2), reveals the sharing of C 18:1 Ɯ 7 c and C 16:1 Ɯ 7 c as the primary fatty acids, with levels of C 18:1 Ɯ 7 c ranging from 52.2 to 75.1 %, and levels of C 16:1 Ɯ 7 c ranging from 6.6 to 17.1 %. There was no difference in the type of secondary fatty acids although there were differences in the level.When compared to previously published cellular fatty acid profiles for the reference strains B. vaviloviae LMG 28367 T, B. lathyri LMG 26379 T, B. lupini LMG 26383 T, B. eneae CCUG 43111 T, B. vestrisii CCUG 43114 T and B. massiliensis CCUG 43117 T, the results from this study have both similarities and differences to previously published results [17, 18, 20]. For example, when compared to the previously published results for B. vaviloviae Vaf-18 T [20], this study found that the two primary fatty acids were C 16:1 Ɯ 7 c and C 18:1 Ɯ 7 c comprising 65.6 % of the total, with each accounting for 13.4 and 52.2 %, respectively of the total. Safronova et al. [20] reported that C 18:1 Ɯ 7 c and C 17:0 cyclo were the two primary fatty acids, comprising 58.8 % of the total with each accounting for 43.4 and 13.5 %, respectively of the total. In addition, an inseparable mixture of C 16:1 Ɯ 7 c / C 15: 0 2-OH accounted for 6.4 % of the total. The present study found that C 17:0 cyclo accounted for 6.0 % of the total. Differences were probably due to differences in growth media, with the present study using PCA while Safronova et al. [20] used BCCM / LMG medium 115, and with incubation time. In the present study, the culture was incubated under standard MIDI conditions while Safronova et al. [20] reported that they deviated from standard MIDI incubation time, but did not report what the deviation was. In addition, it appears that there can be variation between strains in the amount of a particular fatty acid present. For example, La Scola et al. [17] reported variability in the levels of C 16:1 Ɯ 7 c and C 18:1 Ɯ 7 c between various strains of B. vestrisii.Polar lipids, quinones and polyamines were extracted from cells grown on PYE medium [0.3 % peptone from casein (w/ v), 0.3 % yeast extract (w/v), pH 7.2] at 28 Ǫ C. Polar lipids and quinones were extracted from biomass harvested at the stationary growth phase and analysed as described previously [43 – 45]. Polyamines were extracted from biomass harvested at the late exponential growth phase as described by Busse and Auling [46] and analyzed as described by Busse et al. [47]. HPLC analyses were carried out using the equipment described by Stolz et al. [48].The polar lipid profile of strain 1131 T was composed of the major lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, moderate to minor amounts of phosphatidylmonomethylethanolamine, two unidentified aminolipids, one unidentified glycolipid (GL1), and two unidentified polar lipids lacking a functional group (Fig. S3a). This polar lipid profile was highly similar to those of the closely related species B. vaviloviae LMG 28367 T (Fig. S3b) and B. lathyri LMG 26379 T (Fig. S3c). The polar lipid profile of strain 1131 T differed from that of B. vaviloviae LMG 28367 T only in the presence of the minor unidentified aminolipid AL2 and the absence of lipid L2. The most striking difference in the polar lipid profile of B. lathyri LMG 26379 T was the presence of two additional unidentified glycolipids (GL2, GL3).The quinone system of strain 1131 T was composed of ubiquinone Q-10 (97.5 %) and Q-11 (2.5 %). The quinone systems of B. vaviloviae LMG 28367 T and B. lathyri LMG 26379 T, both of which were initially described without providing information regarding the quinone system, showed similar characteristics. The primary quinone for B. vaviloviae LMG 28367 T and B. lathyri LMG 26379 T was Q-10, comprising 98.0 and 97.1 %, respectively, while Q-11 comprised 2.0 and 2.9 %, respectively. These quinone systems resemble those of many Alphaproteobacteria. The polyamine pattern of 1131 T was composed of sym -homospermidine [47.3 µmol (g dry weight) ‒1], spermidine [2.1 µmol (g dry weight) ‒1], putrescine [2.5 µmol (g dry weight) ‒1], cadaverine [0.2 µmol (g dry weight) ‒1] and spermine [0.1 µmol (g dry weight) ‒1]. The polyamine pattern of B. vaviloviae LMG 28367 T was composed of sym -homospermidine [35.9 µmol (g dry weight) ‒1], spermidine [8.0 µmol (g dry weight) ‒1], putrescine [3.9 µmol (g dry weight) ‒1] and spermine [0.6 µmol (g dry weight) ‒1]. The polyamine pattern of B. lathyri LMG 26379 T contained sym -homospermidine [39.5 µmol (g dry weight) ‒1], spermidine [5.7 µmol (g dry weight) ‒1], putrescine [3.2 µmol (g dry weight) ‒1], cadaverine [0.1 µmol (g dry weight) ‒1] and spermine [0.1 µmol (g dry weight) ‒1]. The polyamine patterns of the three strains were very similar and are well in agreement with the affiliation of the three strains to the Alphaproteobacteria.On the basis of the phenotypic (including chemotaxonomic traits) and phylogenetic data, we conclude that strain 1131 T represents a novel species, for which the name Bosea psychrotolerans is proposed.