DOI: 10.15468/efj9av
title: Pannonibacter carbonis sp. nov., isolated from coal mine water

description: This dataset contains the digitized treatments in Plazi based on the original journal article Xi, Lijun, Qiao, Nenghu, Liu, Dejian, Li, Jing, Zhang, Jingjing, Liu, Jianguo (2018): Pannonibacter carbonis sp. nov., isolated from coal mine water. International Journal of Systematic and Evolutionary Microbiology 68 (6): 2042-2047, DOI: 10.1099/ijsem.0.002794, URL: http://dx.doi.org/10.1099/ijsem.0.002794AbstractTwo bacterial strains were isolated from coal mine water in China. Isolates were facultatively anaerobic, Gram-stainnegative, rod-shaped, motile by means of a single polar flagellum, and they did not produce bacteriochlorophyll a. Cells grew in tryptic soy broth with 0–5.5 % (w/v) NaCl, at 4–55 Ǫ C and pH 3.5–10.5. Isolates were positive for catalase, oxidase, urease, Voges–Proskauer test, gelatin hydrolysis and H 2 S production. Analysis of 16S rRNA gene sequences indicated that the closest relatives of strains Q4.6 T and Q2.11 were the type strains Labrenzia suaedae DSM 22153 T (97.4 %), Pannonibacter phragmitetus DSM 14782 T (96.9 and 97.0 %) and Pannonibacter indicus DSM 23407 T (96.8 %). The genomic average nucleotide identity (ANI) value for Q4.6 T and Q2.11 was 100 %; however, this value was less than 77.7 % for the type strains P. phragmitetus and P. indicus, and less than 74.0 % for the type strain L. suaedae. The cellular fatty acid profile of strains Q4.6 T and Q2.11 consisted primarily of C 18: 1 Ɯ 7 c. The principal quinone of the isolates was Q-10. The polar lipid profile consisted of diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine and phosphatidyl choline. On the basis of phylogenetic analysis, genomic ANI analysis, DNA–DNA hybridization results, as well as phenotypic and chemotaxonomic data, strains Q4.6 T and Q2.11 are assigned as a novel species within the genus Pannonibacter. The type strain is Pannonibacter carbonis Q4.6 T (=CGMCC 1.15703 T =KCTC 52466 T).Pannonibacter was first established in 2003 as a novel and independent genus related to the Stappia – Roseibium cluster in the a- subclass of Proteobacteria [1]. Until now, only two recognized species existed [2]. The natural habitats of these species, such as soda lakes and hot springs, suggests that members of this genus are extremophiles; however, strains assigned to Pannonibacter phragmitetus (DSM 14782 T) have been observed in human blood cultures [3], suggesting that some Pannonibacter species may be related to human clinical infections. Pannonibacter species can usually grow in alkaline conditions (up to pH 11), are arsenate- [4] and chromium-tolerant [5 – 9], and most cells produce polyhydroxyalkanoate (PHA) granules [10]. In 2007, the genus Labrenzia was separated from the genera Stappia, Pannonibacter and Roseibium. Reclassification was mainly based on genomic variation, including the presence of bacteriochlorophyll a (BChl a) and pufLM genes, as well as differences in physiological and biochemical characteristics [11]; however, many Labrenzia species do not possess pufLM genes [12]. Furthermore, one recently described species that harbours pufLM genes is not able to produce BChl a [13]. Therefore, the distinction between the genera Pannonibacter and Labrenzia is somewhat obscure. Although some differences exist between the morphological, physiological and biochemical properties of Pannonibacter and Labrenzia, they also share many similar characteristics, including Gramstain reactions, major quinone components, and fatty acid and polar lipid profiles.Many studies demonstrate that coal beds are generally colonized by an array of bacteria, which commonly include members of the phylum Proteobacteria with abundances up to 30 % [14 – 16]. This indicates that Proteobacteria play important roles in the coal mine biosphere. While studying the bacterial diversity of coal mine water samples collected from coal producing wells in Jinchen (110 Ǫ 55′ E, 35 Ǫ 30′ N), Shanxi Province, China, strains Q4.6 T and Q2.11 were isolated and purified. The water sample was mixed well at room temperature, serially diluted and plated on coal mine water agar. After 7 days incubation at 30 Ǫ C, the strain was transferred and purified on International Streptomyces Project medium 2 [17]. Pannonibacter indicus DSM 23407 T and Labrenzia suaedae DSM 22153 T (provided by the Leibniz Institute DSMZ, Germany) were used as references strains in this study.Cell growth under anaerobic and photoorganoheterotrophic conditions was tested according to the method described by Xi et al. [18]. The in vivo absorption spectrum of intact bacterial cells in 60 % sucrose (w/v) and the absorption spectrum of pigments extracted with acetone were recorded using a UV-2450 visible spectrophotometer (Shimadzu). The morphological characteristics of the isolates, Q4.6 T and Q2.11, and reference strains were observed on tryptic soy agar (TSA; Oxoid) or in tryptic soy broth (TSB; Oxoid) following incubation for 2 – 5 days at 30 Ǫ C. Cell morphology was investigated using a transmission electron microscope (JEM-1400, JEOL). Flagella were observed after staining with Ryu solution [19]. Gram stains were conducted as per the normal protocol [20] and observed by light microscopy (Leica, DMI3000B). The growth temperature range was determined in TSB by incubating at 4, 18, 25, 30, 35, 37, 45, 50 and 55 Ǫ C for 5 days. The optimum pH and range was determined by growing the strains at 37 Ǫ C for 5 days in TSB, prepared with buffered solutions, ranging from 3.0 to 11.0. The pH was adjusted in steps of 0.5 pH unit. The composition of the buffer systems were as follows (filtered to remove bacteria): pH 3.0 – 7.0, 0.1 M citric acid/0.2 M Na 2 HPO 4; pH 8.0 – 9.0, 0.2 M Tris/0.2 M HCl; pH 10.0 – 11.0, 0.05 M NaHCO 3 /0.1 M NaOH [21]. Growth at various NaCl concentrations from 0 – 10 % was investigated by incubating cells in amended TSB for 3 – 5 days. The Bojian Gram-negative bacterial identification system (Qingdao) was used to determine the Voges – Proskauer (VP) test, H 2 S production, urease, oxidase and gelatin hydrolysis. Aerobic assimilation and acid production using a number of carbohydrates as the sole carbon source (final concentration: 0.1 % (w/v)) were tested at 30 Ǫ C, according to Dong and Cai [22]. Biomass for quantitative fatty acid analysis was obtained by scraping colonies from TSA plates following incubation at 30 Ǫ C for 3 days. Biomass for additional chemotaxonomic studies was obtained after 3 – 5 days cultivation in TSB at 30 Ǫ C with shaking. Standard analytical procedures were used to extract and analyse isomeric quinones [23 – 25] and polar lipids [26, 27]. Fatty acids were analysed using the standard Microbial Identification System (MIDI, Sherlock version 6.1) and gas chromatography (Agilent GC 6890). The resulting profiles were identified using the TSBA40 database [28].Genomic DNA was extracted as described by Chun and Goodfellow [29]. PCR amplification of the 16S rRNA gene was performed using universal bacterial primers 27F and 1492R [30]. Amplification of the pufLM gene was performed as described by Nagashima et al. [31]. 16S amplicons were purified using the TIANgel midi purification kit DP209 (TIANGEN Bio). Amplicons were then ligated into a PMD19-T vector and transformed into competent Escherichia coli DH5a cells using the PMD19-T cloning kit (Takara Bio). Sequencing was performed by Sangon Bio. The isolates were identified using the EzBioCloud server based on the 16S rRNA sequence data [32]. Phylogenetic trees were reconstructed using MEGA (version 7.0) [33] based on approximately 1400 nucleotides from each related type strain (retrieved from the GenBank/EMBL/DDBJ database) mentioned above. Breoghania corrubedonensis UBF-P1 T was used as an outgroup. The neighbour-joining (NJ) [34], maximum-parsimony (MP) [35] and maximum-likelihood (ML) [36] methods were used for phylogenetic analysis. Phylogenetic tree topologies were determined using bootstrap analysis [37] based on 1000 replicates.DNA – DNA hybridization (DDH) was carried out using the microplate method with biotinylated DNA probes [38, 39]. The reciprocal hybridization experiment (i.e. A×B and B×A) was performed in triplicate for each pair of strains. Hybridization values, measured by a SpectraMax M5e (Molecular Devices), were expressed as the mean of the corresponding reciprocal values. The draft genomes of Q4.6 T and Q2.11 were sequenced on the Illumina MiSeq platform, according to previously published methods [40]. Briefly, genomic DNA was extracted and cut to the appropriate size using g-TUBE (Covaris). A mate-pair fragment library was generated using the TruSeqTM DNA Sample Prep Kit-Set A (Illumina). The mate-paired library was amplified using the TruSeq PE Cluster Kit (Illumina) and sequenced on the Illumina MiSeq platform. Average nucleotide identity (ANI) values were calculated using OrthoANI, an improved ANI algorithm developed to accommodate the concept of orthology values [41], in order to determine the similarity of Q4.6 T to related strains.An almost-full length 16S rRNA gene sequence (1497 bp) was confirmed for strains Q4.6 T and Q2.11. The 16S rRNA gene sequence similarity between strain Q4.6 T and Q2.11 was 99.9 %, with only one differing nucleotide. The closest relatives to Q4.6 T and Q2.11, as determined by 16S rRNA gene sequences, were the type strains L. suaedae DSM 22153 T (97.4 %), P. phragmitetus DSM 14782 T (96.9 and 97.0 %, respectively) and P. indicus DSM 23407 T (96.8 %). Sequence similarity between the isolates and other related species was less than 96.8 % (96.7 % – 93.0 %). The phylogenetic trees [NJ tree (Fig. 1), ML tree (Fig. S1, available in the online version of this article) and MP tree (Fig. S2)] demonstrate that strains Q4.6 T and Q2.11 form a stable and unique, but loose branch within the Pannonibacter genus, indicating that the relationship between the two isolates are not close, yet closer than with the genus Labrenzia. Therefore, according to 16S rRNA phylogenetic analysis, strains Q4.6 T and Q2.11 should assigned to a novel species within the genus Pannonibacter, not the genus Labrenzia. Neither the pufLM nor the BChl a genes were detected in strains Q4.6 T and Q2.11. This further supports classification of these two isolates within Pannonibacter species, as they do not conform to the general species description for the genus Labrenzia.Q4.6 T and Q2.11 colonies grown on TSA plates were circular, smooth, cream-coloured and approximately 1 – 1.6 mm in diameter. Cells were rod shaped, 0.3 – 0.8 µm wide and 1.8 – 3.3 µm long (Fig. S3), which is within the published size range (0.3 – 0.8 µm wide, 1.8 – 4.0 µm long) for Pannonibacter cells [1, 2]. Strains Q4.6 T and Q2.11 were Gram-stainnegative, facultatively anaerobic and motile by means of a single polar flagellum. These characteristics are similar to other members of the genus Pannonibacter, whereas some species within the genus Labrenzia are strictly aerobic and motile by means of several polar flagella. Strain Q4.6 T grew in TSB in the presence of 0 – 5.5 % (w/v) NaCl (optimum growth in 0 – 2 % NaCl), at 4 – 55 Ǫ C (optimum growth at 30 – 35 Ǫ C) and pH 3.5 – 10.5 (optimum growth at 6.5 – 8). Strain Q2.11 grew in TSB in the presence of 0 – 7 % (w/v) NaCl (optimum growth in 0 – 2 % NaCl), at 10 – 45 Ǫ C (optimum growth at 30 – 35 Ǫ C) and pH 3.5 – 10.5 (optimum growth at 6.5 – 8). Similar to other species within Pannonibacter, strains Q4.6 T and Q2.11 were positive for catalase, oxidase and PHA production, and negative for indole production; however, they differ in their urease, acetoin and H 2 S production. Thus, these special characteristics suggest that strains Q4.6 T and Q2.11 should be regarded as a novel species within the genus Pannonibacter. The physiological and biochemical properties of strains Q4.6 T and Q2.11 are discussed in the species description and summarized in Table 1.Fig. 1. Phylogenetic tree showing the relationship of strains Q4.6 T and Q2.11 with strains of closely related species within the genus Pannonibacter as inferred from 16S rRNA gene sequences. Data with gaps and ambiguous nucleotides were removed during alignment for reconstruction of the tree. The tree was generated using the NJ method in MEGA 7.0 based on a comparison of 1497 nt. Bootstrap values are expressed as percentages of 1000 replications. The GenBank/EMBL/DDBJ accession number of each sequence is shown in parentheses.The genome size of Q4.6 T was approximately 4 780 018 bp, the number of contigs was 34, the N50 was 267 537 bp and the sequencing depth of coverage was 100×. According to the reference, the genome of Q4.6 T meets the quality requirement for prokaryotic taxonomic studies [42].The G+C content of the genomic DNA for strains Q4.6 T and Q2.11 was 63.6 mol%, which is inside of the range for within Pannonibacter species (63.0 – 64.6 mol%) [1, 2], but outside of the upper G+C content range for species within Labrenzia (56 – 62.4 mol%) [11, 12]. The genomic OrthoANI value for Q4.6 T and Q2.11 was 100 %. This value was less than 77.7 % with type strains P. phragmitetus and P. indicus, and less than 74.0 % with type strain L. suaedae, which is far lower than the 95 % ANI cut-off value for the species boundary [43]. These result strongly support the designation of Q4.6 T and Q2.11 as a novel species within the genus Pannonibacter. The DDH value between Q4.6 T and Q2.11 was 88.7±3.2 %, whereas the DDH values between L. suaedae DSM 22153 T and the two isolates, Q4.6 T and Q2.11, were 12.3±2.4 % and 10.2±1.7 % respectively. These values were below the 70 % cut-off recommended for assigning strains to the same genospecies [44]. According to standard practice [45], DDH was not used for taxonomic species discrimination between strains that share less than 97.0 % 16S rRNA sequence similarity. Thus, taken with the 16S rRNA similarity, the genomic ANI values and the DDH results strongly support assignment of strains Q4.6 T and Q2.11 to a novel species within the genus Pannonibacter.The principal quinone of strains Q4.6 T and Q2.11 was Q-10, which is the same quinone reported for other Pannonibacter species. The cellular fatty acid profile of strains Q4.6 T and Q2.11 consisted predominantly of C 18: 1 Ɯ 7 c (75.1, 75.7 %) (Table 2), which is within the range previously reported for genus Pannonibacter. Although this profile is similar to related type strains, Q4.6 T and Q2.11 exhibited differences in certain components, particularly with respect to the amount of C 20: 1 Ɯ 9 c and an unknown fatty acid (equivalent chain length, 18.814) (Table 2). The polar lipid profiles of strains Q4.6 T and Q2.11 contained diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline, an unidentified amino lipid, two or three unidentified phospholipids, and one unidentified lipid (Fig. S4). These profiles were similar to those of P. phragmitetus DSM 14782 T and P. indicus DSM 23407 T; however, strains Q4.6 T and Q2.11 could be differentiated from the two Pannonibacter type strains by the presence of several unidentified phospholipids and one unidentified lipid. Therefore, these results further support strains Q4.6 T and Q2.11 as a novel species in the genus Pannonibacter.Characteristic Q4.6 T Q2.11 DSM 23407 T DSM 22153 T Temperature range for growth (Ǫ C) 4 – 55 10 – 45 4 – 55 10 – 40 pH for growth 3.5 – 10.5 3.5 – 10.5 3.5 – 10.5 5.0 – 10.5 Salinity range of growth (%) 0 – 5.5 0 – 7.0 0 – 7.0 0 – 2.5 Cell size (µm) 1.8 – 3.0×0.3 – 0.7 1.8 – 3.3×0.5 – 0.8 1.8 – 2.0×0.6 – 0.8 1.0 – 2.5×0.5 – 1.0 H2S + + + ‒ Tween 80 ‒ ‒ + ‒ Presence of pufLM genes ‒ ‒ ‒ + Oxidase + + + W Nitrate reduction ‒ ‒ ‒ + VP test + + ‒ ‒ Hydrolysis of gelatin + + + ‒ Urease + + + ‒ Carbon source utilization: Trehalose + W W + Sucrose W W + + Arabinose ‒ ‒ ‒ + Mannose ‒ + ‒ + Lactose ‒ ‒ ‒ + Erythritol ‒ ‒ ‒ W Acid production (aerobically): D- Glucose + + W W Mannitol ‒ ‒ + + Maltose ‒ + ‒ + Sucrose ‒ ‒ ‒ + Sorbitol + + + ‒ Trehalose ‒ ‒ + ‒ Xylitol + W + + Mannose + ‒ + + Lactose + ‒ W W Potassium Gluconate + + ‒ + Inositol ‒ + ‒ + DNA G+C content (mol%) 63.6 63.6 63.5 58.7Fatty acid Q4.6 T Q2.11 DSM 23407 T DSM 14782 T DSM 22153 T C16:0 2.0 2.1 6.8 6.5 3.4 10-Me C18:1 ‒ ‒ ‒ 9.9 ‒ C18:1 Ɯ 7 c 75.1 75.7 69.6 75.8 75.5 11-methyl C18:1 Ɯ 7 c 3.0 3.0 10.1 9.9 3.7 C18:0 3.7 4.4 3.1 ‒ 3.4 C18:0 3OH 1.5 1.7 1.4 2.2 2.1 C20:1 Ɯ 9 c 2.3 1.8 ‒ ‒ 1.8 Summed feature 2 4.5 4.0 2.7 ‒ 3.7 Summed feature 2 3.7 3.4 1.9 ‒ 3.0 Unknown 18.814 4.3 2.0 ‒ ‒ 2.2On the basis of phylogenetic analysis and DDH results, as well as the phenotypic and chemotaxonomic data presented herein, strain Q4.6 T is assigned as a novel species within the genus Pannonibacter; as such, the name Pannonibacter carbonis (Q4.6 T) is proposed.