Lysinimonas yzui sp. nov., isolated from cattail root soil from mine tailings

2003 Lysinimonas yzui sp. nov., isolated from cattail root soil from minetailings LijuanMei1, ZhePiao1, JianHu1, LinlinShi2, YanchaoBai1 and ShixueYin1,* TAXONOMIC DESCRIPTION Mei etal., Int. J. Syst. Evol. Microbiol. 2020;70:2003–2007 DOI 10.1099/ijsem.0.004013 Author affiliations: 1College of Environmental Science and Engineering, Yangzhou University, Yangzhou 225127, PR China; 2Research Center of Agricultural Resource and Environment, Institute of Agricultural Science in Taihu Lake District, Suzhou 215000, PR China. *Correspondence: Shixue Yin, sxyin@ yzu. edu. cn Keywords: Lysinimonas yzui; tailings; strain N7XX-4T. Abbreviations: AL, unidentified aminolipids; DPG, diphosphatidylglycerol; GL, unidentified glycolipid; HPLC, highperformance liquid chromatography; PG, phosphatidylglycerol; PL, unidentified phospholipid; UL, unidentified polar lipids. The NCBI accession number for the 16S rRNA gene sequence of strain N7XX-4T is MG934573. The wholegenome sequence is deposited in the Type Strain Genome Database, Global Catalogue of Microorganisms, under the accession number GCM60011600. Two supplementary figures are available with the online version of this article. 004013 © 2020 The Authors Abstract A yellowpigmented, Gramstainnegative, aerobic, nonmotile rod shaped, mesophilic bacterium, designated strain N7XX-4T, was isolated from cattail root grown on the mine tailings of Phoenix mountain, Tongling city, Anhui Province (PR China). Analysis of the 16S rRNA gene sequence revealed that the strain represented a novel member of the family Microbacteriaceae. The nearest phylogenetic neighbour was Lysinimonas kribbensis MSL-13T (97.8 % 16S rRNA gene sequence similarity). The most abundant fatty acid in whole cells of N7XX-4T was anteisoC15 : 0 (29.9 %). The predominant menaquinones were MK-12(H2), MK-13(H2) and MK-11(H2). The peptidoglycan type of the isolate was B1δ with lLys as the diagnostic cellwall diamino acid. On the basis of differences in phenotypic and genotypic characteristics, strain N7XX-4T (=CGMCC 1.16548T=DSM 106791T=JCM 32630T) is designated as the type strain of a novel species of the genus Lysinimonas, for which the name Lysinimonas yzui sp. nov. is proposed. The genus Lysinimonas was recently proposed by Jang et al. [1], based on two species, Lysinimonas soli SGM3-12T(=KACC 13362 T ) and Lysinimonas kribbensis MSL-13 T (=JCM 16015 T ). Phylogenetically, the genus has its evolutionary lineage within the family Microbacteriaceae in the phylum Actinobacteria which is phenotypically diverse and undergoing considerable taxonomic expansion and reorganization. At the time of writing, the genus Lysinimonas comprises two species with validly published names, Lysinimonas soli and Lysinimonas kribbensis. In this paper, we describe the characterization of a strain we isolated, N7XX-4T, and propose that this strain represents the type strain of novel species of the genus Lysinimonas on the basis of a polyphasic approach, including the determination of biochemical, physiological and chemotaxonomic properties and a detailed phylogenetic investigation based on the 16S rRNA gene sequence. Strain N7XX-4T was isolated from cattail root grown on the mine tailings of Phoenix mountain, Tongling city, Anhui Province (PR China), using the standard dilution plating technique on NSC agar medium (pH 7.0) at room temperature (20 °C) after 30 days in mixed gases of 5 % CO2, 2 % O2, 93 % N2 [2]. The isolate was maintained at −80 °C as a suspension in R2A broth supplemented with 20 % (v/v) glycerol. The Gram reaction was performed using Hucker’s method [3]. Catalase activity was examined by observing bubble production in 3 % (v/v) hydrogen peroxide solution and oxidase activity was examined using 1 % tetramethylpphenylenediamine [4]. Cell morphology and motility of N7XX-4 T were examined using a phasecontrast microscope (BX51, Olympus) and by transmission electron microscopy (HT7700, Hitachi) after negative staining with 1 % (w/v) phosphotungstic acid. Optimum conditions for growth were determined by culture for up to 14 days in R2A broth. Growth was determined at 4, 10, 15, 20, 25, 28, 30, 35, 37, 40 and 45 °C and with 0, 1, 3, 5 and 7 % (w/v) NaCl. The pH range for growth was tested in R2A broth adjusted to pH 4.0–10.0 at intervals of 1.0 pH unit by using the buffer systems described by Xu et al. [5]. Casein degradation was tested on R2A agar containing milk powder (5 %, w/v). Starch degradation was tested on R2A agar containing starch (1 %, w/v). Tyrosine degradation was tested on R2A agar containing tyrosine (0.1 %, w/v). carboxymethyl cellulose and Tween 80 were examined on R2A agar amended with 1 % (w/v) substrate [6, 7]. DNase activity was determined 2004 Mei etal., Int. J. Syst. Evol. Microbiol. 2020;70:2003–2007 Table 1. Characteristics that differentiate strain N7XX-4T, Lysinimonas soli SGM3-12T and Lysinimonas kribbensis MSL-13T Strains: 1, N7XX-4T.; 2, Lysinimonas soli SGM3-12T; 3, Lysinimonas kribbensis MSL-13T. Data were taken from this study unless otherwise indicated. Fatty acids were analysed using cells grown on R2A agar at 28 °C for 3 days; fatty acids representing <1.0 % are omitted. All strains are positive for utilization of lrhamnose and dlyxose and weakly positive for utilization of dmannose (API 50 CH), and positive for naphtholASBIphosphate hydrolase, α-galactosidase, β-galactosidase, potassium-5ketogluconate (API 50CH) and trypsase, chymotrypsin, Nacetylglucosamine and β-glycosidase activities (APIZYM). All strains are negative for urease and Dnase activities, utilization of potassium gluconate and anaerobic growth. +, Positive; −, negative; w, weakly positive. *Data from Jang 2013 [1]. DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; GL, unidentified glycolipid; AL, unidentified aminolipids; PL, unidentified phospholipid; UL, unidentified polarlipids. Characteristic 1 2 3 Utilization as carbon source of: larabinose + + − dribose − + − dgalactose w + − dglucose + + − dfructose w + − inositol w w − Nacetylglucosamine w − − cellobiose + + − maltose + + − melibiose + + − trehalose + + − raffinose − + − glycogen + w − lfucose − + − Hydrolysis of: casein − w + starch − − w tyrosine − − + Tween 80 + w + Enzyme activities: alkaline phosphatase + w − cystine aromatase + + − β-glucuronidase w − − β-gluconase + w + α-mannosidase − − + Catalase +− − Oxidase − + − pH range (optimum pH) 6.0–9.0 (7.5) 5.0–9.0 (7.5) 6.0–10.5 (7.0) Growth temperature range (optimum temperature) °C 10–37 (30) 10–37 (28) 20–37 (30) Colony colour yellow brown, yellow* yellow NaCl tolerance range (w/v, %) 0–5 0–3 0–3 Continued 2005 Mei etal., Int. J. Syst. Evol. Microbiol. 2020;70:2003–2007 on DNase test agar (Difco). Anaerobic growth was assessed on R2A agar containing 0.5 % Na 2 SO 4 , 0.5 % NaNO 3 , 0.5 % NaHCO3 or 0.02 % FeCl3 in airtight jars containing a BBL GasPak Anaerobic System (Difco) for 10 days at 28 °C. N7XX4T was also characterized using the whole test spectra of the API ZYM and API 50 CH identification systems (bioMérieux) according to the manufacturer’s instructions, and relative physiological and biochemical properties were tested with Biolog GenIII MicroPlates [by the identification service of Beijing Biogenro Biotechnology (Beijing, PR China)] using cells grown on R2A for 4 days at 28 °C. Cells of N7XX-4T were Gramstain negative, aerobic and nonmotile rods (Fig. S1, available in the online version of this article). Colonies were yellow, round and convex after 4 days of growth at 28 °C on R2A agar. Growth occurred at temperatures ranging from 10 to 37 °C and at pH 6–9. Cells were catalasepositive and oxidasenegative. N7XX-4T showed significant differences from Lysinimonas kribbensis DSM 19272T and Lysinimonas soli NBRC 107106T in terms of carbon source utilization enzyme activities. The detailed physiological and biochemical characteristics of N7XX-4T are summarized in Table1 and in the species description. Genomic DNA of N7XX-4T was extracted and purified using a Bacterial Genomic DNA Extracting kit (TIANGEN). The 16S rRNA was amplified by PCR using the universal primers 27F (5′- AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGCTACCTTGTTACGACTT-3′) [8]. The PCR product was purified with a Gel Extraction kit (TransGen Biotech) and ligated into pMD18T vector (TaKaRa), and then sequenced by Sangon Biotech (Shanghai, PR China). The resultant almostcomplete 16S rRNA gene sequence (1487 bp) of N7XX-4T was obtained, and submitted to EzBioCloud ( www. ezbiocloud. net/) [9] and NCBI for a blast search to obtain the 16S rRNA gene sequence similarity values between N7XX-4T and the related species. Sequences of the closest relatives, including those from N7XX-4T, Lysinimonas kribbensis MSL-13T (EF466129), Lysinimonas soli SGM3-12T (JN378395), Leifsonia psychrotolerans LI1T (GQ406810) and Chryseoglobus frigidaquae CW1T (EF373534) were aligned using clustal W [10] in MEGALIGN (Lasergene software; DNASTAR). Phylogenetic trees were reconstructed using the neighbourjoining and maximumparsimony methods in mega 7 [11]. The topology of the trees was evaluated by bootstrap analysis with 1000 replications. Similarity values indicated that the novel strain represented a member of the family Microbacteriaceae, with Lysinimonas kribbensis MSL-13 T as the closest relative (97.8 % 16S rRNA gene sequence similarity). The isolate exhibited less than 97 % sequence similarity with all other members of the family Microbacteriaceae, such as Herbiconiux flava NBRC 16403T (96.6 %), Chryseoglobus frigidaquae CW1T(96.6 %) and Herbiconiux ginsengi wged11T (96.7 %). According to the neighbourjoining phylogenetic tree, N7XX-4T formed a robust cluster with the two species of the genus Lysinimonas and the three strains were clearly separable from the type strains of the type species of the genus Leifsonia, Agrococcus, Homoserinibacter and Schumannella (Fig.1). The cluster was also recovered in the maximumparsimony tree. The whole genome was sequenced by the Institute of Microbiology of the Chinese Academy of Sciences (IMCAS), Beijing, China and the sequence was deposited in the Type strain Genome Database, Global Catalogue of Microorganisms (http:// gctype. wdcm. org/ sequencing/ GCM10011600), under the accession number GCM60011600. The genome size and DNA G+C content were 3.08 Mb and 69 mol% respectively. Quantitative analysis of wholecell fatty acids was performed by growing N7XX-4T and the reference strain, Lysinimonas kribbensis DSM 19272T, on R2A at 28 °C for 3 days, and analysing the fatty acids according to the instructions of the Microbial Identification System (Sherlock Version6.0; MIDI) by gas chromatography (56890 N; Agilent Technologies) [12]. Profiles of the major fatty acids of N7XX-4T and the two type strains of closely related species of the genus Lysinimonas are shown in Table 1. The major fatty acids of N7XX-4T were anteisoC15 : 0 (29.9%), isoC15 : 0 (26.1%) and C16 : 0 (22.7%). Menaquinones and polar lipids were determined as described by Minnikin et al. [13] and Kroppenstedt [14]. The major menaquinones of N7XX-4T were MK-12(H2)(46.2%), Characteristic 1 2 3 DNA G+C content (mol%) 67.0 67.3* 73.4* Major menaquinone(s) MK-12(H2) MK-11(H2) MK-13(H2) MK-12(H2)* MK-11(H2)* MK-12* MK-12(H2) MK-11(H2) MK-13(H2) Polar lipids DPG, PG, GL, AL DPG, PG, GL, PL* DPG, PG, GL, AL, UL Major fatty acids: isoC15 : 0 26.1 4.2* 4.0 C16 : 0 22.7 -* 0.3 anteisoC15 : 0 29.9 47.8* 44.8 isoC16 : 0 7.2 39.5* 42.9 Table 1. Continued 2006 Mei etal., Int. J. Syst. Evol. Microbiol. 2020;70:2003–2007 MK-13(H2)(31.2%) and MK-11(H2)(22.6%). The polar lipid profile of N7XX-4T consisted of diphosphatidylglycerol, phosphatidylglycerol, two unidentified glycolipids and an unidentified aminolipid; and Lysinimonas kribbensis MSL-13T revealed a polar lipid profile similar to that of N7XX-4 T except for the presence of an unidentified polar lipid (Fig. S2). Preparation of cell walls and determination of peptidoglycan structure were performed as described by Schleifer and Kandler [15], with the modification that TLC on cellulose sheets (Merck) was used instead of paper chromatography. The peptidoglycan of N7XX-4T and the reference strain Lysinimonas kribbensis MSL-13T contained alanine, glutamic acid, lysine, glycine and diaminobutyric acid. These data indicated that the cellwall peptidoglycan of strain N7XX-4T was type B1δ. To date, the cellwall peptidoglycans of the species of the genus Lysinimonas are all type B1δ, in concordance with the report of Jang et al. [1]. DNA–DNA hybridization was carried out according to previously published methods [16]. N7XX-4T showed 41.2 % DNA–DNA relatedness to Lysinimonas kribbensis MSL-13T. The G+C content of the DNA was determined by HPLC according to the method previously described by Tamaoka et al. [17]. The DNA G+C content of N7XX-4 T was 67.0 mol%. The neighbourjoining phylogenetic tree revealed that N7XX4T represents a member of the family Microbacteriaceae and clusters with members of the genus Lysinimonas; this was supported by the maximum parsimony tree. N7XX-4 T can be differentiated from closely related species on the basis of chemotaxonomic characteristics such as abilities to utilize Rhodoglobus vestalii LV3T (AJ459101) Salinibacterium amurskyense KMM 3673T (AF539697) Chryseoglobus frigidaquae CW1T (EF373534) Microcella putealis CV2T (AJ717388) Yonghaparkia alkaliphila KSL-113T (DQ256087) Lysinimonas soli SGM3-12T (JN378395) Lysinimonas kribbensis MSL-13T (EF466129) N7XX-4T (MG934573) Leifsonia ginsengi wged11T (DQ473536) Agrococcus jenensis DSM 9580T (X92492) Microbacterium lacticum DSM 20427T (X77441) Schumannella luteola KHIAT (AB362159) Leifsonia aquatica JCM 1368T (D45057) Okibacterium fritillariae VKM Ac-2059T (AB042094) Plantibacter flavus DSM 14012T (AJ310417) Microterricola viridarii KV-677T (AB282862) Phycicola gilvus SSWW-21T (AM286414) Glaciibacter superstes AHU1791T (AB378301) Subtercola boreus K300T (AF224722) Agreia bicolorata VKM Ac-1804T (AF159363) Amnibacterium kyonggiense KSL51201-037T (FJ527819) Labedella gwakjiensis KSW2-17T (DQ533552) Cryobacterium psychrophilum DSM 4854T (AJ544063) Alpinimonas psychrophila Cr8-25T (GU784868) Klugiella xanthotipulae 44C3T (AY372075) Frondihabitans australicus E1HC-02T (DQ525859) Frigoribacterium faeni 801T (Y18807) Clavibacter michiganensis subsp. michiganensis DSM 46364T (X77435) Compostimonas suwonensis SMC46T (JN000316) Mycetocola saprophilus CM-01T (AB012647) Marisediminicola antarctica ZS314T (GQ496083) Rathayibacter rathayi DSM 7485T (X77439) Leucobacter komagatae JCM 9414T (D45063) Pseudoclavibacter helvolus DSM 20419T (X77440) Gulosibacter molinativorax ON4T (AJ306835) Curtobacterium citreum DSM 20528T (X77436) Gryllotalpicola koreensis RU-16T (HQ832501) Agromyces ramosus DSM 43045T (X77447) Humibacter albus SC-083 T (AM494541) 99 86 69 99 90 84 82 67 79 67 64 63 66 0.01 Fig. 1. Neighbourjoining phylogenetic tree based on 16S rRNA gene sequences, showing positions of N7XX-4T and related taxa within the family Microbacteriaceae. Bootstrap values of over 60 % (based on 1000 replicates) are shown at branching points. Dots indicate that the corresponding branches were also recovered in the maximum parsimony tree. Bar, 0.01 substitutions per nucleotide. 2007 Mei etal., Int. J. Syst. Evol. Microbiol. 2020;70:2003–2007 particular carbon sources, enzyme activities, fatty acids, polar lipids, and menaquinones (Table1). N7XX-4T and the two species of the genus Lysinimonas with validly published names contained the partially saturated menaquinones MK-12(H 2 ), MK-11(H 2 ) and/or MK-13(H 2 ) as the predominant menaquinones, which made these three strains phenotypically differentiable from other members of the family Microbacteriaceae [1]. Results for the Gramstain reaction make N7XX-4T differentiable from the two species of the genus Lysinimonas with validly published names. AnteisoC15 : 0, the major fatty acid in recognized species of the genus Lysinimonas, was also the major fatty acid component of N7XX-4T. N7XX-4T contained higher levels of isoC15 : 0 and C16 : 0 content (>20 %) but had low isoC16 : 0 content (<10 %), while isoC 16 : 0 is the major fatty acid of the two species of the genus Lysinimonas with validly published names. N7XX-4T is the same as Lysinimonas soli SGM3-12T and Lysinimonas kribbensis MSL-13T in having an unusual peptidoglycan structure (type B1δ). A comparison of characteristics of the three strains is shown in Table1. On the basis of the results of phylogenetic analysis its chemotaxonomic characteristics, N7XX-4T represents a novel species, for which the name Lysinimonas yzui sp. nov. is proposed. DESCRIPTION Of Lysinimonas yzui SP. NOv. Lysinimonas yzui ( y. zu′i. N.L. gen. n. yzui pertaining to YZU, Yangzhou University, where the taxonomic studies on the type strain were performed). Cells are Gramstainnegative, nonsporeforming, aerobic and nonmotile rods. Colonies are yellow, round and convex after 4 days of growth at 28 °C on R2A agar. Cells are usually 0.5–0.7 µm wide and 1.0–1.1 µm long. Growth occurs at 10–37 °C (optimum 30 °C) and at pH 6–9 (optimum pH 7.5) and in the presence of 0–5 % (w/v) NaCl. Growth does not occur under anaerobic conditions. Oxidasenegative, ureasenegative and catalasepositive. Negative for hydrolysis of carboxymethyl cellulose, gelatin, DNA, casein, starch and tyrosine and positive for hydrolysis of Tween 80. Does not assimilate erythritol, darabinose, dribose, lxylose, dardonitol, methyl βdxylopyranoside, lsorbose, dsorbitol, raffinose, starch, dlyxose, dtagatose, dfucose, lfucose, potassium gluconate or potassium 5ketogluconate (API 50 CH test strips). Positive for the following enzyme activities (API ZYM test strips): alkaline phosphatase, esterase (C4), esterase lipase (C8), cystine aromatase, acid phosphatase, naphtholASBIphosphate hydrolase, α-galactosidase, β-galactosidase, α-gluconase and β-gluconase. The major fatty acids are anteisoC15 : 0, isoC15 : 0, C16 : 0, isoC16 : 0 and anteisoC17 : 0. The type strain, N7XX-4T (=CGMCC 1.16548T=DSM 106791T=JCM 32630T), was isolated from cattail root grown on mine tailings from Phoenix Mountain, Tongling city, Anhui Province (PR China). The G+C content of the DNA of the type strain is 67.0 mol%(by HPLC) and 69 mol% (by genomebased analysis). Funding information This study was financially supported by the Environmental Protection of Yangzhou City (YHK1414), the National Key Basic Research Program of China (2015CB150503), and the National Natural Science Foundation of China (41071177). Acknowledgements We are grateful to G. -Q. 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