International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3427–3433
DOI 10.1099/ijs.0.063339-0
Description of Micrococcus aloeverae sp. nov., an endophytic actinobacterium isolated from Aloe vera Om Prakash, Yogesh Nimonkar, Hitendra Munot, Avinash Sharma, Venkata Ramana Vemuluri, Mahesh S. Chavadar and Yogesh S. Shouche Correspondence
Microbial Culture Collection, National Centre for Cell Science, Pune, Maharastra 411007, India
Om Prakash [email protected]
A yellow Gram-stain-positive, non-motile, non-endospore -forming, spherical endophytic actinobacterium, designated strain AE-6T, was isolated from the inner fleshy leaf tissues of Aloe barbadensis (Aloe vera) collected from Pune, Maharashtra, India. Strain AE-6T grew at high salt concentrations [10 % (w/v) NaCl], temperatures of 15–41 6C and a pH range of 5–12. It showed highest (99.7 %) 16S rRNA gene sequence similarity with Micrococcus yunnanensis YIM 65004T followed by Micrococcus luteus NCTC 2665T (99.6 %) and Micrococcus endophyticus YIM 56238T (99.0 %). Ribosomal protein profiling by MALDI-TOF/MS also showed it was most closely related to M. yunnanensis YIM 65004T and M. luteus NCTC 2665T. Like other members of the genus Micrococcus, strain AE-6T had a high content of branched chain fatty acids (isoC15 : 0 and anteiso-C15 : 0). MK-8(H2) and MK-8 were the predominant isoprenoid quinones. Cell wall analysis showed an ‘A2 L-Lys-peptide subunit’ type of peptidoglycan and ribose to be the major cell wall sugar. The DNA G+C content was 70 mol%. Results of DNA–DNA hybridization of AE-6T with its closest relatives from the genus Micrococcus produced a value of less than 70%. Based on the results of this study, strain AE-6T could be clearly differentiated from other members of the genus Micrococcus. We propose that it represents a novel species of the genus Micrococcus and suggest the name Micrococcus aloeverae sp. nov., with strain AE-6T (5MCC 2184T5DSM 27472T) as the type strain of the species.
Endophytes are micro-organisms (bacteria and fungi) isolated from the inner tissues of plants. It has been found that most plant species harbour one, or more than one, kind of endophyte and they reside inside the plant without producing any adverse effects (Adams & Kloepper, 1996; James et al., 2002; Sessitsch et al., 2002; Kaga et al., 2009; Johnston-Monje & Raizada, 2011). To date; most of the endophytes that have been isolated are fungi and only a small amount of work has been carried out on bacterial endophytes (Verma et al., 1998; Waller et al., 2005). Although the area of plant-endophyte interaction is not well studied the available data indicates that some endophytes have plant growth promoting activities and protect plants from invasion of pathogens and environmental stresses (Verma et al., 1998; James et al., 2002; Waller et al., 2005). In the present study, we have isolated an endophytic strain, belonging to the genus Micrococcus, from the fleshy inner tissues of the leaf of Aloe vera (Aloe barbadensis) using aseptic sampling and isolation techniques. The results of this Abbreviations: SSC, Saline Sodium Citrate (SSC) Buffer. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of AE-6T is KF524364. Two supplementary figures and one supplementary table are available with the online version of this paper.
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study indicate that the isolated strain has distinct features from species of the genus Micrococcus with validly published names and that it should be described as a novel species. Besides this study, members of the genus Micrococcus have been detected as endophytes on several other plants including the zea (Johnston-Monje & Raizada, 2011), cotton (Mcinroy & Kloepper, 1995), rice seeds (Kaga et al., 2009) and the roots of Polyspora axillaris (Zhao et al., 2009) and Aquilaria sinensis (Chen et al., 2009). The genus Micrococcus was first described by Cohn (1872) for Gram-stain-positive, non-motile, non-endospore-forming, small spherical bacterium. Members of the genus Micrococcus are widely distributed and have been isolated from a variety of habitats including the soil (Zhang et al., 2010), the air (Rieser et al., 2013), activated sludge (Liu et al., 2007), permanently cold samples (Liu et al., 2000), human skin (Kloos et al., 1974), dairy waste (Chittpurna et al., 2011) and the inner tissues of plants (Zhao et al., 2009). At the time of writing there are a total of nine species of the genus Micrococcus with validly published names, including two endophytic species (www.bacterio.net). Stackebrandt et al. (1995) reclassified some members of genus Micrococcus into four different genera Kocuria, Dermacoccus, Kytococcus, and Nesterenkonia, while Wieser et al. (2002) gave an emended description of the genus Micrococcus for the first time. 3427
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Almost all the members of the genus Micrococcus are yellow and grow at high pH (pH 10). The characteristic features of members of the genus Micrococcus are aerobic growth, catalase-positive cells, a high content of branched chain fatty acids (iso-C15 : 0 and anteiso-C15 : 0), a high G+C content (66.3–73.3 mol%) and the presence of MK-8 and MK-8(H2) as the predominant respiratory quinones (Stackebrandt et al., 1995; Wieser et al., 2002). In order to isolate the endophytic bacteria, fresh leaf samples from Aloe barbadensis (Aloe vera) were collected from Pune, Maharashtra, India, in a pre-sterile polyethylene bag. Endophytic strains were isolated using the method of Gayathri et al. (2010). In brief, after collection samples were immediately transported to the laboratory and stored at 4 uC for not longer than two days. Firstly were washed with distilled water and sterilized in 70 % (v/v) ethanol (0.5 min) followed by 0.1 % mercuric chloride (HiMedia, Mumbai, India) (3.0 min). To remove the disinfectant, leaves were again washed thrice with sterile distilled water. After treatment, inner tissues were removed with a sterile scalpel, crushed in normal saline and plated on tryptic soy agar and Luria agar plates. Isolated strains were purified by restreaking on fresh medium plates and maintained with 15 % (v/v) glycerol in the vapour phase of liquid nitrogen. Based on its potential for growth at a wide range of salt concentrations, pH values and temperatures strain AE-6T was selected for further characterization. Extraction and purification of genomic DNA was carried out using the method of Prakash & Lal (2006). The 16S rRNA gene sequence was amplified according to methods described by Prakash et al. (2012) and sequencing was carried out using Sanger’s method (Prakash et al. 2014). An almost complete stretch of 1411 bp good quality 16S rRNA gene sequence was obtained after manual editing of the ABI-sequence file in ChromasPro software (http://www. technelysium.com.au/ChromasPro.html). A sequence similarity search was conducted using the SeqMatch tool of the Ribosomal Database Project (RDP). The 30 most similar reference sequences were retrieved and selected for multiple alignments. The 16S rRNA gene sequence of Cellulomonas flavigena 134T was used as an outgroup. Multiple sequence alignment was performed with CLUSTAL X, version 1.81 (Thompson et al., 1997). To get unambiguous data, alignment was manually edited using DAMBE (Xia, 2013). A phylogenetic tree was reconstructed using software package MEGA version-5 (Tamura et al., 2011) and the Kimura twoparameter model (Kimura, 1980). Bootstrap values were deduced based on 1000 replications (Felsenstein, 1985). In order to evaluate the robustness of the tree topology, clustering was performed using all three different methods: neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MR) (Fitch, 1971; Felsenstein, 1981; Saitou & Nei, 1987; Tamura et al., 2011).
yunnanensis YIM 65004T (99.7 %), M. luteus DSM 20030T (99.6 %) and M. endophyticus YIM 56238T (99.0 %). All three methods of phylogenetic reconstruction provided similar tree topologies and clustering. Strain AE-6T clustered with members of the genus Micrococcus and showed phylogenetic closeness with M. yunnanensis YIM 65004T and M. luteus DSM 20030T with a high bootstrap value (Fig. 1). Micrococcus lactis DSM 23694T and Zhihengliuella aestuarii JCM 16364T formed separate clades and showed distinct positions to other members of the genus Micrococcus and the genus Zhihengliuella. The taxonomic position of M. lactis DSM 23694T and Z. aestuarii JCM 16364T should be discussed separately based on other experimental evidence. Thus, 16S rRNA sequence similarity data and phylogenetic analysis confirmed that strain AE-6T is a member of the genus Micrococcus. In addition, due to high levels of 16S rRNA sequence similarity with some members of the genus Micrococcus other approaches could be used to confirm the phylogenetic position of strain AE-6T and these are discussed below. In order to generate phenotypic data, type strains of the closest relatives of strain AE-6T were procured from DSMZ-Germany and experiments were conducted in similar laboratory conditions. Growth responses of the organisms were tested on tryptic soy agar, Luria agar, nutrient agar and R2A agar (Hi-Media). Colony shape, size and pigmentation were recorded on tryptic soy agar plates after-72 h incubation at 30 uC. Gram-staining was conducted using Hi-Media’s Gram-Stain kit. Catalase and oxidase tests were performed using 3 % (v/v) H2O2 and oxidase discs (Hi-Media), respectively. The pH range for growth was determined in tryptic soy broth by adjusting the pH of the medium with 1M NaOH and 1M HCl. The optimum pH for growth was determined by measuring the optical density (OD600 nm) at different time intervals. Growth at different concentrations of NaCl was tested by supplementing TSB with a range of NaCl concentrations [1–15 % (w/v) with 1 % (w/v) increments]. In order to investigate the temperature range and the optimum temperature for growth inoculated medium was incubated at different temperatures (5–45 uC in increments of 5 uC up to 40 uC, and of 1 uC at 40–45 uC) and the optical densities (OD600 nm) at different time intervals were measured. The use of different carbon sources and enzyme activities were assessed using API 20NE and API ZYM kits (bioMe´rieux). Skimmed-milk-agar and Tween-80 agar were used to assess protease and esterase production, respectively. For the gelatinase test, tryptic soy broth was supplemented with 12 % (w/v) gelatine, as a gelling agent, while urease production was monitored using urea agar with a pH indicator (Smibert & Krieg, 1994).
Sequence similarity calculations revealed that strain AE-6T showed closest similarity to members of the genus Micrococcus and had highest sequence similarity with M.
Strain AE-6T is a Gram-stain-positive, non-motile, nonendospore-forming, small, spherical bacterium of endophytic origin. It had a mesophilic nature and grew in a wide range of salt concentrations [0–10 %(w/v) NaCl], and at pH 5–12 and temperatures of (15–41 uC). Strain AE-6T formed yellow, round, convex colonies, which were 0.5 mm in diameter, on tryptic soy agar plates after 72 h incubation at 30 uC. Strain AE-6T showed positive results for catalase but
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International Journal of Systematic and Evolutionary Microbiology 64
Micrococcus aloeverae sp. nov
Arthrobacter oxydans DSM 20119T (X83408)
99 89
Arthrobacter scleromae DSM 17756T (AF330692) Arthrobacter sulfonivorans DSM 14002T (AF235091)
69 0.01
95
Arthrobacter humicola JCM 15921T (AB279890) 83 Arthrobacter oryzae JCM 15922T (AB279889) Arthrobacter globiformis NBRC 12137T (X80736)
50
33 Arthrobacter pascens DSM 20545T (X80740) Micrococcus lactis DSM 23694T (FN673681)
100
Zhihengliuella aestuarii JCM 16364T (EU939716) Zhihengliuella alba NBRC 109061T (AB778263)
45
42
97
Zhihengliuella flava DSM 26152T (AB778260) Zhihengliuella halotolerans DSM 17364T (DQ372937)
57 98
54
Zhihengliuella salsuginis NBRC 109062T (AB778264)
Micrococcus terreus NBRC 104258T (FJ423763) Micrococcus cohnii DSM 23974T (FR832424) Micrococcus lylae DSM 20315T (X80750) 87
Micrococcus yunnanensis DSM 21948T (FJ214355) 68
52
Micrococcus luteus NCTC 2665T (CP001628) Micrococcus aloeverae AE6T MCC 2184 (KF524364)
42
Micrococcus endophyticus DSM 17945T (EU005372) Micrococcus flavus DSM 19079T (DQ491453)
34 30
Micrococcus antarcticus JCM 11467T (AJ005932)
Citricoccus parietis CCUG 57388T (FM992367) 74
Citricoccus zhacaiensis JCM 15136T (EU305672) Citricoccus nitrophenolicus DSM 23311T (GU797177)
26
Citricoccus alkalitolerans DSM 15665T (AY376164)
71 83
Citricoccus muralis DSM 14442T (AJ344143) Cellulomonas flavigena DSM 20109T (X83799)
Fig. 1. A neighbour-joining phylogenetic tree, reconstructed using 16S rRNA gene sequences, showing the relationships of strain AE-6 with other species of genus Micrococcus. Numbers at the nodes represent confidence levels in terms of bootstrap support generated from 1000 replicates. Bootstrap values ,50 % are not shown. Cellulomonas flavigena DSM 20109Twas used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
was negative for oxidase. The strain produced good amounts of extracellular protease in nutrient broth after 24 h of incubation and gave a positive result for gelatinase, lipase and esterase. Detailed descriptions of strain AE-6T in terms of its morphological, physiological and chemotaxonomic features are given in Table 1 and in the species description. Polar lipids and isoprenoid quinones were extracted from freeze-dried cells and analysed by two dimensional TLC and HPLC, as described in Tindall (1990a, b) respectively. Cell http://ijs.sgmjournals.org
wall sugar and peptidoglycan analyses were performed by the DSMZ Identification Service according to published protocols (Schumann, 2011). For fatty acid methyl ester (FAME) analysis strain AE-6T, M. luteus DSM 20030T and M. yunnanensis YIM 65004T were grown simultaneously on tryptic soy agar plates for 48 h at 30 uC. Extraction and analysis of fatty acids were conducted as described by Prakash et al. (2014). Peaks were identified using the database TSBA6 of MIS (MIDI). 3429
1, Strain AE-6T; 2, M. endophyticus DSM17945T; 3, M. luteus DSM20030T; 4, M. yunnanenisis DSM 21948T; 5, M. lylae DSM 20315T; 6, M. flavus DSM 30824T. Strain AE-6 T, M. endophyticus DSM17945T, M. yunnanensis DSM 21948T, M. luteus DSM20030T, M. lylae DSM 20315T, and M. flavus DSM 30824T showed negative results for potassium nitrate (NO3), L- tryptophan (TRP), Larginine (ADH), 4- nitrophenyl-bD galactopyranoside (PNPG), L-arabinose (ARA), N-acetylglucosamine (NAG), adipic acid (ADI), trisodium citrate (CIT), cystine arylamidase, naphthol-ASBI-phosphohydrolase, a- galactosidase, b- galactosidase, b- glucuronidase, b- glucosidase, N-acetyl- b-glucosaminidase, a-mannosidase, a-fucosidase. All the species were Gram-stain positive, non-motile, non-endospore- forming and showed positive results for catalase and naphthol-AS- BI- phosphohydrolase. +, Positive; 2, negative; W, weakly positive. Characteristics
International Journal of Systematic and Evolutionary Microbiology 64
Colonies Colour Diameter (mm) after 48 h Shape Temperature range for growth (uC) Optimum temperature for growth (uC) pH range for growth Optimum pH for growth Tolerance to NaCl (w/v) Oxidase Protease Glucose(fermentative) Urease Tween-20 hydrolysis (72h) Aesculin hydrolysis Gelatinase D-Glucose D – Mannose D – Mannitol D – Maltose Potassium gluconate Capric acid Malic acid Phenylacetic acid Alkaline phosphtase Esterase Esterase Lipase Lipase Leucine arylamidase Valine arylamidase Trypsin a-Chymotrypsin Acid phosphatase a-Glucosidase
1
2
3
4
5
6
Yellow 0.5 Round convex 15–41 35 5–12 9 10 % 2 +++ 2 2 2 2 + + 2 2 + 2 2 2 2 + + + + ++ + 2 2 + ++
Yellow 0.5 Round convex 15–37 28 6–9 7–8 10 %
Yellow 0.5 Round convex 20–40 37 5–10 7 10 %
Yellow 1 Round convex 4–45 28 6–8 7–8 15 %
White 1 Round convex 20–45 35 6–9 7 10 %* ” ” 2 2 2 2 + + 2 2 + 2 2 + + 2 ++ 2 2 +++ 2 2 2 2 ++
Yellow 0.5 Round convex 26–34 31 5–9 6 10 % + ” 2 2 2 2 + 2 2 2 2 2 2 2 2 + ++ ++ 2 +++ 2 2 2 + +++
W
W
W
+ + 2 + 2 2 2 + + + + 2 2 2 + + + 2 + + 2 2 + ++
++ 2 + 2 + 2 2 + 2 + 2 2 2 2 + + + + + + + + ++ ++
++++ 2 2 2 + + + + 2 + 2 + + + + + + 2 +++ + + 2 + ++
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Table 1. Differential phenotypic traits of strain AE-6T and the closest phylogenetically related species of the genus Micrococcus
2 + 71.4* MK-8(H2), MK-7(H2)* + 2 69* MK-8(H2)* Data were all from this study in comparable laboratory conditions except for: *Data taken from Zhao et al. (2009).
2 2 70* MK-8, MK-8(H2)* 2 + 72.9* MK-8(H2), MK-7(H2)* Simmons citrate test Starch casein test DNA G + C content (mol%) Quinones
2 2 70 MK-8(H2), MK-7(H2), MK-8
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A comparison of the FAMEs present in strain AE-6T and other members of the genus Micrococcus is presented in Table S1 (available in the online Supplementary Material). Data indicated that the iso and anteiso forms of branched chain fatty acids are major components of species of the genus Micrococcus with iso-C15 : 0 and anteiso-C15 : 0 representing the predominant forms (Table S1). The qualitative and quantitative similarities in the fatty acid profiles of strain AE-6T and other members of the genus Micrococcus, means that FAME data support the findings of 16S rRNA gene sequence analysis and further suggest that strain AE-6T is a member of the genus Micrococcus. MK8(H2) and MK-7 (H2) were detected as the predominant menaquinones, while MK-8 and MK-7 were present as minor menaquinones. Polar lipid profiles showed prominent spots of diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG), while an unidentified phospholipid (PL), glycolipid (GL) and a phosphatidylinositol (PI) were detected in minor amounts (Fig. S1). Lysine, glycine, alanine and glutamic acids were detected in the hydrolysate of peptidoglycan. The approximate ratio of amino acids was: 1.4 Ala: 0.6 Gly: 1.4 Glu: 1.0 Lys. The peptides D-Ala – L-Ala, L-Ala – D-Glu, L-Lys – D-Ala and D-Glu – Gly were detected by two dimensional TLC. Dinitrophenylation indicated that alanine represented the N terminus of the interpeptide bridge. Based on the data above we deduced that strain AE-6T has an ‘A2, L-Lys’ type of peptidoglycan. Data on cell wall sugars indicated that strain AE-6T possesses ribose and less glucose, mannose and rhamnose.
2 2 71.7* MK-8(H2), MK-7(H2)*
6 3 2 1 Characteristics
Table 1. cont.
4
5
Micrococcus aloeverae sp. nov
Protein profiling was carried out by MALDI-TOF/MS as described by Prakash et al. (2014). At least two biological replicates of strain AE-6T and its closest relatives were cultivated in similar conditions on TSA agar plates. After growth (48 h) total proteins were extracted and analysed using MALDI-TOF/MS (Bruker Daltonics). The result of MALDI-TOF/MS analysis agreed with 16S rRNA gene sequence analysis and FAME data (Fig. S2). A dendrogram, created using the ribosomal protein data from this study, showed similar topology to the 16S rRNA gene-based phylogenetic tree (Fig. S2). In a similarly way to the 16S rRNA gene-based tree strain AE-6T formed a cluster with M. luteus DSM 20030T and M. yunnanensis YIM 65004T. It has been found that a proposed 97 % cut-off for DDH relatedness is not very stringent (Stackebrandt & Goebel, 1994; Stackebrandt & Ebers, 2006). Stackebrandt & Ebers (2006) proposed a more stringent and higher (98.7–99 %) 16S rRNA gene sequence similarity value for DDH in cases of high-quality almost full-length (.1400 bp) 16S rRNA gene sequences. Based on the findings above we selected five different members of the genus Micrococcus, which were those showing the highest 16S rRNA gene sequence similarity to strain AE-6T, for DDH-experiments. Genomic DNA was extracted and purified using the method of Marmur (1961) with additional RNase treatment. DDH was carried out based on the principles of De Ley et al. 3431
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(1970) with the modifications used by Huss et al. (1983). An optimized fluorimetric procedure, evaluated by LovelandCurtze et al. (2011), using a Step One Plus Real-Time PCR system (Applied Biosystems) fitted with a 96-well thermal cycling block was used to conduct the experiment. DNA suspended in 26Saline Sodium Citrate (SSC) Buffer was used for analysis, which was carried out in triplicate. Reassociation was carried out at optimum renaturation temperatures [Tor50.516(% G+C)+47.0] according to De Ley et al. (1970). DNA G+C content was analysed as described by Prakash et al. (2014). The DNA G+C contents of strain AE-6T was found to be 70 mol%. This was similar to the DNA G+C contents of other members of the genus Micrococcus. Strain AE-6T had the highest DDH relatedness value (57.3 %) with M. endophyticus DSM 17945T, followed by M. luteus NCTC 2665T (53.3 %), M. lylae DSM 20315T (50.8 %) and M. yunnanensis YIM 65004T (41.3 %). The DDH relatedness values determined in the present study were less than the threshold value proposed by Wayne et al. (1987) for the delineation of bacterial species. Thus DDH data also suggested that strain AE-6T represents a novel species of the genus Micrococcus. Phylogenetic closeness with endophytic Micrococcus-like M. yunnanensis YIM 65004T and M. endophyticus DSM 17945T support the endophytic origin of AE-6T. Our phylogenetic, chemotaxonomic, morphological, physiological and genotypic data indicate that strain AE-6T is related to species of the genus Micrococcus with validly published names, and that it deviates from other related genera such as Arthrobacter and Citricoccus, confirming that strain AE-6T is a member of the genus Micrococcus. Comparative studies of the biochemical and physiological traits of strain AE-6T using phylogenetically close members of the genus Micrococcus (Table 1) showed several variations in phenotypic data. In addition, DDH data with other species of the genus Micrococcus showed a value of less than 70 %, which is the threshold for bacterial species delineation. Thus, our data suggest that strain AE-6T should be distinguished from other members of the genus Micrococcus and we propose that it as a novel species of genus Micrococcus with the suggested name Micrococcus aloeverae sp. nov.
for nitrate reductase, urease and amylase. The major cellular fatty acids are anteiso-C15 : 0, iso-C15 : 0 and C16 : 0, followed by iso-C16 : 0, C14 : 0, anteiso-C17 : 0 and iso-C14 : 0. The predominant menaquinones are MK-8 (H2) and MK-7 (H2), while MK-8 and MK-7 are present as minor components. Major polar lipids are DPG and PG, while PL, GL, PI are present in minor quantities. The peptidoglycan is an A2, L-Lys- peptide subunit type and ribose is the predominant cell wall sugar. The type strain, AE-6T (5MCC 2184T5DSM 27472T), was isolated from the inner tissues of leaves of Aloe barbadensis (Aloe vera) collected from Pune, Maharashtra, India. The DNA G+C content is 70 mol%
Acknowledgements This work was supported by the Department of Biotechnology (DBT; grant no. BT/PR/0054/NDB/52/94/2007), the Government of India, under the project ‘Establishment of microbiolculture collection’. We are grateful to Dr Praveen Rahi, Microbial Culture Collection (MCC)-NCCS, Pune for providing the MALDI-TOF, MS data. We are also thankful to Dr Bernhard Schink for correction of the etymology of the strain and Dr R. Pukall for valuable discussion about Micrococcus taxonomy.
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Prakash, O. & Lal, R. (2006). Description of Sphingobium fuliginis sp. nov., a phenanthrene-degrading bacterium from a fly ash dumping site, and reclassification of Sphingomonas cloacae as Sphingobium cloacae comb. nov. Int J Syst Evol Microbiol 56, 2147–2152.
Waller, F., Achatz, B., Baltruschat, H., Fodor, J., Becker, K., Fischer, M., Heier, T., Hu¨ckelhoven, R., Neumann, C. & other authors (2005).
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The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proc Natl Acad Sci U S A 102, 13386–13391. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic
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DOI 10.1099/ijs.0.063339-0
Description of Micrococcus aloeverae sp. nov., an endophytic actinobacterium isolated from Aloe vera Om Prakash, Yogesh Nimonkar, Hitendra Munot, Avinash Sharma, Venkata Ramana Vemuluri, Mahesh S. Chavadar and Yogesh S. Shouche Correspondence
Microbial Culture Collection, National Centre for Cell Science, Pune, Maharastra 411007, India
Om Prakash [email protected]
A yellow Gram-stain-positive, non-motile, non-endospore -forming, spherical endophytic actinobacterium, designated strain AE-6T, was isolated from the inner fleshy leaf tissues of Aloe barbadensis (Aloe vera) collected from Pune, Maharashtra, India. Strain AE-6T grew at high salt concentrations [10 % (w/v) NaCl], temperatures of 15–41 6C and a pH range of 5–12. It showed highest (99.7 %) 16S rRNA gene sequence similarity with Micrococcus yunnanensis YIM 65004T followed by Micrococcus luteus NCTC 2665T (99.6 %) and Micrococcus endophyticus YIM 56238T (99.0 %). Ribosomal protein profiling by MALDI-TOF/MS also showed it was most closely related to M. yunnanensis YIM 65004T and M. luteus NCTC 2665T. Like other members of the genus Micrococcus, strain AE-6T had a high content of branched chain fatty acids (isoC15 : 0 and anteiso-C15 : 0). MK-8(H2) and MK-8 were the predominant isoprenoid quinones. Cell wall analysis showed an ‘A2 L-Lys-peptide subunit’ type of peptidoglycan and ribose to be the major cell wall sugar. The DNA G+C content was 70 mol%. Results of DNA–DNA hybridization of AE-6T with its closest relatives from the genus Micrococcus produced a value of less than 70%. Based on the results of this study, strain AE-6T could be clearly differentiated from other members of the genus Micrococcus. We propose that it represents a novel species of the genus Micrococcus and suggest the name Micrococcus aloeverae sp. nov., with strain AE-6T (5MCC 2184T5DSM 27472T) as the type strain of the species.
Endophytes are micro-organisms (bacteria and fungi) isolated from the inner tissues of plants. It has been found that most plant species harbour one, or more than one, kind of endophyte and they reside inside the plant without producing any adverse effects (Adams & Kloepper, 1996; James et al., 2002; Sessitsch et al., 2002; Kaga et al., 2009; Johnston-Monje & Raizada, 2011). To date; most of the endophytes that have been isolated are fungi and only a small amount of work has been carried out on bacterial endophytes (Verma et al., 1998; Waller et al., 2005). Although the area of plant-endophyte interaction is not well studied the available data indicates that some endophytes have plant growth promoting activities and protect plants from invasion of pathogens and environmental stresses (Verma et al., 1998; James et al., 2002; Waller et al., 2005). In the present study, we have isolated an endophytic strain, belonging to the genus Micrococcus, from the fleshy inner tissues of the leaf of Aloe vera (Aloe barbadensis) using aseptic sampling and isolation techniques. The results of this Abbreviations: SSC, Saline Sodium Citrate (SSC) Buffer. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of AE-6T is KF524364. Two supplementary figures and one supplementary table are available with the online version of this paper.
063339 G 2014 IUMS
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study indicate that the isolated strain has distinct features from species of the genus Micrococcus with validly published names and that it should be described as a novel species. Besides this study, members of the genus Micrococcus have been detected as endophytes on several other plants including the zea (Johnston-Monje & Raizada, 2011), cotton (Mcinroy & Kloepper, 1995), rice seeds (Kaga et al., 2009) and the roots of Polyspora axillaris (Zhao et al., 2009) and Aquilaria sinensis (Chen et al., 2009). The genus Micrococcus was first described by Cohn (1872) for Gram-stain-positive, non-motile, non-endospore-forming, small spherical bacterium. Members of the genus Micrococcus are widely distributed and have been isolated from a variety of habitats including the soil (Zhang et al., 2010), the air (Rieser et al., 2013), activated sludge (Liu et al., 2007), permanently cold samples (Liu et al., 2000), human skin (Kloos et al., 1974), dairy waste (Chittpurna et al., 2011) and the inner tissues of plants (Zhao et al., 2009). At the time of writing there are a total of nine species of the genus Micrococcus with validly published names, including two endophytic species (www.bacterio.net). Stackebrandt et al. (1995) reclassified some members of genus Micrococcus into four different genera Kocuria, Dermacoccus, Kytococcus, and Nesterenkonia, while Wieser et al. (2002) gave an emended description of the genus Micrococcus for the first time. 3427
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Almost all the members of the genus Micrococcus are yellow and grow at high pH (pH 10). The characteristic features of members of the genus Micrococcus are aerobic growth, catalase-positive cells, a high content of branched chain fatty acids (iso-C15 : 0 and anteiso-C15 : 0), a high G+C content (66.3–73.3 mol%) and the presence of MK-8 and MK-8(H2) as the predominant respiratory quinones (Stackebrandt et al., 1995; Wieser et al., 2002). In order to isolate the endophytic bacteria, fresh leaf samples from Aloe barbadensis (Aloe vera) were collected from Pune, Maharashtra, India, in a pre-sterile polyethylene bag. Endophytic strains were isolated using the method of Gayathri et al. (2010). In brief, after collection samples were immediately transported to the laboratory and stored at 4 uC for not longer than two days. Firstly were washed with distilled water and sterilized in 70 % (v/v) ethanol (0.5 min) followed by 0.1 % mercuric chloride (HiMedia, Mumbai, India) (3.0 min). To remove the disinfectant, leaves were again washed thrice with sterile distilled water. After treatment, inner tissues were removed with a sterile scalpel, crushed in normal saline and plated on tryptic soy agar and Luria agar plates. Isolated strains were purified by restreaking on fresh medium plates and maintained with 15 % (v/v) glycerol in the vapour phase of liquid nitrogen. Based on its potential for growth at a wide range of salt concentrations, pH values and temperatures strain AE-6T was selected for further characterization. Extraction and purification of genomic DNA was carried out using the method of Prakash & Lal (2006). The 16S rRNA gene sequence was amplified according to methods described by Prakash et al. (2012) and sequencing was carried out using Sanger’s method (Prakash et al. 2014). An almost complete stretch of 1411 bp good quality 16S rRNA gene sequence was obtained after manual editing of the ABI-sequence file in ChromasPro software (http://www. technelysium.com.au/ChromasPro.html). A sequence similarity search was conducted using the SeqMatch tool of the Ribosomal Database Project (RDP). The 30 most similar reference sequences were retrieved and selected for multiple alignments. The 16S rRNA gene sequence of Cellulomonas flavigena 134T was used as an outgroup. Multiple sequence alignment was performed with CLUSTAL X, version 1.81 (Thompson et al., 1997). To get unambiguous data, alignment was manually edited using DAMBE (Xia, 2013). A phylogenetic tree was reconstructed using software package MEGA version-5 (Tamura et al., 2011) and the Kimura twoparameter model (Kimura, 1980). Bootstrap values were deduced based on 1000 replications (Felsenstein, 1985). In order to evaluate the robustness of the tree topology, clustering was performed using all three different methods: neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MR) (Fitch, 1971; Felsenstein, 1981; Saitou & Nei, 1987; Tamura et al., 2011).
yunnanensis YIM 65004T (99.7 %), M. luteus DSM 20030T (99.6 %) and M. endophyticus YIM 56238T (99.0 %). All three methods of phylogenetic reconstruction provided similar tree topologies and clustering. Strain AE-6T clustered with members of the genus Micrococcus and showed phylogenetic closeness with M. yunnanensis YIM 65004T and M. luteus DSM 20030T with a high bootstrap value (Fig. 1). Micrococcus lactis DSM 23694T and Zhihengliuella aestuarii JCM 16364T formed separate clades and showed distinct positions to other members of the genus Micrococcus and the genus Zhihengliuella. The taxonomic position of M. lactis DSM 23694T and Z. aestuarii JCM 16364T should be discussed separately based on other experimental evidence. Thus, 16S rRNA sequence similarity data and phylogenetic analysis confirmed that strain AE-6T is a member of the genus Micrococcus. In addition, due to high levels of 16S rRNA sequence similarity with some members of the genus Micrococcus other approaches could be used to confirm the phylogenetic position of strain AE-6T and these are discussed below. In order to generate phenotypic data, type strains of the closest relatives of strain AE-6T were procured from DSMZ-Germany and experiments were conducted in similar laboratory conditions. Growth responses of the organisms were tested on tryptic soy agar, Luria agar, nutrient agar and R2A agar (Hi-Media). Colony shape, size and pigmentation were recorded on tryptic soy agar plates after-72 h incubation at 30 uC. Gram-staining was conducted using Hi-Media’s Gram-Stain kit. Catalase and oxidase tests were performed using 3 % (v/v) H2O2 and oxidase discs (Hi-Media), respectively. The pH range for growth was determined in tryptic soy broth by adjusting the pH of the medium with 1M NaOH and 1M HCl. The optimum pH for growth was determined by measuring the optical density (OD600 nm) at different time intervals. Growth at different concentrations of NaCl was tested by supplementing TSB with a range of NaCl concentrations [1–15 % (w/v) with 1 % (w/v) increments]. In order to investigate the temperature range and the optimum temperature for growth inoculated medium was incubated at different temperatures (5–45 uC in increments of 5 uC up to 40 uC, and of 1 uC at 40–45 uC) and the optical densities (OD600 nm) at different time intervals were measured. The use of different carbon sources and enzyme activities were assessed using API 20NE and API ZYM kits (bioMe´rieux). Skimmed-milk-agar and Tween-80 agar were used to assess protease and esterase production, respectively. For the gelatinase test, tryptic soy broth was supplemented with 12 % (w/v) gelatine, as a gelling agent, while urease production was monitored using urea agar with a pH indicator (Smibert & Krieg, 1994).
Sequence similarity calculations revealed that strain AE-6T showed closest similarity to members of the genus Micrococcus and had highest sequence similarity with M.
Strain AE-6T is a Gram-stain-positive, non-motile, nonendospore-forming, small, spherical bacterium of endophytic origin. It had a mesophilic nature and grew in a wide range of salt concentrations [0–10 %(w/v) NaCl], and at pH 5–12 and temperatures of (15–41 uC). Strain AE-6T formed yellow, round, convex colonies, which were 0.5 mm in diameter, on tryptic soy agar plates after 72 h incubation at 30 uC. Strain AE-6T showed positive results for catalase but
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International Journal of Systematic and Evolutionary Microbiology 64
Micrococcus aloeverae sp. nov
Arthrobacter oxydans DSM 20119T (X83408)
99 89
Arthrobacter scleromae DSM 17756T (AF330692) Arthrobacter sulfonivorans DSM 14002T (AF235091)
69 0.01
95
Arthrobacter humicola JCM 15921T (AB279890) 83 Arthrobacter oryzae JCM 15922T (AB279889) Arthrobacter globiformis NBRC 12137T (X80736)
50
33 Arthrobacter pascens DSM 20545T (X80740) Micrococcus lactis DSM 23694T (FN673681)
100
Zhihengliuella aestuarii JCM 16364T (EU939716) Zhihengliuella alba NBRC 109061T (AB778263)
45
42
97
Zhihengliuella flava DSM 26152T (AB778260) Zhihengliuella halotolerans DSM 17364T (DQ372937)
57 98
54
Zhihengliuella salsuginis NBRC 109062T (AB778264)
Micrococcus terreus NBRC 104258T (FJ423763) Micrococcus cohnii DSM 23974T (FR832424) Micrococcus lylae DSM 20315T (X80750) 87
Micrococcus yunnanensis DSM 21948T (FJ214355) 68
52
Micrococcus luteus NCTC 2665T (CP001628) Micrococcus aloeverae AE6T MCC 2184 (KF524364)
42
Micrococcus endophyticus DSM 17945T (EU005372) Micrococcus flavus DSM 19079T (DQ491453)
34 30
Micrococcus antarcticus JCM 11467T (AJ005932)
Citricoccus parietis CCUG 57388T (FM992367) 74
Citricoccus zhacaiensis JCM 15136T (EU305672) Citricoccus nitrophenolicus DSM 23311T (GU797177)
26
Citricoccus alkalitolerans DSM 15665T (AY376164)
71 83
Citricoccus muralis DSM 14442T (AJ344143) Cellulomonas flavigena DSM 20109T (X83799)
Fig. 1. A neighbour-joining phylogenetic tree, reconstructed using 16S rRNA gene sequences, showing the relationships of strain AE-6 with other species of genus Micrococcus. Numbers at the nodes represent confidence levels in terms of bootstrap support generated from 1000 replicates. Bootstrap values ,50 % are not shown. Cellulomonas flavigena DSM 20109Twas used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
was negative for oxidase. The strain produced good amounts of extracellular protease in nutrient broth after 24 h of incubation and gave a positive result for gelatinase, lipase and esterase. Detailed descriptions of strain AE-6T in terms of its morphological, physiological and chemotaxonomic features are given in Table 1 and in the species description. Polar lipids and isoprenoid quinones were extracted from freeze-dried cells and analysed by two dimensional TLC and HPLC, as described in Tindall (1990a, b) respectively. Cell http://ijs.sgmjournals.org
wall sugar and peptidoglycan analyses were performed by the DSMZ Identification Service according to published protocols (Schumann, 2011). For fatty acid methyl ester (FAME) analysis strain AE-6T, M. luteus DSM 20030T and M. yunnanensis YIM 65004T were grown simultaneously on tryptic soy agar plates for 48 h at 30 uC. Extraction and analysis of fatty acids were conducted as described by Prakash et al. (2014). Peaks were identified using the database TSBA6 of MIS (MIDI). 3429
1, Strain AE-6T; 2, M. endophyticus DSM17945T; 3, M. luteus DSM20030T; 4, M. yunnanenisis DSM 21948T; 5, M. lylae DSM 20315T; 6, M. flavus DSM 30824T. Strain AE-6 T, M. endophyticus DSM17945T, M. yunnanensis DSM 21948T, M. luteus DSM20030T, M. lylae DSM 20315T, and M. flavus DSM 30824T showed negative results for potassium nitrate (NO3), L- tryptophan (TRP), Larginine (ADH), 4- nitrophenyl-bD galactopyranoside (PNPG), L-arabinose (ARA), N-acetylglucosamine (NAG), adipic acid (ADI), trisodium citrate (CIT), cystine arylamidase, naphthol-ASBI-phosphohydrolase, a- galactosidase, b- galactosidase, b- glucuronidase, b- glucosidase, N-acetyl- b-glucosaminidase, a-mannosidase, a-fucosidase. All the species were Gram-stain positive, non-motile, non-endospore- forming and showed positive results for catalase and naphthol-AS- BI- phosphohydrolase. +, Positive; 2, negative; W, weakly positive. Characteristics
International Journal of Systematic and Evolutionary Microbiology 64
Colonies Colour Diameter (mm) after 48 h Shape Temperature range for growth (uC) Optimum temperature for growth (uC) pH range for growth Optimum pH for growth Tolerance to NaCl (w/v) Oxidase Protease Glucose(fermentative) Urease Tween-20 hydrolysis (72h) Aesculin hydrolysis Gelatinase D-Glucose D – Mannose D – Mannitol D – Maltose Potassium gluconate Capric acid Malic acid Phenylacetic acid Alkaline phosphtase Esterase Esterase Lipase Lipase Leucine arylamidase Valine arylamidase Trypsin a-Chymotrypsin Acid phosphatase a-Glucosidase
1
2
3
4
5
6
Yellow 0.5 Round convex 15–41 35 5–12 9 10 % 2 +++ 2 2 2 2 + + 2 2 + 2 2 2 2 + + + + ++ + 2 2 + ++
Yellow 0.5 Round convex 15–37 28 6–9 7–8 10 %
Yellow 0.5 Round convex 20–40 37 5–10 7 10 %
Yellow 1 Round convex 4–45 28 6–8 7–8 15 %
White 1 Round convex 20–45 35 6–9 7 10 %* ” ” 2 2 2 2 + + 2 2 + 2 2 + + 2 ++ 2 2 +++ 2 2 2 2 ++
Yellow 0.5 Round convex 26–34 31 5–9 6 10 % + ” 2 2 2 2 + 2 2 2 2 2 2 2 2 + ++ ++ 2 +++ 2 2 2 + +++
W
W
W
+ + 2 + 2 2 2 + + + + 2 2 2 + + + 2 + + 2 2 + ++
++ 2 + 2 + 2 2 + 2 + 2 2 2 2 + + + + + + + + ++ ++
++++ 2 2 2 + + + + 2 + 2 + + + + + + 2 +++ + + 2 + ++
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Table 1. Differential phenotypic traits of strain AE-6T and the closest phylogenetically related species of the genus Micrococcus
2 + 71.4* MK-8(H2), MK-7(H2)* + 2 69* MK-8(H2)* Data were all from this study in comparable laboratory conditions except for: *Data taken from Zhao et al. (2009).
2 2 70* MK-8, MK-8(H2)* 2 + 72.9* MK-8(H2), MK-7(H2)* Simmons citrate test Starch casein test DNA G + C content (mol%) Quinones
2 2 70 MK-8(H2), MK-7(H2), MK-8
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A comparison of the FAMEs present in strain AE-6T and other members of the genus Micrococcus is presented in Table S1 (available in the online Supplementary Material). Data indicated that the iso and anteiso forms of branched chain fatty acids are major components of species of the genus Micrococcus with iso-C15 : 0 and anteiso-C15 : 0 representing the predominant forms (Table S1). The qualitative and quantitative similarities in the fatty acid profiles of strain AE-6T and other members of the genus Micrococcus, means that FAME data support the findings of 16S rRNA gene sequence analysis and further suggest that strain AE-6T is a member of the genus Micrococcus. MK8(H2) and MK-7 (H2) were detected as the predominant menaquinones, while MK-8 and MK-7 were present as minor menaquinones. Polar lipid profiles showed prominent spots of diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG), while an unidentified phospholipid (PL), glycolipid (GL) and a phosphatidylinositol (PI) were detected in minor amounts (Fig. S1). Lysine, glycine, alanine and glutamic acids were detected in the hydrolysate of peptidoglycan. The approximate ratio of amino acids was: 1.4 Ala: 0.6 Gly: 1.4 Glu: 1.0 Lys. The peptides D-Ala – L-Ala, L-Ala – D-Glu, L-Lys – D-Ala and D-Glu – Gly were detected by two dimensional TLC. Dinitrophenylation indicated that alanine represented the N terminus of the interpeptide bridge. Based on the data above we deduced that strain AE-6T has an ‘A2, L-Lys’ type of peptidoglycan. Data on cell wall sugars indicated that strain AE-6T possesses ribose and less glucose, mannose and rhamnose.
2 2 71.7* MK-8(H2), MK-7(H2)*
6 3 2 1 Characteristics
Table 1. cont.
4
5
Micrococcus aloeverae sp. nov
Protein profiling was carried out by MALDI-TOF/MS as described by Prakash et al. (2014). At least two biological replicates of strain AE-6T and its closest relatives were cultivated in similar conditions on TSA agar plates. After growth (48 h) total proteins were extracted and analysed using MALDI-TOF/MS (Bruker Daltonics). The result of MALDI-TOF/MS analysis agreed with 16S rRNA gene sequence analysis and FAME data (Fig. S2). A dendrogram, created using the ribosomal protein data from this study, showed similar topology to the 16S rRNA gene-based phylogenetic tree (Fig. S2). In a similarly way to the 16S rRNA gene-based tree strain AE-6T formed a cluster with M. luteus DSM 20030T and M. yunnanensis YIM 65004T. It has been found that a proposed 97 % cut-off for DDH relatedness is not very stringent (Stackebrandt & Goebel, 1994; Stackebrandt & Ebers, 2006). Stackebrandt & Ebers (2006) proposed a more stringent and higher (98.7–99 %) 16S rRNA gene sequence similarity value for DDH in cases of high-quality almost full-length (.1400 bp) 16S rRNA gene sequences. Based on the findings above we selected five different members of the genus Micrococcus, which were those showing the highest 16S rRNA gene sequence similarity to strain AE-6T, for DDH-experiments. Genomic DNA was extracted and purified using the method of Marmur (1961) with additional RNase treatment. DDH was carried out based on the principles of De Ley et al. 3431
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(1970) with the modifications used by Huss et al. (1983). An optimized fluorimetric procedure, evaluated by LovelandCurtze et al. (2011), using a Step One Plus Real-Time PCR system (Applied Biosystems) fitted with a 96-well thermal cycling block was used to conduct the experiment. DNA suspended in 26Saline Sodium Citrate (SSC) Buffer was used for analysis, which was carried out in triplicate. Reassociation was carried out at optimum renaturation temperatures [Tor50.516(% G+C)+47.0] according to De Ley et al. (1970). DNA G+C content was analysed as described by Prakash et al. (2014). The DNA G+C contents of strain AE-6T was found to be 70 mol%. This was similar to the DNA G+C contents of other members of the genus Micrococcus. Strain AE-6T had the highest DDH relatedness value (57.3 %) with M. endophyticus DSM 17945T, followed by M. luteus NCTC 2665T (53.3 %), M. lylae DSM 20315T (50.8 %) and M. yunnanensis YIM 65004T (41.3 %). The DDH relatedness values determined in the present study were less than the threshold value proposed by Wayne et al. (1987) for the delineation of bacterial species. Thus DDH data also suggested that strain AE-6T represents a novel species of the genus Micrococcus. Phylogenetic closeness with endophytic Micrococcus-like M. yunnanensis YIM 65004T and M. endophyticus DSM 17945T support the endophytic origin of AE-6T. Our phylogenetic, chemotaxonomic, morphological, physiological and genotypic data indicate that strain AE-6T is related to species of the genus Micrococcus with validly published names, and that it deviates from other related genera such as Arthrobacter and Citricoccus, confirming that strain AE-6T is a member of the genus Micrococcus. Comparative studies of the biochemical and physiological traits of strain AE-6T using phylogenetically close members of the genus Micrococcus (Table 1) showed several variations in phenotypic data. In addition, DDH data with other species of the genus Micrococcus showed a value of less than 70 %, which is the threshold for bacterial species delineation. Thus, our data suggest that strain AE-6T should be distinguished from other members of the genus Micrococcus and we propose that it as a novel species of genus Micrococcus with the suggested name Micrococcus aloeverae sp. nov.
for nitrate reductase, urease and amylase. The major cellular fatty acids are anteiso-C15 : 0, iso-C15 : 0 and C16 : 0, followed by iso-C16 : 0, C14 : 0, anteiso-C17 : 0 and iso-C14 : 0. The predominant menaquinones are MK-8 (H2) and MK-7 (H2), while MK-8 and MK-7 are present as minor components. Major polar lipids are DPG and PG, while PL, GL, PI are present in minor quantities. The peptidoglycan is an A2, L-Lys- peptide subunit type and ribose is the predominant cell wall sugar. The type strain, AE-6T (5MCC 2184T5DSM 27472T), was isolated from the inner tissues of leaves of Aloe barbadensis (Aloe vera) collected from Pune, Maharashtra, India. The DNA G+C content is 70 mol%
Acknowledgements This work was supported by the Department of Biotechnology (DBT; grant no. BT/PR/0054/NDB/52/94/2007), the Government of India, under the project ‘Establishment of microbiolculture collection’. We are grateful to Dr Praveen Rahi, Microbial Culture Collection (MCC)-NCCS, Pune for providing the MALDI-TOF, MS data. We are also thankful to Dr Bernhard Schink for correction of the etymology of the strain and Dr R. Pukall for valuable discussion about Micrococcus taxonomy.
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