Antonie van Leeuwenhoek (2015) 107:1599–1606 DOI 10.1007/s10482-015-0455-x
ORIGINAL PAPER
Nocardioides solisilvae sp. nov., isolated from a forest soil Vishnuvardhan Reddy Sultanpuram . Thirumala Mothe . Farooq Mohammed
Received: 4 February 2015 / Accepted: 10 April 2015 / Published online: 19 April 2015 Ó Springer International Publishing Switzerland 2015
Abstract A Gram-stain positive, rod shaped aerobic bacterial strain designated Ka25T was isolated from a forest soil sample near Kasol, Himachal Pradesh, India. Based on 16S rRNA gene sequence analysis it was identified that strain Ka25T belongs to the class Actinobacteria and is most closely related to Nocardioides daejeonensis KCTC 19772T (97.2 %), Nocardioides dubius JCM 13008T (96.9 %), Nocardioides psychrotolerans CGMCC 1.11156T (96.8 %), Nocardioides pacificus DSM 27278T(96.7 %) and Nocardioides daphniae JCM 16608T(96.4 %) sequence similarity respectively. The chemotaxonomic properties of strain Ka25T are consistent with those of the members of the genus Nocardioides: MK-8(H4) is the predominant menaquinone, iso-C16 : 0, iso-C17 : 0 and C18 : 1x9c were identified as the predominant cellular fatty acids and the cell-wall peptidoglycan is based on LL-2,6-diaminopimelic acid. The DNA G?C content of strain Ka25T was determined to be 71.6 mol %. The results of the polyphasic taxonomic analysis allowed a clear differentiation of strain Ka25T from all other
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0455-x) contains supplementary material, which is available to authorized users. V. R. Sultanpuram (&) T. Mothe F. Mohammed Microbial Ecology Lab, Department of Biochemistry, Mahatma Gandhi University, Anneparthy, Yellareddygudem (PO), Nalgonda 508254, Telangana, India e-mail: [email protected]
members of the genus Nocardioides. Strain Ka25T is thus considered to represent a novel member of the genus Nocardioides, for which the name Nocardioides solisilvae sp. nov. is proposed. The type strain is Ka25T (=KCTC39528T =MCC2762T). Keywords Nocardioides Nocardioides solisilvae sp. nov Forest soil Polyphasic taxonomy
Introduction The family Nocardioidaceae originally was described by Nesterenko et al. (1985, 1990), then emended by Stackebrandt et al. (1997) and Zhi et al. (2009). Originally the family included the genus Nocardioides (Prauser 1976) with the species Nocardioides albus(Prauser 1976), Nocardioides luteus (Prauser 1984) and Nocardioides simplex (O’Donnell et al. 1982) and some other related organisms with LLdiaminopimelic acid in their cell wall (To´th and Borsodi 2014). Recent analysis of 16S rRNA gene sequences shows that the genera comprising the current family Nocardioidaceae form at least two separate phylogenetic clusters. The first cluster includes the genera Nocardioides, Aeromicrobium, and Marmoricola whose morphology is relatively simple, with motile rod shaped bacteria. A predominant menaquinone occurs in their profile (with tetra hydrogenated side
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chain of 8 or 9 isoprene units) and significant amounts of straight-chain saturated and unsaturated fatty acids can be found in their cells. The second cluster contains the genera Kribbella, Actinopolymorpha, Flindersiella and Thermasporomyces which have more complex cell morphology and lack motile cells. Their menaquinone profile is more complex, involving longer and more saturated side chains. Their fatty acid profiles are dominated by iso- and anteiso-branched fatty acids and contain only minor amounts of straightchain components (To´th and Borsodi 2014). The genus Nocardioides includes Gram-stain positive, coccus-to-rod-shaped bacteria with LL-diaminopimelic acid as the diagnostic diamino acid of their cell-wall peptidoglycan, MK-8(H4) as the predominant menaquinone and C18 : 1x9c, iso-C16: 0, isoC17 : 0 and 10-methyl C18 : 0 as the major fatty acids (Lee et al. 2012). Originally, the genus contained two species, N. albus and N. luteus (Prauser 1984) and at the time of writing, the genus comprises 75 validly named species (http://www.bacterio.net/nocardioides. html). During a study of the cultivable bacterial diversity of a high altitude forest soil from Kasol, India, a novel bacterial strain designated Ka25T was isolated. This paper focuses on the taxonomic characterisation of strain Ka25T based upon a polyphasic approach.
Materials and methods Isolation of the strain StrainKa25T was isolated from a high altitude (1521 m) forest soil sample from Kasol, Himachal Pradesh, India (Latitude 32°000 , Longitude 77°160 ) on nutrient agarmedium consisting of (l-1) bacteriological peptone (5 g), NaCl (5 g), beef extract (1.5 g), yeast extract (1.5 g), agar (15 g) at pH 7.4 ± 0.2.The isolation was made by means of the standard dilution-plating technique. Pure culture was obtained by repeated streaking on nutrient agar at pH 7.4 ± 0.2 [Himedia, Mumbai, India] andthe isolate was then preserved at 4 °C for further use. Nocardioides daejeonensis KCTC 19772T was obtained from the Korean Collection for Type Cultures and cultured under the same conditions for comparative purposes.
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Morphological, biochemical and physiological characterization Morphological, biochemical and physiological characterisation of strain Ka25T was carried out as described by Yoon and Park (2006).Microscopic observations of strain Ka25T were made by light microscopy (MLX; Olympus-Magnus). The pH (range 6.0–12.0, with an interval of 0.5) were tested with K2HPO4–KH2PO4 buffer for pH 6.0–8.0, NaHCO3– NaOH buffer for pH 8.5–11.0 and Na2CO3–NaOH buffer for pH 11.5–12.0, temperature (0, 4, 10, 16, 20, 28, 30, 35, 37, 40, 50, 55 and 60 °C) and salt concentration (0–30 % w/v, with an interval of 0.5 % w/v) ranges for growth were examined in tryptone soya broth medium at 30°C for 2 days. The Gram reaction was performed using the non-staining (KOH) method, as described by Buck (1982). Catalase activity was assessed by bubble production in 3 % (v/v) H2O2 and oxidase activity was assessed using 1 % (w/v) tetramethyl p-phenylenediamine (Cappuccino and Sherman 2002). Nitrate reduction, urease production, citrate utilisation, gelatine liquefication, indole production and arginine hydrolase activity were determined as previously described (Cowan and Steel 1965; Lanyi 1987). Anaerobic growth was examined in serum bottles using nutrient broth with sodium thioglycolate (1 gl-1) and replacing the air in the bottles with nitrogen. Enzyme activities were tested using API ZYM strips (bioMe´rieux) according to the manufacturer’s instructions. Tests for the hydrolysis of casein, starch, xanthine and hypoxanthine (Atlas 1993) were carried out over 7 days at 30 °C. Utilisation of various substrates as sole carbon and energy sources was determined as described by Shirling and Gottlieb (1966). Susceptibility to antibiotics was tested by placing antibiotic-impregnated discs on TSA plates that were seeded with suspensions of the test strain. Phylogenetic analysis and genetic relatedness Genomic DNA was extracted and purified according to the method of Marmur (1961). The 16S rRNA gene sequence of strain Ka25T was obtained by PCR as described previously (Vishnuvardhan Reddy et al. 2013). Identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarity were achieved using the EzTaxon-e server
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(http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012). The CLUSTAL_W algorithm of MEGA 5 (Tamura et al. 2011) was used for sequence alignments and the phylogenetic analysis of the related 16S rRNA gene sequences was performed using MEGA 5. Distances were calculated by using the Kimura correction in a pair-wise deletion manner (Kimura 1980). Neighbourjoining (NJ) (Saitou and Nei 1987), minimum evolution (ME), maximum likelihood (ML) and maximum parsimony (MP) (Nei and Kumar 2000) methods in the MEGA 5 software were used to construct phylogenetic trees. Percentage support values were obtained using a bootstrap procedure (Felsenstein 1985). The mol% G?C content of the DNA of strain Ka25T was determined by HPLC (Mesbah et al. 1989). To study the genetic relatedness of strain Ka25T and N.daejeonensis KCTC 19772T, the strains were analysed by AP-PCR and BOX-A1R PCR. The APPCR fingerprinting was performed by using the M13F primer [(-20): GTAAAACGACGGCCAGT] and the following PCR program: two cycles of 94 °C for 5 min, 40 °C for 5 min and 72 °C for 5 min; followed by 40 high stringency cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 2 min. Similarly, BOX-A1R PCR was performed by using the BOX-A1R primer (50 -CTACGGCAAGGCGACGCTGACG-30 ). The cycling programme started with denaturation at 95 °C for 7 min, followed by 35 cycles of: 94 °C for 1 min; 1 min at 53 °C and 8 min at 72 °C. After the extension step, the temperature was held at 72 °C for 15 min. Amplified DNA products were resolved by electrophoresis on 2 % w/v agarose gel. Chemotaxonomic characterization Menaquinones, amino acids of whole cell hydrolysates and polar lipids of strains Ka25T and N.daejeonensis KCTC 19772T were analysed from cells grown in tryptone soya broth incubated for 5 days at 30 °C with 7.5 pH and 4 % (w/v) NaCl. Menaquinones were extracted from freeze-dried biomass and purified according to Collins and Jones (1980) and analysed by HPLC (Kroppenstedt 1985). Amino acids of whole-cell hydrolysates were purified by TLC after hydrolysis of whole cells with 6 M HCl at 100 °C for 18 has described previously by Komagata and Suzuki (1987). Polar lipids were extracted, examined by two-dimensional TLC and identified using published procedures (Minnikin et al. 1984). For
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fatty acid analysis, strains Ka25T and N.daejeonensis KCTC 19772T were grown in tryptone soya broth at 30 °C with pH 7.5 and 4 % (w/v) NaCl for 2 days. The fatty acids analysis, which was outsourced to Zeal Biologicals, Secunderabad, was performed by following the procedures of Sasser (1990) according to the standard protocol of MIDI Microbial Identification System.
Results and discussion Morphological, biochemical and physiological characteristics Cells of strain Ka25T were observed to be Gram-stain positive, aerobic, non-spore-forming, and non-motile. Cells were observed to be oval to coccoid with 1.0–2.1 lm length and 0.6–1.2 lm width. Tests for oxidase and catalase gave positive results. Colonies grown on TSA agar plates for 5 days at 30 °C were observed to be smooth, translucent, circular, convex, cream coloured and 1.0–1.5 mm in diameter. Strain Ka25T was found to be able to grow between pH 6.0 and 11.0, with an optimum pH value of 7.5. The strain was found to be able to grow in the range of 20–45° C, with optimum growth temperature at 30 °C.Strain Ka25T was found to grow in the presence of 0–14 % NaCl (w/ v) with an optimum of 4 % NaCl (w/v). The differential characteristics of strain Ka25T compared with N.daejeonensis KCTC 19772T are shown in Table 1, while other additional characteristics are given in the species description. Strain Ka25T was found to be sensitive to nalidixic acid (30 lg), gentamycin (120 lg), vancomycin (30 lg), tetracycline (30 lg), streptomycin (10 lg),kanamycin (30 lg), amikacin (30 lg), erythromycin (15 lg), ciproflaxcin (5 lg)and resistant to penicillin (10 lg) and ampicillin (10 lg). Phylogenetic analysis and genetic relatedness The almost complete 16S rRNA gene sequence (1.435 bp) of strain Ka25T was obtained (Gen-Bank/ EMBL/DDBJ accession numberLN555580).The results of phylogenetic analysis of the 16S rRNA gene sequence showed that strain Ka25T clusters within the genus Nocardioides (a consensus tree is shown as Fig. 1) and the sequence similarities with the nearest phylogenetic members are in agreement with
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Table 1 Characteristics used to distinguish strain Ka25T from Nocardioides daejeonensis KCTC 19772T Characteristic
Nocardioidessolisilvae Ka25Tsp. nov.
Nocardioides daejeonensis KCTC 19772T
Cell length (lm)
1.0–2.1
0.8–2.0
Cell width (lm)
0.6–1.2
0.5–0.8
Colony colour
Cream
Pale yellow
NaCl range(%, w/v)
0–14
0–10
NaCl optima (%, w/v)
4
4
pH range
6.0–11.0
5.5–9.0
pH optima
7.5
7.5
Temp range (°C)
20–45
15–37
Temp optima (°C)
30
30
Oxidase
?
-
Urease
?
-
Casein hydrolysis
?
-
Enzyme activity Esterase (C4)
?
W
a-Glucosidase (starch hydrolysis) b-Glucosidase
? W
?
3-Hydroxybenzoate
-
?
4-Hydroxybenzoate
-
?
DL-3-Hydroxybutyrate
?
-
2-Ketogluconate (a)
-
?
Caprate
-
?
Assimilation of
Itaconate
-
w
Melibiose
?
-
D-Mannitol
?
-
D-Sorbitol
?
-
Data are from this study. ?positive, -negative, w weak positive Both the strains were non-motile, positive for catalase activity, starch and esculin hydrolysis; negative for xanthine, hypoxanthine and cellulose hydrolysis. Both the strains were showing nitrate reduction activities and citrate utilization, but both strains showed negative result for arginine hydrolyase and indole production. Both the strains were showing negative enzyme activity for N-acetyl-bglucosamidase, b-glucuronidase and Lipase (C14). Alkaline phosphatase, acid phosphatase, esterase (C8), leucine arylamidase, napthol-AS-BL-phosphohydrolase, valine arylamidase activity were positive for both the strains. Cystine arylamidase and trypsine activities for both the strains showed weak positive. DL-lactate, maltose, L-rhamnose and citrate were utilised by both the strains, but, propionate, subarate, D-ribose, sucrose, myo-inositol, L-alanine and N-acetyl-D-glucosamine were not utilized
the EzTaxon-e server analysis. EzTaxon-e server search analysis revealed that the strain Ka25T is most closely related to N.daejeonensis KCTC 19772T (97.2 % sequence similarity), Nocardioides dubius JCM 13008T (96.9 %), Nocardioides psychrotolerans CGMCC 1.11156T (96.8 %), Nocardioides pacificus DSM 27278T (96.7 %), Nocardioides daphniae JCM 16608T (96.4 %) and other members of the genus Nocardioides (\96.4 %). The sequence similarities of strain
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Ka25T with N.daejeonensis KCTC 19772T using different clustering algorithms (96.8 % in NJ tree; 96.8 % in ME tree and 97.2 % in ML tree) along with the EzTaxon-e server analysis, consistently indicated that N.daejeonensis KCTC 19772Tis the closest relative. All the clustering algorithms used function differently, hence minor differences in the sequence similarities of the strains were observed when each of them was used. The G?C content of the DNA of strain Ka25T was 71.6 mol%, which is
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Terrabacter tumescens DSM 20308T (AF00502316S) Nocardioides deserti DSM 26045T(KM816582 ) Nocardioides psychrotolerans CGMCC 1.11156 T (JF750425) 90 * 65 Nocardioides szechwanensis CGMCC 1.11147 T (JF750424) * Nocardioides kribbensis JCM 13594T (AY835924) T 67 * Nocardioides lianchengensis DSM 24663 (HQ657322) T (EF633986) Nocardioides dokdonensis JCM 14815 94 T 72 Nocardioides salarius DSM 18239 (DQ401092) * Nocardioides basaltis KCTC 19365T (EU143365) 67 * Nocardioides marinisabuli JBRI 2003 T (AM422448) Nocardioides daphniae JCM 16608T (AM398438) # 51 Nocardioides solisilvae sp. nov. Ka25T (LN555580) # Nocardioides daejeonensis KCTC 19772T (JF937066) 90 * Nocardioides dubius KCTC 9992T (AY928902) Nocardioides pacificus JCM 19260T (KC986355) 55 * 62 Nocardioides echinoideorum JCM 30276T (KM085325) Nocardioides daedukensis KCTC 19601T (FJ842646) * 97 Nocardioides jensenii LMG 16325T (Z78210) Nocardioides alpinus LMG 26053T (GU784866) 67 # 78 Nocardioides terrigena KCTC 19217T (EF363712) Nocardioides sediminis KCTC 19271T (EF466110) * Nocardioides ginsengisegetis KCTC 19469T(GQ339901) Nocardioides dilutus KCTC 19288T (EF466121) 82 * Nocardioides terrae NBRC 104259T (FJ423762) Nocardioides caricicola JCM 17686T (FJ750845) 55 Nocardioides aquiterrae JCM 11813T (AF529063) 67 * * Nocardioides pyridinolyticus JCM 10369T (U61298) Nocardioides soli CCTCC AB 2012934T(JF937914) 85 * Nocardioides hankookensis KCTC 19246T (EF555584) 66 Nocardioides endophyticus JCM 18532T KC878444) 88 * * 93 Nocardioides conyzicola JCM 18531T (KC878445) 0.01
Fig. 1 Phylogenetic analysis of strain Ka25T with other closely related members based on 16S rRNA gene sequences available from the EMBL database (accession numbers are given in parentheses). Multiple alignments, distance calculations (distance options according to the Kimura 2- parameter model) and clustering with the neighbour-joining method, maximumlikelihood method, minimum-evolution method and
maximum-parsimony method were performed by using the software package MEGA version 5 (Tamura et al. 2011). Bootstrap values based on 1000 replications are listed as percentages at the branching points.* Clustering similarly in all the tested methods; # clustering similarly in maximumlikelihood and maximum-parsimony methods. Bar, 0.01 nucleotide substitutions per nucleotide position
similar to that of the nearest phylogenetic neighbours. DNA–DNA hybridizations (DDH) play an important role in species delineation in microorganisms and this is applicable mostly in cases where 16S rRNA gene sequence similarities are 97 % or higher. This threshold value has been reasonably increased to between 98.2 and 99.0 % depending on the taxonomic group which is to be investigated (Stackebrandt and Goebel 1994; Stackebrandt and Ebers 2006; MeierKolthoff et al. 2013). Moreover, an increased value of 16S rRNA gene sequence similarity can be applied for Actinobacteria 99.0 % (Meier-Kolthoff et al. 2013).As the nearest neighbours of strain Ka25T share only 97.2 % 16S rRNA gene sequence identity, DNA– DNA hybridization was not carried out in the present
study; instead strains Ka25T and N.daejeonensis KCTC 19772T were compared by AP-PCR and BOX-A1R PCR fingerprinting. The AP-PCR with M13F primer and BOX-A1R primer PCR showed notable differences between the two strains (Supplementary Fig S1). Since band profiles generated from using the BOX A1R primers are powerful for determining and clustering intraspecies relationships (Lee and Wong, 2009), these profiles support the conclusion that strain Ka25T should be considered to represent a novel species of the genus Nocardioides. Chemotaxonomic characteristics The menaquinone composition of strain Ka25T was found to beMK-8(H4) (97.0 %) and MK-8 (H6) (3 %).
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Table 2 Fatty acid compositions of strain Ka25T and Nocardioides daejeonensis KCTC 19772T Peak name
Nocardioidessolisilvae Ka25Tsp. nov.
Nocardioides daejeonensis KCTC 19772T
revealed that iso-C16:0(30.2 and 33.6 %, respectively), C18:1 x9C (13.8 and 10.5 %, respectively), iso-C17:0 (12.4 and 16.7 %, respectively) and C18:010methyl (6.2 and 7.6 %, respectively) are the major fatty acids present (Table 2).
Saturated C16:0
3.9
4.3
C17:0
Tr
1.7
C18:0
2.2
3.5
C17:010 methyl
2.4
3.2
C18:010 methyl Unsaturated
6.2
7.6
C17:1
ND
2.1
C17:1x6C
ND
4.6
C17:1x8C
2.1
2.4
C18:1x9C
13.8
10.5
iso-C16:1H
3.4
ND
iso-C14:0
3.6
ND
iso-C15:0
4.6
1.3
iso-C16:0
30.2
33.6
iso-C17:0
12.4
16.7
iso-C18:0
1.1
4.2
anteiso-C17:0
3.5
1.2
Summed feature 4*
ND
1.3
Summed feature 3*
3.7
ND
Summed feature 9*
4.9
ND
Branched
Major fatty acids ([5 %) are shown in bold type. Fatty acids that accounted for 1.0 % of the total in each strain are not shown. (Data are percentages of the total fatty acid content, all the data were from this study. ND not detected) * Summed feature 3 contains iso-C15 : 0 2-OH and/or C16 : 1x7c; summed feature 4comprises iso-C17: 1 I and/or anteiso-C17: 1 B; summed feature 9 comprises iso-C17:1x9c and/or 10-methyl C (16:0)
The menaquinone profile of strain Ka25T issimilar to that of other members of the genus Nocardioides,with MK-8 (H4) as the predominant menaquinone component. The cell walls of strain Ka25T and N.daejeonensisKCTC 19772T were found to contain LL-2,6, diaminopimelic acid and glycine. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and an unidentified lipid were the polar lipids detected in strain Ka25T, whereas N. daejeonensis KCTC 19772T contained two additional unidentified lipids distinct from the polar lipids of strain Ka25T (Supplementary Fig. S2).Whole-cell fatty acid analysis of strains Ka25T and N.daejeonensisKCTC 19772T
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Conclusion The phenotypic, chemotaxonomic and phylogenetic data showed that the novel bacterium, strain Ka25T, should be assigned to the genus Nocardioides. Strain Ka25T showed esterase and a glucosidase activities and can assimilate DL-3-hydroxybuytarate, citrate, melibiose, mannitol and sorbitol, differentiating it from its nearest relative N. daejeonensis KCTC 19772T. Hence, strain Ka25T can be designated as the type strain of a novel species, for which the name Nocardioides solisilvae sp. nov., is proposed.
Description of Nocardioides solisilvae sp. nov Nocardioides solisilvae(L. n. solum, soil; L. n. silva, forest; N.L. gen. n. solisilvae, of/from forest soil) Gram-stain positive, aerobic, non-motile and coccoid to rod shaped. Colonies grown on TSA for 5 days are circular, convex and cream coloured. Growth occurs at 20–45 °C (optimum at 30 °C), at pH values of 6.0–11.0 (optimum at pH 7.5) and in presence of 0–14 % (w/v) NaCl (optimum 4 %).Oxidase and catalase are positive. Positive for casein, starch and esculin hydrolysis but negative for xanthine, hypoxanthine and cellulose hydrolysis. Positive for nitrate reduction test, urease production, citrate utilisation and gelatin liquefication. Negative for indole production and arginine dihydrolase. Dextrose, mannose, cellobiose, mannitol, lactose, fructose, xylose, trehalose, inositol, maltose, rhamnose, raffinose, inulin, arabinose, sorbitol and melibiose are utilised as carbon sources. Sucrose, salicin and xylose utilisation as carbon sources is negative. Tyrosine, nitrate, urea and glycine are utilised as nitrogen sources whereas nitrite, aspartate, ammonium chloride and glutamate as nitrogen sources do not support growth. Positive (API ZYM) for esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase (weak), cystine
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arylamidase (weak), trypsin (weak), b-glucosidase (weak) and naphthol-AS-BI-phosphohydrolase activities but negative for alkaline phosphatase, lipase (C14), a-chymotrypsin, acid phosphatase, a-galactosidase, b-galactosidase, b-glucuronidase, N-acetyl-bglucosaminidase, a-mannosidase and a-fructosidase. The cell wall amino acids, whole cell wall sugars, major menaquinones, major phospholipids and major fatty acid components are typical of the members of the genus Nocardioides. The DNA G?C content of the type strain is 71.6 mol%. The type strain is Ka25T (=KCTC39528T = MCC2762T), which was isolated from a forest soil sample from Kasol, India. The 16S rRNA gene sequence of strain Ka25Thas been deposited in GenBank/EMBL/ DDBJ with accession numberLN555580. Acknowledgments SVR and MT thank Department of Science & Technology, Government of India, for providing FTSYS project grants (SB/FT/LS-115/2012 and SB/FT/LS-320/ 2012, respectively). FM thanks Department of Science & Technology, Government of India, for providing PA fellowship.
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1606 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis (MEGA) using maximum likelihood, evolutionary distance and maximum parsimony methods. Mol Biol Evol 28:2731–2739 To´th E, Borsodi A (2014) The Family Nocardioidaceae. In: Rosenberg E, Delong EF, Stackebrandt E, Lory S, Thompsin F (eds) The prokaryotes. Springer, Heidelberg, pp 651–694
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ORIGINAL PAPER
Nocardioides solisilvae sp. nov., isolated from a forest soil Vishnuvardhan Reddy Sultanpuram . Thirumala Mothe . Farooq Mohammed
Received: 4 February 2015 / Accepted: 10 April 2015 / Published online: 19 April 2015 Ó Springer International Publishing Switzerland 2015
Abstract A Gram-stain positive, rod shaped aerobic bacterial strain designated Ka25T was isolated from a forest soil sample near Kasol, Himachal Pradesh, India. Based on 16S rRNA gene sequence analysis it was identified that strain Ka25T belongs to the class Actinobacteria and is most closely related to Nocardioides daejeonensis KCTC 19772T (97.2 %), Nocardioides dubius JCM 13008T (96.9 %), Nocardioides psychrotolerans CGMCC 1.11156T (96.8 %), Nocardioides pacificus DSM 27278T(96.7 %) and Nocardioides daphniae JCM 16608T(96.4 %) sequence similarity respectively. The chemotaxonomic properties of strain Ka25T are consistent with those of the members of the genus Nocardioides: MK-8(H4) is the predominant menaquinone, iso-C16 : 0, iso-C17 : 0 and C18 : 1x9c were identified as the predominant cellular fatty acids and the cell-wall peptidoglycan is based on LL-2,6-diaminopimelic acid. The DNA G?C content of strain Ka25T was determined to be 71.6 mol %. The results of the polyphasic taxonomic analysis allowed a clear differentiation of strain Ka25T from all other
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0455-x) contains supplementary material, which is available to authorized users. V. R. Sultanpuram (&) T. Mothe F. Mohammed Microbial Ecology Lab, Department of Biochemistry, Mahatma Gandhi University, Anneparthy, Yellareddygudem (PO), Nalgonda 508254, Telangana, India e-mail: [email protected]
members of the genus Nocardioides. Strain Ka25T is thus considered to represent a novel member of the genus Nocardioides, for which the name Nocardioides solisilvae sp. nov. is proposed. The type strain is Ka25T (=KCTC39528T =MCC2762T). Keywords Nocardioides Nocardioides solisilvae sp. nov Forest soil Polyphasic taxonomy
Introduction The family Nocardioidaceae originally was described by Nesterenko et al. (1985, 1990), then emended by Stackebrandt et al. (1997) and Zhi et al. (2009). Originally the family included the genus Nocardioides (Prauser 1976) with the species Nocardioides albus(Prauser 1976), Nocardioides luteus (Prauser 1984) and Nocardioides simplex (O’Donnell et al. 1982) and some other related organisms with LLdiaminopimelic acid in their cell wall (To´th and Borsodi 2014). Recent analysis of 16S rRNA gene sequences shows that the genera comprising the current family Nocardioidaceae form at least two separate phylogenetic clusters. The first cluster includes the genera Nocardioides, Aeromicrobium, and Marmoricola whose morphology is relatively simple, with motile rod shaped bacteria. A predominant menaquinone occurs in their profile (with tetra hydrogenated side
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chain of 8 or 9 isoprene units) and significant amounts of straight-chain saturated and unsaturated fatty acids can be found in their cells. The second cluster contains the genera Kribbella, Actinopolymorpha, Flindersiella and Thermasporomyces which have more complex cell morphology and lack motile cells. Their menaquinone profile is more complex, involving longer and more saturated side chains. Their fatty acid profiles are dominated by iso- and anteiso-branched fatty acids and contain only minor amounts of straightchain components (To´th and Borsodi 2014). The genus Nocardioides includes Gram-stain positive, coccus-to-rod-shaped bacteria with LL-diaminopimelic acid as the diagnostic diamino acid of their cell-wall peptidoglycan, MK-8(H4) as the predominant menaquinone and C18 : 1x9c, iso-C16: 0, isoC17 : 0 and 10-methyl C18 : 0 as the major fatty acids (Lee et al. 2012). Originally, the genus contained two species, N. albus and N. luteus (Prauser 1984) and at the time of writing, the genus comprises 75 validly named species (http://www.bacterio.net/nocardioides. html). During a study of the cultivable bacterial diversity of a high altitude forest soil from Kasol, India, a novel bacterial strain designated Ka25T was isolated. This paper focuses on the taxonomic characterisation of strain Ka25T based upon a polyphasic approach.
Materials and methods Isolation of the strain StrainKa25T was isolated from a high altitude (1521 m) forest soil sample from Kasol, Himachal Pradesh, India (Latitude 32°000 , Longitude 77°160 ) on nutrient agarmedium consisting of (l-1) bacteriological peptone (5 g), NaCl (5 g), beef extract (1.5 g), yeast extract (1.5 g), agar (15 g) at pH 7.4 ± 0.2.The isolation was made by means of the standard dilution-plating technique. Pure culture was obtained by repeated streaking on nutrient agar at pH 7.4 ± 0.2 [Himedia, Mumbai, India] andthe isolate was then preserved at 4 °C for further use. Nocardioides daejeonensis KCTC 19772T was obtained from the Korean Collection for Type Cultures and cultured under the same conditions for comparative purposes.
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Morphological, biochemical and physiological characterization Morphological, biochemical and physiological characterisation of strain Ka25T was carried out as described by Yoon and Park (2006).Microscopic observations of strain Ka25T were made by light microscopy (MLX; Olympus-Magnus). The pH (range 6.0–12.0, with an interval of 0.5) were tested with K2HPO4–KH2PO4 buffer for pH 6.0–8.0, NaHCO3– NaOH buffer for pH 8.5–11.0 and Na2CO3–NaOH buffer for pH 11.5–12.0, temperature (0, 4, 10, 16, 20, 28, 30, 35, 37, 40, 50, 55 and 60 °C) and salt concentration (0–30 % w/v, with an interval of 0.5 % w/v) ranges for growth were examined in tryptone soya broth medium at 30°C for 2 days. The Gram reaction was performed using the non-staining (KOH) method, as described by Buck (1982). Catalase activity was assessed by bubble production in 3 % (v/v) H2O2 and oxidase activity was assessed using 1 % (w/v) tetramethyl p-phenylenediamine (Cappuccino and Sherman 2002). Nitrate reduction, urease production, citrate utilisation, gelatine liquefication, indole production and arginine hydrolase activity were determined as previously described (Cowan and Steel 1965; Lanyi 1987). Anaerobic growth was examined in serum bottles using nutrient broth with sodium thioglycolate (1 gl-1) and replacing the air in the bottles with nitrogen. Enzyme activities were tested using API ZYM strips (bioMe´rieux) according to the manufacturer’s instructions. Tests for the hydrolysis of casein, starch, xanthine and hypoxanthine (Atlas 1993) were carried out over 7 days at 30 °C. Utilisation of various substrates as sole carbon and energy sources was determined as described by Shirling and Gottlieb (1966). Susceptibility to antibiotics was tested by placing antibiotic-impregnated discs on TSA plates that were seeded with suspensions of the test strain. Phylogenetic analysis and genetic relatedness Genomic DNA was extracted and purified according to the method of Marmur (1961). The 16S rRNA gene sequence of strain Ka25T was obtained by PCR as described previously (Vishnuvardhan Reddy et al. 2013). Identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarity were achieved using the EzTaxon-e server
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(http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012). The CLUSTAL_W algorithm of MEGA 5 (Tamura et al. 2011) was used for sequence alignments and the phylogenetic analysis of the related 16S rRNA gene sequences was performed using MEGA 5. Distances were calculated by using the Kimura correction in a pair-wise deletion manner (Kimura 1980). Neighbourjoining (NJ) (Saitou and Nei 1987), minimum evolution (ME), maximum likelihood (ML) and maximum parsimony (MP) (Nei and Kumar 2000) methods in the MEGA 5 software were used to construct phylogenetic trees. Percentage support values were obtained using a bootstrap procedure (Felsenstein 1985). The mol% G?C content of the DNA of strain Ka25T was determined by HPLC (Mesbah et al. 1989). To study the genetic relatedness of strain Ka25T and N.daejeonensis KCTC 19772T, the strains were analysed by AP-PCR and BOX-A1R PCR. The APPCR fingerprinting was performed by using the M13F primer [(-20): GTAAAACGACGGCCAGT] and the following PCR program: two cycles of 94 °C for 5 min, 40 °C for 5 min and 72 °C for 5 min; followed by 40 high stringency cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 2 min. Similarly, BOX-A1R PCR was performed by using the BOX-A1R primer (50 -CTACGGCAAGGCGACGCTGACG-30 ). The cycling programme started with denaturation at 95 °C for 7 min, followed by 35 cycles of: 94 °C for 1 min; 1 min at 53 °C and 8 min at 72 °C. After the extension step, the temperature was held at 72 °C for 15 min. Amplified DNA products were resolved by electrophoresis on 2 % w/v agarose gel. Chemotaxonomic characterization Menaquinones, amino acids of whole cell hydrolysates and polar lipids of strains Ka25T and N.daejeonensis KCTC 19772T were analysed from cells grown in tryptone soya broth incubated for 5 days at 30 °C with 7.5 pH and 4 % (w/v) NaCl. Menaquinones were extracted from freeze-dried biomass and purified according to Collins and Jones (1980) and analysed by HPLC (Kroppenstedt 1985). Amino acids of whole-cell hydrolysates were purified by TLC after hydrolysis of whole cells with 6 M HCl at 100 °C for 18 has described previously by Komagata and Suzuki (1987). Polar lipids were extracted, examined by two-dimensional TLC and identified using published procedures (Minnikin et al. 1984). For
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fatty acid analysis, strains Ka25T and N.daejeonensis KCTC 19772T were grown in tryptone soya broth at 30 °C with pH 7.5 and 4 % (w/v) NaCl for 2 days. The fatty acids analysis, which was outsourced to Zeal Biologicals, Secunderabad, was performed by following the procedures of Sasser (1990) according to the standard protocol of MIDI Microbial Identification System.
Results and discussion Morphological, biochemical and physiological characteristics Cells of strain Ka25T were observed to be Gram-stain positive, aerobic, non-spore-forming, and non-motile. Cells were observed to be oval to coccoid with 1.0–2.1 lm length and 0.6–1.2 lm width. Tests for oxidase and catalase gave positive results. Colonies grown on TSA agar plates for 5 days at 30 °C were observed to be smooth, translucent, circular, convex, cream coloured and 1.0–1.5 mm in diameter. Strain Ka25T was found to be able to grow between pH 6.0 and 11.0, with an optimum pH value of 7.5. The strain was found to be able to grow in the range of 20–45° C, with optimum growth temperature at 30 °C.Strain Ka25T was found to grow in the presence of 0–14 % NaCl (w/ v) with an optimum of 4 % NaCl (w/v). The differential characteristics of strain Ka25T compared with N.daejeonensis KCTC 19772T are shown in Table 1, while other additional characteristics are given in the species description. Strain Ka25T was found to be sensitive to nalidixic acid (30 lg), gentamycin (120 lg), vancomycin (30 lg), tetracycline (30 lg), streptomycin (10 lg),kanamycin (30 lg), amikacin (30 lg), erythromycin (15 lg), ciproflaxcin (5 lg)and resistant to penicillin (10 lg) and ampicillin (10 lg). Phylogenetic analysis and genetic relatedness The almost complete 16S rRNA gene sequence (1.435 bp) of strain Ka25T was obtained (Gen-Bank/ EMBL/DDBJ accession numberLN555580).The results of phylogenetic analysis of the 16S rRNA gene sequence showed that strain Ka25T clusters within the genus Nocardioides (a consensus tree is shown as Fig. 1) and the sequence similarities with the nearest phylogenetic members are in agreement with
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Table 1 Characteristics used to distinguish strain Ka25T from Nocardioides daejeonensis KCTC 19772T Characteristic
Nocardioidessolisilvae Ka25Tsp. nov.
Nocardioides daejeonensis KCTC 19772T
Cell length (lm)
1.0–2.1
0.8–2.0
Cell width (lm)
0.6–1.2
0.5–0.8
Colony colour
Cream
Pale yellow
NaCl range(%, w/v)
0–14
0–10
NaCl optima (%, w/v)
4
4
pH range
6.0–11.0
5.5–9.0
pH optima
7.5
7.5
Temp range (°C)
20–45
15–37
Temp optima (°C)
30
30
Oxidase
?
-
Urease
?
-
Casein hydrolysis
?
-
Enzyme activity Esterase (C4)
?
W
a-Glucosidase (starch hydrolysis) b-Glucosidase
? W
?
3-Hydroxybenzoate
-
?
4-Hydroxybenzoate
-
?
DL-3-Hydroxybutyrate
?
-
2-Ketogluconate (a)
-
?
Caprate
-
?
Assimilation of
Itaconate
-
w
Melibiose
?
-
D-Mannitol
?
-
D-Sorbitol
?
-
Data are from this study. ?positive, -negative, w weak positive Both the strains were non-motile, positive for catalase activity, starch and esculin hydrolysis; negative for xanthine, hypoxanthine and cellulose hydrolysis. Both the strains were showing nitrate reduction activities and citrate utilization, but both strains showed negative result for arginine hydrolyase and indole production. Both the strains were showing negative enzyme activity for N-acetyl-bglucosamidase, b-glucuronidase and Lipase (C14). Alkaline phosphatase, acid phosphatase, esterase (C8), leucine arylamidase, napthol-AS-BL-phosphohydrolase, valine arylamidase activity were positive for both the strains. Cystine arylamidase and trypsine activities for both the strains showed weak positive. DL-lactate, maltose, L-rhamnose and citrate were utilised by both the strains, but, propionate, subarate, D-ribose, sucrose, myo-inositol, L-alanine and N-acetyl-D-glucosamine were not utilized
the EzTaxon-e server analysis. EzTaxon-e server search analysis revealed that the strain Ka25T is most closely related to N.daejeonensis KCTC 19772T (97.2 % sequence similarity), Nocardioides dubius JCM 13008T (96.9 %), Nocardioides psychrotolerans CGMCC 1.11156T (96.8 %), Nocardioides pacificus DSM 27278T (96.7 %), Nocardioides daphniae JCM 16608T (96.4 %) and other members of the genus Nocardioides (\96.4 %). The sequence similarities of strain
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Ka25T with N.daejeonensis KCTC 19772T using different clustering algorithms (96.8 % in NJ tree; 96.8 % in ME tree and 97.2 % in ML tree) along with the EzTaxon-e server analysis, consistently indicated that N.daejeonensis KCTC 19772Tis the closest relative. All the clustering algorithms used function differently, hence minor differences in the sequence similarities of the strains were observed when each of them was used. The G?C content of the DNA of strain Ka25T was 71.6 mol%, which is
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Terrabacter tumescens DSM 20308T (AF00502316S) Nocardioides deserti DSM 26045T(KM816582 ) Nocardioides psychrotolerans CGMCC 1.11156 T (JF750425) 90 * 65 Nocardioides szechwanensis CGMCC 1.11147 T (JF750424) * Nocardioides kribbensis JCM 13594T (AY835924) T 67 * Nocardioides lianchengensis DSM 24663 (HQ657322) T (EF633986) Nocardioides dokdonensis JCM 14815 94 T 72 Nocardioides salarius DSM 18239 (DQ401092) * Nocardioides basaltis KCTC 19365T (EU143365) 67 * Nocardioides marinisabuli JBRI 2003 T (AM422448) Nocardioides daphniae JCM 16608T (AM398438) # 51 Nocardioides solisilvae sp. nov. Ka25T (LN555580) # Nocardioides daejeonensis KCTC 19772T (JF937066) 90 * Nocardioides dubius KCTC 9992T (AY928902) Nocardioides pacificus JCM 19260T (KC986355) 55 * 62 Nocardioides echinoideorum JCM 30276T (KM085325) Nocardioides daedukensis KCTC 19601T (FJ842646) * 97 Nocardioides jensenii LMG 16325T (Z78210) Nocardioides alpinus LMG 26053T (GU784866) 67 # 78 Nocardioides terrigena KCTC 19217T (EF363712) Nocardioides sediminis KCTC 19271T (EF466110) * Nocardioides ginsengisegetis KCTC 19469T(GQ339901) Nocardioides dilutus KCTC 19288T (EF466121) 82 * Nocardioides terrae NBRC 104259T (FJ423762) Nocardioides caricicola JCM 17686T (FJ750845) 55 Nocardioides aquiterrae JCM 11813T (AF529063) 67 * * Nocardioides pyridinolyticus JCM 10369T (U61298) Nocardioides soli CCTCC AB 2012934T(JF937914) 85 * Nocardioides hankookensis KCTC 19246T (EF555584) 66 Nocardioides endophyticus JCM 18532T KC878444) 88 * * 93 Nocardioides conyzicola JCM 18531T (KC878445) 0.01
Fig. 1 Phylogenetic analysis of strain Ka25T with other closely related members based on 16S rRNA gene sequences available from the EMBL database (accession numbers are given in parentheses). Multiple alignments, distance calculations (distance options according to the Kimura 2- parameter model) and clustering with the neighbour-joining method, maximumlikelihood method, minimum-evolution method and
maximum-parsimony method were performed by using the software package MEGA version 5 (Tamura et al. 2011). Bootstrap values based on 1000 replications are listed as percentages at the branching points.* Clustering similarly in all the tested methods; # clustering similarly in maximumlikelihood and maximum-parsimony methods. Bar, 0.01 nucleotide substitutions per nucleotide position
similar to that of the nearest phylogenetic neighbours. DNA–DNA hybridizations (DDH) play an important role in species delineation in microorganisms and this is applicable mostly in cases where 16S rRNA gene sequence similarities are 97 % or higher. This threshold value has been reasonably increased to between 98.2 and 99.0 % depending on the taxonomic group which is to be investigated (Stackebrandt and Goebel 1994; Stackebrandt and Ebers 2006; MeierKolthoff et al. 2013). Moreover, an increased value of 16S rRNA gene sequence similarity can be applied for Actinobacteria 99.0 % (Meier-Kolthoff et al. 2013).As the nearest neighbours of strain Ka25T share only 97.2 % 16S rRNA gene sequence identity, DNA– DNA hybridization was not carried out in the present
study; instead strains Ka25T and N.daejeonensis KCTC 19772T were compared by AP-PCR and BOX-A1R PCR fingerprinting. The AP-PCR with M13F primer and BOX-A1R primer PCR showed notable differences between the two strains (Supplementary Fig S1). Since band profiles generated from using the BOX A1R primers are powerful for determining and clustering intraspecies relationships (Lee and Wong, 2009), these profiles support the conclusion that strain Ka25T should be considered to represent a novel species of the genus Nocardioides. Chemotaxonomic characteristics The menaquinone composition of strain Ka25T was found to beMK-8(H4) (97.0 %) and MK-8 (H6) (3 %).
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Table 2 Fatty acid compositions of strain Ka25T and Nocardioides daejeonensis KCTC 19772T Peak name
Nocardioidessolisilvae Ka25Tsp. nov.
Nocardioides daejeonensis KCTC 19772T
revealed that iso-C16:0(30.2 and 33.6 %, respectively), C18:1 x9C (13.8 and 10.5 %, respectively), iso-C17:0 (12.4 and 16.7 %, respectively) and C18:010methyl (6.2 and 7.6 %, respectively) are the major fatty acids present (Table 2).
Saturated C16:0
3.9
4.3
C17:0
Tr
1.7
C18:0
2.2
3.5
C17:010 methyl
2.4
3.2
C18:010 methyl Unsaturated
6.2
7.6
C17:1
ND
2.1
C17:1x6C
ND
4.6
C17:1x8C
2.1
2.4
C18:1x9C
13.8
10.5
iso-C16:1H
3.4
ND
iso-C14:0
3.6
ND
iso-C15:0
4.6
1.3
iso-C16:0
30.2
33.6
iso-C17:0
12.4
16.7
iso-C18:0
1.1
4.2
anteiso-C17:0
3.5
1.2
Summed feature 4*
ND
1.3
Summed feature 3*
3.7
ND
Summed feature 9*
4.9
ND
Branched
Major fatty acids ([5 %) are shown in bold type. Fatty acids that accounted for 1.0 % of the total in each strain are not shown. (Data are percentages of the total fatty acid content, all the data were from this study. ND not detected) * Summed feature 3 contains iso-C15 : 0 2-OH and/or C16 : 1x7c; summed feature 4comprises iso-C17: 1 I and/or anteiso-C17: 1 B; summed feature 9 comprises iso-C17:1x9c and/or 10-methyl C (16:0)
The menaquinone profile of strain Ka25T issimilar to that of other members of the genus Nocardioides,with MK-8 (H4) as the predominant menaquinone component. The cell walls of strain Ka25T and N.daejeonensisKCTC 19772T were found to contain LL-2,6, diaminopimelic acid and glycine. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and an unidentified lipid were the polar lipids detected in strain Ka25T, whereas N. daejeonensis KCTC 19772T contained two additional unidentified lipids distinct from the polar lipids of strain Ka25T (Supplementary Fig. S2).Whole-cell fatty acid analysis of strains Ka25T and N.daejeonensisKCTC 19772T
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Conclusion The phenotypic, chemotaxonomic and phylogenetic data showed that the novel bacterium, strain Ka25T, should be assigned to the genus Nocardioides. Strain Ka25T showed esterase and a glucosidase activities and can assimilate DL-3-hydroxybuytarate, citrate, melibiose, mannitol and sorbitol, differentiating it from its nearest relative N. daejeonensis KCTC 19772T. Hence, strain Ka25T can be designated as the type strain of a novel species, for which the name Nocardioides solisilvae sp. nov., is proposed.
Description of Nocardioides solisilvae sp. nov Nocardioides solisilvae(L. n. solum, soil; L. n. silva, forest; N.L. gen. n. solisilvae, of/from forest soil) Gram-stain positive, aerobic, non-motile and coccoid to rod shaped. Colonies grown on TSA for 5 days are circular, convex and cream coloured. Growth occurs at 20–45 °C (optimum at 30 °C), at pH values of 6.0–11.0 (optimum at pH 7.5) and in presence of 0–14 % (w/v) NaCl (optimum 4 %).Oxidase and catalase are positive. Positive for casein, starch and esculin hydrolysis but negative for xanthine, hypoxanthine and cellulose hydrolysis. Positive for nitrate reduction test, urease production, citrate utilisation and gelatin liquefication. Negative for indole production and arginine dihydrolase. Dextrose, mannose, cellobiose, mannitol, lactose, fructose, xylose, trehalose, inositol, maltose, rhamnose, raffinose, inulin, arabinose, sorbitol and melibiose are utilised as carbon sources. Sucrose, salicin and xylose utilisation as carbon sources is negative. Tyrosine, nitrate, urea and glycine are utilised as nitrogen sources whereas nitrite, aspartate, ammonium chloride and glutamate as nitrogen sources do not support growth. Positive (API ZYM) for esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase (weak), cystine
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arylamidase (weak), trypsin (weak), b-glucosidase (weak) and naphthol-AS-BI-phosphohydrolase activities but negative for alkaline phosphatase, lipase (C14), a-chymotrypsin, acid phosphatase, a-galactosidase, b-galactosidase, b-glucuronidase, N-acetyl-bglucosaminidase, a-mannosidase and a-fructosidase. The cell wall amino acids, whole cell wall sugars, major menaquinones, major phospholipids and major fatty acid components are typical of the members of the genus Nocardioides. The DNA G?C content of the type strain is 71.6 mol%. The type strain is Ka25T (=KCTC39528T = MCC2762T), which was isolated from a forest soil sample from Kasol, India. The 16S rRNA gene sequence of strain Ka25Thas been deposited in GenBank/EMBL/ DDBJ with accession numberLN555580. Acknowledgments SVR and MT thank Department of Science & Technology, Government of India, for providing FTSYS project grants (SB/FT/LS-115/2012 and SB/FT/LS-320/ 2012, respectively). FM thanks Department of Science & Technology, Government of India, for providing PA fellowship.
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