Antonie van Leeuwenhoek (2015) 107:1299–1306 DOI 10.1007/s10482-015-0424-4
ORIGINAL PAPER
Tropicihabitans flavus gen. nov., sp. nov., a new member of the family Cellulomonadaceae Moriyuki Hamada • Chiyo Shibata • Arif Nurkanto Shanti Ratnakomala • Puspita Lisdiyanti • Tomohiko Tamura • Ken-ichiro Suzuki
•
Received: 15 January 2015 / Accepted: 2 March 2015 / Published online: 12 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract Two novel Gram-stain positive actinobacteria, designated PS-14-16T and RS-7-1, were isolated from the rhizosphere of a mangrove and sea sediment, respectively, and their taxonomic positions were investigated by a polyphasic approach. Both strains were observed to form vegetative hyphae in the early phase of growth but the hyphae eventually fragment into short rods to coccoid cells. The peptidoglycan type of both strains was found to be A4a. Their predominant menaquinone was identified as MK9(H4) and the major fatty acid as anteiso-C15:0. The DNA G?C content was determined to be 68.4–68.5 mol%. 16S rRNA gene sequencing revealed that strains PS-14-16T and RS-7-1 were related to members of the family Cellulomonadaceae. Their nearest phylogenetic neighbour was found to be Sediminihabitans luteus, which is currently the only species of the genus Sediminihabitans, with a similarity of 97.94 %. However, strains PS-14-16T
and RS-7-1 were distinguishable from the members of the genus Sediminihabitans and the other genera within the family Cellulomonadaceae in terms of chemotaxonomic characteristics and phylogenetic relationship. The results of DNA–DNA hybridization experiments indicated that strains PS-14-16T and RS7-1 belong to the same species. Strains PS-14-16T and RS-7-1 are concluded to represent a novel genus and species of the family Cellulomonadaceae, for which the name Tropicihabitans flavus gen. nov., sp. nov. is proposed. The type strain of T. flavus is PS-14-16T (=NBRC 110109T = IanCC A 516T). Keywords Actinobacteria Mangrove Cellulomonadaceae Tropicihabitans flavus Polyphasic taxonomy
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0424-4) contains supplementary material, which is available to authorized users. M. Hamada (&) C. Shibata T. Tamura K. Suzuki Biological Resource Center, National Institute of Technology and Evaluation (NBRC), 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan e-mail: [email protected] A. Nurkanto S. Ratnakomala P. Lisdiyanti Indonesian Institute of Sciences (LIPI), Jl, Raya JakartaBogor Km. 46, Cibinong 16911, Indonesia
The family Cellulomonadaceae was proposed for the genera Cellulomonas (Bergey et al. 1923), Oerskovia (Prauser et al. 1970), Promicromonospora (Krasil’nikov et al. 1961) and Jonesia (Rocourt et al. 1987) by Stackebrandt and Prauser (1991). Later, the genera Promicromonospora and Jonesia were transferred to new families Promicromonosporaceae and Jonesiaceae, respectively (Stackebrandt et al. 1997). At that time, the genus Rarobacter (Yamamoto et al. 1988) was included in the family Cellulomonadaceae,
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however, a few years later, the genus was excluded from this family and transferred to a new family, Rarobacteraceae (Stackebrandt and Schumann 2000). Subsequently, three more genera, Actinotalea, Paraoerskovia and Sediminihabitans, were proposed as members of the family Cellulomonadaceae (Yi et al. 2007; Khan et al. 2009; Hamada et al. 2012a). Members of the family Cellulomonadaceae have been isolated from diverse environments, such as soils, composts, decaying plant materials, barks, sea sediments and various clinical specimens. Discovery of novel microorganisms from natural environments is important because it helps to understand the microbial diversity of a given habitat and may also result in isolation of bacteria with possible industrial or other applications. Approximately 99 % of all species in natural environments are not yet cultured and are an untapped source of new bioactive substances (Lewis 2013; Ling et al. 2015). Recently, marine environments have attracted attention as an isolation source and it has been reported that marinederived microorganisms including actinobacteria are useful for screening for novel bioactive substances (Blunt et al. 2007; Lam 2006; Khan et al. 2011). During the course of studies on the isolation and diversity of actinobacteria in seashore environments, we isolated two novel actinobacteria from the rhizosphere of a mangrove and sea sediment collected in Indonesia. Comparative 16S rRNA gene sequence analysis revealed that the isolates are phylogenetically related to members of the family Cellulomonadaceae. The objective of this study was to determine the taxonomic positions of the isolates using a polyphasic approach.
Materials and methods Bacterial strains and isolation Two actinobacteria, strains PS-14-16T and RS-7-1, were isolated from seashore environments in Indonesia. Specifically, strain PS-14-16T was isolated from the rhizosphere of a mangrove (Rhizophora mucronata) growing on Pramuka Island (5°440 72900 S 106°360 92400 E), DKI Jakarta. Strain RS-7-1 was isolated from a seasediment sample collected from the foreshore of Rambut Island (5°580 63400 S 106°410 59600 E), DKI Jakarta. The procedure employed for bacterial isolation was as
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described by Hamada et al. (2013a). NBRC medium 802 (1.0 % polypeptone, 0.2 % yeast extract, 0.1 % MgSO47H2O and 1.5 % agar if required; pH 7.0) was used as basal medium for this study. Biomass for molecular and chemotaxonomic studies, except for cellular fatty acid analysis, was obtained by culturing the strains in shake flasks for 2 days at 28 °C at 100 rpm. Biomass grown on trypticase soy agar (Difco) for 24 h at 28 °C was used for cellular fatty acid analysis. Sediminihabitans luteus NBRC 108568T was used as a reference strain for DNA–DNA hybridization. Morphological, physiological and biochemical tests Colony morphology was examined after incubation at 28 °C for 3 days on an agar plate of NBRC medium 802. Morphological features were observed with age (up to 5 days) under a light microscope (BX-51; Olympus) and a scanning electron microscope (JSM6060; JEOL). Cell motility, growth parameters (temperature, pH and NaCl tolerance), anaerobic growth, Gram staining and oxidase activity were determined using the methods described by Hamada et al. (2010). Catalase activity was determined by production of bubbles after the addition of a drop of 3.0 % H2O2 (vol/vol). Other physiological and biochemical tests were performed using API ZYM, API Coryne and API 50CH systems (bioMe´rieux) according to the manufacturer’s instructions. 16S rRNA Sequence determination and phylogenetic analysis DNA was isolated using PrepMan Ultra Reagent (Applied Biosystems) according to the manufacturer’s instruction. The 16S rRNA gene was amplified by PCR using KOD FX (Toyobo) with the following pair of primers: 9F and 1541R (Hamada et al. 2012b). The amplified 16S rRNA gene was subjected to cycle sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) with the following primers: 9F, 785F, 802R and 1541R (Hamada et al. 2012b). The products were analysed using an automated DNA sequencer (ABI PRISM 3730 Genetic Analyzer; Applied Biosystems). The phylogenetic neighbours were identified and pairwise 16S rRNA gene sequence similarities were calculated using the EzTaxon-e server (Kim et al. 2012). The almost-
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Fig. 1 Scanning electron micrographs of strain PS-14-16T grown on NBRC medium 802 for 1 day (a) and 4 days (b) at 28 °C; bars, 2 lm
complete 16S rRNA gene sequences determined in this study were aligned with reference sequences from the genus Sediminihabitans and some related taxa by using the CLUSTAL_X program (Thompson et al. 1997). Phylogenetic trees were constructed by the neighbour-joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981) and maximum-parsimony (MP) (Fitch 1971) algorithms by using the MEGA 6.0 program (Tamura et al. 2013). The resultant tree topologies were evaluated by bootstrap analysis (Felsenstein 1985) on the basis of 1000 replicates. G?C content of DNA and DNA–DNA hybridization Genomic DNA was obtained using the method of Saito and Miura (1963). The DNA G?C contents were determined by the method of Tamaoka and Komagata (1984). The microplate hybridization method developed by Ezaki et al. (1989) was used to determine DNA–DNA relatedness. Chemotaxonomic tests Cell-wall sugars, isoprenoid quinones and amino acids of peptidoglycan and their isomers were analysed as described previously (Hamada et al. 2012b). Polar lipids were analysed as described by Hamada et al. (2010), with the following chromatographic systems:
chloroform/methanol/water (65:25:4, by vol.) used in the first direction and chloroform/acetic acid/methanol/water (80:18:12:5, by vol.) in the second direction. The preparation and analysis of cellular fatty acid methyl esters were performed using the protocol of the MIDI Sherlock Microbial Identification System (Sasser 1990) and a gas chromatograph (6890N; Agilent Technologies) with Sherlock MIDI software (version 6.2) and the TSBA5 database (version 6.2). Nucleotide sequence accession numbers The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains PS-14-16T and RS7-1 are AB971840 and AB971841, respectively.
Results and discussion Strains PS-14-16T and RS-7-1 were observed to form circular, smooth and pale yellow colonies that were approximately 0.5–1.0 mm in diameter on NBRC medium 802 after 3 days of incubation at 28 °C. The cells of the strains were observed to be Gram-stain positive, non-motile and non-endospore forming. Both strains were found to develop branched vegetative hyphae (0.4–0.5 9 3–10 lm) in the early phase of growth but these hyphae eventually fragmented into short-rods to coccoid cells (0.4–0.5 9 0.4–1.0 lm) (Fig. 1). These strains were also found to be catalase-
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positive and oxidase-negative. The strains were observed to grow at 15–37 °C (optimum 28 °C) but not at 5, 10, 45 or 60 °C. The pH range for growth was found to be 6.0–9.0 (optimum pH 7.0–8.0). The strains were found to grow in the presence of 0–10 % NaCl (w/v) but not in 15 % NaCl (w/v). No growth was observed under anaerobic conditions. The results of other physiological and biochemical analyses are summarized in the species description below. Phylogenetic analyses based on the 16S rRNA gene sequences revealed that strains PS-14-16T and RS-7-1 are related to the members of the family Cellulomonadaceae and the similarity value between them is 100 %. However, the strains did not form a reliable clade with any members of the family Cellulomonadaceae (Figs. 2, S1, S2). The highest similarity value was observed with S. luteus H97-3T (97.94 %), followed by Oerskovia species (96.91–97.05 %), Cellulomonas iranensis OT (97.05 %) and Paraoerskovia marina CTT-37T (96.76 %). The DNA–DNA relatedness value between both strains was determined to be 72 ± 5 % (reciprocal value, 80 ± 7 %) whereas that between strain PS-14-16T and S. luteus NBRC 108568T was 14 ± 3 % (reciprocal value, 21 ± 6 %). The peptidoglycan samples of strains PS-14-16T and RS-7-1 were found to contain alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), serine (Ser) and lysine (Lys) in molar ratios of 2.2:1.0:1.0:1.0:0.7 and 2.2:0.9:1.0:1.0:0.8, respectively. Enantiomeric analysis of the peptidoglycan amino acids revealed the presence of D-Ala, L-Ala, D-Asp, D-Glu, L-Ser and L-Lys. These data suggested that the peptidoglycan type of strains PS-14-16T and RS-7-1 is A4a, with LLys as the diagnostic diamino acid and an interpeptide bridge comprising L-Ser–D-Asp, A11.36 (Schleifer and Kandler 1972; Schumann 2011). No cell-wall sugar was detected in either strain. The predominant menaquinone in both strains was identified as MK9(H4), with MK-9(H6) present as a trace component (99:1). The results of cellular fatty acid analysis are shown in Table 1. The profiles were found to be qualitatively similar to that of S. luteus H97-3T although C20:0 was only observed in the two novel strains and other quantitative differences were found. The major polar lipid of both strains was found to be phosphatidylglycerol (PG), along with minor or trace amounts of diphosphatidylglycerol (DPG), phosphatidylinositol (PI) and two phosphatidylinositol mannosides (PIMs) (Supplementary Fig. S3). The
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Antonie van Leeuwenhoek (2015) 107:1299–1306 Fig. 2 Phylogenetic tree derived from the 16S rRNA gene c sequences of strains PS-14-16T and RS-7-1 and their taxonomic neighbours, reconstructed with the neighbour-joining algorithm. The 16S rRNA gene sequence of Brevibacterium linens DSM 20425T (X77451) was used as the outgroup. Bootstrap values ([70 %) based on 1000 replicates are shown at branch nodes. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and maximum-parsimony algorithms. Bar 0.01 Knuc substitutions per nucleotide position
DNA G?C contents of strains PS-14-16T and RS-7-1 were determined to be 68.5 and 68.4 mol%, respectively. The result of the DNA–DNA hybridization indicated that strains PS-14-16T and RS-7-1 belong to the same species. However, the results of the phylogenetic analyses based on the 16S rRNA gene sequences revealed that the strains did not form a reliable clade with any members of the family Cellulomonadaceae irrespective of the treeing algorithms that were employed (Figs. 2, S1, S2). In addition, the strains were revealed to have some differentiating phenotypic characteristics within the family Cellulomonadaceae (Table 2). Both strains possessed a peptidoglycan interpeptide bridge comprising L-Ser–D-Asp. This structure has not been reported for the members of the family Cellulomonadaceae. Furthermore, the polar lipid profile (PG, DPG, PI and PIMs) and the major fatty acid composition (only anteiso-C15:0 [10 %) also support their phenotypic distinctiveness. Therefore, both strains should not be affiliated to an established genus of the family Cellulomonadaceae. On the basis of the phylogenetic relationships and their distinctive phenotypic characteristics, it is proposed that strains PS-14-16T and RS-7-1 should be classified as representing a novel genus and species of the family Cellulomonadaceae, with the name Tropicihabitans flavus gen. nov., sp. nov., with PS-14-16T as the type strain. Description of Tropicihabitans gen. nov. Tropicihabitans (Tro.pi.ci.ha0 bi.tans. L. adj. tropicus, tropical; L. pres. part. habitans, inhabiting; N.L. masc. n. Tropicihabitans, an inhabitant of tropical area). Cells are Gram-stain positive, aerobic and nonendospore forming, and develop branched vegetative hyphae in the early phase of growth but these hyphae eventually fragment into short-rods to coccoid cells. The cell-wall peptidoglycan is of the A4a type with an
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Myceligenerans xiligouense XLG9A10.2T (AY354285) Promicromonospora citrea DSM 43110T (X83808) 76 Xylanibacterium ulmi XIL08T (AY273185) Xylanimicrobium pachnodae VPCX2T (AF105422) 77 Xylanimonas cellulosilytica XIL07T (AF403541) 91 Isoptericola chiayiensis 06182M-1T (FJ469988) 99 Isoptericola halotolerans YIM 70177T (AY789835) Isoptericola jiangsuensis CLGT (EU852101) 92 Isoptericola dokdonensis DS-3T (DQ387860) 92 Isoptericola variabilis NBRC 104115T (AB489221) Isoptericola nanjingensis H17T (HQ222356) Isoptericola hypogeus HKI0342T (AJ854061) Cellulosimicrobium cellulans DSM 43879T (X79455) Luteimicrobium xylanilyticum W-15T (JQ039191) Luteimicrobium album RI148-Li105T (AB646194) Luteimicrobium subarcticum R19-04T (AB489904) Demequina aestuarii NBRC 106260T (AB639015) 99 Lysinimicrobium mangrovi HI08-69T (AB639012) Actinotalea fermentans NBRC 105374T (AB639014) Cellulomonas bogoriensis 69B4T (X92152) 78 Cellulomonas carbonis T26T (HQ702749) Cellulomonas hominis DMMZ CE40T (X82598) 99 Cellulomonas denverensis W6929T (AY501362) Cellulomonas gelida DSM 20111T (X83800) Cellulomonas uda DSM 20107T (X83801) 83 Cellulomonas iranensis OT (AF064702) Cellulomonas composti TR7-06T (AB166887) Cellulomonas flavigena DSM 20109T (X83799) Cellulomonas marina FXJ8.089T (JF346422) Cellulomonas oligotrophica Kc5T (AB602499) 99 Cellulomonas fimi DSM 20113T (X79460) 90 Cellulomonas biazotea DSM 20112T (X83802) Cellulomonas chitinilytica X.bu-bT (AB268586) Cellulomonas cellasea DSM 20118T (X83804) Cellulomonas soli Kc1T (AB602498) Cellulomonas humilata NCTC 25174T (X82449) 99 Cellulomonas xylanilytica XIL11T (AY303668) Cellulomonas terrae DB5T (AY884570) 99 Tropicihabitans flavus PS-14-16T (AB971840) Tropicihabitans flavus RS-7-1 (AB971841) 75 Oerskovia paurometabola DSM 14281T (AJ314851) 90 Oerskovia jenensis DSM 46000T (AJ314848) 99 Oerskovia turbata DSM 20577T (X83806) Oerskovia enterophila DSM 43852T (X83807) Sediminihabitans luteus H97-3T (AB695376) Sanguibacter keddieii ST-74T (X79450) 95 Sanguibacter antarcticus KOPRI21702T (EF211071) Sanguibacter soli DCY22T (EF547937) Paraoerskovia sediminicola H25-14T (AB695378) Paraoerskovia marina NBRC104352T (AB695379) Brevibacterium linens DSM 20425T (X77451) 80
0.01 Knuc
71
70 99
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PG phosphatidylglycerol, DPG diphosphatidylglycerol, PI phosphatidylinositol, PIMs phosphatidylinositol mannosides, PGL unidentified phosphoglycolipid
Data are from Hamada et al. (2012a) b
a
? positive, - negative, ± variable
Data for reference genera were taken from Hamada et al. (2012a) (Sediminihabitans), Stackebrandt et al. (2002) (Oerskovia), Hamada et al. (2013b) (Paraoerskovia), Schumann et al. (2009), Stackebrandt and Schumann (2012) (Cellulomonas) and Li et al. (2013) (Actinotalea)
DPG
74.7–75.8 69–76 70.8–73.2 73.9–74.9b 74.0 68.5 DNA G?C content (mol%)
or D-Ser–D-Asp D-Asp
ai-C15:0, C16:0, i-C16:0 ai-C15:0, C16:0, i-C15:0, ai-C17:0
PG, DPG, PI, PGL PG, DPG, PI, PIMs
ai-C15:0, i-C15:0 ai-C15:0, i-Cb15:0
PG, DPG, PI
MK-9(H4) MK-9(H4)
ai-C15:0, ai-C17:0, C16:0
PG, PI, PIMs PG, DPG, PI, PIMs Polar lipid(s)
MK-9(H4), MK-8(H4)
L-Orn
ai-C15:0
a
Tropicihabitans flavus (fla0 vus. L. masc. adj. flavus, yellow, referring to the colour of the colonies). Shows the following characteristics in addition to those given in the genus description. Vegetative hyphae are 0.4–0.5 9 3–10 lm and short-rods to coccoid cells are 0.4–0.5 9 0.4–1.0 lm. Cells are non-motile. Colonies are circular, smooth and pale yellow. The temperature range for growth is 15–37 °C
Major fatty acid(s)
Description of Tropicihabitans flavus sp. nov.
MK-9(H4)
interpeptide bridge composed of L-Ser–D-Asp. The predominant menaquinone is MK-9(H4) and the major cellular fatty acid is anteiso-C15:0. The major polar lipid is phosphatidylglycerol, along with minor or trace amounts of diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. Phylogenetically, the genus Tropicihabitans belongs to the family Cellulomonadaceae within the suborder Micrococcineae. The type species is Tropicihabitans flavus.
MK-9(H4)
Strains 1, PS-14-16T; 2, RS-7-1; 3. S. luteus H97-3T. –, not detected; tr, trace component (\1 %). Bold type indicates major components ([10 %)
Major menaquinone
Data for S. luteus are taken from Hamada et al. (2012a) and were obtained from cells cultivated under the same conditions and analysed in the same laboratory
or D-Asp
–
D-Glu
1.52
or Gly–D-Asp2
1.15
L-Ser–D-Glu
anteiso-C19:0
or L-Thr–D-Asp
51.70 21.99
L-Thr–D-Glu
56.09 8.58
L-Ser–D-Asp
58.01 7.06
Interpeptide bridge
–
anteiso-C15:0 anteiso-C17:0
A4b
tr
L-Orn
tr
A4b
–
anteiso-C13:0
L-Lys
–
tr
A4a
tr
tr
L-Lys
tr
iso-C20:0
A4a
iso-C18:0
L-Lys
tr
A4a
tr
A4a
tr
L-Lys
3.21
iso-C17:0
Diamino acid
2.27
2.63
Peptidoglycan type
1.52
2.40
Rods
1.47
iso-C16:0
–
iso-C15:0
±
1.23
–
–
tr
±
5.32
1.01
–
5.04
iso-C14:0
–
C20:0
Motility
–
2
1.10
1.26
Irregular rods
6.49
1.21
23
6.04
C19:0
Hyphae to coccoid
C18:0
2
2.22
Hyphae to rods or coccoid
1.56
4
1.57
Hyphae to coccoid
10.01
C17:0
1
2.98
8.51
1
tr
8.89
Hyphae to coccoid
1.07
C16:0
Morphology
C15:0
Number of species
2.90
Cellulomonas
2.53
Paraoerskovia
3.30
Oerskovia
C14:0
Sediminihabitans
3
PS-14-16T
2
Characteristic
1
Table 2 Differential chemotaxonomic characteristics of strain PS-14-16T and the genera within the family Cellulomonadaceae
Fatty acid
Actinotalea
Table 1 Cellular fatty acid compositions (%) of strains PS-1416T and RS-7-1 and the type strain of Sediminihabitans luteus
MK-10(H4)
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and the optimal temperature is 28 °C. The pH range for growth is 6.0–9.0 and the optimal pH is 7.0–8.0. Growth occurs in NaCl concentrations of 0–10 %. Catalase-positive and oxidase-negative. Acid is produced from D-cellobiose, D-fructose, D-galactose, Dglucose, glycerol, D-lactose, D-maltose, D-mannose, Dsucrose, D-turanose and D-xylose. N-Acetyl-b-glucosaminidase, acid phosphatase, esterase (C4), esterase lipase (C8), a-glucosidase, b-glucuronidase, leucine arylamidase, phosphohydrolase, pyrazinamidase and pyrrolidonyl arylamidase are present, whereas alkaline phosphatase, chymotrypsin, cysteine arylamidase, fucosidase, a-galactosidase, b-galactosidase, b-glucosidase, lipase (C14), mannosidase, trypsin, urease and valine arylamidase are absent. Aesculin is hydrolysed but gelatin is not. Nitrate is reduced. The major cellular fatty acid is anteiso-C15:0, while C16:0, anteiso-C17:0, C18:0, C20:0, C14:0, iso-C16:0, C17:0, iso-C15:0, C19:0, anteiso-C19:0, C15:0 and isoC14:0 are found in minor amounts. The DNA G?C content of the type strain is 68.5 mol%. The type strain PS-14-16T (=NBRC 110109T = InaCC A 516T) was isolated from the rhizosphere of a mangrove (Rhizophora mucronata) growing on Pramuka Island, DKI Jakarta, Indonesia. A second strain, RS-7-1 (=NBRC 110110 = InaCC A 517), was isolated from a sea-sediment sample collected from the foreshore of Rambut Island, DKI Jakarta, Indonesia. Acknowledgments This work was supported by Science and Technology Research Partnership for Sustainable Development (SATREPS) which is a research program in collaborated with the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA).
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1306 Prauser H, Lechevalier MP, Lechevalier HA (1970) Description of Oerskovia gen. n. to harbor Ørskov’s motile Nocardia. Appl Microbiol 19:534 Rocourt J, Wehmeyer U, Stackebrandt E (1987) Transfer of Listeria denitrificans to a new genus, Jonesia gen. nov., as Jonesia denitrificans comb. nov. Int J Syst Bacteriol 37:266–270 Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. MIDI Inc, Newark Schleifer KH, Kandler O (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36:407–477 Schumann P (2011) Peptidoglycan structure. In: Rainey F, Oren A (eds) Taxonomy of prokaryotes, methods in microbiology, vol 38. Academic Press, London, pp 101–129 Schumann P, Ka¨mpfer P, Busse HJ, Evtushenko LI (2009) Proposed minimal standards for describing new genera and species of the suborderer Micrococcineae. Int J Syst Evol Microbiol 59:1823–1849 Stackebrandt E, Prauser H (1991) The family Cellulomonadaceae. In: Balows A, Tru¨per HG, Dworkin M, Harder W, Schleifer KH (eds) The prokaryotes, vol 2, 2nd edn. Springer, New York, pp 1323–1345 Stackebrandt E, Schumann P (2000) Description of Bogoriellaceae fam. nov., Dermacoccaceae fam. nov., Rarobacteraceae fam. nov. and Sanguibacteraceae fam. nov. and emendation of some families of the suborder Micrococcineae. Int J Syst Evol Microbiol 50:1279–1285
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Antonie van Leeuwenhoek (2015) 107:1299–1306 Stackebrandt E, Schumann P (2012) Genus I. Cellulomonas. In: Goodfellow M, Ka¨mpfer P, Busse HJ, Trujillo ME, Suzuki K, Ludwig W, Whitman WB (eds) Bergey’s manual of systematic bacteriology. The Actinobacteria, part A, vol 5, 2nd edn. Springer, New York, pp 702–710 Stackebrandt E, Rainey FA, Ward-Rainey NL (1997) Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47:479–491 Stackebrandt E, Breymann S, Steiner U, Prauser H, Weiss N, Schumann P et al (2002) Re-evaluation of the status of the genus Oerskovia, reclassification of Promicromonospora enterophila (Ja´ger, 1983) as Oerskovia enterophila comb. nov. and description of Oerskovia jenensis sp. nov. and Oerskovia paurometabola sp. nov. Int J Syst Evol Microbiol 52:1105–1111 Tamaoka J, Komagata K (1984) Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25:125–128 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 Yamamoto N, Sato SI, Saito K, Hasuo T, Tadenuma M, Suzuki KI, Tamaoka J, Komagata K (1988) Rarobacter faecitabidus gen. nov., sp. nov., a yeast-lysing coryneform bacterium. Int J Syst Bacteriol 38:7–11 Yi H, Schumann P, Chun J et al (2007) Demequina aestuarii gen. nov., sp. nov., a novel actinomycete of the suborder Micrococcineae, and reclassification of Cellulomonas fermentans Bagnara, 1985 as Actinotalea fermentans gen. nov., comb. nov. Int J Syst Evol Microbiol 57:151–156
ORIGINAL PAPER
Tropicihabitans flavus gen. nov., sp. nov., a new member of the family Cellulomonadaceae Moriyuki Hamada • Chiyo Shibata • Arif Nurkanto Shanti Ratnakomala • Puspita Lisdiyanti • Tomohiko Tamura • Ken-ichiro Suzuki
•
Received: 15 January 2015 / Accepted: 2 March 2015 / Published online: 12 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract Two novel Gram-stain positive actinobacteria, designated PS-14-16T and RS-7-1, were isolated from the rhizosphere of a mangrove and sea sediment, respectively, and their taxonomic positions were investigated by a polyphasic approach. Both strains were observed to form vegetative hyphae in the early phase of growth but the hyphae eventually fragment into short rods to coccoid cells. The peptidoglycan type of both strains was found to be A4a. Their predominant menaquinone was identified as MK9(H4) and the major fatty acid as anteiso-C15:0. The DNA G?C content was determined to be 68.4–68.5 mol%. 16S rRNA gene sequencing revealed that strains PS-14-16T and RS-7-1 were related to members of the family Cellulomonadaceae. Their nearest phylogenetic neighbour was found to be Sediminihabitans luteus, which is currently the only species of the genus Sediminihabitans, with a similarity of 97.94 %. However, strains PS-14-16T
and RS-7-1 were distinguishable from the members of the genus Sediminihabitans and the other genera within the family Cellulomonadaceae in terms of chemotaxonomic characteristics and phylogenetic relationship. The results of DNA–DNA hybridization experiments indicated that strains PS-14-16T and RS7-1 belong to the same species. Strains PS-14-16T and RS-7-1 are concluded to represent a novel genus and species of the family Cellulomonadaceae, for which the name Tropicihabitans flavus gen. nov., sp. nov. is proposed. The type strain of T. flavus is PS-14-16T (=NBRC 110109T = IanCC A 516T). Keywords Actinobacteria Mangrove Cellulomonadaceae Tropicihabitans flavus Polyphasic taxonomy
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0424-4) contains supplementary material, which is available to authorized users. M. Hamada (&) C. Shibata T. Tamura K. Suzuki Biological Resource Center, National Institute of Technology and Evaluation (NBRC), 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan e-mail: [email protected] A. Nurkanto S. Ratnakomala P. Lisdiyanti Indonesian Institute of Sciences (LIPI), Jl, Raya JakartaBogor Km. 46, Cibinong 16911, Indonesia
The family Cellulomonadaceae was proposed for the genera Cellulomonas (Bergey et al. 1923), Oerskovia (Prauser et al. 1970), Promicromonospora (Krasil’nikov et al. 1961) and Jonesia (Rocourt et al. 1987) by Stackebrandt and Prauser (1991). Later, the genera Promicromonospora and Jonesia were transferred to new families Promicromonosporaceae and Jonesiaceae, respectively (Stackebrandt et al. 1997). At that time, the genus Rarobacter (Yamamoto et al. 1988) was included in the family Cellulomonadaceae,
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however, a few years later, the genus was excluded from this family and transferred to a new family, Rarobacteraceae (Stackebrandt and Schumann 2000). Subsequently, three more genera, Actinotalea, Paraoerskovia and Sediminihabitans, were proposed as members of the family Cellulomonadaceae (Yi et al. 2007; Khan et al. 2009; Hamada et al. 2012a). Members of the family Cellulomonadaceae have been isolated from diverse environments, such as soils, composts, decaying plant materials, barks, sea sediments and various clinical specimens. Discovery of novel microorganisms from natural environments is important because it helps to understand the microbial diversity of a given habitat and may also result in isolation of bacteria with possible industrial or other applications. Approximately 99 % of all species in natural environments are not yet cultured and are an untapped source of new bioactive substances (Lewis 2013; Ling et al. 2015). Recently, marine environments have attracted attention as an isolation source and it has been reported that marinederived microorganisms including actinobacteria are useful for screening for novel bioactive substances (Blunt et al. 2007; Lam 2006; Khan et al. 2011). During the course of studies on the isolation and diversity of actinobacteria in seashore environments, we isolated two novel actinobacteria from the rhizosphere of a mangrove and sea sediment collected in Indonesia. Comparative 16S rRNA gene sequence analysis revealed that the isolates are phylogenetically related to members of the family Cellulomonadaceae. The objective of this study was to determine the taxonomic positions of the isolates using a polyphasic approach.
Materials and methods Bacterial strains and isolation Two actinobacteria, strains PS-14-16T and RS-7-1, were isolated from seashore environments in Indonesia. Specifically, strain PS-14-16T was isolated from the rhizosphere of a mangrove (Rhizophora mucronata) growing on Pramuka Island (5°440 72900 S 106°360 92400 E), DKI Jakarta. Strain RS-7-1 was isolated from a seasediment sample collected from the foreshore of Rambut Island (5°580 63400 S 106°410 59600 E), DKI Jakarta. The procedure employed for bacterial isolation was as
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described by Hamada et al. (2013a). NBRC medium 802 (1.0 % polypeptone, 0.2 % yeast extract, 0.1 % MgSO47H2O and 1.5 % agar if required; pH 7.0) was used as basal medium for this study. Biomass for molecular and chemotaxonomic studies, except for cellular fatty acid analysis, was obtained by culturing the strains in shake flasks for 2 days at 28 °C at 100 rpm. Biomass grown on trypticase soy agar (Difco) for 24 h at 28 °C was used for cellular fatty acid analysis. Sediminihabitans luteus NBRC 108568T was used as a reference strain for DNA–DNA hybridization. Morphological, physiological and biochemical tests Colony morphology was examined after incubation at 28 °C for 3 days on an agar plate of NBRC medium 802. Morphological features were observed with age (up to 5 days) under a light microscope (BX-51; Olympus) and a scanning electron microscope (JSM6060; JEOL). Cell motility, growth parameters (temperature, pH and NaCl tolerance), anaerobic growth, Gram staining and oxidase activity were determined using the methods described by Hamada et al. (2010). Catalase activity was determined by production of bubbles after the addition of a drop of 3.0 % H2O2 (vol/vol). Other physiological and biochemical tests were performed using API ZYM, API Coryne and API 50CH systems (bioMe´rieux) according to the manufacturer’s instructions. 16S rRNA Sequence determination and phylogenetic analysis DNA was isolated using PrepMan Ultra Reagent (Applied Biosystems) according to the manufacturer’s instruction. The 16S rRNA gene was amplified by PCR using KOD FX (Toyobo) with the following pair of primers: 9F and 1541R (Hamada et al. 2012b). The amplified 16S rRNA gene was subjected to cycle sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) with the following primers: 9F, 785F, 802R and 1541R (Hamada et al. 2012b). The products were analysed using an automated DNA sequencer (ABI PRISM 3730 Genetic Analyzer; Applied Biosystems). The phylogenetic neighbours were identified and pairwise 16S rRNA gene sequence similarities were calculated using the EzTaxon-e server (Kim et al. 2012). The almost-
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Fig. 1 Scanning electron micrographs of strain PS-14-16T grown on NBRC medium 802 for 1 day (a) and 4 days (b) at 28 °C; bars, 2 lm
complete 16S rRNA gene sequences determined in this study were aligned with reference sequences from the genus Sediminihabitans and some related taxa by using the CLUSTAL_X program (Thompson et al. 1997). Phylogenetic trees were constructed by the neighbour-joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981) and maximum-parsimony (MP) (Fitch 1971) algorithms by using the MEGA 6.0 program (Tamura et al. 2013). The resultant tree topologies were evaluated by bootstrap analysis (Felsenstein 1985) on the basis of 1000 replicates. G?C content of DNA and DNA–DNA hybridization Genomic DNA was obtained using the method of Saito and Miura (1963). The DNA G?C contents were determined by the method of Tamaoka and Komagata (1984). The microplate hybridization method developed by Ezaki et al. (1989) was used to determine DNA–DNA relatedness. Chemotaxonomic tests Cell-wall sugars, isoprenoid quinones and amino acids of peptidoglycan and their isomers were analysed as described previously (Hamada et al. 2012b). Polar lipids were analysed as described by Hamada et al. (2010), with the following chromatographic systems:
chloroform/methanol/water (65:25:4, by vol.) used in the first direction and chloroform/acetic acid/methanol/water (80:18:12:5, by vol.) in the second direction. The preparation and analysis of cellular fatty acid methyl esters were performed using the protocol of the MIDI Sherlock Microbial Identification System (Sasser 1990) and a gas chromatograph (6890N; Agilent Technologies) with Sherlock MIDI software (version 6.2) and the TSBA5 database (version 6.2). Nucleotide sequence accession numbers The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains PS-14-16T and RS7-1 are AB971840 and AB971841, respectively.
Results and discussion Strains PS-14-16T and RS-7-1 were observed to form circular, smooth and pale yellow colonies that were approximately 0.5–1.0 mm in diameter on NBRC medium 802 after 3 days of incubation at 28 °C. The cells of the strains were observed to be Gram-stain positive, non-motile and non-endospore forming. Both strains were found to develop branched vegetative hyphae (0.4–0.5 9 3–10 lm) in the early phase of growth but these hyphae eventually fragmented into short-rods to coccoid cells (0.4–0.5 9 0.4–1.0 lm) (Fig. 1). These strains were also found to be catalase-
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positive and oxidase-negative. The strains were observed to grow at 15–37 °C (optimum 28 °C) but not at 5, 10, 45 or 60 °C. The pH range for growth was found to be 6.0–9.0 (optimum pH 7.0–8.0). The strains were found to grow in the presence of 0–10 % NaCl (w/v) but not in 15 % NaCl (w/v). No growth was observed under anaerobic conditions. The results of other physiological and biochemical analyses are summarized in the species description below. Phylogenetic analyses based on the 16S rRNA gene sequences revealed that strains PS-14-16T and RS-7-1 are related to the members of the family Cellulomonadaceae and the similarity value between them is 100 %. However, the strains did not form a reliable clade with any members of the family Cellulomonadaceae (Figs. 2, S1, S2). The highest similarity value was observed with S. luteus H97-3T (97.94 %), followed by Oerskovia species (96.91–97.05 %), Cellulomonas iranensis OT (97.05 %) and Paraoerskovia marina CTT-37T (96.76 %). The DNA–DNA relatedness value between both strains was determined to be 72 ± 5 % (reciprocal value, 80 ± 7 %) whereas that between strain PS-14-16T and S. luteus NBRC 108568T was 14 ± 3 % (reciprocal value, 21 ± 6 %). The peptidoglycan samples of strains PS-14-16T and RS-7-1 were found to contain alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), serine (Ser) and lysine (Lys) in molar ratios of 2.2:1.0:1.0:1.0:0.7 and 2.2:0.9:1.0:1.0:0.8, respectively. Enantiomeric analysis of the peptidoglycan amino acids revealed the presence of D-Ala, L-Ala, D-Asp, D-Glu, L-Ser and L-Lys. These data suggested that the peptidoglycan type of strains PS-14-16T and RS-7-1 is A4a, with LLys as the diagnostic diamino acid and an interpeptide bridge comprising L-Ser–D-Asp, A11.36 (Schleifer and Kandler 1972; Schumann 2011). No cell-wall sugar was detected in either strain. The predominant menaquinone in both strains was identified as MK9(H4), with MK-9(H6) present as a trace component (99:1). The results of cellular fatty acid analysis are shown in Table 1. The profiles were found to be qualitatively similar to that of S. luteus H97-3T although C20:0 was only observed in the two novel strains and other quantitative differences were found. The major polar lipid of both strains was found to be phosphatidylglycerol (PG), along with minor or trace amounts of diphosphatidylglycerol (DPG), phosphatidylinositol (PI) and two phosphatidylinositol mannosides (PIMs) (Supplementary Fig. S3). The
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Antonie van Leeuwenhoek (2015) 107:1299–1306 Fig. 2 Phylogenetic tree derived from the 16S rRNA gene c sequences of strains PS-14-16T and RS-7-1 and their taxonomic neighbours, reconstructed with the neighbour-joining algorithm. The 16S rRNA gene sequence of Brevibacterium linens DSM 20425T (X77451) was used as the outgroup. Bootstrap values ([70 %) based on 1000 replicates are shown at branch nodes. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and maximum-parsimony algorithms. Bar 0.01 Knuc substitutions per nucleotide position
DNA G?C contents of strains PS-14-16T and RS-7-1 were determined to be 68.5 and 68.4 mol%, respectively. The result of the DNA–DNA hybridization indicated that strains PS-14-16T and RS-7-1 belong to the same species. However, the results of the phylogenetic analyses based on the 16S rRNA gene sequences revealed that the strains did not form a reliable clade with any members of the family Cellulomonadaceae irrespective of the treeing algorithms that were employed (Figs. 2, S1, S2). In addition, the strains were revealed to have some differentiating phenotypic characteristics within the family Cellulomonadaceae (Table 2). Both strains possessed a peptidoglycan interpeptide bridge comprising L-Ser–D-Asp. This structure has not been reported for the members of the family Cellulomonadaceae. Furthermore, the polar lipid profile (PG, DPG, PI and PIMs) and the major fatty acid composition (only anteiso-C15:0 [10 %) also support their phenotypic distinctiveness. Therefore, both strains should not be affiliated to an established genus of the family Cellulomonadaceae. On the basis of the phylogenetic relationships and their distinctive phenotypic characteristics, it is proposed that strains PS-14-16T and RS-7-1 should be classified as representing a novel genus and species of the family Cellulomonadaceae, with the name Tropicihabitans flavus gen. nov., sp. nov., with PS-14-16T as the type strain. Description of Tropicihabitans gen. nov. Tropicihabitans (Tro.pi.ci.ha0 bi.tans. L. adj. tropicus, tropical; L. pres. part. habitans, inhabiting; N.L. masc. n. Tropicihabitans, an inhabitant of tropical area). Cells are Gram-stain positive, aerobic and nonendospore forming, and develop branched vegetative hyphae in the early phase of growth but these hyphae eventually fragment into short-rods to coccoid cells. The cell-wall peptidoglycan is of the A4a type with an
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Myceligenerans xiligouense XLG9A10.2T (AY354285) Promicromonospora citrea DSM 43110T (X83808) 76 Xylanibacterium ulmi XIL08T (AY273185) Xylanimicrobium pachnodae VPCX2T (AF105422) 77 Xylanimonas cellulosilytica XIL07T (AF403541) 91 Isoptericola chiayiensis 06182M-1T (FJ469988) 99 Isoptericola halotolerans YIM 70177T (AY789835) Isoptericola jiangsuensis CLGT (EU852101) 92 Isoptericola dokdonensis DS-3T (DQ387860) 92 Isoptericola variabilis NBRC 104115T (AB489221) Isoptericola nanjingensis H17T (HQ222356) Isoptericola hypogeus HKI0342T (AJ854061) Cellulosimicrobium cellulans DSM 43879T (X79455) Luteimicrobium xylanilyticum W-15T (JQ039191) Luteimicrobium album RI148-Li105T (AB646194) Luteimicrobium subarcticum R19-04T (AB489904) Demequina aestuarii NBRC 106260T (AB639015) 99 Lysinimicrobium mangrovi HI08-69T (AB639012) Actinotalea fermentans NBRC 105374T (AB639014) Cellulomonas bogoriensis 69B4T (X92152) 78 Cellulomonas carbonis T26T (HQ702749) Cellulomonas hominis DMMZ CE40T (X82598) 99 Cellulomonas denverensis W6929T (AY501362) Cellulomonas gelida DSM 20111T (X83800) Cellulomonas uda DSM 20107T (X83801) 83 Cellulomonas iranensis OT (AF064702) Cellulomonas composti TR7-06T (AB166887) Cellulomonas flavigena DSM 20109T (X83799) Cellulomonas marina FXJ8.089T (JF346422) Cellulomonas oligotrophica Kc5T (AB602499) 99 Cellulomonas fimi DSM 20113T (X79460) 90 Cellulomonas biazotea DSM 20112T (X83802) Cellulomonas chitinilytica X.bu-bT (AB268586) Cellulomonas cellasea DSM 20118T (X83804) Cellulomonas soli Kc1T (AB602498) Cellulomonas humilata NCTC 25174T (X82449) 99 Cellulomonas xylanilytica XIL11T (AY303668) Cellulomonas terrae DB5T (AY884570) 99 Tropicihabitans flavus PS-14-16T (AB971840) Tropicihabitans flavus RS-7-1 (AB971841) 75 Oerskovia paurometabola DSM 14281T (AJ314851) 90 Oerskovia jenensis DSM 46000T (AJ314848) 99 Oerskovia turbata DSM 20577T (X83806) Oerskovia enterophila DSM 43852T (X83807) Sediminihabitans luteus H97-3T (AB695376) Sanguibacter keddieii ST-74T (X79450) 95 Sanguibacter antarcticus KOPRI21702T (EF211071) Sanguibacter soli DCY22T (EF547937) Paraoerskovia sediminicola H25-14T (AB695378) Paraoerskovia marina NBRC104352T (AB695379) Brevibacterium linens DSM 20425T (X77451) 80
0.01 Knuc
71
70 99
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PG phosphatidylglycerol, DPG diphosphatidylglycerol, PI phosphatidylinositol, PIMs phosphatidylinositol mannosides, PGL unidentified phosphoglycolipid
Data are from Hamada et al. (2012a) b
a
? positive, - negative, ± variable
Data for reference genera were taken from Hamada et al. (2012a) (Sediminihabitans), Stackebrandt et al. (2002) (Oerskovia), Hamada et al. (2013b) (Paraoerskovia), Schumann et al. (2009), Stackebrandt and Schumann (2012) (Cellulomonas) and Li et al. (2013) (Actinotalea)
DPG
74.7–75.8 69–76 70.8–73.2 73.9–74.9b 74.0 68.5 DNA G?C content (mol%)
or D-Ser–D-Asp D-Asp
ai-C15:0, C16:0, i-C16:0 ai-C15:0, C16:0, i-C15:0, ai-C17:0
PG, DPG, PI, PGL PG, DPG, PI, PIMs
ai-C15:0, i-C15:0 ai-C15:0, i-Cb15:0
PG, DPG, PI
MK-9(H4) MK-9(H4)
ai-C15:0, ai-C17:0, C16:0
PG, PI, PIMs PG, DPG, PI, PIMs Polar lipid(s)
MK-9(H4), MK-8(H4)
L-Orn
ai-C15:0
a
Tropicihabitans flavus (fla0 vus. L. masc. adj. flavus, yellow, referring to the colour of the colonies). Shows the following characteristics in addition to those given in the genus description. Vegetative hyphae are 0.4–0.5 9 3–10 lm and short-rods to coccoid cells are 0.4–0.5 9 0.4–1.0 lm. Cells are non-motile. Colonies are circular, smooth and pale yellow. The temperature range for growth is 15–37 °C
Major fatty acid(s)
Description of Tropicihabitans flavus sp. nov.
MK-9(H4)
interpeptide bridge composed of L-Ser–D-Asp. The predominant menaquinone is MK-9(H4) and the major cellular fatty acid is anteiso-C15:0. The major polar lipid is phosphatidylglycerol, along with minor or trace amounts of diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. Phylogenetically, the genus Tropicihabitans belongs to the family Cellulomonadaceae within the suborder Micrococcineae. The type species is Tropicihabitans flavus.
MK-9(H4)
Strains 1, PS-14-16T; 2, RS-7-1; 3. S. luteus H97-3T. –, not detected; tr, trace component (\1 %). Bold type indicates major components ([10 %)
Major menaquinone
Data for S. luteus are taken from Hamada et al. (2012a) and were obtained from cells cultivated under the same conditions and analysed in the same laboratory
or D-Asp
–
D-Glu
1.52
or Gly–D-Asp2
1.15
L-Ser–D-Glu
anteiso-C19:0
or L-Thr–D-Asp
51.70 21.99
L-Thr–D-Glu
56.09 8.58
L-Ser–D-Asp
58.01 7.06
Interpeptide bridge
–
anteiso-C15:0 anteiso-C17:0
A4b
tr
L-Orn
tr
A4b
–
anteiso-C13:0
L-Lys
–
tr
A4a
tr
tr
L-Lys
tr
iso-C20:0
A4a
iso-C18:0
L-Lys
tr
A4a
tr
A4a
tr
L-Lys
3.21
iso-C17:0
Diamino acid
2.27
2.63
Peptidoglycan type
1.52
2.40
Rods
1.47
iso-C16:0
–
iso-C15:0
±
1.23
–
–
tr
±
5.32
1.01
–
5.04
iso-C14:0
–
C20:0
Motility
–
2
1.10
1.26
Irregular rods
6.49
1.21
23
6.04
C19:0
Hyphae to coccoid
C18:0
2
2.22
Hyphae to rods or coccoid
1.56
4
1.57
Hyphae to coccoid
10.01
C17:0
1
2.98
8.51
1
tr
8.89
Hyphae to coccoid
1.07
C16:0
Morphology
C15:0
Number of species
2.90
Cellulomonas
2.53
Paraoerskovia
3.30
Oerskovia
C14:0
Sediminihabitans
3
PS-14-16T
2
Characteristic
1
Table 2 Differential chemotaxonomic characteristics of strain PS-14-16T and the genera within the family Cellulomonadaceae
Fatty acid
Actinotalea
Table 1 Cellular fatty acid compositions (%) of strains PS-1416T and RS-7-1 and the type strain of Sediminihabitans luteus
MK-10(H4)
Antonie van Leeuwenhoek (2015) 107:1299–1306
L-Ser–D-Glu
1304
Antonie van Leeuwenhoek (2015) 107:1299–1306
and the optimal temperature is 28 °C. The pH range for growth is 6.0–9.0 and the optimal pH is 7.0–8.0. Growth occurs in NaCl concentrations of 0–10 %. Catalase-positive and oxidase-negative. Acid is produced from D-cellobiose, D-fructose, D-galactose, Dglucose, glycerol, D-lactose, D-maltose, D-mannose, Dsucrose, D-turanose and D-xylose. N-Acetyl-b-glucosaminidase, acid phosphatase, esterase (C4), esterase lipase (C8), a-glucosidase, b-glucuronidase, leucine arylamidase, phosphohydrolase, pyrazinamidase and pyrrolidonyl arylamidase are present, whereas alkaline phosphatase, chymotrypsin, cysteine arylamidase, fucosidase, a-galactosidase, b-galactosidase, b-glucosidase, lipase (C14), mannosidase, trypsin, urease and valine arylamidase are absent. Aesculin is hydrolysed but gelatin is not. Nitrate is reduced. The major cellular fatty acid is anteiso-C15:0, while C16:0, anteiso-C17:0, C18:0, C20:0, C14:0, iso-C16:0, C17:0, iso-C15:0, C19:0, anteiso-C19:0, C15:0 and isoC14:0 are found in minor amounts. The DNA G?C content of the type strain is 68.5 mol%. The type strain PS-14-16T (=NBRC 110109T = InaCC A 516T) was isolated from the rhizosphere of a mangrove (Rhizophora mucronata) growing on Pramuka Island, DKI Jakarta, Indonesia. A second strain, RS-7-1 (=NBRC 110110 = InaCC A 517), was isolated from a sea-sediment sample collected from the foreshore of Rambut Island, DKI Jakarta, Indonesia. Acknowledgments This work was supported by Science and Technology Research Partnership for Sustainable Development (SATREPS) which is a research program in collaborated with the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA).
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