IJSEM Papers in Press. Published October 30, 2014 as doi:10.1099/ijs.0.062869-0
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Compostibacillus humi gen. nov., sp. nov., a member of the family Bacillaceae, isolated
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from sludge compost
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Zhen Yu, Junlin Wen, Guiqin Yang, Jing Liu, Shungui Zhou *
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Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650, PR China
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*
Corresponding author:
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Shungui Zhou
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E-mail: [email protected]
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Tel: (86)-20-37300951
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Subject Category: New Taxa- (Firmicutes)
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Running Title: Compostibacillus humi gen. nov., sp. nov.
20 21 22 23 24 25 26 27 28 29 30 * The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains DX-3T and GIESS002 are JX274434 and KJ024967, respectively.
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Two novel Gram-staining-positive, rod-shaped, endospore-forming, and moderately
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thermophilic bacteria, designated strain DX-3T and GIESS002, respectively were isolated
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from sludge composts from Guangdong Province, China. Analysis of 16S rRNA gene
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sequences revealed that the isolates were closely related to each other with extremely high
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similarity (99.6%), and were members of the family Bacillaceae. However, these two isolates
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formed a novel phylogenetic branch within this family. Their closest relatives were the
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members of the genera Ornithinibacillus, Oceanobacillus and Virgibacillus. Cells of both
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strains were facultatively anaerobic and catalase- and oxidase-positive. The cell-wall
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peptidoglycan type was A1γ (meso-DAP direct). The predominant isoprenoid quinone was
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MK-7. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and
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phosphatidylethanolamine. The major cellular fatty acid was iso-C15:0. The DNA G+C
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content was 43.2-43.7 mol%. The polyphasic taxonomic results indicated that strains DX-3T
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and GIESS002 represent a novel species in a new genus in the family Bacillaceae, order
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Bacillales for which the name Compostibacillus humi gen. nov., sp. nov. is proposed. The type
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strain is DX-3T (=KCTC 33104T =CGMCC 1.12360T).
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At the time of writing, the family Bacillaceae in the order Bacillales comprised more than 50
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recognized genera (http://www.bacterio.net/bacillaceae.html) with different physiological features.
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Many of these genera include thermophilic or thermotolerant endospore-forming species, such as
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the genera Bacillus (Han et al., 2013; Yang et al., 2013a; 2013b), Caldalkalibacillus (Xue et al.,
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2006), Geobacillus (Cihan et al., 2011) and Vulcanibacillus (L'Haridon et al., 2006). Members of
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these genera have been isolated from various warm or hot environments. For example, Bacillus
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thermoantarcticus was isolated from a geothermal soil (Nicolaus et al., 1996), Caldalkalibacillus
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thermarum was from a hot spring (Xue et al., 2006), Geobacillus thermodenitrificans was from a
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high temperature well-pipeline sediment sample (Cihan et al., 2011) and Vulcanibacillus
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modesticaldus was from deep-sea hydrothermal vents (L'Haridon et al., 2006). Compost is also a
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very important source for isolation of thermophiles, and many thermophilic or thermotolerant
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species in the family Bacillaceae from compost have been reported (Sung et al., 2002; Yang et al.,
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2013a, 2013b; Han et al., 2013).
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In our previous studies, we have isolated some thermophilic bacteria from a demonstration
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compost plant in Guangdong Province, China and identified four novel Gram-staining-positive,
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endospore-forming, facultative anaerobic species as Bacillus composti (Yang et al., 2013a),
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Bacillus thermocopriae (Han et al., 2013), Bacillus thermophilus (Yang et al., 2013a) and Bacillus
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thermotolerans (Yang et al., 2013b), which are included in the same genus of Bacillus. In this
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paper, we describe two novel moderately thermophilic bacteria, designated strains DX-3T and
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GIESS002, which were isolated from different compost samples obtained in the thermophilic and
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maturation phases of composting, respectively. On the basis of partial 16S rRNA gene sequence
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analyses, the novel isolates were found to be closely related to members of the genera
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Ornithinibacillus, Oceanobacillus, Virgibacillus, Terribacillus, Halobacillus, Sediminibacillus,
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Paucisalibacillus and Lentibacillus. However, the polyphasic taxonomic analysis indicated that
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these two isolates could not be assigned to any of the recognized genera, and represented a novel
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species in a new genus Compostibacillus caeni gen. nov., sp. nov. of the family Bacillaceae.
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Sludge composting was performed in a demonstration compost plant in Guangdong Province,
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China. Compost samples were collected during different fermentation phases of the whole
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composting process. For each sampling, the temperature in the compost pile was also monitored
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and recorded. The sampling procedures were described in detail by Yang et al. (2013a). Isolation
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was carried out by the dilution-plate method. About 5.0 g sub-sample of each compost sample was
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added into 100 ml 0.85 % NaCl solution, stirred for 30 min, serially diluted, and then placed on
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agar plate of TSA (trypticase soya agar, pH 7.2), which contained (in L-1): 17 g tryptone, 3 g
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soytone, 5 g NaCl, 2.5 g K2HPO4, 2.5 g glucose and 15 g agar. After incubation at their respective
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source temperature for 5 d, a single colony was picked and transferred to fresh TSA media for
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further purification. Strain DX-3T was isolated from the compost sample in the thermophilic phase
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of composting at 55 oC, while strain GIESS002 was from the sample in the maturation phase at a
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temperature of 45 oC. These two isolates were preserved at -80 oC in tryptic soy broth (TSB,
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Oxoid Ltd.) supplemented with 15 % (v/v) glycerol for further study.
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Total genomic DNA was prepared using a commercial genomic DNA extraction kit (Aidlab
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Biotechnologies Co., Ltd.). The 16S rRNA genes of strains DX-3T and GIESS002 were
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PCR-amplified using universal primers (27f and 1492r; Baker et al. 2003). After purified using
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Gel Extraction Kit D2500-01 (Omega Bio-tek), the PCR products were cloned into plasmid vector
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using a TA cloning kit (TaKaRa, Dalian, China) and sequenced using an ABI PRISM 3100 Genetic
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Analyser (Applied Biosystems). Sequences closely related to those of the two isolates were
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obtained using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The 16S rRNA gene sequences of
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the novel strains and related strains were aligned using CLUSTAL_X (Thompson et al., 1997) and
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sequence similarities with alignment gaps and ambiguous bases omitted were calculated using
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MEGA version 5.0 (Tamura et al., 2007). Phylogenetic trees were constructed based on the
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aligned 16S rRNA gene sequences using the maximum-likelihood, minimum-evolution and
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maximum parsimony methods. Statistical support for the branches of the phylogenetic trees was
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determined using bootstrap analysis (Felsenstein, 1985) (based on 1200 re-samplings).
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Almost full-length 16S rRNA gene sequences of strains DX-3T (1555 nt) and GIESS002 (1556 nt)
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were obtained. Sequence analysis results revealed that these two isolates were closely related to
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each other with a high sequence similarity of 99.6% and belonged to the family Bacillaceae. For
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both strains, the 16S rRNA gene sequence similarities with members of all genera in the family
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Bacillaceae were equal or lower than 96.2 %, e.g. Ornithinibacillus (≤ 96.2 %), Oceanobacillus (≤
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94.5 %), Virgibacillus (≤ 94.6 %), Terribacillus (≤ 94.0 %), Salinibacillus (≤ 93.7 %) and Bacillus
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(≤ 93.4 %). Except for the above genera, these two isolates did not show more than 93%
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similarities with the other genera of the family Bacillaceae. The phylogenetic tree based on the
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maximum-likelihood method showed that the two novel isolates clustered in a separate clade (Fig.
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1), which was also shown in the minimum-evolution and maximum parsimony trees
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(Supplementary Fig. S2), indicating that these two isolates might represent a novel species in a
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new genus.
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The DNA G+C contents of strains DX-3T and GIESS002 determined by HPLC as described by
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Mesbah et al. (1989) were 43.7 and 43.2 mol%, respectively. These values were significantly
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higher than those of the references as shown in Table 1. The DNA G+C contents also supported
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the view that these two isolates were distinguishable from all members of their closely related
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genera of Ornithinibacillus (36-41 mol%) (Mayr et al., 2006; Bagheri et al., 2012),
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Oceanobacillus (33.6-40.2 mol%) (Namwong et al., 2009) and Virgibacillus (30.7-43 mol%)
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(Carrasco et al., 2009) (Supplementary Table S1). In addition, DNA–DNA hybridization was
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performed using the microplate hybridization method (Ezaki et al., 1989). DNA–DNA
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hybridization results between strains DX-3T and GIESS002 indicated high levels of relatedness
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(100 and 99% reciprocally), suggesting that the two isolates belong to the same species. On the
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other hand, strain DX-3T was a member of a different species from Ornithinibacillus contaminans
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(31.2-32.8% relatedness between DX-3T and Ornithinibacillus contaminans DSM 22953T).
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In order to phenotypically characterize strains DX-3T and GIESS002, standard phenotypic tests
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were performed. The closely related strains, Ornithinibacillus contaminans DSM 22953T,
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Ornithinibacillus bavariensis DSM 15681T, Oceanobacillus profundus DSM 18246T and
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Virgibacillus halophilus JCM 21758T were employed as references. Except that JCM 21758T was
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obtained from Japan Collection of Microorganisms (JCM), the others were purchased from the
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German Collection of Microorganisms and Cell Cultures (DSMZ). Recommended minimal
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standards for describing new taxa of aerobic, endospore-forming bacteria were followed (Logan et
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al., 2009).
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Cell morphology and endospore formation were examined using a JEM 1400 transmission
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electron microscope (TEM; JEOL, Japan) and a light microscope (Olympus BX51, Japan),
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respectively. Motility was tested by observing the growth spread in a plate of semi-solid TSA
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medium (Mayr et al., 2006). The temperature range (10, 15, 20, 25, 30, 37, 40, 45, 50, 55, 57, 60,
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65 and 70 oC) for growth was investigated in TSB for up to 1 week. To test the optimal growth pH,
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the distilled water in TSB was replaced with the following buffers: for pH 5.0-5.5, 0.1 M citric
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acid/0.1 M sodium citrate; for pH 6.0-8.5, 0.1 M KH2PO4/0.1 M NaOH; for pH 9.0-10.0, 0.1 M
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NaHCO3/0.1 M Na2CO3. Tolerance to NaCl was tested in TSB containing 0-15 % NaCl (w/v) with
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increments of 0.5 %. Anaerobic growth was examined in an anaerobic chamber (Sheldon
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Manufacturing Inc.) for 2 weeks. The Gram staining, nitrate reduction, methyl red and
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Voges-Proskauer reactions, indole production, H2S production, and hydrolysis of aesculin, starch,
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gelatin, casein, DNA and Tween 80 were performed as recommended by Smibert & Krieg (1994).
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Catalase was tested with 3 % (v/v) H2O2 and oxidase was determined using an oxidase reagent
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(BioMérieux). Haemolysis was assessed by spot-inoculation on TSA supplemented with 5 %
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ovine blood (Oxoid) after cells were incubated at 37 oC for 1-3 d. Utilization of various substrates
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as sole carbon source was examined as described by Dong & Cai (2001). Utilization of various
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substrates as sole nitrogen source was tested in a basal medium [MgSO4·7H2O 0.2 g, NaCl 5.0 g,
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K2HPO4 0.5 g, K2HPO4 0.5 g, yeast extract 0.02 g and agar 15 g in 1000 ml distilled water, pH 7.5]
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supplemented with D-glucose (1%, w/v). The API 20E and API 50CH systems (bioMérieux) were
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used to determine the acid production and some other physiological and biochemical properties
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according to the manufacturer’s instructions.
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Cells of strains DX-3T and GIESS002 were Gram-staining-positive, facultative anaerobic, motile
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by peritrichous flagella, and rod-shaped (0.6-0.8×1.6-3.0 µm) (Supplementary Fig. S1).
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Cylindrical or ellipsoidal endospores were produced at the terminal position in swollen sporangia.
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Both strains were moderately thermophilic and grew optimally at 50 oC, with the difference that
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strain DX-3T could grow at 60 oC but strain GIESS002 could not. The growth temperature feature
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also separated the two isolates from their related species of Ornithinibacillus contaminans,
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Ornithinibacillus bavariensis, Oceanobacillus profundus and Virgibacillus halophilus which
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cannot grow optimally at a temperature higher than 42 oC (Table 1). The two novel isolates were
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able to grow at pH 7.0-10.0 (optimum at pH 8.0 and pH 8.0-8.5 for strains DX-3T and GIESS002,
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respectively). Strain DX-3T grew at 0.5-11% (w/v) NaCl, with optimum growth at 6% (w/v) NaCl,
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while growth of strain GIESS002 occurred at 0.5-10% (w/v) NaCl (optimum 5.5%). Both strains
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gave positive results for catalase, oxidase, citrate utilization and hydrolysis of gelatin and casein,
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and negative results for methyl red and Voges-Proskauer reactions, nitrate reduction, production of
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H2S and indole, urease and hydrolysis of starch, esculin, DNA and Tween 80. Beta haemolysis
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(β-hemolysis) was observed after 3 days of incubation on blood agar at 37 oC for strains DX-3T
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and GIESS002. Detailed phenotypic features are included in the species description and Table 1
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and supplementary Tables S1 & S2.
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Isoprenoid quinones of the isolates were extracted with methanol from freeze-dried cells and
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purified by thin layer chromatography (TLC). The purified isoprenoid quinones were analyzed
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with HPLC (Agilent 1260) as described by Groth et al. (1997). Strain DX-3T contained a quinone
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system that consisted of the major compound MK-7 (97.1%) and the minor compound MK-8
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(2.9%). In strain GIESS002, the MK-7 and MK-8 accounted for 96.4% and 3.6% of the total
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isoprenoid quinones, respectively. These quinone systems were consistent with those found in the
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reference strains and most of the other species in the family Bacillaceae.
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For analyses of the cell-wall peptidoglycan and polar lipids, cells of strains DX-3T and GIESS002
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were prepared by cultivation on TSA supplemented with 5.5 % of NaCl at 50 °C. After cell-wall
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material was prepared and hydrolyzed, the cell-wall peptidoglycan was isolated and determined by
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TLC on cellulose plates using published protocols (Schumann, 2011). The quantitative ratio of
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amino acids in the peptidoglycan hydrolysate (4 M HCl, 100 °C, 16 h) was then determined by gas
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chromatography-mass spectrometry (GC/MS 320 Singlequad, Varian). Analysis results showed
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that peptidoglycans of strains DX-3T and GIESS002 contained the same predominant amino acids
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of alanine (Ala), glutamic acid (Glu) and meso-diaminopimelic acid (meso-DAP) in molar ratios
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of 1.1:1.0:0.6 and 1.0:1.0:0.5, respectively. In addition to the mentioned amino acids, the partial
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hydrolysates of both peptidoglycans consisted of the peptides L-Ala-D-Glu and meso-DAP-D-Ala.
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Therefore, the cell-wall peptidoglycan types of these two isolates were identified as A1γ
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meso-DAP-direct (http://www.dsmz.de/peptidoglycan-types.info), which were in accordance with
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those reported for their closely related genera in the family Bacillaceae except for the genera
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Ornithinibacillus (A4β L-Orn-D-Asp), Halobacillus (A4β L-Orn-D-Asp) and Paucisalibacillus
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(A4α L-Lys-D-Asp) (Supplementary Table S1).
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Polar lipids were extracted, separated by two-dimensional TLC and identified as described by
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Minnikin et al. (1984). Different detection reagents, including molybdophosphoric acid,
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molybdenum blue, ninhydrin and alpha-naphthol were used to determined total lipids,
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phospholipids, amino-containing lipids and glycolipids, respectively. For strains DX-3T and
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GIESS002,
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diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE)
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(Supplementary Fig. S3). The polar lipid profile features distinguished these two isolates from
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members of their closely related genera of Ornithinibacillus and Virgibacillus, whose members
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seldom contain PE as the predominant polar lipid (Carrasco et al., 2009; Kämpfer et al., 2010;
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Shin et al., 2012).
the
polar
lipid
profiles
consisted
of
predominant
components
of
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In preparation for cellular fatty acid profile analysis, cells of the two novel isolates and the
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reference strains were grown at their respective optimum growth temperatures in TSB till the
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late-exponential growth phase, harvested by centrifugation, washed with distilled water and
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freeze-dried. The fatty acids in whole cells were saponified, methylated and extracted using the
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standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0B). The fatty
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acids were analyzed with GC (Agilent 6890) and identified using the TSBA6.0 database of the
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Microbial Identification System. Twelve fatty acids with abundance of >5 % were detected for
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strain DX-3T: iso-C15:0 (31.2 %), iso-C17:0 (17.6 %), anteiso-C17:0 (15.4 %), anteiso-C15:0 (8.4 %),
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iso-C16:0 (7.5 %) and C16:0 (6.6 %), while the fatty acid profile of strain GIESS002 comprised
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mainly iso-C15:0 (28.8 %), anteiso-C15:0 (17.0 %), anteiso-C17:0 (14.5 %), iso-C17:0 (13.8 %) and
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iso-C16:0 (10.6 %). Fatty acid profiles of the two strains and reference strains are summarized in
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Table 2. In comparison to the references and most members of the family Bacillaceae, the fatty
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acid profiles of these two isolates had certain similar features, i.e. varied and abundant saturated
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branched-chain fatty acids and occurrence of longer chain fatty acids with 15-17 carbons (Nunes
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et al., 2006; Mayr et al., 2006; Carrasco et al., 2008). However, the iso-C15:0/anteiso-C15:0 ratio of
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greater than 1 could distinguish the isolates from most members of the genera Ornithinibacillus,
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Oceanobacillus and Virgibacillus, and all members of other related genera, i.e. Terribacillus,
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Halobacillus, Sediminibacillus, Paucisalibacillus and Lentibacillus (Supplementary Table S1).
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Among existing genera, the mentioned genera in supplementary Table S1 are the candidates to
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accommodate strains DX-3T and GIESS002 without loss of monophyleticity. However,
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phylogenetic analysis of 16S rRNA gene sequences revealed that these two isolates represent a
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novel branch within the Gram-staining-positive, endospore-forming bacteria in the family
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Bacillaceae. The novel isolates also can be distinguished from their related genera by some
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taxonomic characteristics. For instances, temperature for growth (range and optimum) of the
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isolates can separate them from all the mentioned genera above. Being capable of growing under
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anaerobic conditions can distinguish them from the genera Ornithinibacillus, Terribacillus,
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Halobacillus and Paucisalibacillus. The cell-wall peptidoglycan type of strains DX-3T and
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GIESS002 is distinct from the genera Ornithinibacillus, Halobacillus and Paucisalibacillus. And
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the Voges-Proskauer test can separate them from the genus Terribacillus. In addition, these two
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novel isolates can be differentiated from members of their closest genera Ornithinibacillus,
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Oceanobacillus and Virgibacillus by characters of the DNA G+C contents. The higher
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iso-C15:0/anteiso-C15:0 ratios distinguish them from members of the genera Terribacillus,
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Halobacillus, Sediminibacillus, Paucisalibacillus and Lentibacillus. In conclusion, the results of
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polyphasic taxonomic analysis suggest that strains DX-3T and GIESS002 represent a novel species
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of a new genus in the family Bacillaceae, for which the name Compostibacillus humi gen. nov., sp.
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nov. is proposed.
249 250
Description of Compostibacillus gen. nov.
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Compostibacillus (Com.pos.ti.ba.cil'lus. N.L. neut. n. compostum, compost; L. masc. n. bacillus, a
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small rod; N.L. masc. n. Compostibacillus, a rod-shaped bacterium from compost).
253 254
Cells are Gram-staining-positive, motile, endospore-forming and rod-shaped. Cylindrical or
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ellipsoidal endospores are produced at the terminal position in swollen sporangia. Moderately
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thermophilic. Oxidase and catalase positive. Nitrate is not reduced. Phylogenetically related to the
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genus Ornithinibacillus and other genera of the Bacillaceae. The cell-wall peptidoglycan type is
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determined as A1γ (meso-DAP direct). The cellular fatty acids consist mainly of iso- and
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anteiso-branched acids, with iso-C15:0 predominating and anteiso-C15:0, anteiso-C17:0, iso-C17:0 and
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iso-C16:0 at moderate amounts. The major respiratory quinone is MK-7. The DNA G+C contents of
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known strains are 43.2-43.7 mol%. The type species is Compostibacillus humi.
262 263
Description of Compostibacillus humi sp. nov.
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Compostibacillus humi (hu'mi. L. gen. n. humi, of the soil).
265 266
Exhibits the following properties in addition to those given in the genus description. Cells are
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0.6-0.8 µm × 1.6-3.0 µm in size. Motile by peritrichous flagella. Colonies are circular, entire,
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smooth, yellowish brown and with a diameter of 2-3 mm on TSA after 48 h of incubation at 50 oC.
269
Anaerobic growth occurs. Grows at 30-60 oC (optimally at 50 oC), pH 7.0-10.0 (optimally at pH
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8.0-8.5) and 0.5-11 % (w/v) NaCl (optimally 5.5-6 % (w/v) NaCl). Beta haemolysis is observed.
271
Citrate is utilized. The methyl red and Voges-Proskauer reactions, β-galactosidase, arginine
272
dihydrolase, lysine decarboxylase, orinithine decarboxylase, urease and tryptophane deaminase
273
are negative. Indole, H2S and acetoin are not produced. Gelatin and casein are hydrolyzed, but
274
starch, esculin, DNA and Tween 80 are not. D-Ribose, sodium malonate, sodium acetate, lactic
275
acid, L-alanine, glycogen, 3-hydroxybenzoic acid, L-serine, D-mannitol, 3-hydroxybutyric acid,
276
4-hydroxybenzoic acid and L-proline can be utilized as sole carbon sources; D-maltose,
277
D-saccharose, D-glucose, D-sorbitol, L-arabinose, propionic acid and L-histidine cannot be
278
utilized. NH4Cl, (NH4)2SO4 and NH4NO3 can be utilized as sole nitrogen sources, but NaNO3,
279
KNO3, Mg(NO3)2, ammonium acetate and hexamethylene tetramine cannot. Acid is produced
280
from D-ribose, D-fructose, L-sorbose, esculin ferric citrate, D-tagatose and potassium
281
5-ketogluconate, but not from glycerol, D-xylose, D-mannose, methyl-αD-glucopyranoside,
282
N-acetylglucosamine, amygdalin, D-cellobiose, D-lactose, D-maltose, D-saccharose, D-trehalose,
283
starch, xylitol and D-arabitol. The major cellular fatty acids are iso-C15:0, iso-C17:0, anteiso-C17:0
10
284
and anteiso-C15:0. Polar lipid profile is consisted of diphosphatidylglycerol, phosphatidylglycerol
285
and phosphatidylethanolamine. The G+C content of the genomic DNA is 43.2-43.7 mol%.
286 287
The type strain is DX-3T (=KCTC 33104T =CGMCC 1.12360T), isolated from sludge compost
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samples collected from a compost plant in Guangdong Province, China. Strain GIESS002
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(=CCTCC AB 2013108 =KCTK 33158) is a reference strain of the species, isolated from the same
290
source. Acid is produced by the type strain from L-sorbose, but not from D-glucose, D-mannitol,
291
arbutin, salicin and gentiobiose. The DNA G+C content of the type strain is 43.7 mol%.
292 293
Acknowledgements
294
This study was supported by the Agricultural Science & Technology Achievements Transfer
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Project of Guangdong Province, China (2012NL040), the National Natural Science Foundation of
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China (31100353) and the Foundation for Excellent Young Scientist in Guangdong Academy of
297
Sciences (qnjj201401).
298 299
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Groth, I., Schumann, P., Rainey, F. A., Martin, K., Schuetze, B. & Augsten, K. (1997).
326
Demetria terragena gen. nov., sp. nov., a new genus of actinomycetes isolated from compost soil.
327
Int J Syst Bacteriol 47, 1129-1133.
328
Han, L., Yang, G., Zhou, X., Yang, D., Hu, P., Lu, Q. & Zhou, S. (2013). Bacillus
329
thermocopriae sp. nov., isolated from a compost. Int J Syst Evol Microbiol 63, 3024-3029.
330
Kim, Y.G., Choi, D.H., Hyun, S. & Cho, B.C. (2007). Oceanobacillus profundus sp. nov.,
331
isolated from a deep-sea sediment core. Int J Syst Evol Microbiol 57, 409-413.
332
Kämpfer, P., Falsen, E., Lodders, N., Langer, S., Busse, H.J. & Schumann, P. (2010).
333
Ornithinibacillus contaminans sp. nov., an endospore-forming species. Int J Syst Evol. Microbiol
334
60, 2930-2934.
335
Logan, N.A., Berge, O., Bishop, A.H., Busse, H.J., De Vos, P., Fritze, D., Heyndrickx, M.,
336
Kämpfer, P., Rabinovitch, L. & other authors (2009). Proposed minimal standards for
337
describing new taxa of aerobic endospore-forming bacteria. Int J Syst Evol Microbiol 59,
338
2114-2121.
339
L'Haridon, S., Miroshnichenko, M.L., Kostrikina, N.A., Tindall, B.J., Spring, S., Schumann,
340
P., Stackebrandt, E., Bonch-Osmolovskaya, E.A. & Jeanthon, C. (2006). Vulcanibacillus
12
341
modesticaldus gen. nov., sp. nov., a strictly anaerobic, nitrate-reducing bacterium from deep-sea
342
hydrothermal vents. Int J Syst Evol Microbiol 56, 1047-1053.
343
Mayr, R., Busse, H.J., Worliczek, H.L., Ehling-Schulz, M. & Scherer, S. (2006).
344
Ornithinibacillus gen. nov., with the species Ornithinibacillus bavariensis sp. nov. and
345
Ornithinibacillus californiensis sp. nov. Int J Syst Evol Microbiol 56, 1383-1389.
346
Mesbah, M., Premachandran, U. & Whitman, W.B. (1989). Precise measurement of the G+C
347
content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol
348
39, 159-167.
349
Minnikin, D.E., O’Donnell, A.G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. &
350
Parlett, J.H. (1984). An integrate procedure for the extraction of bacterial isoprenoid quinones
351
and polar lipids. J Microbiol Methods 2, 233-241.
352
Namwong, S., Tanasupawat, S., Lee, K.C. & Lee, J.S. (2009). Oceanobacillus kapialis sp. nov.,
353
from fermented shrimp paste in Thailand. Int J Syst Evol Microbiol 59, 2254-2259.
354
Nicolaus, B., Lama, L., Esposito, E., Manca, M.C., Di Prisco, G. & Gambacorta, A. (1996).
355
Bacillus thermoantarcticus sp. nov., from Mount Melbourne, Antarctica: a novel thermophilic
356
species. Polar Biol 16, 101-104.
357
Nunes, I., Tiago, I., Pires, A.L., da Costa, M.S., & Veríssimo, A. (2006). Paucisalibacillus
358
globulus gen. nov., sp. nov., a Gram-positive bacterium isolated from potting soil. Int J Syst Evol
359
Microbiol 56, 1841-1845.
360
Schumann, P. (2011). Peptidoglycan structure. In Taxonomy of Prokaryotes, Methods in
361
Microbiology, vol. 38, pp. 101-129. Edited by F. Rainey & A. Oren. London: Academic Press.
362
Shin, N.R., Whon, T.W., Kim, M.S., Roh, S.W., Jung, M.J., Kim, Y.O. & Bae, J.W. (2012).
363
Ornithinibacillus scapharcae sp. nov., isolated from a dead ark clam. Antonie van Leeuwenhoek
364
101, 147-154.
365
Smibert, R.M. & Krieg, N.R. (1994). Phenotypic characterization. In Methods for General and
366
Molecular Bacteriology, pp. 607-654. Edited by P. Gerhardt, R.G.E. Murray, W.A. Wood and N.R.
367
Krieg. Washington, DC: American Society for Microbiology.
13
368
Sung, M.H., Kim, H., Bae, J.W., Rhee, S.K., Jeon, C.O., Kim, K., Hong, S.P., Lee, S.G., Yoon,
369
J.H., Park, Y.H. & Baek, D.H. (2002). Geobacillus toebii sp. nov., a novel thermophilic
370
bacterium isolated from hay compost. Int J Syst Evol Microbiol 52, 2251-2255.
371
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5:
372
molecular evolutionary genetic analysis using maximum likelihood, evolutionary distance, and
373
maximum parsimony methods. Mol Biol Evol 28, 2731-2739.
374
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. (1997). The
375
CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by
376
quality analysis tools. Nucleic Acids Res 25, 4876-4882.
377
Xue, Y., Zhang, X., Zhou, C., Zhao, Y., Cowan, D.A., Heaphy, S., Grant, W.D., Jones, B.E.,
378
Ventosa, A. & Ma, Y. (2006). Caldalkalibacillus thermarum gen. nov., sp. nov., a novel
379
alkalithermophilic bacterium from a hot spring in China. Int J Syst Evol Microbiol 56, 1217-1221.
380
Yang, G., Chen, M., Yu., Z., Lu, Q. & Zhou, S. (2013a). Bacillus composti sp. nov. and Bacillus
381
thermophilus sp. nov., two thermophilic Fe(III)-reducing bacteria isolated from compost. Int J Syst
382
Evol Microbiol 63, 3030-3036.
383
Yang, G., Zhou, X., Zhou, S., Yang, D., Wang, Y. & Wang, D. (2013b). Bacillus
384
thermotolerans sp. nov., a thermophilic bacterium capable of reducing humus. Int J Syst Evol
385
Microbiol 63, 3672-3678.
14
386
Table 1 Characteristics that distinguish strains DX-3T and GIESS002 from their closely
387
related species.
388
Strains: 1, strain DX-3T; 2, strain GIESS002; 3, Ornithinibacillus contaminans DSM 22953T; 4,
389
Ornithinibacillus bavariensis DSM 15681T; 5, Oceanobacillus profundus DSM 18246T; 6,
390
Virgibacillus halophilus JCM 21758T. Data were taken from this study, except for isolation source,
391
cell size, DNA G+C content, and peptidoglycan type of strains DSM 22953T (Kämpfer et al.,
392
2010), DSM 15681T (Mayr et al., 2006), DSM 18262T (Kim et al., 2007) and JCM 21758T (An et
393
al., 2007). +, positive; -, negative. For spore shape: C, cylindrical; E, ellipsoidal; S, spherical. For
394
spore position: C, central; T, terminal.
15
395 Characteris
1
2
3
Compost in
Compost in
Human
thermophili
maturation
blood
c phase
phase
sample
0.6-0.8×1.6
0.6-0.8×1.6
0.8-1.0×2.0
(μm)
-3.0
-3.0
-3.0
Spore shape
C/E
C/E
S
E
E
E
T
T
C
T
T
T
+
+
-
+
+
+
+
+
-
-
-
-
-
-
-
-
+
+
50
50
30
42
35
25-30
6
5.5
0
0.5-4
1-3
5.5-8
8.0
8.0-8.5
7.0-9.0
8.0-9.0
7.5-8.5
7.0-8.5
25 °C
-
-
+
+
+
+
57 °C
+
+
-
-
-
-
60 °C
+
-
-
-
-
-
+
-
-
-
+
+
pH 6.5
-
-
+
-
+
+
pH 10.0
+
+
-
+
-
+
+
+
-
-
-
-
Esculin
-
-
+
+
+
+
Casein
+
+
-
-
+
+
43.7
43.2
36.6
36.4
40.2
42.6
A1γ
A1γ
A4β
A4β
A1γ
A1γ
(m-DAP
(m-DAP
(L-Orn-D-
(L-Orn-D-
(m-DAP
(m-DAP
tics Isolation source Cell size
Spore position Motility Anaerobic growth Nitrate reduction
4
5
Pasteurized
Deep-sea
milk
sediment
0.4×2.0-6.0
0.2-0.4×0.82.0
6
Field soil
0.5×1.75
Optimal temperature (°C) Optimal salinity (%) Optimal pH Growth at:
10-11% NaCl (w/v)
Citrate utilization Hydrolysis of:
DNA G+C content (mol%) Peptidoglyc *
an type
16
direct)
direct)
Asp)
Asp)
direct)†
396
*
m-DAP, meso-diaminopimelic acid; L-Orn, L-ornithine; D-Asp, D-aspartate.
397
†
Determined in this study for DSM 18246T.
398
17
direct)
399
Table 2 Cellular fatty acid profiles of strains DX-3T and GIESS002 and their closely related
400
species.
401
Strains: 1, strain DX-3T; 2, strain GIESS002; 3, Ornithinibacillus contaminans DSM 22953T; 4,
402
Ornithinibacillus bavariensis DSM 15681T; 5, Oceanobacillus profundus DSM 18246T; 6,
403
Virgibacillus halophilus JCM 21758T. Data were taken from this study. Values were percentages of
404
the total fatty acids; -, fatty acids representing < 1.0 % of the total.
405 Fatty acid (%)
1
2
3
4
5
6
iso-C11:0
1.5
-
1.4
-
-
-
C12:0
2.0
1.2
2.6
-
-
-
C14:0
-
1.3
1.3
1.1
-
1.6
iso-C14:0
-
2.6
-
1.4
10.3
5.8
iso-C15:0
31.2
28.8
17.2
26.7
5.2
24.5
anteiso-C15:0
8.4
17.0
25.5
23.4
48.5
32.4
C16:0
6.6
4.9
4.3
3.9
3.8
4.5
iso-C16:0
7.5
10.6
5.0
6.1
13.1
8.1
C16:0 2-OH
2.2
-
2.8
-
-
-
iso-C17:0
17.6
13.8
6.4
10.4
1.1
4.2
anteiso-C17:0
15.4
14.5
24.8
22.3
11.8
13.6
C18:0
1.2
-
2.1
-
-
1.3
C18:1 ω9c
1.7
2.2
1.8
-
1.0
-
Summed feature 3
*
1.5
-
-
-
2.1
1.2
Summed feature 8
*
-
-
1.5
1.0
-
-
406
*
407
and/or C18:1ω6c.
Summed feature 3 comprises C16:1ω7c and/or C16:1ω6c; Summed feature 8 comprises C18:1ω7c
18
408
Figure legend
409 410
Fig. 1 Phylogenetic tree constructed using the maximum-likelihood method based on 16S rRNA
411
gene sequences.
412
19
413
Legends for the supplementary figures and tables
414 415
Fig. S1 Transmission electron micrograph of a cell of strain DX-3T, showing cell morphology and
416
peritrichous flagella.
417 418
Fig. S2 Phylogenetic trees constructed using the minimum-evolution method (a) and the
419
maximum parsimony method (b) based on 16S rRNA gene sequences.
420 421
Fig. S3 Polar lipid profiles of strain DX-3T after two-dimensional TLC and detection with
422
molybdophosphoric acid (a), molybdenum blue (b), α-Naphthol (c) and ninhydrin (d).
423
424
Table S1 Distinguishing features of the novel genus compared to the members of the closely
425
related genera of the family Bacillaceae.
426
427
Table S2 Acid production characteristics that distinguish strains DX-3T and GIESS002 from their
428
closely related species.
429
20
Halobacillus yeomjeoni MSS-402T (AY881246)
71 100
Halobacillus campisalis ASL-17T (EF486356) Halobacillus faecis IGA7-4T (AB243865)
70
Sediminibacillus albus NHBX5T (DQ989634) 99
Sediminibacillus halophilus EN8dT (AM905297) Bacillus subtilis DSM10T (AJ276351) Bacillus aerius 24KT (AJ831843)
100
Virgibacillus halophilus 5B73CT (AB243851)
85
Virgibacillus soli CC-YMP-6T (EU213011) Virgibacillus halodenitrificans DSM 10037T (AY543169) Virgibacillus litoralis JSM 089168T (FJ425909)
58
Virgibacillus carmonensis LMG 20964T (AJ316302)
73 100
Virgibacillus necropolis LMG 19488T (AJ315056)
Ornithinibacillus bavariensis WSBC 24001T (Y13066) Ornithinibacillus halophilus G8BT (HQ433440) Ornithinibacillus scapharcae TW25 T (AEWH01000025) Ornithinibacillus californiensis MB-9T (AF326365) Ornithinibacillus contaminans CCUG 53201T (FN597064) Oceanobacillus profundus CL-MP28T (DQ386635)
93
Oceanobacillus polygoni SA9T (AB750685) Oceanobacillus picturae LMG 19492T (AJ315060) Oceanobacillus oncorhynchi 20AGT (AJ640134) Salinibacillus xinjiangensis J4T (JX402080) Salinibacillus aidingensis 25-7T (AY321436)
99
Salinibacillus kushneri 8-2T (AY321434)
99
Compostibacillus humi DX-3T (JX274434)
100
Compostibacillus humi GIESS002 (KJ024967) Terribacillus halophilus 002-051T (AB243849) 100
Terribacillus aidingensis YI7-61T (FJ386524) Paenibacillus motobuensis MC10T (AY741810)
0.01
430
431
Fig. 1 Phylogenetic tree constructed using the maximum-likelihood method based on 16S rRNA
432
gene sequences. Bootstrap values, generated from 1200 re-samplings, above 50% are indicated at
433
the branching points. Bar 0.01 substitutions per nucleotide position.
434
21
1
Compostibacillus humi gen. nov., sp. nov., a member of the family Bacillaceae, isolated
2
from sludge compost
3 4
Zhen Yu, Junlin Wen, Guiqin Yang, Jing Liu, Shungui Zhou *
5
Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650, PR China
6 7 8 9
*
Corresponding author:
10
Shungui Zhou
11
E-mail: [email protected]
12
Tel: (86)-20-37300951
13 14 15 16 17
Subject Category: New Taxa- (Firmicutes)
18 19
Running Title: Compostibacillus humi gen. nov., sp. nov.
20 21 22 23 24 25 26 27 28 29 30 * The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains DX-3T and GIESS002 are JX274434 and KJ024967, respectively.
1
31
Two novel Gram-staining-positive, rod-shaped, endospore-forming, and moderately
32
thermophilic bacteria, designated strain DX-3T and GIESS002, respectively were isolated
33
from sludge composts from Guangdong Province, China. Analysis of 16S rRNA gene
34
sequences revealed that the isolates were closely related to each other with extremely high
35
similarity (99.6%), and were members of the family Bacillaceae. However, these two isolates
36
formed a novel phylogenetic branch within this family. Their closest relatives were the
37
members of the genera Ornithinibacillus, Oceanobacillus and Virgibacillus. Cells of both
38
strains were facultatively anaerobic and catalase- and oxidase-positive. The cell-wall
39
peptidoglycan type was A1γ (meso-DAP direct). The predominant isoprenoid quinone was
40
MK-7. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and
41
phosphatidylethanolamine. The major cellular fatty acid was iso-C15:0. The DNA G+C
42
content was 43.2-43.7 mol%. The polyphasic taxonomic results indicated that strains DX-3T
43
and GIESS002 represent a novel species in a new genus in the family Bacillaceae, order
44
Bacillales for which the name Compostibacillus humi gen. nov., sp. nov. is proposed. The type
45
strain is DX-3T (=KCTC 33104T =CGMCC 1.12360T).
2
46
At the time of writing, the family Bacillaceae in the order Bacillales comprised more than 50
47
recognized genera (http://www.bacterio.net/bacillaceae.html) with different physiological features.
48
Many of these genera include thermophilic or thermotolerant endospore-forming species, such as
49
the genera Bacillus (Han et al., 2013; Yang et al., 2013a; 2013b), Caldalkalibacillus (Xue et al.,
50
2006), Geobacillus (Cihan et al., 2011) and Vulcanibacillus (L'Haridon et al., 2006). Members of
51
these genera have been isolated from various warm or hot environments. For example, Bacillus
52
thermoantarcticus was isolated from a geothermal soil (Nicolaus et al., 1996), Caldalkalibacillus
53
thermarum was from a hot spring (Xue et al., 2006), Geobacillus thermodenitrificans was from a
54
high temperature well-pipeline sediment sample (Cihan et al., 2011) and Vulcanibacillus
55
modesticaldus was from deep-sea hydrothermal vents (L'Haridon et al., 2006). Compost is also a
56
very important source for isolation of thermophiles, and many thermophilic or thermotolerant
57
species in the family Bacillaceae from compost have been reported (Sung et al., 2002; Yang et al.,
58
2013a, 2013b; Han et al., 2013).
59 60
In our previous studies, we have isolated some thermophilic bacteria from a demonstration
61
compost plant in Guangdong Province, China and identified four novel Gram-staining-positive,
62
endospore-forming, facultative anaerobic species as Bacillus composti (Yang et al., 2013a),
63
Bacillus thermocopriae (Han et al., 2013), Bacillus thermophilus (Yang et al., 2013a) and Bacillus
64
thermotolerans (Yang et al., 2013b), which are included in the same genus of Bacillus. In this
65
paper, we describe two novel moderately thermophilic bacteria, designated strains DX-3T and
66
GIESS002, which were isolated from different compost samples obtained in the thermophilic and
67
maturation phases of composting, respectively. On the basis of partial 16S rRNA gene sequence
68
analyses, the novel isolates were found to be closely related to members of the genera
69
Ornithinibacillus, Oceanobacillus, Virgibacillus, Terribacillus, Halobacillus, Sediminibacillus,
70
Paucisalibacillus and Lentibacillus. However, the polyphasic taxonomic analysis indicated that
71
these two isolates could not be assigned to any of the recognized genera, and represented a novel
72
species in a new genus Compostibacillus caeni gen. nov., sp. nov. of the family Bacillaceae.
73 74
Sludge composting was performed in a demonstration compost plant in Guangdong Province,
75
China. Compost samples were collected during different fermentation phases of the whole
3
76
composting process. For each sampling, the temperature in the compost pile was also monitored
77
and recorded. The sampling procedures were described in detail by Yang et al. (2013a). Isolation
78
was carried out by the dilution-plate method. About 5.0 g sub-sample of each compost sample was
79
added into 100 ml 0.85 % NaCl solution, stirred for 30 min, serially diluted, and then placed on
80
agar plate of TSA (trypticase soya agar, pH 7.2), which contained (in L-1): 17 g tryptone, 3 g
81
soytone, 5 g NaCl, 2.5 g K2HPO4, 2.5 g glucose and 15 g agar. After incubation at their respective
82
source temperature for 5 d, a single colony was picked and transferred to fresh TSA media for
83
further purification. Strain DX-3T was isolated from the compost sample in the thermophilic phase
84
of composting at 55 oC, while strain GIESS002 was from the sample in the maturation phase at a
85
temperature of 45 oC. These two isolates were preserved at -80 oC in tryptic soy broth (TSB,
86
Oxoid Ltd.) supplemented with 15 % (v/v) glycerol for further study.
87 88
Total genomic DNA was prepared using a commercial genomic DNA extraction kit (Aidlab
89
Biotechnologies Co., Ltd.). The 16S rRNA genes of strains DX-3T and GIESS002 were
90
PCR-amplified using universal primers (27f and 1492r; Baker et al. 2003). After purified using
91
Gel Extraction Kit D2500-01 (Omega Bio-tek), the PCR products were cloned into plasmid vector
92
using a TA cloning kit (TaKaRa, Dalian, China) and sequenced using an ABI PRISM 3100 Genetic
93
Analyser (Applied Biosystems). Sequences closely related to those of the two isolates were
94
obtained using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The 16S rRNA gene sequences of
95
the novel strains and related strains were aligned using CLUSTAL_X (Thompson et al., 1997) and
96
sequence similarities with alignment gaps and ambiguous bases omitted were calculated using
97
MEGA version 5.0 (Tamura et al., 2007). Phylogenetic trees were constructed based on the
98
aligned 16S rRNA gene sequences using the maximum-likelihood, minimum-evolution and
99
maximum parsimony methods. Statistical support for the branches of the phylogenetic trees was
100
determined using bootstrap analysis (Felsenstein, 1985) (based on 1200 re-samplings).
101 102
Almost full-length 16S rRNA gene sequences of strains DX-3T (1555 nt) and GIESS002 (1556 nt)
103
were obtained. Sequence analysis results revealed that these two isolates were closely related to
104
each other with a high sequence similarity of 99.6% and belonged to the family Bacillaceae. For
105
both strains, the 16S rRNA gene sequence similarities with members of all genera in the family
4
106
Bacillaceae were equal or lower than 96.2 %, e.g. Ornithinibacillus (≤ 96.2 %), Oceanobacillus (≤
107
94.5 %), Virgibacillus (≤ 94.6 %), Terribacillus (≤ 94.0 %), Salinibacillus (≤ 93.7 %) and Bacillus
108
(≤ 93.4 %). Except for the above genera, these two isolates did not show more than 93%
109
similarities with the other genera of the family Bacillaceae. The phylogenetic tree based on the
110
maximum-likelihood method showed that the two novel isolates clustered in a separate clade (Fig.
111
1), which was also shown in the minimum-evolution and maximum parsimony trees
112
(Supplementary Fig. S2), indicating that these two isolates might represent a novel species in a
113
new genus.
114 115
The DNA G+C contents of strains DX-3T and GIESS002 determined by HPLC as described by
116
Mesbah et al. (1989) were 43.7 and 43.2 mol%, respectively. These values were significantly
117
higher than those of the references as shown in Table 1. The DNA G+C contents also supported
118
the view that these two isolates were distinguishable from all members of their closely related
119
genera of Ornithinibacillus (36-41 mol%) (Mayr et al., 2006; Bagheri et al., 2012),
120
Oceanobacillus (33.6-40.2 mol%) (Namwong et al., 2009) and Virgibacillus (30.7-43 mol%)
121
(Carrasco et al., 2009) (Supplementary Table S1). In addition, DNA–DNA hybridization was
122
performed using the microplate hybridization method (Ezaki et al., 1989). DNA–DNA
123
hybridization results between strains DX-3T and GIESS002 indicated high levels of relatedness
124
(100 and 99% reciprocally), suggesting that the two isolates belong to the same species. On the
125
other hand, strain DX-3T was a member of a different species from Ornithinibacillus contaminans
126
(31.2-32.8% relatedness between DX-3T and Ornithinibacillus contaminans DSM 22953T).
127 128
In order to phenotypically characterize strains DX-3T and GIESS002, standard phenotypic tests
129
were performed. The closely related strains, Ornithinibacillus contaminans DSM 22953T,
130
Ornithinibacillus bavariensis DSM 15681T, Oceanobacillus profundus DSM 18246T and
131
Virgibacillus halophilus JCM 21758T were employed as references. Except that JCM 21758T was
132
obtained from Japan Collection of Microorganisms (JCM), the others were purchased from the
133
German Collection of Microorganisms and Cell Cultures (DSMZ). Recommended minimal
134
standards for describing new taxa of aerobic, endospore-forming bacteria were followed (Logan et
135
al., 2009).
5
136 137
Cell morphology and endospore formation were examined using a JEM 1400 transmission
138
electron microscope (TEM; JEOL, Japan) and a light microscope (Olympus BX51, Japan),
139
respectively. Motility was tested by observing the growth spread in a plate of semi-solid TSA
140
medium (Mayr et al., 2006). The temperature range (10, 15, 20, 25, 30, 37, 40, 45, 50, 55, 57, 60,
141
65 and 70 oC) for growth was investigated in TSB for up to 1 week. To test the optimal growth pH,
142
the distilled water in TSB was replaced with the following buffers: for pH 5.0-5.5, 0.1 M citric
143
acid/0.1 M sodium citrate; for pH 6.0-8.5, 0.1 M KH2PO4/0.1 M NaOH; for pH 9.0-10.0, 0.1 M
144
NaHCO3/0.1 M Na2CO3. Tolerance to NaCl was tested in TSB containing 0-15 % NaCl (w/v) with
145
increments of 0.5 %. Anaerobic growth was examined in an anaerobic chamber (Sheldon
146
Manufacturing Inc.) for 2 weeks. The Gram staining, nitrate reduction, methyl red and
147
Voges-Proskauer reactions, indole production, H2S production, and hydrolysis of aesculin, starch,
148
gelatin, casein, DNA and Tween 80 were performed as recommended by Smibert & Krieg (1994).
149
Catalase was tested with 3 % (v/v) H2O2 and oxidase was determined using an oxidase reagent
150
(BioMérieux). Haemolysis was assessed by spot-inoculation on TSA supplemented with 5 %
151
ovine blood (Oxoid) after cells were incubated at 37 oC for 1-3 d. Utilization of various substrates
152
as sole carbon source was examined as described by Dong & Cai (2001). Utilization of various
153
substrates as sole nitrogen source was tested in a basal medium [MgSO4·7H2O 0.2 g, NaCl 5.0 g,
154
K2HPO4 0.5 g, K2HPO4 0.5 g, yeast extract 0.02 g and agar 15 g in 1000 ml distilled water, pH 7.5]
155
supplemented with D-glucose (1%, w/v). The API 20E and API 50CH systems (bioMérieux) were
156
used to determine the acid production and some other physiological and biochemical properties
157
according to the manufacturer’s instructions.
158 159
Cells of strains DX-3T and GIESS002 were Gram-staining-positive, facultative anaerobic, motile
160
by peritrichous flagella, and rod-shaped (0.6-0.8×1.6-3.0 µm) (Supplementary Fig. S1).
161
Cylindrical or ellipsoidal endospores were produced at the terminal position in swollen sporangia.
162
Both strains were moderately thermophilic and grew optimally at 50 oC, with the difference that
163
strain DX-3T could grow at 60 oC but strain GIESS002 could not. The growth temperature feature
164
also separated the two isolates from their related species of Ornithinibacillus contaminans,
165
Ornithinibacillus bavariensis, Oceanobacillus profundus and Virgibacillus halophilus which
6
166
cannot grow optimally at a temperature higher than 42 oC (Table 1). The two novel isolates were
167
able to grow at pH 7.0-10.0 (optimum at pH 8.0 and pH 8.0-8.5 for strains DX-3T and GIESS002,
168
respectively). Strain DX-3T grew at 0.5-11% (w/v) NaCl, with optimum growth at 6% (w/v) NaCl,
169
while growth of strain GIESS002 occurred at 0.5-10% (w/v) NaCl (optimum 5.5%). Both strains
170
gave positive results for catalase, oxidase, citrate utilization and hydrolysis of gelatin and casein,
171
and negative results for methyl red and Voges-Proskauer reactions, nitrate reduction, production of
172
H2S and indole, urease and hydrolysis of starch, esculin, DNA and Tween 80. Beta haemolysis
173
(β-hemolysis) was observed after 3 days of incubation on blood agar at 37 oC for strains DX-3T
174
and GIESS002. Detailed phenotypic features are included in the species description and Table 1
175
and supplementary Tables S1 & S2.
176 177
Isoprenoid quinones of the isolates were extracted with methanol from freeze-dried cells and
178
purified by thin layer chromatography (TLC). The purified isoprenoid quinones were analyzed
179
with HPLC (Agilent 1260) as described by Groth et al. (1997). Strain DX-3T contained a quinone
180
system that consisted of the major compound MK-7 (97.1%) and the minor compound MK-8
181
(2.9%). In strain GIESS002, the MK-7 and MK-8 accounted for 96.4% and 3.6% of the total
182
isoprenoid quinones, respectively. These quinone systems were consistent with those found in the
183
reference strains and most of the other species in the family Bacillaceae.
184 185
For analyses of the cell-wall peptidoglycan and polar lipids, cells of strains DX-3T and GIESS002
186
were prepared by cultivation on TSA supplemented with 5.5 % of NaCl at 50 °C. After cell-wall
187
material was prepared and hydrolyzed, the cell-wall peptidoglycan was isolated and determined by
188
TLC on cellulose plates using published protocols (Schumann, 2011). The quantitative ratio of
189
amino acids in the peptidoglycan hydrolysate (4 M HCl, 100 °C, 16 h) was then determined by gas
190
chromatography-mass spectrometry (GC/MS 320 Singlequad, Varian). Analysis results showed
191
that peptidoglycans of strains DX-3T and GIESS002 contained the same predominant amino acids
192
of alanine (Ala), glutamic acid (Glu) and meso-diaminopimelic acid (meso-DAP) in molar ratios
193
of 1.1:1.0:0.6 and 1.0:1.0:0.5, respectively. In addition to the mentioned amino acids, the partial
194
hydrolysates of both peptidoglycans consisted of the peptides L-Ala-D-Glu and meso-DAP-D-Ala.
195
Therefore, the cell-wall peptidoglycan types of these two isolates were identified as A1γ
7
196
meso-DAP-direct (http://www.dsmz.de/peptidoglycan-types.info), which were in accordance with
197
those reported for their closely related genera in the family Bacillaceae except for the genera
198
Ornithinibacillus (A4β L-Orn-D-Asp), Halobacillus (A4β L-Orn-D-Asp) and Paucisalibacillus
199
(A4α L-Lys-D-Asp) (Supplementary Table S1).
200 201
Polar lipids were extracted, separated by two-dimensional TLC and identified as described by
202
Minnikin et al. (1984). Different detection reagents, including molybdophosphoric acid,
203
molybdenum blue, ninhydrin and alpha-naphthol were used to determined total lipids,
204
phospholipids, amino-containing lipids and glycolipids, respectively. For strains DX-3T and
205
GIESS002,
206
diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE)
207
(Supplementary Fig. S3). The polar lipid profile features distinguished these two isolates from
208
members of their closely related genera of Ornithinibacillus and Virgibacillus, whose members
209
seldom contain PE as the predominant polar lipid (Carrasco et al., 2009; Kämpfer et al., 2010;
210
Shin et al., 2012).
the
polar
lipid
profiles
consisted
of
predominant
components
of
211 212
In preparation for cellular fatty acid profile analysis, cells of the two novel isolates and the
213
reference strains were grown at their respective optimum growth temperatures in TSB till the
214
late-exponential growth phase, harvested by centrifugation, washed with distilled water and
215
freeze-dried. The fatty acids in whole cells were saponified, methylated and extracted using the
216
standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0B). The fatty
217
acids were analyzed with GC (Agilent 6890) and identified using the TSBA6.0 database of the
218
Microbial Identification System. Twelve fatty acids with abundance of >5 % were detected for
219
strain DX-3T: iso-C15:0 (31.2 %), iso-C17:0 (17.6 %), anteiso-C17:0 (15.4 %), anteiso-C15:0 (8.4 %),
220
iso-C16:0 (7.5 %) and C16:0 (6.6 %), while the fatty acid profile of strain GIESS002 comprised
221
mainly iso-C15:0 (28.8 %), anteiso-C15:0 (17.0 %), anteiso-C17:0 (14.5 %), iso-C17:0 (13.8 %) and
222
iso-C16:0 (10.6 %). Fatty acid profiles of the two strains and reference strains are summarized in
223
Table 2. In comparison to the references and most members of the family Bacillaceae, the fatty
224
acid profiles of these two isolates had certain similar features, i.e. varied and abundant saturated
225
branched-chain fatty acids and occurrence of longer chain fatty acids with 15-17 carbons (Nunes
8
226
et al., 2006; Mayr et al., 2006; Carrasco et al., 2008). However, the iso-C15:0/anteiso-C15:0 ratio of
227
greater than 1 could distinguish the isolates from most members of the genera Ornithinibacillus,
228
Oceanobacillus and Virgibacillus, and all members of other related genera, i.e. Terribacillus,
229
Halobacillus, Sediminibacillus, Paucisalibacillus and Lentibacillus (Supplementary Table S1).
230 231
Among existing genera, the mentioned genera in supplementary Table S1 are the candidates to
232
accommodate strains DX-3T and GIESS002 without loss of monophyleticity. However,
233
phylogenetic analysis of 16S rRNA gene sequences revealed that these two isolates represent a
234
novel branch within the Gram-staining-positive, endospore-forming bacteria in the family
235
Bacillaceae. The novel isolates also can be distinguished from their related genera by some
236
taxonomic characteristics. For instances, temperature for growth (range and optimum) of the
237
isolates can separate them from all the mentioned genera above. Being capable of growing under
238
anaerobic conditions can distinguish them from the genera Ornithinibacillus, Terribacillus,
239
Halobacillus and Paucisalibacillus. The cell-wall peptidoglycan type of strains DX-3T and
240
GIESS002 is distinct from the genera Ornithinibacillus, Halobacillus and Paucisalibacillus. And
241
the Voges-Proskauer test can separate them from the genus Terribacillus. In addition, these two
242
novel isolates can be differentiated from members of their closest genera Ornithinibacillus,
243
Oceanobacillus and Virgibacillus by characters of the DNA G+C contents. The higher
244
iso-C15:0/anteiso-C15:0 ratios distinguish them from members of the genera Terribacillus,
245
Halobacillus, Sediminibacillus, Paucisalibacillus and Lentibacillus. In conclusion, the results of
246
polyphasic taxonomic analysis suggest that strains DX-3T and GIESS002 represent a novel species
247
of a new genus in the family Bacillaceae, for which the name Compostibacillus humi gen. nov., sp.
248
nov. is proposed.
249 250
Description of Compostibacillus gen. nov.
251
Compostibacillus (Com.pos.ti.ba.cil'lus. N.L. neut. n. compostum, compost; L. masc. n. bacillus, a
252
small rod; N.L. masc. n. Compostibacillus, a rod-shaped bacterium from compost).
253 254
Cells are Gram-staining-positive, motile, endospore-forming and rod-shaped. Cylindrical or
9
255
ellipsoidal endospores are produced at the terminal position in swollen sporangia. Moderately
256
thermophilic. Oxidase and catalase positive. Nitrate is not reduced. Phylogenetically related to the
257
genus Ornithinibacillus and other genera of the Bacillaceae. The cell-wall peptidoglycan type is
258
determined as A1γ (meso-DAP direct). The cellular fatty acids consist mainly of iso- and
259
anteiso-branched acids, with iso-C15:0 predominating and anteiso-C15:0, anteiso-C17:0, iso-C17:0 and
260
iso-C16:0 at moderate amounts. The major respiratory quinone is MK-7. The DNA G+C contents of
261
known strains are 43.2-43.7 mol%. The type species is Compostibacillus humi.
262 263
Description of Compostibacillus humi sp. nov.
264
Compostibacillus humi (hu'mi. L. gen. n. humi, of the soil).
265 266
Exhibits the following properties in addition to those given in the genus description. Cells are
267
0.6-0.8 µm × 1.6-3.0 µm in size. Motile by peritrichous flagella. Colonies are circular, entire,
268
smooth, yellowish brown and with a diameter of 2-3 mm on TSA after 48 h of incubation at 50 oC.
269
Anaerobic growth occurs. Grows at 30-60 oC (optimally at 50 oC), pH 7.0-10.0 (optimally at pH
270
8.0-8.5) and 0.5-11 % (w/v) NaCl (optimally 5.5-6 % (w/v) NaCl). Beta haemolysis is observed.
271
Citrate is utilized. The methyl red and Voges-Proskauer reactions, β-galactosidase, arginine
272
dihydrolase, lysine decarboxylase, orinithine decarboxylase, urease and tryptophane deaminase
273
are negative. Indole, H2S and acetoin are not produced. Gelatin and casein are hydrolyzed, but
274
starch, esculin, DNA and Tween 80 are not. D-Ribose, sodium malonate, sodium acetate, lactic
275
acid, L-alanine, glycogen, 3-hydroxybenzoic acid, L-serine, D-mannitol, 3-hydroxybutyric acid,
276
4-hydroxybenzoic acid and L-proline can be utilized as sole carbon sources; D-maltose,
277
D-saccharose, D-glucose, D-sorbitol, L-arabinose, propionic acid and L-histidine cannot be
278
utilized. NH4Cl, (NH4)2SO4 and NH4NO3 can be utilized as sole nitrogen sources, but NaNO3,
279
KNO3, Mg(NO3)2, ammonium acetate and hexamethylene tetramine cannot. Acid is produced
280
from D-ribose, D-fructose, L-sorbose, esculin ferric citrate, D-tagatose and potassium
281
5-ketogluconate, but not from glycerol, D-xylose, D-mannose, methyl-αD-glucopyranoside,
282
N-acetylglucosamine, amygdalin, D-cellobiose, D-lactose, D-maltose, D-saccharose, D-trehalose,
283
starch, xylitol and D-arabitol. The major cellular fatty acids are iso-C15:0, iso-C17:0, anteiso-C17:0
10
284
and anteiso-C15:0. Polar lipid profile is consisted of diphosphatidylglycerol, phosphatidylglycerol
285
and phosphatidylethanolamine. The G+C content of the genomic DNA is 43.2-43.7 mol%.
286 287
The type strain is DX-3T (=KCTC 33104T =CGMCC 1.12360T), isolated from sludge compost
288
samples collected from a compost plant in Guangdong Province, China. Strain GIESS002
289
(=CCTCC AB 2013108 =KCTK 33158) is a reference strain of the species, isolated from the same
290
source. Acid is produced by the type strain from L-sorbose, but not from D-glucose, D-mannitol,
291
arbutin, salicin and gentiobiose. The DNA G+C content of the type strain is 43.7 mol%.
292 293
Acknowledgements
294
This study was supported by the Agricultural Science & Technology Achievements Transfer
295
Project of Guangdong Province, China (2012NL040), the National Natural Science Foundation of
296
China (31100353) and the Foundation for Excellent Young Scientist in Guangdong Academy of
297
Sciences (qnjj201401).
298 299
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300
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Kim, Y.G., Choi, D.H., Hyun, S. & Cho, B.C. (2007). Oceanobacillus profundus sp. nov.,
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Logan, N.A., Berge, O., Bishop, A.H., Busse, H.J., De Vos, P., Fritze, D., Heyndrickx, M.,
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12
341
modesticaldus gen. nov., sp. nov., a strictly anaerobic, nitrate-reducing bacterium from deep-sea
342
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343
Mayr, R., Busse, H.J., Worliczek, H.L., Ehling-Schulz, M. & Scherer, S. (2006).
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Ornithinibacillus gen. nov., with the species Ornithinibacillus bavariensis sp. nov. and
345
Ornithinibacillus californiensis sp. nov. Int J Syst Evol Microbiol 56, 1383-1389.
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Mesbah, M., Premachandran, U. & Whitman, W.B. (1989). Precise measurement of the G+C
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350
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Namwong, S., Tanasupawat, S., Lee, K.C. & Lee, J.S. (2009). Oceanobacillus kapialis sp. nov.,
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355
Bacillus thermoantarcticus sp. nov., from Mount Melbourne, Antarctica: a novel thermophilic
356
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357
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358
globulus gen. nov., sp. nov., a Gram-positive bacterium isolated from potting soil. Int J Syst Evol
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361
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363
Ornithinibacillus scapharcae sp. nov., isolated from a dead ark clam. Antonie van Leeuwenhoek
364
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Smibert, R.M. & Krieg, N.R. (1994). Phenotypic characterization. In Methods for General and
366
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13
368
Sung, M.H., Kim, H., Bae, J.W., Rhee, S.K., Jeon, C.O., Kim, K., Hong, S.P., Lee, S.G., Yoon,
369
J.H., Park, Y.H. & Baek, D.H. (2002). Geobacillus toebii sp. nov., a novel thermophilic
370
bacterium isolated from hay compost. Int J Syst Evol Microbiol 52, 2251-2255.
371
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372
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373
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375
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376
quality analysis tools. Nucleic Acids Res 25, 4876-4882.
377
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378
Ventosa, A. & Ma, Y. (2006). Caldalkalibacillus thermarum gen. nov., sp. nov., a novel
379
alkalithermophilic bacterium from a hot spring in China. Int J Syst Evol Microbiol 56, 1217-1221.
380
Yang, G., Chen, M., Yu., Z., Lu, Q. & Zhou, S. (2013a). Bacillus composti sp. nov. and Bacillus
381
thermophilus sp. nov., two thermophilic Fe(III)-reducing bacteria isolated from compost. Int J Syst
382
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Yang, G., Zhou, X., Zhou, S., Yang, D., Wang, Y. & Wang, D. (2013b). Bacillus
384
thermotolerans sp. nov., a thermophilic bacterium capable of reducing humus. Int J Syst Evol
385
Microbiol 63, 3672-3678.
14
386
Table 1 Characteristics that distinguish strains DX-3T and GIESS002 from their closely
387
related species.
388
Strains: 1, strain DX-3T; 2, strain GIESS002; 3, Ornithinibacillus contaminans DSM 22953T; 4,
389
Ornithinibacillus bavariensis DSM 15681T; 5, Oceanobacillus profundus DSM 18246T; 6,
390
Virgibacillus halophilus JCM 21758T. Data were taken from this study, except for isolation source,
391
cell size, DNA G+C content, and peptidoglycan type of strains DSM 22953T (Kämpfer et al.,
392
2010), DSM 15681T (Mayr et al., 2006), DSM 18262T (Kim et al., 2007) and JCM 21758T (An et
393
al., 2007). +, positive; -, negative. For spore shape: C, cylindrical; E, ellipsoidal; S, spherical. For
394
spore position: C, central; T, terminal.
15
395 Characteris
1
2
3
Compost in
Compost in
Human
thermophili
maturation
blood
c phase
phase
sample
0.6-0.8×1.6
0.6-0.8×1.6
0.8-1.0×2.0
(μm)
-3.0
-3.0
-3.0
Spore shape
C/E
C/E
S
E
E
E
T
T
C
T
T
T
+
+
-
+
+
+
+
+
-
-
-
-
-
-
-
-
+
+
50
50
30
42
35
25-30
6
5.5
0
0.5-4
1-3
5.5-8
8.0
8.0-8.5
7.0-9.0
8.0-9.0
7.5-8.5
7.0-8.5
25 °C
-
-
+
+
+
+
57 °C
+
+
-
-
-
-
60 °C
+
-
-
-
-
-
+
-
-
-
+
+
pH 6.5
-
-
+
-
+
+
pH 10.0
+
+
-
+
-
+
+
+
-
-
-
-
Esculin
-
-
+
+
+
+
Casein
+
+
-
-
+
+
43.7
43.2
36.6
36.4
40.2
42.6
A1γ
A1γ
A4β
A4β
A1γ
A1γ
(m-DAP
(m-DAP
(L-Orn-D-
(L-Orn-D-
(m-DAP
(m-DAP
tics Isolation source Cell size
Spore position Motility Anaerobic growth Nitrate reduction
4
5
Pasteurized
Deep-sea
milk
sediment
0.4×2.0-6.0
0.2-0.4×0.82.0
6
Field soil
0.5×1.75
Optimal temperature (°C) Optimal salinity (%) Optimal pH Growth at:
10-11% NaCl (w/v)
Citrate utilization Hydrolysis of:
DNA G+C content (mol%) Peptidoglyc *
an type
16
direct)
direct)
Asp)
Asp)
direct)†
396
*
m-DAP, meso-diaminopimelic acid; L-Orn, L-ornithine; D-Asp, D-aspartate.
397
†
Determined in this study for DSM 18246T.
398
17
direct)
399
Table 2 Cellular fatty acid profiles of strains DX-3T and GIESS002 and their closely related
400
species.
401
Strains: 1, strain DX-3T; 2, strain GIESS002; 3, Ornithinibacillus contaminans DSM 22953T; 4,
402
Ornithinibacillus bavariensis DSM 15681T; 5, Oceanobacillus profundus DSM 18246T; 6,
403
Virgibacillus halophilus JCM 21758T. Data were taken from this study. Values were percentages of
404
the total fatty acids; -, fatty acids representing < 1.0 % of the total.
405 Fatty acid (%)
1
2
3
4
5
6
iso-C11:0
1.5
-
1.4
-
-
-
C12:0
2.0
1.2
2.6
-
-
-
C14:0
-
1.3
1.3
1.1
-
1.6
iso-C14:0
-
2.6
-
1.4
10.3
5.8
iso-C15:0
31.2
28.8
17.2
26.7
5.2
24.5
anteiso-C15:0
8.4
17.0
25.5
23.4
48.5
32.4
C16:0
6.6
4.9
4.3
3.9
3.8
4.5
iso-C16:0
7.5
10.6
5.0
6.1
13.1
8.1
C16:0 2-OH
2.2
-
2.8
-
-
-
iso-C17:0
17.6
13.8
6.4
10.4
1.1
4.2
anteiso-C17:0
15.4
14.5
24.8
22.3
11.8
13.6
C18:0
1.2
-
2.1
-
-
1.3
C18:1 ω9c
1.7
2.2
1.8
-
1.0
-
Summed feature 3
*
1.5
-
-
-
2.1
1.2
Summed feature 8
*
-
-
1.5
1.0
-
-
406
*
407
and/or C18:1ω6c.
Summed feature 3 comprises C16:1ω7c and/or C16:1ω6c; Summed feature 8 comprises C18:1ω7c
18
408
Figure legend
409 410
Fig. 1 Phylogenetic tree constructed using the maximum-likelihood method based on 16S rRNA
411
gene sequences.
412
19
413
Legends for the supplementary figures and tables
414 415
Fig. S1 Transmission electron micrograph of a cell of strain DX-3T, showing cell morphology and
416
peritrichous flagella.
417 418
Fig. S2 Phylogenetic trees constructed using the minimum-evolution method (a) and the
419
maximum parsimony method (b) based on 16S rRNA gene sequences.
420 421
Fig. S3 Polar lipid profiles of strain DX-3T after two-dimensional TLC and detection with
422
molybdophosphoric acid (a), molybdenum blue (b), α-Naphthol (c) and ninhydrin (d).
423
424
Table S1 Distinguishing features of the novel genus compared to the members of the closely
425
related genera of the family Bacillaceae.
426
427
Table S2 Acid production characteristics that distinguish strains DX-3T and GIESS002 from their
428
closely related species.
429
20
Halobacillus yeomjeoni MSS-402T (AY881246)
71 100
Halobacillus campisalis ASL-17T (EF486356) Halobacillus faecis IGA7-4T (AB243865)
70
Sediminibacillus albus NHBX5T (DQ989634) 99
Sediminibacillus halophilus EN8dT (AM905297) Bacillus subtilis DSM10T (AJ276351) Bacillus aerius 24KT (AJ831843)
100
Virgibacillus halophilus 5B73CT (AB243851)
85
Virgibacillus soli CC-YMP-6T (EU213011) Virgibacillus halodenitrificans DSM 10037T (AY543169) Virgibacillus litoralis JSM 089168T (FJ425909)
58
Virgibacillus carmonensis LMG 20964T (AJ316302)
73 100
Virgibacillus necropolis LMG 19488T (AJ315056)
Ornithinibacillus bavariensis WSBC 24001T (Y13066) Ornithinibacillus halophilus G8BT (HQ433440) Ornithinibacillus scapharcae TW25 T (AEWH01000025) Ornithinibacillus californiensis MB-9T (AF326365) Ornithinibacillus contaminans CCUG 53201T (FN597064) Oceanobacillus profundus CL-MP28T (DQ386635)
93
Oceanobacillus polygoni SA9T (AB750685) Oceanobacillus picturae LMG 19492T (AJ315060) Oceanobacillus oncorhynchi 20AGT (AJ640134) Salinibacillus xinjiangensis J4T (JX402080) Salinibacillus aidingensis 25-7T (AY321436)
99
Salinibacillus kushneri 8-2T (AY321434)
99
Compostibacillus humi DX-3T (JX274434)
100
Compostibacillus humi GIESS002 (KJ024967) Terribacillus halophilus 002-051T (AB243849) 100
Terribacillus aidingensis YI7-61T (FJ386524) Paenibacillus motobuensis MC10T (AY741810)
0.01
430
431
Fig. 1 Phylogenetic tree constructed using the maximum-likelihood method based on 16S rRNA
432
gene sequences. Bootstrap values, generated from 1200 re-samplings, above 50% are indicated at
433
the branching points. Bar 0.01 substitutions per nucleotide position.
434
21
