Antonie van Leeuwenhoek (2011) 99:221–229 DOI 10.1007/s10482-010-9479-4
ORIGINAL PAPER
Bacillus deserti sp. nov., a novel bacterium isolated from the desert of Xinjiang, China Lei Zhang • Gao-Lin Wu • Yang Wang Jun Dai • Cheng-Xiang Fang
•
Received: 3 March 2010 / Accepted: 15 June 2010 / Published online: 26 June 2010 Ó Springer Science+Business Media B.V. 2010
Abstract A Gram-positive, rod-shaped, motile and spore-forming bacterium, designated ZLD-8T, was isolated from a desert soil sample collected from Xinjiang Province in north-west China, and subjected to a polyphasic taxonomic analysis. This isolate grew optimally at 30°C and pH 7.0. It grew with 0–4% NaCl (optimum, 0–1%). Comparative 16S rRNA gene sequence analysis showed that strain ZLD-8T was closely related to members of the genus Bacillus, exhibiting the highest 16S rRNA gene sequence similarity to Bacillus kribbensis DSM 17871T (98.0%). The
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain ZLD-8T is GQ465041.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-010-9479-4) contains supplementary material, which is available to authorized users. L. Zhang College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China L. Zhang Y. Wang J. Dai C.-X. Fang (&) College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China e-mail:
[email protected] G.-L. Wu State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau of Northwest A&F University, Institute of Soil and Water Conservation of CAS&MWR, Yangling, Shaanxi 712100, People’s Republic of China
levels of 16S rRNA gene sequence similarity with respect to other Bacillus species with validly published names were less than 96.3%. The DNA G ? C content of strain ZLD-8T was 40.1 mol%. The strain contained MK-7 as the predominant menaquinone. The diagnostic diamino acid in the cell-wall peptidoglycan was mesodiaminopimelic acid. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids ([5% of total fatty acids) were anteiso-C15:0 (39.56%), iso-C14:0 (25.69%), C16:1 x7c alcohol (10.13%) and iso-C15:0 (5.27%). These chemotaxonomic results supported the affiliation of strain ZLD-8T to the genus Bacillus. However, low DNA–DNA relatedness values and distinguishing phenotypic characteristics allowed genotypic and phenotypic differentiation of strain ZLD8T from recognized Bacillus species. On the basis of the polyphasic evidence presented, strain ZLD-8T is considered to represent a novel species of the genus Bacillus, for which the name Bacillus deserti sp. nov. is proposed. The type strain is ZLD-8T (=CCTCC AB 207173T = KCTC 13246T). Keywords Bacillus deserti sp. nov Xinjiang Province North-west China
Introduction The genus Bacillus, as defined traditionally, comprises Gram-positive, rod-shaped, endospore-forming
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bacteria with an aerobic or facultatively anaerobic metabolism (Rheims et al. 1999; Ko et al. 2006). More than 200 bacterial species are included in the genus Bacillus, although many species have been transferred into other genera, e.g. Paenibacillus, Brevibacillus, Viridibacillus and Virgibacillus (http:// www.bacterio.cict.fr/b/bacillus.html). Members of the genus Bacillus have been isolated from a wide variety of aquatic and terrestrial environments, ranging from sewage sludge (Demharter and Hensel 1989), ocean sediments (Bae et al. 2005) and hot springs (Derekova et al. 2008) to desert soils (Palmisano et al. 2001). In this study, we report on the taxonomic characterization of a Gram-positive and spore-forming bacterial strain, ZLD-8T, which was isolated from a desert soil sample from Xinjiang Province, north-west China. On the basis of phenotypic characteristics, chemotaxonomic data, phylogenetic analysis and DNA–DNA relatednesses, the isolate is considered to represent a novel species of the genus Bacillus.
Materials and methods Strains and culture conditions Strain ZLD-8T was isolated with the serial dilution plating method, using marine agar 2216 (MA; Difco) at 30°C for 1 week. However, the isolate grew better on nutrient agar (NA; Difco). It was routinely cultivated on NA (Difco) at 30°C and maintained as a glycerol suspension (20%, v/v) at -80°C. The type strains Bacillus kribbensis DSM 17871T, Bacillus benzoevorans DSM 5391T and Bacillus nealsonii DSM 15077T were obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany). Bacillus circulans IAM 12462T and Brevibacillus brevis ATCC 8246T was obtained from the China Center for Type Culture Collection (CCTCC), China. Unless otherwise indicated, morphological, physiological and chemotaxonomic studies were performed with cells grown on NA (Difco) at pH 7.0 and 30°C. Phenotypic characterization Cell morphology, size and motility were examined by using phase-contrast microscopy (BX51 microscope; Olympus) after cells were grown in nutrient broth
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(NB; Difco) at 30°C for 24 h at 200 rpm. Sporangia were observed by phase-contrast microscopy after cells were grown on NA supplemented with MnSO4 (5 mg l-1) at 30°C for 48 h. The Gram reaction was carried out according to the classical Gram procedure described by Doetsch (1981). Flagella were observed from silver-nitrate-stained cells of strain ZLD-8T that were incubated on NA slants at 30°C for 12 h (Rhodes 1958). Growth at different temperatures (4, 7, 10, 17, 25, 30, 37, 42, 45 and 50°C) and NaCl concentrations (0–10%, w/v, using increments of 1%) was investigated on NA (Difco) for up to 1 week. The pH range for growth was determined in nutrient broth (Difco) at pH 4.0–11.0, by using the following buffer system: pH 4.0–5.0, 0.1 M citric acid/0.1 M sodium citrate; pH 6.0–8.0, 0.1 M KH2PO4/0.1 M NaOH; pH 9.0–10.0, 0.1 M NaHCO3/0.1 M Na2CO3; pH 11.0, 0.05 M Na2HPO4/0.1 M NaOH. Growth under anaerobic condition was determined after 1 week incubation in an anaerobic chamber (Oxoid AnaeroJar System, England) on NA at 30°C. Oxidase activity was determined using a 1% solution of tetramethyl-p-phenylenediamine (Kova´cs 1956). Catalase activity was detected by assessing the production of bubbles after the addition of a drop of 3% H2O2. Some conventional biochemical tests were performed as described by Smibert and Krieg (1994), including tests for Voges-Proskauer reaction, starch hydrolysis, casein hydrolysis and egg yolk reaction. Tests were also made for hydrolysis of CM-cellulose (0.1%, w/v), chitin from crab shells (1%, w/v) and tyrosine (0.5%, w/v). Activities of constitutive enzymes and other physiological properties were determined by using the API 20E, API 20NE, API ID 32GN, API 50 CH (with API 50 CH B/E medium) and API ZYM strips (bioMe´rieux) according to the manufacturers’ instructions. Susceptibility to antibiotics was examined as described by Buczolits et al. (2002). 16S rRNA gene sequencing and phylogenetic analysis, Determination of G ? C content of DNA, and DNA–DNA hybridization For 16S rRNA gene sequencing and phylogenetic analysis, DNA was extracted using a commercial genomic DNA extraction kit (ChaoShi-Bio; China). The primer pair 27f (50 -GAGTTTGATCCTGGCT CAG-30 ) and 1527r (50 -AGAAAGGAGGTGATCCA
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GCC-30 ) was used for amplification of the 16S rRNA gene (Lane 1991). PCR and 16S rRNA gene sequencing were carried out as described by Lin et al. (2004). The identification of phylogenetic neighbours and the calculation of pairwise 16S rRNA gene sequence similarities were achieved using the EzTaxon server (http://www.eztaxon.org/; Chun et al. 2007). Phylogenetic analysis was performed by using MEGA, version 4.1 (Tamura et al. 2007), after multiple alignment of the data via CLUSTAL_X (Thompson et al. 1997). A distance matrix method (distance options according to the Kimura two-parameter model), including clustering by neighbour-joining, and a discrete character-based maximum-parsimony method were used. The relationships among taxa were also established by maximum-likelihood algorithm with PHYLIP 3.66 package (Felsenstein 2006). In each case, bootstrap values were calculated based on 1000 replications. DNA for DNA–DNA hybridizations and DNA base composition analysis was prepared according to the procedure of Ausubel et al. (1995). The DNA G ? C content was determined by HPLC according to the method of Mesbah et al. (1989). DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989), with slight modifications. Briefly, DNA was non-covalently adsorbed to polystyrene microplates (black MaxiSorp, FluoroNunc; Nunc) by incubating 100 ll portions of a denatured DNA solution [10 ng DNA per ll phosphate-buffered saline (PBS)/MgCl2 (8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2, 137 mM NaCl, 2.7 mM KCl, 0.1 M MgCl2)] per well at 30°C for 4 h in a hybridization oven. Before incubation, plates were sealed with self-adhesive vinyl tape (Nunc). The plates were then washed once with 300 ll PBS per well with the aid of a multichannel pipette, dried at 45°C for 15 min and stored in a desiccator at 4°C. Probe DNA was labelled by mixing 10 ll DNA solution [0.5 lg ll-1 in 0.1 9 SSC (1 9 SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0 ± 0.2)] plus 10 ll photobiotin solution (Sigma) (0.5 lg ll-1 in water) in a 1.5 ml Eppendorf tube and illuminating the mixture for 30 min under a 400 W mercury-vapour lamp while the open tube was kept upright in a cooling block on ice. The labelled probe DNA was diluted by adding 185 ll 0.1 M Tris/HCl (pH 9.0) and the remaining free photobiotin was removed by extracting twice with 200 ll 1-butanol saturated with 0.1 M Tris/HCl (pH
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9.0). The probe DNA was then sonicated for 30 s at 10 W, denatured at 100°C for 10 min and immediately cooled on ice. A pre-hybridization step was performed by adding 200 ll pre-hybridization solution (2 9 SSC, 5 9 Denhardt’s solution, 50% formamide, 100 lg denatured salmon sperm DNA ml-1) per well, sealing the microplate with vinyl tape and incubating for 30 min at 40°C in the hybridization oven. For the actual hybridization, the pre-hybridization solution was removed and 100 ll hybridization solution (prehybridization solution plus 2.5% dextran sulfate and 1 lg probe DNA ml-1) was added per well. The microplate was sealed again with vinyl tape and incubated for 3 h at 40°C. The microplate was then washed three times with 300 ll 1 9 SSC per well, using the microplate washer. For the enzymic development, 100 ll streptavidin-b-D-galactosidase (Gibco BRL) solution was added per well (0.5 U ml-1 in PBS plus 0.5% BSA) and the microplate was covered with a preheated empty microplate and incubated for 10 min at 37°C. Subsequently, the plate was washed three times with 300 ll 1 9 SSC per well using the microplate washer. Finally, the substrate for b-D-galactosidase, 4-methylumbelliferyl b-D-galactopyranoside (Sigma), was added (100 ll per well, 0.1 mg ml-1 in PBS plus 1 mM MgCl2) and the plate was incubated at 37°C. The reaction product, 4-methylumbelliferone (excitation max., 360 nm; emission max., 465 nm) was quantified using a SpectraMax Gemini XPS microplate reader (Molecular Devices) at 0, 15, 30 and 45 min. Reassociation values were calculated using the fluorescence values at 30 min and the homologous reaction was regarded as representing 100% reassociation. Salmon DNA was immobilized as a background absorbance control. The DNA–DNA hybridization values are the means of two independent experiments. In each of these experiments, all hybridization reactions were done in quadruplicate and calculations were based on the mean fluorescence values (clearly aberrant fluorescence values were omitted). All reciprocal hybridizations (different hybridizations using the same DNAs, A and B, but once with A as the immobilized DNA and once with B as the immobilized DNA) were carried out. Chemotaxonomy The respiratory quinone system was extracted and determined by HPLC as described by Xie and Yokota
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(2003). The isomer type of diaminopimelic acid in the peptidoglycan was determined by the method described by Staneck and Roberts (1974). Polar lipids were extracted and analyzed by two-dimensional TLC according to Tindall (1990). For fatty acid methyl ester analysis, strain ZLD-8T and Bacillus kribbensis DSM 17871T were grown on NA (Difco) at 30°C for 2 days. Fatty acids were extracted, methylated and analysed according to the standard protocol of the Sherlock Microbial Identification System (MIDI 1999).
B. asahii MA001T (95.7%), B. drentensis LMG 21831T (95.6%) and B. nealsonii DSM 15077T (95.5%), respectively. No other recognized bacterial species showed more than 95.5% 16S rRNA gene sequence similarity to the new isolate. In a phylogenetic tree based on the neighbour-joining algorithm, the novel isolate formed a coherent cluster with B. kribbensis BT080T at a bootstrap confidence value of 100% (Fig. 1). Similar topologies were obtained when other treeing methods (maximum parsimony and maximum likelihood) were used (see Supplementary Figs S2 and S3). The DNA G ? C content of strain ZLD-8T was 40.1 mol%.
Results and discussion Chemotaxonomic characteristics Phenotypic characteristics T
Strain ZLD-8 showed the phenotypic characteristics typical of most species of the genus Bacillus. Colonies were cream-white, circular, smooth, slightly raised and non-translucent, 1–2 mm in diameter after incubation at 30°C for 2 days on NA (Difco). Cells are Gram-positive, rod-shaped, strictly aerobic, 1.3– 2.5 lm long and 0.6–0.8 lm wide. Cells are motile by means of peritrichous flagella. Spores are oval, lie subterminally or terminally and may swell the sporangia slightly (see Supplementary Figs S1). The temperature range for growth was 7–45°C, with optimum growth at 30°C. The pH range for growth was pH 6.0–9.0, with optimum growth at pH 7.0. The physiological characteristics of strain ZLD-8T are summarized in the species description and a comparison of selective characteristics with closely related type strains is given in Table 1. Enzyme activities are given in the species description and a comparison of these with Bacillus kribbensis DSM 17871T is given in Table 2. Phylogenetic analysis based on 16S rRNA gene sequence comparison, DNA G ? C content For strain ZLD-8T, 1481 bp of the 16S rRNA gene sequence was determined. Comparative 16S rRNA gene sequence analysis showed that strain ZLD-8T was most closely related to members of the genus Bacillus. Strain ZLD-8T exhibited the highest 16S rRNA gene sequence similarity with respect to Bacillus kribbensis DSM 17871T (98.0%), followed by Bacillus horneckiae 1P01SCT (96.2%), B. circulans IAM 12462T (96.1%), B. muralis LMG 20238T (95.9%), B. benzoevorans DSM 5391T and B. foraminis CV53T (95.8%),
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The chemotaxonomic data for strain ZLD-8T supported its affiliation to the genus Bacillus. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The predominant isoprenoid quinine was MK-7. The polar lipid profile (Fig. 2) consisted mainly of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). An unidentified glycolipid (GL) and an unidentified polar lipids (L3) occurred at moderate levels. Trace amounts of two unidentified polar lipids (L1, 2) and three unidentified aminolipid (AL1, AL2, AL3) were also detected. The fatty acid profile of strain ZLD-8T was similar to that of Bacillus kribbensis DSM 17871T, although there were some quantitative differences in fatty acid contents (Table 3). The major fatty acids of strain ZLD-8T were anteiso-C15:0 (39.56%), iso-C14:0 (25.69%), C16:1x7c alcohol (10.13%) and isoC15:0 (5.27%). Taxonomic conclusion Although strain ZLD-8T exhibited comparatively high 16S rRNA gene sequence similarity with Bacillus kribbensis DSM 17871T (98.0%) and some other related Bacillus species (about 96%), the new isolate could be distinguished from its phylogenetically related Bacillus species on the basis of some phenotypic characteristics and chemotaxonomic distinctness (Tables 1, 2, 3). This organism differed obviously from its closest phylogenetic relative, Bacillus kribbensis DSM 17871T, by some taxonomic markers, such as its NaCl tolerance, acid
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Table 1 Differential phenotypic characteristics of strain ZLD-8T and phylogenetically related Bacillus species Characteristic
1
2
3
4
5
6
7
8
9
10
Spore shape
O
O
O
O
E
O
-
E
E/C
O
Swollen sporangia
W
?
ND
?
W
-
-
-
?
-
Anaerobic growth Growth conditions
-
-
-
?
W
-
-
-
?
?
NaCl 5%
-
?
?
?
?
-
-
-
ND
?
50°C
-
-
-
-
-
-
-
-
?
?
Nitrate reduction
-
-
?
V
?
?
?
W
V
-
Hydrolysis of Casein
?
?
ND
-
-
?
-
?
-
?
Gelatin
?
?
?
?
-
-
?
-
-
-
Starch
-
-
ND
?
?
-
?
W
ND
?
Glycerol
-
W
-
W
?
-
?
-
-
-
D-xylose
-
W
-
?
-
-
?
-
-
W
D-Galactose
-
W
-
?
W
-
?
-
-
-
D-Glucose
?
?
-
?
?
-
?
-
W
?
D-Fructose
-
?
-
?
?
-
?
-
?
?
Acid production from
D-Mannose
-
W
-
?
?
-
?
-
?
W
Inositol D-Mannitol
?
? ?
-
? ?
?
-
? ?
-
-
W ?
Methyl a-D-mannopyranoside
W
-
-
?
-
-
-
ND
-
-
Amygdalin
-
?
-
?
-
-
?
ND
-
W
Salicin
-
W
-
?
?
-
?
ND
?
?
D-Cellobiose
?
?
-
?
?
-
?
-
-
?
D-Maltose
?
?
-
?
?
-
?
-
?
?
D-Lactose
-
?
-
?
?/W
-
?
-
?
?
D-Melibiose
-
?
-
?
W
-
?
-
?
?
Sucrose
-
?
-
?
-
-
?
-
?
?
D-Trehalose
-
?
-
?
?
-
?
-
-
?
Glycogen
-
?
-
?
-
-
?
ND
-
-
40.1
43.3
35.6 ± 0.5
35.7
41.2
41.3 ± 1.1
43.1
39.4
39.4
ND
DNA G ? C content (mol%) T
T
T
Taxa: 1, strain ZLD-8 (present study); 2, B. kribbensis DSM 17871 (Lim et al. 2007); 3, B. horneckiae 1P01SC (Vaishampayan et al. 2009); 4, B. circulans IAM 12462T (Claus and Berkeley 1986; Pettersson et al. 2000); 5, B. muralis LMG 20238T (Heyrman et al. 2005); 6, B. benzoevorans DSM 5391T (Pichinoty et al. 1984; Pettersson et al. 2000; Zhang et al. 2009); 7, B. foraminis CV53T (Tiago et al. 2006); 8, B. asahii MA001T (Yumoto et al. 2004); 9, B. drentensis LMG 21831T (Heyrman et al. 2004); 10, B. nealsonii DSM 15077T (Venkateswaran et al. 2003; Zhang et al. 2009). For acid production, data for columns 1-7, 9 and 10 were determined using API 50 CH test strips (bioMe´rieux) and data for columns 1, 2, 4, 6 and 10 were from this study. Symbols: O oval, E ellipsoidal, C circular; ? positive, - negative, W weakly positive, V variable, ND no data available
production from carbohydrates (Table 1), constitutive enzyme pattern (Table 2) and its discriminative fatty acid profile (Table 3). To establish the precise taxonomic position of strain ZLD-8T, DNA–DNA hybridizations were performed between the new isolate and Bacillus kribbensis DSM 17871T and between the new isolate and Brevibacillus brevis
ATCC 8246T. Strain ZLD-8T shared low levels of DNA relatedness with Brevibacillus brevis ATCC 8246T (8 ± 3%; reciprocal, 11 ± 2%). The reciprocal values for DNA–DNA hybridization between strain ZLD-8T and Bacillus kribbensis DSM 17871T were 28 ± 4 and 35 ± 6%, respectively, which were far below the threshold value of 70% for the
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Table 2 Comparison of enzyme activities of strains ZLD-8T and Bacillus kribbensis DSM 17871T Enzyme activity
Bacillus deserti ZLD-8T
Bacillus kribbensis DSM 17871T
Alkaline phosphatase
?
-
Leucine arylamidase
?
-
Valine arylamidase Cystine arylamidase
?
? -
a-Chymotrypsin
?
Acid phosphatase
?
-
b-Glucuronidase
-
?
a-Glucosidase
-
?
All data are from this study, with API ZYM strips (bioMe´rieux). Both strains are positive for esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase and b-glucosidase, and negative for lipase (C14), trypsin, a-galactosidase, b-galactosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. ?, Positive; -, negative
60 51 99
Bacillus drentensis LMG 21831T (AJ542506) Bacillus soli LMG 21838T (AJ542513) Bacillus bataviensis LMG 21833T (AJ542508)
86
Bacillus novalis LMG 21837T (AJ542512) Bacillus pocheonensis Gsoil 420T (AB245377) Bacillus foraminis CV53T (AJ717382) Bacillus jeotgali YKJ-10 T (AF221061)
99
Bacillus horneckiae 1P01SCT (EU861362) Bacillus benzoevorans DSM 5391T (D78311) Bacillus circulans ATCC 4513T (AY724690)
85 100
Bacillus nealsonii DSM 15077T (EU656111) Strain ZLD-8T (GQ465041) Bacillus kribbensis DSM 17871T (DQ280367)
100
84
Bacillus asahii MA001T (AB109209) Bacillus muralis LMG 20238T (AJ628748) 100
Bacillus simplex NBRC 15720T (AB363738)
Bacillus herbersteinensis DSM16534T (AJ781029)
84
Bacillus megaterium IAM 13418T (D16273)
53 99 62
Bacillus acidicola 105-2T (AF547209) Bacillus shackletonii LMG 18435T (AJ250318) Bacillus subtilis DSM 10T (AJ276351) Bacillus badius ATCC 14574T (X77790) Brevibacillus brevis JCM 2503T (D78457)
0.005
Fig. 1 Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships of strain ZLD-8T and related taxa. Bootstrap percentages (based on
1000 replications) greater than 50% are shown. Brevibacillus brevis JCM 2503T was used as an outgroup. Bar, 0.005 substitutions per nucleotide position
delineation of bacterial species (Wayne et al. 1987). On this basis, together with the distinct position occupied by strain ZLD-8T in the phylogenetic tree (Fig. 1), the results of the polyphasic taxonomic
study strongly suggested that strain ZLD-8T should be placed in the genus Bacillus as a member of a novel species, for which we propose the name Bacillus deserti sp. nov.
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227
Description of Bacillus deserti sp. nov
Fig. 2 Polar lipid profile of strain ZLD-8T after separation in two-dimensional thin layer chromatography. Components were visualized by staining with 5% molybdophosphoric acid in ethanol followed by heating of the plates at 130°C. Abbreviations: DPG diphosphatidylglycerol; PE phosphatidylethanolamine, PG phosphatidylglycerol, GL unidentified glycolipid, AL1-3 unidentified aminolipid, L1-3 unidentified polar lipids Table 3 Cellular fatty acid compositions of strains ZLD-8T and Bacillus kribbensis DSM 17871T Fatty acid
Bacillus deserti ZLD-8T
Bacillus kribbensis DSM 17871T
Straight-chain saturated C14:0
1.83
1.08
C16:0
1.71
2.37
C18:0
0.20
1.06
iso-C14:0
25.69
28.58
iso-C15:0
5.27
8.11
iso-C16:0
2.62
12.20
anteiso-C15:0
39.56
36.17
anteiso-C17:0
2.37
3.14
Branched saturated
Mono-unsaturated C16:1 x11c C16:1 x7c alcohol
3.11
0.60
10.13
3.13
2.17
0.31
Summed featuresa 4
All data from this study. Data are percentages of total fatty acids a
Summed features represent groups of two or three fatty acids that could not be separated by gas liquid chromatography with the MIDI system. Summed feature 4 contains anteiso-C17:1 B and/or iso-C17:1 I
Bacillus deserti (des.er0 ti. L. gen. n. deserti of a desert). Cells are Gram-positive, rod-shaped, strictly aerobic, spore-forming, 1.3–2.5 lm long and 0.6–0.8 lm wide. Cells are motile by means of peritrichous flagella. Endospores are oval in shape, lie subterminally or terminally and may swell the sporangia slightly. Growth occurs at 7–45°C; optimal temperature is 30°C. Growth occurs at pH 6.0–9.0, with optimal growth at pH 7.0. Growth occurs at 0–4% (w/v) NaCl (optimum, 0–1%). Oxidase- and catalase-positive. Does not reduce nitrate to nitrite. Hydrolyses aesculin, casein and gelatin. Does not hydrolyse starch, cellulose, chitin or tyrosine. H2S production, citrate utilization, Voges-Proskauer reaction, egg yolk reaction, indole production and urease are negative. Acid is produced from D-glucose, D-mannitol, methyl a-Dmannopyranoside (weakly), aesculin, D-cellobiose and D-maltose, but not from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, D-galactose, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl a-D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-lactose, D-melibiose, sucrose, D-trehalose, inulin, D-melezitose, D-raffinose, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate or potassium 5-ketogluconate. According to the API 20NE and ID 32GN galleries (bioMe´rieux), the following substrates are not utilized as sole carbon sources: D-glucose, L-arabinose, D-mannose, N-acetyl-D-glucosamine, potassium gluconate, maltose, D-melibiose, sucrose, inositol, D-mannitol, L-fucose, L-rhamnose, D-ribose, caprate, adipate, maleic acid, citrate, phenyl acetate, acetate, lactate, 3-hydroxybutyrate, valerate, salicin, propionate, 3-hydroxybenzoate, 4-hydroxybenzoate, malonate, itaconate, suberate, potassium 2-ketogluconate, potassium 5-ketogluconate, D-sorbitol, glycogen, L-alanine, L-histidine, L-proline and L-serine. According to the API ZYM gallery (bioMe´rieux), produces alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and b-glucosidase, but not lipase (C14), valine arylamidase, trypsin, a-galactosidase, b-galactosidase,
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b-glucuronidase, a-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase or a-fucosidase. Susceptible to bacitracin, chloramphenicol, kanamycin, erythromycin, gentamicin, penicillin G, streptomycin, tetracycline and vancomycin. The cell-wall peptidoglycan contains meso-diaminopimelic acid. The predominant menaquinone is MK-7. The polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid, three unidentified aminolipid and three unidentified polar lipids. The major fatty acids are anteiso-C15:0, iso-C14:0, C16:1x7c alcohol and iso-C15:0. The DNA G ? C content is 40.1 mol%. The type strain, ZLD-8T (=CCTCC AB 207173T= KCTC 13246T), was isolated from the surface layer of a desert soil from Xinjiang Province, north-west China. Acknowledgements This work was supported by the R & D Infrastructure and Facility Development Program from the Ministry of Science and Technology of the People’s Republic of China (Grant No.2005DKA21208) and NWSUAF (Z111020910) China.
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