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1 Department of Soil Environmental Science, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan
2 Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan
3 Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung, Taiwan
Correspondence
Wen-Ming Chen
p62365{at}ms28.hinet.net
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ABSTRACT |
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Neighbour-joining and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showing the position of strain ZLT within the genus Paenibacillus are available as supplementary figures with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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to accommodate 11 species previously assigned to the genus Bacillus. Since then, many novel Paenibacillus species have been described. At the time of writing, more than 60 species are recognized as members of the genus (http://www.bacterio.cict.fr/p/paenibacillus.html). Species belonging to the genus Paenibacillus have been isolated from various ecological niches such as soil, rhizospheres, phyllospheres, water, insects, food, faeces, blood cultures and Antarctic sediments (Saha et al., 2005). A few Paenibacillus species have been isolated from human biological samples and the possibility that these bacteria may cause human infection is of particular concern (Roux & Raoult, 2004). Generally, members of this genus produce endospores, possess anteiso-C15 : 0 as the major cellular fatty acid and have genomic DNA G+C contents in the range 39–54 mol% (Shida et al., 1997a; Saha et al., 2005). The aim of the present study was to determine the taxonomic position of a novel Paenibacillus-like organism, designated strain ZLT, by using a polyphasic taxonomic approach.
In January 2006, strain ZLT was isolated from a water sample collected from Jhonglun warm spring in Chiayi County, Taiwan. The temperature of the spring water ranged between 35 and 45 °C and the pH was between 7 and 8. The water sample was diluted with sterile distilled water and vortexed thoroughly. Appropriate dilutions were then plated on trypticase soy broth agar and incubated at 42 °C for 2 days. The majority of colonies (>99 %) showed identical morphology.
The optimum pH range for growth was examined in trypticase soy broth by using appropriate biological buffers (pH 3–11) (Chung et al., 1995). The optimum temperature for growth (15–60 °C) of the novel strain was examined in trypticase soy broth adjusted to pH 7. Growth was examined by measuring the turbidity (OD600) of cultures grown at various pH values and temperatures. Anaerobic cultivation was performed on nutrient agar and trypticase soy broth agar by using an Oxoid AnaeroGen system.
Strain ZLT showed good growth on complex media, such as trypticase soy broth, nutrient agar and LB medium. Strain ZLT formed visible, cream/white-coloured, semi-transparent colonies of a circular shape with entire edges. Colonies were approximately 0.3–0.5 mm in diameter after 48 h incubation at 42 °C. Strain ZLT showed good growth at temperatures ranging from 20 to 45 °C and pH 7 and 10. Optimal growth was seen at 35–42 °C and pH 8.0–9.0.
Cells were observed in the lag exponential and stationary phases of growth under a phase-contrast microscope to ascertain shape and motility (using the hanging-drop technique). Flagellum staining was performed by using Spot Test Flagella Stain (BD Difco). Gram Stain Set S (BD) and the Ryu non-staining KOH method (Powers, 1995) were used to ascertain the Gram reaction. Poly-
-hydroxybutyrate granule accumulation was observed under light microscopy after staining cells with Sudan black.
Cells of strain ZLT stained Gram-variable and were straight rods (0.8–1.0 µm wide and 2.0–12.4 µm long). Spore-forming cells were rare (endospores). Terminal or subterminal ellipsoidal spores were observed in non-swollen or slightly swollen sporangia. Cells were motile by means of peritrichous flagella. No accumulation of poly--hydroxybutyrate granules was observed.
Extraction of genomic DNA, PCR amplification and sequencing of 16S rRNA genes were carried out as described by Chen et al. (2001). Sequence analysis was performed with a DNA sequencer (ABI PRISM 310; Applied Biosystems) and the Fragment Assembly System program from the Wisconsin Package 9.1 (Genetics Computer Group, 1995) supplied by the National Health Research Institute of Taiwan. The resultant sequence was compared with the corresponding results taken from the EMBL, GenBank and RDP II databases for representatives of the genera classified in the order Bacillales. Multiple-sequence alignment of strain ZLT with its closest relatives was performed by using BioEdit software (Hall, 1999) and MEGA version 3.1 (Kumar et al., 2004). Phylogenetic trees were inferred using the maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) algorithms. Unrooted tree topologies were evaluated by bootstrap analyses of the neighbour-joining data set (Felsenstein, 1993). An almost complete 16S rRNA gene sequence (1464 nt) was obtained for strain ZLT. A comparison of this sequence with those of representatives of the genera classified in the order Bacillales showed that the novel organism fell within the evolutionary radiation occupied by the genus Paenibacillus. Maximum 16S rRNA gene sequence similarity between strain ZLT and the type strains of recognized members of the genus Paenibacillus was 94.3 %.
It is evident from the phylogenetic tree based on the neighbour-joining algorithm that strain ZLT clearly forms a monophyletic branch in the genus Paenibacillus (see Fig. 1 and Supplementary Fig. S1 available in IJSEM Online). The organism was most closely related to Paenibacillus assamensis GPTSA 11T, sharing a 16S rRNA gene sequence similarity of 94.3 %. Strain ZLT also shared comparatively low 16S rRNA gene sequence similarity with Paenibacillus timonensis 2301032T (94.0 %), Paenibacillus macerans ATCC 8244T (93.3 %), Paenibacillus barengoltzii SAFN-016T (93.3 %) and Paenibacillus sanguinis 2301083T (93.2 %). Similar tree topologies were obtained in phylogenetic trees generated with the maximum-parsimony (see Supplementary Fig. S2 in IJSEM Online) and maximum-likelihood algorithms (data not shown).
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for genomic distinction of species.
The DNA G+C content was determined in duplicate as described by Mesbah et al. (1989). The DNA G+C content of strain ZLT was 49.2 (±1.2) mol% (mean±SD).
Chemotaxonomic studies were performed to establish whether strain ZLT had a chemical profile consistent with its assignment within the genus Paenibacillus. Biomass for chemical studies was grown in shake flasks of trypticase soy broth for 5 days at 37 °C, checked for purity, harvested by centrifugation, washed twice in distilled water and freeze-dried. Polar lipids were extracted and analysed by two-dimensional TLC as described by Ventosa et al. (1993). The major cellular phospholipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and one unknown phosphoglycolipid. Respiratory quinones of strain ZLT were extracted, separated and identified as described by Saha et al. (2005). The predominant isoprenologue was unsaturated menaquinone with seven isoprene units (MK-7). The diagnostic cell-wall amino acid of strain ZLT was determined by TLC (Staneck & Roberts, 1974). The isolate contained meso-diaminopimelic acid (wall chemotype III). Cellular fatty acids were analysed as methyl esters by GC according to the instructions of the Microbial Identification System (MIDI; Microbial ID). The predominant fatty acids of strain ZLT were C16 : 0, anteiso-C15 : 0 and iso-C16 : 0. All of these properties are typical of representatives of the genus Paenibacillus (Shida et al., 1997a). The fatty acid profile of strain ZLT was generally comparable with those of the type strains of P. assamensis, P. timonensis, P. macerans and P. sanguinis (Shida et al., 1997b; Roux & Raoult, 2004; Saha et al., 2005), but differed in the proportions of certain fatty acids (Table 1). Strain ZLT contained higher amounts of C16 : 0 (33.5 %) and C14 : 0 (7.5 %), a feature that enabled the novel strain to be clearly distinguished from P. assamensis, P. timonensis, P. macerans and P. sanguinis.
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Detailed results of the biochemical characterization and antibiotic sensitivity tests are given in Table 2 and in the species description below. It is apparent from the results (Table 2) that strain ZLT can be distinguished from representatives of its close phylogenetic relatives by using a combination of phenotypic attributes, especially ONPG hydrolysis, nitrate reduction, the Voges–Proskauer reaction, optimum temperature for growth, acid production from various sugars (such as glucose, mannitol, arabinose, inositol, rhamnose and sucrose) and enzyme activities (catalase, oxidase and gelatinase).
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Description of Paenibacillus fonticola sp. nov.
Paenibacillus fonticola (fon.ti.co'la. L. masc. n. fons fontis a spring, fountain; L. suf. -cola from L. masc. or fem. n. incola an inhabitant of a place, a resident; N.L. n. fonticola an inhabitant of a fountain).
Cells are facultatively anaerobic, Gram-variable, sporulating, motile, rod-shaped, 0.8–1.0 µm in diameter and 2.0–12.4 µm in length. Grows at pH 7–10 and at 20–45 °C. Positive (API 20NE and 20E) for catalase, urease, aesculin hydrolysis, gelatin hydrolysis, -galactosidase, OPNG test, gelatinase and acid production from arabinose. Negative for oxidase, nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, D-maltose, gluconate, caprate, adipate, malate, citrate, phenylacetate, lysine decarboxylase and ornithine decarboxylase, citrate utilization, H2S production, tryptophan deaminase, acetoin production and acid production from glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose and amygdalin. In API ZYM enzyme reactions, positive for C4 esterase, naphthol-AS-BI-phosphohydrolase, 
-galactosidase and -galactosidase, but negative for alkaline phosphatase, C8 lipase, C14 lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, acid phosphatase, -<1?show=[to]>glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. The following carbon sources are oxidized (positive in the Biolog GN2 system): dextrin, Tween 80, D-cellobiose, D-fructose, -D-glucose, D-mannose, D-melibiose, sucrose and D-trehalose. The following substrates are not utilized as carbon sources (negative in the Biolog GN2 system): -cyclodextrin, glycogen, Tween 40, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, D-arabitol, i-erythritol, L-fucose, D-galactose, gentiobiose, myo-inositol, -D-lactose, lactulose, maltose, D-mannitol, methyl -D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, turanose, xylitol, methyl pyruvate, monomethyl succinate, acetic acid, cis-aconitic acid, citrate, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconate, D-glucosaminic acid, D-glucuronic acid, -hydroxybutyric acid, -hydroxybutyric acid, 
-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, -ketovaleric acid, -ketobutyric acid, -ketoglutaric acid, DL-lactate, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, L-alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, D-serine, L-serine, L-threonine, DL-carnitine, -aminobutyric acid, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol, DL--glycerol phosphate, glucose 1-phosphate and glucose 6-phosphate. Sensitive to ampicillin (10 µg), cefotaxime (30 µg), chloramphenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), penicillin G (10 U), rifampicin (5 µg), streptomycin (10 µg) and tetracycline (30 µg). The major fatty acids are C16 : 0, anteiso-C15 : 0 and iso-C16 : 0. The G+C content of the DNA is 49.2 mol%.
The type strain, ZLT (=BCRC 17579T=LMG 23577T), was isolated from a water sample collected from Jhonglun warm spring, Chiayi County, Taiwan.
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