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Supplementary Data
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Identification of strain BK-58
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Methods
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The identification of strain BK-58 was based on its morphological characteristics,
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physiological and biochemical properties, and nucleotide sequence analysis of 16S
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rDNA. The morphological characteristics of BK-58 were observed by an optical
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microscope. To investigate the physiological and biochemical properties of strain BK-58,
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dozens of analyses were performed, such as gram stain, Vogas-Proskauer, liquefaction
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of gelatin, and nitrate reduction. For the sequence analysis, 16S rDNA was amplified by
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PCR using the oligonucleotide primers P16S-8: 5′-AGAGTTTGATCCTGGCTCA-3′
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and P16S-1541: 5′-AAGGAGGTGATCCAGCCGCA -3′ with genomic DNA as the
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template. The PCR products were purified by a DNA Gel Extraction Kit (Axygen) and
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were then ligated with pUCm-T (Sangon Biotech, Shanghai, China) and transformed
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into Escherichia coli DH5α competent cells. A positive clone was selected, sequenced,
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and compared with sequences in the GenBank database using the BLAST program. The
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identification of phylogenetic neighbors was initially carried out by the BLASTN
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program against the database containing type strains with validly published prokaryotic
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names and representatives of uncultured phylotypes. The top 30 sequences with the
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highest scores were then selected for the calculation of pairwise sequence similarity
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using the global alignment algorithm, which was implemented at the EzTaxon-e server
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(http://eztaxon-e.ezbiocloud.net/). To construct the phylogenetic tree, the sequences
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were retrieved from the EzTaxon-e database individually, and the phylogenetic tree was
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constructed by MEGA 5.2 based on the neighbor-joining method with bootstrapping
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1000 times.
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Results
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Single colonies of strain BK-58 appeared within 48 h of incubation on nutrient agar.
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The colonies were smooth, moist, opaque, and circular. Furthermore, the cells were
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gram-negative straight rods and no motility or flagellum was observed. The
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morphological and physiological properties of strain BK-58 (Supplementary table 1)
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indicated that strain BK-58 belonged to the genus Klebsiella and was most similar to
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Klebsiella oxytoca.
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A 16S rDNA fragment containing 1454 bp of strain BK-58 was deposited in the
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GenBank database under accession number KF469213. Multiple alignments revealed
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that the 16S rDNA sequence was closely related to that of K. oxytoca (AB053117,
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99.86 % similarity). Phylogenetic relationships and the approximate phylogenetic
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position of the strain are shown in Supplementary figure 1. By examining
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morphological, physiological, and biochemical properties and comparing the 16S rDNA
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gene sequence, strain BK-58 was assigned to the genus Klebsiella and is most closely
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related to K. oxytoca. The strain was named Klebsiella sp. BK-58 and was deposited in
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the China General Microbiological Culture Collection Center (CGMCC NO.4949,
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Beijing, China).
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Supplementary Table 1 Morphological and physiological properties of Klebsiella sp.
BK-58
Characteristics
Properties
Anaerobic growth
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Glucose O/F
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Catalase
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Cytochrome oxidase
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Citrate utilization
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Formation of H2S
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Vogas-Proskauer
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Liquefaction of gelatin
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Nitrate reduction
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Indole production
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Lysine decarboxylation
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Ornithine decarboxylation
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Malonate utilization
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Urease
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MR test
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Growth at 10 °C /44.5 °C
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Arginine dihydrolase
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Carbon source utilizing
D-Glucose
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Lactose
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Sucrose
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D-Mannitol
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D-Arabinose
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Rhamnose
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D-Sorbitol
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Raffinose
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Supplementary Figure 1 Phylogenetic tree for Klebsiella sp. BK-58 and related strains
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based on the 16S rDNA sequence. The numbers in parentheses are accession numbers of
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published sequences. The accession number of the 16S rDNA sequence of Klebsiella sp.
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BK-58 is KF469213. Pectobacterium chrysanthemi was used as the out-group
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