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Supplementary material
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Materials and methods for isolation and characterization of bacteria
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Media and culture conditions
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The nitrogen-free minimal medium for the enrichment of nitrile-utilizing microorganisms consisted of (g/l): glucose 12,
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KH2PO4 2, K2HPO4 2, MgSO4·7H2O 0.4, NaCl 1, FeSO4·7H2O 0.01, CoCl2 0.01, CaCl2 0.015. The pH of the medium was
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adjusted to 7.0 and autoclaved at 121°C for 20 min, similarly hereafter. The medium plates were prepared by adding
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18 g agar to 1 litre medium.
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The rich medium for preparation of isolates comprised the following components (g/l): glucose 12, yeast extract 5,
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ε-caprolactam 1, KH2PO4 1, K2HPO4 1, MgSO4·7H2O 0.2, NaCl 1, FeSO4·7H2O 0.01, CoCl2 0.01 and CaCl2 0.015 (pH 7.0).
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Enrichments procedures were carried out in the above-mentioned nitrogen-free minimal medium containing 2-
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amino-2,3-dimethylbutyronitrile (2 g/l) as nitrogen source. One gram of each soil sample was suspended in 10 ml
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distilled water. About 1 ml aliquot of the suspension was introduced to a 250-ml flask containing 40 ml enrichment
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medium and incubated at 30°C on a rotary shaker at 150 rpm for 5 days. A 2 ml culture aliquot was then transferred to
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the same fresh medium. After repeating the same procedure for 3 times, the resulting cultures were diluted tenfold
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and spread onto plates.
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The resulting isolated single colonies were grown aerobically at 30°C for 48 h in rich medium. Cells were collected
via centrifugation (9,000 × g, 10 min) and stored at 4°C for further use.
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Microorganism screening by colorimetric method
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The cell pellet (0.1 g) was resuspended in Erlenmeyer flask (50 ml) containing 5 ml distilled water. After incubating at
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30°C, 150 rpm for 5 min, reactions were started by addition of 50 µl ADBN (final concentration 9 g/l). Samples (0.5 ml)
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were withdrawn at regular intervals, followed by centrifugation. Strains capable of hydrating ADBN were rapidly
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screened through a high-throughput colorimetric method (Zheng et al. 2010). The colorimetric screening method was
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performed in a 96-well microplate. Positive samples were marked for further analysis by GC.
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Phenotypic and biochemical characterization
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Strain ZA0707 was cultivated on LB agar medium at 30°C. The cell morphology was observed by light microscope
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(Leica DM 4000 B, Germany) and environmental scanning electron microscope (ESEM-XL30, Netherlands).
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Physiological and biochemical characterization tests were carried out as described in Bergey’s Manual of Systematic
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Bacteriology (Holt 1984). Almost 21 biochemical tests were carried out in 24 h, with the API Coryne strip system as
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described by Freney et al. (1991). Data analysis was performed with the help of APILAB software by using the API
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Coryne (version 2.0) data base.
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16S rRNA sequence determination and phylogenetic analysis
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The chromosomal DNA of strain ZA0707 was extracted according to the procedure by Wilson (Ausubel et al. 1997).
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The 16S rRNA genes were amplified by polymerase chain reaction (PCR) using the universal primers: p16s-8 (5’-
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AGAGTTTGATCCTGGCTCAG-3’) and p16s-1492 (5’-GGCTACCTTGTTACGACTT-3’). PCR reactions were carried out in a
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thermal cycler (Bio-Rad, USA) under the following conditions: 5 min at 94°C, 35 cycles of 50 s at 94°C, 90 s at 52°C, 2
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min at 72°C, and one final step of 10 min at 72°C. The amplified PCR products were sequenced by Invitrogen Biotech
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Co., Ltd. (Shanghai, China). Related sequences were obtained from the GenBank database (National Center for
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Biotechnology Information, NCBI) using the BLAST search program. The ZA0707 16S rRNA sequence and reference
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sequence were aligned employing multiple-sequence alignment software CLUSTAL W version 1.81 (Thompson et al.
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1994). A phylogenetic tree was constructed by molecular evolutionary genetics analysis software version 2 (Kumar et
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al. 2001), using the neighbor-joining method with the Kimura 2-Parameter Distance model (Kimura 1980).
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Results of the isolated strain and its characterization
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Screening for ADBN-converting strains
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After several batches of enrichment, various microbial strains were isolated from soil samples. Two microorganisms
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with nitrile hydratase activity were selected out of 405 isolates which were capable of utilizing ADBN as the sole
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nitrogen source. One of them, strain ZA0707 with the higher nitrile hydratase activity was selected as the best strain
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for further studies and deposited in the China Centre for Type Culture Collection (CCTCC M 2010050).
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Morphological and physiological characteristics
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Colonies of strain ZA0707 were smooth, circular with regular edges, orange in color and opaque on LB agar. Cells were
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non-motile, non-fermentative and Gram-positive. They showed hypha in the early growth phase and developed to
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short rods and then cocci after 2-3 days of incubation. Physiological characteristics of strain ZA0707 were investigated
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by API system analysis and shown in Supplementary Table 1. This data suggested that strain ZA0707 belonged to
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Rhodococcus genus.
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16S rRNA gene sequencing and phylogenetic analysis
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Analysis of the partial 16S rRNA gene sequence of ZA0707 (1464 bp) was found to be 100% identical to that of R.
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qingshengii djl-6T using BLAST program. The phylogenetic relationships between the sequence and those of related
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species of genus Rhodococcus retrieved from GenBank were illustrated in Supplementary Fig. 1. The result of this
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phylogenetic analysis was consistent with the phenotypic tests. Therefore, strain ZA0707 was designated as R.
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qingshengii ZA0707 and the sequence was deposited in the GenBank database under accession no. HQ439600.
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References
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Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1997) Current protocols in molecular
biology. Wiley, New York
Freney J, Duperron MT, Courtier C, Hansen W, Allard F, Boeufgras JM, Monget D, Fleurette J (1991) Evaluation of API
coryne in comparison with conventional methods for identifying coryneform bacteria. J Clin Microbiol 29:38-41
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Holt JG (1984) Bergey's manual of determinative bacteriology. Williams and Wilkins, Baltimore
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Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies
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of nucleotide sequences. J Mol Evol 16:111-120
Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: molecular evolutionary genetics analysis software.
Bioinformatics 17:1244-1245
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Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence
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alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids
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Res 22:4673-4680
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Zheng YG, Lin ZJ, Zheng RC, Lei LH, Dai CL, Shen YC (2010) High-throughput screening method of nitrile invertase. CN
Patent 101838681 A, 31 May 2010
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Supplementary Table 1. Phenotypic characteristics of strain ZA0707
Characteristics
ZA0707
Characteristics
ZA0707
Gram staining
+
Gelatin hydrolysis
-
Nitrate reduction
-
Fermentation
-
Pyrazinamidase
-
D-glucose
-
Pyrrolidonyl arylamidase
-
D-ribose
-
Alkaline phosphatase
+
D-xylose
-
β-Glucuronidase
-
D-mannitol
-
β-Galactosidase
-
Malonate
-
α-Glucosidase
+
Lactose
-
N-acetylglucosaminidase
-
Sucrose
w
Esculin hydrolysis
+
Glycogen
-
Urease
+
Catalase
+
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+, positive; -, negative; w, weakly positively
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Tests were carried out whithin 24 h, with the API Coryne strip system as described by Freney et al.. Data analysis was
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carried out using APILAB software with API Coryne (version 2.0) data base
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Supplementary Fig. 1. Phylogenetic tree based on 16S rRNA sequences, constructed by the neighbor-joining method,
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depicting the relationship between strain ZA0707 and representatives of some related taxa. Numbers in parentheses
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are accession numbers of published sequences. Bar represents 1 nt substitutions per 100 nt. Bootstrap values
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expressed as percentages of 1000 replications are shown at branch points. Streptomyces padanus ATCC 25646 was
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used as outgroup
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