Supplementary material
Materials and methods for isolation and characterization of bacteria
Media and culture conditions
The nitrogen-free minimal medium for the enrichment of nitrile-utilizing microorganisms consisted of (g/l): glucose 12, 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 adjusted to 7.0 and autoclaved at 121°C for 20 min, similarly hereafter. The medium plates were prepared by adding 18 g agar to 1 litre medium.
The rich medium for preparation of isolates comprised the following components (g/l): glucose 12, yeast extract 5, 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).
Enrichments procedures were carried out in the above-mentioned nitrogen-free minimal medium containing 2-amino-2,3-dimethylbutyronitrile (2 g/l) as nitrogen source. One gram of each soil sample was suspended in 10 ml distilled water. About 1 ml aliquot of the suspension was introduced to a 250-ml flask containing 40 ml enrichment 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 the same fresh medium. After repeating the same procedure for 3 times, the resulting cultures were diluted tenfold and spread onto plates.
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.
Microorganism screening by colorimetric method
The cell pellet (0.1 g) was resuspended in Erlenmeyer flask (50 ml) containing 5 ml distilled water. After incubating at 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 Samples (0.5 ml) were withdrawn at regular intervals, followed by centrifugation. Strains capable of hydrating ADBN were rapidly screened through a high-throughput colorimetric method (Zheng et al. 2010). The colorimetric screening method was performed in a 96-well microplate. Positive samples were marked for further analysis by GC.
Phenotypic and biochemical characterization
Strain ZA0707 was cultivated on LB agar medium at 30°C. The cell morphology was observed by light microscope (Leica DM 4000 B, Germany) and environmental scanning electron microscope (ESEM-XL30, Netherlands).
Physiological and biochemical characterization tests were carried out as described in Bergey’s Manual of Systematic Bacteriology (Holt 1984). Almost 21 biochemical tests were carried out in 24 h, with the API Coryne strip system as described by Freney et al. (1991). Data analysis was performed with the help of APILAB software by using the API Coryne (version 2.0) data base.
16SrRNA sequence determination and phylogenetic analysis
The chromosomal DNA of strain ZA0707 was extracted according to the procedure by Wilson (Ausubel et al. 1997). The 16SrRNA genes were amplified by polymerase chain reaction (PCR) using the universal primers: p16s-8 (5’-AGAGTTTGATCCTGGCTCAG-3’) and p16s-1492 (5’-GGCTACCTTGTTACGACTT-3’). PCR reactions were carried out in a 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 min at 72°C, and one final step of 10 min at 72°C. The amplified PCR products were sequenced by Invitrogen Biotech Co., Ltd. (Shanghai, China). Related sequences were obtained from the GenBank database (National Center for Biotechnology Information, NCBI) using the BLAST search program. The ZA070716SrRNA sequence and reference sequence were aligned employing multiple-sequence alignment software CLUSTAL W version 1.81 (Thompson et al. 1994). A phylogenetic tree was constructed by molecular evolutionary genetics analysis software version 2 (Kumar et al. 2001), using the neighbor-joining method with the Kimura 2-Parameter Distance model (Kimura 1980).
Results of the isolated strain and its characterization
Screening for ADBN-converting strains
After several batches of enrichment, various microbial strains were isolated from soil samples. Two microorganisms with nitrile hydratase activity were selected out of 405 isolates which were capable of utilizing ADBN as the sole nitrogen source. One of them, strain ZA0707 with the higher nitrile hydratase activity was selected as the best strain for further studies and deposited in the China Centre for Type Culture Collection (CCTCC M 2010050).
Morphological and physiological characteristics
Colonies of strain ZA0707 were smooth, circular with regular edges, orange in color and opaque on LB agar. Cells were non-motile, non-fermentative and Gram-positive. They showed hypha in the early growth phase and developed to short rods and then cocci after 2-3 days of incubation. Physiological characteristics of strain ZA0707 were investigated by API system analysis and shown in Supplementary Table 1. This data suggested that strain ZA0707 belonged to Rhodococcus genus.
16SrRNA gene sequencing and phylogenetic analysis
Analysis of the partial 16SrRNA gene sequence of ZA0707 (1464 bp) was found to be 100% identical to that of R. qingshengiidjl-6T using BLAST program. The phylogenetic relationships between the sequence and those of related species of genus Rhodococcus retrieved from GenBank were illustrated in Supplementary Fig. 1. The result of this phylogenetic analysis was consistent with the phenotypic tests. Therefore, strain ZA0707 was designated as R. qingshengiiZA0707 and the sequence was deposited in the GenBank database under accession no. HQ439600.
References
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, BoeufgrasJM, Monget D, Fleurette J (1991) Evaluation of API coryne in comparison with conventional methods for identifying coryneform bacteria. J Clin Microbiol 29:38-41
Holt JG (1984) Bergey's manual of determinative bacteriology. Williams and Wilkins, Baltimore
Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111-120
Kumar S, Tamura K, JakobsenIB, Nei M (2001) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244-1245
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673-4680
Zheng YG, Lin ZJ, Zheng RC, Lei LH, Dai CL, ShenYC (2010) High-throughput screening method of nitrile invertase. CN Patent 101838681 A, 31 May 2010
Supplementary Table 1. Phenotypic characteristics of strain ZA0707
Characteristics / ZA0707 / Characteristics / ZA0707Gram 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 / +
+, positive; -, negative; w, weakly positively
Tests were carried out whithin 24 h, with the API Coryne strip system as described by Freney et al.. Data analysis was carried out using APILAB software with API Coryne (version 2.0) data base
Supplementary Fig. 1.Phylogenetic tree based on 16SrRNA sequences, constructed by the neighbor-joining method, depicting the relationship between strain ZA0707 and representatives of some related taxa. Numbers in parentheses are accession numbers of published sequences. Bar represents 1 nt substitutions per 100 nt. Bootstrap values expressed as percentages of 1000 replications are shown at branch points. Streptomyces padanusATCC 25646was used as outgroup
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