Supplemental Informationfor Biotechnology Letters

Supplemental Informationfor Biotechnology Letters

Indigo biosynthesis by Comamonas sp. MQ

Yuanyuan Qua • Xuwang Zhang a•Qiao Maa•Fang Mab,* • Qiang Zhanga•Xinliang Lia• Hao Zhou a•Jiti Zhou a

a State Key Laboratory of Fine Chemicals, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education, China), School of Environmental Science and Technology, DalianUniversity of Technology, Dalian 116024, China

b State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China

* Corresponding author:Fang Ma

E-mail:
Methods

Isolation and identification of indigo-producing bacteria

The indigo-producing bacteria were isolated from activated sludge of a local sewage farm (Chunliu River Wastewater Treatment Plant, Dalian, China).The fresh activated sludge samples were firstly inoculated into 100 ml MSM containing 1,000 mg naphthalene/l, and cultivated at 30 oC under continuous shaking. After about one-week incubation, 10% (v/v) of the broth was inoculated into fresh MSM supplemented with 100 mg naphthalene/l and 50 mg indole/l. Subsequently, the culture was further plated and incubated on naphthalene-indole-MSM agar plate. After about two months of screening and enriching, one of the blue colonies designated as MQ was selected for further characterization.

The 16S rRNA gene of strain MQ was amplified by PCR using the following primers: 8F (5’-AGAGTTTGATCATGGCTCAG-3’) and 1522R (5’-AAGGACGTCATCCAGCCGCA-3’). The amplicons were sequenced by TaKaRa Biotechnology (Dalian) Co., Ltd. (China), and the sequence was compared with those in GenBank using the NationalCenter for Biotechnology Information BLAST network service (blastn). The 16S rRNA gene sequence of strain MQ and related sequenceswere aligned by Clustal X (1.8) and then used to construct a phylogenetic tree using the neighbor-joining method by MEGA (version 4.1) with 1,000 bootstrap replicates.

Results and discussion

Identification of indigo-producing bacterial strain

The sequence of partial 16S rRNA (1,478 bp) from strain MQ was deposited in GenBank database under the accession number HQ176414. Based on the sequence similarity search by blastn, strain MQ exhibited 99% homology to Comamonas testosteroni WDL7 (accession number AF538933), which revealed that strain MQ belonged to genus Comamonas. Neighbor-joining phylogenetic analysis was performed using 16S rRNA gene sequences of strain MQ and the close relatives, together with the sequences of many different indigo-producing bacteria (Fig.S1). Among the indigo-producing bacteria reported previously, most of them belonged to genus Pseudomonas, such as Pseudomonas putidamt-2, Pseudomonas putida F1, Pseudomonas sp. HOB1, etc. (O’Connor and Hartmans1998; Pathak and Madamwar 2010). Meanwhile, Acinetobacter sp. ST-550 and Acinetobacter sp.PP-2could also produce indigo from indole under certain circumstances (Doukyu et al. 2002; Qu et al. 2010). Compared with the strains mentioned above, strain MQ represented a novel indigo-producing bacterium belonging to genus Comamonas (Fig. S1).

To date, very few reports were available concerning Comamonas species strains using for microbial biosynthesis of indigo. Most studies concentrated on cloning genes encoding aromatic dioxygenase from Comamonas spp., and indigo production was used as an indicator for recombinant gene expression since the presence of dioxygenase activity would result in the biotransformation of indole to indigo (Goyal and Zylstra 1996; Ju and Parales 2006). However, further investigation of indigo production by Comamonas species was still limited.

References

Doukyu N, Nakano T, Okuyama Y, Aono R (2002) Isolation of an Acinetobacter sp. ST-550 which produces a high level of indigo in a water-organic solvent two-phase system containing high levels of indole. Appl Microbiol Biotechnol 58:543-546

Goyal AK, Zylstra GJ(1996) Molecular cloning of novel genes for polycyclic aromatic hydrocarbon degradation from Comamonas testosteroni GZ39. Appl Environ Microbiol 62:230-236

Ju K, Parales RE(2006) Control of substrate specificity by active-site residues in nitrobenzene dioxygenase. Appl Environ Microbiol 72:1817-1824

O’Connor KE, Hartmans S (1998) Indigo formation by aromatic hydrocarbon-degrading bacteria. Biotechnol Lett 20:219-223

Pathak H, Madamwar D (2010) Biosynthesis of indigo dye by newly isolated naphthalene-degrading strain Pseudomonas sp. HOB1 and its application in dyeing cotton fabric. Appl Biochem Biotechnol 160:1616-1626

Qu YY, Pi WQ, Ma F, Zhou JT, Zhang XW(2010) Influence and optimization of growth substrates on indigo formation by a novel isolate Acinetobacter sp. PP-2. Bioresour Technol 101:4527-4532

Figures

Fig. S1Phylogenetic tree of the newly isolated MQ (bold) and related species.The strains previously reported for the biosynthesis of indigo are underlined and the accession numbersin the parenthesis correspond to partial 16S rRNA gene sequences

Fig. S2 The mass spectra of indigo (a) and isatin (b) produced by strain MQ (in APCI positive ion mode). The insets showed the UV-visible spectrum and the structural formula of the metabolites

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