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Profiling of biodegradation and bacterial 16S rRNA genes in diverse contaminated ecosystems using 60-mer oligonucleotide microarray

  • Genomics, Transcriptomics, Proteomics
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Abstract

We have developed an oligonucleotide microarray for the detection of biodegradative genes and bacterial diversity and tested it in five contaminated ecosystems. The array has 60-mer oligonucleotide probes comprising 14,327 unique probes derived from 1,057 biodegradative genes and 880 probes representing 110 phylogenetic genes from diverse bacterial communities, and we named it as BiodegPhyloChip. The biodegradative genes are involved in the transformation of 133 chemical pollutants. Validation of the microarray for its sensitivity specificity and quantitation were performed using DNA isolated from well-characterized mixed bacterial cultures also having non-target strains, pure degrader strains, and environmental DNA. Application of the developed array using DNA extracted from five different contaminated sites led to the detection of 186 genes, including 26 genes unique to the individual sites. Hybridization of 16S rRNA probes revealed the presence of bacteria similar to well-characterized genera involved in biodegradation of various pollutants. Genes involved in complete degradation pathways for hexachlorocyclohexane (lin), 1,2,4-trichlorobenzene (tcb), naphthalene (nah), phenol (mph), biphenyl (bph), benzene (ben), toluene (tbm), xylene (xyl), phthalate (pht), Salicylate (sal), and resistance to mercury (mer) were detected with highest intensity. The most abundant genes belonged to the enzyme hydroxylases, monooxygenases, and dehydrogenases which were present in all the five samples. Thus, the array developed and validated here shall be useful in assessing not only the biodegradative potential but also the composition of environmentally useful bacteria, simultaneously, from hazardous ecosystems.

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Acknowledgments

This work was supported by the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India. AP is grateful to the Council of Scientific and Industrial Research, Government of India, for the research fellowship grant. We thank Genotypic Technology Private Limited (GTPL), Bangalore, India for their help in the development of microarray and hybridization experiments at their facility. We thank Dr. Raja Mugasimangalam, Dr. Sudha Narayan Rao, and Mr. Madhavan (GTPL) for their valuable discussions and advice on microarray design. Our sincere thanks to Mohd. Aiyaz (GTPL) for his excellent help in the experimental analysis and help to submit the results to the OMNIBUS-GEO. This article carries the IITR communication number 2803.

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Authors and Affiliations

  1. Environmental Biotechnology Division, Indian Institute of Toxicology Research (CSIR), P.O. Box 80, Mahatma Gandhi Marg, Lucknow, 226 001, Uttar Pradesh, India

    Ashutosh Pathak & Natesan Manickam

  2. Environmental Microbiology Division, Indian Institute of Toxicology Research (CSIR), Lucknow, India

    Rishi Shanker

  3. Centre of Excellence in Microbiology, Dr. R.M.L. Avadh University, Faizabad, India

    Satyendra Kumar Garg

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  1. Ashutosh Pathak
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  2. Rishi Shanker
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  3. Satyendra Kumar Garg
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  4. Natesan Manickam
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Correspondence to Natesan Manickam.

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Fig. S1

Hierarchical gene tree of functional (biodegradation) genes from five contaminated sites based on signal intensity values of Cy3-labeled targets hybridized onto the BiodegPhyloChip (JPEG 583 kb)

Fig. S2

Cluster analysis of 16S rRNA genes from five contaminated sites based on signal intensity values of Cy3-labeled targets hybridized onto the BiodegPhyloChip (JPEG 25 kb)

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Pathak, A., Shanker, R., Garg, S.K. et al. Profiling of biodegradation and bacterial 16S rRNA genes in diverse contaminated ecosystems using 60-mer oligonucleotide microarray. Appl Microbiol Biotechnol 90, 1739–1754 (2011). https://doi.org/10.1007/s00253-011-3268-5

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