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| Status |
Public on Apr 15, 2011 |
| Title |
Development and evaluation of a 60-mer oligonucleotide microarray for profiling of biodegradation and bacterial 16S rRNA genes in diverse contaminated ecosystems |
| Platform organism |
synthetic construct |
| Sample organisms |
Escherichia coli K-12; Rhodococcus jostii RHA1; Sphingomonas sp. NM05; soil metagenome; Escherichia coli BL21; freshwater sediment metagenome; Escherichia coli DH5[alpha]; Bordetella sp. IITR-02 |
| Experiment type |
Genome variation profiling by array
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| Summary |
We have developed a 60-mer oligonucleotide multibacterial microarray for detection and expression profiling of biodegradative genes and bacterial diversity (16S rRNA gene) in different habitats contaminated with varieties of hazardous chemicals. The genes selected were involved in biodegradation and biotransformation of various groups of compounds viz. nitroaromatic compounds (148 genes), chloroaromatic compounds (75 genes), monoaromatic compounds (373 genes), polyaromatic hydrocarbons (174 genes), pesticides/ herbicides (34 genes), alkanes/aliphatics (185 genes) and heavy metals (68 genes), which covered a total number of 133 chemicals. The efficiency (specificity, detection sensitivity) of the developed array was evaluated using the labeled genomic DNA of pure bacterial strains, Escherichia coli DH5α and Sphingomonas sp. strain NM-05 (involved in the biodegradation of γ-hexachlorohexane isolated from IPL, Lucknow) at different concentrations of 300ng, 500ng, 800ng, 1000ng and 1250ng. The specificity of the developed array was further validated using mixed cultures containing three strains (Sphingomonas sp. strain NM-05, Rhodococcus sp. strain RHA1 and Bordetella sp. strain IITR-02) involved in biodegradation of γ-hexachlorohexane, biphenyl and chlorobenzenes respectively. The mixed culture also contained non-target/non-degrader strains (E. coli DHα, E.coli BL21 and E.coli K12 NCTC50192). The developed array was applied for profiling using the total soil DNA in five contaminated habitats of north India, viz. chloroaromatic chemicals contaminated site (India Pesticide Limited, Chinhat, Lucknow), a river sediments (Gomti river sediment, Lucknow), heavy metal industry dump site (Jajmau industrial area Kanpur), a effluent treatment plant (CETP along Ganges river near Kanpur), and an oil refinery (Mathura oil refinery). Hybridization of 16S rRNA probes revealed the presence of bacteria similar to well characterized genera involved in biodegradation of 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 hydroxylases, monooxygenases and dehydrogenases which were present in all the five samples. Many compound specific genes which initiate the degradation pathway were also detected. Thus, the array developed and validated here may be useful in assessing the biodegradative potential and composition of environmentally useful bacteria in hazardous ecosystems.
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| Overall design |
Agilent one-color CGH experiment,Organism: Genotypic designed Agilent-17159 Genotypic designed Agilent Multibacterial 8x15k Array , Labeling kit: Agilent Genomic DNA labeling Kit (Part Number: 5190-0453)
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| Contributor(s) |
Pathak A, Manickam N |
| Citation(s) |
21503758 |
| Submission date |
Sep 24, 2010 |
| Last update date |
Mar 22, 2012 |
| Contact name |
Natesan Manickam |
| E-mail(s) |
nmanickam@iitr.res.in
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| Phone |
+91-522-2620107
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| Fax |
+91-522-2628227
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| Organization name |
Indian Institute of Toxicology Research
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| Department |
Department Environmental Biotechnology Division
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| Lab |
Environmental Biotechnology Lab
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| Street address |
Mahatma Gandhi Marg
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| City |
Lucknow |
| State/province |
Uttar Pradesh |
| ZIP/Postal code |
226001 |
| Country |
India |
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| Platforms (1) |
| GPL10926 |
Agilent-017159 Genotypic custom 60-mer oligonucleotide multibacterial microarray |
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| Samples (17)
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| Relations |
| BioProject |
PRJNA132805 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE24353_RAW.tar |
39.4 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
| Processed data provided as supplementary file |
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