Detection, expression and quantitation of the biodegradative genes in Antarctic microorganisms using PCR

Abstract

In this study, 28 hydrocarbon-degrading bacterial isolates from oil-contaminated Antarctic soils were screened for the presence of biodegradative genes such as alkane hydroxylase (alks), the ISPalpha subunit of naphthalene dioxygenase (ndoB), catechol 2,3-dioxygenase (C23DO) and toluene/biphenyl dioxygenase (todC1/bphA1) by using polymerase chain reaction (PCR) methods. All naphthalene degrading bacterial isolates exhibited the presence of a 648 bp amplicon that shared 97% identity to a known ndoB sequence from Pseudomonas putida. Twenty-two out of the twenty-eight isolates screened were positive for one, two or all three different regions of the C23DO gene. For alkane hydroxylase, all 6 Rhodococcus isolates were PCR-positive for a 194 bp and a 552 bp segment of the alkB gene, but exhibited variable results with primers located at different segments of this gene. Three Pseudomonas spp. 4/101, 19/1, 30/3 amplified 552 bp segment of alkB. Only two Pseudomonas sp. 7/156 and 4/101 amplified a segment of alkB exhibiting 89-94% nucleotide sequence identity with the existing sequence of alkB in the GenBank sequence database. Transcripts of three genes, alkB2, C23DO and ndoB, that were amplified by DNA-PCR in three different bacterial isolates also exhibited positive amplification by reverse transcriptase PCR (RT-PCR) method confirming that these genes are functional. A competitive PCR (cPCR) method was developed for a quantitative estimation of ndoB from pure cultures of the naphthalene-degrading Pseudomonas sp. 30/2. A minimum of 1 x 10(7) copies of the ndoB gene was detected based on the comparison of the intensities of the competitor and target DNA bands. It is expected that the identification and characterization of the biodegradative genes will provide a better understanding of the catabolic pathways in Antarctic psychrotolerant bacteria, and thereby help support bioremediation strategies for oil-contaminated Antarctic soils.