Assessing the role of alkane hydroxylase genotypes in environmental samples by competitive PCR

Abstract

Aims: A molecular tool for extensive detection of prokaryotic alkane hydroxylase genes (alkB) was developed. AlkB genotypes involved in the degradation of short-chain alkanes were quantified in environmental samples in order to assess their occurrence and ecological importance. Methods and Results: Four primer pairs specific for distinct clusters of alkane hydroxylase genes were designed, allowing amplification of alkB-related genes from all tested alkane-degrading strains and from six of seven microcosms. For the primer pair detecting alkB genes related to the Pseudomonas putida GPo1 alkB gene and the one targeting alkB genes of Gram-positive strains, both involved in short-chain alkane degradation (<C 10 ), a quantitative competitive PCR (cPCR) assay was developed and validated on alkB-containing strains. AlkB genes of the two groups were then quantified in hydrocarbon-contaminated and pristine freshwater and soil samples, and their respective frequency was compared to degradation rates of short-chain n-alkanes. Pseudomonas putida-related alkB genes were prevalent in freshwater samples, but Gram-positive alkB-containing strains were more consistently related to alkane degradation activities. The latter genotype was more abundant in soils, although both genotypes increased in the most contaminated soils studied. Conclusions: Predominance of alkB genotypes depends on the ecosystem and environmental conditions, but alkane exposure generally leads to an increase of both studied genotypes. Significance and Impact of the Study: The study illustrates the distribution of two different alkB genotypes in two types of ecosystems, and highlights their respective roles in the environment.

n-Alkane assimilation and tert-butyl alcohol (TBA) oxidation capacity in Mycobacterium austroafricanum strains

“…This result can be partially attributed to the efficiency of the PCR primer pair (Rhose2/ Rhoas1). These primers, previously designed from the alkB sequence of P. putida GPo1 and located between positions Tyr160/Ile166 and Tyr270/Gly275 (Heiss-Blanquet et al 2005;Solano-Serena et al 2004), were less degenerate than the TS2S/deg1RE primer pair (Smits et al 1999) and amplified a smaller DNA fragment (343 bp instead of 525 bp). The polypeptide sequences, deduced from the nucleotide sequences of the PCR amplified fragments, were compared to the corresponding polypeptide fragments of AlkB from M. tuberculosis H37Rv and P. putida GPoI.…”

“…PCR amplification of the partial alkB gene was performed using the forward primer Rhose2 (5′-ACG-GSC-CAY-TTC-TAC-RTC-G-3′) and the reverse primer Rhoas1 (5′-CCG-TAR-TGY-TCG-AGR-TAG-3′), previously designed (Heiss-Blanquet et al 2005;Solano-Serena et al 2004). Rhose2 and Rhoas1 were located between positions Tyr160/Ile166 and Tyr270/Gly275, respectively, in the alkB sequence of P. putida GPo1.…”

Bioremediation Approaches for Treating Low Concentrations of N-Nitrosodimethylamine in Groundwater

“…PCR-based screening for the presence of monooxygenases capable of propane oxidation was performed using GoTaq Green Master Mix (Promega Inc., Madison, WI) following the manufacturer's guidelines with altered annealing temperatures and extension times as dictated by the DNA sequence being amplified. PCR primer sets designed to amplify the DNA encoding soluble diiron monooxygenases (SDMO; including PMO), AlkB type monooxygenases (AlkB), and P450-type monooxygenases (P450), were used to detect the presence of these enzymes in ENV425 (Heiss-Blanquet et al, 2005;Lisista et al, 2003;Coleman et al, 2006). Real-time quantitative reverse transcriptase PCR (QRT-PCR) was carried out with a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) following the manufacturer's protocol.…”

A new method for the detection of oil degrading genes in Pseudomonas aeruginosa based on transformation and PCR hybridization

“…Therefore, comprehensive analysis of environmental samples required the use of multiple primer and probe sets targeting the respective subgroups (Luz et al, 2004;Heiss-Blanquet et al, 2005;Kloos et al, 2006).…”