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.

Soil type affects plant colonization, activity and catabolic gene expression of inoculated bacterial strains during phytoremediation of diesel

“…In bioremediation studies, the quantitative analysis of functional genes such as alkane hydroxylase genes has provided a valuable tool for studying the relationship between specific microbial populations or strains and the performance of degradation processes [30][31][32]. The abundance of alkane degrading genes has been assessed in the soil and plant interior [33][34][35], but the activity of alkane degrading bacteria in situ and under natural conditions by e.g.…”

“…The abundance of alkane degrading genes has been assessed in the soil and plant interior [33][34][35], but the activity of alkane degrading bacteria in situ and under natural conditions by e.g. gene expression has been rarely addressed [31,36]. However, for efficient degradation not only the abundance of plantassociated bacteria and/or applied inoculants strains but also the degrading activity is highly important [37].…”

Draft Genome Sequence of Pseudomonas sp. Strain Ant30-3, a Psychrotolerant Bacterium with Biodegradative Attribute Isolated from Antarctica

“…strain Ant30-3, isolated from fuel-contaminated soil near the former Vanda Station located in the McMurdo Dry Valleys, Antarctica, manifests growth at near subzero temperatures, as well as freeze tolerance, expression of a capB- encoded cold acclimation protein (1, 2), the secretion of a copious amount of extracellular polymeric substance (EPS) (3), and the antifreeze property of cellular proteins that promote the growth of stable hexagonal water crystals at subzero temperatures (4). Also, aromatic hydrocarbon-degrading genes were detected in Ant30-3 by PCR (5). Thus, Ant30-3 manifests multifaceted cold-adaptive genetic determinants with the potential for applications in the biodegradation of hydrocarbon compounds in cold ecosystems.…”

“…The 133 genes related to the metabolism of aromatic compounds included genes for two catechol 1,2-dioxygenases, one catechol 2,3-dioxygenase (5), three ferredoxin-related coding genes (12), two protocatechuate 3,4-dioxygenases (12), two alkanesulfonate monooxygenases (13), and a gene cluster for the degradation of phenols, cresols, and catechol (12). Six genes belonging to the cold shock family of proteins, including CspA, CspC, CspD, and CspG, chaperonin GroEL and GroES, and the RecA protein, were found on the Ant 30-3 genome.…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…-identification of alkanes bio-degradative genes of Rhodococcus strains in microbial communities from contaminated soil in different geographical areas such as deep sea environments [300][301][302] or arctic soils [187,303];…”