Distribution of alkB genes within n-alkane-degrading bacteria

Abstract

Fifty‐four bacterial strains belonging to 37 species were tested for their ability to assimilate short chain and/or medium chain liquid n‐alkanes. A gene probe derived from the alkB gene of Pseudomonas oleovorans ATCC 29347 was utilized in hybridization experiments. Results of Southern hybridization of PCR‐amplificates were compared with those of colony hybridization and dot blot hybridization. Strongest signals were received only from Gram‐negative bacteria growing solely with short n‐alkanes (C10). Hybridization results with soil isolates growing with n‐alkanes of different chain lengths suggested as well that alkB genes seem to be widespread only in solely short‐chain n‐alkane‐degrading pseudomonads. PCR products of Rhodococcus sp., Nocardioides sp., Gordona sp. and Sphingomonas sp. growing additionally or solely with medium‐chain n‐alkane as hexadecane had only few sequence identity with alkB though hybridizing with the gene probe. The derived amino acid sequence of the alkB‐amplificate of Pseudomonas aureofaciens showed high homology (95%) with AlkB from Ps. oleovorans. alkB gene disruptants were not able to grow with decane.

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

“…M. smegmatis mc2 155 was able to grow only on dodecane. This narrow range of substrate use was previously found in P. putida P1, which grew on octane (Smits et al 1999) and in P. aureofasciens RWTH 529, which only grew on decane (Vomberg and Klinner 2000). Johnson and Hyman (2006) recently demonstrated a new capacity of P. putida GPo1 to also grow on the gaseous n-alkanes, propane and butane, confirming the broad alkane spectrum of the alkane hydroxylase in this strain.…”

Diversity of Alkane Hydroxylase Systems in the Environment

“…While in some studies alkB homologs were detected in 10-40% of the bacterial population [97,101,102], other groups did not detect alkB homologs at all [99,100]. AlkB homologs that are closely related to the P. putida GPo1 enzyme appear to be common in gram-negative strains only (probably pseudomonads) [76]. This is supported by the observation that genes almost identical to the P. putida GPo1 alkB gene were found in several P. putida, P. aeruginosa, and P. mendocina strains [67,87], but not in other strains.…”

“…Alaskan sediments Colony hybridization with alkB 39% of viable heterotrophs from uncontaminated soil contain alkB gene probe 67% of viable heterotrophs from contaminated soil contain alkB [97] Contaminated soil PCR followed by Southern blot Detection and quantification of alkB (and other genes) in soil: 1-10 with alkB gene probe gene copies per gram of soil can be detected [98 Variety of cold ecosystems PCR, and Southern blots None of the psychrophilic alkane degraders possess genes with alkB gene probe with high homology to P. putida alkB [99] Fuel oil-contaminated site Dot-blots DNA extracted from soil shows no significant hybridization with an alkB-gene probe [100] Shallow aquifer Southern blots 10-20% of the total bacterial community hybridizes with alkB gene probe with an alkB gene probe [101] Various sources PCR with highly degenerate Most alkane-degrading strains contain distantly related alkB homologs (54 bacterial strains) primers for alkB homologs (homology not sufficient for Southern or dot-blots) [50] Shallow aquifer Dot-blots with DNA extracted alkB genotypes start at 11% of total community, and peak at (natural attenuation site) from aquifer samples 52% after injection of synthetic jet fuel in the aquifer [102] Various sources Southern, colony and dot-blots, alkB genes (close homologs) are widespread only in short-chain n-alkane (54 bacterial strains) PCR followed by Southern degrading pseudomonads [76] Rhizosphere vs. bulk soil Multiplex PCR At a contaminated site alkB was 10 times more prevalent in the endophytic community compared to the bulk soil community [103] Propane and butaneDot-blots 8 of 15 strains (including pseudomonads and rhodococci) utilizing bacteria gave a strong signal, and 7 a weak signal with the IMT37 gene [37] Land treatment unit (LTU) PCR, terminal restriction alkB increased in abundance during the first 3 weeks of LTU operation, fragment length polymorphism and comprised > 80% of the total PCR products [104] Arctic and Antarctic soil PCR-hybridization Rhodococcal alkB genes occur in contaminated and pristine soils, and colony hybridization P. putida alkB occurs more frequently in contaminated soils [105] similar genes in other butane degraders (pseudomonads, rhodococci and unidentified bacteria). In a dot-blot experiment, 8 out of 15 propane or butane-degrading bacteria gave a strong signal with the gene-probe, while the remaining 7 gave a weak but detectable signal [37].…”

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

“…Hybridization probes derived from various organisms have been used for the detection of microbial communities after isolation of the bacteria (Sotzsky et al, 1994;Whyte et al, 1995;Vomberg and Klinner, 2000). Investigations with PCR-based methods were also mostly performed with cultured isolates (Smits et al, 1999;Vomberg and Klinner, 2000;van Beilen et al, 2002).…”

“…Investigations with PCR-based methods were also mostly performed with cultured isolates (Smits et al, 1999;Vomberg and Klinner, 2000;van Beilen et al, 2002). The high sequence divergence found in Pseudomonas Aeruginosa bacteria from different taxonomic groups (Smits et al, 1999;van Beilen et al, 2003) caused a group-specificity in the detection of alkane degraders with the PCR and hybridization methods described in the literature.…”