Cloning and Heterologous Expression of a Gene Encoding an Alkane-Induced Extracellular Protein Involved in Alkane Assimilation from Pseudomonas aeruginosa (original) (raw)
Related papers
Differential Expression of the Components of the Two Alkane Hydroxylases from Pseudomonas aeruginosa
Oxidation of n-alkanes in bacteria is normally initiated by an enzyme system formed by a membrane-bound alkane hydroxylase and two soluble proteins, rubredoxin and rubredoxin reductase. Pseudomonas aeruginosa strains PAO1 and RR1 contain genes encoding two alkane hydroxylases (alkB1 and alkB2), two rubredoxins (alkG1 and alkG2), and a rubredoxin reductase (alkT). We have localized the promoters for these genes and analyzed their expression under different conditions. The alkB1 and alkB2 genes were preferentially expressed at different moments of the growth phase; expression of alkB2 was highest during the early exponential phase, while alkB1 was induced at the late exponential phase, when the growth rate decreased. Both genes were induced by C 10 to C 22 /C 24 alkanes but not by their oxidation derivatives. However, the alkG1, alkG2, and alkT genes were expressed at constant levels in both the absence and presence of alkanes.
Alkane hydroxylase systems in Pseudomonas aeruginosa strains able to grow on n-octane
Studies in Organic Chemistry, 1998
Pseudomonas strains able to grow on n-octane were selected from different strain collections, and compared with P. oleovorans GPol with respect to the genetics of alkane metabolism in these strains. We found that the enzymes involved in alkane oxidation in four P. aeruginosa strains are virtually identical to the alkane hydroxylase system of P. oleovorans GPol. PCR-cloning and sequencing showed that the differences are limited to a few nucleotide substitutions outside the openreading frames.
2011
The potential for two overlapping fragments of DNA from a clone of newly isolated alkanes degrading bacterium Pseudomonas frederiksbergensis encoding sequences with similar homology to two parts of functional proteins is described. One strand contains a sequence with high homology to alkanes monooxygenase (alkB), a member of the alkanes hydroxylase family, and the other strand contains a sequence with some homology to alcohol dehydrogenase gene (alkJ). Overlapping of the genes on opposite strands has been reported in eukaryotic species, and is now reported in a bacterial species. The sequence comparisons and ORFS results revealed that the regulation and the genes organization involved in alkane oxidation represented in Pseudomonas frederiksberghensis varies among the different known alkane degrading bacteria. The alk gene cluster containing homologues to the known alkane monooxygenase (alkB), and rubredoxin (alkG) are oriented in the same direction, whereas alcohol dehydrogenase (al...
Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants
Journal of Bacteriology, 1991
We isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up 14C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C12 to C19. However, growth on these alkanes and uptake of [14C]hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media contain...
Pseudomonas oleovorans GPo1 can metabolize medium-chain-length alkanes by means of an enzymatic system whose induction is regulated by the AlkS protein. In the presence of alkanes, AlkS activates the expression of promoter PalkB, from which most of the genes of the pathway are transcribed. In addition, expression of the first enzyme of the pathway, alkane hydroxylase, is known to be influenced by the carbon source present in the growth medium, indicating the existence of an additional overimposed level of regulation associating expression of the alk genes with the metabolic status of the cell. Reporter strains bearing PalkB-lacZ transcriptional fusions were constructed to analyze the influence of the carbon source on induction of the PalkB promoter by a nonmetabolizable inducer. Expression was most efficient when cells grew at the expense of citrate, decreasing significantly when the carbon source was lactate or succinate. When cells were grown in Luria-Bertani rich medium, PalkB was strongly down-regulated. This effect was partially relieved when multiple copies of the gene coding for the AlkS activator were present and was not observed when the promoter was moved to Escherichia coli, a heterologous genetic background. Possible mechanisms responsible for PalkB regulation are discussed. The genetics and enzymology of bacterial metabolism of n-alkanes have been well characterized for Pseudomonas oleo-vorans GPo1, which harbors a large plasmid, named OCT (9), encoding the enzymes required to oxidize medium-chain-length (C 6 to C 12) n-alkanes to the corresponding terminal acyl coenzyme A derivatives, which then enter the-oxidation cycle (see reference 50 for a review) (Fig. 1). The genes coding for these enzymes are clustered in two operons, alkBFGHJKL and alkST (50) (Fig. 1). The alkS gene codes for a transcriptional regulator which, in the presence of alkanes, activates expression of the alkBFGHKJL operon (53). This operon is transcribed from a single promoter, PalkB (28). The first enzyme of the pathway, alkane hydroxylase, has attracted much attention due to its ability to oxidize alkanes, alkenes, and related products , yielding alcohols or epoxides (for reviews, see references 51 and 52). Its use as a biocatalyst requires the development of strains harboring the alkane hydroxylase but not the subsequent enzymes of the pathway. This enzyme is composed by three different subunits: a membrane-bound hydroxylase and two soluble proteins, rubredoxin and rubredoxin reductase, which act as electron carriers between NADH and the hydrox-ylase (36, 38, 49). Alkane hydroxylase is expressed at high levels upon induction, which has been shown to affect the membrane lipid fatty acids (10, 37). The amount of newly synthesized alkane hydroxylase in P. oleovorans varies depending on the carbon source present in the growth medium, which indicates that its expression may be modulated by catabolite repression (20, 46). The term catab-olite repression describes a number of regulatory processes that ensure that when the cell is exposed to a preferred carbon source, the catabolic pathways for other, nonpreferred sub-strates are not induced, even if the appropriate inducers are present (31). Previous analyses of the expression of alkane hydroxylase in cells growing on different carbon sources had been done mainly by measuring enzyme activity in cells harboring either the complete OCT plasmid or the complete alk pathway cloned into a broad-host-range plasmid (20, 46). To identify the minimum determinants that lead to catabolic repression of the alk operon, we have constructed reporter strains containing exclusively the AlkS regulator and the PalkB promoter fused to a reporter gene. Expression of the PalkB promoter was analyzed when these strains were grown at the expense of different carbon sources. We conclude that PalkB promoter activity is modulated by the carbon source used and that its expression in a rich medium is strongly repressed. Our results suggest that repression in Luria-Bertani (LB) rich medium occurs by an interference with AlkS function.
Frontiers in Marine Science
Indigenous bacterial populations play an important role in the restoration of crude oilpolluted marine environments. The identification and characterization of these bacteria are key in defining bioremediation strategies for the mitigation of possible future oil spills. In this work, we characterized Pseudomonas aeruginosa strain GOM1, which was isolated from the water column in the southwestern Gulf of Mexico. Phylogenetic analysis revealed that GOM1 strain was most closely related to P. aeruginosa WC55, a strain isolated from the northern Gulf of Mexico after the Deepwater Horizon oil spill. The hydrocarbon-degrading capacity of P. aeruginosa GOM1 was investigated using various approaches. This strain degraded 96% of the aliphatic fraction (C12-C38) of crude oil during a 30-day incubation period, exhibiting a high activity on long-chain alkanes, and expressing alkane hydroxylases AlkB1, AlkB2 and AlmA. Addition of nitrogen and phosphate to seawater culture medium enhanced hexadecane degradation by GOM1. Additionally, the strain exhibited high surfactant/rhamnolipid production and emulsifying activity when grown in a complex medium in the presence of hexadecane. Comparisons of growth kinetics, hydrocarbon degradation and gene expression between GOM1 and the closely related P. aeruginosa laboratory strain PAO1 revealed that the marine isolate is better adapted to degrade alkanes. Taken together, our results place P. aeruginosa GOM1 as a potentially effective candidate to be included in a consortium for use in the bioremediation of oil-polluted sites.
Cloning and Expression of Pseudomonas aeruginosa AlkB Gene in E. coli
Journal of Pure and Applied Microbiology, 2020
Pre identified hydrocarbons degrading bacteria were used in this study, specific primer was conducted to amplification of AlkB gene, approximately 1206bp band size of this gene for Pseudomonas aeruginosa was detected and proofed by sequence and alignment analysis with NCBI database. The AlkB gene was inserted in PET-21a(+) plasmid vector as expression vector, then transformed in BL21(DE3) competent E. coli and confirmed by colony PCR technique using the T7 promoter and T7 terminator primers. The expression of the inserted gene was checked by determined the concentration of AlkB protein for multiple periods by Bradford assay method and the SDS-polyacrylamide gel electrophoresis method was revealed band of ~46 KD molecular weight of the concerned protein. The gene amplification and cloning strategy was lay out before the practical part of the study by SnapGene software, this study was conducted to introduce cloned bacteria which facilitate the first step (key step) of alkane's biodegradation and propose an appropriate strategy to construct genetically engineered microorganisms with multiple recombinant plasmid for enhance the degradation of the aliphatic fraction of hydrocarbon
Environmental and hospital-sampling allowed us to isolate and identify one hundred of Pseudomonas aeruginosa (P. aeruginosa) strains. The study of the degradation capacity of alkanes has shown that almost all isolates grew in the presence of long chain n-alkanes, while no growth was recorded in the presence of short chain n-alkanes, isoalkanes or cycloalkanes. The kinetics of growth in the presence of hexadecane, as a sole carbon source, enabled recording various optical densities (OD) depending on the strain of P. aeruginosa. The polymerase chain reaction (PCR) in the presence of ERIC (Enterobacterial repetitive intergenic consensus) primers has shown genetic diversity within isolates. The search for alkB and alkB1 genes, which are respectively responsible for the degradation of short chain n-alkanes and long chain n-alkanes, has shown the absence of alkB gene; however, the alkB1 gene, strongly present within the population of P. aeruginosa isolated, is absent in a few strains despite their ability to degrade long chain n-alkanes. The nucleotide sequencing of a alkB1 gene fragment for 4 P. aeruginosa strains as well as the reference strain P. aeruginosa PAO-1 has shown a highly conserved nucleotide sequences in spite of their heterogeneity origin.
Biotechnology Letters, 2014
Pseudomonas citronellolis UAM-Ps1 cometabolically transforms methyl tert-butyl ether (MTBE) to tert-butyl alcohol with n-pentane (2.6 mM), n-octane (1.5 mM) or dicyclopropylketone (DCPK) (4.4 mM), a gratuitous inducer of alkane hydroxylase (AlkB) activity. The reverse transcription quantitative real-time PCR was used to quantify the alkane monooxygenase (alkB) gene expression. The alkB gene was expressed in the presence of n-alkanes and DCPK and MTBE oxidation occurred only in cultures when alkB was transcribed. A correlation between the number of alkB transcripts and MTBE consumption was found (LSBE consumption in lmol = 1.44e-13 x DNA copies, R 2 = 0.99) when MTBE (0.84 mM) was added. Furthermore, alkB was cloned and expressed into Escherichia coli and the recombinant AlkB had a molecular weight of 42 kDa. This is the first report where the expression of alkB is related to the cometabolic oxidation of MTBE.