Biodegradation of petroleum hydrocarbons by psychrotrophic Pseudomonas strains possessing both alkane (alk) and naphthalene (nah) catabolic pathways.
ABSTRACT: Three hydrocarbon-degrading psychrotrophic bacteria were isolated from petroleum-contaminated Arctic soils and characterized. Two of the strains, identified as Pseudomonas spp., degraded C5 to C12 n-alkanes, toluene, and naphthalene at both 5 and 25 degrees C and possessed both the alk catabolic pathway for alkane biodegradation and the nah catabolic pathway for polynuclear aromatic hydrocarbon biodegradation. One of these strains contained both a plasmid slightly smaller than the P. oleovorans OCT plasmid, which hybridized to an alkB gene probe, and a NAH plasmid similar to NAH7, demonstrating that both catabolic pathways, located on separate plasmids, can naturally coexist in the same bacterium.
Project description:The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.
Project description:Pseudomonas putida G7 exhibits chemotaxis to naphthalene, but the molecular basis for this was not known. A new gene, nahY, was found to be cotranscribed with meta cleavage pathway genes on the NAH7 catabolic plasmid for naphthalene degradation. The nahY gene encodes a 538-amino-acid protein with a membrane topology and a C-terminal region that resemble those of chemotaxis transducer proteins. A P. putida G7 nahY mutant grew on naphthalene but was not chemotactic to this aromatic hydrocarbon. The protein NahY thus appears to function as a chemoreceptor for naphthalene or a related compound. The presence of nahY on a catabolic plasmid implies that chemotaxis may facilitate biodegradation.
Project description:The sequence of a 2,437-bp DNA segment from the naphthalene upper catabolic pathway operon of plasmid NAH7 was determined. This segment contains three large open reading frames designated nahQ', nahE, and nahD. The first of these is the 3' end of an open reading frame that has no known function, the second (993 bp) encodes trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (deduced molecular weight, 36,640), and the third (609 bp) encodes 2-hydroxychromene-2-carboxylate isomerase (deduced molecular weight, 23,031). This DNA has a high degree of sequence homology (greater than 91% for the first 2161 bp) with a DNA segment from the dox (dibenzothiophene oxidation) operon of Pseudomonas sp. strain C18, which encodes a pathway analogous to that encoded by NAH7. However, 84 bp downstream from nahD, the last gene in the nah operon, this homology ends. This 84-bp sequence at the downstream end of nah and dox homology has 76% homology to a sequence that occurs just upstream of the nah promoter in NAH7. These directly repeated 84-bp sequences thus encompass the upper-pathway nah operon and constitute the ends of a highly conserved region.
Project description:Rhodococcus erythropolis X5 is a psychrotrophic (cold-adapted) hydrocarbon-degrading bacterium, as it showed effective n-alkane destruction at low positive temperatures. Here, the genome of strain X5 was completely sequenced; it consists of a 6,472,161-bp circular chromosome (62.25% GC content) and a 526,979-bp linear plasmid, pRhX5-526k (62.37% GC content).
Project description:NAH7 and pWW0 from gammaproteobacterial Pseudomonas putida strains are IncP-9 conjugative plasmids that carry the genes for degradation of naphthalene and toluene, respectively. Although such genes on these plasmids are well-characterized, experimental investigation of their conjugation systems remains at a primitive level. To clarify these conjugation systems, in this study, we investigated the NAH7-encoded conjugation system by (i) analyzing the origin of its conjugative transfer (oriT)-containing region and its relaxase, which specifically nicks within the oriT region for initiation of transfer, and (ii) comparing the conjugation systems between NAH7 and pWW0. The NAH7 oriT (oriTN) region was located within a 430-bp fragment, and the strand-specific nicking (nic) site and its upstream sequences that were important for efficient conjugation in the oriTN region were identified. Unlike many other relaxases, the NAH7 relaxase exhibited unique features in its ability to catalyze, in a conjugation-independent manner, the site-specific intramolecular recombination between two copies of the oriTN region, between two copies of the pWW0 oriT (oriTW) region (which is clearly different from the oriTN region), and between the oriTN and oriTW regions. The pWW0 relaxase, which is also clearly different from the NAH7 relaxase, was strongly suggested to have the ability to conjugatively and efficiently mobilize the oriTN-containing plasmid. Such a plasmid was, in the presence of the NAH7?nic derivative, conjugatively transferable to alphaproteobacterial and betaproteobacterial strains in which the NAH7 replication machinery is nonfunctional, indicating that the NAH7 conjugation system has a broader host range than its replication system.Various studies have strongly suggested an important contribution of conjugative transfer of catabolic plasmids to the rapid and wide dissemination of the plasmid-loaded degradation genes to microbial populations. Degradation genes on such plasmids are often loaded on transposons, which can be inserted into the genomes of the recipient bacterial strains where the transferred plasmids cannot replicate. The aim was to advance detailed molecular knowledge of the determinants of host range for plasmids. This aim is expected to be easily and comprehensively achieved using an experimental strategy in which the oriT region is connected with a plasmid that has a broad host range of replication. Using such a strategy in this study, we showed that (i) the NAH7 oriT-relaxase system has unique properties that are significantly different from other well-studied systems and (ii) the host range of the NAH7 conjugation system is broader than previously thought.
Project description:Unculturable polycyclic aromatic hydrocarbon (PAH)-degrading bacteria are a significant reservoir of the microbial potential to catabolize low-molecular-weight PAHs. The population of these bacteria is larger than the population of nah-like bacteria that are the dominant organisms in culture-based studies. We used the recently described phn genes of Burkholderia sp. strain RP007, which feature only rarely in culture-based studies, as an alternative genotype for naphthalene and phenanthrene degradation and compared this genotype with the genotypically distinct but ubiquitous nah-like class in different soils. Competitive PCR quantification of phnAc and nahAc, which encode the iron sulfur protein large (alpha) subunits of PAH dioxygenases in nah-like and phn catabolic operons, revealed that the phn genotype can have a greater ecological significance than the nah-like genotype.
Project description:Pseudomonas putida G7 carries a naphthalene-catabolic and self-transmissible plasmid, NAH7, which belongs to the IncP-9 incompatibility group. Adjacent to the putative origin of conjugative transfer (oriT) of NAH7 are three genes, traD, traE, and traF, whose functions and roles in conjugation were previously unclear. These three genes were transcribed monocistronically and thus were designated the traD operon. Mutation of the three genes in the traD operon resulted in 10- to 10(5)-fold decreases in the transfer frequencies of the plasmids from Pseudomonas to Pseudomonas and Escherichia coli and from E. coli to E. coli. On the other hand, the traD operon was essential for the transfer of NAH7 from E. coli to Pseudomonas strains. These results indicated that the traD operon is a host-range modifier in the conjugative transfer of NAH7. The TraD, TraE, and TraF proteins were localized in the cytoplasm, periplasm, and membrane, respectively, in strain G7 cells. Our use of a bacterial two-hybrid assay system showed that TraE interacted in vivo with other essential components for conjugative transfer, including TraB (coupling protein), TraC (relaxase), and MpfH (a channel subunit in the mating pair formation system).
Project description:Pseudomonas putida G7 is one of the most studied naphthalene-degrading species. The nah operon in P. putida, which is present on the 83 kb metabolic plasmid NAH7, codes for enzymes involved in the conversion of naphthalene to salicylate. The enzyme NahF (salicylaldehyde dehydrogenase) catalyzes the last reaction in this pathway. The nahF gene was subcloned into the pET28a(TEV) vector and the recombinant protein was overexpressed in Escherichia coli Arctic Express at 285 K. The soluble protein was purified by affinity chromatography followed by gel filtration. Crystals of recombinant NahF (6×His-NahF) were obtained at 291 K and diffracted to 2.42 Å resolution. They belonged to the hexagonal space group P6(4)22, with unit-cell parameters a = b = 169.47, c = 157.94 Å. The asymmetric unit contained a monomer and a crystallographic twofold axis generated the dimeric biological unit.
Project description:Crude oil pollution has consistently deteriorated all environmental compartments through the cycle of activities of the oil and gas industries. However, there is a growing need to identify microbes with catabolic potentials to degrade these pollutants. This research was conducted to identify bacteria with functional degradative genes. A crude oil-polluted soil sample was obtained from an aged spill site at Imo River, Ebubu, Komkom community, Nigeria. Bacteria isolates were obtained and screened for hydrocarbon degradation potential by turbidometry assay. Plasmid and chromosomal DNA of the potential degraders were further screened for the presence of selected catabolic genes (C230, Alma, Alkb, nahAC, and PAHRHD(GP)) and identified by molecular typing. Sixteen (16) out of the fifty (50) isolates obtained showed biodegradation activity in a liquid broth medium at varying levels. Bacillus cereus showed highest potential for this assay with an optical density of 2.450 @ 600?nm wavelength. Diverse catabolic genes resident in plasmids and chromosomes of the isolates and, in some cases, both plasmid and chromosomes of the same organism were observed. The C230 gene was resident in >50% of the microbial population tested, while other genes occurred in lower proportions with the least observed in nahAC and PAHRHD. These organisms can serve as potential bioremediation agents.
Project description:The 11-kb sequence encompassing the alkylbenzene upper pathway in Pseudomonas putida 01G3, a psychrotrophic strain able to degrade alkylbenzenes at low temperatures, was characterized. Together with a potential regulator (EbdR), six putative enzymes (EbdAaAbAcAdBC) were identified, and they exhibited highly significant similarities with enzymes implicated in the equivalent pathway in P. putida RE204. ebd genes appeared to be preferentially induced by ethylbenzene. Multiple-alignment data and growth rate measurements led us to classify 01G3 and closely related strains in two groups with distinct substrate specificities. Close to identified genes, remnants of IS5-like elements provided insight into the evolution of catabolic sequences through rearrangements from a less complex ancestral cluster.