Real-Time PCR quantification of PAH-ring hydroxylating dioxygenase (PAH-RHDalpha) genes from Gram positive and Gram negative bacteria in soil and sediment samples

Real-Time PCR based assays were developed to quantify Gram positive (GP) and Gram negative (GN) bacterial populations that are capable of degrading the polycyclic aromatic hydrocarbons (PAH) in soil and sediment samples with contrasting contamination levels. These specific and sensitive Real-Time PCR assays were based on the quantification of the copy number of the gene that encodes the alpha subunit of the PAH-ring hydroxylating dioxygenases (PAH-RHDalpha), involved in the initial step of the aerobic metabolism of PAH. The PAH-RHDalpha-GP primer set was designed against the different allele types present in the data base (narAa, phdA/pdoA2, nidA/pdoA1, nidA3/fadA1) common to the Gram positive PAH degraders such as Rhodococcus, Mycobacterium, Nocardioides and Terrabacter strains. The PAH-RHDalpha-GN primer set was designed against the genes (nahAc, nahA3, nagAc, ndoB, ndoC2, pahAc, pahA3, phnAc, phnA1, bphAc, bphA1, dntAc and arhA1) common to the Gram negative PAH degraders such as Pseudomonas, Ralstonia, Commamonas, Burkholderia, Sphingomonas, Alcaligenes, Polaromonas strains. The PCR clones for DNA extracted from soil and sediment samples using the designed primers showed 100% relatedness to the PAH-RHDalpha genes targeted. Deduced from highly sensitive Real-Time PCR quantification, the ratio of PAH-RHDalpha gene relative to the 16S rRNA gene copy number showed that the PAH-bacterial degraders could represent up to 1% of the total bacterial community in the PAH-contaminated sites. This ratio highlighted a positive correlation between the PAH-bacterial biodegradation potential and the PAH-contamination level in the environmental samples studied.     

Insights into the genetic diversity of initial dioxygenases from PAH-degrading bacteria

Alpha subunit genes of initial polyaromatic hydrocarbon (PAH) dioxygenases were used as targets for the PCR detection of PAH-degrading strains of the genera Pseudomonas, Comamonas and Rhodococcus which were obtained from activated sludge or soil samples. Sequence analysis of PCR products from several Pseudomonas strains showed that alpha subunits (nahAc allele) of this genus are highly conserved. PCR primers for the specific detection of alpha subunit genes of initial PAH dioxygenases from Pseudomonas strains were not suitable for detecting the corresponding genes from the genera Comamonas and Rhodococcus. Southern analysis using a heterologous gene probe derived from the P. putida OUS82 PAH dioxygenase alpha subunit identified segments of the PAH-degradation gene cluster from C. testosteroni strain H. Parts of this gene cluster containing three subunits of the initial PAH dioxygenase were isolated. These three subunits [ferredoxin (pahAb), alpha (pahAc) and beta (pahAd) subunit] were amplified by PCR as one fragment and expressed in Escherichia coli DH5alpha, resulting in an active initial dioxygenase with the ability to transform indole and phenanthrene. The DNA sequence alignment of alpha subunits from C. testosteroni H and various PAH-degrading bacteria permitted the design of new primers and oligonucleotide probes which are useful for the detection of the initial PAH dioxygenases from strains of Pseudomonas, Comamonas and Rhodococcus.     

Detection of catabolic genes in indigenous microbial consortia isolated from a diesel-contaminated soil

Bioremediation is often used for in situ remediation of petroleum-contaminated sites. The primary focus of this study was on understanding the indigenous microbial community which can survive in contaminated environment and is responsible for the degradation. Diesel. toluene and naphthalene-degrading microbial consortia were isolated from diesel-contaminated soil by growing on selective hydrocarbon substrates. The presence and frequency of the catabolic genes responsible for aromatic hydrocarbon biodegradation (xylE, ndoB) within the isolated consortia were screened using polymerase chain reaction PCR and DNA DNA colony hybridization. The diesel DNA-extract possessed both the xy/E catabolic gene for toluene, and the nah catabolic gene for polynuclear aromatic hydrocarbon degradation. The toluene DNA-extract possessed only the xylE catabolic gene, while the naphthalene DNA-extract only the ndoB gene. Restriction enzyme analysis with HaeIII indicated similar restriction patterns for the xylE gene fragment between toluene DNA-extract and a type strain, Pseudomonas putida ATCC 23973. A substantial proportion (74%) of the colonies from the diesel-consortium possessed the xylE gene, and the ndoB gene (78%), while a minority (29%) of the toluene-consortium harbored the xylE gene. 59% of the colonies from the naphthalene-consortium had the ndoB gene, and did not have the xylE gene. These results indicate that the microbial population has been naturally enriched in organisms carrying genes for aromatic hydrocarbon degradation and that significant aromatic biodegradative potential exists at the site. Characterization of the population genotype constitutes a molecular diagnosis which permits the determination of the catabolic potential of the site to degrade the contaminant present.     

Molecular characterization of dioxygenases from polycyclic aromatic hydrocarbon-degrading Mycobacterium spp

Polycyclic aromatic hydrocarbon (PAH)-degrading genes nidA and nidB that encode the alpha and beta subunits of the aromatic ring-hydroxylating dioxygenase have been cloned and sequenced from Mycobacterium vanbaalenii PYR-1 [Khan et al., Appl. Environ Microbiol. 67 (2001) 3577-3585]. In this study, the presence of nidA and nidB in 12 other Mycobacterium or Rhodococcus strains was investigated. Initially, all strains were screened for their ability to degrade PAHs by a spray plate method, and for the presence of the dioxygenase Rieske center region by polymerase chain reaction (PCR). Only Mycobacterium sp. PAH 2.135 (RJGII-135), M. flavescens PYR-GCK (ATCC 700033), M. gilvum BB1 (DSM 9487) and M. frederiksbergense FAn9T (DSM 44346), all previously known PAH degraders, were positive in both tests. From the three positive strains, complete open reading frames of the nidA and nidB genes were amplified by PCR, using primers designed according to the known nidA and nidB sequences from PYR-1, cloned in the pBAD/Thio-TOPO vector and sequenced. The sequences showed >98% identity with the M. vanbaalenii PYR-1 nidA and nidB genes. Southern DNA-DNA hybridization using nidA and nidB probes from PYR-1 revealed that there is more than one copy of nidA and nidB genes in the strains PYR-1, BB1, PYR-GCK and FAn9T. However, only one copy of each gene was observed in PAH2.135.     

Genetic diversity of dioxygenase genes in polycyclic aromatic hydrocarbon-degrading bacteria isolated from mangrove sediments

To investigate the diversity of dioxygenase genes involved in polycyclic aromatic hydrocarbon (PAH)-degradation, a total of 32 bacterial strains were isolated from surface mangrove sediments, from the genera Mycobacterium, Sphingomonas, Terrabacter, Sphingopyxis, Sphingobium and Rhodococcus. Two sets of PCR primers were constructed to detect the nidA-like and nahAc-like sequences of the alpha subunit of the PAH ring-hydroxylating dioxygenase. PCR amplified the DNA fragments from all Gram-positive bacteria by using nidA-like primers and from all Gram-negative bacteria, except two, by using nahAc-like primers. The nidA-like primers showed three subtypes of nidA-like gene: (i) fadA1, clustering with nidA3 from M. vanbaalenii PYR-1, (ii) nidA, clustering with nidA from PYR-1, and (iii) fadA2 clustering with dioxygenase from Arthrobacter sp. FB24. The amplicons detected by nahAc-like primers had high sequence homologies to phnA1a from Sphingomonas sp. CHY-1 and were amplifiable from 8 of the 16 Gram-negative isolates. The primer also generated amplicons that had a 32-36% similarity to phnA1a and 53-93% identity to p-cumate dioxygenase. These results suggest that the nidA-like and nahAc-like genes are prevalent in the PAH-degrading bacteria and that they are useful for determining the presence of PAH-dioxygenase genes in environmental samples.     

Characterization of hydrocarbon-degrading microbial populations in contaminated and pristine Alpine soils

Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).     

Distribution of alkB genes within n-alkane-degrading bacteria

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.     

不同富集方法分离多环芳烃降解菌的比较研究

多环芳烃是一类普遍存在的环境污染物。本研究探讨了普通富集法,固定化富集法以及巴斯德消毒后富集法三种途径从相同红树林土壤中分离菲降解茵的差异。通过平板培养和变性梯度凝胶电泳两种方法分析分离结果。上述方法分别获得以鞘氨醇单胞茵、分枝杆菌以及红球茵为优势菌群的群落,表明分离方法对多环芳烃降解菌多样性的研究是一种重要的影响因素。     

Genetic and phenotypic diversity of carbofuran-degrading bacteria isolated from agricultural soils

Thirty-seven carbofuran-degrading bacteria were isolated from agricultural soils, and their genetic and phenotypic characteristics were investigated. The isolates were able to utilize carbofuran as a sole source of carbon and energy. Analysis of the 16S rRNA gene sequence indicated that the isolates were related to members of the genera Rhodococcus, Sphingomonas, and Sphingobium, including new types of carbofuran-degrading bacteria, Bosea and Microbacterium. Among the 37 isolates, 15 different chromosomal DNA patterns were obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences. Five of the 15 representative isolates were able to degrade carbofuran phenol, fenoxycarb, and carbaryl, in addition to carbofuran. Ten of the 15 representative isolates had 1 to 8 plasmids. Among the 10 plasmid-containing isolates, plasmid-cured strains were obtained from 5 strains. The cured strains could not degrade carbofuran and other pesticides anymore, suggesting that the carbofuran degradative genes were on the plasmid DNAs in these strains. When analyzed with PCR amplification and dot-blot hybridization using the primers targeting for the previously reported carbofuran hydrolase gene (mcd), all of the isolates did not show any positive signals, suggesting that their carbofuran hydrolase genes had no significant sequence homology with the mcd gene.     

Molecular characterization of Pseudomonas aeruginosa 2NR degrading naphthalene

Three naphthalene-degrading strains were isolated from compost, characterized by morphological and physiological properties and differentiated by 16S rDNA RFLP. During growth on naphthalene, Pseudomonas aeruginosa 2NR produced ortho catechol pathway intermediates and gentisic acid. The ability to accumulate and degrade gentisic acid shows that Ps. aeruginosa 2NR has a different salicylate pathway to that of the intensely studied Ps. putida NCIB 9816. Molecular analysis showed the presence both of genes of the upper naphthalene pathway and genes of the ortho and meta catechol pathways. The insertion of nagH and nagG, coding for salicylate 5-hydroxylase in Pseudomonas sp. U2, was absent in Ps. aeruginosa 2NR, as in Ps. putida NCIMB 9816.     

Polycyclic Aromatic Hydrocarbon Degradation by a New Marine Bacterium, Neptunomonas naphthovorans gen. nov., sp. nov.

Two strains of bacteria were isolated from creosote-contaminated Puget Sound sediment based on their ability to utilize naphthalene as a sole carbon and energy source. When incubated with a polycyclic aromatic hydrocarbon (PAH) compound in artificial seawater, each strain also degraded 2-methylnaphthalene and 1-methylnaphthalene; in addition, one strain, NAG-2N-113, degraded 2,6-dimethylnaphthalene and phenanthrene. Acenaphthene was not degraded when it was used as a sole carbon source but was degraded by both strains when it was incubated with a mixture of seven other PAHs. Degenerate primers and the PCR were used to isolate a portion of a naphthalene dioxygenase iron-sulfur protein (ISP) gene from each of the strains. A phylogenetic analysis of PAH dioxygenase ISP deduced amino acid sequences showed that the genes isolated in this study were distantly related to the genes encoding naphthalene dioxygenases of Pseudomonas and Burkholderia strains. Despite the differences in PAH degradation phenotype between the new strains, the dioxygenase ISP deduced amino acid fragments of these organisms were 97.6% identical. 16S ribosomal DNA-based phylogenetic analysis placed these bacteria in the gamma-3 subgroup of the Proteobacteria, most closely related to members of the genus Oceanospirillum. However, morphologic, physiologic, and genotypic differences between the new strains and the oceanospirilla justify the creation of a novel genus and species, Neptunomonas naphthovorans. The type strain of N. naphthovorans is strain NAG-2N-126.     

Impact of Inoculation Protocols, Salinity, and pH on the Degradation of Polycyclic Aromatic Hydrocarbons (PAHs) and Survival of PAH-Degrading Bacteria Introduced into Soil

Degradation of polycyclic aromatic hydrocarbons (PAHs) and survival of bacteria in soil was investigated by applying different inoculation protocols. The soil was inoculated with Sphingomonas paucimobilis BA 2 and strain BP 9, which are able to degrade anthracene and pyrene, respectively. CFU of soil bacteria and of the introduced bacteria were monitored in native and sterilized soil at different pHs. Introduction with mineral medium inhibited PAH degradation by the autochthonous microflora and by the strains tested. After introduction with water (without increase of the pore water salinity), no inhibition of the autochthonous microflora was observed and both strains exhibited PAH degradation.     

Phylogenetic and physiological comparisons of PAH-degrading bacteria from geographically diverse soils

The diversity of bacteria isolated from creosote- contaminated soils in the United States, Norway, and Germany was determined by comparing their ability to degrade polycyclic aromatic hydrocarbons (PAHs), their phospholipid ester-linked fatty acid (GC-FAME) profiles, sole carbon source utilization patterns (Biolog assays (Use of trade names or specific products does not imply endorsement by the U.S. EPA.), and 16S rRNA sequences. Bacteria were initially obtained by enrichment with phenanthrene and fluoranthene. Many were capable of degrading a broad range of the PAHs found in creosote. Phenanthrene- or fluoranthene- degraders were abundant in most of the soils tested. Several of the fluoranthene-degrading isolates clustered with Sphingomonas (formerly Pseudomonas) paucimobilis strain EPA505 in the GC-FAME and Biolog analyses and three of the isolates examined by 16S rRNA sequence comparisons showed a close relationship with Sphingomonas. In addition, the Sphingomonas strains showed the most extensive degradation of 4- & 5-ring PAHs in creosote. Burkholderia cepacia strains isolated on phenanthrene from PAH-contaminated soils had limited ability to attack higher molecular weight PAHs either individually or in creosote. Thus, PAH degradation capabilities appeared to be associated with members of certain taxa, independent of the origin of the soils from which the bacteria were isolated.     

Isolation of Marine Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Cycloclasticus Strains from the Gulf of Mexico and Comparison of Their PAH Degradation Ability with That of Puget Sound Cycloclasticus Strains

Phenanthrene- and naphthalene-degrading bacteria were isolated from four offshore and nearshore locations in the Gulf of Mexico by using a modified most-probable-number technique. The concentrations of these bacteria ranged from 10² to 10⁶ cells per ml of wet surficial sediment in mildly contaminated and noncontaminated sediments. A total of 23 strains of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were obtained. Based on partial 16S ribosomal DNA sequences and phenotypic characteristics, these 23 strains are members of the genus Cycloclasticus. Three representatives were chosen for a complete phylogenetic analysis, which confirmed the close relationship of these isolates to type strain Cycloclasticus pugetii PS-1, which was isolated from Puget Sound. PAH substrate utilization tests which included high-molecular-weight PAHs revealed that these isolates had similar, broad substrate ranges which included naphthalene, substituted naphthalenes, phenanthrene, biphenyl, anthracene, acenaphthene, and fluorene. Degradation of pyrene and fluoranthene occurred only when the strains were incubated with phenanthrene. Two distinct partial PAH dioxygenase iron sulfur protein (ISP) gene sequences were PCR amplified from Puget Sound and Gulf of Mexico Cycloclasticus strains. Phylogenetic analyses of these sequences revealed that one ISP type is related to the bph type of ISP sequences, while the other ISP type is related to the nah type of ISP sequences. The predicted ISP amino acid sequences for the Gulf of Mexico and Puget Sound strains are identical, which supports the hypothesis that these geographically separated isolates are closely related phylogentically. Cycloclasticus species appear to be numerically important and widespread PAH-degrading bacteria in both Puget Sound and the Gulf of Mexico.     

Dust-borne bacteria in animal sheds, schools and children's day care centres

A total of 316 bacterial strains, including psychrophiles, mesophiles and thermophiles, were isolated and identified from indoor dusts in schools, children's day care centres and animal sheds. Several species which had not previously been reported from indoor environments were found: Sphingomonas, Brevibacterium, Nocardiopsis, Deinococcus and Rhodococcus/Gordona. A new psychrophilic actinomycete genus was also found in animal sheds, representing a new undescribed peptidoglycan type and an unusual whole-cell fatty acid composition. The indoor dusts of animal sheds contained mainly the Gram-negative genera Pseudomonas, Pantoea, Flavobacterium and Xanthomonas early in the indoor feeding season, but changed to a composition dominated by Bacillus, Micrococcus and mesophilic and thermophilic actinomycetes towards the end of the season. The dust contained, and air-borne bacterial flora in schools and day care centres were dominated by, Gram-positive bacilli and actinomycetes, notably Bacillus cereus, Brevibacillus brevis, B. licheniformis, B. subtilis and species of Arthrobacter, Corynebacterium, Rhodococcus/Gordona, Nocardiopsis sp., Deinococcus, Staphylococcus and Micrococcus. Indoor air and dust contained Klebsiella oxytoca, Acinetobacter calcoaceticus, Ac. lwoffi, Bacillus cereus and Nocardiopsis dassonvillei with the status of hazard group II. Indoor dusts of animal sheds contained eight different 3-hydroxy fatty acids, the 2-hydroxy fatty acid 14:0 and two 10-methyl fatty acids, whereas in dusts from schools and day care centres, these were below the detection level (< 3.5 ng mg-1). The 3-and 2-hydroxy fatty acids could be assigned to one or more of the dust-contained cultivable strains, but 10-methyl C16:0 was not present in any of the strains isolated. The dusts from schools and children's day care centres contained 0.2-0.3 ng of endotoxin mg-1 and 0.5-1.4 ng of beta-D-glucan mg-1, whereas the dusts from animal sheds contained more 0.3-41 ng mg-1 and 8-35 ng mg-1, respectively.     

一株多环芳烃降解菌的鉴定及GST基因克隆和序列分析

由石油污染土壤中分离到一株能以多环芳烃（菲、芴、萘）为唯一碳源的细菌,经形态观察、生理生化（BiologGN）和 G+C mol%分析,鉴定该菌为少动鞘氨醇单胞菌(Sphingomonas paucimobilis)。与16S rDNA序列同源性的比较进一步确证了鉴定结果。经菲诱导后的细菌谷胱甘肽S转移酶（Glutathione Stransferase, GST）酶活明显高于未诱导前,表明谷胱甘肽S转移酶可能与多环芳烃的降解有关。根据该酶基因的同源性序列设计引物,PCR扩增出编码谷胱甘肽S转移酶基因片段,进一步证实在该菌中有GST的存在。测序后基于编码GST的基因所进行的系统发育分析表明,该多环芳烃降解菌与其它多环芳烃降解菌在进化上亲缘关系较近。     

Evolution of a degradative bacterial consortium during the enrichment of naphtha solvent

A microbial mixed culture able to degrade naphtha solvent, a model of hydrocarbon aromatic mixture, was isolated from a hydrocarbon-polluted soil. Composition of the population was monitored by phenotypic and molecular methods applied on soil DNA, on whole enrichment culture DNA, and on 85 isolated strains. Strains were characterized for their 16S rDNA restriction profiles and for their random amplified polymorphic DNA profiles. Catabolic capabilities were monitored by phenotypic traits and by PCR assays for the presence of the catabolic genes methyl mono-oxygenase ( xylA, M), catechol 2,3 dioxygenase (xylE) and toluene dioxygenase (todC1) of TOL and TOD pathways. Different haplotypes belonging to Pseudomonas putida, Ps. aureofaciens and Ps. aeruginosa were found to degrade aromatic compounds and naphtha solvent. The intrinsic catabolic activity of the microbial population of the polluted site was detected by PCR amplification of the xylE gene directly from soil DNA.     

Characterization of aerobic polycyclic aromatic hydrocarbon-degrading bacteria from Bizerte lagoon sediments, Tunisia

Aims: To characterize polycyclic aromatic hydrocarbon (PAH)‐degrading bacteria from sediments of the Bizerte lagoon, and to determine their ability to resist other pollutants such as antibiotics and heavy metals. Methods and Results: More than 100 strains were isolated for their ability to use fluoranthene as the sole carbon and energy source. Most of them showed antibiotic and heavy metal resistance; 20 representative strains were selected for further analysis. 16S rRNA coding sequences analysis showed that the majority of the selected bacteria (75%) were affiliated to the Gammaproteobacteria. The selected strains also utilized high molecular weight PAHs containing up to four benzene rings and showed different profiles of PAH substrate usage suggesting different PAH degradation pathways. These results are consistent with the fact that nah‐like genes and idoA‐like genes, involved in PAH degradation, were detected in 6 and 1 strains respectively. Conclusions: The Bizerte lagoon, polluted by many human activities, leads to the co‐selection of strains able to cope with multiple contaminants. Significance and Impact of the Study: Polluted areas are often characterized by the concomitant presence of organic pollutants, heavy metals and antibiotics. This study is one of the first showing bacterial strains adapted to multiple contaminants, a promising potential for the development of bioremediation processes.