Dynamic changes in nahAc gene copy numbers during degradation of naphthalene in PAH-contaminated soils

Many bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) contain the nahAc gene that encodes a component of multimeric naphthalene dioxygenases. Because the nahAc gene is highly conserved, this gene serves as a potential biomarker for PAH degradation activity. The aim of this research was to examine the relationship between the rate of naphthalene degradation and the copy number of the nahAc gene in soils using conventional and real-time PCR. Four sets of degenerate primers for real-time PCR were designed based on the nahAc DNA sequences of 33 bacterial species. Before addition of naphthalene, copy numbers of the nahAc gene were below the detection limits of the assay at 5×10³ copy numbers per gram of soil, but increased by over a thousand fold to 10⁷ copies after 6 days of exposure to naphthalene vapors (approximately 30 ppm soil water concentration). Two unreported naphthalene dioxygenase homologs were found in the naphthalene-spiked soil by cloning and sequencing of the PCR products from the nahAc primers. Results of these experiments demonstrate the highly dynamic changes that occur in soil microbial communities after exposure to naphthalene and suggest that there is a direct relationship between gene copy numbers and degradation rates for naphthalene in PAH-contaminated soils.     

Abundance of dioxygenase genes similar to Ralstonia sp. strain U2 nagAc is correlated with naphthalene concentrations in coal tar-contaminated freshwater sediments

We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-microl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 +/- 0.7) x 10(3) to (2.9 +/- 0.3) x 10(5) copies of nagAc-like dioxygenase genes per microg of DNA extracted from sediment samples. These values corresponded to (1.2 +/- 0.6) x 10(5) to (5.4 +/- 0.4) x 10(7) copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene.     

Naphthalene-degrading bacteria of the genus <Emphasis Type="Italic">Rhodococcus</Emphasis> from the Verkhnekamsk salt mining region of Russia

Eight moderately halotolerant naphthalene-degrading strains of the genus Rhodococcus isolated from soil samples and slime pit bottom sediment of the Verkhnekamsk salt mining region of Russia were characterized by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S ribosomal RNA gene sequence analysis. The diversity of their dioxygenase (nar-like) genes was investigated as these genes are known to be involved in naphthalene-degradation. The analysis of the nar-like genes identified revealed their heterogeneity in the strains under study and identity to the known sequences of nar-like genes of previously characterized from members of the genus Rhodococcus.     

Abundance of Dioxygenase Genes Similar to Ralstonia sp. Strain U2 nagAc Is Correlated with Naphthalene Concentrations in Coal Tar-Contaminated Freshwater Sediments

We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-μl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 ± 0.7) × 10³ to (2.9 ± 0.3) × 10⁵ copies of nagAc-like dioxygenase genes per μg of DNA extracted from sediment samples. These values corresponded to (1.2 ± 0.6) × 10⁵ to (5.4 ± 0.4) × 10⁷ copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene.     

Development of Catechol 2,3-Dioxygenase-Specific Primers for Monitoring Bioremediation by Competitive Quantitative PCR

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10² to 10³ gene copies, which was lowered to 10⁰ to 10¹ gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.     

Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry

The effect of microbial inoculation on the mineralization of naphthalene in a bioslurry treatment was evaluated in soil slurry microcosms. Inoculation by Pseudomonas putida G7 carrying the naphthalene dioxygenase (nahA) gene resulted in rapid mineralization of naphthalene, whereas indigenous microorganisms in the PAH-contaminated soil required a 28 h adaptation period before significant mineralization occurred. The number of nahA-like gene copies increased in both the inoculated and non-inoculated soil as mineralization proceeded, indicating selection towards naphthalene dioxygenase producing bacteria in the microbial community. In addition, 16S rRNA analysis by denaturing gradient gel electrophoresis (DGGE) analysis showed that significant selection occurred in the microbial community as a result of biodegradation. However, the indigenous soil bacteria were not able to compete with the P. putida G7 inoculum adapted to naphthalene biodegradation, even though the soil microbial community slightly suppressed naphthalene mineralization by P. putida G7.     

应用TaqMan荧光定量PCR检测土拉弗朗西斯菌

目的：利用Roche LightCycler实时定量PCR系统建立一种快速、灵敏、特异的检测土拉弗朗西斯菌的方法。方法：基于TaqMan荧光探针实时定量PCR技术,选择土拉弗朗西斯菌染色体上的特异序列[醇醛酮还原酶（AKR）和外膜蛋白FopA基因]作为检测靶序列,建立土拉弗朗西斯菌实时定量PCR检测方法;评价该检测方法的特异性和灵敏性;采用克隆菌株污染环境土壤来模拟实际样品,评价该检测方法在快速检测与现场检测等实际应用中的表现。结果：优化筛选基因组中的FT-AKR和FT-fopA片段作为检测靶序列,所建立的土拉弗朗西斯菌实时定量PCR检测方法检测克隆菌株质粒的灵敏度均为10个拷贝/每个反应体系;以其他非土拉弗朗西斯菌为模板未出现非特异扩增;模拟环境土壤样品检测灵敏度2个引物对分别为440和960CFU/g土壤;盲测实验结果显示对于灵敏度范围内的阳性样本均能正确识别,并能正确检测出不同浓度的阳性样本。以FT-fopA片段为靶序列的扩增效率不及基于FT-AKR引物对的扩增。结论：基于FT-AKR片段的引物对扩增效率高,检测土拉弗朗西斯菌具有特异、灵敏的特点,对临床诊断、环境污染监测、防治生物突发事件等具有重要意义。     

Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with<Emphasis Type="Italic">HMG I/Y</Emphasis> as an endogenous reference gene

In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T₀) were calculated by comparing threshold cycle (C _T) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsC _T value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.     

Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5°C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 × 10² copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.     

Evaluating the biodegradation of aromatic hydrocarbons by monitoring of several functional genes

Various microbial activities determine the effectiveness of bioremediation processes. In this work, we evaluated the feasibility of gene array hybridization for monitoring the efficiency of biodegradation processes. Biodegradation of ¹⁴C-labelled naphthalene and toluene by the aromatic hydrocarbon-degrading Pseudomonas putida F1, P. putida mt-2 and P. putida G7 was followed in mixed liquid culture microcosm by a preliminary, nylon membrane-based gene array. In the beginning of the study, toluene was degraded rapidly and increased amount of toluene degradation genes was detected by the preliminary gene array developed for the study. After toluene was degraded, naphthalene mineralization started and the amount of naphthalene degradation genes increased as biodegradation proceeded. The amount of toluene degradation genes decreased towards the end of the study. The hybridization signal intensities determined by preliminary gene array were in good agreement with mineralization of naphthalene and toluene and with the amount of naphthalene dioxygenase and toluene dioxygenase genes quantified by dot blot hybridization. The clear correlation between the results obtained by the preliminary array and the biodegradation process suggests that gene array methods can be considered as a promising tool for monitoring the efficiency of biodegradation processes.     

Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10(2) to 10(3) gene copies, which was lowered to 10(0) to 10(1) gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2, 3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2, 3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.     

萘降解细菌的分离及其降解基因的分子检测

从污水处理厂的活性污泥和石油工业废水中各分离到24个降解萘的细菌分离株,提取这些分离株的总DNA,然后与各种萘降解基因杂交。结果表明,这2个来源的分离株在萘降解基因的种类上有明显不同。来自工业废水的分离株含有萘双加氧酶的铁硫蛋白大亚基基因nahAc,水杨醛脱氢酶基因nahF及其重复基因nahV,水杨酸羟化酶基因nahG及其重复基因nahU,儿茶酚2,3-双加氧酶基因nahH和儿茶酚1,2-双加氧酶基因catA,以及萘趋化蛋白基因nahY。来自活性污泥的分离株只含有nahAc、nahF、nahG和catA,不含有nahY、nahV、nahU和nahH。     

Identification of the Key Genes of Naphthalene Catabolism in Soil DNA

The key genesnahAc and xylEof the naphthalene catabolism of fluorescent Pseudomonas spp. in total soil DNA samples were detected by the polymerase chain reaction (PCR) technique. The collection of fluorescent Pseudomonas spp. was screened for the occurrence of these genes. The results obtained show the possibility of using this approach in the goal-directed search for plasmid-containing naphthalene-degrading fluorescent pseudomonads in soil. The distribution of the naphthalene catabolism genes in soils contaminated with creosote and petroleum products was also studied.     

Microbial dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.     

Microbial Dioxygenase Gene Population Shifts during Polycyclic Aromatic Hydrocarbon Biodegradation

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.     

Effects of the inoculant strain Pseudomonas putida KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community

The naphthalene-degrading activity of a Pseudomonas sp. strain isolated from a creosote-contaminated soil was shown to be encoded by the IncP9 plasmid pNF142 by transfer to Pseudomonas putida KT2442. The effects of the inoculant strain KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community were studied in microcosms with the following treatments: (I) soil, (II) soil with naphthalene, (III) soil with naphthalene and inoculated with KT2442 (pNF142). The inoculant became the dominant bacterial population in treatment (III) as evidenced by cultivation and denaturing gradient gel electrophoresis (DGGE) analysis. The bacterial DGGE profiles revealed drastically reduced complexity due to the numerical dominance of the inoculant. However, group-specific fingerprints (beta-proteobacteria, actinobacteria) that excluded KT2442 (pNF142) showed less severe changes in the bacterial community patterns. A major effect of naphthalene on the soil bacterial community was observed in treatment (II) after 21 days. Two dominant bands appeared whose sequences showed the highest similarity to those of Burkholderia sp. RP007 and Nocardia vinaceae based on 16S rRNA gene sequencing. These bands were less intense in treatment (III). The increased abundance of RP007-like populations due to naphthalene contamination was also confirmed by PCR amplification of the phnAc gene. The nahAc and nahH genes were detected in DNA and cDNA only in treatment III. Although the inoculant strain KT2442 (pNF142) showed good survival and expression of genes involved in naphthalene degradation, this study suggests that KT2442 (pNF142) suppressed the enrichment of indigenous naphthalene degraders.     

Detection and enumeration of aromatic oxygenase genes by multiplex and real-time PCR

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5 degrees C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 x 10(2) copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.     

Degradation of substituted naphthalenesulfonic acids by Sphingomonas xenophaga BN6

Sphingomonas xenophaga BN6 was isolated from the river Elbe as a member of a multispecies bacterial culture which mineralized 6-aminonaphthalene-2-sulfonate. Pure cultures of strain BN6 converted a wide range of amino- and hydroxynaphthalene-2-sulfonates via a catabolic pathway similar to that described for the metabolism of naphthalene to salicylate by Pseudomonas putida NAH7 or Pseudomonas sp NCIB 9816. In contrast to the naphthalene-degrading pseudomonads, S. xenophaga BN6 only partially degraded the naphthalenesulfonates and excreted the resulting amino- and hydroxysalicylates in almost stoichiometric amounts. Enzymes that take part in the degradative pathway of the naphthalenesulfonates by strain BN6 were purified, characterized and compared with the isofunctional enzymes from the naphthalene-degrading pseudomonads. According to the enzyme structures and the catalytic constants, no fundamental differences were found between the 1,2-dihydroxynaphthalene dioxygenase or the 2′-hydroxybenzalpyruvate aldolase from strain BN6 and the isofunctional enzymes from the naphthalene-degrading pseudomonads. The limited available sequence information about the enzymes from strain BN6 suggests that they show about 40–60% sequence identity to the isofunctional enzymes from the pseudomonads. In addition to the gene for the 1,2-dihydroxynaphthalene dioxygenase, the genes for two other extradiol dioxygenases were cloned and sequenced from strain BN6 and the corresponding gene products were studied. S. xenophaga BN6 has also been used as a model organism to study the mechanism of the non-specific reduction of azo dyes under anaerobic conditions and to establish combined anaerobic/aerobic treatment systems for the degradation of sulfonated azo dyes. Furthermore, the degradation of substituted naphthalenesulfonates by mixed cultures containing strain BN6 was studied in continuous cultures and was described by mathematical models. Received 02 April 1999/ Accepted in revised form 09 July 1999