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Applied and Environmental Microbiology, November 2007, p. 7392-7399, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01099-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Diversity of ndo Genes in Mangrove Sediments Exposed to Different Sources of Polycyclic Aromatic Hydrocarbon Pollution

Newton C. Marcial Gomes,¹ Ludmila R. Borges,² Rodolfo Paranhos,³ Fernando N. Pinto,³ Ellen Krögerrecklenfort,¹ Leda C. S. Mendonça-Hagler,⁴ and Kornelia Smalla^1*

Federal Biological Research Centre for Agriculture and Forestry (BBA), Braunschweig, Germany,¹ Laboratory of Ecology and Biotechnology of Yeast, Department of Microbiology, ICB, UFMG, Belo Horizonte, Brazil,² Laboratory of Hydrobiology, Institute of Biology, CCS, UFRJ, Rio de Janeiro, Brazil,³ Laboratory of Taxonomy and Ecology of Microorganisms, Institute of Microbiology, CCS, UFRJ, Rio de Janeiro, Brazil⁴

Received 16 May 2007/ Accepted 20 September 2007

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Polycyclic aromatic hydrocarbon (PAH) pollutants originating from oil spills and wood and fuel combustion are pollutants which are among the major threats to mangrove ecosystems. In this study, the composition and relative abundance in the sediment bacterial communities of naphthalene dioxygenase (ndo) genes which are important for bacterial adaptation to environmental PAH contamination were investigated. Three urban mangrove sites which had characteristic compositions and levels of PAH compounds in the sediments were selected. The diversity and relative abundance of ndo genes in total community DNA were assessed by a newly developed ndo denaturing gradient gel electrophoresis (DGGE) approach and by PCR amplification with primers targeting ndo genes with subsequent Southern blot hybridization analyses. Bacterial populations inhabiting sediments of urban mangroves under the impact of different sources of PAH contamination harbor distinct ndo genotypes. Sequencing of cloned ndo amplicons comigrating with dominant DGGE bands revealed new ndo genotypes. PCR-Southern blot analysis and ndo DGGE showed that the frequently studied nah and phn genotypes were not detected as dominant ndo types in the mangrove sediments. However, ndo genotypes related to nagAc-like genes were detected, but only in oil-contaminated mangrove sediments. The long-term impact of PAH contamination, together with the specific environmental conditions at each site, may have affected the abundance and diversity of ndo genes in sediments of urban mangroves.

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Polycyclic aromatic hydrocarbons (PAHs) are often accidentally released into the environment via oil spills or as a result of wood and fuel combustion. Despite the oil-catabolic versatility of environmental microorganisms, terrestrial and aquatic environments, e.g., mangrove forests, are still highly susceptible to PAH contamination. Mangrove forests are important natural ecosystems, located in tropical and subtropical countries, which are endangered due to the increased growth of urban areas and due to the constant anthropogenic inputs of chemical contaminants (19). Sediment contamination with PAHs is especially dangerous for mangrove forests, since PAHs can be phytotoxic and can affect plants at all stages of plant growth (20, 26, 28, 38). Furthermore, several studies have shown that contamination with petroleum hydrocarbons might affect the structural and functional diversity of bacterial populations in contaminated soils and sediments (5, 15, 16, 44), which may have large ecological implications. Aerobic PAH degradation in mangrove sediments might occur only in the surface layer and in microsites with sufficient O₂ exposure. Due to the low mobility of PAHs, the microbial community inhabiting this layer is assumed to be crucial for PAH degradation. Thus, the sediment surface is thought to be the dominant site of bacterial activity for carbon mineralization in mangrove forests (3). However, due to the enormous complexity of mangrove ecosystems, we are still far from understanding the biological processes involved in PAH decontamination in such environments. Molecular approaches aiming to unravel the organisms and genes involved in in situ degradation are still needed.

During the last decade, several studies explored the diversity of genes coding for enzymes involved in aerobic PAH degradation and their roles in bacterial adaptation to PAH pollution (6, 30, 33, 50). The multicomponent enzyme system called naphthalene dioxygenase (NDO) initiates the metabolism of low-molecular-weight PAHs (10) and is frequently described as a dominant enzyme group involved in the aerobic degradation of PAHs in the environment (24, 30, 33). Several genes coding for the alpha large subunit of the Rieske-type iron-sulfur protein of this enzyme system have been characterized (2, 22, 32). The alpha subunit confers substrate specificity to the NDO, and the gene coding for this protein makes up part of the gene cluster encoding the complete NDO multicomponent enzyme. However, very often the ndo genes exhibit low levels of sequence similarity and different gene orders (25, 31, 32). This variability makes it a difficult task to design generic primers targeting ndo genes, particularly when these primers also need to be suitable for denaturing gradient gel electrophoresis (DGGE), as primer degeneracies need to be avoided. Therefore, to overcome this problem, it is important to focus the analyses on specific groups of genes which share more sequence similarities. On the basis of the classification system proposed by Nam et al. (35), we designed primers which target the ndo alpha subunits belonging to the main clade of group III, which comprises several ndo genes well known for their environmental importance, including the nahAc and phnAc genes. The main goals of this work were to develop a PCR-DGGE approach based on the newly designed ndo primers and their application to comparison of the relative abundance and diversity of ndo genes (group III) in total community DNA (TC-DNA) from mangrove sediments from different sites. This study provides baseline information about the composition and relative abundance of ndo genes in surface sediments of urban mangroves. Chemical analyses of sediment samples were performed to characterize each sampling site for its PAH contamination.

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Sampling sites and sample processing.
The mangroves investigated in this work were located in Guanabara Bay (Rio de Janeiro, Brazil), an urban area suffering from long-term impacts of hydrocarbon pollution (34a, 40). Sediment samples were taken from three selected urban mangrove forests during the period of low tide at the following coordinates: site 1, 22°46'53"S, 43°04'16"W; site 2, 22°49'25"S, 43°10'35"W; and site 3, 22°44'08"S, 43°13'55"W. The distance between each of the sampling sites was in the range of 10 to 20 km. TC-DNA was extracted from four composite sediment samples taken from each of the three sites. Each of the four samples consisted of four cores (20 cm of top sediment with a 4-cm diameter) randomly collected with at least a 1-m distance from each other and thoroughly mixed in the laboratory before processing. The extraction of microbial cells from the sediment was done by shaking (100 rpm), for 30 min, 5 g of sediment sample in an Erlenmeyer flask containing 5 g of sterile glass beads (4-mm diameter) and 45 ml extraction solution containing Tween 80 (0.1%) and sodium pyrophosphate (0.1%). After the microbial cells were dislodged from the sediment matrices, 15 ml of the supernatant of each sample was centrifuged and the pellet resuspended in absolute ethanol (99.8%) up to a final volume of 1.5 ml and frozen at –60°C. For PAH analyses, three composite samples consisting of three sediment cores were randomly taken from the top sediment layer (20 cm) from each sampling site.

TC-DNA extraction.
For TC-DNA extraction, 1 ml of the microbial cell suspension previously fixed in ethanol (which corresponded to the amount of bacterial cell pellet in 1 g of sediment) was transferred to a lysing matrix E tube containing a mixture of ceramic and silica particles (Q Biogene) and centrifuged for 10 min at 16,000 x g. The supernatant was discarded, and the TC-DNA extraction was performed by using a BIO-101 DNA extraction kit (Q Biogene) according to the manufacturer's recommendations. Mechanical lysis was performed by using a FastPrep FP120 bead-beating system (Q Biogene) two times for 30 s at a maximum vertical velocity of 5.5 m per s.

PCR amplification of ndo gene fragments for DGGE analyses.
A nested PCR-DGGE approach was developed to fingerprint ndo gene fragments amplified from TC-DNA. On the basis of the classification system proposed by Nam et al. (35), we designed new primers targeting ndo genes coding for the alpha subunits belonging to the main clade of group III. The new primers were designed to target conserved regions within the ndo gene sequences (longer than 900 bp) available in the GenBank database under the following accession numbers: AB004059, AB024945, AB066446, AF004284, AF010471, AF036940, AF039533, AF061751, AF252550, AY048759, AY154358, AY154359, AY154360, AY154361, AY154362, AY154365, BSU62430, D84146, M60405, M83949, and PSU49496. The retrieved sequences were aligned using GeneCompar software (version 1.3, Applied Maths), and candidate primers were selected. The primers were optimized with the program Oligo 4.0 (National Biosciences Inc.) and empirically tested against target and nontarget strains. The target strains were the naphthalene-degrading strains Pseudomonas putida G7 (9), Pseudomonas fluorescens OS18P and OS19P (21), P. putida ARS 9, Pseudomonas sp. ARS 10 (I. Kosheleva, unpublished data), P. putida KT2442(pNF142) (13), and Escherichia coli carrying a cloned fragment (994 bp) of a gene closely related to the phnAc gene of Burkholderia sp. strain RP007 (14). The nontarget strains were the toluene-degrading strains Pseudomonas mendocina KR1 (47), Burkholderia cepacia G4 (36), and P. putida mt2 (1). The primer pair NAPH-1F (5'-TGGCTTTTCYTSACBCATG-3') and NAPH-1R (5'-DGRCATSTCTTTTTCBAC-3') was used in a nested PCR approach (amplified fragment size of approximately 896 bp). For the first PCR, a reaction mixture of 25 µl was prepared containing 1x Stoffel PCR buffer II (Applied Biosystems, Foster, CA), 0.2 mM deoxynucleoside triphosphates, 3.75 mM MgCl₂, 2.5 µg bovine serum albumin, 4% (vol/vol) dimethyl sulfoxide, 0.2 µM primers NAPH-1F and NAPH-1R, 1.25 U AmpliTaq gold polymerase (Applied Biosystems), and template DNA (ca. 10 ng). After 7 min of denaturation at 95°C, 35 thermal cycles of 1 min at 94°C, 1 min at 53°C, and 2 min at 72°C were carried out. A final extension step at 72°C for 10 min was performed to finish the reaction. One µl of the product of the first PCR was used as the template for a second PCR using the primers NAPH-2F (5'-TATCACGGCTGG-3') and NAPH-2R GC (5'-ATSTCTTTTTCBAC-3', with a GC clamp attached to the 5' end). These primers were specially designed to target short regions within ndo gene sequences amplified in the first PCR round. The aim of this approach was to enhance the number of environmental ndo gene amplicons and simultaneously attach a GC clamp to the amplified sequences to prevent the complete melting of double-strand DNA during the DGGE analyses (fragment size of approximately 740 bp). The GC-clamp sequence was published elsewhere (37). The reaction mixtures (25 µl) consisted of 1 µl template, 1x Stoffel buffer (Applied Biosystems), 0.2 mM deoxynucleoside triphosphates, 2.5 mM MgCl₂, 1% (vol/vol) dimethyl sulfoxide, 0.2 µM primers, and 2.5 U Amplitaq DNA polymerase (Stoffel fragment; Applied Biosystems). Denaturation was carried out for 5 min at 94°C, after which 32 thermal cycles of 1 min at 95°C, 1.5 min at 51°C, and 2 min at 72°C were performed. A final extension step of 10 min at 72°C finished the reaction.

DGGE analysis of ndo genes.
DGGE of the amplified ndo gene sequences was performed by means of a DCode System (universal mutation detection system; Bio-Rad). The GC-clamped amplicons were applied to a double-gradient polyacrylamide gel containing 6 to 9% acrylamide (7) with a gradient of 26 to 58% of denaturant. The run was performed in 1x Tris-acetate-EDTA buffer at 58°C at a constant voltage of 160 V for 15 h. The DGGE gels were silver stained according to the method of Heuer et al. (18).

PCR targeting nahAc and phnAc genes.
PCR amplifications of the genes nahAc and phnAc were performed with primer sets described by Wilson et al. (49) and Lloyd-Jones et al. (34), respectively. The PCR conditions were according to Gomes et al. (14). A touchdown PCR program was used for the amplification of nahAc-like genes, as described by Wilson et al. (49).

Southern blot hybridization (SBH).
Probes were generated from PCR products of nahAc from P. putida KT2442(pNF142) (14), of phnAc from a cloned phnAc gene fragment (14), and of cloned ndo genes from dominant bands 3NDO-S1, 5NDO-S2, and 4NDO-S3 (this study). The PCR products were excised from the agarose gel and digoxigenin (DIG)-labeled dUTP was added as recommended by the manufacturer (Roche). Southern blotting onto Hybond N nylon membranes (Amersham Pharmacia Biotech) was done according to the method of Sambrook et al. (42). Hybridization was performed under conditions of medium stringency, following the protocol published by Fulthorpe et al. (13). The hybridization of the DIG-labeled probes was detected by using a DIG luminescent detection kit (Roche) as specified by the manufacturer and by exposure to X-ray film (Roche).

Cloning and sequencing.
The ndo gene sequence fragments for cloning were retrieved directly from the second PCR (GC-ndo PCR). Before being cloned, the amplicons obtained from the composite samples from each sampling site were combined, ligated into pGEM-T vectors (Promega), and transformed into competent cells (Escherichia coli JM109; Promega) following the supplier's instructions. Only clones containing inserts which shared electrophoretic mobility with matched dominant bands, as determined by DGGE, were selected for sequencing. BLAST-N and TBLASTX (http://www.ncbi.nlm.nih.gov/BLAST/) were used for identity searches for ndo gene sequences (740 bp). Deduced amino acid sequences corresponding to cloned large alpha subunits of ndo genes were aligned to their closest relatives, and the tree was calculated according to the neighbor-joining method and bootstrapping analysis by using Molecular Evolutionary Genetics Analysis (MEGA3) integrated software.

PAH analysis.
Frozen sediment samples were homogenized, and subsamples (10 g) were treated as described in the Environmental Protection Agency (EPA) (http://www.epa.gov) standard method 3540. The sediment extracts were fractionated by gas chromatography, based on EPA method 3630. The concentrated extract (1 ml) was further analyzed as described in EPA standard method 8270C.

PCA and pairwise permutation tests of significance.
Principal component analysis (PCA) was performed to group sediment sampling sites according to their PAH composition by using the software package Canoco 4.5 (Microcomputer Power, Ithaca, NY). The PAH data were log transformed and analyzed by PCA to reduce the dimensionality. The pairwise permutations to test for significant differences between DGGE fingerprints from different sampling sites were performed on the similarity matrices obtained from a dendrogram constructed with the Pearson correlation coefficients calculated for each pair of lanes (27). The Pearson correlation coefficient was calculated based on the array of densitometric curves of each fingerprint using the software package GelCompar 4.0 (Applied Maths) as described by Smalla et al. (43).

Nucleotide sequence accession numbers.
Representative ndo gene sequences obtained in this study were deposited in the GenBank database under accession numbers EF455674 to EF455679.

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PAH analysis.
The PAH sediment analyses indicated that the compositions and concentrations of PAH contamination were highly different between sampling sites (Table 1). Sampling site 1, which was located close to an environmentally protected area (Guapimirim), had the lowest level of PAH contamination. Sampling site 2, with the innermost location in the city, showed values for total PAH concentration that were about 4.5 times higher than the values for site 1. Sampling site 3, which was close to the petrochemical complex of Duque de Caxias, had the highest level of total PAH contamination, more than 10 times higher than that at site 1. A wide spectrum of alkylated (C_n) and nonalkylated PAH compounds was detected in the samples from all three sites. For all PAHs (except for nonalkylated naphthalene), the lowest concentrations were measured for site 1, while the highest, in general, were measured for the samples from site 3. For three PAH compounds (phenanthrene, C₁ phenanthrene, and fluoranthrene) the concentrations were higher for samples from site 2 than for samples from site 3. Very high concentrations of C₃ dibenzothiaphene, C₃ and C₄ phenanthrene, C₁ and C₂ chrysene, and C₂ pyrene were determined for site 3. PCA was performed to ordinate the sediment sampling sites according to their compositions and levels of PAH contamination (Fig. 1). The ordination biplot of sampling sites and PAH compounds generated by PCA showed clearly that homologous alkylated PAH groups of phenanthrene, dibenzothiophene, fluorene, and chrysene, which are typical aromatic compounds in crude oil, had a strong impact on the spatial ordination of sampling site 3. In contrast, higher levels of homologous nonalkylated PAH groups had a greater impact on the ordination of site 2. Moreover, based on the PAH ratios calculated by the index (other three- to six-ring PAHs)/(series of five alkylated PAHs), known as the pyrogenic index (45, 46), the sources of PAH contamination in the sediments from the three sites could be classified. While sampling site 3 gave values typical of PAH contamination from both petrogenic and pyrogenic sources (0.27 ± 0.12 [mean ± standard deviation]), sampling sites 1 and 2 had values typical for pyrogenic PAHs (0.89 ± 0.45 and 1.84 ± 0.49, respectively). The calculations based on other indices [Fl/(Fl + Py), Fl/Py, IP/(IP + Bghi), and C₀/(C₀ + C₁)F/Py, where Fl is fluoranthrene, Py is pyrene, IP is indeno (1,2,3-cd)pyrene, Bghi is benzo(ghi)perylene, and F is fluorene] (39, 51) gave similar results (data not shown). However, these indices revealed that petrogenic pollution was the main PAH contamination at site 3.

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TABLE 1. Concentrations of individual PAHs in sediment samples from urban mangrove forests in Guanabara Bay

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FIG. 1. Ordination biplot of sampling sites and PAH compounds generated by PCA according to different levels of contamination in the sediment. The PAHs with less impact on sampling site ordination were suppressed from the graphic. PC, principal component.

Detection of ndo genes.
In this study, we used DGGE for ndo gene fingerprinting. The novelty in this approach was the development of an ndo PCR-DGGE system for ndo gene diversity assessment based on a nested PCR strategy. The nested ndo PCR yielded ndo gene amplicons of the expected size for all target strains tested, and no PCR product was detected for nontarget strains (data not shown). The GC-ndo PCR products from all target strains were analyzed by DGGE to check their electrophoretic mobilities. In general, only one strong band per strain was obtained. However, for some strains, additional faint bands were detected (see marker lane in Fig. 2). Therefore, when analyzing the data, it is necessary to take into consideration that this approach might amplify some noise signals along with the targeted sequences. GC-ndo PCR amplicons were retrieved from TC-DNA of all sampling sites. Thus, the ndo DGGE approach enabled us to analyze the relative abundance and diversity of ndo genes in sediment samples from urban mangrove forests (Fig. 2). The ndo DGGE profiles of the samples revealed a few equally abundant dominant bands and several less intense bands, indicating the dominance of a limited number of ndo genotypes in each mangrove site studied. The pairwise permutation tests performed using similarity matrices of ndo DGGE profiles revealed significant differences among the sampling sites (P < 0.05).

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FIG. 2. Sampling site comparisons of DGGE fingerprints of ndo gene fragments amplified from sediment DNA templates. The band positions indicated in the gel correspond to the melting behavior of selected representative ndo gene clones which matched dominant genotypes. From top to bottom, the ndo gene fragments used as markers (M) are phnAc (environmental clone) (13), nagAc (environmental clone) (unpublished data), nahAc [P. putida KT2442(pNF142)] (13), and nahAc (Pseudomonas sp. strain ARS 10) (I. Kosheleva, unpublished data).

Despite the low numbers of bands, the distribution of specific genotypes was sampling site dependent. Genotypes 1NDO-S1 and 3NDO-S1 were detected only at site 1. While genotype 5NDO was detected only as a weak band in one sample from site 1, it was dominant in all four samples from site 2 and in two samples from site 3. Genotypes 2NDO-S3 and 4NDO-S3 were detected in three and two of the four samples from site 3, respectively, but not at any of the other sites. Selected dominant genotypes from each sampling site were chosen for cloning and subsequent sequencing. The sequences obtained from cloned ndo gene fragments matching dominant ndo DGGE bands were aligned with other, closely related alpha subunits retrieved from GenBank by using BLAST-N and TBLASTX searches. The phylogenetic tree of amino acid sequences deduced from ndo gene fragments from cloned sequences and their closest relatives is shown in Fig. 3. The ndo gene sequences retrieved from band types 1NDO-S1, 3NDO-S1, 5NDO-S2, and 5NDO-S3 showed the closest phylogenetic relationship to the phnAc gene of Burkholderia sp. strain Eh1-1 (AY367787). However, these sequences formed a cluster distant from the sequences of their closest relatives. The cloned sequences retrieved from 2NDO-S3 and 4NDO-S3 were phylogenetically closely related to nagAc of Polaromonas naphthalenivorans CJ2 (22) (DQ167474) and Ralstonia sp. strain U2 (12) (AF036940), respectively. The TBLASTX search also revealed that both sequences were highly similar (96 to 100% identity) to ndo alpha subunits described for members of the family Comamonadaceae (Polaromonas, Delftia, and Comamonas) (data not shown).

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FIG. 3. Phylogenetic relationships of the amino acid sequences of the large alpha subunits deduced from the ndo genes. The sequences were aligned with related sequences retrieved from GenBank. The narAa gene from a PAH-degrading gram-positive bacterium (Rhodococcus sp. strain NCIMB12038) was used as the outgroup. The tree was constructed by using the neighbor-joining method and bootstrapping analysis (1,000 repetitions). In parentheses are the numbers of cloned ndo gene sequences matching the selected dominant genotypes. The numbers on the branches indicate percentages of bootstrap values. The scale bar represents the percentage of amino acid divergence.

SBH analyses.
To confirm the ndo DGGE results, ndo genes were PCR amplified from sediment TC-DNA by means of the three primer systems targeting ndo (first PCR of the ndo DGGE approach), nahAc, and phnAc and were subsequently hybridized (Table 2). While no PCR amplicons or SBH signals of the predicted sizes were obtained with the nahAc primers, phnAc PCR products and SBH signals were detected in two samples each from sampling sites 1 and 3. PCR products of the predicted size were generated with ndo-specific primers (first PCR) for all samples from all sampling sites. The Southern-blotted amplicons did not hybridize with the phnAc-derived probe or the nahAc probe (Table 2). A good correlation with the ndo genotypes detected by ndo DGGE was found for SBH with probes generated from representative ndo clones (3NDO-S1, 5NDO-S2, and 4NDO-S3). As was already demonstrated by the ndo DGGE profiles, the SBH analyses with a different set of ndo probes showed that the distribution of ndo genes was sampling site dependent.

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TABLE 2. PCR-SBH analysis of naphthalene dioxygenase genes of DNA samples from urban mangrove sediment in Guanabara Bay

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The results from sediment PAH analyses suggested that the composition and level of PAH contamination at each sampling site correlated with the expected extent and source of PAH contamination. Indeed, based on the PCA, alkylated PAHs had the greatest impact on the spatial ordination of sampling site 3, which was located in the vicinity of the petrochemical complex of Duque de Caxias. All five indicator compounds for crude oil pollution (alkylated series of naphthalene, phenanthrene, dibenzothiophene, fluorene, and chrysene) were detected at high levels in sediments from site 3 (46). Calculations of PAH ratios have frequently been used to distinguish between PAH contamination due to combustion and to petroleum sources (4, 51). The assumption that the main source of PAH contamination in sampling site 3 was oil contamination was strengthened by calculating the pyrogenic index for each sampling site. Furthermore, our results revealed that fuel combustion was the main source of PAH contamination at site 2, which is the innermost site in the city. Although a large number of different PAH compounds was also detected at site 1, the levels determined were one to two orders of magnitude lower. The PAH analyses indicated that different sources of PAH contamination resulted in a characteristic composition and level of PAH pollutants in the mangrove sediments of each sampling site. Langworthy et al. (30) showed that moderate to high PAH concentrations in sediment altered the microbial community structure and increased the frequencies of nahA and alkB genes in river sediments. Laurie and Lloyd-Jones (33) have shown that nah-type genes typically associated with Pseudomonas spp. are not always dominant in the environment and that the phn-type genes may have a greater ecological importance than the nah-like genes. In agreement with their findings, we have shown more recently that the result of naphthalene contamination on the soil bacterial community was an increased abundance of a population closely related to Burkholderia sp. strain RP007. The increased abundance of the RP007-like population correlated with the detection of the phnAc gene (14) in naphthalene-contaminated soil, which was described first for Burkholderia sp. strain RP007. However, in the mangrove sediments which were investigated in this study, neither phn nor nah genotypes seemed to be abundant in the sampling sites with higher levels of PAH contamination. In contrast, while the novel ndo genotypes 1NDO-S1 and 3NDO-S1 were detected in urban mangrove forests with low levels of PAH contamination (site 1), genotype 5NDO was enhanced in sediment from mangrove forests contaminated with high levels of PAHs (sites 2 and 3). The phylogenetic relationship of the 1NDO-S1, 3NDO-S1, and 5NDO-S2 and -S3 genotypes indicated that they belong to a new divergent cluster of ndo genes (Fig. 3) whose products share more than 60% identity at the amino acid sequence level to that of the closest relative (phnAc of Burkholderia sp. strain EH1). Genotypes 2NDO-S3 and 4NDO-S3, which were closely related to nagAc-like genes, were only enriched in sediments of sampling site 3, where oil was determined to be the main source of PAH contamination. These genotypes showed high similarities to the ndo gene encoding the alpha subunits found in members of the family Comamonadaceae. Many genera belonging to this family have been described as degrading aromatic compounds, including low-molecular-weight PAHs (23, 29, 41). The nagAc genotype has been detected in strains isolated from oil-contaminated sites from different parts of the world (12, 22, 24, 48). Recently, Dionisi et al. (8) have shown that the abundance of dioxygenase genes closely related to the nagAc gene in freshwater sediments in the vicinity of coal tar may be an indication of naphthalene contamination. In a cultivation-dependent study performed by Zhou et al. (52), ring-hydroxylating dioxygenase genes were detected in PAH-degrading bacteria isolated from mangrove sediments in China. In their work, nahAc and phnAc were not detected among the isolates, while the phnA1 gene was the prevalent genotype detected instead. However, no in situ information about the diversity of dioxygenase genes in mangrove sediments was provided.

The higher variability of the ndo gene patterns might be the result of a higher spatial heterogeneity of bacterial populations carrying ndo genes, which might mirror scattered PAH pollution. Interestingly, when 16S rRNA gene fragments were amplified from the same DNA samples, very stable DGGE patterns were observed (N. C. M. Gomes, L. Borges, R. Paranhos, F. Pinto, E. Krogerrecklenfort, L. Mendonca-Hagler, and K. Smalla, submitted for publication). While samples from the two most polluted sites revealed an increased abundance of a few ndo genotypes, the highest number of bands (ndo genotypes) was detected in the sediments with the lowest pollution. It is tempting to speculate that, in addition to other environmental variables, the characteristic level and composition of PAH pollution may have influenced the site-specific bacterial community composition (Gomes et al., submitted) and the abundance and diversity of ndo genes in the bacterial community. Although the role of the detected novel ndo genotypes in the aerobic degradation of low-molecular-weight PAHs in contaminated mangroves remains to be demonstrated, these genotypes were more abundant than the nah and phn genotypes frequently detected in other studies.

In conclusion, this study provides new insights into the abundance and diversity of ndo genes in sediments of urban mangrove forests by combining a newly developed fingerprinting approach and PCR-Southern blot analysis. Despite the variability of the patterns, the ndo DGGE approach allowed us to compare multiple samples from three different mangrove forests which were located a few kilometers apart and to detect site-specific ndo genotypes. The long-term impact of PAH contamination, together with the specific environmental conditions at each site, may have affected the ndo gene diversity in sediments of urban mangroves. Sequencing analyses of more-abundant ndo types provided insights into the kinds of ndo genes that were present in surface sediments and allowed us to detect novel genotypes. The importance of the detected catabolic genotypes and the bacterial populations carrying these genes for in situ degradation of PAHs in contaminated mangrove sediments still needs to be investigated.

 
This study was funded by Deutsche Forschungsgemeinschaft SM59/4-1.

We thank I. Kosheleva for kindly providing the reference degrading strains used in this work.

 
* Corresponding author. Mailing address: BBA, Plant Virology, Microbiology and Biosafety, Messeweg 11-12, D-38104 Braunschweig, Germany. Phone: 49-531-2993814. Fax: 49-531-2993013. E-mail: k.smalla{at}bba.de

Published ahead of print on 28 September 2007.

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Applied and Environmental Microbiology, November 2007, p. 7392-7399, Vol. 73, No. 22
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