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Applied and Environmental Microbiology, April 2005, p. 2036-2045, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2036-2045.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genetic Diversity of Benzoyl Coenzyme A Reductase Genes Detected in Denitrifying Isolates and Estuarine Sediment Communities

Bongkeun Song^1* and Bess B. Ward²

Department of Biological Sciences, University of North Carolina, Wilmington, North Carolina,¹ Department of Geosciences, Princeton University, Princeton, New Jersey²

Received 1 June 2004/ Accepted 12 November 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Benzoyl coenzyme A (benzoyl-CoA) reductase is a central enzyme in the anaerobic degradation of organic carbon, which utilizes a common intermediate (benzoyl-CoA) in the metabolism of many aromatic compounds. The diversity of benzoyl-CoA reductase genes in denitrifying bacterial isolates capable of degrading aromatic compounds and in river and estuarine sediment samples from the Arthur Kill in New Jersey and the Chesapeake Bay in Maryland was investigated. Degenerate primers were developed from the known benzoyl-CoA reductase genes from Thauera aromatica, Rhodopseudomonas palustris, and Azoarcus evansii. PCR amplification detected benzoyl-CoA reductase genes in the denitrifying isolates belonging to -, ß-, or -Proteobacteria as well as in the sediment samples. Phylogenetic analysis, sequence similarity comparison, and conserved indel determination grouped the new sequences into either the bcr type (found in T. aromatica and R. palustris) or the bzd type (found in A. evansii). All the Thauera strains and the isolates from the genera Acidovorax, Bradyrhizobium, Paracoccus, Ensifer, and Pseudomonas had bcr-type benzoyl-CoA reductases with amino acid sequence similarities of more than 97%. The genes detected from Azarocus strains were assigned to the bzd type. A total of 50 environmental clones were detected from denitrifying consortium and sediment samples, and 28 clones were assigned to either the bcr or the bzd type of benzoyl-CoA reductase genes. Thus, we could determine the genetic capabilities for anaerobic degradation of aromatic compounds in sediment communities of the Chesapeake Bay and the Arthur Kill on the basis of the detection of two types of benzoyl-CoA reductase genes. The detected genes have future applications as genetic markers to monitor aromatic compound degradation in natural and engineered ecosystems.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organic compounds are sources of electrons and carbon for microbial communities in the environment. Aromatic compounds, as oxidized intermediates from naturally produced lignin compounds as well as direct inputs from industrial and agricultural activities (13), are the second-most-abundant kind of organic carbon in the environment (1, 14). Aromatic compounds are primarily utilized by oxygen-respiring bacteria utilizing specific mono- or dioxygenase systems (for a recent review, see reference 9). Aerobic degradation requires molecular oxygen as a cosubstrate for the initial attack and ring cleavage. Aromatic compounds introduced into anoxic environments cannot be metabolized by the oxygenase systems due to the absence of molecular oxygen. However, these compounds are utilized by various anaerobic bacteria, such as denitrifying, iron-reducing, selenate-reducing, sulfate-reducing, fermentative, and anaerobic phototrophic bacteria, via pathways that do not require oxygen (for a review, see references 4, 11, and 25).

Some enzymes involved in the anaerobic degradation of aromatic compounds are highly sensitive to oxygen, have novel characteristics, and are quite distinct from those enzymes at work in aerobic metabolic systems (2, 10). Biochemical and genetic studies have detected novel catabolic mechanisms and pathways for the degradation of aromatic acids, amino acids, and hydrocarbons (for a review, see reference 10). Various aromatic acids are initially converted to aromatic coenzyme A (CoA) derivatives by aromatic acid-specific CoA ligases and further converted to benzoyl-CoA by specific enzymes. Aromatic amino acids are metabolized by deamination and oxidoreduction to benzoyl-CoA. In addition, anaerobic degradation of toluene and xylene is initiated by the addition of fumarate to methyl groups and further oxidized by modified ß oxidation to produce benzoyl-CoA. Benzoyl-CoA is thus the central intermediate for the anaerobic degradation of many aromatic compounds. Several novel reductases are involved in the complete degradation of benzoyl-CoA.

Anaerobic degradation of the benzoyl-CoA pathway was studied in detail for Thauera aromatica and Rhodopseudomonas palustris (for a review, see reference 8). Benzoyl-CoA is reduced to cyclohexa-1,5-diene-1-carbonyl-CoA by benzoyl-CoA reductase, and this product is further metabolized in two different pathways represented by T. aromatica and R. palustris. Benzoyl-CoA reductase is the essential enzyme for reducing the benzene ring structure. It is a heterotetramer with , ß, , and subunits. Two subunits ( and ) have two ATP binding sites, and the remaining subunits (ß and ) are involved in binding benzoyl-CoA (3). The biochemical mechanisms of benzene ring reduction were proposed in detail elsewhere (24).

The genomic organizations of the benzoyl-CoA reductase genes in T. aromatica and R. palustris are bcrCBAD and badFEDG, respectively, which encode the , ß, , and subunits (Fig. 1). The benzoyl-CoA reductases of these two bacteria have 82 and 42% amino acid similarity, respectively, to the 2-hydroxyglutaryl-CoA dehydratase in Acidaminococcus fermentans (Table 1), which is involved in glutamate fermentation in these anaerobic bacteria (7, 15). Recently, another benzoyl-CoA reductase gene (bzdNOPQ) was found in the Azoarcus evansii and Azoarcus sp. strain CIB (8, 16). The activity of benzoyl-CoA reductase in Azoarcus sp. strain CIB was demonstrated in cell extracts incubated with radioactively labeled benzoyl-CoA, which was converted to nonaromatic cyclic intermediates (16). The bzdNOPQ gene has 43% amino acid sequence similarity to the benzoyl-CoA reductases of T. aromatica and R. palustris and 44% similarity to 2-hydroxyglutaryl-CoA dehydratase (Table 1). Furthermore, the operonic organizations of anaerobic benzoate degradation pathways in the denitrifying bacteria T. aromatica and A. evansii are quite different, although both of these species are closely related in terms of 16S rRNA gene sequences (8).

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FIG. 1. Primer-targeting locations on the benzoyl-CoA reductase genes. The sequences of primers 1 to 8 are listed in Table 3.

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TABLE 1. Amino acid sequence comparison of benzoyl-CoA reductases

The detection of benzoyl-CoA reductase genes from bacterial pure cultures and environmental samples can be used to determine the genetic capability for anaerobic degradation of aromatic compounds and to monitor the anaerobic degradation of many different aromatic compounds in the environment. Sequence divergence of benzoyl-CoA reductase genes could be used to identify and distinguish among different bacterial populations degrading aromatic compounds in various environments. Microarray or real-time PCR amplification with specific primers for different types of benzoyl-CoA reductase genes could be applicable in environmental studies to determine which types are dominant and activated in particular environmental conditions and to evaluate the population response to variation in environmental factors. Here we describe the first step in this approach; benzoyl-CoA reductase genes were targeted to determine the distribution and the capability for anaerobic degradation of aromatic compounds in bacterial isolates and in the environment.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and growth conditions.
Twenty-eight denitrifying isolates (Proteobacteria) capable of degrading aromatic compounds and one denitrifying bacterial consortium degrading 4-chlorobenzoate were examined in this study. Ecological and geographical origins of each isolate and environmental sample are listed in Table 2. The strains in the genus Thauera were cultivated anaerobically in a minimal salts medium (19) with succinate as a carbon source and nitrate as an electron acceptor. All other strains were grown on M-R2A medium as described previously (26). A stable bacterial consortium from estuarine sediments was obtained as described previously (20).

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TABLE 2. Ecological and geographical origins of isolates and environmental samples

Environmental samples.
A freshwater river sediment sample was obtained by boxcoring from station CT1 (38°48.36'N and 75°54.63'W) in the Choptank River in Maryland in July 2000. Estuarine sediment samples from the upper Chesapeake Bay in Maryland (station CB1, 39°20.83'N and 76°10.95'W) were obtained in April 2001. The sediment samples were stored at –80°C until analyzed. Highly polluted estuarine sediment samples were obtained from the Arthur Kill in New Jersey in 1998 and were stored at 4°C for 4 years before the analysis.

Genomic bacterial DNA isolation from pure cultures and environmental samples.
Genomic DNAs of the bacterial isolates and a bacterial consortium were extracted using a modified phenol-chloroform method (12). The purity of the DNA was determined by measuring absorbance at 230, 260, and 280 nm. Environmental DNAs from sediment samples were extracted using a FastDNA spin kit (Bio 101, Inc.) following the manufacturer's instructions.

Primer design and PCR amplification of benzoyl-CoA reductase genes.
Four different sets of primers for benzoyl-CoA reductase genes were designed using amino acid sequences of Bcr, Bad, and Bzd (Table 3 and Fig. 1). The primers bzA1F and bzD1R were designed by comparing the amino acid sequences of the and subunits of the benzoyl-CoA reductase genes in T. aromatica and R. palustris, respectively. The primers bzQ1F and bzQ1R were based on the DNA sequences of the bzdQ gene in A. evansii. Primers bzCN1F and bzCN1R were designed using the Codehop program (http://bioinformatics.weizmann.ac.il/blocks/codehop.html) with the amino acid sequences of the subunits of T. aromatica, R. palustris, and A. evansii. Primers bzAQ4F and bzAQ4R were designed using the Codehop program with the amino acid sequences of the subunits of benzoyl-CoA reductase. PCR amplification with each set of bzCN1F-bzCN1R primers, bzQ1F-bzQ1R primers, or bzAQ4F-bzAQ4R primers was performed in a total volume of 50 µl containing 5 µl of 10x PCR buffer (500 mM KCl, 200 mM Tris-HCl [pH 8.4]), 1.5 mM MgCl₂, 20 µM of each deoxyribonucleoside triphosphate, 1 µM of each primer, 1 U of Taq polymerase, and 100 ng of genomic or total DNA. The PCR cycle was started with a 5-min denaturation step at 95°C, followed by 30 cycles of denaturation for 30 s at 95°C and primer annealing for 30 s at 60°C, and concluded by a 1-min extension at 72°C. The PCR conditions for reactions with primers bzA1F and bzD1R were as described above except that the extension at 72°C was 3 min. The amplified products were examined in 1.0% agarose gels by electrophoresis and then were purified using a Qiaquick gel extraction kit (QIAGEN, Bothell, Wash.) according to the manufacturer's instructions. DNAs from T. aromatica, A. evansii, and R. plaustris were used as controls for the PCR amplifications.

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TABLE 3. PCR primers for benzoyl-CoA reductase genes

Cloning and sequencing PCR products.
The cleaned PCR products were used for direct cloning with the TOPO-TA cloning system (Invitrogen, Carlsbad, Calif.) following the manufacturer's instructions. The ligated plasmids were transformed in high-transforming-efficiency Escherichia coli TOP10F' (Invitrogen) following the manufacturer's instructions. The transformed cells were plated on Luria agar plates containing 50 µg of kanamycin ml^–1. The inserts were sequenced using BigDye Terminator chemistry (Applied Biosystems, Foster City, Calif.) and a model 310 automated DNA sequencer (Applied Biosystems).

Restriction fragment length polymorphism was used to screen 48 clones from each consortium and sediment clone library. Each amplified PCR product was digested with CfoI restriction enzyme, and the digested samples were visualized on 3% agarose gels. Only unique patterns were sequenced for further analysis.

Phylogenetic analysis of benzoyl-CoA reductase genes.
The amino acid sequences of benzoyl-CoA reductase from T. aromatica (AJ224959) and R. palustris (U75363) as well as putative benzoyl-CoA reductase sequences from A. evansii (AJ428529) and Magnetospirillum magnetotacticum (NZ_AAAP01003789) were obtained from the GenBank database. In addition, the 2-hydroxyglutaryl-CoA dehydratase gene in Acidaminococcus fermentans (X14252) was used as an out-group for comparison. The amino acid sequences of each subunit were aligned using the ClustalW program (http://www.ebi.ac.uk/clustalw/). The phylogenetic analyses were performed with PAUP* 4.0 (23). Phylogenetic trees were reconstructed using the neighbor-joining method (18) with mean character difference and bootstrap values obtained by use of PAUP* 4.0.

Signature sequence (indel) analysis.
The nucleotide sequences of the cloned genes were translated and added to multiple alignments of known benzoyl-CoA reductases and 2-hydroxyglutaryl-CoA dehydratases. Alignments were carried out with ClustalW. The conserved indels were determined according to the method described by Gupta and Griffiths (5, 6).

Nucleotide sequence accession numbers.
Sequences obtained from this study were deposited in GenBank under the accession numbers AY956841 to AY956907.

Top Abstract Introduction Materials and Methods Results Discussion References

 
PCR amplification of benzoyl-CoA reductase genes.
A total of 28 bacterial isolates, one bacterial consortium, and three environmental samples were investigated by detecting benzoyl-CoA reductase genes (Table 2) with four different sets of primers (Table 3). Two sets of primers were specific for the benzoyl-CoA reductase genes in Thauera, Rhodopseudomonas, or Azoarcus strains. Two others were comprised of degenerate primers used to amplify the genes from various bacteria as well as from sediment samples. Primers bzA1F and bzD1R, designed from the and subunits of the benzoyl-CoA reductase genes in T. aromatica and R. palustris, yielded 2.1-kb fragments from the strains belonging to the species T. aromatica, T. chlorobenzoica, and T. selenatis (Table 4). R. palustris and T. aromatica K172 were used as positive controls for PCR amplification. In addition, PCR products were obtained from a denitrifying bacterial consortium capable of degrading 4-chlorobenzoate, which contained Thauera strains as the dominant group in its mixed bacterial population (20). The primers bzQ1F and bzdQ1R, designed to be specific for the bzdQ gene ( subunit) in A. evansii, generated 888-bp fragments from the strains belonging to the species A. tolulyticus, A. toluvorans, and A. toluclasticus (Table 4).

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TABLE 4. PCR amplification of benzyol-CoA reductase genes with various primers

PCR with the degenerate primers bzCN1F and bzCN1R designed from the subunit of benzoyl-CoA reductases from T. aromatica, R. palustris, and A. evansii detected the subunit of benzoyl-CoA reductase genes in strains of the genera Thauera and Azoarcus (Table 4). However, it was not possible to detect benzoyl-CoA reductase genes in other denitrifying bacteria capable of degrading aromatic compounds with these primers. By a comparison of the amino acid sequences of the subunits of benzoyl-CoA reductases from R. palutris, Thauera strains, and Azoarcus strains, the degenerate primers bzAQ4F and bzAQ4R were designed to target a smaller fragment of the subunit of benzoyl-CoA reductase genes (Fig. 2). PCR amplification with these degenerate primers generated 485-bp fragments from various bacteria belonging to the -, ß-, and -Proteobacteria (Table 4). In addition, PCR with primers bzAQ4F and bzAQ4R amplified the subunit of benzoyl-CoA reductase genes from a bacterial consortium and from sediment samples from the Choptank River, the Chesapeake Bay, and the Arthur Kill. The identities of the PCR products were verified by sequencing. However, benzoyl-CoA reductase genes were not detected in strains belonging to the genera Ochrobactrum, Mesorhizobium, or Pseudomonas with any of the primers used in this study.

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FIG. 2. Amino acid sequence alignments of subunits of benzoyl-CoA reductases from Rhodopseudomonas, Azoarcus, and Thauera. Ta, T. aromatica; Tc, T. chlorobenzoica; Ts, T. selenatis; Mma, M. magnetotacticum; Rp, R. palustris; Atl, A. tolulyticus; Atv, A. toluvorans; Atc, A. toluclasticus; Aev, A. evansii; Af, Acidaminococcus fermentans. Asterisks, colons, and periods respectively indicate identical amino acid residues, conserved substitutions of amino acid residues, and semiconserved substitutions of amino acid residues in the aligned sequences.

Phylogenetic analysis of and subunits of benzoyl-CoA reductases.
The translated amino acid sequences of the and subunits in Thauera strains and of the subunits in Azoarcus strains were used for phylogenetic analysis (Fig. 3). Two subunits ( and ) of benzoyl-CoA reductase in strains of the genus Thauera were 30% similar to each other. The sequences of the subunit (BzdQ) in Azoarcus shared overall 37 and 47% amino acid similarities with the - and -subunit sequences, respectively, in Thauera strains. The subunits found in Thauera strains shared more than 98% amino acid sequence similarity with each other; the subunits shared more than 97% amino acid sequence similarity. The BzdQ sequences within the Azoarcus strains shared more than 99% similarity. The 2-hydroxyglutaryl-CoA dehydratase (Hgd) from Acidaminococcus fermentans had only one subunit (HgdC) related to the and subunits of benzoyl-CoA reductases, which had 33 and 49% similarities with the and subunits in Thauera strains, respectively, and 57% similarity with the BzdQ sequences in Azoarcus strains (Fig. 3). M. magnetotacticum had 82 and 80% amino acid sequence similarities with the and subunits of Thauera strains (Fig. 3). The sequence alignments showed the presence of highly conserved amino acids on both of the subunits from Thauera and Azoarcus strains. These regions were targeted to design PCR primers (bzAQ4F and bzAQ4R) for further analysis (Fig. 2).

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FIG. 3. Phylogenetic analysis of the and subunits of benzoyl-CoA reductases in the genera Azoarcus and Thauera. Four hundred thirty-one amino acids were used in each sequence for comparison. BcrA and BzdQ were subunits of benzoyl-CoA reductases, and BcrD was the subunit. The scale bar represents a difference in 5 of 100 amino acids. Bootstrap values greater than 50 are shown. The gene sequences cloned in this study are shown in boldface.

Phylogenetic analysis of subunits of benzoyl-CoA reductases.
PCR products amplified with the primers bzCN1F and bzCN1R, which targeted the subunits of benzoyl-CoA reductase genes, were sequenced and translated to amino acid sequences. Sequence analysis showed that the subunits in Thauera and Azoarcus strains shared overall 32% similarities at the amino acid level and clearly separated into two distinct clusters of bcr and bzd types (Fig. 4). The sequences from T. aromatica and T. chlorobenzoica had 99% amino acid sequence similarities and shared 97% similarity with T. selenatis and 81% similarity with R. palustris. In addition, benzoyl-CoA reductase found in the genome sequence of M. magnetotacticum had 80 and 35% similarities with the subunits of Thauera and Azoarcus strains, respectively (Fig. 4). Thus, benzoyl-CoA reductase genes detected from Thauera strains, R. palustris, and M. magnetotacticum were assigned to the bcr type.

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FIG. 4. Phylogenetic analysis of subunits of benzoyl-CoA reductases in the genera Azoarcus and Thauera. Three hundred nine amino acids were used in each sequence for comparison. The benzoyl-CoA reductases were assigned to either the bzd or the bcr type. The scale bar represents a difference in 5 of 100 amino acids. Bootstrap values greater than 50 are shown. The gene sequences cloned in this study are shown in boldface.

The sequences from A. tolulyticus strains grouped together and shared similarities of 92% with A. evansii, A. toluclasticus, and A. toluvorans. They were assigned to the bzd type. The ß subunit of 2-hydroxyglutaryl-CoA dehydratase (HgdB) from Acidaminococcus fermentans was used as an out-group for phylogenetic analysis and shared 45% similarities with both types.

Phylogenetic analysis of subunits from bacterial isolates and environmental samples.
The PCR products (485 bp) amplified with the degenerate primers bzAQ4F and bzAQ4R from Bradyrhizobium sp. strain 2FB3, Acidovorax sp. strain 2FB7, Ensifer sp. strain 2FB8, Pseudomonas sp. strain 4FB3, and Paracoccus sp. strain 4FB8 were assigned to the bcr type of benzoyl-CoA reductase on the basis of sequence homology and conserved indel analysis (see Fig. 6). Phylogenetic analysis showed that those sequences were related to subunits of benzoyl-CoA reductases found in T. aromatica and R. palustris (Fig. 5). The sequences from Bradyrhizobium sp. strain 2FB3, Acidovorax sp. strain 2FB7, and Paracoccus sp. strain 4FB8 were closely related to the sequence of R . palustris, with 97% amino acid sequence similarities, but they clustered separately from that of R. palustris. The sequences from Ensifer sp. strain 2FB8 and Pseudomonas sp. strain 4FB3 grouped with Thauera strain sequences, with 99% similarities.

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FIG. 6. Conserved indel analysis of benzoyl-CoA reductases of the bcr type. The conserved deletions and insertions of amino acids are highlighted. Ta, T. aromatica; Tc, T. chlorobenzoica; Ts, T. selenatis; Mma, M. magnetotacticum; Rp, R. palustris; Aev, A. evansii; Af, Acidaminococcus fermentans; Ac, Acidovorax sp.; Pa, Paracoccus sp.; Br, Bradyrhizobium sp.; En, Ensifer sp.; Ps, Pseudomonas sp.; CB1, north Chesapeake Bay sediment, CT1, Choptank River sediment; AK, Arthur Kill sediment. Asterisks, colons, and periods respectively indicate identical amino acid residues, conserved substitutions of amino acid residues, and semiconserved substitutions of amino acid residues in the aligned sequences.

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FIG. 5. Phylogenetic analysis of subunits of benzoyl-CoA reductases detected in denitrifying isolates and the environmental samples. One hundred sixty-one amino acids were used in each sequence for comparison. The benzoyl-CoA reductases were assigned to either the bzd or the bcr type. The scale bar represents a difference of 5 in 100 amino acids. Bootstrap values greater than 50 are shown. The gene sequences cloned in this study are shown in boldface. CB1, north Chesapeake Bay sediment; CT1, Choptank River sediment; AK, Arthur Kill sediment.

Analysis of benzoyl-CoA reductase genes from the bacterial consortium capable of degrading 4-chlorobenzoate under denitrifying conditions produced five unique restriction fragment patterns (data not shown). Two clones (4CB2_A2 and 4CB2_A3) were closely assigned to the bcr type and shared 99% amino acid similarities with Thauera strain sequences. Three clones were not closely related to either the bcr or the bzd type of benzoyl-CoA reductases (data not shown).

A total of 45 environmental clones were detected from sediment samples of the Choptank River (CT), the Chesapeake Bay (CB), and the Arthur Kill (AK). On the basis of sequence similarity and conserved indel detection, 26 clones were assigned to either the bcr or the bzd type of benzoyl-CoA reductase genes (Fig. 6 and 7). Phylogenetic analysis of the environmental clones confirmed their assignments and relationships to the bcr and bzd types of benzoyl-CoA reductases (Fig. 5). Three clones (cluster 1) from Chesapeake Bay and one clone (CT1_D7) from the Choptank River were assigned to the bcr type. Cluster I grouped with the gene in R. palustris, with approximately 96% amino acid similarities. Clone CT1_D7 had more than 91% amino acid sequence similarity with other bcr-type sequences. In addition, four clones shared the same deletion and insertion of amino acid signatures with the bcr-type benzoyl-CoA reductases detected in pure cultures (Fig. 6).

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FIG. 7. Conserved indel analysis of benzoyl-CoA reductases of the bzd type. The conserved deletions and insertions of amino acids are highlighted. Ta, T. aromatica; Rp, R. palustris; Atl, A. tolulyticus; Atv, A. toluvorans; Atc, A. toluclasticus; Aev, A. evansii; Af, Acidaminococcus fermentans; CB1, North Chesapeake Bay sediment; CT1, Choptank River sediment; AK, Arthur Kill sediment. Asterisks, colons, and periods respectively indicate identical amino acid residues, conserved substitutions of amino acid residues, and semiconserved substitutions of amino acid residues in the aligned sequences.

A total of 22 clones from sediment samples were assigned to the bzd type (Fig. 5 and 7). Cluster II and cluster III, containing 15 clones from Choptank River and Chesapeake Bay sediments, were closely related to the bzd genes of the Azoarcus strains, with more than 84% amino acid similarities. Clone CB1_E9 had 98% similarity with the known bzd genes. Cluster IV, with 3 clones from the Arthur Kill, shared more than 91% sequence similarities among themselves and had 83% similarities with the Azoarcus sequences. Two clones from the Choptank River and the Arthur Kill clustered together (cluster V) and shared 85% similarities with the known bzd sequences. Clone AK_B3 had 86% similarities with cluster IV and the Azoarcus sequences. All the clones assigned to the bzd type shared two conserved deletions and three conserved insertions on their amino acid sequences (Fig. 7).

Twenty-two additional clones obtained from the sediment samples and a denitrifying consortium were not assigned to either the bcr or the bzd type of benzoyl-CoA reductases due to lower sequence similarities and a lack of conserved indels in their amino acid sequences. Furthermore, sequence analysis with the known subunit of 2-hydroxygluatryl-CoA dehydratase indicated that the 22 unknown sequences could not be identified as 2-hydroxygluatryl-CoA dehydratase genes (data not shown). Thus, the degenerate primers bzAQ4F and bzAQ4R are able to detect at least two types of benzoyl-CoA reductase genes from pure cultures and environmental samples.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Benzoyl-CoA reductase genes were detected in proteobacterial isolates which were previously characterized as aromatic-compound-degrading denitrifiers. Furthermore, the presence of bacterial communities capable of degrading aromatic compounds was demonstrated by the presence of benzoyl-CoA reductase genes that were detected in the sediment samples from freshwater and estuarine sediments. These results suggest that the capability for anaerobic degradation of aromatic hydrocarbons is widespread, but perhaps unrecognized, in the culture collection and is also common in the environment. Thus, using the primers described here to target benzoyl-CoA reductase genes, we can begin to determine the distribution and abundance of anaerobic-bacterium-degrading aromatic compounds as well as monitor the metabolic capabilities for aromatic compound degradation in natural and engineered communities.

Genetic diversity of benzoyl-CoA reductase genes in bacterial isolates.
Sequences of benzoyl-CoA reductase genes in the three previously known examples, T. aromatica, R. palustris, and A. evansii (8), were quite divergent, which made it difficult to design good degenerate primers capable of amplifying benzoyl-CoA reductase genes in various bacterial isolates. Two sets of primers (bzA1F and bzD1R; bzQ1F and bzQ1R) amplified only the benzoyl-CoA reductase genes in Thauera and Azoarcus strains, respectively. Phylogenetic analysis showed that the PCR products amplified with these primers were tightly clustered with the genes of T. aromatica or A. evansii, as was expected based on the primer specificities. Degenerate PCR primers (bzCN1F and bzCN1R) designed by comparing the conserved amino acid residues of the subunits from T. aromatica, R. palustris, and A. evansii detected only the genes from the genera Azoarcus and Thauera. Thus, benzoyl-CoA reductase genes in other bacteria must be more divergent than those found in the genera Azoarcus and Thauera.

New degenerate primers (bzAQ4F and bzAQ4R), designed by comparing the subunits, amplified the correct benzoyl-CoA reductase gene fragments in the genera Azoarcus and Thauera as well as in other denitrifying bacteria. The genes amplified from other denitrifying bacteria were assigned to the bcr type and were closely related to Thauera and Rhodopseudomonas sequences, although they were not detected with the other more specific primer sets.

The genes found in Ensifer and Pseudomonas strains were almost identical to bcr genes in Thauera strains, which implies horizontal gene transfer among these strains. The denitrifying isolates assigned to the genera Acidovorax, Bradyrhizobium, and Paracoccus had the bcr-type benzoyl-CoA reductase with close relatedness to the Rhodopseudomonas sequence. None of the isolates contained the bzd-type benzoyl-CoA reductase found in Azoarcus strains. Thus, two types of benzoyl-CoA reductase genes were found in bacterial isolates, and they appear to have been transferred horizontally across generic lines in different bacteria. However, benzoyl-CoA reductase genes in the strains belonging to the genera Ochrobactrum and Mesorhizobium and in other Pseudomonas strains were not detected with any of the primers used in this study, although the strains tested have been shown to metabolize benzoate (16). There are two possibilities related to the divergence of anaerobic benzoate degradation. First, benzoyl-CoA reductase genes in these bacteria must be highly divergent from those detected with the primers used here. Southern blotting with gene probes might be an alternative method to detect benzoyl-CoA reductase genes in those isolates. Second, these isolates might have alternative pathways of degrading benzoate under anaerobic conditions. Indirect evidence for alternative pathways was found in a benzoate-degrading sulfate reducer which required selenium and molybdenum for benzoate degradation. These elements are not essential in the benzoate degradation of Thauera, Azoarcus, and Rhodopseudomonas strains (17). Therefore, we conclude that the genes involved in anaerobic benzoate degradation are probably even more diverse than previously reported and more diverse than we could document in this study. Further investigation is required to detect benzoyl-CoA reductase genes or alternative genes in other denitrifying bacteria capable of degrading aromatic compounds.

Genetic diversity of benzoyl-CoA reductase genes in environmental samples.
The stable denitrifying bacterial consortium used in this study has been described previously in terms of its ability to degrade 4-chlorobenzoate and its composition in terms of 16S rRNA and dissimilatory nitrite reductase genes (nirS) (20, 22). Both of those studies showed the presence of Thauera strains in this consortium, which was also consistent with the detection in the consortium of bcr-type benzoyl-CoA reductase genes in this study. The sequences from consortium-derived clones 4CB_A2 and 4CB_A3 clustered with the sequences from the genus Thauera, which we conclude are derived from the Thauera strains present in this consortium. Three additional clones (4CB_A1, 4CB_A4, and 4CB_B2), distantly related to the bcr and bzd types (data not shown), might be derived from the other components of this consortium (20).

Environmental clones from the sediment samples of the Choptank River, the Chesapeake Bay, and the Arthur Kill showed the genetic diversity of the bcr and bzd types of benzoyl-CoA reductase genes. A large number of clones (clusters II, III, IV, and V and clones AK_B3 and CB1_E9) were assigned to the bzd type and had close relatedness to the genes of Azoarcus strains. Four clones (cluster I and CT_D7) from Chesapeake Bay and Choptank River sediments were assigned to the bcr type. Amino acid sequence alignments showed the presence of conserved signature amino acids (indels) in both bcr and bzd types of benzoyl-CoA reductase. Among 45 environmental clones, 22 clones were considered to be benzoyl-CoA reductase genes belonging to either the bcr or the bzd type on the basis of conserved indel analysis. The benzoyl-CoA reductase genes detected from the environmental samples are much more diverse than those found in pure cultures. The genetic diversity of benzoyl-CoA reductase genes in environmental samples implies the presence of diverse anaerobes capable of degrading aromatic compounds in sediment communities. Isolating bacteria containing these genes and cloning complete genes from the environmental samples using cosmid or fosmid vectors will be very important for further investigation. Both of these approaches will improve our understanding of which groups of bacteria play major roles in aromatic compound degradation, which types of functional genes represent the activities occurring in various environments, and how aromatic compounds are metabolized.

Ecological significance.
The environmental clones detected from three different sediment samples did not cluster exclusively by their geographical and ecological origins, although geographical patterns were evident. Cluster II contains several clones from sediments from the Choptank River and the Chesapeake Bay, which are similar freshwater environments, although they are geographically separated and likely to receive quite different organic carbon inputs. The detection of similar sequences in both sites was observed in studies of dissimilatory nitrite reductase genes (nirS) (C. Francis and B. Ward, unpublished data). Both studies imply the presence of similar types of denitrifying bacteria in these freshwater reaches of the Choptank River and the Chesapeake Bay. However, most of the clones obtained from three different sites were not closely related, which indicated the presence of different denitrifying communities capable of degrading aromatic compounds.

The genera Azoarcus and Thauera have been highlighted for the studies of aromatic compound degradation under denitrifying conditions. Many bacterial isolates enriched and selected for anaerobic degradation of aromatic compounds coupled to denitrification were assigned to these genera, which implies that they are major bacterial groups for aromatic compound degradation under denitrifying conditions. However, the study of halobenzoate-degrading denitrifying isolates showed that many additional groups of bacteria are involved in aromatic compound degradation under denitrifying conditions (21). The present results from sediment samples also imply that many different bacteria are involved in anaerobic degradation of aromatic compounds in the environment. Most of the environmental clones did not cluster closely with the benzoyl-CoA reductase genes from bacterial isolates. Thus, the isolates obtained from various studies related to aromatic compound degradation under denitrifying conditions are not representative of the bacterial populations capable of degrading aromatic compounds in the environment. This disparity between the culture collection and the environment has been detected for nearly every functional gene investigated, and it is thus not surprising in the case of benzoyl-CoA reductase. But this disparity does imply that the ability for anaerobic metabolism of aromatic compounds, once thought to be rare and uncommon in natural systems, is widespread among cultivated isolates and commonly present in natural environments.

 
Jeffrey Corwell provided the box cores from which sediment samples were taken. We thank Caroline Harwood for providing the cells of R. palutris.

This research was supported by a microbial biology postdoctoral research fellowship to Bongkeun Song and by NSF grant OCE-9981482 to Bess B. Ward.

 
* Corresponding author. Mailing address: Department of Biological Sciences, University of North Carolina, Wilmington, NC 28403-5915. Phone: (910) 962-2326. Fax: (910) 962-2410. E-mail: songb{at}uncw.edu.

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Applied and Environmental Microbiology, April 2005, p. 2036-2045, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2036-2045.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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