INTRODUCTION

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Hydrolytic dehalogenation by haloalkane dehalogenases is the most important step in the biodegradation of 1-halo-n-alkanes and ,-dihalo-n-alkanes. Haloalkane dehalogenases active against long-chain haloaliphatic compounds are present in the gram-positive haloalkane-utilizing bacterial strains Y2, isolated in the United Kingdom (16), m15-3, isolated in Japan (25), HA1, isolated in Switzerland (20), GJ70, isolated in The Netherlands (7), and NCIMB13064, isolated in the United Kingdom (2). These enzymes are different from the extensively studied haloalkane dehalogenase (DhlA) found in several gram-negative 1,2-dichloroethane-utilizing bacteria of the genera Xanthobacter and Ancylobacter (8, 23). DhlA is able to dehalogenate 1,2-dichloroethane, which is a poor substrate for other haloalkane dehalogenases, whereas the enzymes isolated from the gram-positive strains exhibit higher activity toward long-chain mono- and dihalogenated substrates.

Rhodococcus sp. strain NCIMB13064 has been used to study the genetics of haloalkane metabolism in gram-positive bacteria. It is host to the self-transmissible 100-kb plasmid pRTL1 (11), which carries the genes (dhaA, adhA, and aldA) for the initial steps in the aerobic degradation of 1-chlorobutane and several other 1-halo-n-alkanes (Fig. 1). The dhaA gene codes for a haloalkane dehalogenase that hydrolytically converts 1-chlorobutane to n-butanol (12). Based on amino acid sequence similarities, the adhA and aldA genes, which are located downstream of dhaA (Fig. 1B), were proposed to encode the alcohol and aldehyde dehydrogenases that catalyze the oxidative conversion of n-butanol to n-butyric acid (15a). The latter compound is further metabolized by -oxidation to acetate, which can serve as a growth substrate for many bacteria (2). The 13-kb DNA fragment of pRTL1 that has been sequenced harbors two additional genes, dhaR and invA, and the insertion sequence IS2112 (Fig. 1B). The dhaR gene product putatively acts as a repressor-type regulator for dhaA expression (15a), and IS2112 was shown to be involved in several genome rearrangements that resulted in the loss of haloalkane dehalogenase activity in strain NCIMB13064 (10). The invA gene encodes a putative protein that shares extensive similarity with the DNA invertase Pin of Escherichia coli (50%) and Hin of Salmonella enterica serovar Typhimurium (48%) (15a). The Pin and Hin invertases are responsible for the inversion of a specific DNA fragment that can serve as a genetic switch between the expression of alternative sets of genes (4). However, it is not known whether the putative invertase gene invA plays an analogous role in regulating haloalkane metabolism in Rhodococcus sp. strain NCIMB13064.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   (A) Initial enzymatic steps in the degradation of 1-chlorobutane by Rhodococcus sp. strain NCIMB13064. Below each conversion step, the responsible enzyme is indicated (DhaA, haloalkane dehalogenase; AdhA, alcohol dehydrogenase; AldA, aldehyde dehydrogenase). (B) Genetic organization of the haloalkane-degradative genes dhaA, adhA, and aldA, the regulatory gene dhaR, the putative invertase gene invA, and insertion element IS2112 on plasmid pRTL1 in Rhodococcus sp. strain NCIMB13064 (as determined by DNA sequencing [15a]; accession no. L49435). Arrows indicate the direction of transcription. Solid line, noncoding pRTL1 DNA. The positions of BamHI, ClaI, and EcoRI restriction sites are important for explaining the hybridization results obtained in this study and are indicated by B, C, and E, respectively.

Although the haloalkane dehalogenases of strains Y2, m15-3, HA1, and GJ70 have been characterized (7, 16, 20, 25), nothing is known about the genetics of haloalkane degradation in these strains. The biochemical characteristics of the haloalkane dehalogenases isolated from these strains, however, closely resemble those of the haloalkane dehalogenase (DhaA) from Rhodococcus sp. strain NCIMB13064. Furthermore, the amino-terminal sequences of the dehalogenases from strains Y2 and HA1 are identical to that of DhaA. This apparent homology raises interesting questions about the evolution and distribution of these enzymes since the organisms were isolated from geographically separate locations. To determine the degree of homology between the haloalkane dehalogenases and to obtain insight in their evolution and distribution, we analyzed the genetic organization and genomic location of the haloalkane dehalogenase gene region in each of the strains Y2, m15-3, HA1, and GJ70 and in another gram-positive haloalkane-degrading bacterium, strain TB2, newly isolated from an industrial site in the United States. In addition, we determined the phylogenetic affiliation of the different haloalkane degraders by 16S rRNA gene sequence analysis. The results indicate that the analyzed gram-positive haloalkane degraders are members of the genus Rhodococcus, and despite the fact that they were isolated independently from different continents, they all possess a haloalkane catabolic gene cluster highly similar to the one found on plasmid pRTL1 in Rhodococcus sp. strain NCIMB13064.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Haloalkane-utilizing bacteria used in this study. The bacterial strains screened for the presence of the dhaA gene are listed in Table 1. Strains Y2, m15-3, HA1, and GJ70 were isolated from widely separated sites by different research groups (6, 16, 19, 24). Strains NCIMB13064 and 170 (formerly known as Pseudomonas cichorii 170) have been investigated previously and were shown to possess identical dhaA genes (12, 14). Strain TB2 was isolated in our laboratory from a soil sample obtained from a trichloropropane-polluted site in the United States and is capable of growth with 1-chlorobutane as the sole carbon and energy source.

PCR amplification, cloning, and sequencing of putative dhaA genes. Total genomic DNA was isolated from nutrient broth-grown cells by a phenol extraction procedure described elsewhere (14) and was directly used as the template for PCR amplification. Putative dhaA genes were amplified with the same primers that were successfully used to amplify the dhaA gene from Pseudomonas pavonaceae 170 (14): 5'-AAAATCGCCATGGCAGAAATCGGTA-3' and 5'-TGGACATCGGACCATGGCGTGAACC-3' (NcoI sites are underlined). The amplification reaction mixture (100 µl) contained standard Taq amplification buffer, 250 µM (each) deoxyribonucleotide triphosphate, 100 ng of each primer, 100 ng of genomic DNA, and 2 U of Taq DNA polymerase (Boehringer GmbH, Mannheim, Germany). The cycling parameters were 94°C for 5 min followed by 30 cycles of 94°C for 60 s, 58°C for 60 s, and 72°C for 90 s, with a final elongation step of 72°C for 10 min. Samples (10 µl) of the reaction mixtures were subjected to electrophoresis in 0.8% agarose gels, and PCR products were stained with ethidium bromide.

16S rRNA gene sequence analysis. The 16S rRNA genes were amplified with the oligonucleotide primers that were described by Marchesi et al. (13): 63f (5'-CAGGCCTAACACATGCAAGTC-3') and 1387r (5'-GGGCGG(A/T)GTGTACAAGGC-3') (numbering based on the E. coli 16S rRNA gene [1]). The amplification reaction mixture contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl₂, 0.01% bovine serum albumin, 0.01% gelatin, 0.05% Triton X-100, 200 µM (each) deoxyribonucleotide triphosphate, 0.5 µM (each) primer, 100 ng of genomic DNA, and 1 to 2.5 U of Taq DNA polymerase. The cycling parameters were 94°C for 2 min followed by 30 cycles of 92°C for 30 s, 55°C for 30 s, and 75°C for 45 s, with a final elongation step of 75°C for 5 min.

Plasmid visualization. Rhodococcus strains were grown in 10 ml of yeast extract-peptone medium (11) at 28°C to an optical density at 600 nm of 0.9 to 1.0. Cultures were diluted (1:1) with 0.9% NaCl, after which cells from 2 ml of culture were collected in 1.5-ml tubes. Cell pellets were resuspended in 100 µl of 20% Ficoll (approximate molecular weight, 400,000) in 1× Tris-acetate (TAE) buffer (17). Resuspended cells were mixed with 5 µl of fresh lysozyme solution (18% Ficoll, 0.18% bromophenol blue, lysozyme [2.4 mg/ml], and RNase [0.24 mg/ml] in TAE) and incubated for 10 min at room temperature. Then, 15 µl of 2% sodium dodecyl sulfate (SDS)-10% Ficoll in TAE was added, and preparations were incubated at 60°C for 20 to 30 min. The mixtures were subsequently transferred into the wells of a 0.9% vertical agarose slab, from which the TAE buffer was carefully removed. Mixtures were overlaid with 50 µl of 2% SDS-5% Ficoll in TAE, after which the wells were sealed with melted agarose. Electrophoresis was performed in TAE buffer for 40 min at 1 V/cm and then at 5 to 6 V/cm until completion. Plasmid DNA was stained with ethidium bromide.

Southern hybridizations. DNA fragments or intact plasmids were separated on an agarose gel and subsequently transferred to a Hybond-N+ membrane (Amersham Pharmacia Biotech) or to a positively charged nylon membrane (Boehringer GmbH) by diffusion blotting (17). The membranes were treated according to the instructions of the manufacturer. Parts of the dhaA gene or of IS2112 were amplified by PCR and were purified using the Qiaquick PCR purification kit (Qiagen) or by using the GFX PCR DNA and gel band purification kit (Amersham). These PCR fragments were labeled with digoxigenin-11-dUTP using the nonradioactive DNA labeling and detection kit from Boehringer GmbH or with fluorescein-11-dUTP by using the gene images random prime labeling module of Amersham and were used as hybridization probes. Hybridization was carried out at 62 to 68°C, and detection of the hybridization signals was performed according to the manufacturer's protocol.

Cloning and sequencing of the dhaA gene regions of strains GJ70 and HA1. To obtain the dhaA gene regions of strains GJ70 and HA1, genomic libraries of these strains were constructed using the cosmid vector pLAFR3 (21). Total DNA was partially digested with Sau3A, and fragments of 15 to 25 kb in size were isolated and ligated into the BamHI site of pLAFR3. Ligation mixtures were packaged in vitro by using the DNA packaging kit of Boehringer GmbH. E. coli HB101 was transduced with these packaging mixtures (17), and colonies were selected on LB agar plates without NaCl (LBZ) (15) containing tetracycline (12.5 µg/ml). Tetracycline-resistant colonies were screened for dehalogenase activity by monitoring halide production upon incubation with 1,2-dibromoethane as described before (15).

Nucleotide sequence accession numbers. The 16S rRNA gene sequences of strains TB2, m15-3, HA1, Y2, NCIMB13064, and GJ70 have been submitted to the DDBJ/EMBL/GenBank databases under accession no. AJ250924, AJ250925, AJ250926, AJ250927, AJ250928, and AJ250929, respectively. The nucleotide sequence data of the regions upstream of the invA gene in strains GJ70 and HA1 have been submitted under accession no. AJ250982 and AJ250983, respectively.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

16S rRNA gene sequence analysis. The haloalkane-utilizing bacterial strains Y2, NCIMB13064, m15-3, HA1, GJ70, and TB2 were isolated by different research groups from geographically distinct locations (Table 1). The first four strains were previously identified as Rhodococcus erythropolis Y2 (16), Rhodococcus rhodochrous NCIMB13064 (2), Corynebacterium sp. strain m15-3 (25), and Arthrobacter sp. strain HA1 (19). Strain GJ70 was previously identified as a gram-positive actinomycete-like organism (7). In order to establish precisely the phylogenetic affiliation of these haloalkane-utilizing bacteria, we determined their 16S rRNA gene sequences.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Phylogenetic tree based on 16S rRNA gene sequence analysis, illustrating the relationships of the six haloalkane-degrading strains GJ70, HA1, NCIMB13064, Y2, m15-3, and TB2 to the most closely related bacteria. Base positions 54 to 1368 (numbering based on the E. coli 16S rRNA gene) were included in the analysis. Scale bar, 0.02 fixed mutation per site. Bootstrap values were derived from 500 analyses. A sequence from Arthrobacter globiformis was used as the outgroup.

Sequence identity among dhaA genes of haloalkane-utilizing bacteria. Haloalkane dehalogenases active against long-chain haloaliphatic compounds were previously isolated from strains Y2 (16), m15-3 (25), HA1 (20), and GJ70 (7). On the basis of their substrate specificities and their identical N-terminal amino acid sequences, these four dehalogenases have been classified as one group of related enzymes (3). The biochemical characteristics and amino-terminal sequences of these dehalogenases suggest that they closely resemble the haloalkane dehalogenase (DhaA) that had been found in strain NCIMB13064 (12) and recently also in P. pavonaceae 170 (14). To determine the extent of sequence similarity between the haloalkane dehalogenase genes of strains Y2, m15-3, HA1, and GJ70 and the dhaA genes that are present in strains NCIMB13064 and 170, the putative dhaA genes of the first four strains were amplified with primers designed for the 5' and 3' ends of the dhaA gene (12). In addition, we also screened the newly isolated strain TB2 for the presence of the dhaA gene.

Genetic organization of the dhaA gene regions in haloalkane-utilizing Rhodococcus strains. Southern blot hybridization analysis under high-stringency conditions with dhaA- and IS2112-specific probes was used to determine the extent of the conserved dhaA segments in strains m15-3, HA1, Y2, TB2, and GJ70, compared to the sequenced dhaA gene region of strain NCIMB13064 (Fig. 1B). When the complete dhaA gene was used as the probe, BamHI-digested DNA of strains HA1, m15-3, NCIMB13064, Y2, and TB2 revealed single hybridization signals at about 5.6 kb (data not shown). For BamHI-digested DNA of strain GJ70, a single hybridization signal at about 9 kb was found. We examined the regions flanking the dhaA gene by digesting the genomic DNAs with EcoRI or ClaI. Since the dhaA gene has two restriction sites for EcoRI close to each other (Fig. 1B), only two hybridizing bands were expected, and these were found. EcoRI-digested DNA gave hybridization signals at about 9 and 2 kb for all strains (data not shown). The 2-kb DNA band was identical in size to the EcoRI restriction fragment that includes the dhaR gene in strain NCIMB13064 (Fig. 1B). These results suggest that the six haloalkane degraders possess a conserved DNA segment of at least 11 kb, including a 1.5-kb region upstream, and an 8.5-kb region downstream, of the dhaA gene. Since the dhaA gene has one cutting site for ClaI (Fig. 1B), two DNA bands hybridized with the dhaA gene probe (Fig. 3). One fragment (0.9 kb) was found in DNA from all six strains, which is in agreement with a conserved region downstream of the dhaA gene (Fig. 1B). The size of the second DNA fragment, however, was different for some strains: 5.4 kb for strains m15-3, NCIMB13064, and TB2; 4 kb for strains HA1 and Y2; and 3.5 kb for strain GJ70. These results imply that the DNA regions upstream of the conserved 11-kb dhaA gene segment (upstream of the EcoRI site in the invA gene) in strains HA1, Y2, and GJ70 are different from those in strains NCIMB13064, m15-3, and TB2.

View larger version (75K): [in this window] [in a new window]   FIG. 3.   Results of a Southern hybridization experiment with a dhaA gene probe and ClaI-digested total DNA of strains GJ70 (lane 2), HA1 (lane 3), m15-3 (lane 4), NCIMB13064 (lane 5), and Y2 (lane 6). ClaI-digested total DNA of the dhaA-carrying gram-negative bacterium P. pavonaceae 170 (14) was used as a positive control (lane 1). Lane 7, digoxigenin-labeled DNA markers of 23.1, 9.4, 6.7, 4.4, 2.3, and 2.0 kb. Hybridization signals identical in size to the hybridization signals obtained for strains NCIMB13064 and m15-3 were also observed for strain TB2 (result not shown).

View larger version (12K): [in this window] [in a new window]   FIG. 4.   Schematic overview of the sequenced DNA regions upstream of the EcoRI site in the invA gene of strains NCIMB13064, GJ70, and HA1. DNA segments in strain HA1 that differ from the corresponding region in strain NCIMB13064 in about 0 to 1 (X), 10 (Z), or 35% (Y) of their positions are indicated. Open boxes, regions in strains HA1 or GJ70 that show no sequence similarity to the corresponding region in strain NCIMB13064; B, C, and E, BamHI, ClaI, and EcoRI restriction sites, respectively.

View larger version (106K): [in this window] [in a new window]   FIG. 5.   Results of a Southern hybridization experiment with an IS2112-specific probe and EcoRI-digested total DNA of strains GJ70 (lane 3), HA1 (lane 4), m15-3 (lane 5), NCIMB13064 (lane 6), Y2 (lane 7), and TB2 (lane 8). Lane 1 contained the same markers as lane 7 in Fig. 3. EcoRI-digested total DNA of P. pavonaceae 170 was used as a negative control (lane 2).

View larger version (15K): [in this window] [in a new window]   FIG. 6.   Physical maps of the dehalogenase gene regions of the six Rhodococcus strains isolated from widely separated geographical locations. Arrows indicate the direction of transcription. B, C, and E, BamHI, ClaI, and EcoRI restriction sites, respectively.

Genomic localization of the dhaA gene region. Since the haloalkane catabolic gene cluster of strain NCIMB13064 is located on a self-transmissible plasmid (pRTL1) (11, 15a), it is reasonable to propose that plasmid transfer has played a role in the dissemination of this gene cluster among the Rhodococcus strains. Using a direct lysis method, which prevents shearing of plasmid DNA, plasmids were detected in all analyzed strains (Fig. 7A). Plasmids of the same size, slightly smaller than 80-kb plasmid pRTL2 (11) of strains NCIMB13064 and S92 (a derivative of strain NCIMB13064) (11), were detected in strains Y2, TB2, m15-3, GJ70, and HA1. In strains TB2 and HA1, plasmids comparable in size to the 100-kb plasmid pRTL1 of strains NCIMB13064 and S92 were also present. In strain GJ70, additional smaller plasmids were detected (Fig. 7A).

View larger version (62K): [in this window] [in a new window]   FIG. 7.   Visualization of plasmids of haloalkane-degrading Rhodococcus strains. Agarose gel (A) and autoradiogram (B) of the same gel after Southern hybridization with the dhaA gene as the probe. Lane 1, strain Y2; lane 2, strain TB2; lane 3, strain m15-3; lane 4, strain GJ70; lane 5, strain HA1; lane 6, strain NCIMB13064; lane 7, strain S92 (derivative of strain NCIMB13064) (11). C and P, chromosomal and plasmid DNA, respectively.