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Previous Article | Next Article 
Applied and Environmental Microbiology, April 2005, p. 1754-1764, Vol. 71, No. 4
0099-2240/05/$08.00+0 doi:10.1128/AEM.71.4.1754-1764.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
The Questor Centre and School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast, United Kingdom
Received 18 August 2004/ Accepted 22 October 2004
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ABSTRACT |
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INTRODUCTION |
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For a long time studies of the biochemistry and genetics of naphthalene utilization have served as a paradigm for research conducted on various groups of bacteria. Indeed, naphthalene is one of the most common xenobiotic compounds present in the environment. Not surprisingly, naphthalene-degrading bacteria are widely distributed in nature (4), and genetic control of the pathways involved in naphthalene degradation has been studied in detail for the gram-negative Pseudomonas species and related species. Both the naphthalene- and salicylate-utilizing phenotypes of Pseudomonas putida G7 have been attributed to genes located on the NAH7 catabolic plasmid (6). Naphthalene catabolism has been studied in most detail in P. putida strains G7 and NCIB9816-4 (reviewed in reference 34), and it occurs via dihydroxylation and cleavage of the first ring and subsequent production of salicylate. Salicylate is then converted into catechol, which is utilized by a route used for a number of aromatic compounds, such as phenol, toluene, etc. The naphthalene degradation genes (nah) of NAH7 are correspondingly organized into two operons encoding the conversion of naphthalene to salicylate (upper operon) and the conversion of salicylate to pyruvate and acetyl coenzyme A (lower operon). The two operons are closely genetically linked to each other and to their common regulatory gene, nahR. The nahR gene encodes a positive regulator protein belonging to the LysR family of transcriptional regulators that are widely distributed in bacteria (35). A different route of naphthalene degradation via the conversion of salicylate to gentisate has also been described (1, 11, 36, 42). The genetic regulation of this pathway was recently studied in detail for Ralstonia sp. strain U2 (11). All of the associated genes were organized in a single operon spanning an 18-kb region and were also regulated by a LysR-type transcriptional regulator (11). In all of these cases salicylate appeared to be the main inducer of the expression of the catabolic genes (11, 34).
In contrast to the cases described above, Rhodococcus sp. strain NCIMB12038 degrades naphthalene via salicylate and gentisate, and naphthalene appears to be the sole inducer (1). The narAa and narAb genes encoding the 
and ß subunits of the naphthalene dioxygenase (NDO) catalytic component iron-sulfur protein (ISPNAR) have been cloned and characterized. Both subunits of the NCIMB12038 NDO showed approximately 30% amino acid identity to the corresponding P. putida NDO subunits, with conservation of the key catalytic residues (19). Rhodococcus strains P200 and P400 were shown to degrade naphthalene via catechol and catechol-2,3-dioxygenase activity and not via the gentisate route (16).
Here we describe and compare the organization of genes (nar region) involved in naphthalene catabolism in three Rhodococcus strains, strains NCIMB12038, P200, and P400. We found that genes in the nar region are not organized in a single operon. Instead, there are several transcription units which differ in the strains investigated but are activated by growth on naphthalene. The primary operon contains only the narAa, narAb, and narB genes. Genes encoding other components shown to be essential for NDO function in Pseudomonas, such as a reductase and ferredoxin, are not located in this operon. An analysis of the gene organization and transcription patterns in the nar regions of different strains and an analysis of the homology of related loci led to the conclusion that genetic exchange and rearrangements played a major part in the evolution of the region of the Rhodococcus genome analyzed.
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MATERIALS AND METHODS |
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and plasmid vector pBluescript KS(+) were used for the cloning experiments.
Media and growth conditions.
Rhodococcus and E. coli strains were propagated in a rich 2YT medium (25). When required, Difco agar (1.8%, wt/vol) was added to the medium. Rhodococcus cells were also grown in minimal M9 medium (33) with naphthalene (1 g/liter), salicylate (3 mM), or pyruvate (15 mM) as a carbon and energy source. Ampicillin (100 µg ml–1), isopropyl-ß-D-thiogalactopyranoside (IPTG) (50 µg ml–1), and 5-bromo-4-chloro-3-indolylphosphate (X-Gal) (50 µg ml–1) were used for detection of recombinant plasmids. YEP medium was used for growth of Rhodococcus prior to DNA isolation (17).
Nucleic acid techniques.
Total DNA from Rhodococcus was isolated as described previously (17). Standard methods of DNA and RNA manipulation were used throughout this work (33). For recovery and purification of DNA fragments from agarose, GFX PCR DNA and a gel band purification kit (Amersham-Pharmacia Biotech) were used. For Southern blot hybridization experiments DNA fragments or whole plasmids were transferred to a Hybond-N+ membrane (Amersham) as described by Sambrook et al. (33). Hybridization was carried out as described previously (15). The partial narAa narAb sequence (1,500 bp) was amplified with primers F200 (5'-GACGTSAATWSSGACTGGAC-3'; nucleotides [nt] 4168 to 4187 of the NCIMB12038 sequence) and R202 (5'-TCTGCCTCCATGTASAGCCA-3'; nt 5609 to 5590) and was used as a hybridization probe.
Pulsed-field gel electrophoresis.
Pulsed-field gel electrophoresis was performed with a clamped homogeneous electrical field electrophoresis system (Bio-Rad, Hercules, Calif.). Agarose-embedded Rhodococcus DNA was prepared as recommended by the manufacturer. Agarose plugs were incubated in 5 ml of lysozyme solution (10 mM Tris [pH 7.2], 50 mM NaCl, 0.2% sodium deoxycholate, 0.5% sodium lauryl sarcosine, 8 mg of lysozyme per ml, 10 U of mutanolysin per ml) at 37°C for 2 h. Plugs were then rinsed with water and incubated in lysis buffer (7% sodium dodecyl sulfate, 1 mg of proteinase K per ml, 100 mM EDTA [pH 8.0], 0.2% sodium deoxycholate) at 55°C for 2 days. The plugs were washed three times (1 h each) in Tris-EDTA buffer at room temperature and stored at 4°C.
Plasmid separation was performed by using 1% pulsed-field-certified agarose in 0.5x Tris-borate-EDTA buffer at 4°C. The gels were electrophoresed for 22 h at 6 V/cm with a switch time of 40 to 80 s and at a field angle of 120°. 
ladder (Bio-Rad) was used as a molecular weight marker. To distinguish between linear and circular forms, the mobilities of the plasmids were analyzed by using two different switch times, 40 to 80 s and 30 to 60 s (12).
Reverse transcription-PCR.
For isolation of total RNA from Rhodococcus, cells were harvested in the mid-exponential phase of growth. Approximately 2 x 1010 cells were used for RNA isolation with a FastRNA BLUE kit (Bio 101, La Jolla, Calif.). To remove DNA contamination, 50-µl RNA samples were treated with 10 to 50 U of RNase-free DNase I (Promega) and 20 U of placental RNase inhibitor (Amersham) at 37°C for 30 min. The samples were then purified by extraction with acid phenol and chloroform, and the RNA was subsequently precipitated.
Reverse transcription was carried out with the ImProm-II reverse transcription system (Promega). Reactions were conducted in 20-µl mixtures by using 2 µg of total RNA and 15 pmol of each primer at 42°C for 1 h. The reactions were stopped by heating the mixtures at 72°C for 15 min, and 2.5-µl aliquots were used for PCR amplification. PCRs were conducted in 50-µl mixtures with 5 U of Taq polymerase (Amersham) as recommended by manufacturer. Thirty-two cycles of 30 s at 96°C, 30 s at 55°C, and 2 min at 72°C were performed. To ensure the absence of contamination with bacterial DNA, the same PCRs were performed with total RNA preparations (without reverse transcription) and all of the primer pairs analyzed. PCRs were also performed with the same primers and with the total DNA as positive controls.
Mapping of the narA transcription start site.
A modification of the previously described method (27) was used for automated fluorescent primer extension analysis. Total RNA preparations of naphthalene-grown Rhodococcus strains were obtained as described above. Specific primer IC36A (5'-TGGAGGGTCTGCCGGAGTTCGTTGCTC-3'), which was complementary to the antisense strand of the narAa gene (nt 31 to 5 downstream from the initiation codon), was 5' end labeled with WellRED dye (Invitrogen). The same primer was used for mapping transcription start sites in all three strains. Reverse transcription was carried out in 25-µl mixtures with 200 U of Q-Thermol reverse transcriptase (Qbiogene, Livingston, United Kingdom) and Thermo buffer supplied by the manufacturer. The reaction mixtures contained 20 µg of RNA, 3.5 mM labeled primer, 1 M betaine, 0.5 mM dATP, 0.5 mM dTTP, 0.5 mM dCTP, 0.5 mM 7-deaza-dGTP, 50 µg of actinomycin D (Amersham), and 40 U of RNase inhibitor (Amersham). The reaction mixtures were incubated as follows: 30 min at 42°C, 30 s at 70°C, 30 min at 42°C, 30 s at 70°C, and then 10 min at 42°C. Aliquots (2 µl) of the reaction mixtures together with 0.5 µl of CEQ DNA Size Standard-400 (BeckmanCoulter) were mixed with 37.5 µl of deionized formamide and used for analysis with a CEQ8000 sequencer (BeckmanCoulter) under the following conditions: capillary temperature, 50°C; denaturation temperature, 90°C for 120 s; injection voltage, 2.0 kV; injection time, 30 s; and separation at 6.0 kV for 35 min.
DNA sequencing.
Fragments containing the narA and narB genes of Rhodococcus sp. strains P200 and P400 were obtained by using primers designed for NCIMB12038 (19). A GenomeWalker kit (Clontech, Palo Alto, Calif.) and inverse PCR were employed to obtain DNA fragments representing the rest of the regions. The nucleotide sequences of both strands were determined. Alignment of sequences was performed by using CLUSTALW (37) with parameters set at default values. Searches for nucleotide and amino acid sequence similarities were carried out by using the FASTA and BLAST programs (29) in the EMBL and GenBank databases. Secondary structure prediction was performed by analyzing the results of SCRATCH server (igb.uci.edu/tools/scratch/) analysis of the NarR1 sequence. The helix-turn-helix motifs were also analyzed by the HTH method (http://npsa-pbil.ibcp.fr/). The potential transmembrane helix structures were predicted by using the PHD transmembrane helix prediction method (32; http://npsa-pbil.ibcp.fr/).
Nucleotide sequence accession numbers.
The nucleotide sequences determined in this work have been deposited in the GenBank database under accession numbers AF082663 (NCIMB12038), AY392423 (P400), and AY392424 (P200).
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RESULTS |
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narC sequences were found in only P200 and P400. The translated amino acid sequences of these genes showed strong similarity to trans-2'-carboxybenzalpyruvate hydratase-aldolase from phenanthrene-degrading Nocardioides (10) and to a hydratase/aldolase PhnE protein involved in polycyclic aromatic hydrocarbon catabolism in Burkholderia (22). However, no complete sequence of this gene was found in NCIMB12038 (see below).
Six open reading frames (orf1 to orf6) were found downstream of the cis-dihydrodiol dehydrogenase gene inNCIMB12038, and one open reading frame (orf7) was found between rub2 and narAa in all strains analyzed (Fig. 1). The coding regions of orf1 and orf2 overlapped, as did those of orf4 and orf5 and those of orf5 and orf6 (Table 1). ORF4 and ORF5 showed some similarity to several methyl-accepting chemotaxis proteins; the most important of these proteins is NahY, which is involved in chemotaxis to naphthalene in P. putida (Table 1). A putative ORF6 protein showed similarity to several outer membrane proteins, including the benzoate-specific porin-like protein of P. putida, BenF (Table 1). A putative oxiA gene was found in P400 but not in the other two strains analyzed. The translated product of this gene showed a significant level of identity with oxidoreductases from Streptomyces and flavin adenine dinucleotide/flavin mononucleotide-containing dehydrogenases from Corynebacterium and other bacteria (Table 1).
rub1 genes were found in all three strains, and they had orientations opposite those of the narA genes. These genes encoded putative proteins that showed high levels of similarity to several well-characterized rubredoxins, which are small noneheme iron proteins involved in electron transfer (3, 26). A second rubredoxin gene (rub2), which encodes a putative 8,968-Da protein, was found only in strain NCIMB12038; it was located upstream of the NDO genes (Fig. 1), and its putative product showed a high degree of similarity to Rub1 (89% amino acid identity).
narR1 genes were found in all of the strains analyzed (Table 1 and Fig. 1); these genes encode a putative protein with a molecular mass of about 25 kDa. NarR1 sequences showed similarity to a number of helix-turn-helix transcriptional regulators belonging to the GntR family (9, 31). The highest degrees of similarity were found with the transcriptional regulator GntR (accession no. NP_718424) from Shewanellaoneidensis MR-1 (32.5% amino acid identity) and the putative regulatory protein VanR (accession no. AAC27105) fromAcinetobacter sp. strain ADP1 (28.9% amino acid identity). The levels of similarity with the other, well-characterized representatives of the family, such as the Bacillus subtillis gluconate operon repressor (GntR) or the E. coli fatty acid metabolism regulator (31), were somewhat lower (24 to 26% amino acid identity). An analysis of the structure-based sequence alignments of NarR1 with various family members showed that NarR1 should be assigned to the FadR subfamily (31). A second putative regulatory gene (narR2) was found in all of the strains studied immediately downstream of narR1. The start codons of narR2 overlapped the stop codons of narR1 (Table 1). The putative NarR2 protein (25 kDa) showed similarity to regulators belonging to the NtrC family of prokaryotic enhancer-binding proteins. This family includes the regulator of the upper TOL operon in P. putida (XylR), which showed the highest level of similarity to NarR2 (Table 1). It is important to note that NarR2 is at least two times smaller than most of the other NtrC family proteins and that the similarity is limited almost exclusively to the amino-terminal, signal response domain (A domain) (5). The last 20 C-terminal residues of NarR2 showed some similarity to domain B, which may represent the Q-linker interdomain sequence (40). Both the narR1 and narR2 genes are preceded by ribosome binding sequences.
In NCIMB12038, the narR1 and narR2 genes were found to be surrounded by perfect direct repeat sequences that were 125 nt long. A region of iteron-like repeats was also found upstream of the narR1 gene (Table 1 and Fig. 1). A partial transposase sequence (
Tn) was found in the same region of NCIMB12038. The Tn sequence appeared to be part of the transposase gene, and a putative product showed similarity to Streptomyces (accession no. NP_821294 and AA45539) and Rhodococcus erythropolis (accession no. NP_898790) trans-posases.
Comparison of Rhodococcus nar genes.
The nucleotide sequences of the nar regions analyzed (strains NCIB12038, P200, and P400) were compared to each other and to the nucleotide sequences of related genes of indene-converting Rhodococcus sp. strain I24 (38) and naphthalene-degrading Rhodococcus sp. strain CIR2 (GenBank accession no. AF121905 andAB024936). The results of this analysis are shown in Fig. 2. All of the related genes found in the five sequences analyzed exhibited significant levels of homology, which varied for different pairs from 88 to 100%. However, a number of intervening nonhomologous regions and possible inserted or deleted sequences were also identified. Several putative genes were found only in some of the strains analyzed; orf1 to orf6 were found in NCIMB12038 and CIR2, and oxiA was present only in P400. Only strains NCIMB12038 and CIR2 had rub2 genes. The results of the comparison of the nar regions of the five Rhodococcus strains are shown in Fig. 2. The following conclusions were drawn from the analysis described above. (i) The narAa, narAb, and narB genes were present in the same order in all five strains. These genes comprise the central module of structural genes essential for the initial stages of naphthalene degradation. Based on the homology levels of narA and narB and the naphthalene degradation routes used (studied in NCIMB12038, P200, and P400), the strains could be divided into two distinct groups (Fig. 2); the first group (group I) includes P200 and P400 (which degrade naphthalene via catechol), as well as I24, and the second group (group II) includes NCIMB12038 (which degrades naphthalene via gentisate) and CIR2. (ii) Putative aldolase genes (narC) in strains P400, P200, and I24 were found to be more than 99% homologous (Fig. 2). The homology between strains belonging to groups I and II ends 35 nt downstream of narB; however, short sequences (37 nt) that appeared to be the 5' part of the narC gene were found in both NCIMB12038 and CIR2. (iii) The sequence homology of group II strains ends abruptly after the narC stop codon. (iv) Alignment of the P200 sequence with the P400 and I24 sequences resulted in identification of long nonhomologous stretches in the 5' parts of the region analyzed (Fig. 2). (v) It is important to note that while group I (P200, P400, and I24) was defined as a group of sequences showing at least 98% homology in the narA-narB region, in the rub1-narR1 region strain P200 showed a higher level of homology with strains NCIMB12038 and CIR2 (98%) and a lower level of homology with P400 and I24 (92%) (Fig. 2). (vi) Nucleotide sequence alignment of strains NCIMB12038 and CIR2 (group II) revealed a high level of homology (97 to 99%) in the 3' region and no homology in the 5' regions (Fig. 2). Nonhomologous parts of both NCIMB12038 and CIR2 contained sequences resembling various transposases; these parts were orf1 (CIR2) and Tn (NCIMB12038; nt 2551 to 2907). (vii) The I24, P400, P200, and NCIMB12038 sequences did not show any homology at their 3' ends (Fig. 2).
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Identification of the narA region transcripts.
To analyze transcription of the naphthalene degradation genes, total RNA preparations from cells grown on naphthalene, salicylate, and pyruvate (NCIMB12038) and on naphthalene and pyruvate (P200 and P400, as these strains do not grow on salicylate) were obtained and subjected to reverse transcription-PCR analysis as described in Materials and Methods. The oligonucleotide primer pairs were designed to cover all of the genes identified, as well as the intergenic regions (we used a total of 36 primer pairs, whose sequences are not shown). The data obtained identified several distinct transcriptional units in the regions of NCIMB12038, P200, and P400 investigated (Fig. 1). In all cases transcription was either constitutive or induced by growth on naphthalene; no induction by growth on salicylate was observed in any of the strains. The genes encoding the catalytic component of NDO (narAa and narAb) and cis-naphthalene dihydrodiol dehydrogenase (narB) in all three strains were transcribed as a single unit that was induced by growth on naphthalene. However, in NCIMB12038 transcription stopped immediately after the narB gene (Fig. 1A), and a putative Rho-independent terminator was found at nt 7168 to 7267. The structure included a potential hairpin with a predicted free energy (G) of –81.4 kcal mol–1 and a short stretch of A residues. In contrast, in P200 and P400 transcription continued beyond narB to include narC and stopped before the oxiA gene (Fig. 1B and C). The other two transcripts induced by growth on naphthalene in strain NCIMB12038 were found to include orf1 to orf3 and orf4 to orf6 (Fig. 1A). Analysis of the rubredoxin genes revealed that they also were transcribed as separate units, but notably, only transcription of the rub2 gene (NCIMB12038) (Fig. 1A) was induced by growth on naphthalene. Transcription of the rub1 gene appeared to be constitutive in the three strains analyzed. These results suggest that Rub2 may be involved in electron transport associated with NDO in Rhodococcus sp. strain NCIMB12038.
Two overlapping transcriptional units that were different sizes were found in the location of the putative regulatory genes narR1 and narR2. The narR2 gene was transcribed constitutively, yet both narR1 and narR2 were found on a longer transcript that was induced only by growth on naphthalene. It was notable that the same pattern of narR1 and narR2 transcription was seen for strains NCIMB12038, P200, and P400 (Fig. 1).
Mapping of NDO transcript initiation sites.
The homology of the sequences analyzed extended upstream of the narAa genes (Fig. 2). To more accurately define the narA-narB modules, mapping of the transcriptional start sites for these genes was undertaken for strains NCIMB12038, P200, and P400. Precise mapping of the narA 5' transcriptional termini was performed by primer extension analysis as described in Materials and Methods. The same primer (IC36A) was used for all three strains. The results are shown in Fig. 3. The primer-specific cDNA products were synthesized only with RNA obtained from naphthalene-grown cells.
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70 promoters (Fig. 3B). For each of the other two strains (P200 and P400) two major and three minor peaks were detected, which identified possible 5' ends of the narAa transcript (Fig. 3C and D). The minor products corresponding to the T (nt 3282; strain P200) (Fig. 3C), G (nt 3265; strain P200) (Fig. 3C), G (nt 3213; strain P400) (Fig. 3D), and C (nt 3201; strain P400) (Fig. 3D) could be generated by 5' end processing of the corresponding major transcripts. Transcription start sites corresponding to the major peaks are shown in the nucleotide sequence of the region in Fig. 3E. No consensus sequences typical for E. coli or Streptomyces promoters were identified at the appropriate positions. It is important that although the narA promoter regions of P200 and P400 differ by a single nucleotide substitution, the corresponding transcripts have different start sites (Fig. 3E). The specificity of transcriptional regulator proteins and/or RNA polymerases might be responsible for the differences in the transcription start sites of P200 and P400. The directly repeated 17-nt sequence was shown to be present in the region analyzed (Fig. 3E), which might influence the precision of the promoter recognition by polymerase and/or regulatory proteins.
Localization of the naphthalene degradation genes in the Rhodococcus genome.
Pulsed-field gel electrophoresis analysis revealed the presence of various plasmids in strains NCIMB12038, P200, and P400 (Fig. 4A). It is well established that Rhodococcus species harbor circular as well as linear plasmids (21). The distinction between linear and circular DNA molecules was analyzed by the method of Kalkus et al. (12), as described in Materials and Methods (results not shown). A single linear 380-kbp plasmid (designated p2SL1) was detected in Rhodococcus sp. strain NCIMB12038 (Fig. 4A). One linear 300-kb plasmid (P20L1) and one circular 150-kb plasmid (P20C1) were found in P200. Four plasmids were found in the P400 strain, a 420-kb linear plasmid (P40L1), a 180-kb circular plasmid (P40C1), a 45-kb linear plasmid (P40L2), and a 20-kb linear plasmid (P40L3) (Fig. 4A). A partial narAa-narAb sequence was employed as a hybridization probe (see Materials and Methods). The results of the hybridization (Fig. 4B) clearly demonstrated the plasmid (p2SL1) location of the nar genes in NCIMB12038. This was confirmed by hybridization of various restriction products of p2SL1 with the same probe (results not shown). Two hybridization bands were observed with the P400 DNA (Fig. 4B). The upper band corresponded to plasmid P40C1, and the lower band may have represented either of the two smaller plasmids. The results described above may be explained by the presence of two independent copies of NDO sequences in P400 or by the structural instability of the P40C1 plasmid in the bacterial population. In contrast to NCIMB12038 and P400, the hybridization with P200 DNA indicated a chromosomal location for the nar genes (Fig. 4B).
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DISCUSSION |
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Naphthalene degradation genes of Rhodococcus.
Although the narAa and narAb genes of Rhodococcus exhibit little homology with the Pseudomonas nah genes, it has been shown that key catalytic residues are conserved in both ISPNAR and ISPNAP enzymes (19). In contrast, the organization of the genes was significantly different from that of gram-negative bacteria. LysR-type transcriptional regulators regulate the majority of the naphthalene degradation systems that have been described, and salicylate is usually required to induce operon expression (11, 28, 34, 41). In Rhodococcus sp. strain NCIMB12038 transcription of the naphthalene degradation structural genes identified was induced only in the presence of naphthalene, and no LysR-type regulator gene was found in the nar region. Instead, we identified two genes which encode putative regulatory proteins belonging to the GntR family (narR1) and the NtrC family of prokaryotic enhancer-binding proteins (narR2). The narR1 and narR2 genes overlap at the 3' and 5' ends, suggesting that there is transcriptional and translational coupling. A similar type of cognate response regulator organization has been reported previously for bpdST genes in biphenyl-degrading Rhodococcus sp. strain M5 (18). The deduced amino acid sequence of narR2 showed significant similarity to the A domain of XylR-like regulators, which is considered to be a target for specific regulatory molecules. Also, a helix-turn-helix motif is found in the C terminal part of NarR2. It may be speculated that the constitutively expressed product of narR2 activates expression of the narR1-narR2 operon after binding with naphthalene. In turn, NarR1 may activate transcription of the required structural operons. No sequences that resembled an operator consensus for GntR-like proteins (31) were found, however. It is important to note that although many GntR regulators bind to palindromic sequences, there are representatives of this family that recognize boxes with no clearly defined symmetrical properties (2, 31). Probably the most important operon induced by naphthalene includes genes encoding ISPNAR and cis-naphthalene dihydrodiol dehydrogenase. This operon was found in all of the strains analyzed. However, –10 and –35 sequences typical of 70 promoters are present only in NCIMB12038. A sequence similar to the integration host factor (IHF) recognition site (WATCAAN4TTR) (8) was also found in this region (Fig. 3B). It is worth noting that the presence of an IHF binding site often indicates that there is a complex mechanism for operon regulation (39). IHF, which binds to the minor groove of DNA to create a sharp 180° bend, is involved in the regulation of various transcriptional units, where it facilitates the formation of nucleoprotein complexes (e.g., 70 and 54) essential for transcription initiation (30).
In Pseudomonas and other gram-negative bacteria all of the structural genes required for naphthalene utilization are usually clustered. In contrast, only three structural genes required for naphthalene utilization (narAa, narAb, and narB) were identified in the region of Rhodococcus strains NCIMB12038, P200, and P400 analyzed (Fig. 1). The electron transport components of NDO in Pseudomonas were identified as reductase and ferredoxin (34); however, no genes corresponding to genes encoding such proteins were found in the Rhodococcus strains analyzed. Variations in the electron transport components of Rhodococcus oxygenases were reported previously. In the biphenyl degradation operon of Rhodococcus sp. strain RHA1 genes encoding reductase and ferredoxin are located immediately downstream of the genes encoding and ß ISPNAR subunits (23). It is also known that a flavin reductase subunit encoded by genes in different pathways is involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1 (24). A two-component electron transport chain consisting of rubredoxin and rubredoxin reductase was reported previously for Pseudomonas alkane hydrolase (13). In the Rhodococcus strain NCIMB12038 nar region a rubredoxin gene (rub2) was found, which was transcribed as a separate mRNA molecule in cells induced by naphthalene. It seems reasonable to suggest that Rub2 forms part of the Rhodococcus NDO complex. However, no naphthalene-inducible rubredoxin genes were found in the regions of strains P200 and P400 analyzed, and no reductase component for the NDO was found in any of the strains studied.
The absence of a number of genes essential for naphthalene utilization in the nar clusters suggests that these genes are located in other parts of the Rhodococcus genome. The differences among Rhodococcus strains NCIMB12038, P200, and P400 described above also lead to the suggestion that genomic rearrangements were responsible for evolution of the corresponding nar regions. To investigate this hypothesis, five Rhodococcus strains were compared.
Comparison of the NDO gene modules.
All of the strains analyzed show homology in the narAa, narAb, and narB genes. The DNA homology in the Rhodococcus strains analyzed extends upstream of narAa, covering the promoter region and orf7. We mapped the relevant transcription start sites for NCIMB12038, P200, and P400 and demonstrated not only that the NCIMB12038 promoter is different from the promoters of the two other strains but also that the transcription starts are different in strains P200 and P400. These results imply that the narAa-narB module may have evolved in Rhodococcus independently from the adjacent regulatory sequences and was at some stage recombined with the latter sequences. The deletions (NCIMB12038) and insertions (I24) in the narAa promoter region (Fig. 2) are also indicative of this mode of evolution. The narC gene was shown to be cotranscribed with narAa-narB in P200 and P400; however, this gene has not been found in NCIMB12038. A short nucleotide sequence homologous to the 5' end of narC was, however, detected in both NCIMB12038 and CIR2. This suggests that the ancestral strain probably possessed the narC gene and that more than one recombination event was involved in the formation of the corresponding region of NCIMB12038 (Fig. 2). Several putative genes (orf1 to orf6) were found downstream of narAa-narB in NCIMB12038, transcription of which was induced in the presence of naphthalene. However, these genes do not form a continuous transcription unit with the narA and narB genes (Fig. 1). These genes apparently represent separate modules, and their fusion with the narA and narB genes led to the formation of the present NCIMB12038 nar region.
Comparison of the narR1-narR2 regions.
The narR1 and narR2 genes represent another important cluster. These genes were found in all strains analyzed except CIR2. Similar to the levels of homology of narA and narB, there were high levels of homology (90% or more) between the narR genes of different strains. However, detailed analysis of the levels of homology between the genes in this region revealed a pattern that is quite different from that of narA and narB. The narR2 genes of the strains belonging to group I (P200, P400, and I24) were found to be almost identical to each other (at least 98% homology) and showed 90 to 92% homology to narR2 of NCIMB12038. In contrast, narR1 of P200 (group I) was identical to narR1 of NCIMB12038 (group II) and exhibited 92 to 93% homology with the counterparts in members of its group (P400 and I24) (Fig. 2). The data can be explained by the assumption that narR1 and narR2 descended from different immediate ancestors and recombined with each other in the course of evolution. This recombination event(s) was apparently independent of the events involved in the formation of the narA-narB regions. The homology of the rub1 genes exhibited the same pattern as the homology of the narR1 genes, and therefore these genes were probably included in the same recombination module.
rub2 genes.
The rub2 (rnoA1 in CIR2) genes were present only in the group II strains. These genes were probably present in a common ancestral strain and were deleted from all group I sequences together with part of the DR1 sequence and Tn (Fig. 2).
Nonhomologous sequences.
Comparison of the nar regions demonstrated that homologous genes in various strains are separated and surrounded by nonhomologous sequences (Fig. 2). These sequences were found in the rub1-narR1 (strains P200 and P400), narR2-orf7 (NCIMB12038, P200, and P400), and narA promoter (NCIMB12038, P400, P200, and I24) regions. The sequences of the group II strains (NCIMB12038 and CIR2) lack homology in their 5' halves (Fig. 2). Strains P200, P400, NCIMB12038, and I24 do not exhibit any homology downstream of narC.
It is essential to note that the mechanisms of genetic rearrangement in Rhodococcus species remain generally unknown. Also, our data indicate that in all three strains studied the nar gene regions appear to have different genomic locations.
Conclusion.
A hypothetical sequence of events leading to formation of the Rhodococcus nar regions analyzed is shown in Fig. 5. This scheme highlights key events essential for the formation of the diversity investigated. The plasmid locations of the naphthalene degradation determinants analyzed (in strains NCIMB12038 and P400) are consistent with the apparent widespread exchange of genetic material in nar regions. The data clearly demonstrate that a web-type evolution was involved in the creation of genetic regions responsible for naphthalene utilization in Rhodococcus.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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54-dependent promoter Pu of Pseudomonas putida. Proc. Natl. Acad. Sci. USA 92:7277-7281.
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