Applied and Environmental Microbiology, May 2003, p . 2748-2754, Vol . 69, No . 5

PA or 2,4-DNP degradation occurs by initial reduction (hydrogenation) of the aromatic ring, producing the respective hydride and dihydride Meisenheimer complexes of PA (H^--PA and 2H^--PA) or 2,4-DNP (H^--2,4-DNP) . The PA-degrading bacterium R. (opacus) erythropolis HL PM-1 possesses a npd gene cluster, containing npdC (encoding hydride transferase I [HTI]), npdG (encoding the NADPH-dependent F₄₂₀ reductase [NDFR]), and npdI (encoding HTII) (9, 17, 30) . HTII performs the first hydride transfer to PA, forming H^--PA, and HTI catalyzes the second hydride transfer, giving rise to 2H^--PA . Both enzymes require the NDFR to supply the hydride ions in the form of F₄₂₀H₂ .

We have found here homologous genes for npdG and npdI in several 2,4-DNP-degrading strains of the genus Rhodococcus . Further, it was shown that the npdG and npdI genes have the same function as the homologous genes in R. (opacus) erythropolis HL PM-1 . Hence, they are involved in the degradation of 2,4-DNP . We have also detected these genes in Rhodococcus sp . strain PN1, which was originally enriched on p-nitrophenol (4NP) . The strain degrades 4NP by an oxygenolytic pathway via 4-nitrocatechol, which has nothing in common with the 2,4-DNP pathway (33) . Several bacteria are known to degrade 4NP by oxygenolytic removal of the nitro group, producing hydroquinone or via 4-nitrocatechol into 1,2,4-benzenetriol (20, 25, 26, 32) .

We propose that the NDFRs form a new group within the family of F₄₂₀-dependent NADPH reductases (FDNRs) . Similarly, we suggest that the HTIIs form a new group within a protein family, which we refer to here as the F₄₂₀-dependent glucose-6-phosphate dehydrogenases-F₄₂₀-dependent N⁵,N¹⁰-methylenetetrahydromethanopterin reductases-hydride transferases (FGD-MER-HT) .

 
Purification, manipulation, and transformation of DNA.
Plasmid DNA from E . coli was isolated by using Qiagen columns (Qiagen GmbH) . Genomic DNA was purified according to Eulberg et al . (11) . DNA fragments were purified from agarose gels by using the Easypure DNA purification kit (Biozym Diagnostics GmbH, Hess-Oldendorf, Germany) . DNA manipulations were accomplished by using standard methods (2, 24, 31) . E . coli was transformed with plasmid DNA according to Inoue et al . (19) . Electroporation of R . rhodochrous ATCC 12674 was performed according to the method of Hashimoto et al . (16) . The conditions were as follows: 1-mm electrode gap electroporation cuvettes, a voltage of 1.8 to 2 kV, a capacity of 25 µF, a resistance of 400 , and a time constant of 6.8 to 9.5 ms .

Amplification and cloning of DNA.
PCR was performed in a T Gradient Thermocycler 96 (Biometra GmbH, Göttingen, Germany) . Primers were purchased from MWG Biotech AG (Ebersberg, Germany) . Reaction mixtures (total volume of 25 µl) contained 50 pmol of each primer, 1.5 mM MgCl₂, 4% dimethyl sulfoxide, 0.2 mM of each deoxynucleoside triphosphate, 0.5 U of Taq polymerase (Gibco/Life Technologies GmbH), 1x PCR buffer (Gibco/Life Technologies GmbH), and 10 to 100 ng of genomic or plasmid DNA . The cycling parameters were as follows: initial denaturation at 95°C for 3 min, followed by 20 cycles consisting of denaturation at 95°C for 30 s, annealing at 55 to 64°C for 30 s, and elongation at 72°C for 2 min .

The primers used for amplification of the npdG genes from the Rhodococcus strains RB1, PN1, HL 24-1, CB 24-1, DNP 14-5, and DNP 14-9 were 5'-ATG AAG TC(GC) TC(GC) AAG ATC GC(GC) GTC-3', 5'-ATG AAG AGC AGC AAG ATC GC(GC) GTC-3', or 5'-CGC (AC)GC (CT)CG CGG ATC GTG (AG)AC-3' . npdI genes were amplified with the primers 5'-ATG ATC AA(AG) GGC ATC CAG CT(GC) CA(CT) GG-3' and 5'-(GCT)GC (GC)AG CTC CGG (GC)AG GAC CTG-3' .

To produce a His-Tag fusion protein of the npdG genes from strains CB 24-1, HL 24-1, and DNP 14-5, the primers used were 5'-TTT TCA TAT GAA GAG CAG CAA GAT CGC-3' and 5'-TTT TGG ATC CTC AGG CGG CGC GTG GGT C-3' (NdeI and BamHI restriction sites are underlined) . The PCR products were restricted with NdeI and BamHI and ligated into the NdeI and BamHI sites of pAC28 .

To produce a His-Tag fusion protein of the npdG gene from strain PN1, the primers employed were 5'-GAGGGATCCTTGAAGAGCAGCAAGATTGCC-3' and 5'-GGTGGTACCCTCGGCACGGCCCGGCAGAGCACGG-3' (BamHI and KpnI sites are underlined) . The PCR product was restricted with BamHI and KpnI and ligated into the BamHI and the KpnI site of pQE-80L .

For expression of npdG and npdI of strain CB 24-1 in R . rhodochrous ATCC 12674, npdG was amplified with the primers 5'-TTT TCT GCA GAT GAA GAG CAG CAA GAT CGC-3' and 5'-TTT TGG ATC CTC AGG CGG CGC GTG GGT C-3'), and npdI was amplified with the primers 5'-TTT TGG ATC CGT CGC CGT GTT CTG CCC TTA AC-3' and 5'-TTT TCT GCA GTC AGG CGA GCT CCG GGA C-3' (the PstI and BamHI sites are underlined) . The PCR products were digested with BamHI and subsequently ligated to produce a single PstI fragment with npdG and npdI juxtaposed to one another . The PstI fragment was finally restricted with PstI and ligated into the PstI site of pK4 .

For expression of npdG and npdI of strain PN1 in R . rhodochrous ATCC 12674, npdG or npdI were amplified with the primers 5'-TCGGGTACCTCCCGTCCCAACGT GTAGGAGACAG-3' and 5'-GGTGGTACCCTCGGCACGGCCCGGCAGAGCACGG-3' for npdG and the primers 5'-TATGGTACCTCGAGTTCAACATCATGAAGAGAAGTC-3' and 5'-TTCGGTACCGCACAGGTCCCGCGTTGCCTGCGTG-3' for npdI (the KpnI sites are underlined) . The products were cut with KpnI and inserted together into the KpnI site of pK4 .

For expression of npdG and npdI from Rhodococcus sp . strain HL 24-1, the primers used for amplification were 5'-TCATGCGAGCTCCGGCAGGACCTGG-3' and 5'-ATGAAGAGCAGCAAGATCGCCGTCGTC-3' . For expression of npdG and npdI from Rhodococcus sp . strain DNP 14-5, the primers used for amplification were 5'-CCCAAGCTTGGGTCATGCGAGCTCCGGCAGGAC-3' and 5'-TTTTCTGCAGATGAAGAGCAGCAAGATCGC-3' . The PCR fragments were ligated into the PstI site of pK4, which had been cut with PstI and blunt ends created with T4 DNA polymerase (MBI Fermentas) .

Sequence and phylogenetic analyses.
Sequencing was performed commercially (MWG Biotech AG) . Homology searches were performed with BLASTN, BLASTP, and BLASTX . Motif searches were performed with the Motif tool (http://motif.genome.ad.jp) . Pairwise and multiple alignments were carried out by using BLAST2 (http://www.ncbi.nlm.nih.gov/gorf/bl2.html) and CLUSTALW (http://www.ebi.ac.uk/clustalw/[1, 35, 36]) . Translations were achieved by using the Translation Machine (http://www2.ebi.ac.uk/; EMBL Outstation European Bioinformatics Institute) . Phylogenetic trees were constructed by using PROML (maximum likelihood), SEQBOOT, CONSENSE, and DRAWTREE of PHYLIP 3.6a (12) .

Enzyme purification, enzyme assays, and SDS-PAGE.
NDFRs and HTIIs were purified as His-Tag fusion proteins as previously described (17) . Preparation of cell extracts, determination of protein concentrations, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were done as previously described . Reduction of F₄₂₀ to F₄₂₀H₂ by the purified NDFR was monitored by using the method previously described . The concentration of F₄₂₀ was 10 to 21.4 µM . The amount of pure protein added was 0.25 to 0.7 µg . The reaction time was 12 s . Conversion of PA to the hydride Meisenheimer complex of PA was performed as described earlier .

Resting-cell experiments.
Cells (exponential phase) were resuspended in 50 mM KH₂PO₄-K₂HPO₄ buffer (pH 7.5) to obtain an optical density at 595 nm (OD₅₉₅) of 2.0 . PA or 2,4-DNP were added to a final concentration of 0.5 or 0.7 mM . Cell suspensions were shaken at 30°C, and culture supernatants were analyzed by high-pressure liquid chromatography (HPLC) at regular time intervals .

HPLC analyses.
Metabolites were detected and analyzed as previously described (17) .

Nucleotide sequence accession numbers.
The sequences have been submitted to GenBank . The accession numbers are AY027571, AY027572, AY027573, AY027574, are AY027575 for the npdG genes and AY027566, AY027567, AY027568, AY027569, and AY027570 for the npdI genes . The accession number for the genes from strain PN1 is AB090357 .

The npdG or npdI gene sequences show high sequence similarities to one another.
Pairwise alignments performed with the npdG and npdI genes from the Rhodococcus spp . (Table 1) and npdG and npdI from the reference strain R. (opacus) erythropolis HL PM-1 showed high sequence identities (86 to 99%), suggesting that the genes are homologous . The npd genes of Rhodococcus sp . strain RB1 showed the highest sequence identities (99%), and the npd genes of Rhodococcus sp . strains CB 24-1 and HL 24-1 had the lowest sequence identities (86%) . The sequences of strains DNP 14-5 and DNP 14-9 were identical to one another .

The npdG genes are involved in the conversion of F₄₂₀ to F₄₂₀H₂.
In order to prove the involvement of the npdG genes in 2,4-DNP degradation, we cloned the genes from strains CB 24-1, PN1, HL 24-1, and DNP 14-5 . npdG was amplified from strains CB 24-1, HL 24-1, or DNP 14-5 and cloned into pAC28 to produce pNTG29, pNTG31, or pNTG32, respectively . The genes were expressed as His-Tag fusion proteins in E . coli BL21(DE3) containing the respective plasmids . npdG was amplified from strain PN1 and cloned into pQE-80L to produce pQEG1 . The gene was expressed as a His-Tag fusion protein in E . coli JM109(pQEG1) . Cell extracts were prepared from induced cultures, and the proteins purified to 95% homogeneity . All of the NDFRs converted F₄₂₀ to F₄₂₀H₂ . Hence, we may surmise that the npdG genes are involved in the degradation of 2,4-DNP .

The specific activity was determined for the NDFRs from strains CB 24-1 and PN1 . NDFR from strain CB 24-1 converted F₄₂₀ to F₄₂₀H₂ with a specific activity of 23.5 ± 2 U/mg . This was comparable to the activity of NDFR from strain HL PM-1 (17) . The NDFR from strain PN1 had an activity of 57.3 ± 0.6 U/mg for F₄₂₀ . This was about 2.5 times higher than the activity of NDFR from strains CB 24-1 or HL PM-1 . This may be a reflection of the differences in sequence .

The npdI genes encode hydride transferases (HTs) involved in the initial hydride transfer.
Our aim was to prove the role of the npdI genes in 2,4-DNP degradation . According to Ebert et al . (9) and Heiss et al . (17), HTII requires NDFR for converting 2,4-DNP to the H^--2,4-DNP or PA to the H^--PA . Hence, npdI and npdG were cloned from Rhodococcus spp . CB 24-1, DNP 14-5, HL 24-1, or PN1 into pK4 to create pKCB2, pKDNP1, pKHLI, or pKPNGI1, respectively . The plasmids were transformed into R . rhodochrous ATCC 12674 to create R . rhodochrous ATCC 12674(pKCB2), R . rhodochrous ATCC 12674(pKDNP1), R . rhodochrous ATCC 12674(pKHLI), and R . rhodochrous ATCC 12674(pKPNGI1) .

Resting cells of R . rhodochrous ATCC 12674(pKCB2), R . rhodochrous ATCC 12674(pKPNGI1) (Fig . 1), R . rhodochrous ATCC 12674(pKDNP1), and R . rhodochrous ATCC 12674(pKHLI) (data not shown) converted 2,4-DNP to the hydride Meisenheimer complex of 2,4-DNP (H^--2,4-DNP) . They also converted PA to the hydride Meisenheimer complex of PA (H^--PA), indicating the transfer of a hydride ion to the aromatic ring of PA or 2,4-DNP . Both the spectra and the retention volumes of the products were identical to the authentic Meisenheimer complexes described above . This finding shows that the HTIIs are involved in the hydride transfer to 2,4-DNP or PA and that the npdI genes are involved in the degradation of 2,4-DNP .

 
Notably, resting cells of R . rhodochrous ATCC 12674(pKCB2) almost completely converted PA to the H^--PA within 25 min, whereas 2,4-DNP was not completely converted to the H^--2,4-DNP under the same conditions . Resting cells of R . rhodochrous ATCC 12674(pKPNGI1) did not convert either compound completely within the time frame measured .

R . rhodochrous ATCC 12674(pK4) did not convert either PA or 2,4-DNP . Further, cell extracts prepared from strain R . rhodochrous ATCC 12674 showed no NDFR activity . To show that the strain does not possess a functional HTII, an enzyme assay was performed with PA as a substrate, cell extracts from strain ATCC 12674, and purified NDFR [from R. (opacus) erythropolis HL PM-1] . PA was not converted . This shows that strain ATCC 12674 does not possess a functional NDFR or HTII .

The NDFRs (NpdGs) form a new protein group.
BLASTX searches performed with the npdG genes detected similarities with FDNRs or oxidoreducates from Streptomyces spp., Nocardioides simplex FJ2-1A, and Archaea strains . The NDFRs had the highest sequence similarities with the NADPH-dependent F₄₂₀ reductase of N . simplex FJ2-1A (similarity, 65 to 68%; identity, 57 to 58%; expect value, 9e^-62; score 237) .

Multiple alignments revealed five conserved domains across the entire length of the proteins . The consensus sequences of the most highly conserved domains were as follows: (i) [I,L,V][A,G][I,F,V,L][L,I,V]GGTGX(2)GXG[L,M][A,V]X(2)[A,G]X(2)[G,N] at positions 6 to 27 of NpdG of strain HL PM-1; (ii) [V,I][V,I][V,I,L]GSRX(2)EXAX(3)A at positions 30 to 44; (iii) [V,I,L]X[G,A]X(2)NX(3)[A,V]X(4)[V,I]X[V,I,L,F][V,I,L,F]X[VIL] at positions 57 to 76; (iv) GS[A,V][A,T]X(13)V[AV][A,G,S]A at positions 119 to 137; and (v) DX(2)[V,I]X[G,S][E,D,N]X(7)[V,A]X(2)L[A,T]X(2)[I,V,M]X(5)[V,I,L]X(2)GX[L,V]X(2)[A,S]X(2)[V,L,I,M]EX[L,I][V,T][A,P]X[L,I][L,I][S,G,N][V,L] at positions 155 to 204 .

Several motifs were detected across the NpdG sequence from the Blocks database . Three signatures were identified in the first conserved domain (positions 5 to 28): (i) an aromatic-ring hydroxylase (flavoprotein monooxygenase) signature (score 1003), (ii) an isoflavone reductase signature (score 1195), and (iii) an NAD-dependent epimerase/dehydratase family signature (score 1082) . All three of these enzyme families require NAD(P)H or FAD as cofactors . Interestingly, this consensus region is glycine-rich .

Further signatures were detected at positions 56 to 80 (conserved region number 3) . These included a signature of the aspartate-semialdehyde dehydrogenase protein family (score 1040) and a GTP-binding domain (score 1029) . Both protein families possess NADP- or GTP-binding domains, respectively . Lastly, an oxidoreductase FAD and NAD(P)-binding domain at positions 156 to 167 were found . This motif was contained within the fifth conserved region identified from the multiple alignment . Since all of the motifs were contained in NAD(P)- or flavin-binding proteins, the conserved regions may be required for binding of NAD(P)H or F₄₂₀ .

A phylogenetic tree showed that the NDFRs from the Rhodococcus spp . clustered as a separate group from the FDNRs (Fig . 2) . For simplicity, we propose to collectively call the NDFRs and FDNRs the F₄₂₀-NADP-oxidoreductases .

 
The HTIIs (NpdIs) form a new protein group.
Homology searches performed with the npdI genes identified N⁵,N¹⁰-methylenetetrahydromethanopterin reductases (MERs) from Archaea, glucose-6-phosphate dehydrogenases (FGDs) from Mycobacterium, and an HT from N . simplex FJ2-1A . The HTIIs had the highest sequence similarities with the latter (similarity, 65 to 77%; identity, 55 to 67%; expect value, 6e^-130; score 464; AAK38742) . As for the FDNRs, all of these enzymes were dependent on the coenzyme F₄₂₀ or flavins .

Multiple alignments with the HTIIs, the HTI (from strain HL PM-1), the MERs, and FGDs revealed four conserved domains, particularly in the NH₂-terminal two-thirds of the proteins . The FGD-MER-HT consensus sequences were very similar to the FGD-MER consensus sequences previously identified by Purwantini et al . (28) . They were as follows: (i) AX(4)[F,M][D,E]X(3)[M,V,I,L][T,S]D[H,Q] at positions 23 to 35 of NpdI of strain HL PM-1; (ii) TX(4)[L,V]G[T,P][A,G,S]X(13)A[S,Q,K][T,A,S][M,I,F,L]X[S,T][I,L,M]X(2)[M,I,F,L]X(6)[V,L]X[M,I,L,V]GXG at positions 52 to 93; (iii) EX[V,I]X(3)RX[M,L,F]X(3)[D,E,K]X[V,I]X(3)[G,D]X(9)[M,I,L] at positions 114 to 142; (iv) [I,V]X[I,V]X[F,I,M,V)[A,G][A,G]X[S,G][T,P]X(3)[R,K,E,Q]X(2)[A,G]X(2)[G,A,S]DG at positions 158 to 179 .

Motif searches identified the following signature sequences at positions 159 to 181 (conserved region number 4) from the Blocks and Prints databases: aromatic ring hydroxylase (flavoprotein mononoxygenase) (score 1146), FAD-dependent pyridine nucleotide reductase (score 1047), NAD/NADP octopine/nopaline dehydrogenase (score: 1049), and flavin-containing amine oxidase (score 1006) . This region may be significant for binding of NAD(P), flavins, or F₄₂₀ .

A phylogenetic tree calculated from the HTII, FGD, and the MER sequences showed that the HTIIs and the HT of N . simplex FJ2-1A constituted a new group separate from the FGDs, the MERs, and the HTI of R. (opacus) erythropolis HL PM-1 (Fig . 3) . Because of the similarity of the HTIIs with the HT of N . simplex FJ2-1A, we propose to call of the HT of N . simplex FJ2-1A, HTII (NpdI) .

 
The majority of the bacteria known up to date with the ability to mineralize 2,4-DNP or PA by initial reduction of the aromatic ring are gram-positive actinomycetes (3, 5, 9, 23, 29) . In contrast, gram-negative bacteria do not seem to possess productive 2,4-DNP degradation capabilities (13, 18, 14) . The present study has shown that six Rhodococcus spp . all have the capacity to grow on 2,4-DNP as sole nitrogen source and contain homologous enzymes (NDFR and HTII) involved in the initial reductive attack on 2,4-DNP or PA . It is interesting that Rhodococcus sp . strain PN1, which was initially enriched on 4NP (33), also possesses the ring hydrogenation capacity of 2,4-DNP and the responsible npdG and npdI genes . In contrast, the 2,4-DNP degraders HL PM-1 and HL 24-1 do not grow on 4NP or other mononitrophenols (22) .

Homology searches with both the NDFRs and the HTIIs identified F₄₂₀-dependent enzymes (FDNRs, MERs, and FGDs) from Archaea or actinomycetes . No homologous enzymes from gram-negative bacteria were detected . This coincides with the absence of the coenzyme F₄₂₀ in these bacteria . Hence, gram-negative bacteria may lack the ability to reduce the aromatic nuclei of 2,4-DNP or PA because they lack the HTs and the coenzyme F₄₂₀ for it .

Functionally, F₄₂₀-dependent oxidoreductases shuttle electrons between F₄₂₀ and NADPH and have primarily been shown to play a role in methanogenesis in Archaea . The MERs in Archaea also play a role in methanogenesis (4) . FGDs convert glucose-6-phosphate to 6-phosphogluconolactone in the presence of F₄₂₀ and have been detected in the actinomycetes Streptomyces, Corynebacterium, and Mycobacterium (10, 27) . That F₄₂₀-dependent enzymes play a role in degradation of nitroaromatic compounds was demonstrated for the first time in N . simplex FJ2-1A, and R. (opacus) erythropolis HL PM-1 (9, 30) . In the present work, homologous HTIIs and NDFRs were detected in several closely related Rhodococcus spp . and shown to play a role in 2,4-DNP or PA hydrogenation .

Consensus sequences were identified in the NDFRs and the FDNRs . The FGD-MER-HT consensus regions were comparable to the FGD-MER consensus sequences described previously (28) . The authors of that study proposed that these regions may be involved in the binding of F₄₂₀ . Hence, the conserved domains may reveal structurally important motifs and should assist in future predictions on potential regions involved in the binding of coenzymes .

The phylogenetic analyses showed that the NDFRs form a new group within the group of FDNRs (Fig . 2) . Similarly, the HTIIs formed a new group within the MERs and FGDs (Fig . 3) . The FGD-MER-HT tree shows different groups of proteins with differing functions derived from different phylogenetic groups of microorganisms: the FGDs from mycobacteria, the MERs from the Archaea and the HTIIs from the 2,4-DNP degrading Rhodococcus spp . The three protein groups may have originated from a common ancestor, followed by independent evolution in each taxon subsequent to lateral gene transfer .

The HTI from strain HL PM-1 was shown to be involved in the hydride transfer to H^--PA to form 2H^--PA (17) . The phylogenetic tree showed that this enzyme did not cluster with the HTIIs . However, the FGD-MER-HT consensus sequences were also found in the HTI . This coincides with the enzymes' similar functions: both the HTII and the HTI are involved in hydride transfer reactions, and both require F₄₂₀H₂ . Only their substrate specificities differ: the HTII takes PA, whereas the HTI takes only the hydride Meisenheimer complexes (H^--PA or the H^--2,4-DNP) as its substrates .

The npdG and npdI gene showed very high sequence similarities and clustered separately from related enzymes . Hence, they may be suitable for use as gene probes for finding bacteria in the environment with the capacity to hydrogenate electron deficient aromatic ring systems . Further, the ability to assess the maintenance of a PA-degrading population in continuous culture is important for efficient bioelimination of PA from industrial effluents . We plan to examine more 2,4-DNP/PA utilizers (and bacteria lacking this ability) for the presence of npdG, npdI, and npdC for a statistically significant sample to confirm that the genes are specific to 2,4-DNP or PA degraders .

This work was supported by the German Research Foundation (DFG) and the Hyogo Prefecture Government .

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