Degradation of 2-Nitrobenzoate by Burkholderia Terrae Strain KU-15

Biosci. Biotechnol. Biochem., 71 (1), 145–151, 2007

Degradation of 2-Nitrobenzoate by Burkholderia terrae Strain KU-15

y Hiroaki IWAKI and Yoshie HASEGAWA

Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan

Received July 25, 2006; Accepted September 17, 2006; Online Publication, January 7, 2007 [doi:10.1271/bbb.60419]

Bacterial strain KU-15, identified as a Burkholderia tified and characterized. This information has contrib- terrae by 16S rRNA gene sequence analysis, was one of uted to understanding of the nature of the biodegradation 11 new isolates that grew on 2-nitrobenzoate as sole of nitroaromatic compounds and the development of source of carbon and nitrogen. Strain KU-15 was also bioremediation solutions. Very little research, however, found to grow on anthranilate, 4-nitrobenzoate, and 4- has been done on the degradation of 2-nitrobenzoate aminobenzoate. Whole cells of strain KU-15 were found (2-NBA). to accumulate ammonia in the medium, indicating that The degradation pathway of 2-NBA has been estab- the degradation of 2-nitrobenzoate proceeds through a lished from only two microorganisms: Pseudomonas reductive route. Metabolite analyses by high-perform- fluorescens strain KU-7 and Arthrobacter protophor- ance liquid chromatography indicated that 3-hydrox- miae strain RKJ100 (Fig. 5).13–15) These strains share the yanthranilate, anthranilate, and catechol are intermedi- same 2-NBA degradation pathway, including the for- ates of 2-nitrobenzoate metabolism in strain KU-15. mation of 2-hydroxylaminobenzoate (2-HABA) by an Enzyme studies suggested that 2-nitrobenzoate degra- NADPH-dependent nitroreductase. 2-HABA is further dation occurs via the formation of 2-hydroxylamino- converted to 3-hydroxyanthranilate (3-HAA) by a mu- benzoate and that the pathway branches at this point to tase. Gram-positive bacterium A. protophormiae strain form two different aromatic intermediates: anthranilate RKJ100 has a branch route after the formation of 2- and 3-hydroxyanthranilate. PCR amplifications and HABA to form anthranilate (AA) by an NADPH- DNA sequencing revealed DNA fragments encoding a dependent 2-HABA reductase. Besides the reductive polypeptide homologous to 2-amino-3-carboxymuconate pathway, Cain and Cartwright have suggested that a 6-semialdehyde decarboxylase and anthranilate 1,2- Nocardia has an oxidative pathway including the cat- dioxygenase. echol formation directly from 2-NBA with the removal of the nitro group as nitrite, but there have been no Key words: biodegradation; 2-nitrobenzoate; anthrani- confirmatory data for this putative pathway.16) late; 3-hydroxyanthranilate; Burkholderia On a molecular view of 2-NBA metabolism, only the genetic locus consisting of nbaEXHJIGFCD, which is Nitroaromatic compounds such as nitrobenzene, ni- responsible for the conversion of 3-HAA to the Krebs trophenols, nitrotoluenes, and nitrobenzoates are used cycle intermediate in strain KU-7, has been deter- in the synthesis of pesticides, plasticizers, dyes, phar- mined.17) In A. protophormiae, it has been suggested maceuticals, and explosives.1–3) These compounds are that the genes responsible for 2-NBA degradation are found as contaminants in waste water, rivers, and located on a 65-kb plasmid,14) but the sequences have groundwater, and in the atmosphere.4–6) Given this wide not been determined. spectrum of pollution, the ability of bacteria to degrade In this paper, we report the isolation and identification nitroaromatic compounds is of considerable interest and of novel 2-NBA degrading bacteria from agricultural environmental importance.7–12) Recent research on the soils. A selected strain, KU-15, identified as a Burkhol- biodegradation of nitroaromatic compounds has yielded deria terrae by 16S rRNA gene sequence, was further a wealth of information on the microbiological, bio- investigated. The study afforded the first example in a chemical, and genetic aspects of the process.7–12) Novel gram-negative bacterium of a 2-NBA degrading branch metabolic pathways have been discovered and genes and pathway that is identical with the one in gram-positive enzymes responsible for the pathways have been iden- A. protophormiae. The pathway in strain KU-15 con-

y To whom correspondence should be addressed. Tel: +81-6-6368-0909; Fax: +81-6-6388-8609; E-mail: [email protected] Abbreviations: 2-NBA, 2-nitrobenzoate; 3-HAA, 3-hydroxyanthranilate; 2-HABA, 2-hydroxylaminobenzoate; AA, anthranilate; ACMSD, 2- amino-3-carboxymuconate 6-semialdehyde decarboxylase; AADO, anthranilate 1,2-dioxygenase; ACMS, 2-amino-3-carboxymuconate 6-semialdehyde 146 H. IWAKI and Y. HASEGAWA tains a novel reductase system that utilizes NADH as Growth condition and whole-cell studies. The MSM well as NADPH as an electron donor. We also indicate used in the preparation of whole cells and cell extracts that this strain has at least partial genes for 3-HAA and was essentially the same as described above. 2-NBA AA degradation. was added to the MSM at 20 mM. Cells grown on 2-NBA were harvested in mid-exponential phase by centrifuga- Materials and Methods tion at 10;000 g (10 min at 4 C), and washed twice with a 21 mM sodium-potassium phosphate buffer (pH Chemicals. 2-HABA was chemically synthesized as 7.1). The cells were resuspended in the same buffer to described by Bauer and Rosenthal.18) 2-NBA, AA, 3- adjust the optical density at 600 nm of 1.0, and incubated HAA, and catechol were obtained from Wako Pure with 1.0 mM 2-NBA, AA, or catechol in a rotary shaker Chemical Industries (Osaka, Japan). All other chemicals at 30 C. were the highest purity commercially available. Preparation of cell extracts. Whole cells, grown on Isolation of 2-NBA-degrading bacteria. 2-NBA-de- 20 mM 2-NBA or 20 mM sodium succinate, were re- grading strains were isolated by aerobic enrichment suspended in a 21 mM sodium-potassium phosphate cultures from cultivated soils contaminated by pesti- buffer (pH 7.1). Crude cell extracts were obtained by cides in Japan. A mineral salt medium (MSM) was ultra-sonication with a Sonifire model 250 apparatus used, containing (per liter) 1.5 g of KH2PO4, 1.5 g of (Branson, Danbury, CT) for six 20-s bursts on ice, Na2HPO4, 0.2 g of MgSO4.7H2O, 0.01 g of CaCl2. followed by centrifugation (48;000 g, 1 h, 4 C) to 2H2O, 0.005 g of FeSO4.7H2O, 0.002 g of MnSO4. remove unbroken cells and debris. 4H2O, and 0.002 g of ZnSO4.7H2O. It also contained 0.3% (w/v) 2-NBA as a carbon and nitrogen source. Enzyme studies. The conversion of 2-NBA or 2- Enrichments were begun by placing 1 g of soil collected HABA was tested in reaction mixtures that contained from cultivated fields into 20 ml of MSM in 100 ml 2-NBA (0.1 mmol) or 2-HABA (0.1 mmol), sodium-po- flasks; 100 ml of each incubated culture was transferred tassium phosphate buffer (21 mmol, pH 7.1), and cell into 20 ml of fresh medium after 2–3 d. The cultures extracts in a final volume of 1 ml. When necessary, were incubated with agitation (120 rpm) at 30 C. After the mixtures were supplemented with NAD(P)H at a enrichment culturing (three reinoculation steps), sam- concentration of 0.15 mM. The reaction products were ples of the culture were plated to the same medium identified by HPLC. The consumption of NAD(P)H was solidified with 1.5% (w/v) agar. To purify the strain, we measured by monitoring the decrease in absorbance at transferred the colonies several times onto fresh agar 340 nm using a U-3010 spectrophotometer (Hitachi plates. High-Technologies, Tokyo). The conversion of 3-HAA was assayed by recording Identification of 2-NBA-degrading bacteria by 16S enzyme-catalyzed changes in the spectra or absorbance rRNA gene sequencing. 16S rRNA gene analysis was maxima of 3-HAA and products over time using a U- performed as previously described.13,19) The sequences 3010 spectrophotometer. Reaction mixtures contained determined in this study and obtained from the DNA 3-HAA (0.1 mmol), sodium-potassium phosphate buffer 20) databases were aligned using Clustal W in the DNA (21 mmol, pH 6.5), Fe(NH4)2(SO4)2.6H2O (0.3 mmol), Data Bank of Japan web site (http://www.ddbj.nig.ac.jp/ and cell extracts in a final volume of 1 ml. Welcome-e.html). A neighbor-joining tree21) was constructed using TreeView software. The 16S rRNA gene Analytical methods. Nitrite and ammonia releases sequences determined in this study have been deposited were measured by standard methods.22,23) Intermediates in DNA Data Bank of Japan under the following and enzyme reaction products were identified by high- accession numbers: AB212049 and AB266607 to performance liquid chromatography (HPLC). HPLC AB266612. analysis was carried out with a Shimadzu instrument (model LC-6AD) equipped with a UV detector operating Utilization of aromatic compounds for growth. The at a wavelength of 254 nm. Separations were performed ability of strain KU-15 to grow on different aromatic on a Capcell Pak C18 UG120 column (column size, compounds was determined by colony formation on 4:6 250 mm; particle size, 5 mm; Shiseido, Tokyo) MSM plates solidified with 1.5% (w/v) agar. Catechol, with a methanol–H2O mixture (20:80, v/v) containing protocatechuate, three isomers of hydroxybenzoate, and 0.07% perchloric acid at a flow rate of 1.0 ml/min as a benzoate were added to the MSM plates as carbon solvent. Quantitative data for 2-NBA were obtained by sources at 5 mM with 0.1% (w/v) NH4NO3 as nitro- comparing the peak heights of the query compounds gen sources. 4-nitrobenzoate, AA, 4-aminobenzoate, with standards of known concentrations. 3-HAA, 3-nitrobenzoate, mononitrophenols, or dinitrophenols were added to the MSM plates as nitrogen PCR amplification of the 2-amino-3-carboxymuconate source at 0.5 mM, with 10 mM sodium succinate as 6-semialdehyde decarboxylase-encoding gene and the carbon source. AA 1,2-dioxygenase-encoding gene. To amplify the par- Degradation of 2-Nitrobenzoate by Burkholderia 147 tial gene for 2-amino-3-carboxymuconate 6-semialde- tree: four strains were identified as Burkholderia spp., hyde decarboxylase (ACMSD), two degenerate primers and seven were identified as Cupriavidus spp., which (ACMSD-F: 50-GTNCAYCCiTGGGAYATGATG-30, belongs to the family of Burkholderiaceae in -pro- ACMSD-R: 50-CCiRGNGGRAAiGGRTARTC-30) were teobacteria. Genus Cupriavidus have been identified as designed from two conserved amino acid sequence genus Ralstonia.28,29) Burkholderia spp. and Ralstonia regions of the ACMSD from P. fluorescens strain KU- spp. have frequently been reported as nitroaromatic 717) and its bacterial homolog in the database, with the compound degraders.30–39) Our study suggests that following accession numbers: CP000317 (ABE47007), Burkholderia spp. and Ralstonia spp. are also major CP000150 (ABB06684), CP000353 (ABF12071), types of culturable 2-NBA degraders in agricultural CP000091 (AAZ64848). In the case of the AA 1,2-di- soils. It is interesting to observe that P. fluorescens oxygenase (AADO) gene, two primers (50-TGGGT- strain KU-7 isolated from a petrochemical-contaminated GAACCGCTGCGCGCA-30 and 50-TGCAGCATGCT- soil did not belong to this family or to the -proteo- CGCGTGATA-30) were designed from two conserved bacteria (Fig. 1). But these results were obtained using a nucleotide sequence regions of the -subunit of AADO culture-dependent method, and the possibility that the from Burkholderia cepacia DBO124) and its homolog isolates were selectively isolated by strong bias in the with the following accession numbers: CP000125 cultivation steps cannot be excluded. It would also be (ABA52832), BX571966 (CAH39366), CP000011 interesting to test whether Burkholderia spp. and (AAU46052), CP000085 (ABC35661), CP000379 Ralstonia spp. are the main contributors to the degra- (ABF79469), CP000152 (ABB12436), CP000272 dation of 2-NBA under environmental conditions using (ABE36987). Genomic DNA from strain KU-15 was a culture-independent method. isolated by the method of Wilson.25) We selected strain KU-15 for further study. Besides PCR amplifications were carried out in 50-ml reaction 2-NBA, this strain grew on 4-nitrobenzoate, AA, 4- mixtures containing 0.1 mg of strain KU-15 genomic aminobenzoate, 3-HAA, catechol, protocatechuate, three DNA as a template, 50 pmol of each primer, each dNTP isomers of hydroxybenzoate, and benzoate. AA, 3-HAA, at a concentration of 0.2 mM,5mlofTaq DNA buffer, and catechol are known intermediates of 2-NBA and 2.5 units of Taq DNA polymerase (New England metabolism in other bacteria.13–16) No growth occurred Biolabs, Ipswich, MA). After 3 min of incubation at with 3-nitrobenzoate, mononitrophenols, dinitrophenols, 94 C, the reaction mixture was subjected to 30 cycles of or mononitrotoluenes. The 1,432-bp 16S rRNA gene 15 s at 94 C, 30 s at 50 C, and 1 min at 72 C. The PCR sequence from strain KU-15 was 99.9% and 99.8% products were directly cloned in vector pXcmkn1226) identical to that of B. terrae strains KMY01 and and introduced into E. coli XL-1 blue. DNA sequencing KMY0240) respectively. Phylogenetic analysis of 16S of the inserts was determined using the ABI PRISM rRNA gene sequences also showed that strain KU-15 BigDye Terminator (v. 3.1) cycle-sequencing kit (Ap- was clustered together with B. terrae (Fig. 1). Based on plied Biosystems, Foster City, CA) and the Applied the 16S rRNA gene sequence analysis, strain KU-15 was Biosystems PRISM 310 Genetic Analyzer. Nucleotide identified as B. terrae, which belongs to the -proteo- and protein sequence similarity searches were done with bacteria. the BLAST program.27) Degradation of 2-NBA by whole cells of strain KU-15 Results and Discussion Whole-cell studies were performed to characterize the degradation pathway of 2-NBA in strain KU-15. 2-NBA Isolation and identification of 2-NBA-degrading bac- was rapidly degraded by whole cells with the concom- teria itant release of available nitrogen as ammonia. Further- 2-NBA-degrading strains were isolated by aerobic more, no nitrite was detected. In strain KU-15, the enrichment cultures from agricultural soils in Japan release of ammonia but not nitrite suggests the involve- contaminated by pesticides. As a result, 11 bacterial ment of reductive steps in the degradation of 2-NBA, as strains that grew on 2-NBA as their sole source of reported for 2-NBA degrading P. fluorescens stain KU- carbon and nitrogen were isolated. These strains grew on 713) and A. protophormiae strain RKJ100.14,15) both agar plates and liquid media containing 0.3% (w/v) To further characterize the pathway, intermediates 2-NBA within 3 d. HPLC analyses of supernatant media converted from 2-NBA were identified by HPLC by after 3 d of incubation detected no aromatic compounds comparison of retention times with authentic standards. in the cultures, indicating that 2-NBA was degraded HPLC studies showed the presence of peaks with the through ring cleavage (data not shown). following retention times: 5.35 min, 8.24 min, 9.58 min, Analysis of the partial 16S rRNA gene sequences and 16.50 min, which exactly matched those of stand- (about 550 bp) of the isolated strains indicated that the ards 3-HAA, catechol, AA, and 2-NBA respectively 11 isolates included seven 16S rRNA gene types. We (Fig. 2A). The detection of both 3-HAA and AA constructed an NJ tree using the nucleotide and related suggests that the 2-NBA degrading pathway branches. sequences obtained from databases (Fig. 1). The isolated Branching of 2-NBA degradation pathways has been strains fell into only two lineages of the phylogenetic found in gram-positive A. protophormiae strain RKJ100 148 H. IWAKI and Y. HASEGAWA

γ-Proteobacteria Pseudomonas aeruginosa LMG1242 Pseudomonas putida ATCC17527 100 Pseudomonas fluorescens KU-7 100 Pseudomonas fluorescens ATCC17573 Pseudomonas putida KT2440 Burkholderia hospita LMG20598

100 60 Burkholderia terrae KMY01 isolate KU-15 86 62 isolate KU-27 isolate KU-28 Burkholderia terrae KMY02 Burkholderia terricola LMG20594 94 54 Burkholderia glathei ATCC29195 69 isolate KU-25 Burkholderia cepacia DSM50181

73 Burkholderia mallei ATCC23344 100 Burkholderia pseudomallei 1026b β-Proteobacteria 98 Cupriavidus campinensis LMG20568 100 80 Cupriavidus campinensis LMG20579 Cupriavidus basilensis DSM11853 51 74 isolate KU-21 isolate KU-41 100 isolate KU-29 87 isolate KU-20 100 Cupriavidus necator ATCC17697

98 isolate KU-26 74 isolate KU-23 52 Cupriavidus oxalaticus DSM1105 isolate KU-22 Cupriavidus taiwanensis LMG19425 Cupriavidus gilardii LMG5886 54 Cupriavidus pauculus LMG3413 99 Cupriavidus metallidurans CH34 0.01

Fig. 1. Phylogenetic Analysis of 16S rRNA Gene Sequences of 2-NBA-Degrading Bacteria. The tree was constructed by the neighbor-joining method. Bootstrap analysis was performed with 100 trials.

only at a high concentration (20 mM) of 2-NBA. In A. protophormias strain RKJ100, except for -ketoadi- A pate, no other intermediate from AA in the 2-NBA COOH COOH degradation pathway was detected, and the mechanism NH OH 2 NO by which AA is cleaved to form -ketoadipate is not OH 2 OH COOH 15) 3-HAA Catechol NH2 2-NBA known. In our experiments using whole cells, catechol 5.35 (min) 8.24 (min) was found to be an intermediate of 2-NBA metabolism 16.50 (min) AA in strain KU-15. To conﬁrm the formation of catechol 9.58 (min) from AA, AA was used as a substrate for whole-cell study. As for the results, catechol and cis, cis-muconate B Intensity COOH were identiﬁed as intermediates of AA degradation COOH COOH cis, cis-Muconic acid NH (Fig. 2B). This suggests that AA is degraded via the 2 8.63 (min) formation of catechol as a terminal aromatic intermedi- OH AA OH ate. 9.58 (min)

Catechol Degradation of 2-NBA by cell extracts of strain KU- 8.24 (min) 15 0 51015 To further characterize the degradation pathway, the Retention time (min) assumed enzyme reactions were investigated using cell extracts from 20 mM 2-NBA-grown KU-15 cells. When Fig. 2. HPLC Proﬁles of Metabolites of 2-NBA and AA Degradation cell extracts were incubated with 2-NBA and NADPH, by Whole Cells of B. terrae Strain KU-15. A, conversion of 2-NBA to 3-HAA, AA, and catechol. B, the reaction products were identiﬁed as AA and 3-HAA conversion of AA to catechol and cis, cis-muconic acid. HPLC (Fig. 3A). Unlike the 2-NBA nitroreductase of strain was carried out under the conditions described in ‘‘Materials and KU-7,13) NADH replaced NADPH in the reaction (data Methods.’’ Degradation of 2-Nitrobenzoate by Burkholderia 149

A COOH 0.8 NH 2 COOH

NO2 OH 3-HAA COOH 4 5.35 (min) NH 2-NBA 2 16.50 (min) 0.6 AA 3 9.58 (min) 2

0.4 B COOH COOH 1

NH2 3-HAA NHOH

5.35 (min) Absorbance OH 2-HABA 18.00 (min) 0.2

Intensity 0 C 0 COOH COOH NH COOH 2 NHOH 250 300 350 400 NH2 OH AA Wavelength 3-HAA 2-HABA (nm) 5.35 (min) 9.58 (min) 18.00 (min) Fig. 4. Spectral Changes during Metabolism of 3-HAA by Crude Cell Extracts of KU-15. The reaction was initiated by the addition of substrate (0.1 mmol) 0 5101520 to the cell extracts in phosphate buffer (21 mmol, pH 6.5). The Retention time (min) reaction mixture was scanned at intervals of 2 min (spectra 0–4). Arrow indicates direction of spectral change. Fig. 3. HPLC Analysis of Enzyme Reaction Products. A, conversion of 2-NBA to AA and 3-HAA by cell extracts of 2- NBA-grown cells with NADPH at 30 C. B, conversion of 2-HABA to 3-HAA by cell extracts of 2-NBA-grown cells at 30 C. C, semialdehyde (ACMS), which showed maximum ab- conversion of 2-HABA to AA and 3-HAA by cell extracts of 2- 41) NBA-grown cells with NADPH at 30 C. sorbance at 360 nm (Fig. 4). This result indicates crude extracts containing 3-HAA 3,4-dioxygenase. Based on these results, we proposed the pathway for 2-NBA degradation of strain KU-15 presented in Fig. 5. not shown). No decrease in 2-NBA was observed In the first step, 2-NBA is reduced to 2-HABA by without NAD(P)H (data not shown). Also, cell extracts NAD(P)H-dependent 2-NBA nitroreductase. Then the of succinate-grown cells failed to decrease NAD(P)H in 2-NBA degradation pathway branches at 2-HABA. a presence of 2-NBA. This result clearly indicates that 2- 2-HABA is a substrate for 2-HABA mutase and NBA nitroreductase is induced by growth of strain KU- NAD(P)H-dependent 2-HABA reductases. This pathway 15 on 2-NBA. is identical to the one in A. protophormiae, which is a In the pathway of strain RKJ100, 2-HABA was found gram-positive bacterium.15) With a gram-negative bac- to be one of the first metabolites, and AA and 3-HAA terium, this is the first report in which the presence of were formed from 2-HABA by the action of a reductase both AA and 3-HAA is indicated in 2-NBA degradation. and a mutase respectively.15) We synthesized 2-HABA The pathway in strain KU-15 contains a novel reductase chemically to confirm 2-HABA as one of the first system that utilizes NADH as well as NADPH as an metabolites in KU-15. 2-HABA was rapidly degraded in electron donor. the presence of cell extracts, and 3-HAA was identified as the reaction product (Fig. 3B). The expected product Screening of genes encoding ACMSD and anthrani- of the reaction, AA, was identified by the addition of late 1,2-dioxygenase in strain KU-15 NADPH to the reaction mixture (Fig. 3C). The produc- To gain a better understanding of 2-NBA degradation tion of AA and 3-HAA from 2-HABA indicates that the in bacteria, we screened possible genes encoding pathway has a branch point at 2-HABA. In the reaction, ACMSD that play a key role in the degradation of 2- NADH replaced NADPH. Also, cell extracts of succi- NBA and 3-HAA,42,43) and AADO, which is the first nate-grown cells were found to decrease NADPH in the enzyme of AA degradation from strain KU-15 by PCR. presence of 2-HABA. Interestingly, however, NADH PCR amplification using ACMSD primers produced did not replace NADPH in the reaction of succinate- about 400 bp intensive DNA bands as expected on an grown cell extracts. This suggests the existence of agarose gel (data not shown). DNA sequencing revealed at least two 2-HABA reductases: 2-NBA-inducible a 374 base sequence which codes for a 124 amino acid NADH-dependent reductase and constitutive NADPH- polypeptide. This exhibited high identity to the ACMSD dependent reductase. of P. fluorescens strain KU-7 (84% identity)17) by To confirm the ring cleavage of 3-HAA, we checked BLASTP search. This result suggests that ACMS is the conversion of 3-HAA by cell extracts of 2-NBA- decarboxylated to 2-aminomuconic-6-semialdehyde by grown cells of strain KU-15. The extracts catalyzed the ACMSD. In the case of AADO primers, PCR amplifi- cleavage of 3-HAA to 2-amino-3-carboxymuconate 6- cation also produced DNA bands (about 450 bp) as 150 H. IWAKI and Y. HASEGAWA

COOH COOH

O2 NH2 NH2 NH2

COOH COOH COOH COOH OH CHO CHO 3-HAA CO2 2NAD(P)H ACMS 2-Aminomuconic- NO2 NHOH 6-semialdehyde NAD(P)H COOH OH

+ O O 2NAD(P) NH2 2 OH 2 2-NBA 2-HABA COOH NAD(P)+ COOH AA NH3 Catechol cis,cis-Muconic acid

Fig. 5. Proposed Pathway for Degradation of 2-NBA by B. terrae Strain KU-15.

expected on an agarose gel (data not shown). DNA Nitrated phenols in rain: atmospheric occurrence of sequencing revealed a 413 base sequence that codes for phytotoxic pollutants. Chemosphere, 17, 511–515 a 137 amino acid polypeptide. This exhibited high (1988). identity to the -subunit of the AADO of B. cepacia 6) Rippen, G., Zietz, E., Frank, R., Knacker, T., and strain DBO1 (92% identity)24) by BLASTP search. Kloppffer, W., Do airborne nitrophenols contribute to forest decline? Environ. Technol. Lett., 8, 475–482 These results suggest that strain KU-15 has a known 17) (1987). 3-HAA meta-cleavage pathway and an AADO sys- 7) Marvin-Sikkema, F. D., and de Bond, J. A. M., 24) tem, and that 3-HAA and AA were not by-products Degradation of nitroaromatic compounds by microor- but intermediates of the catabolic energy-yielding path- ganisms. Appl. Microbiol. Biotechnol., 42, 499–507 way. These results are consistent with Fig. 5. 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