JOURNAL FERMENTATION AND OF Vol. 85, No. 5, 514-517.

1998

BIOENGINEERING

Cloning and Sequencing of a Gene Cluster for the Meta-Cleavage Pathway of Aniline Degradation in Acinetobacter sp. Strain YAA
MASAHIRO TAKEO, TOSHIKI FUJII, KENTARO TAKENAKA,
AND

YOSHIMICHI

MAEDA*

Dept. of Applied Chemistry, Himeji Institute of Technology, 2167 Shosha, Himeji, Hyogo 671-2201, Japan Received 17 November 1997/Accepted 21 January 1998 A 17.0-kb Sac1 fragment, which contains a large region just downstream of the aniline dioxygenase genes (a&&42-As) of Acinetobacter sp. strain YAA, was cloned from YAA plasmid DNA. We determined the nucleotide sequence of a 10.5kb segment within the fragment, in which eleven genes were found. Eight of the genes, designated atdSBCDEFGH, were homologous to the meta-cleavage pathway genes dmpQBCDEFGH and xylTEGFJQKl the products of which are involved in the phenol and toluene degradation pathways, respectively. The G+C content of the atdSBCDEFGH genes ranges from 51.1% to 59.5% and is unusual for Acinetobacter genes (38-47x), indicating that the genes originated from other bacteria. In addition, these values are quite different from those of the upstream at&II-AS genes (40.1-47.7x). This result suggests that this aniline degradation gene cluster is a hybrid structure. [Key words: me&-cleavage Acinetobacter] pathway genes, aniline degradation, hybrid structure, G+C content,

Aromatic amines can be generated by the breakdown of chemical products such as azo-dyes and herbicides in the environment (l-3). In Japan, aniline, chloroanilines and toluidines have been frequently detected in water, sediment and fish samples at concentrations of up to 20ppb (4). They are considered to be a health risk even when present at low levels in the environment, because of their strong toxicity and mutagenicity with respect to living organisms (5). The major route for their breakdown in the environment has been found to be by biodegradation (5). To elucidate the biodegradation mechanism of aromatic amines, we previously isolated several aniline-assimilating bacteria from activated sludge and successfully cloned aniline dioxygenase genes (atdAl-A5) and a catechol 2,3dioxygenase gene (atdB) from the plasmid DNA of one isolate, strain YAA (6). This result indicated that the aniline degradation pathway of strain YAA was encoded on a plasmid and involved in the meta-cleavage pathway of the benzene ring. We designated this aniline degrading plasmid pYA1. Subsequently, we sequenced a 6.3-kb region of pYA1 containing atdAI-AS, and deduced that aniline dioxygenase is a multicomponent enzyme consisting of a glutamine synthetase-like protein, a glutamine amidotransferase-like protein and three subunits of benzene ring-hydroxylating oxygenase (7). In this study, to further characterize this degradation pathway, we cloned and sequenced the remaining metacleavage pathway genes. First, plasmid DNA was extracted from YAA using the alkaline extraction method (8), digested by the restriction endonuclease Sac1 under the conditions recommended by the supplier (Takara Shuzo, Kyoto), and ligated to SacI-digested pUC19 (Takara Shuzo) using a DNA Ligation Kit Ver. 2 (Takara Shuzo). The resultant library was introduced into competent E. coli JM109 (Takara Shuzo) and transformants were selected on LB plates (9) containing 100 mg. Z-l of am* Corresponding author. 514

picillin. When colonies appearing on the plates were sprayed with 0.1 M catechol in 10 mM phosphate buffer (pH 7.0), the color of one colony turned brilliant yellow, indicating catechol 2,3-dioxygenase activity. As shown in Fig. 1, the recombinant plasmid extracted from the clone, designated pAC200, had a 17.0-kb Sac1 insert fragment containing a large region just downstream of the atdAl-A5 genes. Various fragments of the insert were ligated into pUC18 (Takara Shuzo) or pUC19 to construct recombinant plasmids for sequencing. Sequencing and sequence analysis were carried out as described previously (7). The nucleotide sequence of a 10.5-kb (10,498-bp) segment within the pAC200 insert was determined and the data have been submitted to the DDBJ data bank under accession number ABOO 1. Computer analysis showed that this segment contained eight open reading frames (ORFs) in addition to three genes (atdA4, atdA5 and atdB) previously identified, and the ORFs were designated atdRSCDEFGH (Fig. 1). However, the TnlOOO sequence (10) previously found between atdA5 and atdB could not be found in this segment (6). Southern hybridization studies using TnrOOO internal probes confirmed that TnlOOO is absent in YAA plasmid DNA (data not shown). Thus, we concluded that TnlOOO was inserted into this gene cluster from an E. coli host genome or episome during the previous cloning experiment. Homology searches based on the determined nucleotide sequence showed that the atdSBCDEFGH genes were homologous to the meta-cleavage pathway genes dmpQBCDEFGH and xylTEGFJQKI in the phenol and toluene degradation pathways, respectively (Table 1) (11-14). The amino acid sequence of each predicted gene product also shared 46.0-80.9x identities with those of the corresponding proteins in the phenol and toluene degradation pathways (Table 1). Thus, the atdSBCDEFGH genes are presumed to encode the metacleavage pathway enzymes of this aniline degradation pathway (Table 1). Based on the amino acid sequence homologies, the proposed aniline degradation pathway

VOL.85, 1998

NOTES

515

TnZaor,insertion point 3.0kh ,____~__.__,~_. A1 .. . . -I .____ . . . .ii~~~(AbW] .. 55.5 47.4 41.8 43.5 40.5 40.1 0.6kb

R
41.7 55.5 53.4 58.5

1D 1E 59.5 58.7

(~~~+-w
56.0 54.7 51.1 488

pAC200 pAFR900
c

pAKlOOASm2 pAK17

;~06 pAC41 pAC40

pAS16

_ pAKlOOA Sml pAKlOOASal pASm23 1

pAKlO0

pAC18

pAC17

FIG. 1. Restriction map of the aniline degradation gene cluster of Acinetobacter sp. strain YAA. The dotted box indicates the region of which the nucleotide sequence was determined in this study. Arrowheads indicate the transcriptional direction of the lac promoter on plasmids. Although TnlOOOwas previously found at the indicated site, we could not detect it in this study. Numbers below open boxes and arrows show the GS C
content of each gene or region.

and the corresponding genes are shown in Fig. 2. At this stage, this pathway seems to lack a gene encoding 4oxalocrotonate tautomerase, corresponding to the dmpl and xylH genes, because we could not detect any ORF within the 0.6-kb region downstream of the atdH gene. The atdR gene, not likely to be a member of the metacleavage pathway genes, was Iocated between atdA5 and atdS, and may encode a LysR-type transcriptional activator (15), since the amino acid sequence of the predicted gene product showed weak but overall homology to sequences of LysR-type transcriptional activators (15) such as AmpR (21.0%) (16) and MauR (21.9%) (17). However, the helix-turn-helix motif common to LysRtype transcriptional activators (PROSITE accession num-

ber: PSOO044) is not completely conserved. This gene is now under investigation. The G+C content of each gene of this pathway is presented in Fig. 1. The G+C content of the atdSBCDEFGH genes ranges from 51.1% to 59.5X, which is unusual for Acinetobacter genes (38-47%) (18) and Iower than those of the Pseudomonas meta-cIeavage genes DmpQBCDEFGH and xylTEGFJQKI (57.3-66.8% and 57.5-67.1%, respectively). Accordingly, this metacleavage pathway may have originated from other bacteria. In addition, these values are quite different from those of atdAI-A5 and atdR (40.1-47.7% and 41.7%, respectively) just upstream of the meta-cleavage pathway genes. The GfC contents of the 3.0-kb and 0.6-kb sp. strain YAA

TABLE 1. Characterization of the meta-cleavage pathway genes involved in the aniline degradation pathway of Acinetobacter
and sequence identities with the corresponding genes in the dmp and xyl systems

Sequence identities (%) withh Nucleotide number (position on a 10.5-kb segment)

atdS atdB atdC atdD atdE atdF atdG atdH 336 921 1458 855 666 906 1020

Number of amino acids (estimated molecular mass)

IWative function Of product

dmp system

xyl system nt 53.0 69.0 76.8 69.4 76.8 62.2 60.0 67.2 (T) (E) (G) (F) (J) (K) aa 46.0 73.0 78.4 63.4 79.3 51.4 (Q) 53.3 (I) 65.2

nt (nt (nt (nt (nt (nt (nt (nt 804 (nt

2888323241875655651672903223) 4152) 5644) 6509) 7181) 8195)

aa 74.3 80.9 70.8 68.6 51.7 52.8 67.2

kD) 307 (35.0 kD) 486 (52.1 kD)

112 (12.0

Small ferredoxin-like protein

Catechol2,3-dioxygenase 2-HMSa dehydrogenase 2-HMS hydrolase 2-Oxopent-4-dienoate hydratase Acetoaldehyde dehydrogenase 4-Hydroxy-2-oxovalerate aldolase 4-Oxalocrotonate decarboxylase

8214- 9233) 9236-10039)

285 (31.9 kD) 222 (23.9 kD) 302 (32.4 kD) 340 (36.2 kD) 268 (29.4 kD)

58.0 70.4 75.9 72.0 65.0 59.8 62.4 68.5

(Q) 54.0
(B) (C) (D) (E) (F) (G) (H)

a 2-Hydroxymuconic semialdehyde. b The corresponding gene designation is presented in parentheses.

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TAKE0 ET AL.

J . FERMENT.BIOENG ..

FIG. 2. Proposed aniline degradation pathway and the corresponding aniline degradation genes of Acinetobacter sp. strain YAA. The corresponding me&cleavage pathway genes in the phenol and toluene degradation pathways, along with the aniline degradation genes, are also shown. This meta-cleavage pathway lacks a gene corresponding to the dmpI and xylH genes.

regions flanking this gene cluster (atdAZ-H) are 55.5% and 48.8X, respectively (Fig. 1). Therefore, these data suggest that this aniline degradation gene cluster is a hybrid structure. Previously, GUMS analysis of aniline and o-toluidine metabolites using recombinant E. coli harboring the complete aniline dioxygenase gene cluster (atdAZ-A5) revealed that catechol and 3-methylcatechol were produced and accumulated from aniline and o-toluidine, respectively (6). However, 4-methylcatechol and 4-chlorocatechol could not be detected from any toluidine or chloroaniline (data not shown). To determine the degradability of catechols in the meta-cleavage pathway, extradiol dioxygenase activities for catechols were measured by the method of Hirose et al. (19) with a modification (substrate concentration was 1 mM instead of 100 PM). The cell extract of the recombinant E. coli harboring pAS16 (Fig. l), which contained the complete catechol 2,3-dioxygenase gene (atdB), showed high extradiol dioxygenase activities for catechol (4000 pmol min-l g-protein: lOO%), 4-methylcatechol (134%), 4-chlorocatechol (125%) and 2,3_dihydroxybiphenyl (96X), but little for 3-methylcatechol (24%). Therefore, this meta-cleavage pathway has not completely adapted to the substrate specificity of the aniline dioxygenase. This pattern was unique in that the activity for 2,3_dihydroxybiphenyl, in addition to 4-substituted derivatives, was high (19, 20). These results may also support the hybrid structure of this gene cluster.
We thank Dr. H. Hirata, Himeji Institute of Technology, for the use of his facilities for sequencing. REFERENCES of synthetic organic colorants, 1. Meyer, U.: Biodegradation p. 371-385. In Leisinger, T., Hutter, R., Cook, A.M., and Nuesch, N. (ed.), Microbial degradation of xenobiotics and recalcitrant compounds. Academic Press Inc., London (1981). 2. Kearney, P. C., Isensee, A. R., and Kontson, A.: Distribution and degradation of dinitroaniline herbicides in an aquatic svstem. Pestic. Biochem. Physiol., 7, 242-248 (1977). 3. Bartha, R.: Fate of herbicide-derived chloroanilines in soil. J. Agric. Fd Chem., 19, 385-387 (1971). 4. Environmental Health and Safety Division, Environmental Health Department, Environment Agency of Japan: Kagaku-

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