Differential organization and transcription of the cat2 gene cluster in aniline-assimilating Acinetobacter lwoffii K24

JOURNAL OF BIOSCIENCE Vol.

88, No.

3, 250-257.

AND BIOENGINEERING 1999

Differential Organization and Transcription of the cat2 Gene Cluster in Aniline-Assimilating Acinetobacter Zwofii K24 SEUNG IL KIM,’

KWON-SO0

HA,’

AND

SUN-HEE LEEM2*

Biomolecule Research Group, Korea Basic Science Institute, Taejon 305-333l and Department of Biology, Dong-A University, Pusan 604-714,2 Korea Received 1 March 1999/Accepted 28 May 1999

CatABC genesencode proteins that are responsible for the first three steps of one branch of the Bketoadipate pathway involved in the degradation of various aromatic compound by bacteria. Aniline-assimilating Acinetobacter hvofii K24 is known to have the two-catABC gene clusters (cat1 and cat& on the chromosome (Kim et al., J. Bacterial., 179: 5226~5231,1997). The order of the cat2 gene cluster is catB&& which has not been found in other bacteria. In this report, we analyzed the transcriptional pattern of the cat2 gene cluster and completely sequenced a 5.8 kbp fragment containing the compactly clustered catBzA2C2genes and four ORFs. Similar to the ORFRl of the cat1 gene cluster, an ORF highly homologous with the catR gene was found 102 bp upstream of the catB2 gene and was designated as ORF Rz. Three ORFs, one putative reductase component (ORF& and two putative LysR family regulatory proteins (ORFn, ORF.& were located next to the c&C2 gene in the opposite direction of the cat2 gene cluster. Two ORFs, ORF= and ORFn, were significantly homologous with tdnB and tdnR of the aniline oxygenase complex of Pseudomonas putida UCC22. RT-PCR analysis and Northern blotting revealed that the catB2 gene is independently transcribed and that the catA2C2 genes are cotranscribed. A primer extension assayrevealed that transcription of the catA2C2 gene starts in the C-terminal region of the cafB2 gene. These results suggest that the cat2 gene cluster may be under a different gene adaptation from other cat gene clusters. [Key words: /3-ketoadipate pathway, cat gene, Acinetobacter, biodegradation] The cat genes encode proteins that are involved in one branch of the P-ketoadipate pathway (ortho-cleavage pathway) for converting catechol to succinyl-CoA and acetyl-CoA through ,9-ketoadipate (1). Seven genes (catA, B, C, 0, IJ and fl participate in six steps of this pathway (2). Usually, the genes of the ortho-cleavage pathway are found on the chromosome but the genes of the modified ortho-cleavage pathway originate from degradative plasmids (3-6). The facts that the amino acid sequences of chlorocatechol-degradative genes (modified ortho-cleavage pathway), such as clc, tcb and tfd genes, were reported to be distantly related to the cat genes and that the modified ortho-pathway uses both the catIJ and catF genes for chlorocatechol degradation, suggest that these genes may have originated from cat genes and may have undergone differential adaptation (7). The cat genes are found in diverse aerobic bacteria exhibiting the ability to use various aromatic compounds as energy sources. The cat genes are highly conserved but their organization and regulation are diverse. The diversity of cat genes was demonstrated in two well-studied bacteria, Pseudomonas putida and Acinetobacter calcoaceticus (2, 8-10). P. putida possesses the catRBCA genes in a close cluster and uses the pcaDIJF genes for the last three steps of catechol degradation since the cat operon does not contain the catDIJF genes in this organism (11). However, A. calcoaceticus contains a complete set of cat

genes including the regulatory gene catM, in one operon. The differentiation of cat genes was also found in Acinetobacter Iwofii K24 and Rhodococcus erythropolis ICP (12, 13). In the case of the aniline-assimilating A. Iwo@ K24, two different structures of catABC genes were reported. The ORFR1-catBICIA1 gene cluster of A. lwofii K24 (14) was found to be similar to the catRBCA gene cluster of P. putida (8) and Frateuria sp. ANA-18 (15), and the ORFW-catBzA2C2 gene cluster was found to be similar to the ORFR2-catB2A2C2 of Frateuria sp. ANA-18 (15). In this study, we report the transcriptional characteristics of the cat2 gene cluster and a 5.8 kbp sequence of DNA fragment containing the cat:! gene cluster and four ORFs. MATERIALS

AND METHODS

Bacterial strains, plasmids and growth conditions The bacterial strains used in this study were A. IwofJii K24 and E. coli DHSa. A. lwofii K24 is a natural isolate, which can grow on aniline as the sole carbon and nitrogen source. Total RNA preparations were obtained from A. lwofli K24 which was cultivated up to early exponential phase in an aniline-containing medium at 27°C as previously described (12). E. coli DHSa, which harbors plasmids, was grown aerobically with constant shaking at 37°C in LB broth containing 5Opg of Ap per ml. Plasmids pCD2 and pCD24 were selected by colony hybridization, using a PCR product of the catAa structural gene as a probe (12). Plasmids pCD21, pCD22 and pCD23 were subcloned from pCD2. The analyzed DNA sequence of the EcoRV-PstI fragment of the subclone pCD21 contained partial catBz and catA2-catC, genes (12). Plasmid pUC118 was used in this subcloning. DNA manipulations Plasmid DNAs were prepared

* Corresponding author. Abbreviations: aa, amino acid(s); ABS, activation binding site; Ap, ampicillin; bp, base pair(s); cal, gene(s) for conversion of catechol to succinate and acetyl CoA; Da, dalton(s); kbp, kilobase or 1000 bp; LB, Luria-Bertani (medium); ORF, open reading frame; pa, gene(s) for conversion of protocatechuate to succinate and acetyl CoA; P, plasmid; PCR, polymerase chain reaction: RBS, repression binding site; RT-PCR, reverse transcription-polymerase chain reaction. 250

VOL.

TRANSCRIPTIONAL

88, 1999

BGAT

CAC CGC AGG CAG CGC GCC ATA CAG IVAPLAGYLTSPVFGIVIREALQALRR GGT AAG CGG CGC GAG TTC GTC TCX 'l-K TLPALEDAEDVLRRAQTYFIRGAETLR CAG CGG CCG TGA ACC GCG l-X GAA CAG LPRSGREFLALGVSEEIQQIQRSLPPQ CGT CAT ATG CAG GCG G-,-T GGC GGC CCG T M HLRNAARTINMEEAVAIFYRLQRLE

+ OmR2

CAT M AAACXAGITCAGGRGGGCAAGGA

OF CA Tz GENE CLUSTER IN A. L WOFFIZK24

CHARACTERISTICS

CDT GGA CGG CAC AAA GCC GAT CAC GAT GCG CTC GGC CAG T-I% CGC GAG GCG CCG

81

ATC GAC GAG GCG CCG CGC CTG CGT ATA GAA AAT CCG GCC GGC TK

162

GGi- CAG ACG

CGC GAG GCC GAC GCl' TTC CTC GAT Cl-G CTG GAT Cl-G GCG GCT GAG CGG CGG TM;

243

CGT AAT GIT

324

CAT TTC 'IX

GGC GAC CGC GAT GAA ATA GCG GAG CTG GCG AAG Tpc

GCATA~~~A~~A~~~~~CT~~~A~A~A~C~A~

catB2

406

ATO ATA GCA ACA CCC GP.2 AAG ATC GAG ACX GTG GAG ACG ATT C-i-C GTC GAC

480

MIATPVKIESVETILVD 'XC GCGACGATGAACTGC

CCC

561

GAAAGC CCG GAAAGT AX AAG GTC E S P E S I K V GCG GCA ATG GCC ACG CTG CGC GGC

642

CAG GCG CAG CGC CTC GGC G-I-C CCT

804

GCGAGC

885

GTACCGACG ATT CGTCCG CAC CGG CTGTCG CAGACG CTC VPTIRPHRLSVATMNCQTLVLVRIRCA GAC GGC GTG GX GGT GTG GGC GAG GGC! ACG ACC ATC GGC GGG C'X GCC TAC GGT GAA D G V" G V G E G T T I G G L A Y G E AAC ATC GAT ACG TAT TTC GCA CCG CTG C-X AAG GCX CTC GAC GCG ACC CGA CCA GGC NIDTYFAPLLKGLDATRPGAAMATLRG 'IT-G 'ITI CAG GGC AAC CGC CGG TTT GCC TCG GCG GTT GAA ACC GC-T TTG 7-X GAT GCC LF3GNRFARSAVETALFDAQAQRLGVP TTG'KGGAA CTG ',-IT GGC CGGCGCATC CGC GATTCGG'XcXC GTGGCG'IGGACC CTC LSELFGGRIRDSVDVAWTLASGDTTRD ATC GAC GAA GCC GAG CGC GlT TTC GAA GCG AAG CGG CAT CGC Gi-G TTC AAG C?G AAG IDEAERVFEAKRHRVFKLKIGSRALAD GAC GTGGCG CAC GIT GTGGCGA'I'TCAG AAGGCGCTGCAAGGGCGC CGTGARGTGCGGGW DVAHVVAIQKALQGRGEVRVDVNQAWT GAGWC GAG GCGATC 'IGGGCC GGTAAA CGGITC GCC GATGCGAGC GITGCGCTGATC ESEAIWAGKRFADASVALIEQPIAAEN CGC GCG GGT CTC AAA CGT CTGACG GAT cTC GCT CAG GTG CCG ATC ATG CXG GAC GAA RAGLKRLTDLAQVPIMADEALHGPADA 'IT.? GCACTG GCG AC% GCGCGTGCC G4X GAC GTGTTC GCCGTGAAGATCGCGCAA'TCGGGC FALASARAADVFAVKIAQSGGLSGAAN GTG GCG GCG ATT GCG CTC GCC GCG AAT ATC GAC CTG TAC GGC GX ACGATG CTC GAA VAAIALAANIDLYGGTMLEGAVGTIAS GCGCAA CTC TIT AGC ACC ?TtGGC GAA Tl-GAAGTGGGGCACC GAACTGTTCGCGCCGlTGcn: AQLFSTFGELKWGTELFGPLLLTEEIL ACT GAG CCG cn; CGP TAC GAG AAT TIT GlTTl'G CAT CTT CCA CAA GGA CCG GGT CTG TEPLRYENFVLHLPQGPGLGITLDWDK

GIY: CK;

GTGCGC

ATT

GGTGACACG

CGC 'XC

ACC CGC GAT

ATC GGP TCG CGA GCG TX; GAC G'TGAAC

966

CAG GCG TGG ACC

1047

GCC GCG GAA PAT

1128

CAT GGG CCG GCC GAT GCC

1209

GAGCAGCCGA7T CCC l-l%

GCC GAC

723

GGC CTGAGCGGC

GCC GCG AAC

1290

GGC GCG GTG GGC ACG ATC GCC TCG

1371

CTC

1452

GGC A'l'Z ACG CTC GAC TGG GAC AAG

1533

+1 -3 ATC GAC CGC TTA CGG CGC GAT ACG CGC AAG GGC GCAAGC ATC ACC ATG AAC %A CGG'TTCA'I"IUXTAACCGCCAAGAACAAACG IDRLRRDTRKGASITMN" WTACCCC ATGAAC AAG CAA GCC Al'? GAC GCG CTG CTG CAAAAA ATC AAC GAT AGC GCC ATC AAT GAA GGC AAT CCG

1621

cat@

M

N

K

Q

CGC ACG AAG CAG A?C GTC AAC CGC RTKQIVNRIVRDLFYTIEDLDVQPDEF TGG ACC GCG CTG AAT TAT CFC GGC WTALNYLGDAGRSGELGLLAAGLGFEH T-IT CTC GAT CT.2 CGC ATG GAC GAA FLDLRMDEAEAKAGVEGGTPRTIEGPL TAT GTG GCG GGC GCG CCG GTT TCC YVAGAPVSDGHARLDDGTDPGQTLVMR CGC CGC GTG ITC GGC GAA GAC GGC GRVFGEDGKPLANALVEVWHANHLGNY TCG TX ?-K GAC RAG TCG CAG CCG SYFDKSQPAFNLRRSIRTDAEGKYSFR XC GTG GIG CCG GTC GGI TAC TCG S V V P V G Y S CCX CCG GCG CAT ATT CAC 'ITC TX! RPAHIHFFVSAPGFRKLTTQINIDGDP TAT CTG TGGGAC GAC TIT GCGm YLWDDFAFATRDGLVPAVRQAEVRKAN CGP ACG GCG TGG ACG GTC AGT TCG RTAWTVSSR' CGCCGAAGCGATAAGAAAA~CMGC cat-

A

I

D

AT-AGG

A

L

L

Q

GAT CTG TIT

K

I

TAT ACG AX

CTGACC

N

D

S

m

GAC C-X

A

I

N

GAA GAAAlT

E

G

N

GAC GM: CAG CCG GAC GAG ?TC

GAC GCG GGC AGG AGC GGC GAG CTC GGT CTG TTG GCC GCC GGT CTC GGC 7-X

1702

P 1?84

GAG CAT

1865

GCC GAA GCG AAG GCC GGC G-K GA?, GGC GGC ACG CCG CGC ACC ATC GAA GGA CCG TTG

1946

GAC GGC CAC GCG CGG 'XC

GPG ATG CGC

202?

GGC AAC TAC

2108

GAT GAC GGC ACC GAT CCG GGT CAG ACG cn;

AAG CCG CTC GCG AAT GCG CT2 GTT GAG GTG 'EG GCT 'IT'2

AAT CTG CCC CGC TCG ATT

CAC GCG AAT CAC C-X

CGC ACC GAT GCC GAA GGC AAG TAC AGC TTC CGC

2189

GTG CCG CCG CAA GGG CAG ACG CAG TM; CX CTC GAT CAG 'ITG GX CGC CAT GGG CAT V P P Q G Q T Q L L L D Q L G R H G H G?T TCG GCG CCG GGT TTC CGC AAG CM: ACC ACC CAG ATC AAC Ai-C GAC GGC GAT CCG

2270

GCCACG

2432

CGC GAC GGG CTC Drc: CCC GCC GTC AGG CAG GCC GAG GTG CGGAAG

CGT TGA TCGATT-XGACTTCAC AT13 CT% 'ITT

GCAAAC

-~CC~~~CAAT~C~GCCGAA-GCEGWG

CAC GTA GAG ATG ACT CXC AAT Cn:

MLFHVEMTVNLPSDMDAE

FIG. 1

CCG 'ICC GAT An:

2351

2529 GAC GCG GAG

2617

251

252

KIM

ET AL.

J. B~oscr.

CGc GCC GCC CGT TM: AAG 'TCC GAT GAG AAA GCG A'TG 'KG CAA AAG CTG CAG CAG GAG CGC Gv.2 TGG CG(: CAC TIY: m cm RAARLKSDEKAMSQKLQQEGVWRHLWF AT? CL32 CGC TAC GCGAAC AT AGC GPG TX GAC GTG GAAAGC CCA GCG CAT CTG CAT GAC GTG CM: AGC CAG'r?Y: I‘CG I A G R Y A N I S V F D V E S P A H L H D" L S Q L i' CTG TTT CCG TAT ATG GAC GTC GAA GTG CGC GCG C-X TGC CGG CAT GCT TCA TCG ATC CAC GAC GAC GAT CGC TAA GGGCGC LFPYMDVEVRALCRHASSIHDDDR' WGCAAGCCGGTGCXC GRATGCGGCGCCG AAAGATIGAAGAlcGGcTG ?TAAAGA?r GAG CAC CAG CAG CGG CGA ACE AGZ * L D L" L L P S A A GCG CGA GAC ACA GCA GCA GAT CAC CM GCC GCT CGC GCG TIT GGC TI-I'ACT CAG GCA ATG ATC GCG ATG CTC rcxG CGT GCC r. RSVSCCIVKGSAREAKSLCHDRHE~T CAC CX Al-C GAC CATGCAGT GCC GCAGAC GCCTTCACC GCA TGACGTATC CAC l-IC AATG'X GAT CGACGC AAGCGC SVVDVMCTGCVGEGCSTDVEIGI :; A L A CGC GAC GAT CGT CGP G-IT CT-I A'K CAC AX GAC TGT 'XC GCC GCT GCT GGC AAT ACG CAC GTC GAA TGA A% CAG “GT G'm A V I T T H K D V H VT A G S S A I R V D F S D L T N CGA TK GTC GCC AGC CGC AGC AGC CGG CTC GGC G!X AAAACG TIT GAG GTG AAT CGC ATC GGC GGC AAC GCG CTG TTC &.c SEDGAAAAPEAAFRELHIADAA"RQEG TAT TGC CAC CAC ACG Tpc CAT GAA AGG CGC GCG CCC ACA GGT ATA AAG GTG CGA GCC GTC GCG CGC ATT C%X GAC GCA AI‘C I A V V R E M F P A P G C T Y L H S G D R A N A" c G C%X TAG CC% CCC GTC GAG TI'G G-X CCG 'I-K GAC GCC GAA ATG AAA CCG CAC GAA A'TC ATI GAA CGG CGC GCG CGA GAG CAA ALAADLQDREVGFHFRVFDNFPARSLL GGG CAG GAA AGC CGC ATG TTC CGC GC-I GCG CGC GAA ATA An; CAG CAC GA&. GCG CTG TX CTG CT?' GAG CAG GCG ;TA TGC P L F A A H E A S R A F Y H L V F R Q E Q K L L R Y A CAT GCT CAG GAG CGG CGT CAC ACC GAT ACC CGC CGC AAT CAG AAT GTG TX GGT GGC GCC GGG CGC GAG Cn; G&A CAG ATT M S L L P T V G I G A A I L I H E T A G P A L 'j F i N GCG CGG CGC GCC GAT CGI CAA 'II-C CXA GCC GAC CGT CAC GTC CTC ATG CAG CGA GCG CGA ‘XX GCC GCG CGA C!X 1?y' m“ RPAGITLECGVTVDEHLSRSGGRSAEE ACG CIT GAC CGC AAA CAG GTG 'PX 'I-X GCG An; GTC CGG GTC GCC CXA CAGAGAATA TTG CCC, CGT GAC GCC AGG AGG GGC R K V A F L H T E R H D P D G C L S Y Q R T V G P P ,? CGG TGA CGT CG4 TAT GCG CGC CGG GTT CGT AGC GCT CGA ACG GTT GTC CGT CCA GAC GCG ATA CGC TGA ACG AGC GCA CGC PSTSIRAPNTASSRNDTWVRYASRACA CGT GCG CTI'CAT CAC GCA CGC TGT CGACGC GTACGC TGAAGGTGG TTC T-IT CCA TGATCTTGG CGG CCAACTGAG CAA AAA TRKMVCATSAYASPPEKWSRPPWSLLF w;A CAG AGA GGG CXA GAG CGGACG CCA CAT GCAATT CGC GGC GCC CGG ITG TCX G'TC AAG GATAAGAAA CCA CCA ACA ATC SLSPCLPRWMCNAAGPQADLILFWWC" AGG CAA ATC GAT TAA AAA TCG Al-l' ATA GAA TAA ATG TGA CTG ATA ATA CGG CGC GCT CAT CG-GTCATUTCCACAT M t omm TIT TTC CGG CCG TTT CAG CGC A?T CAG K E P R K L A N L CGT GCG AAT ACC CGG CGT GCC GCG ATA T R I G P T G R Y GCA GCA GAC GCT TKGAA GGT TCG CAT

PLDILFRNYFLHSQYYPAS GCGITATITCAACCTXG

TCA TTC CCA CAT CCC GGC CAG GIY; ACG CAC CAG CCC * E W M G A L H R V L A CXC GCG CAT GCA GAT CAG CAA CM; ACG TGT GGC CCA ACX GIT CGT CAA CTC GAT D R M C I L L Q R T A W S D T L E I GCG CTI'CGC GGC GCX GGC CGGAAC GAT GGC GAT GCC CGC GCC C'E T-K GAC CAT RKAATAPVIAIGAGQEVMCCVSEFTRM G'TG AGC CCG AAC GCT GAG CGG 'IT.2 'ITC GGC AAG CAG TGA GCG 'MC GCC GAG GTA HARVSLPQGALLSREGLYAHLASST GCC AAT GAA 'I-K ACG C-X GGC CAC CTC TGC GAG CGT CAC GCG GCG GCG TGC GCC GIFEREAVEALTVRRRAGLPDDR CAC GAG TIT GTC GAT GGC GAA GCG CAG CGT C'IG CAA KX GCC An: CTC GAC GGC " L K D I A F P L T Q L A G H E V A GCC GGC CAG CAC TGC T-I-I' GAT GGT 'IT'? GCT ACT 'I-l-G CCG CTC c?T CAG ATC GAT G A L V A K I T E S S Q R E K L D I CAG GCC GAC CGC GGA CC& CAA GAA CTC GGT GAT CGC GGC GGT ATT GGT CCA CAA LGVASPLFETIAATNTWLRICAKRGA; ATT 'ITC ACC GAG TIT GCC C-K CAT CCG C7I-C GAT CTG GCC GAG CAC GAG CCG TGC HEGLEGEMRDIQGLVLRAHHALT CCX CGA CTC TAC GCC GCG CCG GCC GCG C'K CAG CAA 'KC CAC GCC CAG GGC TI-C T S E V G R R G R E L L P V G L A E CGAAGG CAG CGA CAT GTT CGC GCG GGC CGC GCC ATG CGP GAT GCT GCC 'XT TIT S P L S M N A R A A G H T I S G T E GAT CAG GTC GAA GCG CAT GGGGTGAGRGTGGAGCGGGTGTTG'ITATtGIY;GCCGGG~~? I

L

D

F

R

M

t

2698

2860 2911

311; 3194

3356 3437 151x 1599

3842 3923 4004 4085 4166 4247 4J2h

CAA i. CAG i. CGC A GAA F L‘TG

4409

ATG CGC GAG CGT ATC GCC CXT 'XX‘ E G 7‘ A TTC CAT ACC GCG CAA ACG TGC X:r AGC EM G R I. R A S A GAG AAT GTC CAG AA,? AAG TX% GAG u"l‘C L I H L F L R :. L

‘JR14

CGC GTG CAA CGC K-T GCT CGT GCT s CAG CGG GIT GTC ACG GCT CGT G&T s T I GIT CGA AAT GAT GCC GAT TTC Cc;c D S 1 : G 1 E A CTG TAC GIT ffiG GX AX GCG ZAG Q V N P H" P II GCG GAT GCA GG(' T-I-I GCG ACC GC*‘ An:

BIOEI+(,..

4430 411, 4651 47 : ‘

4895 4978 SS.'I

own 51'18 5285 5392

TCA GCG CGG CGC AAG CCG 'I-K AGG l RPALREPLAYRQMTREMGDLFAQEAA G-I-7 CAT cfl GCG CCC GCG CAG CCA NMKRARLWLLDIDVDVLGEEPPLRWLF T-E TTG CGC GAG An: GTC GCG CAC QQALDDRVIHEPLCGIGYGAFILRRVE ATC GAG GCT CGG AGA GGC CGC CAC D L S P S A A V GCC GAT CIY; CXC GCT GGI GAA TGA G

I

Q

D

S

T

F

S

TAG CGC DTA ACG TIG CAT CG'i' GCG CTC CAT GCC GK

GAG AAA GGC CT% ‘TC

Gcic GW

5473

TAG CAG ATC GAT ATC GAC GTC GAC TAA TCC CTC TTC GGG CGG CAA ACG CCA CAG GCG

5554

GAT GTG c?r

GGG CAG CCA GCC GAT GCC GTA TCC GGC GAA GAT CAG CCG ACG CAC TTC

5635

GAT GCG TC< GGT GAA GCC GCG CTG ATC GCG GAA CAC CGT GAG CGG CGA GAG GCT LTC D I R G T F G R Q D R F V T L P S I, S CAC GAA GIT TIT CGC CAG CAA CPG ATC

5liFi

V

F

N

E

A

L

L

Q

D

t

i-lb’

ORFn

FIG. 1. (A) Restriction map of the 5.8 kbp DNA fragment containing cat2 gene clusters. Plasmids pCD21, pCD22 and pCD23 were subcloned from pCD2. Plasmid pCD24 is another positive clone (12). The enzyme activity of catA and c&B was assayed by following the method previously reported (14). Plasmid pCD22 possesses catA and catB activity but plasmid pCD21 shows only catA activity. (B) Nucleotide sequence and deduced aa sequence of the 5767 bp fragment of plasmid pCD2 and pCD24. The 198 bp nucleotide (bp 5559-5767) was obtained from pCD24. The putative ribosome-binding sites are underlined. The arrows at ORFR2, ORF,,, ORFn and ORFzz indicate the direction of transcription. The transcription start site of c&AZ is indicated by + 1 and the sequence for primer extension analysis is underlined. The putative 2Fe-2S binding motif of ORFxL is also underlined. The nucleotide sequence reported in this paper has been deposited in the GenBank database under the accession number U77659.

VOL.

88. 1999

TRANSCRIPTIONAL

CHARACTERISTICS

40

20

t0

I

I

20

253

OF CA r, GENE CLUSTER IN A. L WOFFZZ K24

60

80

100

0’

I Homology (70)

40

60

80

100 CatA.mro

B

I

catAR..,+m

clcB tcbD tfdD

Catz‘hyhmaw.,

Cat&m

&A tcbC tfdC

2000

catBRsl catBz * CatBz-M catB~.rol+ catBr CatBI-M CatBR.eq~hmlcP

1

FIG. 2. Dendrogram of the cat2 genes of A. Iwo@ K24 with other related genes. (A) ORFR2 with LysR regulatory genes of the P-ketoadipate pathway. Homology analysis was performed only with 109 aa residues of the N-terminal region. (B) Muconate lactonizing enzymes. (C) Muconolactone isomerases. (D) Catechol 1,2-dioxygenases. Arrows indicate two cat genes (cat1 and catJ of A. Iwofii K24. Sources of genes are as follows: P. putida plasmid pAC27, C/CR (31), clcAB (3); Pseudomonas sp. strain P51, tcbR (5), tcbDC (4); A. eutrophus JMP134, tfdS (22), tfdCD (6); P. putida PRS2000, catRBCA PRS2000 (8); P. putida RBl, catRRsl (32), catBCRsl (33); Frateuria sp. ANA-18, ORFs,.u, catAI.M, catBBI.M,catC1.M, (ABCO9343) and ORFR2.M, cutAz.M, catB 2_M, catCz.M, (ABO09373) (15); A. calcoaceticus, catM (20), catBC*.& (AFOO9224), catAA.,ti (10); R. erythropolis, catRR.erytt,rolCP (X996221, cutABCR.erphram (13); Acinetobacter sp. strain ADPl, benM (34); R. eutropha JMP 134, (35); Pseudomonas sp. strain ESTlOOl, pheB (36); Arthrobacter sp. strain mA3, catAArthro (37); and R. erythropolis AN-13, ~m&t.eutropha catA KerythroAN-I3 (38).

of Hitachi (SanBruno, CA, USA) analyzed the sequencing data and the dendrograms. RNA preparation and RT-PCR Total RNA from A. Iwofli K24 was prepared using the RNesay total RNA kit (Qiagen, Chatsworth, CA, USA), followed by an RNase-free DNase I (Takara, Japan) treatment to remove the DNA contaminant. RT-PCR was performed using the Promega Access RT-PCR system (Promega, Madison, WI, USA) involving reverse transcription (48”C, 45 min), 40 cycles of denaturing (95”C, 1 min), annealing (SS’C, 2min), and polymerization (68°C 3 min, 9 min extension in the last cycle). Six oligonucleotides were used as RT-PCR and PCR primers: primer 2-

using a QIAGEN plasmid kit (Chatsworth, CA, USA). DNA inserts for ligation were isolated from an agarose gel using the j-agarase I (NEB, Beverly, MA, USA) or prep-A clean kit (Bio-Rad, Hercules, CA, USA). Most DNA manipulations such as restriction analysis, ligation and transformation were performed as described by Sambrook et al. (16). DNA sequence determination and sequence analysis The DNA sequence was determined by Prism dyedeoxy terminator cycles sequencing kit (ABI, Foster City, CA, USA) with subsequent electrophoresis and analysis using an Applied Biosystem 313A DNA sequencer. MacDNASIS DNA and the Protein Sequence Analysis System RBS P. putiaiz ml P. putida PRS2000 catBz

of A. lwoffii

catB

ABS

k

AoCTccATc-AGACCTfCAOOOTATQOT ,~,AGCTCCATC-A~TWC3QGAGATTCATTCQATA~CGQCTATCA~TCTCGC~Ml’CCl’T~CAAG

K24

II IIIII I 32oAoT’l’eCAT~A III

I

catB1 of A. lwoffii K24 ,&~~TCToTA

II

IIIII

I

lllllll

I

II

I

III

I

IIIIII

III

IIII

I

I

TQGAAGTAQTCQGAATCGQTG~CTGCTTCQCQQQTTATTTCTACTATGGTGCTCTC

1-1 UT-TCCT~

I I

IIIIIII

I

IIII

I II

I

I

I

IUAAAAQQTO~CGTC--GCAGQTGQCCGCAGCGTAT-CCTCA I

FIG. 3. Alignment of the ORFRz-catB2, the ORFR1-catB, and the c&R-catB intergenic regions of P. putida PRS2000 and P. putidu RBl (8, 14, 33). Bars indicate the identical nucleotide sequence of the cat, and cut2 genes to that of P. putida PRS2000. The sequences of RBS and ABS in P. putidu PRS2000 were previously reported (19). The LysR type transcriptional activator-binding motif, G-N,,-A sequence in P. putidu and T-Nil-A sequence in A. Iwo@ K24 are underlined.

254

KIM ET AL.

1, 5’-GATGAACTGCCAGACGCTCG-3’ (position 5 19 to 538); primer 2-2, 5’-GATCTTCAGCTTGAACACG3’ (position 945 to 927); primer 2-3, 5’-GATCTCGCT CAGGTGCC-3’ (position 1153 to 1169); primer 2-4, 5’GTGCAGCCGGACGAGTTCTGG-3’ (position 1767 to 1787); primer 2-5, 5’-CGATGTTGATCTGGGTGGTC-3’ (position 2340 to 2321); primer 2-6, 5’-TGCTGCAGCT TTTGCGAC-3’ (position 2670 to 2653). PCR was performed as a control under the same conditions as RTPCR minus the reverse transcription, using plasmid pCD2 as a template, thus confirming the PCR products (430 bp, 570 bp, 900 bp, 1200 bp and 1500 bp). Northern blotting and primer extension Total RNA for Northern blotting and primer extension was prepared by the modified method of Simpson et al. (17). Primer extension was carried out as previously described (14), using the AMV Reverse Transcriptase Primer Extension System (Promega, Madison, WI, USA). The oligonucleotide primer (28 mer) was 5’-TTCGATCGTATAGAACAGATC CCTGACG-3’ (position 1756 to 1729). Northern blotting was performed as previously described (14). The 32Plabeled 570 bp PCR product of the catA gene (primer 2-4 and primer 2-5) was used as a probe in hybridization (12). RESULTS AND DISCUSSION DNA sequence analysis of the 5.8 kbp cat2 gene cluster In this study, we completed the sequence analysis of the 5.8 kbp insert using plasmids pCD22, pCD23 & pCD24 and obtained the complete catB2 gene and four additional ORFs (Fig. 1B). The catB2 gene was composed of 1155 bp with the ATG initiation codon (bp 430) and the TGA termination codon (bp 1587). The catB2 gene encoded a protein with a deduced molecular weight of 41,136 Da, containing 385 aa. The aa sequence alignment showed that the catB2 gene shared 97.4% homology (highest homology) with the catBz-M gene of Frateuria sp. ANA18 (ABO09373) and approximately 50% homology with other catB genes, however approximately 40% homology was observed with chloromuconate cycloisomerase (clcB, tfdD and tcbD) (Fig. 2B). Similarly in the cat, gene cluster, an ORF showing characteristics (molecular weight, helix-turn-helix motif in N-terminal) similar to the LysR family regulatory protein (14, 18), was found in the upper region of the catB2 gene. We designated this ORF as ORFR2. ORFRz was initiated at ATG (bp 327) in the reverse direction of the catBAC genes and showed sequence homology with other LysR family regulator proteins of the ,0-ketoadipate pathway; ORFRl of A. Iwojii K24 (54.5% identity), the catR genes of P. putida (51.4% identity), the catM gene of A. calcoaceticus (47.7% identity) and the benM gene of A. calcoaceticus (50.5% identity) (Fig. 2A). The ORFR2-catBz intergenic region was showed more homology with the catR-catB intergenic region of P. putida (19) than that of the cat1 gene cluster and showed 65.5% homology (36 of 55 residues) with the deduced repression binding site (RBS) and activation binding site (ABS) (Fig. 3). However the putative LysR-type transcriptional activator-binding motif of the catB2 promoter was ATAC-N-/-GTAT, which was homologous to the sequence of genes catSI (14), catB of A. calcoaceticus (20), clcABD (21) and tdfCDEF (22). The LysR-type transcriptional activator-binding motif of P. putida was specifically GTAC-N,-GTAT (Fig. 3).

J. Bmscr. BIOEN~ j

A

M

a

12345

1

GACT

/r

1566 CQ AT AT

DC. CO AT TA

/

1.7 kb W T

CO

AT CO CO AT TA

\ 1560

FIG. 4. Transcriptional analysis of the caf2 genes. (A) M, Molecular weight standard. RT-PCR (lanes l-5), about 1 /lg of total RNA was used as a template. Transcriptional pattern of the cat2 genes and the direction of primers are represented by thick arrows and thin arrows, respectively. (B) Northern blot analysis using a 570 bp PCR product of the calA2 gene as a probe. (C) Mapping of the 5’ mRNA start codon of the cutAl gene transcript by primer extension. Lane 1, The arrow indicates the primer extension product.

Three ORFs, ORFx2, ORFn and ORFz2, were located downstream of the cat& gene (Fig. lB), which were in the reverse direction of the catBAC gene cluster. ORF,ZQ was initiated at ATG (bp 4064) and terminated at TAA (bp 2919). ORFx2 encoded 381 aa with a deduced molecular weight of 41,375 Da. However a homology search revealed that the aa sequence of ORFm beginning at 71Met showed significant homology with those of various IA reductases (more than 30%) such as vanB (highest homology of 38.8%), phthalate dioxygenase reductase (23), phenoxybenzoate dioxygenase p-subunit (X78823) and 3-chlorobenzoate-3,4-dioxygenase (24). The putative 2Fe-2S binding motif, CX4CX2CX&,

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88, 1999 Acinetobacter

CHARACTERISTICS

255

calcoaceticus

Peudomonas putida

PRS2000

catR

catB 134hp

4

catA

catC 43hp

Zlhp --

Rhodococcus erythropolis

1CP catA

catR 208hp

cats 29hp

catC 15hp

4

Acinetobacter

OF CA 7., GENE CLUSTER IN A. L WOFFZZ K24

w

lwoffii K24 ORFRl

catB I 224bp

catA 1

catCi 29hp

103hp

FIG. 5. Organization of the cat gene clusters from various bacteria (2, 8, 12-14). Arrows indicate the directions of transcription of the cut genes. Dotted lines show only the transcriptional direction of ORF,, ORFII, and ORFZ2, since their final transcriptional analysis has not yet been investigated. Homologous genes are shaded in the same manner and intergenetic spacing is indicated by base pairs.

was

found

at

position

C&X 4 C 335X 2C 338X 29C 368).

330-368

(Fig.

1B;

VanB encodes one of the

two subunits of vanilate demethylase, a monooxygenase involved in the conversion of vanillic acid to protocatechuic acid (25). Interestingly, ORFxz also shared significant homology with the reductase (tdnB) (23.1%) of the aniline oxygenase gene complex from the catabolic plasmid pTND1 discovered in P. putidu UCC22 (26) and with the aniline dioxygenase reductase gene component (22.3%) from the plasmid pYA1 of Acinetobacter sp. strain YAA (27). ORFyz was initiated at ATG (bp 4994) and terminated at TGA (bp 4104). This ORF encodes 296 aa with a deduced molecular weight of 32,316 Da. ORFn showed characteristics similar to the LysR family regulator genes and shared 24.9% homology with the transcriptional regulator protein of the acetolactate operon, alsR of Bacillus subtilis (28), 21.3% homology with the transcriptional regulator protein cynR, of the cyn operon of E. coli (29) and 18.2% homology with tdnR, one component of the aniline oxygenase gene complex of P. putida UCC22. ORFzz was truncated (58 aa) and terminated at TGA (position 5393). It was homologous with the C-terminal region of some of the LysR family proteins in 58 aa; ywqA4 of B. subtilis (292952) and ptxR of P. aeruginosa (U35068). On the basis of the analysis of the aa sequence homology and the clustered gene arrangement of ORFxz and ORFyz with cat2 genes, it is possible that these two ORFs are involved in the degradation of aromatic compounds such as aniline. The order of the aniline oxygenase complex in P. putida UCC22 (26) was reported to be tdnQ-tdnT-tdnA1 (large subunit of aniline oxygenase)- tdnA2 (small subunit of

aniline oxygenase)- tdnB (reductase) - tdnR (LysR-type regulatory protein). Thus we need to further ascertain whether these genes are involved in aniline oxygenase activity or not. Aniline oxygenase gene complex present on the chromosome is not yet known. The GC content of the 5.8 kbp insert of A. lwofJii K24 is 62.2% and the GC content in the wobble position is 75.7X, which is slightly higher than those of the cat, gene cluster (71.4%) and P. putida (72.4%) (14). Transcription analysis of the cat2 gene cluster in A. lwofii K24 RT-PCR was used to study the transcriptional pattern of the catB2A& gene cluster (Fig. 4A). Six oligonucleotides were synthesized on the basis of nucleotide sequences of the catB2A2C2 genes and used in RT-PCR. We detected catBz, catA and catA2C2 gene products (430 bp, 570 bp and 900 bp, respectively) in RTPCR, but could not detect catBzA2 and catB2A2Cz gene products (about 1200 bp and 1500 bp, respectively). These results suggest that the catB2 gene was independently transcribed and the catAzC2 genes were cotranscribed. To confirm the RT-PCR data, Northern blotting was performed using the catA PCR product (570 bp) as the probe and approximately a 1.7 kb mRNA band, which can span the catA2C2 genes, was detected (Fig. 4B). To determine the transcriptional start site of the catA gene, primer extension analysis was performed using a primer which specifically hybridized to the catA gene. The extension product band of the catA gene corresponded to T at position 1574, which is the C-terminal region of the catB2 gene (Fig. 4C). The cat gene clusters have been reported in A. calcoaceticus, P. putida, A. Iwo@ K24, P. aeruginosa PA0

256

KIM ET AL.

.J. BIOSCI. BIOEM,. .

and R. erythroplis (2, 8, 12, 13, 30). Even though these genes have the same function in bacteria, the organization of the cat genes differs with respect to the bacteria (Fig. 5), more specifically, the location of catA genes are diverse. In the case of the catB and the catC genes, these two genes are very closely linked (15-29 bp) and are cotranscribed in all reported cat genes (8, 9, 13). The cat, gene organization in A. Iwo& K24 is also consistent with these results. However, the cat* genes of A. Iwo@ K24 revealed a different gene order. The catAz gene not only intervened between the catBz and the catC, genes but was also cotranscribed with the catC, gene. The gene order of the catBAC cluster and the cotranscription of the catAC genes have not been observed in other cat genes. The aa sequence analysis of the proteins encoded by cat2 genes revealed that the relationship of the cat2 genes with other cat genes including catI genes was heterogeneous; catAz, catBz, catCz and ORFRz of A. IwoJEi K24 shared the highest homology with those of Frateuria sp. ANA-18 (99.2x, 97.4x, 99.0% and 95.8x, respectively) (Fig. 2). In addition to differential gene organization and transcription, these results suggest that the cat2 genes of A. Iwo@i K24 have undergone their own adaptation process from ancestor genes. In conclusion, the cat2 gene cluster of A. IwoJii K24 has a different organization and transcriptional pattern from other cat gene clusters. ACKNOWLEDGMENTS

We thank S.-Y. Lee for the technical support in DNA sequencing analysis. This research was supported by grants from the Korea Basic Science Institute to S. I. Kim and the Genetic Engineering Research Program, Ministry of Education, Republic of Korea to S.-H. Leem. REFERENCES

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