JOURNAL OF BACTERIOLOGY, Dec. 1994, p. 7574-7586 Vol. 176, No. 24 0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology The Biosynthetic Gene Cluster for Coronamic Acid, an Ethylcyclopropyl Amino Acid, Contains Genes Homologous to Amino Acid-Activating Enzymes and Thioesterases MATHIAS ULLRICH AND CAROL L. BENDER* Department ofPlant Pathology, Oklahoma State University, Stillwater, Oklahoma 74078-9947 Received 18 August 1994/Accepted 11 October 1994

Coronamic acid (CMA), an ethylcyclopropyl amino acid derived from isoleucine, functions as an interme- diate in the biosynthesis of coronatine, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. The DNA required for CMA biosynthesis (6.9 kb) was sequenced, revealing three distinct open reading frames (ORFs) which share a common orientation for transcription. The deduced amino acid sequence of a 2.7-kb ORF designated cmaA contained six core sequences and two conserved motifs which are present in a variety of amino acid-activating enzymes, including synthetases. Further- more, CmaA contained a spatial arrangement of histidine, aspartate, and arginine residues which are conserved in the ferrous active site of some nonheme iron(ll) enzymes which catalyze oxidative cyclizations. The deduced amino acid sequence of a 1.2-kb ORF designated cmaT was related to thioesterases of both procaryotic and eucaryotic origins. These data suggest that CMA assembly is similar to the thiotemplate mechanism of nonribosomal peptide synthesis. No significant similarities between a 0.9-kb ORF designated cmaU and other database entries were found. The start sites of two transcripts required for CMA biosynthesis were identified in the present study. pRG960sd, a vector containing a promoterless glucuronidase gene, was used to localize and study the promoter regions upstream of the two transcripts. Data obtained in the present study indicate that CMA biosynthesis is regulated at the transcriptional level by temperature.

Coronatine (COR) is a chlorosis-inducing, non-host-specific The biosynthesis of CMA initially involves the isomerization phytotoxin produced by several Pseudomonas syringae patho- of L-isoleucine to L-alloisoleucine, which is presumably fol- vars and functions as an important virulence factor in the lowed by an oxidative cyclization to form the cyclopropane ring diseases incited by these bacteria (36, 37, 64). COR induces a (41, 42) (Fig. 1). Recently, CMA was shown to be a distinctive number of responses in plants which can be reproduced by intermediate in the COR biosynthetic pathway. The biosyn- ethylene or indoleacetic acid, suggesting that the toxin alters thetic block to COR in several mutants of P. syringae pv. host metabolism in a manner analogous to that of plant growth glycinea PG4180 was eliminated when CMA was exogenously hormones (13, 24, 49). Recently, striking structural and func- supplied (4, 38). Furthermore, an excess of unlabelled CMA tional homologies between COR, methyl jasmonate, and 12- reduced the incorporation of [14C]isoleucine into COR, indi- oxo-phytodienoic acid were found, suggesting that COR mim- cating that CMA is a free intermediate of COR biosynthesis ics the octadecanoid signalling molecules of higher plants (15, (38). 19, 63). Obvious differences in the structures of the two moieties COR consists of two distinct moieties, a polyketide compo- which form COR prompt questions regarding the mechanistic nent, coronafacic acid (CFA), and a cyclized amino acid, and evolutionary origins of CFA and CMA. Moreover, the coronamic acid (CMA) (2-ethyl-1-aminocyclopropane 1-car- disclosure of a unique reaction cascade which utilizes a boxylic acid). These are derived from separate biosynthetic branched-chain amino acid as a substrate and results in a pathways and coupled via amide bond formation (Fig. 1) (42). cyclopropyl compound could be of immense biochemical in- Both moieties were previously shown to be required for the terest. To further our understanding of CMA biosynthesis, the phytotoxic effects of COR (53, 63). nucleotide sequence required for CMA production was deter- The genes for COR biosynthesis in P. syringae pv. glycinea mined and compared with other DNA and protein sequences. PG4180 are encoded within a 30-kb region of a 90-kb plasmid CMA biosynthesis was previously shown to require two designated p4180A (67). Tn5 mutagenesis, phenotypic charac- transcripts (59), and the start sites for these transcripts were terization, and feeding studies with exogenous CFA and CMA identified in the present study. DNA fragments containing the resulted in the recovery of mutants blocked in distinct biosyn- promoter sequences for each transcript were localized in a thetic steps (4). Genetic complementation studies with CMA- vector containing a promoterless P-glucuronidase gene, and defective mutants and expression of CMA synthesis in another the constructs were used to investigate the effects of temper- bacterium were used to localize a DNA region (6.9 kb) specifi- ature on transcription. cally required for CMA biosynthesis (59). MATERIALS AND METHODS * Corresponding author. Mailing address: 110 Noble Research Bacterial strains, plasmids, and growth conditions. The Center, Oklahoma State University, Stillwater, OK 74078-9947. Phone: bacterial strains and plasmids used in this work are listed in (405) 744-9945. Fax: (405) 744-7373. Electronic mail address: uO7O5AA@ Table 1. Pseudomonas strains were routinely cultured on VMS.UCC.OKSTATE.EDU. King's medium B (25) or mannitol-glutamate (MG) medium 7574 VOL. 176, 1994 CORONAMIC ACID BIOSYNTHESIS 7575

0

H v- H / COOH K Coronafacic Acid (CFA)

H H HH H2N CH3CHH2N _ H H2N C-NH

HOOC HOOC )HOOC HOOC

L-isoleucine L-alloisoleucine Coronamic Acid Coronatine (CMA) (COR) FIG. 1. Biochemical pathway involved in CMA synthesis and incorporation of CMA into COR by P. syringae pv. glycinea PG4180. COR consists of a polyketide component, CFA, coupled via amide bond formation (step 3) to CMA, a cyclopropyl amino acid. CFA is synthesized from acetate and pyruvate, but the precise steps of assembly remain obscure. CMA biosynthesis involves isomerization of L-isoleucine to L-alloisoleucine (step 1) and oxidative cyclization by an unknown mechanism (step 2) (42).

(23) at 24 to 26°C. Escherichia coli HB101 and DH5ot (51) were utilized for generating subclones containing the mutated DNA. used as hosts in cloning experiments and were cultured in All oligonucleotide primers used in this study were synthesized Luria-Bertani medium at 37°C. For RNA isolation, P. syringae by the Recombinant DNA/Protein Resource Core Facility at pv. glycinea PG4180 was cultivated in Hoitink-Sinden medium Oklahoma State University. All materials for 8% polyacryl- optimized for COR production (HSC) (40) at 18°C. The follow- amide gels were purchased from National Diagnostics (Atlan- ing were added to media in the indicated concentra- ta, Ga.) and used as directed by the manufacturer. Sequence tions (in micrograms per milliliter): tetracycline, 12.5; kanamycin, data were aligned and processed with the software program 10; , 40; spectinomycin, 25; and streptomycin, 25. MacVector 4.1 (International Biotechnologies Inc., New Ha- DNA procedures. Agarose gel electrophoresis, polyacryl- ven, Conn.). DNA and protein sequence homology searches of amide gel electrophoresis, DNA restriction digests, ligation the GenBank, EMBL, PIR, and SWISSPROT databases were reactions, small-scale plasmid DNA preparations, and trans- performed by using the University of Wisconsin Genetics formations were performed by standard procedures (51). DNA Computer Group (UWGCG) programs BLASTX, FAST fragments were excised from agarose gels and residual agarose EMBL, and BESTFIT. was removed with the GeneClean DNA extraction kit (Bio RNA isolation and primer extension reactions. Total RNA 101, La Jolla, Calif.). Large-scale preparations of plasmid was isolated from strain PG4180 by the method of Salmeron DNA from E. coli were isolated by alkaline lysis and purified and Staskawicz (50). The 5' ends of the two transcriptional with Qiagen columns (Qiagen, Chatsworth, Calif.). Plasmid units were determined by primer extension using the following DNA was isolated from Pseudomonas strains as described two oligonucleotides: 5'ATTGGCTC'1TGATCACCC, the re- previously (3, 12). Selected clones were mobilized from E. coli verse complement of nucleotides 890 to 907, and 5'CTCCA into P. syringae by using the helper plasmid pRK2013 (5), and GACCGCAGCAGCA, the reverse complement of nucleoti- transconjugants were verified by agarose gel electrophoresis of des 5904 to 5921 (primers Prl and Pr2, respectively; see Fig. 3). plasmid DNA preparations. RNA (20 ,ug) and primer (20 ng) were mixed in 5 ,ul of water, DNA sequencing and analysis. Sequencing reactions were boiled for 60 s, and cooled on ice. The mixture was incubated performed by the dideoxynucleotide method (51) with Seque- with 30 U of avian myeloblastosis virus reverse transcriptase nase version 2.0 (U.S. Biochemicals, Cleveland, Ohio) and (U.S. Biochemicals) in 10 ,ul of reaction buffer (100 mM Tris a-35S-dATP (DuPont-NEN, Boston, Mass.). For DNA se- HCl, pH 8.3; 140 mM KCl; 50 mM MgCl; 10 mM dithioeryth- quencing, a series of subclones (0.5 to 1.5 kb) was generated in ritol; 50 ,uM [each] dATP, dGTP, and dTTP; and 3,000 Ci of pBluescript SK+ (Stratagene, La Jolla, Calif.) and sequenced [32P]dCTP per mmol) at 42°C for 30 min. After adding 3 [lI of by using T3 and T7 primers. Sequencing gaps were filled by chase solution (deoxynucleoside triphosphates at 25 mM), the synthesizing internal primers (18 bp) or by generating nested mixture was incubated at 30°C for 30 min. The reaction was deletions with the Promega Erase-a-base kit (Promega, Mad- stopped by phenol-chloroform extraction, and the labelled ison, Wis.). The locations of TnS insertions in CMA-defective cDNA was precipitated with ethanol at -20°C for 1 h. The mutants of PG4180 were determined by sequencing the DNA pellet was redissolved in 5 ,ul of Tris-EDTA and 5 ,ul of stop flanking the transposon by using the oligonucleotide 5'GGT buffer (95% formamide, 20 mM EDTA, 0.1% bromophenol TCCGTTCAGGACGCTAC, which is derived from the border blue). Primer extension products (1 RI) were boiled for 2 min region of IS50 (47). The internal BamHI site in TnS was and electrophoresed in 8% polyacrylamide gels. The sequenc- 7576 ULLRICH AND BENDER J. BAcrERIOL.

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Relevant characteristics Reference(s)or source E. coli HB101 51 DHSa 51 P. syringae pv. glycinea PG4180 COR+ CMA+ CFA+ CPL~' 4, 67 PG4180.N53 COR+ CMA+ CFA+ CPL+ Kmr 59 PG4180.N1O COR- CMA- CFA+ CPL+ Kmr 59 PG4180.N7 COR- CMA- CFA+ CPL+ Kmr 59 PG4180.N13 COR- CMA- CFA+ CPL+ Kmr 59 PG4180.C14 COR- CMA- CFA+ CPL+ Kmr 4 PG4180.CO COR- CMA- CFA+ CPL+ Kmr 67 PG4180.C1 COR- CMA- CFA+ CPL+ Kmr 4 PG4180.C14 COR- CMA- CFA+ CPL+ Kmr 4 p4180A Contains COR genes 4 pBluescript SK+ Apr; ColEl origin; cloning vehicle Stratagene pRG960sd Sm' Spr; contains promoterless uidA4 with start codon and Shine-Dalgarno sequence 60 pRK2013 Kmr Mob+ Tra+ 16 pMU2 Tcr; contains a 6.45-kb insert derived from p4180A; required for complementation of COR- 59 CMA- CFA+ CPL+ PG4180 mutants pKTX30 Smr; contains a 3.0-kb XhoI insert derived from p4180A; contains part of the CMA 59 biosynthetic gene cluster pRGMU1 Smr Spr; contains a 2.9-kb PstI insert derived from pMU2 in pRG960sd This study pRGMU2 Smr Spr; contains a 2.9-kb PstI insert derived from pMU2 in pRG960sd; insert has This study orientation opposite to that in pRGMU1 pRGMU3 Smi Sp'; contains a 1.4-kb SalI-EcoRV insert derived from pMU2 in pRG960sd (SalI- This study 'EcoRV-uid4) pRGMU4 Smr Sp,; contains a 1.4-kb SalI-EcoRV insert derived from pMU2 in pRG960sd (EcoRV- This study SalI-uidA) pRGMU5 Smi Spr; contains a 1.9-kb Sall insert derived from pMU2 in pRG960sd This study pRGMU6 Smr Spr contains a 1.9-kb Sall insert derived from pMU2 in pRG960sd; insert has This study orientation opposite to that in pRGMU5 pRGMU7 Smr spr; contains a blunt-ended 1.5-kb PstI-AatI fragment derived from pMU2 and cloned This study into the SmaI site of pRG960sd (PstI-AatI-uid4) pRGMU8 Smr Spr; contains a 1.3-kb EcoRV-AatI insert derived from pMU2 in pRG960sd (EcoRV- This study AatI-uidA4) pRGMU9 Smr spr; contains a 1.1-kb SmaI-AatI insert derived from pMU2 in pRG96Osd (SmaI-AatI- This study uidA) a CPL+, CFA and CMA coupled via amide bond formation. ing ladders used to size the primer extension products were pounds, chemical modification procedures, and quantitative derived by using the oligonucleotide primers Prl and Pr2 in analysis of CMA have been described previously (59). nucleotide sequencing reaction mixtures containing the appro- Nucleotide accession number. The reported nucleotide se- priate DNA templates. quence was deposited with GenBank/EMBL under accession Construction of promoter probes and glucuronidase assay. no. U14657. Plasmid pRG960sd contains a promoterless glucuronidase gene (uidA) downstream of a multiple cloning site (60) and was RESULTS used to identify promoter sequences upstream of cmaA and cmaU. Subcloned DNA fragments were ligated into pRG Nucleotide sequence of the CMA biosynthetic region. Ap- 960sd, orientations were determined by restriction digests, and proximately 6.95 kb was previously shown to be required for constructs were mobilized into PG4180. Promoter activities genetic complemnentation of CMA- mutants and for CMA were initially screened by spotting bacterial suspensions (A590 biosynthesis in another bacterium (59). The complete nucle- of 0.05) onto MG agar plates containing 20 jig of X-Gluc otide sequence of the CMA biosynthetic region is shown (see (5-bromo-4-chloro-3-indolyl glucuronide) and incubating them Fig. 3). Three open reading frames (ORFs) sharing a common at 18 or 280C for 5 to 7 days. Promoter strength was quantified orientation for transcription were found and designated cmaA by fluorometric analysis of glucuronidase activity (66) after (2,705 bp), cmaT (1,364 bp), and cmaU (915 bp) (Fig. 2 and 3). cells were incubated in 10 ml of HSC broth at 18 or 280C for 5 The existence of three ORFs was validated by codon prefer- days. Fluorescence in 96-well microtiter plates was monitored ence analyses using the entire sequenced region, the UWGCG with a Fluoroscan II version 4.0 microplate reader (K&M program CODONPREFENCE in default mode, and a Pseudo- Company, Torrance, Calif.). The protein content in cell lysates monas codon usage table provided by J. M. Cherry (8). To was determined with the Bio-Rad (Richmond, Calif.) protein validate the location of the CMA biosynthetic gene cluster, assay kit as recommended by the manufacturer. DNAs flanking the 5' end of cmaA and the 3' end of cmaU Isolation, detection, and quantitation of CMA. Conditions were also sequenced. Sequence analysis indicated the location for bacterial incubations, subsequent isolation of amino com- of another ORF immediately downstream of cmaU and ori- VOL. 176, 1994 CORONAMIC ACID BIOSYNTHESIS 7577 A I t'%EAMICA tSlUbYN\ I Mt~blb I I WILD TYPE 11 CMA BIOSYNTHESIS I CPL WILD TYPEI -1 kb

I1 1 2 3 1 44 6 I1 s I IBamHI ¶11 2 3 I 4 56 S 7 X 0 SaI I~~~~~ I~~~~~~ I %~~~~~~~ " I ft.~~~~~~ I~~~~~~~~~~~

B I S 1 kb

s ~~~s s N V VP PXbH Sa V EH PSaBKHXhK A Sm V

I I I ~ II II1, ' I1 I 1ii§ I I 1- lI )RF-tnp1 c K' cmaA I LA A*A A A A AL a 2 a o Vvt*u FIG. 2. (A) Partial restriction map of plasmid p4180A from P. syringae pv. glycinea PG4180 showing the COR biosynthetic cluster and functional regions. (B) Physical map of the CMA biosynthetic region and location of cmaA, cmaT, and cmaU. The direction of translation (arrows) and locations of TnS insertions in CMA- (filled triangles) and CMA+ (open triangle) mutants (59) are indicated. The locations of TnS insertions were determined by nucleotide sequencing. CPL, amide bond formation. Restriction enzymes used: A, AatI; B, BamHI; E, EcoRI; H, HindIII; K, KpnI; N, NdeI; P, PstI; S, SstI; Sa, SalI; Sm, SMaI; V, EcoRV; Xb, XbaI; Xh, XhoI.

ented in the same direction (Fig. 2 and 3). The sequence of this EMBL, SWISSPROT, and PIR database entries by using the ORF, designated ORF-tnp, shared striking DNA and amino FASTEMBL and BLASTX programs. A region within CmaA acid sequence homology with a variety of previously charac- encompassing 500 amino acid residues showed significant terized transposases from gram-negative bacteria (58), indicat- homology to various enzymes which activate amino acids by ing an unlikely role in CMA biosynthesis. No distinct ORF was adenylation and bind them as thioesters (Table 2; Fig. 4A). detected in the region upstream of cmaA and extending to the Homology within CmaA to these enzymes corresponded to the SstI site. functional domains of 500 to 600 amino acids which are Location of Tn5 insertions in CMA- PG4180 mutants. conserved in the family of amino acid-activating enzymes (1, Nucleotide sequencing of subclones containing TnS insertions 32). CmaA showed the highest degree of homology to func- was used to determine the exact locations of the insertions in tional domains in surfactin synthetase (10) and selected mutants (Fig. 2 and 3). The TnS insertion in PG synthetase GrsB (57), which specifically activate the branched- 4180.N53, a CMA+ mutant (59), established a left border for chain amino acids valine and leucine, respectively. Within the CMA biosynthetic cluster and was located in ORF-tnp 185 CmaA, we observed the conserved spatial arrangement of six bp downstream of the cmaU translational stop (Fig. 2). All core sequences previously identified in enzymes which activate other TnS mutants localized in the present study resulted in the amino acids by adenylation (18, 32, 57) (Fig. 4). Core sequence CMA- phenotype. Mutant PG4180.N10 carried a TnS inser- 2, SGTTGKPKG, and core sequence 4, YRTGD (Fig. 4B), tion in cmaU, whereas mutants PG4180.N7 and PG4180.N13 have presumed roles in ATP binding and hydrolysis, respec- contained TnS insertions in cmaT. The TnS insertions in tively (57), and were more strongly conserved in CmaA than PG4180.C1 and PG4180.CO were located in cmaA. The muta- core sequences 1, 3, 5, and 6. Core sequence 6, LGGHSL (32), tion in PG4180.C14 occupied an intergenic position between was previously shown to be the site for thioester formation in cmaT and cmaA, whereas PG4180.C9 was located upstream of various nonribosomal peptide synthetases; the serine residue the cmaA translational start site (Fig. 2 and 3). within this motif was reported to function as a phospho- Sequence homology analysis. To identify enzymes with pantetheine binding site (52). Interestingly, CmaA contains a homology to the proteins encoded by cmaA, cmaT, and cmaU, serine residue in the spatial arrangement reported for core nucleotide sequence data were compared with GenBank, sequence 6, suggesting the possible involvement of 4'-phos- 7578 ULLRICH AND BENDER J. BACTERIOL.

Sstl BgtlII EcofV GAGCTCAGCAAACTCGATCAGATCTTCGAGGCCATTTTTCAGCGTCCGGTGGGAAATGACGAAJTATCTTCGACCTGGGTGCCAATTCTCTTACCGCT 101 ATCCAGTTGATTGGTCAGGTCAACGAGGCGTTTGGCGCAACATCAATATGGAGCAGTTCTTCTTGACGCCCTGCAAGCAAACGGTGCTTGCGCAGTTGCA 201 AGTCGCCCCTGCGGCGGATAAAGCCTGAGAGGGTTGCCATGCACAGCCTGTTCACACTAAACCTGGACCTAGAGCGAGCCGGCAAGGTGATCTGTGCCCA * sC9 Sina 301 GAGCTTGCCGTTCGACACCG~gWACCCTCCTATTGTTATCGCCCGTGGGACCCAGTGTGCTACATGAAGAACGCTGCACTATTTCTGATCAGCCA 401 TTTCAATCTGATAATCCTGGAAAGCACACCTGGCTGGCGTACCGAACGAAGCCGGGGTCAATCCCGAAGAGGCGTTGCGGACTTCATCCGCCAGTTCAA 501 TGCAGCGCTGCCAGAAGCCCGTGCGTGTAGATGCACTGGTGGGCTATTGCTCCTCAGCGCCTtTGGCACTCTTGGCAGCCAACCAAGGTGCTTGTCGCAC 601 TTGCTACTGCTTAATGGCGCGTACTTCTCAAGACGACGGGTGATCAGCCATTTCAATCTGATAATCCTGGAAAGCGACACCTGGCTGGCGTGCGAACGAA 701 GCCGGGGTCAATCCCGAAGAAGGCGTTGCGGACTTCATCCGCCAGTTCAATGCAGCGCTGCCAGMACCCGTGCGTGTAGATGCACTGGTGGGCTATTGCT ___m4 PRI 801 CCTCAGCGCCTTTGCACTCTTGGCAGCCAACCAAGGTGCTTGTCGCACCTTGCTACTGCTTAATGGCGCGTACTTTCTCAAAGACGACGGGGTGATCAAG 901 AGCCAATFAGCGCGACGTGGAGCCGATGATGCAGTCCATTCC CAG^TTGCGCCCAAGTCTACGAAGCCGTGAGTCTGTTGCACACCCAAAGCACCT cmaA Ns- Q S I P Q G N C A Q V Y E A V S L L H T Q S T Y 25 1001 ATACCCCGAGTGACTATCGCTACCAGCAGGTTCGCCCTCTGCGAGAACTGTCTGCGTTCCGCAGTACCTTACGTTCCCTCAACAACCTGGCGAGCTTGGA T P S D Y R Y Q Q V R P L R E L S A F R S T L R S L N N L A S L E 58 1101 ATTGGTCAGATTGCGCAGGCCGTAAGACCCCCACGCTGGTGTGGTGCGGCACGCAGGACCGTTATACCGACACCGCATCTTCCCGGTACATCGCCCAG L V R I A Q A V K T P T L V W C G T Q D R Y T D T A S S R Y I A Q 91 1201 CTGCTGCCTCACAGTGAACTGGTGGAAGATCCTGATGGCCAGCATCACGATTTTGTCGATGGCCACGAACGTTTGTACCTGACGATGACCCGTTTTCTGG L L P H S E L V E D P D G Q H H a F V D G H E R L Y L T N T R F L D 125 1301 ACCCGTCACAAGCAGAGGGCCATTCAATGACCTCCTACCATTCACATCCTCCCAAAGCCTACCGCCGATTTGAGTCGGTCTGCACGCAAGCGCCAAACGC P S Q A E G H S N T S Y H S H P P K A Y R R F E S V C T Q A P N A 158 AetI 1401 AATTGCGGTGGTGCACGAAGGAAAACCAGTCACTTATCAACAGCTCCAGACGCAGGTGTTGGAACGTAGCGAACGACTGATACGTCe§5S£GGCAGAC I A V V H E G K P V T Y Q Q L Q T Q V L E R S E R L I R Q G L A D 191 1501 CATCCTTACATGCCGCTGATGGCCAACCGGTGCCTTGAGTATCTGATAACGATGCTGGCCTGCT CTGATTACCTACGTCTCCATCGAGCCAA H P Y N P L N A N R C L E Y L I T N L A C > ....MWV S I E P S 225 core 1601 GCAAGCCAAGCAAACGCCTGATCGCCGTGCTGGAACAATTGGGCTGCAATCATTTGCTA--I GTCGCMGACTTGCGCCCTGACCCAACGCT K P S K R L I A V L E Q L G C N H I L L L G Q P T D L R P D P T L 258 ,C1 1701 AACATCTTTCGCCTGGACGACTGCGCGACTCTGTGCTCCGACGGCCCTGCCCTACGCCAGCCTATCCGACGGCGTCTGGATGATGCTTCGGGTGACCGTG T S F A W T T A R L C A P T A L P Y A S L S D G V W N N L R V T V 291

1801 ATGTTTACCTCTGGCACTACCGGCGTCCCCAAAGGTGTACGCATCAGCCAAGACGGGTTGCTGATCCTAGTAGACAACGTACAGCAACAGGTCCACAGCAQ H F T ---V R I S Q D GL I L V D N V Q Q V S K 325 Kpnl 1901 AGCCCCGCAGCTACGTACATCACTCGTCCATCGGCTTCGATGCTGCATTGTTTGAAGTGTGGGAUaGCTGCTGACAGGCGCCTGCGTCACCCTGCAACC P R S Y V H H S S I G F D A A L F E V W V P L L T G A C V T L 0 P 358 2001 TAGCGAGTTCAATATAGATGCACTGGATCACTGCCGTACGGCGGCCAGCTGCGATGTGCTGTTACTGACCACCTCACTGTTCCACCTGGTGGCGCAACAC S E F N I D A L D H C R T A A S C D V L L L T T S L F H L V A Q H 391 Xhol 2101 CGCCTTTCAATGCTCGAGGCTGTGCCGTGCTCTATGTCGGCGGTGAAGTACTTAAACCGGTGCTGCCCGCCGCTGCTGTTGGCCAATCCTCGCATTACCC R L S N L E A V P C S N S A V K Y L NfR C C P P L L L A N P R I T L 425 2201 TGGTGAATGGCTACGGCCCGACCGAGATACAGTGTTCTCCACTTGGTACAGTTTGAACAAGCCTGAGGACGCCGAGCGAGACGTAATTCCTATCGGGCA V N G N T V F S T W Y S L N K P E D A E R D V I P I G Q 458 2301 GTTCCTACACCAGGTGCACGGCAAAATCGTCGATGCCAAACTGCAAGAGGTGGAGGTCGGCACGCCC GTTGCTCTCACAGGTGCGAATCTAGCC F L H Q V H G K I V D A K L Q E V E V G T P __liiiiiiiii A L A 491 Hindull!oorel 3l 2401 CTAGGTTACCTCGACGAAGCTTTGACCCCAACCCGCTTCTACAACTGCCGGAACGCCTATTACCG ACCGC TATGTCATCCAAGATGAGCACGGCA LG~ ~ ~ ~ ~ ~ ~ 1CR1~ I A Y Y V I Q D E H G N 525 2501 TGCTTTTCTACCAAGGCCGCATTGACGAACAGGTAAAATCAAAGGCTTCCGGGTGGAGATCGCCGAGGTCGAACATGCGCTGACCCAACTGCCTGGCGT L F Y Q G R I D E 0 V KIt 1K G F H A L T Q L P G V 558

2601 CGCCACGTGTGGTGCAGGCGCATGTCATGAACGATTTGGAACAGCCTACACGGTTTCATCGTGTTTCATCGTGTTCCGGCATGGCTCGCCAACTATCGA A T C G A G A C H E R F G 11 S L H G f I V F H R V P A W L A N Y R 591 FIG. 3. Nucleotide sequence of a 6.95-kb region required for CMA biosynthesis, containing genes cmaA, cmaT, and cmaU with deduced amino acid sequences indicated below the nucleotides. Nucleotides and amino acid residues are numbered on the left and right, respectively. Restriction sites and putative Shine-Dalgarno sequences (underlined) are indicated. The locations of Prl and Pr2, the oligonucleotide primers used to determine the transcriptional start sites, are indicated. Transcriptional start sites for TUC1 and TUC2 (solid and open circles, respectively, above the appropriate nucleotide), the orientation of transcription (arrows), locations of TnS insertions described in the text (inverted triangles), and the locations of six core sequences found in amino acid-activating enzymes (32) (shaded areas in CmaA) are shown. The location of a potential substrate-binding region,(YGPTE) (10) in CmaA (shaded), the location within CmaA of a conserved sequence previously implicated in aminoacyl adenylation (56) (underlined), the locations of two core sequences common to thioesterases (11, 65) (shaded areas in CmaT), the putative iron-binding motif of nonheme iron(II) enzymes (6) (double-underlined amino acids in CmaA), and NtrC-like activator binding sites upstream of cmaA (asterisks) are indicated. ORF-tnp is a transposase-like ORF flanking the left side of the CMIA biosynthetic region. phopantetheine in CMA biosynthesis. A conserved cysteine sequence YGPTE within CmaA (residues 429 to 433; Fig. 3) is residue in core sequence 3 (Fig. 4B)was previouslyreported in identical to a conserved motif contained in the domains of domains of nonribosomal peptide synthetases (57) but was not nonribosomal peptide synthetases specific for the activation of detected in core sequence 3 of CmaA. The amino acid the branched-chain amino acids L-valine, L-leucine, and D- VOL. 176, 1994 CORONAMIC ACID BIOSYNTHESIS 7579

KpnI BaMHI 2701 AGAAGCAGCTCATGAGCCTATTGGGCACCGGCTTCCTCATTACATGGTACCGAGGCGGATCCACTACCTGGCGGACTTCCCGTGACGGCCATGGCA R K Q A H E P I G H R L P H Y N V P R R I H Y L A E L P V T A N G K 625 satan& AI BqlI ZOU13an4 A&nTrn&r&&CrnTTrrTTArAr.rrr.r-Cr.r.ACA"GrAGCGGTGGTGTCCCCTCAGGCCGGTTCGGCAGTGCTAGAGATCTGGTCCGGCATCCTCGGCACAIIjukMFIUU I G I I I Wu ..--I I --. - V D K R S L Q P P E K A A V V S P Q A G S A V L E I W S G I L G T 658 Patl 2901 CCGCAACCTGC TTGGAACACTCAATTTACGGCTATGGCGCCTCATCTTTGAGCGTGGTCATGGCCCATAGCCGCATCAACGAAATACTTGGCAGAACG R N L Q L E H S I Y G SVM V A H S R I N E I L- G R T 691 c---lorc 3001 u 3001 ACCCCCTTTCGACGAAGTCGCCAGGCTGAGCACCTTTCAAGAGTGGGTGCAGTACTACGCAACCGATCGAGACCCAGTAACTTCTCTCAGGAGGCCAACA~~ ~ ~ ~ ~~~~~---- .-- ..- .Au " T P F R R S R C A E H L S R V GA V L R N R S R P S N F S Q E A N M 7Z5 3101 TGGAATCACTGACTTTTCGTTGACGACAGGAACTTCAGCTTTGATCGAGATGGGTTCATCGGTCCCTTCACGCTCTATGAGCCTGAAGAGATGAA E I T D F S L T K C E L Q CF D R D G F I G P F T L Y E P E E N K 758 3201 ACAGATGCATAAGACCATCCGAGCACAACTGTTCAACCGCACCCATGCTCCCTACGACGCGCCACTGGATGTCGCGATCACCAACTACGACCGGCACCTG 0 M H K T I R A C L F N R T H A P Y D A P L D V A I T N Y D R H L 791 33"1 rZATGTrGACTTGCT~eGAATCATGTGTTTCeCAAGGAAATCGTOCATCGAGTGCGAAGCATCCTCCsCCCCGATGTAATCTOCOCAGCGAGTTCATCsC D V D L L T S H V F R K E I V H R V R S I L P P D V I C A A S S S P 825 sC0 3401 CAAATACCCAGGCAACGAAGGCACTGACTGGCACCAGGCAGACACTTTCGCTCACGCTCCGGCCGACCGCAACTGGTGTGCCCGAGGGCGACGCTTCGGC N T Q A T K A L T C T R C T L S L T L R R A A T C V P E G D A S A 858 3501 GGCGCTATTAATGTGTGGACGGGTTTCACTGATGCCTCCCAAGAACGGCTGCATGCAGTTCATCCCCGGCACCGAAGGCAATGAACTACGACGMC A L L M C G R V S L N P P K K T A A C S S S P A P K A N E L R R N 891 3601 CAGGGGCATGACCTTCGACCCGGTCACCAACAATAATGTGGTCAAGGGGCAGTATGCACGTGGATTCAACGGCTACGACTACACCACTCTGCMGGAC Q G H D L R P G H Q Q Hindlll 3701 AAGTCATGGAAGCCGGATGUM^I.CGGCAGTACCGATCGTGATGAAAGCGGGCAATTTGTAATCTTCGGCTCGATGCTTATGCCCTCCTCGCTACCCAAEcoRI 3801 TTCGCAACCGACAAGACCGCCTCGGCTATGTCGCCCGCTACGTACCCGGACGGTCAGGTTTATCCAGATACAGATTACGTTAGGMTTCGGGCGCGAG 3901 TATCGACTTGATCGGTTCGCCGTGGTGCAGGTGGCCGGTACACCACCGACCCCAAGAACAAAGTGGCCATCAAG AGCCTGGCATGGACCTG^AGCCC 4001 CTAGTCATTTATTGAGGATTGATCATGGAACAGCCCTCCTACCTGGCAATCGATCACATCGCGTATGCACGCGGTCTACGGAGMMACCTCGGCGTTCTT 4101 CACGGCCTTGGGGTTCGAGGTGCTGTTCCATAACACCCATCGAAAGTTTGGCGTGTTGATACCACGTCCGCTGCCAGCAGGCGAATCGTGGAG vC14 EcoRV 4201 TGGTCGACCCTTTCGACCTGACAGCGTGGTCAGCCGGCTGCTGGTGAATGTGTGGGCGTGTATATACCATGCGGCCTTTAGGGTCCGCIMI6CAGAC 4301 TACTCAGACGTCTCTGGCCAGTATCGGAGGCGTCTCGGTAACCAAACCCATGACCATTCCGTACCCGGCCACCGAGGAACATCGCTCGCTGACACCTCG 4401 CACATGTTCCACCCCGCAGTGGGCCTGTTCGAGATCACCGGTTAAGG.AGCCGCCATGGCCGATCCTTTTGTGGTGCAACGGCCCTGGCTTGCACCACTGC cmT- N A D P F V V Q R P W L A P L P 16 4501 CAGGCGCCCCTATGAACGCATGTCGCCTGCTGCTGTGTTTCCCCTACGGAGGAGTGCCCTCAGCGTTCATGTTTGAGCCGGCTAGCGAGGTGGTTGCGAC V T 49 G A P N N A C R L L L C F P Y G G V P S A F N F E P A S E V A 4601 ATGGGCAGTGAAACTACCTGGACGCGAGGATCGAAGTATGGAAGGGCCGGTCAGCGGGTCGCGCATGTTATCGGAGGCGATCCTCGATGCACTGCAzAGC W A V K L P G R1 E D R S N E G P V S G S R M L S E A I L D A L Q S 82 Sa~l 4701 GTAGGCATGCCGTACGCGTTTTACGGTCACAICTTCGGTGCCGGGTTGGCGCTTGATGTACCCACGCGCTAG~e;GCGGCGCGCTGCCGACGATT V G N P Y ^A F Y S 7A G L A L D V T H A L V D A T R A A D D L 116 4801 TGGTCCTGTCGGATGCGCCCCATACCGGCTACTCACGCTACTGGGTGCGATGGACGCAACCAATTGTGGCAGAAAGTATGCTCAGAGAGCCTCTTGCCAC V L S D A P H T G Y S R Y W V R W T Q P I V A E S N L R E P L A T 149 4901 TGCCAACGGATGCCTCGTGCAGTTTCGCCAGGCATGCGCTGGGCAGCTCAAGCCGACCTAGCGCCTCATGCACACTCACTTATCGCTTTATCCGGC A N G C L V Q F R Q A C A G Q A Q S R P S A S N H N S L I A L S G 182 SStI HindIII zWU]%.1 rrnrTnrrsnTnrrrrTTTArnTTrTrAAGGACGACsCGTTGCTTGAGCTCCATTGCCTAGACGAATGGCAGCGCTACACCAGCAMAAATTCC P L P V P L Y V L NI G Q D D P L L E L H C L D E W R Y T S K S F H 216 XXbaI 5101 ATAGCGAGTACGTACCCGGCGGACATTTTTTCTTCAACATCAATTTTCAGCTTTTGTACTTGCCTCTGTTAACCGCTCTAGATIGAACAAGGCAGTGlAGCG~ S E Y V P 8 F N I N F Q L L Y L P L L T A L D E Q G S E R 249 %M TW13 5201 TTATGGAACGTGTATTTTGGAACACCCTTATCTTTCGTCCCTAGACGCTTGCGCGCTAAGTATTGACGGGCTCGACGTGGTGATCACGCCGACGATTTTC_ . . _~~~~~~~~~~~~~~~~~~~~~~~~~~5 Y G T C I L E H P Y L S S L D A C A L S I D G L D V V I T P T I F 282 5301 TTCGCCGAATACCGCCAAGAATCGGATAAGGGTTGCATCAACCGTATCGAAGTCACCAGGCCACGGTTGTTGACGAGCGCATCATCTATCGGCCTTCCG F A E Y R Q E S D K G C I N R I E V T R P R L L T S A S S I G L A E 316 FIG. 3-Continued.

leucine (10). Furthermore, CmaA contained a sequence, GRI N synthetases (6, 33, 39). The arrangement of these DEQVKIKGFR (Fig. 3, CmaA residues 530 to 542), which amino acids was recently shown to be essential for the oxida- shows similarity to a conserved motif, GRXDXQXKXXGXR, tive cyclization activity of isopenicillin N synthetase (9). This in the proline-activating domain of GrsB; this motif was found possible activity within CmaA was further investigated by essential for the activity of enzymes which adenylate amino subjecting the amino acid sequence to hydropathic index acids (56). calculations by using PEPPLOT (UWGCG software) and the In the N-terminal region of CmaA, the spatial arrangement method of Kyte and Doolittle (27). Although the central and of amino acid residues His-106, Asp-108, His-163, and Arg-180 C-terminal regions of CmaA showed a uniform distribution of (Fig. 3) resembled the ferrous active site of various nonheme hydrophilic and hydrophobic regions, the N terminus con- iron(II) proteins, including hydroxylases, oxidases, and iso tained more hydrophilic zones (Fig. 5). The histidine, aspar- 7580 ULLRICH AND BENDER J. BACTERIOL.

5401 D P D F A V G D L V S V E I D W P R R Y A L M R L H F A A L K L C 349 5501 CTGGAGTAAATTCCCGGACTTGGAAAAGATCGGTGCCCATATCGGCCAACACAAAGCGCGGATCGACTTCTCGCTGGAGGGTAATGTCAGCCAGTACTTT W S K F P D L E K I G A H I G Q H K A R I D F S L E G N V S Q Y F 382 PstI vN7 5601 CCGGAAATCCTGCAGGGTGTAGAGCACCTCGTAGCCGCCTACCGATTGAAAGCCGCTTTACGGAAACCGAGTGCTTGCGCCGTTGCTGGTACATCAAGGG P E I L Q G V E H L V A A Y R L K A A L R K P S A C A V A G T S R G 416 0>>> 5701 GTTTTCAGATGTGTACTGTTTCGGTACACATCTGCATACCACGGGCGAGGTCGGGCGTATTCGGCTCAAGCGCAATACATCGGCACGCACAAAGAGCGT F Q M C T V S V H I C I P R A R S G V F G S S A I T S A R T K S V 449 5801 GTCGAAATCTACCTAGTGGATTGACCGCCCAGGAGAGCTATCATGTTCAACATCATGTCTGTACTTGGGTTGCTGCTCTTGATGCCAGGGCCACCGAATA S K S T catl - M F 1 I M S V L G L L L L M P G P P N T 20 ---m4mPR2-- Pstl 5901 CATTGCTG TGCGGTCTGGAGCACTGAGCGGATTCCGACACGCCTGGTCTTTGAGCCTGCTGGAATACTCGGCCTACCTGCTGCAGATTTCCTTTTGGGG L L L R S G A L S G F R H A WI S L S L L E Y S A Y L L Q I S F W G 53 6001 GTATCTGCTCAGACATATCGGTGACAGCGCTCCCTGGTGCCTCAAGGTGGTACAACTGGCGTCGATGGCTTATCTGTTCAAAACCTCACACCGGCTATGG Y L L R H I G D S A P W C L K V V Q L A S M A r L F K T S H R L W1 86 EcoRV 6101 CGCMACCCCGATAACCCTTGCCATTCCTCACCCAACGTGCAGATATCCGGAATGTACTTTTTGGGGCTGACCCTGATCAACCCCAAGGGTCTACTGGTGG R N P D N P C H S S P N V Q I S G M Y F L G L T L I N P K G L L V V 120 EcoRV 6201 TTTCGTTCATCGTCCGAGCAGCACCCTTTACCGATATCAAGTTGTACCTGTATTTTGTCGCGCAACTCAGTATGGTGGTAATCCCGATTGGCTGTGCCTG S F I V R A A P F T D I K L Y L Y F V A Q L S M V V I P I G C A WJ 153 6301 GGTGCTGCTTGGCCTGACAACGCTGAATATTAATCGCACAGCCTCGGTGATCATTTGCTGCTTCGCGGTGGCGATCCTTTCCCAATTGACTGACTCACTG V L L G L T T L N I N R T A S V I I C C F A V A I L S Q L T D S L 186 Aat I 6401 ATCAAGAGCCAAATTGGCATTTTAGGTGTTAGGGAGCCGCTGATTTATTCGATTTTTCGTCCCGCAACGCTCTGGAGGCCTGGATTTATCTGGGGGCAAC I K S Q I G I L G V R E P L I Y S I F R P A T L W R P G F I W G Q Q 220 TN10 6501 AGCTGGATTTTAGAATAAATCAGCGGCTACTTAGGTCCCGTGAAGTAGTGGTTTGGATAATGCTTGCATCGTTCGGGGTCTCTCTGCCACTGCTCTTGGA L D F R I N Q R L L R S R E V V V W I N L A S F G V S L P L L D 253 6601 TAACCCAAGGAGGATATTTTGTTGCAGCAGCATGGGCCGCATAAAGGCAACAGGTTAGTACGAGGATCTTCTTATAGCGTCAATACTTTAGATATT K P K G G Y F V A A S N G R I K A N R L V R G S S Y S V N T L D I 286 Sstl 6701 TTCCGAAGTGGCATTTTTCTTACTCATGATTATATTAAGAGCTCATGGCGTAAATTCTAGTGTGCTGTCTCAAAAATAAGCTCTCTGTAAATCGTCTAT F R S G I F L T H D Y I K S S W R K F* EcoRV 6801 GCTTGCTCGTTTACCACACTGATGGAGACGAAGAAATGAGCGCCCAAGATATCCTCCTGAGTCCTGAAGAACACGCCGAACTGAGCCGTCGCACACGATC orfTnp- M S A Q D I L L S P E E H A E L S R R T R S vN53 6901 AGCCACCATCCGTCAGCGTGACGTCCGCCGCGCGAGGGTCATCCTGTTAG 6950 A T I R Q R D V R R A R V I L L FIGURE 3-Continued.

tate, and arginine residues mentioned above were positioned No significant similarities between the nucleotide and de- within these hydrophilic zones, suggesting a location on the duced amino acid sequences of cmaU and current entries in protein surface or within hydrophilic pockets, thereby allowing the DNA and protein databases were found. Calculation of access to the water and ions needed for proper formation of hydropathy plots for CmaU by the method described above the iron binding site (6) (Fig. 5). revealed a hydrophobic region of approximately 100 amino The deduced protein sequence encoded by cmaT shared acids, suggesting a possible membrane association (data not striking sequence homology with thioesterases of procaryotic shown). and eucaryotic origins (Table 3). Four conserved regions Transcriptional organization of the CMA biosynthetic re- located between amino acid residues 10 and 240 of CmaT were gion. Previous genetic complementation experiments using also found in other thioesterases (Fig. 6A). GHSFG and CMA- mutants indicated the existence of a large transcrip- GGHFF, two conserved motifs previously shown to be essen- tional unit spanning SstI fragment 2 and part of SstI tial for thioesterase function, were also present in CmaT in the fragment 1 (59) (Fig. 2). Data obtained in the same study conserved spatial arrangement (Fig. 6B) (11, 54, 65). The C suggested that a second, smaller transcript originated in SstI terminus of CmaT (amino acid residues 250 to 454) showed no fragment 1 and extended leftward. To test these hypotheses, similarities to any existing database entries. selected DNA fragments were subcloned into plasmid

TABLE 2. Proteins with sequence homology to CmaA Gene Protein (portion and function) Organism Reference % Identitya % Similaritya acvA AcvA synthase (domain B) Aspergillus nidulans 30 28.0 50.5 grsB Gramicidin S synthetase B (domain 4; leucine activating) Bacillus brevis 57 27.5 50.4 srfA Surfactin synthetase (domain 4; valine activating) Bacillus subtilis 17 27.2 49.7 dae D-Alanine-activating enzyme Lactobacillus casei 22 26.6 49.5 tycA Tyrocidine synthase I Bacillus brevis 62 26.1 49.6 pvdD Pyoverdine synthetase D Pseudomonas aeruginosa 28 27.4 47.8 entF Enterobactin synthetase F Escherichia coli 48 25.1 45.5 acsA Acetyl coenzyme A synthetase Bacillus subtilis 20 19.4 40.3 a Determined by using BESTFIT analysis and the entire sequence of CmaA. VOL. 176, 1994 CORONAMIC ACID BIOSYNTHESIS 7581

pRGMU1) and PRO2 (subcloned on pRGMU3). DNA se- A 0 200 400 600 800 902 AAI quences within these two promoter regions participate in the -1-1 activation of two transcriptional units, TUCi and TUC2, respectively, which share a common orientation (Fig. 7). 1 2 345 6 Nucleotide sequence data indicate that cmaA and cmaT are cotranscribed on TUC1, whereas cmaU is situated on the CORE SEQUENCES: second transcript, TUC2. B Determination of transcriptional start sites. Primer exten- C L I T CaNA 1 4 sion L A GA wre core analyses were performed to define the transcriptional start sites for TUCi and TUC2. For transcript TUC1, primer Prl (Fig. 3) was used for primer extension and generation of a I . M. .= sequencing ladder. A single extension product (Fig. 8) was 5 L6-51PMVI detected when total RNA of strain PG4180 was used as a template, indicating an mRNA start site 133 nucleotides up- stream of the predicted Shine-Dalgarno sequence and 143 nucle- 3 -LLT 6 YEASTP otides upstream of the translational start codon for cmaA (Fig. 3). _ ~C I GG cor GH ce The start site for TUC2 was defined by using primer Pr2 (Fig. 3); primer extension (data not shown) revealed a single FIG. 4. (A) Organization of CmaA. Regions with homology to product enzymes which activate amino acids by adenylation (shaded bars) and mapping 33 nucleotides upstream of the putative Shine-Dal- the locations of six core sequences in CmaA which are conserved in garno sequence and 45 nucleotides upstream of the transla- adenylate-forming enzymes that activate amino acids (black bars) are tional start codon of cmaU (Fig. 3). These results confirm the indicated. AA, amino acid. (B) Alignment of amino acids in CmaA initiation sites for the two transcripts involved in CMA biosyn- with six core sequences which are conserved in enzymes that activate thesis. Careful examination of the nucleotide sequences up- amino acids by adenylation and bind them as thioesters (32). stream of the two transcriptional start sites did not reveal any significant homologies to known promoter sequences. Upstream activating sequences in promoter region PROL. pRG960sd, which contains a promoterless glucuronidase To further define the promoter region designated PROl, gene. On the basis of previous information regarding tran- several additional scriptional units (59) and nucleotide sequence data, three subclones were created in pRG960sd. These restriction fragments were subcloned in both orientations new constructs, pRGMU7, pRGMU8, and pRGMU9 (Fig. 7), into pRG960sd, generating the subclones pRGMU1, pRG were conjugated into PG4180, and promoter activities were MU2, pRGMU3, pRGMU4, pRGMU5, and pRGMU6 (Fig. measured after growth in HSC at 18 and 280C for 5 days (Table 7). The six promoter probe plasmids were conjugated into 4, experiment 2). Strains containing pRGMU7 (1.5-kb insert) PG4180, and transconjugants were assayed for glucuronidase and pRGMU8 (1.3-kb insert) produced significantly more activity after incubation in HSC at 18 or 280C for 5 days (Table glucuronidase than pRGMU9, which contained a 1.167-kb 4, experiment 1). These two temperatures were chosen because SmaI-AatI fragment. These results indicate an essential role in prior results indicated that temperature has a significant effect transcriptional activation for the DNA sequence located be- on COR biosynthesis (40). Plasmids pRGMU1 and pRGMU3 tween the EcoRV and SmaI sites (Fig. 3 and 7). A search for showed strong promoter activity at 18'C and substantially less homologies to known transcription factor or enhancer binding activity at 280C (Table 4, experiment 1). The four remaining sites in this region revealed the sequences TGCA-9 bp-TGCT constructs (pRGMU2, pRGMU4, pRGMU5, and pRGMU6) and TGCA-9 bp-TGCG, which were 613 and 588 bp upstream did not promote transcription of the glucuronidase gene, of the TUC1 transcriptional start site, respectively (Fig. 3). regardless of temperature. Therefore, temperature-dependent Both motifs resemble the consensus sequence of the NtrC transcription of CMA biosynthesis genes is likely to proceed binding site in gram-negative bacteria, TGCA-9 bp-TGCA from two independent promoter regions, PRO1 (subcloned on (14), and are absent in pRGMU9. These two regions could

0 200 400 600 800 902 AAL I I I

I I 2> CmaA Asp Args His ,,S0, ,106, (1018J

0 His Sao I (163i IPhob lc Kyte-Doollttle 0 . IUPhIlIc

0 Soo FIG. 5. Hydropathy plot of CmaA made by using the program PEPPLOT (UWGCG) and the method of Kyte and Doolittle (27). The direction of translation (arrow), the region of CmaA with a possible role in amino acid activation (hatched region), and the locations of individual amino acids in hydrophilic regions of CmaA which form a putative iron binding site (6) (vertical lines) are shown. AA, amino acid. 7582 ULLRICH AND BENDER J. BAcrERIOL. function as putative binding sites for a transcriptional activator similar to NtrC. A Effect of temperature on CMA biosynthesis. Strain PG4180 0 200 400453 AA I produces maximal amounts of COR at 18'C but significantly j less at 240C and negligible amounts at 30'C (40). In the present .>CmaT study, PG4180 was incubated at 18 and 280C in 500 ml of HSC 1 2 for 5 days and CMA was quantified as described previously (59). PG4180 produced 1.2 to 1.9 mg of CMA per liter at 180C but virtually no CMA when incubated at 280C (three replicates B CORE SEQUENCES: per temperature). These data were consistent with those ob- tained for transcriptional activity in response to temperature as described above. mu T

DISCUSSION

cr Nucleotide sequence analysis of the CMA biosynthetic gene 2 cluster revealed three genes, cmaA, cmaT, and cmaU, sharing a common orientation on two different transcripts. Physical FIG. 6. (A) Organization of conserved sequences in CmaT. Re- mapping of TnS insertion sites in CMA- mutants indicated gions with homology to known thioesterases (shaded bars) and the their location within the three genes or at intergenic positions. locations of motifs which are highly conserved among thioesterases The TnS insertion in CMA+ strain PG4180.N53 was located in (black bars) are shown. AA, amino acid. (B) Sequences found in CmaT ORF-tnp and thus delimited the left border of the CMA gene and conserved motifs present in several thioesterases (11, 65). The two cluster (Fig. 2 and 3). This confirmed previous findings regard- motifs and their spatial arrangement are believed to be essential for ing the dimensions of the CMA biosynthetic region (59). The thioesterase function. intergenic location of TnS insertions C9 and C14 probably interfered with the transcription of downstream genes (cmaA and cmaT, respectively) because of the polar effects of TnS be essential for aminoacyl adenylation in the proline-activating (46). Therefore, our data indicate that cmaA and cmaT are domain of gramicidin S synthetase. Interestingly, this motif and organized on a 5-kb transcript, whereas the size of the tran- a conserved glycine residue were found in CmaA in the present script containing cmaU is unknown. study but were not detected in adenylate-forming enzymes Sequence analyses performed in the present study revealed which specifically activate carboxylic acids (56). valuable insights into the possible enzymatic mechanisms that Conservation of three essential core sequences (sequences 2, lead to CMA formation. The deduced protein product of 4, and 6) in CmaA suggests that a substrate amino acid could cmaA showed significant homology to functional domains of be adenylated and bound to CmaA as a thioester utilizing the nonribosomal peptide synthetases (10, 32, 57, 61). Common to cofactor 4'-. This cofactor of multifunc- multifunctional peptide synthetases are the adenylation of tional peptide synthetases is assumed to facilitate transport of amino acid substrates and formation of a thioester bond amino acid substrates between domains to facilitate amide between enzyme and substrate. A functional domain usually bond formation between two substrate amino acids or between contains highly conserved core sequences of 3 to 10 amino acid the progressing peptide and a new amino acid (32). However, residues which are thought to be essential for amino acid we suspect that a putative 4'-phosphopantetheine arm at- activation and thioester formation (32). Three of these core tached to CmaA might function to transport an intermediate in sequences were highly conserved in CmaA: the ATP binding the CMA biosynthetic pathway or CMA itself to the putative sequence (core 2), an ATP hydrolysis site (core 4), and the thioesterase encoded by cmaT. Subsequently, CmaT might core sequence containing a serine (core 6) previously shown to function to release an intermediate or the final product (CMA) be the 4'-phosphopantetheine binding site (52). The latter from CmaA. Support for these hypotheses results primarily motif has been reported only for amino acid-adenylating from the fact that CMA should not require activation prior to enzymes that utilize the thiotemplate mechanism and not for ligation to CFA; instead, existing data suggest that the carboxyl similar enzymes which activate carboxylic acids (57). The group of CFA is likely to be activated prior to the ligation of importance of specific amino acid residues within several core CFA and CMA (29). Similar functions for the cofactor 4'- sequences was recently demonstrated by site-directed mu- phosphopantetheine have been discussed for 5-(L-o-aminoad- tagenesis of tycA, the gene encoding tyrocidine synthetase I, a ipyl)-L-cysteinyl-D-valine (ACV) synthetase (30). Thus, the multifunctional enzyme which both activates and epimerizes central region of CmaA could represent a functional domain L-phenylalanine (18). Furthermore, Tokita and coworkers (56) that activates and binds a specific amino acid. Although the recently described a conserved glycine residue within a motif nature of the substrate amino acid remains to be determined, common to all peptide synthetases; this was shown to it is tempting to speculate that the substrate might be either

TABLE 3. Proteins with sequence homology to CmaT Gene Protein (function) Organism Reference % Identity' % Similarity" grsT Gramicidin S synthase T Bacillus brevis 26 26.8 51.6 ORF5 Thioesterase (erythromycin biosynthesis) Saccharopolyspora erythrea 21 26.8 49.6 ORF-1 Thioesterase (candicidin biosynthesis) Streptomyces griseus 11 28.4 49.3 ORF1 Thioesterase (3' of bah) (bialaphos biosynthesis) Streptomyces hygroscopicus 45 28.8 48.7 sasT Thioesterase (S-acyl fatty acid synthetase) Anas platyrhynchos 44 23.6 48.9 a Determined by using BESTFIT analysis and the entire sequence of CmaT. VOL. 176, 1994 CORONAMIC ACID BIOSYNTHESIS 7583

s s s 1 kb

N V VP PXb H Sa V EH P Sa BKH Xh K A SmV .1 1 1 I1 I(f, I I I I I fORF-icmaU |(cmaT <, cmaA

S..t>i>PRORO I |iW>>R--=- '. >Pa R 2

IUiidA d pRGMU3 I uidA pRGMU1

pRGMU4 I pr Vuimi e F idA fIuidA I I uidA 'm pRGMU5 pRGMU6 -+iuidA I

S riWdA '4 7

V

Sm PGU udA -4 FIG. 7. Promoter analysis of the CMA biosynthetic gene cluster. The physical map and organization of CMA biosynthetic genes are outlined at the top. The locations of promoter probe constructs used in this study and their transcriptional orientations with respect to uidA, a promoterless glucuronidase gene, are shown (open rectangles and adjacent arrows). Promoter activities are presented in Table 4. TUC1 and TUC2, transcriptional units 1 and 2; PRO1 and PRO2, promoter regions 1 and 2. Abbreviations for restriction enzymes are defined in the legend to Fig. 2.

L-isoleucine or L-alloisoleucine, one of the two precursors for hydropathy analysis indicated that these amino acid residues CMA (42). Furthermore, it is significant that CmaA is most form a hydrophilic environment believed to be required for similar to peptide synthetase domains which bind and activate iron binding (6). Although the existence of an iron-binding the branched-chain amino acids valine and leucine (10). motif in CmaA and its possible involvement in oxidative The N-terminal region of CmaA contains a pattern of cyclization remain speculative, Parry and coworkers (42) pre- solitary amino acid residues which was recently found con- viously predicted an oxidative cyclization reaction for CMA served in several nonheme iron(II) enzymes (6, 33, 39). Our biosynthesis utilizing an active ferrous site. In the same study, they proposed that CMA synthesis might resemble the oxida- tive cyclization reactions utilized in P-lactam ring formation. TABLE 4. Glucuronidase activity in response to temperature Interestingly, iron was previously shown to significantly in- crease COR yields when amended to PG4180 cultures in vitro Mean GUS activity' (U/mg of because iron is for CMA Expt and PG4180 protein) at: (40), possibly required biosynthesis. transconjugant" Biochemical investigations have also indicated the isomer- 180C 28'C ization of L-isoleucine to L-alloisoleucine as an essential reac- 1 tion in CMA biosynthesis (41, 42). Known mechanisms for the pRG960sd 1.3 0.9 conversion of L-isoleucine to L-alloisoleucine are thought to pRGMU1 412.7 99.5 employ pyridoxal 5'-phosphate (PLP)-dependent enzymes pRGMU2 1.1 0.2 (31). Two possible reactions would be characterized either by pRGMU3 304.6 135.7 (i) an intermediacy of PLP bound to L-isoleucine followed by pRGMU4 0.2 0.9 nonenzymatic tautomerization and release of L-alloisoleucine pRGMU5 0.5 1.2 or by (ii) deamination of L-isoleucine to form 2-keto-(3S)- pRGMU6 1.2 0.6 methylvaleric acid followed by keto-enol tautomerization, sub- 2 sequent amination, and release of L-alloisoleucine (31). The pRG960sd 0.4 1.1 cofactor PLP is known to play an important role in a variety of pRGMU1 396.8 86.7 amino acid-modifying enzymes and is bound to them via a pRGMU7 402.1 112.6 conserved lysine residue (2, 34). Although our efforts to pRGMU8 360.0 101.2 identify a putative PLP binding site in the three CMA genes pRGMU9 42.5 56.8 have not yet succeeded, such a site may not be readily detected Locations and sizes of inserts are indicated in Fig. 7 and Table 1. by conventional homology searches. b Average of two experiments with three replicates per temperature. One unit Like COR biosynthesis (40), CMA biosynthesis is also equals 1 pmol of methylumbelliferone formed per min. GUS, glucuronidase. temperature dependent, and reporter gene studies further 7584 ULLRICH AND BENDER J. BACrERIOL.

DNA ACKNOWLEDGMENTS We thank Ron Parry, Robin Mitchell, Gerald Cohen, Dennis Gross, 3. and Hans von Dohren for stimulating discussions relevant to this study. A M.U. is supported by a postdoctoral fellowship from the German Academic Exchange Service (DAAD). This work was supported by the C GA TC Oklahoma Agricultural Experiment Station Hatch project 2009 and by C NSF grants EHR-9108771 and MCB-9316488. C 0 REFERENCES T 1. Aharonowitz, Y., J. Bergmeyer, J. M. Cantoral, G. Cohen, A. L. 0 Demain, U. Fink, J. Kinghorn, H. Kleinkauf, A. MacCabe, H. C Palissa, E. Pfeifer, T. Schwecke, H. van Liempt, H. von Ddhren, S. Wolfe, and J. Zhang. 1992. b-(L-a-Aminoadipyl)-L-cysteinyl-D- 0 valine synthetase, the multienzyme integrating the four primary T reactions in P-lactam biosynthesis, as a model peptide synthetase. Bio/Technology 11:807-811. a 2. Alexander, F. W., E. Sandmeier, P. K. Mehta, and P. Christen. A 1994. Evolutionary relationships among pyridoxal-5'-phosphate- T dependent enzymes. 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