Research Paper 927 The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases Dirk Konzl, Andrea Klensl, Kurt Schbrgendorfer* and Mohamed A Marahiell Background: The branched cyclic dodecylpeptide antibiotic bacitracin, Addresses: ‘Philipps-Universittit Marburg, produced by special strains of Bacillus, is synthesized nonribosomally by a Fachbereich ChemielBiochemie, Hans-Meerwein- StraOe, 35032 Marburg, Germany. *Biochemie large multienzyme complex composed of the three bacitracin synthetases BAl , GmbH, 6330 Kufstein/Schaftenau, Austria. BA2 and BA3. These enzymes activate and incorporate the constituent amino acids of bacitracin by a thiotemplate mechanism in a pathway driven by a Correspondence: Mohamed A Marahiel protein template. The biochemical features of these enzymes have been studied E-mail: [email protected] intensively but little is known about the molecular organization of their genes. Key words: Bacillus licheniformus, bacitracin, DNA sequence, peptide antibiotics, peptide Results: The entire bacitracin synthetase operon containing the genes synthetases, thiazoline ring bacA-bacC was cloned and sequenced, identifying a modular structure typical of peptide synthetases. The bacA gene product (BAI , 598 kDa) contains five Received: 30 September 1997 Accepted: 28 October 1997 modules, with an internal epimerization domain attached to the fourth; bacB encodes BA2 (297 kDa), and has two modules and a carboxy-terminal Chemistry & Biology December 1997,4:927-937 epimerization domain; bacC encodes BA3, five modules (723 kDa) with http://biomednet.comlelecref/1074552100400927 additional internal epimerization domains attached to the second and fourth. A 0 Current Biology Ltd ISSN 1074-5521 carboxy-terminal putative thioesterase domain was also detected in BA3. A putative cyclization domain was found in BAl that may be involved in thiazoline ring formation. The adenylation/thioester-binding domains of the first two BAl modules were overproduced and the detected amino-acid specificity coincides with the first two amino acids in bacitracin. Disruption of chromosomal bacB resulted in a bacitracin-deficient mutant. Conclusions: The genes encoding the bacitracin synthetases BAl , BA2 and BA3 are organized in an operon, the structure of which reflects the modular architecture expected of peptide synthetases. In addition, a putative thiazoline ring formation domain was identified in the BAl gene. Introduction repeat subunits of peptidoglycan. Recycling of IPP Bacitracins are antibiotic polypeptides produced by involves its dephosphorylation to C,,-isoprenyl phos- certain strains of Bacillus lic/leniformis [1,2] and Bacillus phate (IP) by a phosphatase at the end of each cycle; this suMis [3]. Crudely purified bacitracin contains at least 10 step is blocked by the bacitracin-MZ+-IPP-complex distinct dodecapeptides that differ by one or two amino (where M2+ is a divalent metal ion), resulting in an accu- acids [4]. The most abundant and best characterized of mulation of uridine diphosphate-acetylmuramyl pen- these peptides is bacitracin A (Figure la), a branched tapeptide. This model for bacitracin action is supported cyclic dodecapeptide. It contains an amino-terminal by recently described mechanisms for bacitracin resis- linear pentapeptide moiety with an isoleucine-cysteine tance involving the production of increased IPP [7,8]. thiazoline condensation product and a carboxy-terminal Besides this primary mode of action, bacitracin also heptapeptide ring, in which the free a-carboxy group of seems to affect membrane functions [9], the action of the carboxy-terminal Asn is bound to the E-amino group certain hydrolytic enzymes [lo], and the biosynthesis of of lysine. As well as proteinogenic amino acids, four ubiquinone precursors [ 111. amino acids in the D-configuration (Glu4,Orn7, Phe9 and Aspll) and the nonproteinogenic residue ornithine (Orn) Like many other peptide antibiotics from Bacdus [12], are incorporated into bacitracin A. Bacitracin A is most bacitracin is produced nonribosomally by a large multi- active against Gram-positive bacteria and acts by inhibit- enzyme complex [13-151 utilizing a thiotemplate mecha- ing bacterial cell-wall biosynthesis: bacitracin A forms a nism [16-201. In this mechanism, large multifunctional tight ternary complex with C,,-isoprenyl pyrophosphate enzymes, designated peptide synthetases, activate their (IPP) and a divalent metal cation [5,6]. IPP serves as a substrate amino acids as aminoacyl adenylates using ATP membrane-associated carrier during the synthesis of the hydrolysis. These unstable intermediates are subsequently 928 Chemistry & Biology 1997, Vol 4 No 12 Figure 1 (a) BAl BA2 (b) I Ile CYS Thloesterase HP\H-CH+~- Leu - D-Leu - Ile - Lys - D-Om - Ile - D-Phe Asn -D-Asp -His Thiazollne Thioester formatlon (T) w 0 2 4 6 6 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 kb 1) I I I II I 11 11 11 11 I ” 1 ’ 1 ’ 1 ’ 11 81 11 1 11 11 11 11 11 11 I I bacA bacB bacC PCROS (d) 1 h.A14.3 (e) m POE-BacAi i@i-&$// pBAC-BA POE-BacAZ Chemistry & Biolog!I (a) Primary structure of bacitracin A. Brackets embrace the residues domain organization of the peptide synthetases is illustrated within the incorporated by the three bacitracin synthetases BAl , BA2 and BA3. genes. (d) PCR fragment 05 used for the screening of the h-EMBL3 The thiazoline ring, a product of isoleucine and cysteine cyclization, is genomic library and h clones isolated in this work. Probes A and B used highlighted in yellow. (b) The color code of the patterns used for for chromosomal walking are marked in light blue. (e) Gene fragments, different domains in the remainder of the figure. (c) Physical corresponding to AT domains, were overexpressed and biochemically organization of the bat operon. Three genes, bacA, 6acB and bacC analyzed. Plasmid pBAC-BA was used for the construction of a bacB coding for the bacitracin synthetases BAl -BA3 were identified. The gene disruption mutant. Abreviations: Orn, ornithine; E, EcoRl; S, WT. transferred to a covalently enzyme-bound 4’-phospho- belongs to the large family of adenylate-forming enzymes pantetheinyl (4’-PP)-cofactor as thioesters. The peptide that includes firefly luciferase [28] and the acetyl-CoA syn- product is generated by the stepwise incorporation of the thetases [ZZ]. The adjacent thioester-binding (T) domain thioesterified amino acids in a series of amino- to carboxy- (80 amino acids) shares high homology with the acyl carrier terminal-directed transpeptidations. Thorough sequence protein (ACP) that is involved in fatty acid and polyketide analysis of several genes encoding peptide synthetases has synthesis. It contains the highly conserved sequence motif unveiled a modular organization principle for this class of LGGxJ’I (using single-letter amino-acid code, with x as enzymes [l&21-231. The detected modules (around 1000 any amino acid) with an invariant serine residue (itali- amino acids each) are responsible for the activation and cized), which is the 4’-PP-attachment site [29-311. In con- incorporation of a single amino acid and thus are arranged trast to the original model of the thiotemplate mechanism, in a colinear fashion with the product peptide [B-26]. If a which suggested that a single 4’-PP-cofactor would act as a biosynthetic complex consists of several peptide syn- ‘swinging arm’ to transfer the growing peptide chain from thetases, the colinearity of the modules is conserved one module to the next, the presence of such a cofactor at within each single enzyme [17,18]. Each module can be each module of a multimodular PPS has recently been subdivided into several specific domains [20,22,27]. The shown [32]; this finding established the ‘multiple carrier adenylation (A) domain (550 amino acids) is the elemen- model’ for nonribosomal peptide synthesis. The T domain tary domain of each module and catalyzes the specific acti- is followed by a recently reported condensation (C) vation of a given substrate amino acid. This module domain (3.50 amino acids), which is thought to catalyze Research Paper Bacitracin biosynthesis operon Konz et a/. 929 peptide bond formation [33]. Within peptide synthetases stage [37], so an unusual organization of the first two BAl with a single polypeptide chain, the C domain is always modules is to be expected. found between two A domains. If the peptide biosynthesis complex is composed of several peptide synthetases, the In this study we report the cloning and sequencing of the subunit that activates the first amino acid of the peptide entire bat operon from B. l&enzj&nis. Three genes, bacA, lacks a C domain. The C domain is found instead at the bacB and bad‘, coding for the three biochemically identified amino-terminal end of subunits that catalyze activation bacitracin synthetases were sequenced. The genes reflect and transfer of the distal amino acids of the peptide. In the typical structure of a peptide synthetase, with five modules that catalyze the incorporation of N-methylated modules in bacA, two in bacB and five in bad’. The chromo- amino acids, an additional N-methylase domain (350 amino somal arrangement of the genes is in the same order as their acids) is inserted between the A and T domains [24,25]. If biochemical action. Complete sequence analysis of bacA the module introduces D-amino acids, the T domain is fol- and bacC revealed that they contain modules with internal lowed by an epimerization (E) domain (400 amino acids) E domains, the first example of these domains in prokary- [27,34]. In many bacterial systems the module that cat- otic peptide synthetases. In addition, a strongly modified C alyzes the incorporation of the carboxy-terminal amino domain was identified between the first two A domains of acid of the product peptide contains an additional putative bacA; this region shares highest homologies with proteins thioesterase (Te) domain; Te domains are thought to play involved in the nonribosomal synthesis of thiazoline-con- a role in peptide chain release or cyclization [ZO].