Department of Botany, The University of Queensland, Brisbane 4072, Australia1
Institute of Molecular Agrobiology, The National University of Singapore, Singapore1176042
Author for correspondence: Robert G. Birch. Tel: +61 7 33653347. Fax: +61 7 33651699. e-mail: r.birch{at}botany.uq.edu.au
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ABSTRACT |
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Keywords: leaf scald phytotoxin, nonribosomal peptide synthetase, polyketide synthase
Abbreviations: A, adenylation; ACP, acyl carrier protein; AL, acyl-CoA ligase; AMT, aminotransferase; AT, acyltransferase; C, condensation; Cy, cyclization; DH, dehydratase; KR, ß-ketoacyl reductase; KS, ß-ketoacyl synthase; MT, methyltransferase; NRPS, nonribosomal peptide synthetase; PCP, peptidyl carrier protein; PKS, polyketide synthase; TE, thioesterase
The GenBank accession number for the sequence determined in this work is AF239749.
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INTRODUCTION |
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The major antimicrobial component of the family of albicidins produced in culture by X. albilineans has been partially characterized as a low-Mr compound with several aromatic rings (Birch & Patil, 1985a ). Low yields have slowed studies into the chemical structure of albicidin, its application as a tool to study prokaryote DNA replication and its development as a clinical antibiotic (Zhang et al., 1998
). Genetic analysis of albicidin biosynthesis is likely to indicate approaches to increase yields, probable structural features, and opportunities for engineering novel antibiotics in this family.
Transposon mutagenesis revealed that at least two gene clusters spanning more than 60 kb in the genome of X. albilineans are involved in albicidin production (Rott et al., 1996 ; Wall & Birch, 1997
). Recently, a gene (xabA) required for albicidin biosynthesis in X. albilineans has been identified and found to encode a phosphopantetheinyl transferase, which may activate albicidin synthetases in the albicidin biosynthetic pathway (Huang et al., 2000b
). Here, we show that a second gene (xabB) required for albicidin biosynthesis encodes a large, multifunctional enzyme, with unusual polyketide synthase (PKS) modules fused to a non-ribosomal peptide synthetase (NRPS).
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METHODS |
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Routine genetic procedures.
Bacterial genomic and plasmid DNA isolation, gel electrophoresis, DNA restriction digests, ligation reactions, transformation and colony hybridization were performed by routine procedures (Sambrook et al., 1989 ). DNA fragments were excised from agarose gels and residual agarose was removed with the BRESAclean DNA purification kit (GeneWorks).
Construction of a X. albilineans partial genomic library.
Genomic DNA from X. albilineans Xa13 was digested with EcoRI and size-fractionated. DNA fragments of 1520 kb were ligated to dephosphorylated EcoRI-cleaved pBluescript II SK. The ligated DNA was electroporated into E. coli TOP10. Transformants were selected on LB agar medium containing ampicillin, and stored in LB broth with 15% glycerol at -70 °C.
DNA sequencing and analysis.
Sequencing reactions were performed by dideoxynucleotide chain termination (Sanger et al., 1977 ) using a BigDye Terminator Cycle Sequencing kit and a 373A DNA sequencer (PE Applied Biosystems) through the Australian Genome Research Facility. Oligonucleotide primers were purchased from GeneWorks. Analyses of DNA and protein sequences in the GenBank, EMBL, PIR and SWISS-PROT databases were carried out by using the University of Wisconsin Genetics Computer Group (UWGCG) programs BLASTP, FASTA, PILEUP and BESTFIT through WebANGIS version 2.0.
PCR amplification.
BamHI-digested genomic DNA from X. albilineans LS157 was religated at low concentration (0·5 µg ml-1) to generate circular DNA molecules as templates for inverse PCR. Three primers, one from the IS terminal region of Tn5 (IR2: 5'-CGGGATCCTCACATGGAAGTCAGATCCTG-3'), and two flanking the unique BamHI restriction site of Tn5 (BL: 5'-GGGGACCTTGCACAGATAGC-3' and BR: 5'-CATTCCTGTAGCGGATGGAGATC-3'), were used to amplify the sequences flanking the Tn5 insertion in the genome of LS157. The amplified fragments (1·4 kb and 6·0 kb) were cloned into pZErO-2, yielding pZIL and pZIR (Fig. 1).
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Construction of promoter probes and glucuronidase assay.
Plasmid pRG960sd contains a promoterless ß-glucuronidase gene (uidA) downstream of a multiple cloning site (Van den Edde et al., 1992 ). Sequence upstream of xabB (nucleotide residues 10051210 or 5211210) was amplified from pXABB by PCR. Forward primer P1F1 (5'-ACGCGGATCCCAGCAGGGTGTCATACACG-3') or P1F2 (5'-TCGCGGATCCGCGCGATTGAAGTAGTCC-3') contained a BamHI restriction site (underlined). Reverse primer P1R (5'-TCCCCCGGGCGGCCAGCGTGGTGCTACTAC-3') introduced a XmaI restriction site (underlined). PCR fragments were ligated into BamHI/XmaI-cut pRG960sd, yielding pRG960p1 and pRG960p2. These constructs were mobilized from E. coli DH5
into X. albilineans LS155 as described below.
Promoter strength was quantified by fluorometric analysis of glucuronidase (GUS) activity (Jefferson, 1987 ; Xiao et al., 1992
). The protein content in cell lysates was determined by the dye-binding method (Bradford, 1976
) using a Bio-Rad protein assay kit.
Bacterial conjugation.
DNA transfer between E. coli donor JM109(pLAFR3±insert) or DH5(pRG960sd±insert) and X. albilineans recipient (LS157, LS-JP2 or LS155), was accomplished by triparental transconjugation with helper strain pRK2013. Mid-exponential-phase cultures of the recipient were spotted onto agar plates containing YEB medium with no antibiotics (20 µl per spot). After the liquid had been absorbed by the agar, 20 µl of mid-exponential-phase culture of the helper was added to each spot. The liquid was again allowed to absorb, and 20 µl of mid-exponential-phase culture of the donor was added to each spot. After incubation of the mating plates overnight at 28 °C, transconjugants were selected on SP plates supplemented with ampicillin, and tetracycline or spectinomycin.
Assay and quantification of albicidin production.
Albicidin was quantified by a microbial plate bioassay as described previously (Birch & Patil, 1985b ), except that the 10 ml basal layer of LB agar and the 5 ml overlayer of 50% LB with 1% agar were supplemented with tetracycline or spectinomycin, and E. coli DH5
(pLAFR3) or DH5
(pRG960sd) was used as the indicator strain. This change avoided interference by tetracycline or spectinomycin, which were added to some cultures to ensure retention of pLAFR3 or pRG960sd derivatives in X. albilineans. Inhibition zone widths in the bioassay were converted to albicidin concentrations by interpolation on a dose-response plot produced under the same assay conditions. The plot fitted the formula log[Alb]=0·3 W-0·92, where [Alb] is units of albicidin per 20 µl sample and W is the width in millimeters of the zone of growth inhibition surrounding each well.
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RESULTS |
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The double-strand sequence of the 16511 bp EcoRI genomic fragment in pXABB was obtained by a primer-walking approach, using subclones pBC157, pZIL, pZIR, pSEBL and pSEBR. The Tn5 insertion in the genome of LS157 is accompanied by a 9 bp perfect repeat sequence (GTCCTGAAG), commencing at bp 2490 in the sequence deposited in GenBank with accession number AF239749.
The only ORF longer than 900 bp within the 16·5 kb fragment is disrupted by the Tn5 insertion. This ORF (designated xabB) encodes a protein of 4801 amino acids (Mr 525695). It commences at bp 1230 in the above GenBank sequence with a TTG codon, 6 bp downstream from a RBS, GAGG. There is an alternative start codon (ATG) a further 15 bp downstream. Of the codons in this ORF, 8·5% are rarely used in E. coli. The closest match (TTGAGC-14x-TATAAC) to the consensus -35 (TTGACA) and -10 (TATAAT) sequences for E. coli 70 promoters occurs 117 bp upstream of the translation initiation codon (Fig. 2
).
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Sequence homology analyses
Comparison of XabB with available protein sequence databases reveals an N-terminal region (from Met-1 to Asp-3235) similar to many microbial modular PKSs, and a C-terminal region (from Pro-3236 to Asp-4801) similar to NRPSs.
Recognizable PKS domains commencing at the N terminus of XabB are an acyl-CoA ligase (AL), acyl carrier protein (ACP1), ß-ketoacyl synthase (KS1) and ß-ketoacyl reductase (KR), followed by two consecutive ACPs and one KS (Fig. 1). The motifs characteristic of these domains are aligned with those from other organisms in Fig. 3
. The AL domain contains the conserved adenylation core sequence (SGSSG) and the ATPase motif (TGD), and shows 2230% identity and 5060% similarity to prokaryotic and eukaryotic aromatic-acid CoA ligases and long-chain fatty-acid CoA ligases (protein symbols and GenBank accession nos: MycA, AF184956; LCFA, P46450; PksJ, P40806; ComL2, P14913; FkbB, AF082099; RifA, X86780; RapA, AF040570). The three ACP domains show up to 39·2% identity and 78·6% similarity to ACPs (MycA, AF184956; HMWP1, Y12527; Ta1, AJ006977; PksX6, U11039; EryA1, M63676; MAS, M95808), and all contain a 4'-phosphopantetheinyl-binding cofactor box GxDS(I/L) (Hopwood & Sherman, 1990
), except that A replaces G in ACP1 (Fig. 3
). The two KS domains show up to 56·1% identity and 80·8% similarity to prokaryotic ß-ketoacyl synthases (KSs) (MycA, AF184956; HMWP1, Y12527; Ta1, AJ006977; PksX6, U11039; PksM, O31781; EryA1, M63676; PpsA, G3261605). Both contain motif GPxxxxxxxCSxSL around the active site Cys, and two His residues downstream of the active site Cys, in motifs characteristic of these enzymes (Donadio et al., 1991
; Hopwood, 1997
; Huang et al., 1998
). The KR domain shows up to 27·9% identity and 61·8% similarity to other ß-ketoacyl reductases (HMWP1, Y12527; Ta1, AJ006977; PksX6, U11039; EryA1, M63676; MAS, M95808), and contains the NAD(P)H-binding site GGxGxLG (Scrutton et al., 1990
).
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DISCUSSION |
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The N-terminal region of the predicted XabB protein resembles a multifunctional PKS. Bacterial multifunctional PKSs operate as biochemical assembly lines, composed of a series of catalytic domains involved in sequential assembly and modification of acyl groups on the growing polyketide chain (Cane et al., 1998 ; Keating & Walsh, 1999
). The catalytic domains are arranged in modules, punctuated by acyl carrier protein (ACP) domains that tether the nascent polyketide while it undergoes the catalytic modifications programmed in the associated module. For each polyketide there is an initiation module, a series of elongation modules that define the length and structure of the polyketide chain, and a termination module to release the product from the final tether. The initiation module typically comprises an acyltransferase (AT) domain that couples the initial acyl group from an acyl-CoA substrate to the phosphopantetheinyl tether of the first ACP domain. Each elongation module typically comprises at least a KS, AT and ACP. The AT selects and loads an acyl group (typically malonate) onto the ACP. The KS removes the growing polyketide unit from the upstream ACP and couples it by decarboxylation to this next tethered acyl group (typically extending the chain by one acetyl group). Other catalytic domains (e.g. KR, or dehydratase, DH) within an elongation module can modify the newly elongated polyketide before it is transferred to the next module. A thioesterase (TE) domain in the termination module accomplishes release of the assembled polyketide from the last ACP (Cane et al., 1998
; Keating & Walsh, 1999
).
Biosynthesis of a polyketide can involve the sequential action of several PKS proteins, each with one to six elongation modules (MacNeil et al., 1992 ; Aparicio et al., 1996
). There are variations on the modular PKS design, including participation by some loading domains across modules or in trans from separate proteins (Keating & Walsh, 1999
), and several examples of hybrid PKS/NRPS proteins (Albertini et al., 1995
; Gehring et al., 1998
; Duitman et al., 1999
; Paitan et al., 1999
). Subsequent modification of the polyketide by dedicated tailoring enzymes is generally required to complete the biologically active product (Cane et al., 1998
).
XabB commences with an AL domain (residues 1629) followed by an ACP domain (ACP1, residues 630731). In other PKS systems, an N-terminal AL is involved in activation and incorporation of 3,4-dihydroxycyclohexane carboxylic acid, 3-amino-5-hydroxybenzoic acid (AHBA) or a long-chain fatty acid as a starter (Aparicio et al., 1996 ; Motamedi & Shafiee, 1998
; Tang et al., 1998
; Duitman et al., 1999
). It may be possible to determine the substrate specificity of the AL domain by heterologous expression and purification of the module 1 protein containing AL plus ACP1 domains, and ATP-pyrophosphate exchange assays of the purified fusion protein with potential substrates, as applied to NRPS internal A domains (Guenzi et al., 1998
; Stachelhaus et al., 1998
, 1999
).
The second module in XabB contains a KS (residues 7321165) and a KR (residues 18111971) upstream of two ACPs (residues 24572522 and 25442613), but lacks any discernable AT domain (Fig. 1). Tandem ACPs are unusual within PKS modules. Other instances include WA for a green pigment formation and ST for sterigmatocystin biosynthesis in Aspergillus nidulans (Mayorga & Timberlake, 1992
; Yu & Leonard, 1995
), the PKS1 product for melanin biosynthesis in Colletotrichum lagenarium (Takano et al., 1995
) and the pksX7 product for an unknown polyketide in Bacillus subtilis (Albertini et al., 1995
). The significance of the tandem ACPs in these systems has not been resolved. ST performs the iterative addition of seven acetate units (from malonyl-CoA donors) to a hexanoate primer, to form an octaketide intermediate that is stabilized by cyclization, yielding a product with three aromatic rings (Yu & Leonard, 1995
). This is interesting in the context of the apparent aromatic structure of albicidin. Although most aromatic polyketides are synthesized by iterative type I or type II PKSs (Shen, 2000
), there is an example of synthesis by a non-iterative type I PKS in the case of pyoluteorin in Pseudomonas fluorescens (Nowak-Thompson et al., 1997
).
Possibilities in the case of XabB include use of either or both of ACP2 and ACP3 in chain elongation. One domain could be non-functional, as observed for modification domains in various PKSs (Aparicio et al., 1996 ; Nowak-Thompson et al., 1997
; Tang et al., 1998
). Selection between ACP2 and ACP3 could depend on which has been loaded with an appropriate acyl group by a separate AT enzyme. If they have different loading specificities (e.g. for malonyl-CoA versus methylmalonyl-CoA), this could contribute to the production of multiple, structurally related albicidins by the same synthetase. The evidence that domains typically found within a module can in some instances act in trans (Keating & Walsh, 1999
), including a trans-acting KS in the deduced type I mycobactin PKS (Quadri et al., 1998
), supports the possibility that ACP2 and ACP3 could act sequentially in chain elongation. Finally, ACP2 and ACP3 could accomplish the iterative biosynthesis of a longer ketide chain if ACP3 serves as a waiting position allowing polymerization of successive products from ACP2, as for the tandem PCP domains in enniatin synthetase (Zocher & Keller, 1997
). As discussed below, it will be easier to distinguish between these possibilities when the complete structure of albicidin is elucidated.
The third module in XabB contains a KS (residues 26303046) followed by a PCP (residues 32213307) at the start of the NRPS region. Four other fused PKS/NRPS systems (Albertini et al., 1995 ; Gehring et al., 1998
; Duitman et al., 1999
; Paitan et al., 1999
) are known, three of which lack recognizable AT domains (Fig. 6
). Yersinia pestis HMWP1 contains a typical PKS elongation module (including AT) and an NRPS module with a terminating thioesterase (TE) domain. It is the third protein, following an AL (YbtE) and NRPS (HMWP2), in the biosynthetic apparatus for yersiniabactin (Gehring et al., 1998
). B. subtilis MycA bears the closest resemblance to XabB, showing PKS initiation and elongation modules linked via an aminotransferase (AMT) domain to the NRPS region. In B. subtilis PksX6 and Myxococcus xanthus Ta1, the NRPS region precedes the PKS region. Separate AT enzymes encoded elsewhere in the genome may operate in trans to load the appropriate acyl groups onto the ACPs in the elongation modules of these PKSs. Candidates are a malonyl-CoA transacylase gene (fenF) located immediately upstream of mycA (Duitman et al., 1999
) and an AT gene located 20 kb upstream of ta1 (Paitan et al., 1999
). We have not yet identified an AT gene in the xab gene cluster.
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The step to link polyketide and peptide moieties in the nascent antibiotic is of particular interest. In other keto-peptide synthetases, the first PCP is preceded by a C domain, rather than a KS domain as in XabB. The absence of an A domain upstream of PCP1 may indicate involvement of a trans-acting A domain as in HMWP1 (Gehring et al., 1998 ), or transfer without chain extension as in MycA (Duitman et al., 1999
).
NRPSs typically show a modular architecture and tethered biosynthetic strategy analogous to PKSs (Cane et al., 1998 ; Keating & Walsh, 1999
). In NRPSs a C domain removes the growing peptide unit from the upstream PCP domain and couples it to the next amino acid in the chain, which has already been selected and loaded by an A domain onto the PCP in the same module (Marahiel et al., 1997
; Stachelhaus et al., 1998
). Other catalytic domains (e.g. epimerase or N-methyltransferase) within an elongation module can modify the newly elongated polypeptide before it is transferred to the next module in the biochemical assembly line (Marahiel et al., 1997
).
The A domain in the NRPS region of XabB contains ten conserved sequences (A1A10, Table 2) identified as AMP-binding, ATP-Mg2+-binding, adenine-binding or ATPase sites (Turgay et al., 1992
; Marahiel et al., 1997
). In other NRPS systems, A domains select and load a particular amino-, hydroxy- or carboxy-acid unit (Marahiel et al., 1997
). Substrate specificity is determined at the binding pocket, consisting of a stretch of about 100 amino acid residues between highly conserved motifs A4 and A5 (Conti et al., 1997
). Based on sequence analysis of known A domains, in relation to the crystal structure of the GrsA substrate-binding pocket, models have been developed to predict substrate specificity from eight or ten amino acids lining the pocket (Stachelhaus et al., 1999
; Challis et al., 2000
). Based on alignment of complete sequences between A4 and A5 motifs, the A domain from XabB falls in a diverse cluster involved in loading of aromatic amino acids (mostly Tyr), Leu or His.
The model using eight critical residues (http://jhunix.hcf.jhu.edu/~ravel/nrps/) gives no convincing prediction, because the XabB signature sequence AVKYVAND diverges from the strongly conserved residues D and V/I/L at the start and end of this eight-residue signature. Too few A domains have yet been characterized to allow reliable prediction by the model of nonproteinogenic substrates. However, an initial aspartic acid residue is highly conserved in amino-acid-specific A domains, where it is believed to electrostatically stabilize the amino group of the substrate at the binding pocket. The initial alanine residue in the case of XabB is consistent with a hydroxy-acid substrate, by analogy with the initial glycine in the signature sequence (GALHVVGI) of the hydroxyisovaleric-acid loading domain of enniatin synthetase (Haese et al., 1993 ). Incorporation of a hydroxy acid would involve an ester bond in the product chain, which is consistent with the cleavage and inactivation of albicidin by the AlbD esterase (Zhang & Birch, 1997
).
Based on the architecture of the XabB NRPS region, we infer that the polyketide intermediate tethered on PCP1 is accepted by C1 and coupled to the amino-, hydroxy- or carboxy-acid unit preloaded by A onto PCP2. There are no evident peptide-modifying domains (e.g. epimerase or N-methyltransferase) in the NRPS module. The final C domain at the carboxyl-terminus of XabB may transfer the intermediate product assembled by XabB to a second synthetase protein required for albicidin production as in the mycosubtilin synthetase (Duitman et al., 1999 ). Alternatively, the final C domain may be involved in peptide-chain termination and cyclization, as in the enniatin, HC-toxin, rapamycin and FK506 systems (Konz & Marahiel, 1999
), which also lack a TE motif for chain termination. The ORF downstream of XabB encodes a MT involved in albicidin biosynthesis (Huang et al., 2000a
), not a functionally linked NRPS.
The full chemical structure of albicidin has not yet been determined. Albicidin is insensitive to proteases, but it is cleaved and detoxified by the AlbD esterase (Birch & Patil, 1985b ; Zhang & Birch, 1997
). Despite its relatively large size and lack of an apparent nucleoside motif in spectroscopic data, albicidin is actively accumulated in E. coli by illicit uptake through the nucleoside-specific Tsx outer-membrane pore (Birch et al., 1990
; Fsihi et al., 1993
). Partial chemical characterization of the major antimicrobial component of the albicidin family, by methods including proton- and C13-NMR and mass spectroscopy, indicates features including: an Mr of 842000; approximately 38 carbon atoms; three or four aromatic rings; at least one COOH group; two OCH3 groups, including a probable O-methyl tyrosine; a trisubstituted double bond; and a CN linkage (Birch & Patil, 1985a
; and unpublished analysis by Richard Moore at University of Hawaii and Mary Garson at University of Queensland).
The molecular logic of PKS and NRPS systems has generally been interpreted in the light of known product structures. We anticipate that the biosynthetic possibilities indicated by the architecture of XabB will assist in the interpretation of chemical data to solve the structure of albicidin. It is therefore interesting to consider the likely structure of the intermediate in albicidin biosynthesis produced by XabB, based on the domain structure of this unusual synthetase (Fig. 7). The O-methyl tyrosine component in NMR data could arise by action of the downstream XabC MT (Huang et al., 2000a
) on an intermediate like the one shown in Fig. 7(b)
.
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The characterization of XabB as a multi-modular hybrid enzyme provides new insights into the mechanism of albicidin biosynthesis and possible approaches to engineer the overproduction of albicidins. For example, the complementation experiments (Fig. 4) indicate that increased copy number of xabB stimulates early production of albicidin, but other factors become limiting during idiophase. It may be possible to increase expression of the albicidin synthetase by modifications to the promoter and TTG start codon, or to improve albicidin yields by supplying candidate substrates. The unusual enzyme organization also contributes to the emerging understanding of how microbes generate structural diversity of antibiotics, which will open possibilities for combinatorial engineering of antibiotics of mixed polyketide/peptide origin.
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ACKNOWLEDGEMENTS |
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Received 14 June 2000;
revised 26 December 2000;
accepted 8 November 2000.