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Antimicrobial Agents and Chemotherapy, June 2006, p. 2167-2177, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01545-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Biosynthetic Pathway for Mannopeptimycins, Lipoglycopeptide Antibiotics Active against Drug-Resistant Gram-Positive Pathogens

Nathan A. Magarvey,{dagger} Brad Haltli, Min He, Michael Greenstein, and John A. Hucul*

Wyeth Research, Chemical and Screening Sciences, Natural Products Discovery, 401 North Middletown Road, Pearl River, New York 10965

Received 4 December 2005/ Returned for modification 30 December 2005/ Accepted 7 February 2006


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ABSTRACT
 
The mannopeptimycins are a novel class of lipoglycopeptide antibiotics active against multidrug-resistant pathogens with potential as clinically useful antibacterials. This report is the first to describe the biosynthesis of this novel class of mannosylated lipoglycopeptides. Included here are the cloning, sequencing, annotation, and manipulation of the mannopeptimycin biosynthetic gene cluster from Streptomyces hygroscopicus NRRL 30439. Encoded by genes within the mannopeptimycin biosynthetic gene cluster are enzymes responsible for the generation of the hexapeptide core (nonribosomal peptide synthetases [NRPS]) and tailoring reactions (mannosylation, isovalerylation, hydroxylation, and methylation). The NRPS system is noncanonical in that it has six modules utilizing only five amino acid-specific adenylation domains and it lacks a prototypical NRPS macrocyclizing thioesterase domain. Analysis of the mannopeptimycin gene cluster and its engineering has elucidated the mannopeptimycin biosynthetic pathway and provides the framework to make new and improved mannopeptimycins biosynthetically.


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INTRODUCTION
 
Multidrug-resistant gram-positive bacterial pathogens now exist, and their rising numbers are a major concern. The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) exemplifies the seriousness of this concern (22, 29). Finding novel antibiotics active against such drug-resistant gram-positive pathogens, however, is problematic (32, 42). The mannopeptimycins (Fig. 1), which are produced by Streptomyces hygroscopicus NRRL 30439, represent a new class of lipoglycopeptide antibiotics with exceptional in vitro and in vivo antibacterial activities against MRSA, VRE, and penicillin-resistant Streptococcus pneumoniae (17, 36). Studies with mannopeptimycin-{delta} demonstrated that this antibiotic acts by blocking the transglycosylation reaction in cell wall biosynthesis mediated by lipid II binding. Mannopeptimycin-{delta} inhibits gram-positive cell wall biosynthesis through a mechanism which does not compete or render it ineffective due to cross-resistance with the vancomycin-type antibiotics (33, 36).


Figure 1
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  		FIG. 1. Structures of natural mannopeptimycins.

The unique structure, mode of action, and bioactivity of the mannopeptimycins have generated much interest in identifying a therapeutic candidate from this new class of molecules (17, 33, 36). One semisynthetic analog, AC98-6446, of the natural mannopeptimycins is significantly more potent and effective than the natural mannopeptimycins, with MICs in the 15- to 60-ng/ml range against MRSA, VRE, penicillin-resistant Streptococcus pneumoniae, and glycopeptide-intermediate Staphylococcus aureus (10, 31). The success in improving on the natural mannopeptimycins argues strongly for broadening the effort of generating additional new mannopeptimycins through promising strategies such as combinatorial biosynthesis, mutasynthesis, and chemoenzymatic synthesis (4, 28). The establishment and success of such approaches hinge on the cloning of the mannopeptimycin biosynthetic gene cluster and elucidation of the steps involved in mannopeptimycin biosynthesis.

The mannopeptimycins have a cyclic hexapeptide core containing a unique combination of two proteinogenic and four nonproteinogenic amino acids, including a tandem arrangement of the epimers of ß-hydroxy-enduracididine (ßhEnd) and ß-methyl-phenylalanine (ßmPhe). Assembly of these amino acids into a cyclic peptide is consistent with synthesis by a nonribosomal peptide synthetase (NRPS). The mannopeptimycin hexapeptide is tailored with one N-linked mannose and an O-linked di-mannose ({alpha}-D-mannopyranosyl-{alpha}-1,4-D-mannopyranoside). The terminal O-linked mannose is modified with an isovaleryl group at one of three positions (Fig. 1). Other lipid II-binding agents, ramoplanin and enduracidin, have components in common with the mannopeptimycins (O-linked di-mannose and enduracididine, respectively), but published information regarding the formation of such moieties does not exist (27). Therefore, cloning and sequencing of the mannopeptimycin biosynthetic cluster not only will provide information necessary to make unnatural mannopeptimycin analogs by metabolic engineering but also should lead to identification of novel enzymes, reveal new mechanisms, and lead to a better understanding as to how mannopeptimycin is biosynthesized. Here, the cloning, sequencing, and engineering of the mannopeptimycin biosynthetic gene cluster are presented. The mannopeptimycin NRPS is atypical in having a proposed iterative-acting adenylation (A) domain and no prototypical NRPS thioesterase (TE). The information contained within the mannopeptimycin biosynthetic gene cluster, bioinformatics analysis, and biosynthetic investigations of gene functions (through gene knockout and chemical analyses) provide critical information on advancing the mannopeptimycins as effective agents against drug-resistant pathogens.


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MATERIALS AND METHODS
 
Chemicals, bacterial strains, and culture conditions. All chemicals were purchased from Sigma-Aldrich. Stocks of S. hygroscopicus NRRL 30439 and its derivatives were maintained as spore suspensions in 20% glycerol at –70°C. For genomic DNA isolation, the strains were grown for 2 days in 25 ml tryptone soy broth (Oxoid) at 28°C and shaken at 200 rpm. Antibiotic production strains were grown under the same conditions except in 50 ml PharmaMedia (Traders Protein) and for longer incubation periods (3 to 7 days). For isolation and propagation of recombinant S. hygroscopicus strains, growth media were supplemented with the appropriate antibiotics for selection (apramycin at 25 µg/ml and kanamycin at 50 µg/ml). DNA manipulations were routinely performed in Escherichia coli DH5{alpha} (Invitrogen) and ET123567/pUZ8002 used as a donor strain in intergeneric conjugation experiments as described previously (14). Desmethyl-mannopeptimycins were obtained by fermentation using S. hygroscopicus strain BD-20, a strain generated from the mannopeptimycin producer through multiple rounds of N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis.

Mannopeptimycin isolation and detection. Mannopeptimycins and related compounds were extracted by passage of S. hygroscopicus fermentation broths (2 to 10 ml) through a 3-ml carboxylic acid ion-exchange solid phase extraction column (J. T. Baker). An increasing acetonitrile gradient was used to elute compounds, and mannopeptimycins were collected in the 70% acetonitrile-0.5% trifluoroacetic acid-H2O fraction. Samples were concentrated in vacuo and resuspended in 200 µl of 50% methanol for analysis. Mannopeptimycins were detected by high-pressure liquid chromatography and liquid chromatography-mass spectrometry (LC-MS) (electrospray system with an acetonitrile-H2O gradient, diode array detector {lambda} = 226 nm; Hewlett-Packard API) as described previously (17).

Genomic DNA isolation and cosmid library construction. High-molecular-weight genomic DNA was harvested from wild-type S. hygroscopicus NRRL 30439 according to established protocols (20). The DNA was partially digested using Sau3AI, dephosphorylated, and ligated into BamHI-digested cosmid pWE15 (Stratagene). The ligated mixture was packaged using a Gigapack III XL packaging extract kit (Stratagene), and the resulting library was amplified and titers were determined according to the manufacturer's instructions.

Library screening and annotation of the mannopeptimycin gene cluster. Primer synthesis and cosmid sequencing were done by MWG Biotech (Highpoint, N.C.). Degenerate PCR primers for the A domain were designed based on the highly conserved core motifs of A3 and A8 and consisted of the following sequences: for the A3 forward primer, 5'-ACG/CTCG/CGGCT/ACGCACCGGCCIGCCG/CAAG-3', and for the A8 reverse primer, 5'AGCTCG/CAT/CG/CCGG/CTAGCCG/CCGG/CAT/CCTTG/CACCTG-3' (26). DNA fragments of approximately 800 bp in length were synthesized by PCR (30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min) with S. hygroscopicus genomic DNA as a template. End sequencing of fragments cloned into a sequencing vector (pCR2.1) was accomplished using an ABI3700 sequencer (Applied Biosystems, Foster City, CA). Analysis of sequences confirmed that DNA fragments encoded adenylation domains. DNA fragments were radiolabeled using a RadPrime labeling kit (Pharmacia) with [{alpha}-32P]dCTP (Amersham) according to the manufacturer's directions. The radiolabeled fragments were used to probe the genomic library by standard colony hybridization protocols (34). Overlapping cosmids containing contiguous DNA sequences were identified by chromosomal walking (34) (Fig. 2). The GCG Wisconsin Package was accessed through SeqWeb (Accelrys, SanDiego, CA) and used to identify individual open reading frames (ORFs) and their putative functions.


Figure 2
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  		FIG. 2. Cosmid map and gene cluster for mannopeptimycin. ORFs have been set in color to indicate function (green, NRPS; red, nonproteinogenic amino acid formation; blue, tailoring; yellow, regulatory; black, transporters and unknown function).

Intergeneric conjugation and construction of gene replacement vectors. The transfer of DNA from E. coli ET12567 to S. hygroscopicus NRRL 30439 was accomplished with the use of a bifunctional conjugation vector. The vector pFD666 (8) was modified by insertion of a 0.76-kb PstI fragment containing oriT derived from plasmid RK2 (8, 23) at the PstI site within the multiple cloning site, resulting in the intergeneric conjugation vector pNWA200. A second conjugation vector, pBWA218, was constructed by inserting PCR-amplified oriT from pNWA200 into the SspI site of pUWL218 (44). Intergeneric conjugation with the described vectors followed previously published protocols using an R6 medium supplemented with 10 mM MgCl2 (18a, 20). To inactivate the mannopeptimycin NRPS, a 3.5-kb EcoRI fragment of mppA containing a putative NRPS initiation module was subcloned into the EcoRI site of pNWA200. An apramycin resistance (Amr) gene [aac(3)IV] was removed from pJV176 (N. Magarvey, unpublished) as a BamHI fragment and inserted into the internal BamHI site of the EcoRI fragment. To inactivate mppJ, a 3.9-kb NotI fragment containing the gene was isolated from pNWA117 and subcloned into the NotI site of pNWA200. The Amr gene was removed from pJV176 as an EcoRI fragment and inserted into a unique EcoRI site internal to mppJ. To inactivate mppK and mppL, a 5-kb BamHI/BglII fragment containing the gene was isolated from pNWA117 and cloned into pCR2.1 (Invitrogen), resulting in plasmid pBWA2. pBWA2 was cut with AccI and blunt ended. The 872-bp AccI fragment internal to mppK removed by this procedure was replaced with a 1.5-kb EcoRV fragment containing the Amr gene in both the same and opposite orientations relative to mppK (pBWA14 and pBWA15, respectively). For conjugation, pBWA14 was digested with NotI and a 3.9-kb fragment containing the construct was cloned into the NotI site of pNWA200 whereas pBWA15 was digested with StuI and EcoRV and a 3.9-kb fragment containing the construct was cloned into the NruI site of pNWA200. To inactivate mppL, pBWA2 was digested with AscI and a 1.7-kb Amr gene fragment with BssHII ends was inserted in the opposite orientation relative to mppL. For conjugation, the construct was removed as a 4.7-kb SphI fragment and inserted into the SphI site of pBWA218. To inactivate mppM, a 2-kb BglII/SmaI fragment containing the gene was isolated from pNWA117 and cloned into pUC18. The cloned mppM gene was cut with BstEII and blunt ended. A 1.5-kb EcoRV fragment containing the Amr gene was inserted at this site in the same orientation as mppM. For conjugation, a 3.5-kb XbaI/SmaI fragment containing the construct was isolated and cloned into pNWA200.

Adenylation domain cloning, expression, and ATP-pyrophosphate exchange assay. The A domain of MppB-M2 (the second elongation module of MppB) was isolated by PCR, with pNWA117 as the template and primers with the following sequences: for the forward primer, 5'-AACATATGGACCTCCCGCTGCTCGATG-3', and for the reverse primer, 5'-AACTCGAGGAGCAGCAGCTCGGTGGC-3'. The amplified product was cloned into the His tag expression vector pET22b (Novagen) as an NdeI/XhoI fragment. The resulting clone, pBWA24, was used to transform Rosetta (DE3) pLysS (Novagen). Expression and purification of the protein were conducted according to protocols detailed in the pET expression system manual (Novagen). Specificity of the MppB-M2 A domain was tested using an ATP-pyrophosphate exchange assay as described previously (37).


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RESULTS AND DISCUSSION
 
Cloning and identification of the mannopeptimycin gene cluster. NRPS-derived A domain amplicons from S. hygroscopicus NRRL 30439 were synthesized using degenerate PCR primers (26, 40). One A domain, determined by sequence analysis to contain a serine A domain (ASer) specificity code, was considered as a probable candidate for the mannopeptimycin serine-activating domain (5, 37). A DNA fragment containing this domain was used to screen an S. hygroscopicus NRRL 30439 cosmid library. Four hybridizing cosmids were identified, and within each cosmid, homology to the probe was shown by Southern hybridization analysis with a common 3.5-kb EcoRI DNA fragment. Sequencing this fragment from cosmid pNWA117 revealed an NRPS initiation module containing the ASer domain followed by a peptidyl carrier protein (PCP) domain. To prove that these domains were part of the mannopeptimycin NRPS, the 3.5-kb EcoRI fragment was insertionally inactivated with the Amr gene [acc(3)IV] within a bifunctional intergeneric conjugation vector, pNWA200. The resulting plasmid, pNWA201, was transferred into E. coli strain ET12567/pUZ8002 (14), and the derived strain was used as a donor to transfer the construct to S. hygroscopicus NRRL 30439 via intergeneric conjugation (14). One S. hygroscopicus exconjugant resistant to apramycin but sensitive to kanamycin (Kms) was selected and determined by Southern hybridization analyses of restricted genomic DNAs to be a double crossover mutant [ASer::acc(3)IV] (data not shown). Fermentation with the isolate WNP100 resulted in an extract devoid of antibacterial activity (data not shown). LC-MS analysis confirmed the absence of the mannopeptimycins in the extract. Primers specific to the ends of the pNWA117 insert were used to synthesize specific DNA probes. Screening of an S. hygroscopicus NRRL 30439 cosmid library with these probes identified cosmids with overlapping DNA inserts that resulted in the cloning and sequencing of 67 kb of chromosomal DNA (Fig. 2).

Sequencing and annotation of the mannopeptimycin gene cluster. The analysis of sequence data totaling 67 kb from two cosmids, pNWA117 and pNWA119, has localized the mannopeptimycin gene cluster to 48 kb of contiguous DNA. The sequenced DNA flanking the putative gene cluster included 4 kb upstream and 15 kb downstream. The upstream DNA included ORFs encoding products predicted to be associated with housekeeping functions and primary metabolism rather than secondary metabolism (data not shown). Downstream of the last mannopeptimycin biosynthetic gene, mppZ1, is a 2-kb region of noncoding DNA followed by a series of ORFs with homologies to type II polyketide synthase (PKS) and deoxy-sugar biosynthesis genes. These genes form a putative type II PKS gene cluster with no apparent relationship to mannopeptimycin formation.

The 48.2-kb mannopeptimycin gene cluster (GenBank accession no. AY735112) contains 27 ORFs (mppA to mppZ1) encoding proteins with functions predicted to be involved in precursor biosynthesis, generation of the cyclic peptide core, tailoring reactions, product export, and transcriptional regulation (Fig. 2). The predicted functions of mannopeptimycin genes, based on homology searches, are presented in Table 1, and the results of mutational analysis of the mannopeptimycin gene cluster are presented in Table 2.


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  		TABLE 1. Summary of proteins encoded by the mannopeptimycin biosynthetic gene cluster


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  		TABLE 2. Summary of mutational analysis of the mannopeptimycin biosynthetic gene cluster

Mannopeptimycin NRPS assembly line. (i) Hexapeptide core. The mannopeptimycin gene cluster contains two large genes encoding two multifunctional modular NRPSs that are predicted to be responsible for hexapeptide biosynthesis. The first, mppA (8,244 nucleotides), contains the sequence of the 3.5-kb EcoRI fragment encoding the predicted mannopeptimycin serine adenylation domain (ASer) described above. At the 3' end of mppA and overlapping the predicted mppA stop codon is an ATG start codon for the second large NRPS gene, mppB (11,007 nucleotides) (see Fig. 2 and Table 1). The presence of a loading module in MppA and the relative positioning of the two NRPSs (mppA upstream of mppB) indicate that the amino terminus of MppA is the initiation point for hexapeptide assembly. Based on colinearity rules and the hexapeptide core of mannopeptimycin, the NRPS was expected to contain six A domains within six modules for incorporation of the six different amino acids (4). Sequence analysis of MppA determined that it contains the following domains: two A domains, three PCP domains, and two condensation (C) domains. These domains form three modules, one loading module (MppA-L) and two elongation modules (MppA-M1 and MppA-M2), with each containing an A domain in the order A-PCP-C-A-PCP-C-A-PCP, consistent and colinear with synthesis of the serinyl, glycinyl, and phenylalanyl portion of mannopeptimycin. MppA ends with a PCP domain, consistent with it being an NRPS enzyme whose product is off-loaded onto another multidomain NRPS or PKS enzyme (26, 35). MppB also contains three modules, but with only two A domains. The domain organization of MppB is C-A-PCP-E-C-A-PCP-C-PCP-E, where E represents an epimerization domain. The presence of three C domains and two E domains is consistent with three elongation modules catalyzing three peptide bonds and incorporating two D- amino acids. The first elongation module within MppB (MppB-M1) is comprised of C-A-PCP-E. The domain composition of MppB-M1 is colinear and consistent with addition of D-tyrosine to the tripeptide off-loaded from MppA. Following MppB-M1 is a second complete elongation module, MppB-M2 (C-A-PCP), predicted to be responsible for addition of the penultimate amino acid, ßh-L-End. The third and final elongation module, MppB-M3 (C-PCP-E), is incomplete due to the lack of an A domain. According to colinearity rules, these two terminal modules (MppB-M2 and -M3) should be responsible for the addition of the two epimeric ßhEnd residues in mannopeptimycin (Fig. 3A).


Figure 3
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  		FIG. 3. (A) Mannopeptimycin NRPS assembly line. Nonribosomal peptide synthetase genes (mppA and mppB) are represented by green arrows, and the modules encoded by the respective genes are shown underneath, with domains in colored circles. (B) Substrate specificity of the MppB-M2 NRPS adenylation domain.

To determine the specificity of the MppB-M2 A domain within the mannopeptimycin NRPS, a His-tagged version of the domain was generated and purified. The recombinant His-tagged A domain was analyzed for its specificity by use of an in vitro ATP-pyrophosphate exchange assay as described by Stachelhaus et al. (37). An array of amino acids was tested in this assay. Of the five amino acids shown in Fig. 3B, ßhEnd was the preferred substrate.

We propose that this unexpected NRPS architecture allows for the sequential addition of two ßhEnd residues using a single ßh-L-End-specific A domain, which acts iteratively, aminoacylating two PCPs (Fig. 3A). Differences in ßhEnd stereochemistry would result from the second PCP-tethered ßhEnd being in the necessary proximity to the terminal E domain for its selective epimerization (Fig. 3A).

(ii) Assembly line termination and hexapeptide cyclization. PKS and NRPS chain-terminating TEs (Pfam no. PF00975) are often found as integrated domains within modular PKSs and NRPSs but also are frequently found as a distinct protein encoded by a gene flanking the NRPS and/or PKS gene(s) (21). No TE could be identified from the sequenced mannopeptimycin biosynthetic gene cluster; however, an enzyme encoded by a cluster-associated gene is likely responsible for chain termination and macrocyclization reactions. Immediately downstream of the second mannopeptimycin NRPS gene, mppB, is mppK, whose deduced protein has homology to PBP4 and PBP4a proteins from a variety of bacteria. Among the closest matches were cmcPBP (32% identical and 44% similar) from the Amycolatopsis lactamdurans cephamycin biosynthetic gene cluster, ORF1 (45% identical and 56% similar) from a proposed Saccarothrix mutabilis subsp. capreolus cryptic NRPS gene cluster (GenBank accession no. AAM47271), and a 2,457-amino-acid protein, SdenDRAFT_0529, annotated as an amino acid adenylation protein, identified from the Shewanella denitrificans OS217 genome sequencing project. Closer inspection revealed that SdenDRAFT_0529 is a multimodular NRPS with the domain organization A-PCP-C-A-PCP-C-X. The X domain (347 amino acids) referred to here is actually the region of SdenDRAFT_0529 which has sequence homology (22% identical and 42% similar) to MppK.

PBP4 and PBP4a proteins are members of the alpha/beta hydrolase fold clan of proteins, and both are carboxypeptidases/transpeptidases. Both the well-characterized PBP4 from Actinomadura sp. strain R39 (7) and the PBP4a from Bacillus subtilis (9) have been shown to have TE activity. Thus, it seems possible, based on homology relationships and the relative positioning of mppK within the mannopeptimycin gene cluster, that MppK is the mannopeptimycin chain termination enzyme. Another possible function of MppK could be self-resistance. Aberrant expression of PBP4 (DD-carboxypeptidases) is a characteristic of intermediary glycopeptide-resistant Staphylococcus aureus strains (13, 46). Further, VanY, a DD-carboxypeptidase/carboxyesterase and a relative of PBP4, is encoded by the high-level vancomycin resistance gene cluster contained within Tn1546 of VRE. VanY cleaves off the terminal D-Ala residue of the pentapeptide (UDP-MurNAc-L-Ala-{gamma}-D-Glu-L-Lys-D-Ala-D-Ala) (47) and is proposed to act in series with VanX (D-Ala D-Ala dipeptidase) to prevent accumulation of the MurNAc D-Ala D-Ala pentapeptide, the substrate for the vancomycin-type glycopeptides (1, 47). Since the mode of action of mannopeptimycin is inhibition of cell wall biosynthesis, it is possible that a change in the pentapeptide or structural changes within the cell wall stemming from the increased presence of DD-carboxypeptidase/carboxyesterase within S. hygroscopicus may render the host resistant to its own antibiotic.

To determine if MppK is involved in mannopeptimycin biosynthesis or resistance, a gene knockout strategy was employed. The mppK gene was insertionally inactivated with the Amr marker, and the resulting null form of mppK [{Delta}mppK::aac(3)IV] was used to replace wild-type mppK within S. hygroscopicus by homologous recombination. The resulting strain, WNP102, was tested for its antibiotic sensitivity by use of an agar disk diffusion assay where disks were spotted with increasing amounts of mannopeptimycin-{delta} (0, 10, 25, 50, and 100 µg/ml). Streptomyces hygroscopicus NRRL 30439 and Streptomyces lividans strain 66 were also subject to the same concentrations of mannopeptimycin-{delta} for comparative purposes. Streptomyces hygroscopicus NRRL 30439 was, not surprisingly, resistant to the highest concentration of mannopeptimycin-{delta} tested (100 µg/ml), whereas S. lividans strain 66 was sensitive at 10 µg/ml, indicating that resistance is not intrinsic to Streptomyces spp. (data not shown). WNP102 also was not inhibited by 100 µg/ml of mannopeptimycin-{delta} and grew at rates similar to those of S. hygroscopicus NRRL 30439. WNP102 also grew well in mannopeptimycin-producing fermentation conditions (data not shown). These results demonstrate that MppK is not required for mannopeptimycin self-resistance. Extraction and analysis of WNP102 fermentation broths revealed no mannopeptimycins or intermediates. The absence of mannopeptimycin production from WNP102 and the homology of MppK to proteins (PBP4 and PBP4a) known to have thioesterase activity lead us to suggest that MppK is the mannopeptimycin chain termination/cyclization protein.

Nonproteinogenic, ß-modified amino acids. (i) ß-Methyl-phenylalanine. The mannopeptimycin gene cluster contains a single methyltransferase gene, mppJ, and its protein product (MppJ) contains motifs involved in binding the methyl-donating cofactor S-adenosyl methionine (SAM). The closest homology match to MppJ is LmbW (27% identical and 43% similar), a protein of unassigned function encoded within the lincomycin gene cluster in Streptomyces lincolnensis (30). Lincomycin, an alkaloid antibiotic, contains the modified amino acid propyl-L-proline. Stable isotope experiments determined that the terminal carbon of the propyl-L-proline portion of lincomycin is derived from SAM (2). Although the lincomycin gene cluster has been sequenced, no candidate C-methyltransferase gene has been described.

To determine if MppJ catalyzes the ß-methylation of the phenylalanine residue of mannopeptimycin, a plasmid construct containing an insertionally inactivated form of mppJ [mppJ::aac(3)IV] was made (Fig. 4A). The plasmid containing the mutated allele, mppJ::aac(3)IV, was transferred to the wild-type mannopeptimycin producer by intergeneric conjugation. One exconjugant strain, WNP101 (Apr Kms), was determined by Southern hybridization to contain a double crossover mutation of mppJ and was chosen for further analysis (data not shown). Extracts from fermentation broths of WNP101 were analyzed by LC-MS and were compared to authentic mannopeptimycin standards (mannopeptimycin-{alpha}, -ß, -{gamma}, -{delta}) (Fig. 4E) and desmethyl-mannopeptimycin-{alpha} (Fig. 4B). No natural mannopeptimycins or desmethyl-phenylalanine derivatives were present in the extract. Examination of all ionizing masses by use of LC-MS (positive and negative modes) within a range of 1,200 to 1,420 average mass units (amu), which encompasses all natural mannopeptimycins, revealed the presence of a compound (1,300 amu) not found within extracts derived from the wild-type strain (Fig. 4C). This compound (Fig. 4C) had a significantly shorter retention time (5.76 min) than the mannopeptimycin standards (Fig. 4E), but the chromophore demonstrated an absorbance pattern consistent with that of the mannopeptimycins ({lambda}max end absorbance UV, 220, 278 shoulder). A mannopeptimycin with an ionizing mass of 1,300 amu has not been described previously. This mass, however, is consistent with the addition of 18 mass units (1 O; 2 H) to cyclic desmethyl-mannopeptimycin-{alpha} (1,282 amu), introduced during hydrolytic cleavage of the cyclic peptide, resulting in a linear desmethyl-mannopeptimycin structure. The presence of linear mannopeptimycin analogs has been observed previously when nonmethylated antimetabolites of phenylalanine were substituted for ßmPhe by precursor-directed biosynthesis (D. Abbanat, personal communication).


Figure 4
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  		FIG. 4. Insertional inactivation of mannopeptimycin biosynthetic genes (mppJ and mppM) and chemical analysis of fermentation extracts. (A) Orientation of the apramycin resistance gene [aac(3)IV] within mppJ. (B) Chromatographic separation of an authentic standard of desmethyl-mannopeptimycin-{alpha} (solid line) and fermentation extract derived from WNP101 (dashed line). (C) Electrospray ionization mass spectrum of mannopeptimycin compound eluting at 5.76 min. (D) Orientation of the apramycin resistance gene [aac(3)IV] within mppM. (E) Chromatographic separation of mannopeptimycins produced by Streptomyces hygroscopicus NRRL 30439 (solid line) and the mppM mutant WNP104 (dashed line).

Additional evidence for the role of mppJ in ßmPhe biosynthesis was obtained by comparison of the wild-type mppJ sequence to that of a mutant mppJ allele (obtained by NTG treatment of S. hygroscopicus) from a high-titer cyclic desmethyl-phenylalanine mannopeptimycin producer, BD-20 (J. Lotvin, personal communication). Sequencing of the mppJ mutant from BD-20 and comparison with the wild-type mppJ allele sequence revealed a single point mutation (G -> A at nucleotide position 497) which leads to a missense mutation (G166 -> E166) in the SAM binding motif I (G166 SGSG170). We suggest this is a loss-of-function mutation and is the basis for the loss of methylated mannopeptimycins in BD-20.

It is not obvious why WNP101 would produce linear desmethyl-mannopeptimycins given that it has only the mppJ gene disrupted, whereas BD-20 does accumulate cyclic desmethyl-mannopeptimycins (Table 2). Further work is under way to determine the exact structure of the predicted linear desmethyl product from WNP101. Given that BD-20 was obtained following multiple rounds of NTG mutagenesis, it is possible that BD-20 contains a lesion in a second gene (e.g., a protease) outside the mannopeptimycin biosynthetic gene cluster that allows the strain to accumulate the cyclic desmethyl-phenylalanyl product. From our characterization, we propose that MppJ may be the first described methyltransferase for methylating the ß-position of phenylalanine.

(ii) ß-Hydroxy-enduracididine. The mannopeptimycin cluster contains one oxygenase gene, mppO, with a deduced amino acid sequence displaying homology (46% similarity and 31% identity) with clavaminate synthase I (CSI) and CSII from Streptomyces clavuligerus (3). Clavaminate synthases are trifunctional {alpha}-ketoglutarate-dependent enzymes involved in clavulanic acid synthesis. CSI and CSII catalyze the ß-hydroxylation of deoxyguanidinoproclavaminic acid and then cyclize a pathway intermediate to afford a bicyclic clavam ring (3, 39). MppO also exhibited homology to a viomycin biosynthetic oxygenase (VioC) suggested to be involved in capreomycidine (a six-membered cyclic arginine) formation (38, 48). The VioC oxygenase was recently shown to be a nonheme {alpha}-ketoglutarate-dependent arginine ß-hydroxylase (49). Finally, MppO has homology to SttL, an oxygenase from the streptothricin gene cluster suspected to be involved in forming streptolidine, an arginine-based amino acid sharing a common pathway with capreomycidine (12, 19). Recently we completed a full characterization of MppO and found it is in fact an {alpha}-ketoglutarate-dependent hydroxylase which acts on L-enduracididine as a free amino acid (16).

Addition and acylation of mannose units. Homology-based searching yielded a set of three mannosyltransferase gene candidates (mppG, mppH, and mppI). MppG displayed similarity (29% identical and 45% similar) to SC6D7.16, a suspected polyprenyl mannose synthase from Streptomyces coelicolor (6). MppG also showed similarity to the characterized mycobacterial nucleoside diphosphate-dependent mannosyltransferase, Ppm1, responsible for transfer of mannose from GDP-mannose to C35 and C50 polyprenyls (15). MppH and MppI, which show 70% amino acid identity to each other, had similarities to mycobacterial membrane-associated phosphomannose transferase enzymes (pfam02366) which use mannosylated C35 and C50 polyprenyls as sugar donors in lipoarabinomannan biosynthesis (45). Other low-level homologies were to deduced proteins from the gene clusters of other mannose-containing antibiotics: Dbv20 (dalbavancin), Tcp15 (teicoplanin), and Ram29 (ramoplanin). Topological models (using the TM-Base algorithm [18]) of the deduced proteins revealed each had several transmembrane-spanning domains (MppH, 8 domains; MppI, 9; Ram29, 14; Dbv20, 12; and Tcp15, 12) with the same relative positioning of such domains (data not shown). From this, we propose that MppG generates mannosylated polyprenyls that serve as mannose donors for MppH and MppI to transfer mannoses to the mannopeptimycin aglycon (Fig. 5). The presence of only two genes with similarities to mannosyltransferases is interesting since there are three mannose residues in the mannopeptimycins. Iterative use of glycosltransferases, however, is not unprecedented in natural product biosynthesis. In landomycin A biosynthesis, LanGT1 (an olivosyltransferase) and LanGT4 (an rhodinosyltransferase) act twice, with each transferring two of the sugars (olivose and rhodinose) found in the hexasaccharide portion of the landomycin A structure (24). Ramoplanin, like mannopeptimycin, contains an O-linked di-mannose, and in the ramoplanin gene cluster there is only one glycosyltransferase (Ram29) (11). It is likely that in both ramoplanin and mannopeptimycin biosynthesis a di-mannose moiety is transferred by the action of a single mannosyltransferase. The addition of the third mannopeptimycin mannose to one of the secondary amines of the ßhEnd side chain would presumably be the action of the second cluster-encoded mannosyltransferase (either MppH or MppI).


Figure 5
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  		FIG. 5. Proposed two-compartment scheme for biosynthesis of the mannopeptimycins. Structures and enzymes proposed to be membrane associated are shown in gray.

Two isovaleryltransferase genes, mppM and mppN, were detected from sequence homology searches. MppM and MppN exhibit 72% amino acid similarity to each other and have extensive homology to macrolide 3-O-acyltransferases from the carbomycin and megalomicin biosynthetic clusters (41, 43). Like the predicted mannopeptimycin mannosyltransferases, several transmembrane-spanning domains were found within these predicted isovaleryltransferases (MppM, 10 domains, and MppN, 9). To begin investigating the role of these proteins in mannopeptimycin biosynthesis, a mutant, WNP104, containing a disrupted mppM allele [mppM::aac(3)IV] was made (Fig. 4D). Fermentation of WNP104 and subsequent LC-MS examination of broth-derived extracts revealed a 90% reduction in acylated mannopeptimycins (mannopeptimycin-{gamma}, -{delta}, and -{varepsilon}) (Fig. 4E). The reduction of acylated mannopeptimycins from WNP104 and accumulation of mannopeptmycin-{alpha} demonstrate that MppM is a mannopeptimycin isovaleryltransferase (Table 2). The failure to completely abolish the production of acylated mannopeptimycins implies that a second isovaleryltransferase, likely MppN, is functioning in the WNP104 strain.

Based on the physical nature of the mannosylation and isovalerylation enzymes, we propose a two-compartment biosynthetic strategy for mannopeptimycin, with the aglycon produced within the cytoplasm and tailoring of that aglycon at the membrane (intracellular or extracellular side) (Fig. 5).

Transport, resistance, regulatory, and functionally uncharacterized genes. Localized together within the mannopeptimycin cluster are mppD, mppE, and mppF. MppD has homology to several members of the Pfam family PF01547, which contains the maltose-binding protein MalE and other sugar-binding proteins. MppE and MppF have homology to various ABC transporters involved in sugar uptake pathways. MppD may be responsible for extracellular mannose recognition and binding and MppE and MppF for facilitating the transport of this mannopeptimycin precursor across the membrane.

To investigate whether mannopeptimycin resistance is either intrinsic to the genus Streptomyces or the result of a specific mechanism associated with the biosynthetic pathway, we first compared the MIC of mannopeptimycin-{delta} for a model culture, Streptomyces lividans 1326, to the MIC for S. hygroscopicus NRRL 30439. An agar plate (Bennett's agar) disk diffusion assay was used to determine the sensitivities of the streptomycete strains to mannopeptimycin-{delta} (10, 25, 50, and 100 µg/ml). The MIC of mannopeptimycin-{delta} against S. hygroscopicus NRRL 30439 was greater than 100 µg/ml, while S. lividans 1326 was sensitive to 10 µg/ml, comparable to the MICs against other gram-positive bacteria (31). Thus, mannopeptimycin resistance does not appear to be intrinsic to Streptomyces spp., and therefore S. hygroscopicus NRRL 30439 must have a mechanism of self-protection.

There are two genes (mppL and mppX) whose deduced products have homology to previously described members of the major facilitator superfamily (MSF) permeases. MSF permeases are known to be involved in antibiotic resistance. MppL was similar to the spectinomycin resistance pump SpcT from Streptomyces flavopersicus (a spectinomycin producer) and the putative viomycin self-resistance determinant VioE from Streptomyces sp. strain ATCC 11861 (25, 38). To determine the role of MppL, a strain (WNP103) carrying a null form of mppL [mppL::aac(3)IV] was constructed. WNP103 grew well under mannopeptimycin-producing conditions and produced all of the natural mannopeptimycins, but at a drastically reduced level (10% of the wild type [data not shown]). Given that the target of mannopeptimycin is outside of the cell, efflux would not constitute a viable self-protection strategy. For this reason, we do not propose that MppL plays a role in mannopeptimycin self-resistance nor do we suggest a self-resistance role for other mannopeptimycin gene cluster genes (e.g., mppX). The marked decrease in mannopeptimycin production from WNP103 does, however, imply that MppL is linked to production. One possibility is that it exports the mannopeptimycin aglycone to the extracellular side of the cell membrane to ensure the aglycon is modified by the proposed membrane-associated tailoring enzymes (mannosyltransferases and isovaleryltransferases). Figure 5 shows how MppL could fit with the proposed two-compartment biosynthesis strategy for mannopeptimycin.

At the 3' end of the gene cluster, a potential two-component regulatory system encoded by mppU (response regulator) and mppV (sensor kinase) was found. Another gene, mppZ1, had similarity to a group of small lactone-dependent transcriptional regulators. A series of other protein sequences encoded by genes mppR, mppT, mppW, mppY, and mppZ in the cluster had no significant homologies or their function could not be predicted based on homology to characterized proteins.

Conclusions. This investigation into nature's logic for assembling the mannopeptimycin-type lipoglycopeptides reveals several unique features that deviate from previously studied nonribosomal peptide biosynthesis pathways. First, at the core of the mannopeptimycin pathway is a hexapeptide assembled by an NRPS possessing only five A domains. Second, a prototypical PKS/NRPS macrocyclizing thioesterase is lacking and in its place is a PBP-4a (an alpha/beta hydrolase) homolog (MppK) which is essential for mannopeptimycin biosynthesis. Third, the mannopeptimycin tailoring enzymes phenylalanine ß-methyltransferase, mannosyltransferases, and isovaleryltransferases are new, and predicted membrane association of the latter two implies a two-compartment strategy for assembly of mannopeptimycins. Taken together, this work on mannopeptimycin assembly will serve as an essential foundation for generating unnatural analogs within this exciting antibiotic class with improved potency against multidrug-resistant bacterial pathogens.


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ACKNOWLEDGMENTS
 
We are indebted to M. Zabriskie, Oregon State University, and E. Graziani (Wyeth Research) for providing authentic samples of ß-hydroxyenduracididine and enduracididine. We also thank Pam Fink for providing the sequence of mppJ from BD-20. We are also grateful to Haiyin He and Melissa Wagenaar (Wyeth Research) for assistance with extraction and analysis of mannopeptimycins from fermentation broths.


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FOOTNOTES
 
* Corresponding author. Mailing address: Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965. Phone: (845) 602-2449. Fax: (845) 602-5687. E-mail: huculj{at}wyeth.com. Back

{dagger} Present address: Harvard University, Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, 240 Longwood Avenue, Armenise Building, Boston, MA 02115. Back


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Antimicrobial Agents and Chemotherapy, June 2006, p. 2167-2177, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01545-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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