Noursamycins, Chlorinated Cyclohexapeptides Identified from

Article Cite This: J. Nat. Prod. 2019, 82, 1478−1486

pubs.acs.org/jnp

Noursamycins, Chlorinated Cyclohexapeptides Identified from Molecular Networking of Streptomyces noursei NTR-SR4 Cynthia M. Mudalungu,† Wipert J. von Törne,† Kerstin Voigt,‡ Christian Rückert,§ Stefan Schmitz,∥,# Olga N. Sekurova,⊥ Sergey B. Zotchev,*,⊥ and Roderich D. Süssmuth*,† †

Technische Universität Berlin, Institut für Chemie, Straße des 17. Juni 124, 10623 Berlin, Germany Jena Microbial Resource Collection, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Beutenbergstraße 11a, 07745 Jena, Germany § Centrum für Biotechnologie (CeBiTec), Universität Bielefeld, Universitätsstraße 25, 33615 Bielefeld, Germany ∥ Department of Biotechnology, Norwegian University of Science and Technology, Trondheim NO-7491, Norway ⊥ Department of Pharmacognosy, University of Vienna, Althanstraße 14, 1090 Wien, Austria

Downloaded via GUILFORD COLG on July 18, 2019 at 06:30:36 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: The noursamycins A−F are chlorinated cyclic hexapeptides, which were identified and isolated from the strain Streptomyces noursei NTRSR4 overexpressing a LuxR-like transcriptional activator. The molecules were structurally characterized by mass spectrometric analyses and 1D and 2D NMR spectroscopic techniques. The enzymatic machinery involved in the biosynthesis of these peptides is represented by a modular nonribosomal peptide synthetase (NRPS), and the corresponding gene cluster was identified in the S. noursei genome. The latter suggested the biosynthetic pathway for the noursamycins. Spectral networking analysis uncovered noursamycin derivatives that were later found to result from a relaxed substrate specificity of the A3 and A4 adenylation domains of the NRPS. The stereochemistry of the amino acid constituents of the noursamycins was resolved by chemical derivatization, subsequent enantiomer analytics by GCEIMS, and in silico data analyses. Noursamycins A and B exhibited antibacterial activity against Gram-positive and Gramnegative bacteria, while no apparent cytotoxicity was observed.

N

linking them to the corresponding biosynthetic genes a challenging task. Application of molecular networking for dereplication and identification of secondary metabolites gives a global view on the molecular composition of a sample through its mass fragmentation (MS/MS), thus aiding in the elucidation of their biosynthesis pathways.10 Herein, we report on the structures of various noursamycins that were partially identified through molecular networking, with their biosynthetic gene cluster and bioactivity profiling.

aturally occurring peptides are known to exhibit a variety of biological activities, which include antibacterial, antifungal, cytotoxic, and antiviral properties.1,2 Their biosynthesis follows either ribosomal3 or nonribosomal pathways4 in both bacteria and fungi. Peptides of nonribosomal origin show structural diversity originating from the large pool of nonproteinogenic amino acids used by nonribosomal peptide synthetases (NRPS), which assemble them into peptides that are then often modified by post-NRPS tailoring enzymes. Examples of NRPS peptides are the antibacterial compounds vancomycin5 and daptomycin,6 which are marketed antibiotics, and teixobactin,7 lugdunin,8 and albicidin,9 which have recently raised scientific interest. The biosynthesis of nonribosomal peptides depends on the selection and activation of amino acids by adenylation (A) domains and their conversion into the corresponding aminoacyl adenylates at the expense of adenosine triphosphate (ATP). The aminoacyl adenylate is then covalently attached to the thiolation (T/PCP domain) domain awaiting the formation of the peptide bond, which is mediated by the condensation (C) domain. Very often, microorganisms capable of producing secondary metabolites generate a complex mixture of peptides, making the process of identification of individual components and © 2019 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION The genome of Streptomyces noursei ATCC 11455, an actinomycete bacterium known to produce the antifungal polyene macrolide nystatin, has recently been sequenced (GenBank accession number NZ_CP011533), and its analysis using antiSMASH11 software revealed over 40 secondary metabolite biosynthesis gene clusters. One of the gene clusters apparently encompassed genes that would be responsible for the production of a tetraene macrolide other than nystatin. In an attempt to activate this gene cluster, a regulatory gene Received: November 16, 2018 Published: June 7, 2019 1478

DOI: 10.1021/acs.jnatprod.8b00967 J. Nat. Prod. 2019, 82, 1478−1486

Journal of Natural Products

Article

encoding the LuxR-like transcriptional activator physically linked to the biosynthetic genes was cloned and expressed under control of the ermE*p promoter in the S. noursei NDA59 mutant deficient in nystatin biosynthesis. The resulting recombinant strain, S. noursei NTR-SR4, produced a tetraene compound that could not be detected in NDA59, as determined by spectrophotometric analysis of a butanol extract. However, the corresponding compound could not be purified for structure elucidation due to its instability. The LC-MS analysis of a methanol extract from the mycelia of the strain NTR-SR4 grown in M5288 medium revealed the presence of two compounds with an isotopic pattern characteristic of halogenated peptides. The same compounds could also be identified in the extract of strain NDA59, albeit in ca. 3-fold lower abundance. The HRESIMS analysis of the molecules resulted in the determination of the accurate molecular masses of compounds 1 and 2 with molecular ions [M + H]+ at m/z 824.3850 and m/z 790.4009, respectively (see Supporting Information Figure S2). The difference in the low-resolution molecular mass determination between the two peptides was 34 Da (unit mass), which suggested an additional chlorination. The determined molecular formula of 1 was C40H54N9O8Cl with 18 double-bond equivalents (DBE), while 2 was resolved to be C37H56N9O8Cl with 14 DBE and was indeed indicative of a monochlorination. In order to obtain an estimate of the number of compounds related to 1 and 2 and synthesized by S. noursei, the extract was subjected to spectral MS/MS network analysis. The analysis revealed 65 nodes in 21 clusters (see Figure 1). Each node represents one or more MS/MS spectra that are identical according to the algorithms used. Nodes are connected to one another through edges when their MS/MS spectra appear similar, thus forming clusters. Among the identified clusters, a five-node molecular family (MF) contained molecular masses in the range of 803 to 823 Da. Further structural information was gained from the tandem MS/MS experiments on a MALDI-TOF/TOF mass spectrometer. The mass spectrum of 1 was characteristic of a peptide with a sequence motif fitting Val-Phe-Ile/Leu-Orn. Compound 2 showed a similar fragmentation pattern, but with Phe replaced by Leu/Ile (see Figure 2). An improved production in a modified medium gave ∼4.2 mg of (1) and 4.0 mg of (2), respectively. The identity of 1 as a peptide was finally confirmed by 1D and 2D NMR experiments. The 1D NMR spectrum of noursamycin A (1) revealed the presence of characteristic amide protons in the downfield region. Furthermore, the signals in the aromatic region indicated the presence of Phe, and a highly deshielded shift at 11 ppm was interpreted as the NH proton of the indole moiety present in Trp (see Supporting Information Figure S5). The homonuclear couplings in the TOCSY spectrum confirmed the spin system of each amino acid, whereas the 2 J couplings were assigned through the COSY spectrum (see Supporting Information Table S1). This set of correlations indicated 5-Cl-Trp, Val, Phe, IIe, and Orn as the amino acids present in noursamycin A (1). Moreover, COSY couplings were observed between δH 4.58 and δH 4.45 and suggested the presence of a β-hydroxy asparagine (OH-Asn) with a characteristic downfield δCβ value at 71.6 ppm (OH proton not visible). Apart from the Phe signals (δH 7.16, 7.17, and 7.22 ppm), four other 1H chemical shifts in the aromatic region were indicative of a monosubstituted Trp (δH 7.04, 7.14, 7.31, and 7.54 ppm) that inferred the substitution of the

Figure 1. Spectral network of an S. noursei extract revealing noursamycin and congeners as a molecular family. A 5-day culture was used for the LC-MS/MS analyses. The identified spectral subnetwork revealed noursamycins as a compound family of five members (highlighted in the bottom of the figure and enlarged at the top of the figure). Nodes are connected by edges of varying thickness illustrating the cosine similarity score. The mass difference between the nodes is indicated in bold on the connecting edges.

indole moiety at position 5. All heteronuclear HSQC and HMBC correlations supported the structural connectivities with their respective data as summarized (see Supporting Information Table S1). Finally, the NOESY correlations confirmed the cyclic head-to-tail connection in 1 by means of a sequential walk of the NOE contacts (Figure 3). The configuration of the amino acids of 1 was determined by amino acid analysis on a chiral GC column with EI mass spectrometric detection (see Supporting Information Figure S16). The stereochemistry of lle was determined through comparison of its retention time to that of the four standard isomers (see Supporting Information Figure S18). The relative stereochemistry of the Cβ in OH-Asn was deciphered from the NOE between δH 4.45 and δH 4.58 with an existing spatial coupling to each other that is highly suggestive of a threo configuration. It was therefore concluded that compound 1 contains 5-Cl-L-Trp, D-Val, D-Phe, L-allo-lle, L-Orn, and a threoβ-hydroxyasparagine configuration. The structure elucidation of noursamycin B (2) revealed its structural identity to curacomycin and showed strong resemblance to nicrophorusamide A.12,13 For the latter, the authors determined a D-stereo configuration for the α-position in OH-Asn, i.e., D-threo-β-hydroxyasparagine through the synthesis of different OH-Asp stereoisomers, representing the hydrolyzed form of OH-Asn followed by Marfey analytics. Since only minute amounts of compounds 3 and 4 could be obtained, comparative MS/MS experiments were performed. These ions helped to identify the modified amino acid 1479



Article

Figure 2. Tandem MS/MS spectra of noursamycins A (1), B (2), C (3), and D (4) obtained from MALDI-TOF/TOF-MS.

methylated Leu or Ile moiety (see Figure 2). Varying the cultivation time of the strain in the Strept-M medium from 5 days to 7 days revealed further metabolites with similar fragmentation patterns. Analysis of the data led to the identification of compounds with the following molecular masses: 776.49 [M + H]+, 808.47 [M + H]+, 840.49 [M + H]+, and 856.48 [M + H]+. Among these, peptide 4 (775.49 Da) displayed a fragmentation pattern similar to that of compound 2 with notable variation being manifested at the IIe fragment exchanged to Val (see Figure 2). The structure elucidation of noursamycin C (3) was guided by its MS/MS fragmentation indicating structural variations in position 3 with a mass difference of Δm = 127 Da (Figure 2), which likely corresponded to a methylated Leu or Ile residue. The partial homonuclear COSY correlations of 3 revealed six amide protons indicating the absence of N-methylations. This suggested the presence of an additional methylene group in the side chain (see Supporting Information Figure S19). Therefore, compound 3 is thought to possess either a sec-butyl group or a homoleucine residue in contrast to homohomoleucine (hhLeu) contained in longicatenamycin A.14,15 Similarly, these properties featured by compound 3 at position 3 were noted for ulleungmycin A.16 Due to limited amounts recovered, the structure of noursamycin D (4) was characterized only by MS/

Figure 3. Observed NOE contacts in noursamycin A. The sequential walk through the NOESY spectrum indicated the illustrated couplings.

residues. Mass fragmentation of 1 carried out on an ESI-LTQ Orbitrap XL MS system gave the characteristic “b” and “y” ion series (Supporting Information Figure S4). Peptide 3 ([M + H]+ = 804.41) was characterized by its fragmentation pattern, which showed a variation at position 3 as previously determined in peptides 1 (Phe) and 2 (Leu). In this case, the calculated mass difference Δm = 127 Da indicated a 1480



Article

Figure 4. Tandem MS/MS fragmentation of noursamycin E (5) performed on an ESI-LTQ Orbitrap XL MS system.

in steric interactions that could play a significant role in the binding specificity embodying the entire substrate.17 Further annotations of the domains revealed three epimerase domains in the modules 2, 3 and 6, corresponding to the previously identified D-configurations of Val2, Phe/Leu3 and at the α position in OH-Asn6 (see Figure 6). Further confirmation for the biosynthetic route to noursamycins was obtained from the predicted enzymatic functions for the amino acid biosynthesis, such as the anthranilate phosphoribosyltransferase gene (nsm2), which is involved in the tryptophan biosynthesis.18 The 2-isopropylmalate synthase gene (nsm4) on the other hand is involved in the biosynthesis of leucine.19 In addition, the origin of Val, Leu, and Ile residues in these molecules is likely to be mediated by the branched-chain aminotransferase gene (nsm10).20,21 The hydroxylation of the asparagine moiety in the β-position could be explained by the presence of genes encoding putative TfdA-family dioxygenase (nsm13) and flavin reductase (nsm6). The latter protein may provide a reduced flavin to the former, which can then act as a hydroxylase. The putative function of the adjacent gene encoding an MbtH-like protein (nsm13) in this gene cluster is suggested to be linked to the promotion of the NRPs’ production. This has been proven in previous work by means of gene knockout experiments and in vitro studies, whereby different MbtH-like proteins could complement each other.22−25 To release the peptide from the NRPS assembly line, it seems plausible that a beta-lactamase-like protein encoded by nsm16 is responsible. This can be rationalized by the similarities of the beta-lactamase structural motif to other proteins such as the D-alanyl-D-alanine carboxypeptidase/ transpeptidase and the esterase (EstB) known to catalyze hydrolysis.26 Further, an example of a TE-less fungal PKS from Aspergillus terreus was previously reported, where the successful release of the product was only achieved by the inclusion of an ACTE protein (atrochrysone carboxyl ACP thioesterase) belonging to the β-lactamase superfamily.27 Alternatively, a putative thioesterase encoded by a gene located ca. 6 kb from the presumed right border of the noursamycin cluster (data not shown) may be responsible for this reaction. However, this

MS fragmentation data, which suggested a Val moiety in position 4 instead of Ile as found in peptide 2. Additionally, noursamycin E (5; 809.37 Da) was identified through molecular networking. Its MS/MS fragmentation pattern indicated the sequence 5-Cl-Trp-Val-Phe-Val-Orn-OH-Asn, which fit its exact molecular mass (see Figure 4). Compound F (6; 821.39 Da) revealed a fragmentation pattern that partially resembled 2 (X-Val-Leu-Ile-Orn-X) with exceptions of the alterations at the 5-Cl-Trp and OH-Asn moieties (see Supporting Information Figure S20). The proposed structures of noursamycin A−E are shown in Figure 5. The noursamycin structural features indicated a probable nonribosomal origin, and this guided the search for the putative biosynthetic gene cluster. From the biosynthetic gene clusters in the S. noursei genome identified by antiSMASH, only two clusters encoded the nonribosomal peptide synthetases. The biosynthetic gene cluster (BGC) of peptides 1−5 (see Figure 6) is suggested to span 21 open reading frames (ORFs) and with 10 “core” ORFs being similar in organization to those of the ulleungmycins BGC.16 In the noursamycin gene cluster, two NRPS synthetases, nsm15 and nsm14, encompassing four and two modules, respectively, are presumed to be responsible for the assembly of the hexapeptides. Most importantly, the presence of a halogenase gene (nsm8) with similarities to a tryptophan 7-halogenase (WP_078875718.1) points toward the authenticity of the assigned cluster. Annotations of the NRPS were refined manually using the Basic Local Alignment Search Tool (BLASTp) to identify the A-domain specificities of the individual modules. The substrate specificity conferring codes of the A domains were extracted, revealing the residues surrounding the binding pockets (see Table 1). However, the prediction of the signatures at the A3 and A4 domains indicated substrate promiscuity, a factor conferring the structural diversity found. Amino acid sequence alignment by simulating the A3 binding pocket revealed the likelihood of both the Leu and Phe residues to be accommodated (see Figure 7). One of the contributing factors could be their hydrophobic nature. Moreover, the surrounding residues might lead to differences 1481



Article

Figure 5. Proposed structures of the noursamycin derivatives.

constant value of 32 μg/mL for all the test organisms investigated (see Table 3). Using molecular networking and genomics, this study revealed new head-to-tail cyclized peptides from S. noursei strain NTR-SR4. The characterization of noursamycins A (1) and B (2) was achieved through tandem MS2 and 1D/2D NMR spectroscopy experiments, whereas the structures of noursamycin C (3) to F (6) were deduced mainly through their MS/MS spectra and in silico genomic data. Structurally similar cyclic hexapeptides from various Streptomyces strains have been reported, e.g., the desotamides and the wollamides.30−32 They displayed antibacterial and antimycobacterial activities with no cytotoxic effects on mammalian cells. Additionally, a number of chlorinated cyclic hexapeptides have been reported including ulleungmycins A and B from Streptomyces diastaticus,16 curacomycin A (Streptomyces curacoi),12 and nicrophorusamide A (from the symbiotic gut bacterium of the carrion beetle Nicrophorus concolor).13

protein is a type II thioesterase, and these enzymes usually have an editing function of removing the undesired substrates (misprimed or misacylated) that block the NRPS and polyketide assembly.28,29 Several other genes in the cluster could not be assigned to a specific function in the biosynthesis of noursamycins, e.g., nsm11 (SnoaL domain protein) and nsm9 (aminoacyl-tRNA editing protein). Further studies are needed to investigate the roles of these genes but are not required to obtain a basic understanding of noursamycin biosynthesis. Noursamycins A (1) and B (2) were found to possess antibacterial activity against the Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and Mycobacterium vaccae (see Table 2). Compound 1 appeared to be more potent than 2 (see Tables 2 and 3). This trend was even more apparent as 1, in contrast to 2, showed moderate activity against Gram-negative Escherichia coli (see Table 2) and Salmonella typhimurium (see Table 3). With the exception of a moderate activity against the fungus Sporobolomyces salmonicolor, a strain responsible for opportunistic infections, no antifungal activity was observed. A similar trend was seen in MIC determinations; the lowest MIC value observed was 4 μg/mL for 1 against Bacillus subtilis, whereas 2 indicated a

■

EXPERIMENTAL SECTION

General Experimental Procedures. The 1D and 2D NMR spectra of the compounds were acquired on an Avance III 700 MHz spectrometer (700 MHz for 1H and 175 MHz for 13C; Bruker, 1482



Article

Figure 6. Proposed biosynthesis of the noursamycins. (a) Genes responsible for the synthesis of the identified molecules. (b) NRPS machinery encompassing adenylation (A), thiolation (T), condensation (C), and epimerization domains. The amino acids taken up by the A3 and A4 domains are annotated as substituent groups R1 and R2. The 5-tryptophan halogenase which acts in trans is encoded by nsm8.

Table 1. Substrate Specificity of the A Domains Obtained from the Noursamycin Gene Clustera NRPS signature position of the residues domain

235

236

239

278

299

301

322

330

331

517

predicted substrate

GrsA A1 A2 A3 A4 A5 A6

D D D D D D D

A V A A A T L

W A F I L Eb T

T M W M W D E

I A L L M M V

A G G G G G G

A A G G G F E

I V T I V V V

C T F A F D G

K K K K K K K

Phe Trp Val Leu, Phe Val Orn Asn

a

The 10 residues that participate in the formation of the binding pocket are indicated with their respective positions. The NRP code of GrsA that gives rise to the binding pocket of the Phe-activating A domain is also shown for comparison. bManually aligned residue.

Figure 7. Schematic model of the A3-domain binding pocket. The 10 residues surrounding the activated amino acid (marked in red) form the amino acid recognition pocket. Asp and Lys highlighted in blue are the substrate gatekeeping residues: (a) binding with Phe; (b) the case with Leu. Scientific, Bremen, Germany) coupled to an Agilent 1200 HPLC

Karlsruhe, Germany) equipped with a TXI 5.0 mm probe head. The peptides were dissolved in ∼500 μL of d6-dimethyl sulfoxide (DMSO). Chemical shifts are given in δ (ppm), referenced to tetramethylsilane (TMS) at 310 K. The HRESIMS experiments were carried out on an Exactive Orbitrap mass spectrometer (Thermo

system (Agilent Technologies, Waldbronn, Germany). A Grom-Sil ODS-4 HR 3 μm, 50 mm × 2.0 mm column was used at flow rate of 300 μL/min. 1483



Article

Table 2. Growth Inhibitory Antibacterial and Antifungal Activities of Noursamycin A (1) and B (2)a noursamycin strain Bacteria Bacillus subtillis Staphylococcus aureus Staphylococcus aureus (MRSA) Escherichia coli Pseudomonas aeruginosa Pseudomonas aeruginosa Enterococcus faecalis (VRE) Mycobacterium vaccae Fungi Sporobolomyces salmonicolor Candida albicans Penicillium notatum

control drug

JMRC no.

A

B

Cip

STI:10880 STI:10760 ST:33793 ST:33699 ST:33772 ST:337721 ST:33700 STI:10670

18 21 18 10 0 10 18F 21/26p

16 18 13/15p 0 0 10 16F 22

29 19 0 23/31p 26 28/36p 17F 21p

ST:35974 STI:25000 STI:50164

12p 0 0

12p 0 0

Amp

solvent 0 0 0 0 0 0 0 0

18p 20 19p

10 0 10

a

The inhibition zones were measured (in mm) and the values recorded with reference as to whether there were colonies in the inhibition zone (p) or there was the promotion of inhibition zone (F). The values represented without a prefix letter indicate clear inhibition zones. Ciprofloxacin (Cip) and amphotericin B (Amp) were used as the positive control drugs for the bacterial and fungal tests, respectively. Methanol as a solvent was used as the negative control. and an equilibration phase at 39% B until t20, with a flow rate of 15 mL/min. The respective compounds of interest were pooled and evaporated in vacuo, and the residual aqueous phase was freeze-dried. Final purification steps were carried out with an Acclaim C30 column (3 μm, 4.6 × 100 mm) by application of a nearly isocratic gradient defined by 38−39% CH3CN in 10 min of the gradient run time for compound 1 and 5−100% for compounds 3. About 4.2, 4.0, and 1.2 mg of A, B, and C were obtained, respectively. Noursamycin A (1): white powder; NMR data, see Supporting Information Table S1; HRESIMS m/z 824.3850 [M + H]+ (calcd for C40H55N9O8Cl, 824.3857). Noursamycin B (2): white powder; NMR data, see Supporting Information; HRESIMS m/z 790.4009 [M + H]+ (calcd for C37H56N9O8Cl, 790.4013). Noursamycin C (3): white powder; HRESIMS m/z 804.4152 [M + H]+ (calcd for C38H59N9O8Cl, 804.4170). Noursamycin D (4): HRESIMS m/z 776.3321 [M + H]+ (calcd for C36H55N9O8Cl, 776.3330). Noursamycin E (5): HRESIMS m/z 810.3697 [M + H]+ (calcd for C39H53N9O8Cl, 810.3700). Noursamycin F (6): HRESIMS m/z 822.3878 [M + H]+ (calcd for C37H57N9O10Cl, 822.3911). MS2 Experiments and Molecular Networking. Tandem MS2 experiments for identification of the peptidic compounds were carried out on a matrix-assisted laser desorption ionization (MALDI) device coupled to a time-of-flight (TOF) mass analyzer (ultrafleXtreme MALDI-TOF/TOF, Bruker Corporation, MA (USA)) with dihydroxybenzoic acid (DHB) as the matrix in the ratio 1:1 with the analyte. For molecular networking analysis, the MS/MS data were acquired on an ESI-LTQ Orbitrap XL (Thermo Fisher Scientific GmbH, Bremen, Germany) MS spectrometer coupled to the Agilent 1260 HPLC system (Agilent Technologies, Waldbronn, Germany). An IDA (information-dependent acquisition) method with collision-induced dissociation (CID) of 35 eV was used for fragmentation of the three most abundant ions detected at a time point. The obtained MS2 spectral information were converted to mzXML file format by MSConvert (ProteoWizard).33 Spectral correlation and clustering was performed by MS-cluster following the GNPS workflow (https:// gnps.ucsd.edu).34 The input algorithms assumed a precursor mass tolerance and a fragment mass tolerance of 0.5 Da while setting the cosine threshold at 0.7 with matching peaks in a range of 3 to 10. Thereby, a minimum of six matching peaks was set for the final molecular network visualized using Cytoscape.35,36 Enantiomer Analytics of Amino Acids. Hydrolysis of the purified peptides (∼0.2 mg) and amino acid standards (Sigma) was

Table 3. Comparison of the Minimum Inhibitory Concentrations of Noursamycin A (1) and B (2) MIC value (μg/mL)a test organism

A

B

ciprofloxacin

Bacillus subtilis DSM10b Escherichia coli DSM 1116c E.coli BW25113c Salmonella typhimurium TA100c Micrococcus luteus DSM 1790b Mycobacterium phlei DSM 750b

4 ≥64 ≥64 16 8 8

32 ≥64 ≥64 32 32 32

Noursamycins, Chlorinated Cyclohexapeptides Identified from

Recommend Documents