Analysis of the Mycosamine Biosynthesis and Attachment Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455

The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the β-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.     

In vivo analysis of the regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455 reveals their differential control over antibiotic biosynthesis

Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.     

Site-specific mutagenesis and domain substitutions in the loading module of the nystatin polyketide synthase,and their effects on nystatin biosynthesis in Streptomyces noursei

The loading module for the nystatin polyketide synthase (PKS) in Streptomyces noursei is represented by the NysA protein composed of a ketosynthase (KS(S)), acyltransferase, dehydratase, and an acyl carrier protein. The absolute requirement of this protein for initiation of nystatin biosynthesis was demonstrated by the in-frame deletion of the nysA gene in S. noursei. The role of the NysA KS(S) domain, however, remained unclear, since no data on the significance of the "active site" serine (Ser-170) residue in the loading modules of type I PKSs were available. Site-specific mutagenesis of Ser-170 both in the wild-type NysA and in the hybrid loading module containing malonyl-specific acyltransferase domain from the extender module had no effect on nystatin biosynthesis. A second mutation (S413N) of the NysA KS(S) domain was discovered that completely abolished the ability of the hybrids to restore nystatin biosynthesis, presumably by affecting the ability of the resulting proteins to catalyze the required substrate decarboxylation. In contrast, NysA and its Ser-170 mutants bearing the same S413N mutation were able to restore nystatin production to significant levels, probably by using acetyl-CoA as a starter unit. Together, these data suggest that the KS(S) domain of NysA differs from the KS(Q) domains found in the loading modules of several PKS type I systems in that the active site residue is not significant for its activity.     

An unexpected role for the putative 4'-phosphopantetheinyl transferase-encoding gene nysF in the regulation of nystatin biosynthesis in Streptomyces noursei ATCC 11455

The nysF gene encoding a putative 4'-phosphopantetheinyl transferase (PPTase) is located at the 5' border of the nystatin biosynthesis gene cluster in Streptomyces noursei. PPTases carry out post-translational modification of the acyl carrier protein domains on the polyketide synthases (PKS) required for their full functionality, and hence NysF was assumed to be involved in similar modification of the nystatin PKS. At the same time, DNA sequence analysis of the genomic region adjacent to the nysF gene revealed a gene cluster for a putative lantibiotic biosynthesis. This finding created some uncertainty regarding which gene cluster nysF functionally belongs to. To resolve this ambiguity, nysF was inactivated by both insertion of a kanamycin (Km) resistance marker into its coding region, and by in-frame deletion. Surprisingly, the nystatin production in both the nysF::Km(R) and DeltanysF mutants increased by ca. 60% compared to the wild-type, suggesting a negative role of nysF in the nystatin biosynthesis. The expression of xylE reporter gene under control of different promoters from the nystatin gene cluster in the DeltanysF mutant was studied. The data obtained clearly show enhanced expression of xylE from the promoters of several structural and regulatory genes in the DeltanysF mutant, implying that NysF negatively regulates the nystatin biosynthesis.     

Characterization of the P450 monooxygenase NysL, responsible for C-10 hydroxylation during biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of K(m) and k(cat) and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.     

Candicidin biosynthesis in <Emphasis Type="Italic">Streptomyces griseus</Emphasis>

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.     

Study of the carbohydrate metabolic enzyme activity of Act. noursei]

Activity of aldolase and threosophosphate dehydrogenase, transketolase and phosphogluconate dehydrogenase in Act. noursei, strain 153 and its inactive mutant 149 was studied comparatively. The enzyme activity of the inactive mutant was investigated in the absence of the antibiotic production and under conditions of reduced biosynthesis of nystatin in this strain after addition of the fermentation broth filtrate of the inactive mutant 369 to the medium. The activity of the enzymes of the hexosomonophosphate metabolic pathway in the active strain 153 of Act. noursei was 2-4 times higher than that of the inactive mutant 149. The activity of the enzymes of the hexosomonophosphate metabolic pathways increased and reached the level of the enzyme of the active mutant. The high level of the enzyme activity of the hexosomonophosphate glycolysis pathway is probably one of the necessary conditions for nystatin production.     

A Bifunctional Enzyme in Pseudomonas aeruginosa: a New Pattern in the Organization of Enzymes Concerned with Phenylalanine and Tyrosine Biosynthesis

Two isozymes of chorismate mutase (CA mutase(1) and CA mutase(2)) and two isozymes of prephenate dehydratase (PPA dehydratase(1) and PPA dehydratase(2)) have been found in Pseudomonas aeruginosa. The activities CA mutase(2)-PPA dehydratase(2) catalyzing phenylalanine biosynthesis have been purified almost 40-fold and were found to be associated as a bifunctional enzyme or an enzyme complex. The enzymes specific for tyrosine biosynthesis did not appear to manifest such physical association. Thus, the organization of enzymes concerned with phenylalanine and tyrosine biosynthesis in P. aeruginosa is unique and is unlike most other organisms. Single site mutants have been isolated which have lost both CA mutase(2)-PPA dehydratase(2) activities resulting in a requirement for phenylalanine for growth. Single site revertants of these mutants regained both these activities simultaneously and were able to grow on minimal medium. A mutant, r(6), was also isolated which had normal CA mutase(2) but lacked PPA dehydratase(2) activity.     

Initiation of polyene macrolide biosynthesis: interplay between polyketide synthase domains and modules as revealed via domain swapping, mutagenesis, and heterologous complementation

Polyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT(0) in the nystatin PKS loading module NysA with the propionate-specific AT(1) from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT(0) domain by site-directed mutagenesis were accomplished. However, none of the NysA mutants constructed was able to initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids were functional, providing for different levels of nystatin biosynthesis. An interplay between certain residues in AT(0) and an active site residue in the ketosynthase (KS)-like domain of NysA in initiation of nystatin biosynthesis was revealed. Some hybrids between the NysA and RimA loading modules carrying the NysA AT(0) domain were able to prime rimocidin PKS with both acetate and butyrate units upon complementation of a rimA-deficient mutant of the rimocidin/CE-108 producer Streptomyces diastaticus. Expression of the PimS0 loading module from the pimaricin producer in the same host, however, resulted in production of CE-108 only. Taken together, these data indicate relaxed substrate specificity of NysA AT(0) domain, which is counteracted by a strict specificity of the first extender module KS domain in the nystatin PKS of Streptomyces noursei.     

The mtmVUC genes of the mithramycin gene cluster in Streptomyces argillaceus are involved in the biosynthesis of the sugar moieties

Mithramycin is a glycosylated aromatic polyketide produced by Streptomyces argillaceus, and is used as an antitumor drug. Three genes (mtmV, mtmU and mtmC) from the mithramycin gene cluster have been cloned, and characterized by DNA sequencing and by analysis of the products that accumulate in nonproducing mutants, which were generated by insertional inactivation of these genes. The mtm V gene codes for a 2,3-dehydratase that catalyzes early and common steps in the biosynthesis of the three sugars found in mithramycin (D-olivose, D-oliose and D-mycarose); its inactivation caused the accumulation of the nonglycosylated intermediate premithramycinone. The mtmU gene codes for a 4-ketoreductase involved in D-oliose biosynthesis, and its inactivation resulted in the accumulation of premithramycinone and premithramycin A , the first glycosylated intermediate which contains a D-olivose unit. The third gene, mtmC, is involved in D-mycarose biosynthesis and codes for a C-methyltransferase. Two mutants with lesions in the mtmC gene accumulated mithramycin intermediates lacking the D-mycarose moiety but containing D-olivose units attached to C-12a in which the 4-keto group is unreduced. This suggests that mtmC could code for a second enzyme activity, probably a D-olivose 4-ketoreductase, and that the glycosyltransferase responsible for the incorporation of D-olivose (MtmGIV) shows some degree of flexibility with respect to its sugar co-substrate, since the 4-ketoanalog is also transferred. A pathway is proposed for the biosynthesis of the three sugar moieties in mithramycin.     

Comparative analysis of in vitro antifungal activity and in vivo acute toxicity of the nystatin analogue S44HP produced via genetic engineering]

New polyene macrolide S44HP was purified from the culture of recombinant Streptomyces noursei strain with engineered nystatin polyketide synthase. S44HP, nystatin (NYS), and amphotericin B (Amph-B) were tested against 19 clinical fungal isolates in agar diffusion assay, which demonstrated clear differences in antifungal activities of these antibiotics. Sodium deoxycholate suspensions of all three antibiotics were subjected to acute toxicity studies in vivo upon intravenous administration in mice. NYS exhibited the lowest acute toxicity in mice in these experiments, while both Amph-B and S44HP were shown to be 4 times more toxic as judged from the LD50 values. While the acute toxicity of S44HP was higher than that of Amph-B, the data analysis revealed a significantly increased LD10 to LD50 dose interval for S44HP compared to Amph-B. The data revealed structural features of polyene macrolides, which might have an impact on both the activity and toxicity profiles of these antibiotics. These results represent the first example of preclinical evaluation of an "engineered" polyene macrolide, and can be valuable for rational design of novel antifungal drugs with improved pharmacological properties.     

Identification of functionally clustered nystatin-like biosynthetic genes in a rare actinomycetes, <Emphasis Type="Italic">Pseudonocardia autotrophica</Emphasis>

The polyene antibiotics, including nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very valuable antifungal polyketide compounds, and they are typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain genes potentially encoding polyene biosynthesis. Here, sequence information of an approximately 125.7-kb contiguous DNA region in five overlapping cosmids isolated from the P. autotrophica KCTC9441 genomic library revealed a total of 23 open reading frames, which are presumably involved in the biosynthesis of a nystatin-like compound tentatively named NPP. The deduced roles for six multi-modular polyketide synthase (PKS) catalytic domains were found to be highly homologous to those of previously identified nystatin biosynthetic genes. Low NPP productivity suggests that the functionally clustered NPP biosynthetic pathway genes are tightly regulated in P. autotrophica. Disruption of a NPP PKS gene completely abolished both NPP biosynthesis and antifungal activity against Candida albicans, suggesting that polyene-specific genome screening may constitute an efficient method for isolation of potentially valuable previously identified polyene genes and compounds from various rare actinomycetes widespread in nature.     

Structural analysis and biosynthetic engineering of a solubility-improved and less-hemolytic nystatin-like polyene in Pseudonocardia autotrophica

Polyene antibiotics such as nystatin are a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain an approximately 125.7-kb region of contiguous DNA with a total of 23 open reading frames, which are involved in the biosynthesis and regulation of a structurally unique polyene natural product named NPP. Here, we report the complete structure of NPP, which contains an aglycone identical to nystatin and harbors a unique di-sugar moiety, mycosaminyl-(α1-4)-N-acetyl-glucosamine. A mutant generated by inactivation of a sole glycosyltransferase gene (nppDI) within the npp gene cluster can be complemented in trans either by nppDI-encoded protein or by its nystatin counterpart, NysDI, suggesting that the two sugars might be attached by two different glycosyltransferases. Compared with nystatin (which bears a single sugar moiety), the di-sugar containing NPP exhibits approximately 300-fold higher water solubility and 10-fold reduced hemolytic activity, while retaining about 50% antifungal activity against Candida albicans. These characteristics reveal NPP as a promising candidate for further development into a pharmacokinetically improved, less-cytotoxic polyene antifungal antibiotic.     

Mutations in lipopolysaccharide biosynthetic genes impair maize rhizosphere and root colonization of Rhizobium tropici CIAT899

Three transposon mutants of Rhizobium tropici CIAT899 affected in lipopolysaccharide (LPS) biosynthesis were characterized and their maize rhizosphere and endophytic root colonization abilities were evaluated. The disrupted genes coded for the following putative products: the ATPase component of an O antigen ABC-2 type transporter ( wzt ), a nucleotide-sugar dehydratase ( lpsβ2 ) and a bifunctional enzyme producing GDP-mannose ( noeJ ). Electrophoretic analysis of affinity purified LPS showed that all mutants lacked the smooth LPS bands indicating an O antigen minus phenotype. In the noeJ mutant, the rough LPS band migrated faster than the parental band, suggesting a truncated LPS core. When inoculated individually, the wzt and noeJ mutants colonize the rhizosphere and root to a lower extent than the parental strain while no differences were observed between the lpsβ2 mutant and the parental strain. All mutants were impaired in competitive rhizosphere and root colonization. Pleiotropic effects of the mutations on known colonization traits such as motility and growth rate were observed, but they were not sufficient to explain the colonization behaviours. It was found that the LPS mutants were sensitive to the maize antimicrobial 6-methoxy-2-benzoxazolinone (MBOA). Only the combined effects of altered growth rate and susceptibility to maize antimicrobials could account for all the observed colonization phenotypes. The results suggest an involvement of the LPS in protecting R. tropici against maize defence response during rhizosphere and root colonization.     

Construction of an expression system for site-directed mutagenesis of the lantibiotic mersacidin

The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.     

Phosphomannose isomerase and phosphomannomutase gene disruptions in Streptomyces nodosus: impact on amphotericin biosynthesis and implications for glycosylation engineering

Streptomycetes synthesise several bioactive natural products that are modified with sugar residues derived from GDP-mannose. These include the antifungal polyenes, the antibacterial antibiotics hygromycin A and mannopeptimycins, and the anticancer agent bleomycin. Three enzymes function in biosynthesis of GDP-mannose from the glycolytic intermediate fructose 6-phosphate: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMPP). Synthesis of GDP-mannose from exogenous mannose requires hexokinase or phosphotransferase enzymes together with PMM and GMPP. In this study, a region containing genes for PMI, PMM and GMPP was cloned from Streptomyces nodosus, producer of the polyenes amphotericins A and B. Inactivation of the manA gene for PMI resulted in production of amphotericins and their aglycones, 8-deoxyamphoteronolides. A double mutant lacking the PMI and PMM genes produced 8-deoxyamphoteronolides in good yields along with trace levels of glycosylated amphotericins. With further genetic engineering these mutants may activate alternative hexoses as GDP-sugars for transfer to aglycones in vivo.     

The influence of carbon sources and morphology on nystatin production by Streptomyces noursei

Carbon source nutrition and morphology were examined during cell growth and production of nystatin by Streptomyces noursei ATCC 11455. This strain was able to utilise glucose, fructose, glycerol and soluble starch for cell growth, but failed to grow on media supplemented with galactose, xylose, maltose, sucrose, lactose and raffinose. Utilisation of glucose had a negative influence on production of nystatin independent of the specific growth rate when phosphate and ammonium was in excess. Consumption of carbon sources was related to the specific growth rate. S. noursei ATCC 11455 formed mainly mycelial clumps during cultivation, while pellet growth dominated the culture of the morphologically altered high producing mutant S. noursei NG7.19. When the pellet size increased above a critical size, cell growth and nystatin production terminated. Fluorescent staining of hyphae revealed that this coincided with loss of activity inside the core of the pellets, probably due to diffusion limitation of oxygen or other nutrients.     

Engineered biosynthesis and characterisation of disaccharide-modified 8-deoxyamphoteronolides

Several polyene macrolides are potent antifungal agents that have severe side effects. Increased glycosylation of these compounds can improve water solubility and reduce toxicity. Three extending glycosyltransferases are known to add hexoses to the mycosaminyl sugar residues of polyenes. The Actinoplanes caeruleus PegA enzyme catalyses attachment of a D-mannosyl residue in a β-1,4 linkage to the mycosamine of the aromatic heptaene 67-121A to form 67-121C. NppY from Pseudonocardia autotrophica adds an N-acetyl-D-glucosamine to the mycosamine of 10-deoxynystatin. NypY from Pseudonocardia sp. P1 adds an extra hexose to a nystatin, but the identity of the sugar is unknown. Here, we express the nypY gene in Streptomyces nodosus amphL and show that NypY modifies 8-deoxyamphotericins more efficiently than C-8 hydroxylated forms. The modified heptaene was purified and shown to be mannosyl-8-deoxyamphotericin B. This had the same antifungal activity as amphotericin B but was slightly less haemolytic. Chemical modification of this new disaccharide polyene could give better antifungal antibiotics.

     

Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin

The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.