Previously Uncharacterized Variants, OCF-E-OCF-J, of the Antifungal Occidiofungin Produced by Burkholderia contaminans MS14.

The rise of multidrug resistant fungal infections highlights the need to identify and develop novel antifungal agents. Occidiofungin is a nonribosomally synthesized glycolipopeptide that has a unique mechanism of action, disrupting actin-mediated functions and inducing cellular apoptosis. Antifungal activity has been observed in vitro against various fungal species, including multidrug resistant Candida auris, and in vivo efficacy has been demonstrated in a murine vulvovaginal candidiasis model. Occidiofungin, a cyclic glycolipopeptide, is composed of eight amino acids and in previous studies, an asparagine residue was assigned at position 7 (ASN7). In this study, new structural variants of occidiofungin have been characterized which have aspartic acid (ASP7), glutamine (GLN7), or glutamic acid (GLU7) at position 7. The side chain of the ASP7 variant contains the only terminal carboxylic acid in the peptide and provides a useful site for selective chemical modifications. Analogues were synthesized at the ASP7 position and tested for antifungal activity. These analogues were shown to be more active as compared to the ASP7 variant against a panel of Candida species. The naturally occurring variants of occidiofungin with a side chain containing a carboxylic acid at the seventh amino acid position can be used to develop semisynthetic analogues with enhanced therapeutic properties.

Intravaginal Gel for Sustained Delivery of Occidiofungin and Long-Lasting Antifungal Effects.

Fungal infections are caused by opportunistic pathogens that can be life threatening or debilitating. Candida spp. are becoming increasingly resistant to current clinically approved antifungal therapeutics. Candida infections afflict not only immunosuppressed but also immunocompetent individuals. Recurrent vulvovaginal candidiasis (RVVC) is a disease that afflicts 5-9% of women. Occidiofungin is a novel cyclic peptide that has a broad spectrum of antifungal activity with a novel fungicidal mechanism of action. A gel formulation containing occidiofungin (OCF001) is being developed for use to treat vulvovaginal candidiasis. The formulated gel for intravaginal application used hydroxyethyl cellulose as the primary gelling agent and hydroxypropyl ß-cyclodextrin as a solubilizing agent for occidiofungin. Franz cells and LC-MS/MS were used to determine the rate of drug substance diffusion in the gel formulation. The formulation was tested in an ex vivo mouse skin efficacy study, and the safety was tested following repeat intravaginal administration in rabbits. In this study, the gel formulation was shown to reduce the drug substance rate of diffusion across a skin memetic membrane. The study showed that the formulation extends exposure time to inhibitory concentrations of occidiofungin over a 24-h period and supports a single daily application for the treatment of RVVC.

Discovery of phosphorylated lantibiotics with proimmune activity that regulate the oral microbiome.

Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.

A Polyketide Synthetase Gene Cluster Is Responsible for Antibacterial Activity of Burkholderia contaminans MS14.

Burkholderia contaminans MS14, isolated from a soil sample in Mississippi, is known for producing the novel antifungal compound occidiofungin. In addition, MS14 exhibits a broad range of antibacterial activities against common plant pathogens. Random mutagenesis and gene complementation indicate that four genes are required for antibacterial activity of strain MS14 against the fire blight pathogen Erwinia amylovora. With the aim of finding the biosynthetic gene cluster for the unknown antibacterial compound, we used RNA-seq to analyze the transcriptome of MS14 wild type and mutants lacking antibacterial activity. The twofold lower expressed genes in all mutants were studied, and a polyketide synthase (PKS) gene cluster was predicted to be directly involved in MS14 antibacterial activities. The nptII-resistance cassette and CRISPR-Cas9 systems were used to mutate the PKS gene cluster. Plate bioassays showed that either insertion or frame-shifting one of the PKS genes resulted in a loss of antibacterial activity. Considering that the antibacterial-defective mutants maintain the same antifungal activities as the wild-type strain, the results suggest that this PKS gene cluster is highly likely to be involved in or directly responsible for the production of MS14 antibacterial activity. Purification efforts revealed that the antibacterial activity of the compound synthesized by the gene cluster is sensitive to UV radiation. Nevertheless, these findings have provided more insights to understand the antibacterial activity of strain MS14.

Synthesis and characterization of semisynthetic analogs of the antifungal occidiofungin.

Occidiofungin is a broad-spectrum antifungal compound produced by Burkholderia contaminans MS14. It is a cyclic glycol-lipopeptide with a novel beta-amino acid (NAA2) containing a hydroxylated C18 fatty acid chain with a xylose sugar. This study reports a strategy to produce semisynthetic analogs of occidiofungin to further explore the structure activity relationships of this class of compounds. Oxidative cleavage of the diol present on carbons five C(5) and six C(6) removes the xylose and twelve carbons of the fatty acid chain. The resulting cyclic peptide product, occidiofungin aldehyde, is devoid of antifungal activity. However, the free aldehyde group on this product can be subjected to reductive amination reactions to provide interesting semisynthetic analogs. This chemistry allows the quick generation of analogs to study the structure activity relationships of this class of compounds. Despite restoring the length of the aliphatic side chain by reductive amination addition with undecylamine or dodecylamine to the free aldehyde group, the obtained analogs did not demonstrate any antifungal activity. The antifungal activity was partially restored by the addition of a DL-dihydrosphingosine. The dodecylamine analog was demonstrated to still bind to the cellular target actin, suggesting that the diol on the side chain of native occidiofungin is important for entry into the cell enabling access to cellular target F-actin. These results show that the alkyl side chain on NAA2 along with the diol present on this side chain is important for occidiofungin's antifungal activity.

Occidiofungin: Actin Binding as a Novel Mechanism of Action in an Antifungal Agent.

The identification and development of natural products into novel antimicrobial agents is crucial to combat the rise of multidrug-resistant microorganisms. Clinical fungal isolates have been identified, which have shown resistance to all current clinical antifungals, highlighting a significant need to develop a novel antifungal agent. One of the natural products produced by the bacterium Burkholderia contaminans MS14 is the glycolipopeptide occidiofungin. Occidiofungin has demonstrated in vitro activity against a multitude of fungal species, including multidrug-resistant Candida auris strains, and in vivo effectiveness in treating vulvovaginal candidiasis. Characterization of occidiofungin revealed the mechanism of action as binding to actin to disrupt higher-order actin-mediated functions leading to the induction of apoptosis in fungal cells. Occidiofungin is the first small molecule capable of disrupting higher-order actin functions and is a first-in-class compound that is able to circumvent current antifungal resistant mechanisms by fungal species. Anticancer properties and antiparasitic activities, against Cryptosporidium parvum, have also been demonstrated in vitro. The novel mechanism of action and wide spectrum of activity highlights the potential of occidiofungin to be developed for clinical use.

Evaluation of analogs of mutacin 1140 in systemic and cutaneous methicillin-resistant Staphylococcus aureus infection models in mice.

Mutacin 1140 (Mu1140) is a potent antibiotic against Gram-positive bacteria, such as Staphylococcus aureus. The antibiotic is produced by the oral bacterium Streptococcus mutans and is a member of the epidermin family of type AI lantibiotics. The antibiotic exerts its inhibitory activity by binding to the cell wall precursor lipid II, blocking cell wall synthesis, and by disrupting bacterial membranes. In previous studies, the novel K2A and R13A analogs of Mu1140 have been identified to have superior pharmacokinetic properties compared to native Mu1140. In this study, the use of a combined formulation of the Mu1140 K2A and R13A analogs was shown to be more effective at treating MRSA bacteremia than the native Mu1140 or vancomycin. The analogs were also shown to be effective in treating an MRSA skin infection. The use of K2A and R13A analogs may provide a future alternative for treating serious Gram-positive bacterial infections. In a previous study, the Mu1140 analogs were shown to have significantly longer drug clearance times, leading to higher plasma concentrations over time. These properties warranted further testing to determine whether the analogs are effective for the treatment of systemic MRSA and acute skin infections. In this study, Mu1140 analogs were shown to be more effective than currently available treatments for systemic and skin MRSA infections. Further, the study clearly shows that the new analogs are superior to native Mu1140 for the treatment of a systemic MRSA infection. These findings support continued drug product development efforts using the K2A and R13A Mu1140 analogs, and that these analogs may ameliorate the outcome of serious bacterial infections.

Drug discovery through the isolation of natural products from Burkholderia.

Introduction: The increasing threat of antibiotic-resistant pathogens makes it imperative that new antibiotics to combat them are discovered. Burkholderia is a genus of Gram-negative, non-sporulating bacteria. While ubiquitous and capable of growing within plants and groundwater, they are primarily soil-dwelling organisms. These include the more virulent forms of Burkholderia such as Burkholderia mallei, Burkholderia pseudomallei, and the Burkholderia cepacia complex (Bcc).Areas covered: This review provides a synopsis of current research on the natural products isolated from the genus Burkholderia. The authors also cover the research on the drug discovery efforts that have been performed on the natural products derived from Burkholderia.Expert opinion: Though Burkholderia has a small number of pathogenic species, the majority of the genus is avirulent and almost all members of the genus are capable of producing useful antimicrobial products that could potentially lead to the development of novel therapeutics against infectious diseases. The need for discovery of new antibiotics is urgent due to the ever-increasing prevalence of antibiotic-resistant pathogens, coupled with the decline in the discovery of new antibiotics.

Formulation, Pharmacological Evaluation, and Efficacy Studies of Occidiofungin, a Novel Antifungal.

Occidiofungin is a nonribosomally synthesized cyclic lipopeptide that possesses broad-spectrum antifungal properties at submicromolar concentrations. This report explores multiple routes of administration and formulations of occidiofungin, as well as its toxicity in mice. Further, infection studies were performed in mice to assess the application of occidiofungin for treating systemic and intravaginal yeast infections. Formulations for intravenous and intravaginal administration of occidiofungin were prepared. Pharmacokinetic analyses were performed in a murine model, and a liquid chromatography-mass spectrometry (LC-MS) method was developed and used to quantify occidiofungin in mouse plasma samples. Toxicological and histopathological analyses of two repeat-dose studies using occidiofungin were performed. In these animal models, following intravenous administration, a liposomal formulation of occidiofungin improved the half-life and peak plasma drug concentration over that with a liposome-free formulation. Two long-term repeat-dosing toxicity studies of occidiofungin indicated the absence of toxicity in organ tissues. Murine models of a systemic yeast infection and a vulvovaginal yeast infection were performed. The findings of the systemic infection study revealed limitations in the use of occidiofungin that may be alleviated with the development of novel structural analogs or with further formulation studies. The gel formulation of occidiofungin demonstrated improved efficacy over that of the commercial product Monistat 3 in a vulvovaginal candidiasis study. This report outlines the optimal routes of administration of occidiofungin and demonstrates minimal toxicity following chronic exposure. Further, the results of these studies provide a clear indication for the use of occidiofungin for the treatment of recurrent vulvovaginal candidiasis (RVVC), which is a serious and clinically relevant issue.

Evolution of Lantibiotic Salivaricins: New Weapons to Fight Infectious Diseases.

Lantibiotic salivaricins are polycyclic peptides containing lanthionine and/or ß-methyllanthionine residues produced by certain strains of Streptococcus salivarius, which almost exclusively reside in the human oral cavity. The importance of these molecules stems from their antimicrobial activity towards relevant oral pathogens which has so far been applied through the development of salivaricin-producing probiotic strains. However, salivaricins may also prove to be of great value in the development of new and novel antibacterial therapies in this era of emerging antibiotic resistance. In this review, we describe the biosynthesis, antimicrobial activity, structure, and mode of action of the lantibiotic salivaricins characterized to date. Moreover, we also provide an expert opinion and suggestions for future development of this important field of microbiology.

Novel Antiparasitic Activity of the Antifungal Lead Occidiofungin.

Novel antiparasitic activity was observed for the antifungal occidiofungin. It efficaciously and irreversibly inhibited the zoonotic enteric parasite Cryptosporidium parvumin vitro with limited cytotoxicity (50% effective concentration [EC50] = 120 nM versus 50% cytotoxic concentration [TC50] = 988 nM), and its application disrupted the parasite morphology. This study expands the spectrum of activity of a glycolipopeptide named occidiofungin. Occidiofungin has poor gastrointestinal tract absorption properties, supporting future investigations into its potential activities on other enteric parasites.

Draft Genome Sequence of the Lantibiotic-Producing Strain Streptococcus salivarius HS0302.

Streptococcus salivarius is a prevalent commensal species of human oral mucosal surfaces. S. salivarius strain HS0302 produces the type AII lantibiotic salivaricin A2. Here, we report its draft genome sequence, revealing its potential to produce a variety of bacteriocins.

A Novel Actin Binding Drug with In Vivo Efficacy.

Occidiofungin is produced by the soil bacterium Burkolderia contaminans MS14 and is structurally similar or identical to the burkholdines, xylocandins, and cepacidines. This study identified the primary cellular target of occidiofungin, which was determined to be actin. The modification of occidiofungin with a functional alkyne group enabled affinity purification assays and localization studies in yeast. Occidiofungin has a subtle effect on actin dynamics that triggers apoptotic cell death. We demonstrate the highly specific localization of occidiofungin to cellular regions rich in actin in yeast and the binding of occidiofungin to purified actin in vitro Furthermore, a disruption of actin-mediated cellular processes, such as endocytosis, nuclear segregation, and hyphal formation, was observed. All of these processes require the formation of stable actin cables, which are disrupted following the addition of a subinhibitory concentration of occidiofungin. We were also able to demonstrate the effectiveness of occidiofungin in treating a vulvovaginal yeast infection in a murine model. The results of this study are important for the development of an efficacious novel class of actin binding drugs that may fill the existing gap in treatment options for fungal infections or different types of cancer.

Efficacious Analogs of the Lantibiotic Mutacin 1140 against a Systemic Methicillin-Resistant Staphylococcus aureus Infection.

Mutacin 1140, a member of the epidermin family of type AI lantibiotics, has a broad spectrum of activity against Gram-positive bacteria. It blocks cell wall synthesis by binding to lipid II. Although it has rapid bactericidal effects and potent activity against Gram-positive pathogens, its rapid clearance and short half-life in vivo limit its development in the clinic. In this study, we evaluated the effect of charged and dehydrated residues on the pharmacokinetics of mutacin 1140. The dehydrated residues were determined to contribute to the stability of mutacin 1140, while alanine substitutions for the lysine or arginine residues improved the pharmacological properties of the antibiotic. Analogs K2A and R13A had significantly lower clearances, leading to higher plasma concentrations over time. They also had improved bioactivities against several pathogenic bacteria. In a murine systemic methicillin-resistant Staphylococcus aureus (MRSA) infection model, a 10-mg/kg single intravenous bolus injection of the K2A and R13A analogs (1:1 ratio) protected 100% of the infected mice, while a 2.5-mg/kg dose resulted in 50% survival. The 10-mg/kg treatment group had a significant reduction in bacteria load in the livers and kidneys compared to that in the vehicle control group. The study provides lead compounds for the future development of antibiotics used to treat systemic Gram-positive infections.

Modifying the Lantibiotic Mutacin 1140 for Increased Yield, Activity, and Stability.

Mutacin 1140 belongs to the epidermin family of type AI lantibiotics. This family has a broad spectrum of activity against Gram-positive bacteria. The binding of mutacin 1140 to lipid II leads to the inhibition of cell wall synthesis. Pharmacokinetic experiments with type AI lantibiotics are generally discouraging for clinical applications due to the short half-life of these compounds. The unprotected dehydrated and protease-susceptible residues outside the lanthionine rings may play a role in the short half-life in physiological settings. Previous mutagenesis work on mutacin 1140 has been limited to the lanthionine-forming residues, the C-terminally decarboxylated residue, and single amino acid substitutions at residues Phe1, Trp4, Dha5, and Arg13. To study the importance of the dehydrated (Dha5 and Dhb14) and protease-susceptible (Lys2 and Arg13) residues within mutacin 1140 for stability and bioactivity, each of these residues was evaluated for its impact on production and inhibitory activity. More than 15 analogs were purified, enabling direct comparison of the activities against a select panel of Gram-positive bacteria. The efficiency of the posttranslational modification (PTM) machinery of mutacin 1140 is highly restricted on its substrate. Analogs in the various intermediate stages of PTMs were observed as minor products following single point mutations at the 2nd, 5th, 13th, and 14th positions. The combination of alanine substitutions at the Dha5 and Dhb14 positions abolished mutacin 1140 production, while the production was restored by substitution of a Gly residue at one of these positions. Analogs with improved activity, productivity, and proteolytic stability were identified.IMPORTANCE Our findings show that the efficiency of mutacin 1140 PTMs is highly dependent on the core peptide sequence. Analogs in various intermediate stages of PTMs can be transported by the bacterium, which indicates that PTMs and transport are finely tuned for the native mutacin 1140 core peptide. Only certain combinations of amino acid substitutions at the Dha5 and Dhb14 dehydrated residue positions were tolerated. Observation of glutamylated core peptide analogs shows that dehydrations occur in a glutamate-dependent manner. Interestingly, mutations at positions outside rings A and B, the lipid II binding domain, would interfere with lipid II binding. Purified mutacin 1140 analogs have various activities and selectivities against different genera of bacteria, supporting the effort to generate analogs with higher specificity against pathogenic bacteria. The discovery of analogs with improved inhibitory activity against pathogenic bacteria, increased stability in the presence of protease, and higher product yields may promote the clinical development of this unique antimicrobial compound.

Covalent Structure and Bioactivity of the Type AII Lantibiotic Salivaricin A2.

Lantibiotics are a class of lanthionine-containing, ribosomally synthesized, and posttranslationally modified peptides (RiPPs) produced by Gram-positive bacteria. Salivaricin A2 belongs to the type AII lantibiotics, which are generally considered to kill Gram-positive bacteria by binding to the cell wall precursor lipid II via a conserved ring A structure. Salivaricin A2 was first reported to be isolated from a probiotic strain, Streptococcus salivarius K12, but the structural and bioactivity characterizations of the antibiotic have remained limited. In this study, salivaricin A2 was purified and its covalent structure was characterized. N-terminal analogues of salivaricin A2 were generated to study the importance for bioactivity of the length and charge of the N-terminal amino acids. Analogue salivaricin A2(3-22) has no antibacterial activity and does not have an antagonistic effect on the native compound. The truncated analogue also lost its ability to bind to lipid II in a thin-layer chromatography (TLC) assay, suggesting that the N-terminal amino acids are important for binding to lipid II. The creation of N-terminal analogues of salivaricin A2 promoted a better understanding of the bioactivity of this antibiotic and further elucidated the structural importance of the N-terminal leader peptide. The antibacterial activity of salivaricin A2 is due not only to the presence of the positively charged N-terminal amino acid residues, but to the length of the N-terminal linear peptide.IMPORTANCE The amino acid composition of the N-terminal linear peptide of salivaricin A2 is crucial for function. Our study shows that the length of the amino acid residues in the linear peptide is crucial for salivaricin A2 antimicrobial activity. Very few type AII lantibiotic covalent structures have been confirmed. The characterization of the covalent structure of salivaricin A2 provides additional support for the predicted lanthionine and methyl-lanthionine ring formations present in this structural class of lantibiotics. Removal of the N-terminal Lys1 and Arg2 residues from the peptide causes a dramatic shift in the chemical shift values of amino acid residues 7 through 9, suggesting that the N-terminal amino acids contribute to a distinct structural conformer for the linear peptide region. The demonstration that the bioactivity could be partially restored with the substitution of N-terminal alanine residues supports further studies aimed at determining whether new analogues of salivaricin A2 for novel applications can be synthesized.

Improving the attrition rate of Lanthipeptide discovery for commercial applications.

INTRODUCTION: Lanthipeptides are a class of ribosomally synthesized and post-translationally modified peptides. Lanthipeptides with antimicrobial activity are referred to as lantibiotics. Lantibiotics are generally active against Gram-positive bacteria. However, some modifications have expanded their activity toward Gram-negative bacteria. Furthermore, additional functions aside from antibacterial activities have been reported for lanthipeptides. Areas covered: This review provides a synopsis of current anthipeptide research for potential therapeutics. The review highlights the current tools used for identifying lanthipeptides from genomic sequencing data. It also describes the current approaches that have been used to overcome the limitations in the purification and isolation of lanthipeptides. The status of lanthipeptides in terms of potential applications and approaches that are currently being done to promote the development of lanthipeptides as novel therapeutics are also discussed. Expert opinion: Significant improvements have been made to promote the discovery of new lanthipeptides, while, simultaneously, tools have been developed to promote their production and isolation. Lanthipeptides are showing significant promise for treating bacterial infections, as well as for new applications as anticancer and antiviral agents, or as a novel treatment for pain management. At the current rate of lanthipeptide discovery and isolation of the products, it is likely several new applications will be discovered.

Carboxyl Analogue of Mutacin 1140, a Scaffold for Lead Antibacterial Discovery.

Mutacin 1140 belongs to the epidermin group of lantibiotics. Epidermin class lantibiotics are ribosomally synthesized and posttranslationally modified antibiotics with potent activity against Gram-positive bacteria. In particular, this class is effective at targeting drug-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium tuberculosis, and Clostridium difficile A C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) residue is derived from a decarboxylation of a terminal cysteine that is involved in lanthionine ring formation. Studies on mutacin 1140 have revealed new insight into the structural importance of the C-terminal AviCys residue. A C-terminal carboxyl analogue of mutacin 1140 was engineered. Capping the C-terminal carboxyl group with a primary amine restores bioactivity and affords a novel opportunity to synthesize new analogues. A C-terminal fluorescein-labeled mutacin 1140 analogue traps lipid II into a large lipid II lantibiotic complex, similar to that observed in vivo for the lantibiotic nisin. A C-terminal carboxyl analogue of mutacin 1140 competitively inhibits the activity of native mutacin 1140 and nisin. The presence of a C-terminal carboxyl group prevents the formation of the large lipid II lantibiotic complexes but does not prevent the binding of the lantibiotic to lipid II.IMPORTANCE This study addressed the importance of the C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) residue for antibacterial activity. We have learned that the posttranslational modification for making the AviCys residue is presumably important for the lateral assembly mechanism of activity that traps lipid II into a large complex. The C-terminal carboxyl analogue of this class of lantibiotics is agreeable to the addition of a wide variety of substrates. The addition of fluorescein enabled in vivo visualization of the epidermin class of lantibiotics in action. These results are significant because, as we demonstrate, the presence of the AviCys residue is not essential for bioactivity, but, more importantly, the removal of the carboxyl group is essential. The ability to make a C-terminal carboxyl analogue that is modifiable will facilitate the synthesis of novel analogues of the epidermin class of lantibiotics that can be developed for new applications.

The Siderophore Product Ornibactin Is Required for the Bactericidal Activity of Burkholderia contaminans MS14.

Burkholderia contaminans MS14 was isolated from soil in Mississippi. When it is cultivated on nutrient broth-yeast extract agar, the colonies exhibit bactericidal activity against a wide range of plant-pathogenic bacteria. A bacteriostatic compound with siderophore activity was successfully purified and was determined by nuclear magnetic resonance spectroscopy to be ornibactin. Isolation of the bactericidal compound has not yet been achieved; therefore, the exact nature of the bactericidal compound is still unknown. During an attempt to isolate the bactericidal compound, an interesting relationship between the production of ornibactin and the bactericidal activity of MS14 was characterized. Transposon mutagenesis resulted in two strains that lost bactericidal activity, with insertional mutations in a nonribosomal peptide synthetase (NRPS) gene for ornibactin biosynthesis and a luxR family transcriptional regulatory gene. Coculture of these two mutant strains resulted in restoration of the bactericidal activity. Furthermore, the addition of ornibactin to the NRPS mutant restored the bactericidal phenotype. It has been demonstrated that, in MS14, ornibactin has an alternative function, aside from iron sequestration. Comparison of the ornibactin biosynthesis genes in Burkholderia species shows diversity among the regulatory elements, while the gene products for ornibactin synthesis are conserved. This is an interesting observation, given that ornibactin is thought to have the same defined function within Burkholderia species. Ornibactin is produced by most Burkholderia species, and its role in regulating the production of secondary metabolites should be investigated.IMPORTANCE Identification of the antibacterial product from strain MS14 is not the key feature of this study. We present a series of experiments that demonstrate that ornibactin is directly involved in the bactericidal phenotype of MS14. This observation provides evidence for an alternative function for ornibactin, aside from iron sequestration. Ornibactin should be further evaluated for its role in regulating the biosynthesis of secondary metabolites in other Burkholderia species.

Two Flavoenzymes Catalyze the Post-Translational Generation of 5-Chlorotryptophan and 2-Aminovinyl-Cysteine during NAI-107 Biosynthesis.

Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides containing thioether rings. In addition to these cross-links, the clinical candidate lantibiotic NAI-107 also possesses a C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) and a unique 5-chloro-l-tryptophan (ClTrp) moiety linked to its potent bioactivity. Bioinformatic and genetic analyses on the NAI-107 biosynthetic gene cluster identified mibH and mibD as genes encoding flavoenzymes responsible for the formation of ClTrp and AviCys, respectively. The biochemical basis for the installation of these modifications on NAI-107 and the substrate specificity of either enzyme is currently unknown. Using a combination of mass spectrometry, liquid chromatography, and bioinformatic analyses, we demonstrate that MibD is an FAD-dependent Cys decarboxylase and that MibH is an FADH2-dependent Trp halogenase. Most FADH2-dependent Trp halogenases halogenate free Trp, but MibH was only active when Trp was embedded within its cognate peptide substrate deschloro NAI-107. Structural comparison of the 1.88-Å resolution crystal structure of MibH with other flavin-dependent Trp halogenases revealed that subtle amino acid differences within the MibH substrate binding site generates a solvent exposed crevice presumably involved in determining the substrate specificity of this unusual peptide halogenase.