scholarly journals Self-Resistance during Muraymycin Biosynthesis: a Complementary Nucleotidyltransferase and Phosphotransferase with Identical Modification Sites and Distinct Temporal Order

2018 ◽  
Vol 62 (7) ◽  
Author(s):  
Zheng Cui ◽  
Xia-Chang Wang ◽  
Xiaodong Liu ◽  
Anke Lemke ◽  
Stefan Koppermann ◽  
...  
Keyword(s):  
Temporal Order ◽  
Biochemical Characterization ◽  
Antibacterial Activities ◽  
Biosynthetic Gene Cluster ◽  
Resistance Mechanisms ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Inhibitory Activities ◽  
Nucleoside Inhibitors

ABSTRACTMuraymycins are antibacterial natural products fromStreptomycesspp. that inhibit translocase I (MraY), which is involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5′-C-glycyluridine (GlyU) appended to a 5″-amino-5″-deoxyribose (ADR), forming a disaccharide core that is found in several peptidyl nucleoside inhibitors of MraY. For muraymycins, the GlyU-ADR disaccharide is further modified with an aminopropyl-linked peptide to generate the simplest structures, annotated as the muraymycin D series. Two enzymes encoded in the muraymycin biosynthetic gene cluster, Mur29 and Mur28, were functionally assignedin vitroas a Mg·ATP-dependent nucleotidyltransferase and a Mg·ATP-dependent phosphotransferase, respectively, both modifying the 3″-OH of the disaccharide. Biochemical characterization revealed that both enzymes can utilize several nucleotide donors as cosubstrates and the acceptor substrate muraymycin also behaves as an inhibitor. Single-substrate kinetic analyses revealed that Mur28 preferentially phosphorylates a synthetic GlyU-ADR disaccharide, a hypothetical biosynthetic precursor of muraymycins, while Mur29 preferentially adenylates the D series of muraymycins. The adenylated or phosphorylated products have significantly reduced (170-fold and 51-fold, respectively) MraY inhibitory activities and reduced antibacterial activities, compared with the respective unmodified muraymycins. The results are consistent with Mur29-catalyzed adenylation and Mur28-catalyzed phosphorylation serving as complementary self-resistance mechanisms, with a distinct temporal order during muraymycin biosynthesis.

scholarly journals A Two-Step Mechanism for the Activation of Actinorhodin Export and Resistance in Streptomyces coelicolor

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ye Xu ◽  
Andrew Willems ◽  
Catherine Au-yeung ◽  
Kapil Tahlan ◽  
Justin R. Nodwell
Keyword(s):  
Secondary Metabolites ◽  
Pathogenic Bacteria ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistance Mechanisms ◽  
Biosynthetic Gene ◽  
Content Type

ABSTRACT Many microorganisms produce secondary metabolites that have antibiotic activity. To avoid self-inhibition, the producing cells often encode cognate export and/or resistance mechanisms in the biosynthetic gene clusters for these molecules. Actinorhodin is a blue-pigmented antibiotic produced by Streptomyces coelicolor. The actAB operon, carried in the actinorhodin biosynthetic gene cluster, encodes two putative export pumps and is regulated by the transcriptional repressor protein ActR. In this work, we show that normal actinorhodin yields require actAB expression. Consistent with previous in vitro work, we show that both actinorhodin and its 3-ring biosynthetic intermediates [e.g., (S)-DNPA] can relieve repression of actAB by ActR in vivo. Importantly, an ActR mutant that interacts productively with (S)-DNPA but not with actinorhodin responds to the actinorhodin biosynthetic pathway with the induction of actAB and normal yields of actinorhodin. This suggests that the intermediates are sufficient to trigger the export genes in actinorhodin-producing cells. We further show that actinorhodin-producing cells can induce actAB expression in nonproducing cells; however, in this case actinorhodin is the most important signal. Finally, while the “intermediate-only” ActR mutant permits sufficient actAB expression for normal actinorhodin yields, this expression is short-lived. Sustained culture-wide expression requires a subsequent actinorhodin-mediated signaling step, and the defect in this response causes widespread cell death. These results are consistent with a two-step model for actinorhodin export and resistance where intermediates trigger initial expression for export from producing cells and actinorhodin then triggers sustained export gene expression that confers culture-wide resistance. IMPORTANCE Understanding the links between antibiotic resistance and biosynthesis is important for our efforts to manipulate secondary metabolism. For example, many secondary metabolites are produced at low levels; our work suggests that manipulating export might be one way to enhance yields of these molecules. It also suggests that understanding resistance will be relevant to the generation of novel secondary metabolites through the creation of synthetic secondary metabolic gene clusters. Finally, these cognate resistance mechanisms are related to mechanisms that arise in pathogenic bacteria, and understanding them is relevant to our ability to control microbial infections clinically.

scholarly journals Biosynthesis of cittilins, unusual ribosomally synthesized and post-translationally modified peptides from Myxococcus xanthus

2020 ◽  
Author(s):  
Joachim J. Hug ◽  
Jan Dastbaz ◽  
Sebastian Adam ◽  
Ole Revermann ◽  
Jesko Koehnke ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Biochemical Characterization ◽  
Biosynthetic Gene Cluster ◽  
Cytochrome P450 Enzyme ◽  
Biosynthetic Gene ◽  
Aryl Ether ◽  
Precursor Peptide ◽  
Carbon Storage Regulator

AbstractCittilins are secondary metabolites from myxobacteria comprised of three L-tyrosines and one L-isoleucine forming a bicyclic tetrapeptide scaffold with biaryl and aryl-oxygen-aryl ether bonds. Here we reveal that cittilins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products, for which only the crocagins have been reported from myxobacteria. A 27 amino acid precursor peptide harbors a C-terminal four amino acid core peptide, which is enzymatically modified and finally exported to yield cittilins. The small biosynthetic gene cluster responsible for cittilin biosynthesis also encodes a cytochrome P450 enzyme and a methyltransferase, whereas a gene encoding a prolyl endopeptidase for the cleavage of the precursor peptide is located outside of the cittilin biosynthetic gene cluster. We confirm the roles of the biosynthetic genes responsible for the formation of cittilins using targeted gene inactivation and heterologous expression in Streptomyces. We also report first steps towards the biochemical characterization of the proposed biosynthetic pathway in vitro. An investigation of the cellular uptake properties of cittilin A connected it to a potential biological function as an inhibitor of the prokaryotic carbon storage regulator A (CsrA).Abstract Figure

scholarly journals Hawaiian Bobtail Squid Symbionts Inhibit Marine Bacteria via Production of Specialized Metabolites, Including New Bromoalterochromides BAC-D/D′

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Andrea M. Suria ◽  
Karen C. Tan ◽  
Allison H. Kerwin ◽  
Lucas Gitzel ◽  
Lydia Abini-Agbomson ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Gene Cluster ◽  
Marine Bacteria ◽  
Pathogenic Fungus ◽  
Biosynthetic Gene Cluster ◽  
No Production ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Egg Defense

ABSTRACT The Hawaiian bobtail squid, Euprymna scolopes, has a symbiotic bacterial consortium in the accessory nidamental gland (ANG), a female reproductive organ that protects eggs against fouling microorganisms. To test the antibacterial activity of ANG community members, 19 bacterial isolates were screened for their ability to inhibit Gram-negative and Gram-positive bacteria, of which two strains were inhibitory. These two antibacterial isolates, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, were subjected to further genomic characterization. Genomic analysis of Leisingera sp. ANG59 revealed a biosynthetic gene cluster encoding the antimicrobial compound indigoidine. The genome of Pseudoalteromonas sp. JC28 had a 14-gene cluster with >95% amino acid identity to a known bromoalterochromide (BAC) cluster. Chemical analysis confirmed production of known BACs, BAC-A/A′ (compounds 1a/1b), as well as two new derivatives, BAC-D/D′ (compounds 2a/2b). Extensive nuclear magnetic resonance (NMR) analyses allowed complete structural elucidation of compounds 2a/2b, and the absolute stereochemistry was unambiguously determined using an optimized Marfey’s method. The BACs were then investigated for in vitro antibacterial, antifungal, and nitric oxide (NO) inhibitory activity. Compounds 1a/1b were active against the marine bacteria Bacillus algicola and Vibrio fischeri, while compounds 2a/2b were active only against B. algicola. Compounds 1a/1b inhibited NO production via lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells and also inhibited the pathogenic fungus Fusarium keratoplasticum, which, coupled with their antibacterial activity, suggests that these polyketide-nonribosomal peptides may be used for squid egg defense against potential pathogens and/or fouling microorganisms. These results indicate that BACs may provide Pseudoalteromonas sp. JC28 an ecological niche, facilitating competition against nonsymbiotic microorganisms in the host’s environment. IMPORTANCE Animals that deposit eggs must protect their embryos from fouling and disease by microorganisms to ensure successful development. Although beneficial bacteria are hypothesized to contribute to egg defense in many organisms, the mechanisms of this protection are only recently being elucidated. Our previous studies of the Hawaiian bobtail squid focused on fungal inhibition by beneficial bacterial symbionts of a female reproductive gland and eggs. Herein, using genomic and chemical analyses, we demonstrate that symbiotic bacteria from this gland can also inhibit other marine bacteria in vitro. One bacterial strain in particular, Pseudoalteromonas sp. JC28, had broad-spectrum abilities to inhibit potential fouling bacteria, in part via production of novel bromoalterochromide metabolites, confirmed via genomic annotation of the associated biosynthetic gene cluster. Our results suggest that these bacterial metabolites may contribute to antimicrobial activity in this association and that such defensive symbioses are underutilized sources for discovering novel antimicrobial compounds.

scholarly journals Characterization of the coformycin biosynthetic gene cluster in Streptomyces kaniharaensis

2020 ◽  
Vol 117 (19) ◽  
pp. 10265-10270
Author(s):  
Daan Ren ◽  
Mark W. Ruszczycky ◽  
Yeonjin Ko ◽  
Shao-An Wang ◽  
Yasushi Ogasawara ◽  
...  
Keyword(s):  
Adenosine Deaminase ◽  
Gene Cluster ◽  
Branch Point ◽  
Biosynthetic Pathway ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Histidine Biosynthesis ◽  
Nucleoside Inhibitors

Coformycin and pentostatin are structurally related N-nucleoside inhibitors of adenosine deaminase characterized by an unusual 1,3-diazepine nucleobase. Herein, the cof gene cluster responsible for coformycin biosynthesis is identified. Reconstitution of the coformycin biosynthetic pathway in vitro demonstrates that it overlaps significantly with the early stages of l-histidine biosynthesis. Committed entry into the coformycin pathway takes place via conversion of a shared branch point intermediate to 8-ketocoformycin-5′-monophosphate catalyzed by CofB, which is a homolog of succinylaminoimidazolecarboxamide ribotide (SAICAR) synthetase. This reaction appears to proceed via a Dieckmann cyclization and a retro-aldol elimination, releasing ammonia and D-erythronate-4-phosphate as coproducts. Completion of coformycin biosynthesis involves reduction and dephosphorylation of the CofB product, with the former reaction being catalyzed by the NADPH-dependent dehydrogenase CofA. CofB also shows activation by adenosine triphosphate (ATP) despite the reaction requiring neither a phosphorylated nor an adenylated intermediate. This may serve to help regulate metabolic partitioning between the l-histidine and coformycin pathways.

scholarly journals Antibiotic Resistance Mechanisms Inform Discovery: Identification and Characterization of a Novel Amycolatopsis Strain Producing Ristocetin

2014 ◽  
Vol 58 (10) ◽  
pp. 5687-5695 ◽  
Author(s):  
Andrew W. Truman ◽  
Min Jung Kwun ◽  
Jinhua Cheng ◽  
Seung Hwan Yang ◽  
Joo-Won Suh ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Natural Product ◽  
Gene Cluster ◽  
Wall Stress ◽  
Biosynthetic Gene Cluster ◽  
Resistance Mechanisms ◽  
Biosynthetic Gene ◽  
Single Strain ◽  
Content Type

ABSTRACTDiscovering new antibiotics is a major scientific challenge, made increasingly urgent by the continued development of resistance in bacterial pathogens. A fundamental understanding of the mechanisms of bacterial antibiotic resistance will be vital for the future discovery or design of new, more effective antibiotics. We have exploited our intimate knowledge of the molecular mechanism of glycopeptide antibiotic resistance in the harmless bacteriumStreptomyces coelicolorto develop a new two-step cell wall bioactivity screen, which efficiently identified a new actinomycete strain containing a previously uncharacterized glycopeptide biosynthetic gene cluster. The screen first identifies natural product extracts capable of triggering a generalized cell wall stress response and then specifically selects for glycopeptide antibacterials by assaying for the induction of glycopeptide resistance genes. In this study, we established a diverse natural product extract library from actinomycete strains isolated from locations with widely varying climates and ecologies, and we screened them using the novel two-step bioassay system. The bioassay ultimately identified a single strain harboring the previously unidentified biosynthetic gene cluster for the glycopeptide ristocetin, providing a proof of principle for the effectiveness of the screen. This is the first report of the ristocetin biosynthetic gene cluster, which is predicted to include some interesting and previously uncharacterized enzymes. By focusing on screening libraries of microbial extracts, this strategy provides the certainty that identified producer strains are competent for growth and biosynthesis of the detected glycopeptide under laboratory conditions.

scholarly journals Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

2013 ◽  
Vol 79 (7) ◽  
pp. 2349-2357 ◽  
Author(s):  
Li Li ◽  
Jun Wu ◽  
Zixin Deng ◽  
T. Mark Zabriskie ◽  
Xinyi He
Keyword(s):  
Gene Cluster ◽  
Genetic Manipulation ◽  
Biosynthetic Gene Cluster ◽  
Streptomyces Lividans ◽  
Dna Uptake ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Blasticidin S

ABSTRACTBlasticidin S is a peptidyl nucleoside antibiotic produced byStreptomyces griseochromogenesthat exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesisin vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome ofStreptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, forS. lividansblasticidin S deaminase) was identified inS. lividansand shown to govern thisin vivoconversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin Sin vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene inS. lividansLL2 led to successful production of active blasticidin S in the resultant mutant,S. lividansWJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster,blsE,blsF, andblsL, encoding a predicted radicalS-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis.

scholarly journals In VitroActivities of Daptomycin-, Vancomycin-, and Teicoplanin-Loaded Polymethylmethacrylate against Methicillin-Susceptible, Methicillin-Resistant, and Vancomycin-Intermediate Strains of Staphylococcus aureus

2011 ◽  
Vol 55 (12) ◽  
pp. 5480-5484 ◽  
Author(s):  
Yuhan Chang ◽  
Wen-Chien Chen ◽  
Pang-Hsin Hsieh ◽  
Dave W. Chen ◽  
Mel S. Lee ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
High Performance ◽  
Low Dose ◽  
Antibacterial Activities ◽  
High Dose ◽  
Bone Cements ◽  
Antibacterial Effects ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACTThe objective of this study was to evaluate the antibacterial effects of polymethylmethacrylate (PMMA) bone cements loaded with daptomycin, vancomycin, and teicoplanin against methicillin-susceptibleStaphylococcus aureus(MSSA), methicillin-resistantStaphylococcus aureus(MRSA), and vancomycin-intermediateStaphylococcus aureus(VISA) strains. Standardized cement specimens made from 40 g PMMA loaded with 1 g (low-dose), 4 g (middle-dose) or 8 g (high-dose) antibiotics were tested for elution characteristics and antibacterial activities. The patterns of release of antibiotics from the cement specimens were evaluated usingin vitrobroth elution assay with high-performance liquid chromatography. The activities of broth elution fluid against differentStaphylococcus aureusstrains (MSSA, MRSA, and VISA) were then determined. The antibacterial activities of all the tested antibiotics were maintained after being mixed with PMMA. The cements loaded with higher dosages of antibiotics showed longer elution periods. Regardless of the antibiotic loading dose, the teicoplanin-loaded cements showed better elution efficacy and provided longer inhibitory periods against MSSA, MRSA, and VISA than cements loaded with the same dose of vancomycin or daptomycin. Regarding the choice of antibiotics for cement loading in the treatment ofStaphylococcus aureusinfection, teicoplanin was superior in terms of antibacterial effects.

scholarly journals In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

2015 ◽  
Vol 59 (4) ◽  
pp. 2113-2121 ◽  
Author(s):  
U. Malik ◽  
O. N. Silva ◽  
I. C. M. Fensterseifer ◽  
L. Y. Chan ◽  
R. J. Clark ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Cyclic Peptide ◽  
Sequence Similarity ◽  
Infection Model ◽  
Content Type ◽  
Wide Range ◽  
Inhibitory Activities ◽  
The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

scholarly journals In VitroEfficacies and Resistance Profiles of Rifampin-Based Combination Regimens for Biofilm-Embedded Methicillin-Resistant Staphylococcus aureus

2013 ◽  
Vol 57 (11) ◽  
pp. 5717-5720 ◽  
Author(s):  
Hung-Jen Tang ◽  
Chi-Chung Chen ◽  
Kuo-Chen Cheng ◽  
Kuan-Ying Wu ◽  
Yi-Chung Lin ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Fusidic Acid ◽  
Antibacterial Activities ◽  
Methicillin Resistant ◽  
Content Type ◽  
Biofilm Model ◽  
Rifampin Resistance ◽  
Combination Regimens ◽  
Resistant Mutation

ABSTRACTTo compare thein vitroantibacterial efficacies and resistance profiles of rifampin-based combinations against methicillin-resistantStaphylococcus aureus(MRSA) in a biofilm model, the antibacterial activities of vancomycin, teicoplanin, daptomycin, minocycline, linezolid, fusidic acid, fosfomycin, and tigecycline alone or in combination with rifampin against biofilm-embedded MRSA were measured. The rifampin-resistant mutation frequencies were evaluated. Of the rifampin-based combinations, rifampin enhances the antibacterial activities of and even synergizes with fusidic acid, tigecycline, and, to a lesser extent, linezolid, fosfomycin, and minocycline against biofilm-embedded MRSA. Such combinations with weaker rifampin resistance induction activities may provide a therapeutic advantage in MRSA biofilm-related infections.

scholarly journals Identification and Predictions Regarding the Biosynthesis Pathway of Polyene Macrolides Produced by Streptomyces roseoflavus Men-myco-93-63

2021 ◽  
Vol 87 (10) ◽  
Author(s):  
Xing Han ◽  
Jiao Wang ◽  
Lianna Liu ◽  
Fengying Shen ◽  
Qingfang Meng ◽  
...  
Keyword(s):  
Fermentation Broth ◽  
Pathogenic Fungi ◽  
Biosynthetic Gene Cluster ◽  
Inhibitory Effect ◽  
Plant Diseases ◽  
Active Metabolites ◽  
Content Type ◽  
Polyene Macrolides ◽  
Alternative Agent

ABSTRACT A group of polyene macrolides mainly composed of two constituents was isolated from the fermentation broth of Streptomyces roseoflavus Men-myco-93-63, which was isolated from soil where potato scabs were repressed naturally. One of these macrolides was roflamycoin, which was first reported in 1968, and the other was a novel compound named Men-myco-A, which had one methylene unit more than roflamycoin. Together, they were designated RM. This group of antibiotics exhibited broad-spectrum antifungal activities in vitro against 17 plant-pathogenic fungi, with 50% effective concentrations (EC50) of 2.05 to 7.09 μg/ml and 90% effective concentrations (EC90) of 4.32 to 54.45 μg/ml, which indicates their potential use in plant disease control. Furthermore, their biosynthetic gene cluster was identified, and the associated biosynthetic assembly line was proposed based on a module and domain analysis of polyketide synthases (PKSs), supported by findings from gene inactivation experiments. IMPORTANCE Streptomyces roseoflavus Men-myco-93-63 is a biocontrol strain that has been studied in our laboratory for many years and exhibits a good inhibitory effect in many crop diseases. Therefore, the identification of antimicrobial metabolites is necessary and our main objective. In this work, chemical, bioinformatic, and molecular biological methods were combined to identify the structures and biosynthesis of the active metabolites. This work provides a new alternative agent for the biological control of plant diseases and is helpful for improving both the properties and yield of the antibiotics via genetic engineering.

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