Bo Li

Peptides that contain 5-thiooxazole post-translational modifications have recently been identified from various bacteria and implicated in copper homeostasis and virulence. However, knowledge of their copper-binding affinities is limited, and their roles in virulence are not well understood. In this work, we discovered three new 5-thiooxazole-containing peptides from two opportunistic human pathogens, Pseudomonas aeruginosa and Bordetella bronchiseptica. We heterologously produced and structurally characterized two different peptides that originate from P. aeruginosa, pseudomonin and pseudomonin-efh, the latter of which also contains two EF-hand domains for calcium binding. We show that 5-thiooxazole modification in both peptides enables strong Cu(I) binding at 10–16–10–17 M affinity and weaker Cu(II) binding at 10–7 M affinity. Pseudomonin-efh undergoes calcium-dependent changes of overall secondary structure that are negated by copper. Genetic studies revealed the 5-thiooxazole modification of pseudomonin-efh is required for calcium-dependent virulence regulation in a lettuce infection model for P. aeruginosa. Further, we identified and characterized a related 5-thiooxazole-containing peptide, bordetellin, secreted by its native producer, B. bronchiseptica, in the virulent phase. Our work demonstrates a strong preference for Cu(I) binding by 5-thiooxazole-containing peptides and uncovers a role of these peptides in Ca/Cu-dependent virulence regulation. Identifying 5-thiooxazole-containing peptides from pathogenic bacteria and detecting their secretion in the native producer is a critical step toward understanding the functions of these peptides in virulence and pathogen–host interactions.

Pathogenic bacteria interfere with plant metabolism through specialized metabolites and mechanisms. Mangotoxin was identified in 2003 from Pseudomonas syringae pv. syringae , which causes devastating diseases globally in various crops, including mango. Mangotoxin was found to inhibit arginine biosynthesis in tomato and Escherichia coli ; however, its chemical structure remained unknown. We determined the structure of mangotoxin from the native bacterial producer P. syringae to be an alanine‐citrulline dipeptide that contains an internal alkyne on citrulline. We showed that mangotoxin also inhibits the growth of a multidrug‐resistant human pathogen Klebsiella pneumoniae strain. In vitro characterization of mangotoxin biosynthesis revealed that alkyne formation is catalyzed by a heme oxygenase‐like domain‐containing oxidase with a redox partner on a tripeptide intermediate. We discovered that the mangotoxin biosynthetic enzymes can couple various canonical and noncanonical amino acids into tripeptides and install two‐ or four‐electron oxidations on several tripeptides, providing a foundation for bioengineering of the pathway. Our work answered a two‐decade‐old question on the structure of an elusive toxin and will facilitate the elucidation of the molecular mechanism used by mangotoxin to inhibit amino acid metabolism in plants and bacteria.

Pathogenic bacteria interfere with plant metabolism through specialized metabolites and mechanisms. Mangotoxin was identified in 2003 from Pseudomonas syringae pv. syringae , which causes devastating diseases globally in various crops, including mango. Mangotoxin was found to inhibit arginine biosynthesis in tomato and Escherichia coli ; however, its chemical structure remained unknown. We determined the structure of mangotoxin from the native bacterial producer P. syringae to be an alanine‐citrulline dipeptide that contains an internal alkyne on citrulline. We showed that mangotoxin also inhibits the growth of a multidrug‐resistant human pathogen Klebsiella pneumoniae strain. In vitro characterization of mangotoxin biosynthesis revealed that alkyne formation is catalyzed by a heme oxygenase‐like domain‐containing oxidase with a redox partner on a tripeptide intermediate. We discovered that the mangotoxin biosynthetic enzymes can couple various canonical and noncanonical amino acids into tripeptides and install two‐ or four‐electron oxidations on several tripeptides, providing a foundation for bioengineering of the pathway. Our work answered a two‐decade‐old question on the structure of an elusive toxin and will facilitate the elucidation of the molecular mechanism used by mangotoxin to inhibit amino acid metabolism in plants and bacteria.

Pseudomonas virulence factor (pvf) produces an autoinducing small-molecule signal that regulates bacterial cell-to-cell communication and virulence. While genes like pvf have been linked to the production of small molecules containing a diazeniumdiolate group, the specific chemical signal produced by pvf had not been identified. In this study, we reveal that (S)-valdiazen is the autoinducer produced by pvf in Pseudomonas entomophila, a model for pathogen-host interactions. The (S)-configuration is crucial for the signaling activity of valdiazen at physiological concentrations. We also define the (S)-stereochemistry of leudiazen, a similar signal produced by the plant pathogen Pseudomonas syringae. Using pvf genes needed for (S)-valdiazen signaling and production in P. entomophila, we bioinformatically identified 5383 bacterial organisms that may produce diazeniumdiolate signals. Signaling activity of valdiazen can be quenched by potassium permanganate, which oxidizes the diazeniumdiolate moiety. Identification of (S)-diazeniumdiolates from two bacterial species suggests stereospecific biosynthesis and transduction of these signals. Our findings set the stage for discovering diazeniumdiolate signals from other bacteria.

Dithiolopyrrolone (DTP) natural products are broad-spectrum antimicrobial and anticancer prodrugs. The DTP structure contains a unique bicyclic ene-disulfide that once reduced in the cell, chelates metal ions and disrupts metal homeostasis. In this work we investigate the intracellular activation of the DTPs and their resistance mechanisms in bacteria. We show that the prototypical DTP holomycin is reduced by several bacterial reductases and small-molecule thiols in vitro. To understand how bacteria develop resistance to the DTPs, we generate Staphylococcus aureus mutants that exhibit increased resistance to the hybrid DTP antibiotic thiomarinol. From these mutants we identify loss-of-function mutations in redox genes that are involved in DTP activation. This work advances the understanding of how DTPs are activated and informs development of bioreductive disulfide prodrugs.

Pseudomonas syringae infects a wide variety of crops. The mangotoxin-generating operon ( mgo) is conserved across many P. syringae strains and is responsible for producing an extracellular chemical signal, leudiazen. Disruption of the mgoA gene in P. syringae pv. syringae ( Pss) UMAF0158 alleviated tomato chlorosis caused by this bacterium. We showed that deletion of the entire mgo reduced Pss UMAF0158 population in tomato leaflets. Leudiazen restored the signaling activity of the deletion mutant at a concentration as low as 10 nM. Both the diazeniumdiolate and isobutyl groups of leudiazen are critical for this potent signaling activity. Transcriptional analysis showed that mgo and leudiazen induce the expression of mangotoxin biosynthetic operon as well as an uncharacterized gene cluster, RS17235–RS17245. We found that this cluster enhances the survival of Pss UMAF0158 in planta and is widely distributed in P. syringae strains. Our results demonstrate that mgo plays prominent roles in the virulence and growth of P. syringae. The mgo and mgo-like signaling systems in different bacteria likely regulate diverse microbe-host interactions. [ ] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Heme oxygenase-like domain-containing oxidases (HDOs) are a rapidly expanding enzyme family that typically use dinuclear metal cofactors instead of heme. FlcD, an HDO from the opportunistic pathogen Pseudomonas aeruginosa, catalyzes the excision of an oxime carbon in the biosynthesis of the copper-containing antibiotic fluopsin C. We show that FlcD is a dioxygenase that catalyzes a four-electron oxidation. Crystal structures of FlcD reveal a mononuclear iron in the active site, which is coordinated by two histidines, one glutamate, and the oxime of the substrate. Enzyme activity, Mössbauer spectroscopy, and electron paramagnetic resonance spectroscopy analyses support the usage of a mononuclear iron cofactor. This cofactor resembles that of mononuclear non-heme iron-dependent enzymes and breaks the paradigm of dinuclear HDO cofactors. This study begins to illuminate the catalytic mechanism of methine excision and indicates convergent evolution of different lineages of mononuclear iron-dependent enzymes.

Combination therapy is an effective strategy to combat antibiotic resistance. Multiple synergistic antimicrobial combinations are produced by enzymes encoded in biosynthetic gene clusters (BGCs) that co-localize on the bacterial genome. This phenomenon led to the hypothesis that mining co-localized BGCs will reveal new synergistic combinations of natural products. Here, we bioinformatically identified 38 pairs of co-localized BGCs, which we predict to produce natural products that are related to known compounds, including polycyclic tetramate macrolactams (PoTeMs). We further showed that ikarugamycin, a PoTeM, increases the membrane permeability of Acinetobacter baumannii and Staphylococcus aureus, which suggests that ikarugamycin might be an adjuvant that facilitates the entry of other natural products. Our work outlines a promising avenue to discover synergistic combinations of natural products by mining bacterial genomes.

D-Allulose has become a promising alternative sweetener due to its unique properties of low caloric content, moderate sweetness, and physiological effects. D-Allulose 3-epimerase (DAEase) is a promising enzyme for D-Allulose production. However, the low catalytic efficiency limited its large-scale industrial applications. To obtain a more effective biocatalyst, a putative DAEase from Christensenellaceae bacterium (CbDAE) was identified and characterized. The recombinant CbDAE exhibited optimum activity at pH 7.5°C and 55°C, retaining more than 60% relative activity from 40°C to 70°C, and the catalytic activity could be significantly increased by Co²⁺ supplementation. These enzymatic properties of purified CbDAE were compared with other DAEases. CbDAE was also found to possess desirable thermal stability at 55°C with a half-life of 12.4 h. CbDAE performed the highest relative activity towards D-allulose and strong affinity for D-fructose but relatively low catalytic efficiency towards D-fructose. Based on the structure-guided design, the best double-mutation variant G36N/W112E was obtained which reached up to 4.21-fold enhancement of catalytic activity compared with wild-type (WT) CbDAE. The catalytic production of G36N/W112E with 500 g/L D-fructose was at a medium to a higher level among the DAEases in 3.5 h, reducing 40% catalytic reaction time compared to the WT CbDAE. In addition, the G36N/W112E variant was also applied in honey and apple juice for D-allulose conversion. Our research offers an extra biocatalyst for D-allulose production, and the comprehensive report of this enzyme makes it potentially interesting for industrial applications and will aid the development of industrial biocatalysts for D-allulose.

An impermeable outer membrane and multidrug efflux pumps work in concert to provide Gram-negative bacteria with intrinsic resistance against many antibiotics. These resistance mechanisms reduce the intracellular concentrations of antibiotics and render them ineffective. The natural product thiomarinol A combines holothin, a dithiolopyrrolone antibiotic, with marinolic acid A, a close analogue of mupirocin. The hybridity of thiomarinol A converts the mupirocin scaffold from inhibiting Gram-positive bacteria to inhibiting both Gram-positive and -negative bacteria. We found that thiomarinol A accumulates significantly more than mupirocin within the Gram-negative bacterium Escherichia coli, likely contributing to its broad-spectrum activity. Antibiotic susceptibility testing of E. coli mutants reveals that thiomarinol A overcomes the intrinsic resistance mechanisms that render mupirocin inactive. Structure-activity relationship studies suggest that the dithiolopyrrolone is a privileged moiety for improving the accumulation and antibiotic activity of the mupirocin scaffold without compromising binding to isoleucyl-tRNA synthetase. These studies also highlight that accumulation is required but not sufficient for antibiotic activity. Our work reveals a role of the dithiolopyrrolone moiety in overcoming intrinsic mupirocin resistance in E. coli and provides a starting point for designing dual-acting and high-accumulating hybrid antibiotics.