Bacterial Strains and Plasmids
The bacterial strains and plasmids utilized in this study can be found in Supplementary Table 7.
Screening of WAC Library for Antimicrobial Activity
The pre-fractionation library from the WAC6 was tested against a hyperpermeable, efflux-deficient strain of E. coli BW25113 ΔtolCΔbamB, using 384-well microtitre plates (Corning 3701). Each well contained 49 μl of inoculated Mueller–Hinton broth (MHB) and 1 μl of crude methanolic extract, fractions, or conditioned medium. A Biomek FXP Integrated Liquid Handler was employed to dispense the solutions into the plates, which were then incubated at 37 °C for 20 hours. The growth of cells was quantified by measuring optical density at 600 nm, using microtitre plate readers such as EnVision, SpectraMax, or Biotek Neo.
Purification of MKMs
S. rimosus WAC 7405 was typically cultured in tryptic soy broth (TSB) for 16 hours, then inoculated into ASM medium58 for 4 days at 30 °C with shaking. The active compound was identified from conditioned medium combined with 5% Diaion HP-20 resin, mixed for 2.5 hours, filtered through a milk filter, and extracted with methanol. The extract was then dried and reconstituted for further separation using a Sephadex LH-20 column. Active fractions were analyzed via LC–MS/MS; mass ranges were set from 150 to 2,500 m/z with a scan rate of 1 spectrum per second. A gradient method with H2O and acetonitrile on a specific column was used, yielding various active fractions.
The later phases of purification were refined with seed cultures grown for 2 days before being subjected to HP-20 extraction, followed by cation-exchange chromatography. The details of the optimization processes, such as buffer adjustments and elution conditions, were carefully controlled. Each analog of MKM was resolved on distinct columns, and their purity was confirmed to be over 95%.
Structural Characterization of MKMs
High-resolution mass spectra were collected using an Agilent UPLC and mass detector assembly. For NMR, the compounds were dissolved in deuterated water. Chemical shifts were reported with specific notations outlining coupling constants and peak patterns. The analysis included treating MKM-A with HCl and conducting reactions using Marfey’s reagent to ascertain configurations of critical amino acids involved.
Using the predicted structure from NMR and HRMS/MS data, specific amino acids were modified for further analysis. A combination of methods including LC–MS and chromatographic techniques were employed to clarify the configuration of the amino acids, confirming their identities.
Whole-Genome Sequencing and BGC Analysis
Genomic DNA extraction and sequencing were performed with protocols outlined61. The sequencing utilized kits and tools that were carefully selected for quality. The software for analysis was also chosen to ensure accuracy in trimming and merging reads.
Heterologous Expression of MKM
The MKM biosynthetic gene cluster (BGC) was captured through a specialized cloning method. Precise boundary sequences were selected for target genes, and linearized plasmids were transformed into yeast cells to capture MKM variants. These clones were subsequently screened and transferred into E. coli for further validation.
Overexpression of ManE rRNA Methyltransferase
The ManE gene was amplified and appropriately cloned into a designated plasmid. This plasmid was transformed into particular E. coli strains to analyze its effects on MKM susceptibility.
MIC Determination
The minimum inhibitory concentration (MIC) was assessed through broth microdilution methods across various media types, while specific conditions were employed for distinct bacterial strains. The effectiveness of MKM against various mycobacterial strains was also evaluated under controlled conditions.
Testing Cytotoxicity of MKM
For cytotoxic assessments, mammalian cell cultures were set up, and exposure to MKM compounds was analyzed with specific reagents that measured viability based on luminescence. Control conditions were established to ensure reliability in results.
Haemolysis Assay
Human red blood cells were separated and washed to establish a reliable testing medium. Various concentrations of the compounds were tested in comparison to controls, utilizing a serial dilution strategy to properly quantify results.
Time-Dependent Killing Assay
E. coli BW25113 and K. pneumoniae were utilized to monitor the effects of MKM over designated time intervals, with samples tested at various time points to track bacterial viability.
Propidium Iodide Uptake Assay
Cells in their mid-exponential phase were treated with propidium iodide for permeability testing. The effects of different compounds were examined at varying concentrations, with fluorescence readings taken consistently.
Assessing Outer Membrane Permeability by NPN Assay
The experimental setup involved growing cells and assessing the permeability in response to varying compounds. Sequential measurements provided insights into the dynamics of outer membrane changes.
Scanning Electron Microscopy
E. coli cells were subjected to MKM treatments to investigate structural changes via electron microscopy, confirming methods that facilitated the visualization of impacts at a cellular level.
Resistance Studies
Experiments aimed at developing resistance through sustained exposure to MKM were conducted. The analysis involved genomic studies on resistant mutants to identify genetic changes.
In Vitro Transcription–Translation Assay
The effects of MKM-A on protein synthesis were analyzed through an in vitro system. Concentrations were tested to determine the inhibitory effects on translation, revealing essential insights into its function.
Ribosome Profiling
Ribo-seq experiments tracked translation in response to MKM treatment, providing crucial data on ribosomal function and interactions under experimental conditions.
Statistics and Reproducibility
Statistical analyses were conducted to determine significance, utilizing proper methods to ensure validity and reliability in the results obtained during the research.
Pharmacokinetic Studies in Mice
Mouse studies were intricately designed to maintain controlled conditions throughout the process. Blood samples were carefully collected and analyzed to assess pharmacokinetics post MKM administration.
Identification of ManE-Containing BGCs
Protein sequences were utilized to map homologues across various strains, employing bioinformatics tools to analyze and compare genomic structures.
