*4.7. Molecular Networking*

The MS/MS data were converted from raw to mzXML file format using Proteowizard MSConvert [83] and the data were uploaded to the GNPS server [23]. A molecular network was created with the feature-based molecular networking workflow on the GNPS website ( http://gnps.ucsd.edu (accessed on 9 November 2020)). The data were filtered by removing

all MS/MS fragment ions within +/−17 Da of the precursor *m*/*z*. MS/MS spectra were window filtered by choosing only the top 6 fragment ions in the +/−150 Da window throughout the spectrum. The precursor ion mass tolerance was set to 0.2 Da and a MS/MS fragment ion tolerance of 0.2 Da. A network was then created where edges were filtered to have a cosine score above 0.6 and at least 1 matched peak. Further, edges between two nodes were kept in the network if and only if each of the nodes appeared in each other's respective top 10 most similar nodes. Finally, the maximum size of a molecular family was set to 100, and the lowest scoring edges were removed from molecular families until the molecular family size was below this threshold. The spectra in the network were then searched against GNPS' spectral libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.6 and at least 1 matched peak (https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=124de327f32f47429 1a5037f41ac991d (accessed on 9 November 2020)). For molecular network visualisation, Cytoscape version 3.6.1 was utilised [84] where each node corresponds to a consensus spectrum and each edge represents a modified cosine similarity score between nodes. The data used for the molecular networking analysis were deposited in the MassIVE Public GNPS database under access number MSV000086584.

#### *4.8. MolNetEnhancer Workflow Description for Chemical Class Annotation of Molecular Networks*

To enhance chemical structural information within the molecular network, information from in silico structure annotations from GNPS Library Search and Network Annotation Propagation (NAP) were incorporated into the network using the GNPS MolNetEnhancer workflow (https://ccms-ucsd.github.io/GNPSDocumentation/molnetenhancer/ (accessed on 9 November 2020)) on the GNPS website [31]. Chemical class annotations were performed using the ClassyFire chemical ontology [85]. (https://gnps.ucsd.edu/ ProteoSAFe/status.jsp?task=adbadc0707e7449dbe4de1562ecd7bd3 (accessed on 9 November 2020)).

#### *4.9. Genomic DNA Extraction*

All 25 Polar strains were cultured in ISP2 medium (5 mL, 30 ◦C, 200 rpm for 3 days). High quality genomic DNA was isolated using an in-house protocol based on chemical cell lysis followed by phenol/chloroform extraction [86]. The *Pseudonocardia* sp. strains underwent cell lysis by vigorous vortexing (10 min) of the bacterial cultures with zirconium oxide beads (~0.5 g) (Sigma-Aldrich Ltd., Dorset, UK). The purity and concentration of the obtained genomic DNA was determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) followed by measurements on Qubit 2.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).
