DOI: 10.5281/zenodo.17780843
title: Aerobic catabolism of isobutylene and diethyl ether by Mycolicibacterium sp. ELW1 requires plasmid-borne ibc genes

description: File: 20251029_PC1545-Miller_Results_UNC_proteinID.xlsx

LC-MS/MS identification of fluorescently-labeled proteins; respective bands excised from Coomassie stained gel. Band designations: Band 01 (sample 1), Band 02 (sample 2), Band 03 (sample 3), Band 04 (sample 4), Band 05 (sample 5). 

Batch cultures of Mycolicibacterium sp. ELW1 were grown on isobutylene (IB) (10%, v/v, gas phase) and diethyl ether (DEE) (5 mM final concentration) in MSM media until late log phase (in darked out shaking incubator; 150 RPM; 30C). After centrifugation and washing steps, we split the cultures into two aliquots. On one aliquot we treated the cells with 1,5-hexadiyne (15HD) and 1,6-hexadiyne (16HD) for 1 hr, for IB-grown and DEE-grown cells respectively, while the other aliquot was not subjected to probing or fluorescent labeling. Treated cells were then washed and subjected to fluorescent labeling as previously described (https://doi.org/10.1007/978-1-0716-4502-4_16). Both aliquots were run on 10% Mini-PROTEAN® TGX™ Precast Gels (BIO RAD, USA) for SDS-PAGE, being first visualized by fluorescent NIR and then stained with Coomassie Brilliant Blue R-250. 

We submitted the Coomassie stained gel to the UNC Metabolomics and Proteomics (MAP) Core for band excision, in-gel tryptic digestion, and protein identification and analysis. The samples were analyzed by LC-MS/MS using a Thermo UltiMate 3000-Exploris 480 and a 90-minute Data Dependent Acquisition method. Data were searched in Proteome Discoverer (Thermo Scientific; v3.1) against the Mycobacterium sp. ELW1 reference proteome from Uniprot (https://www.uniprot.org/proteomes/UP000324606) (5,850 sequences) and a common contaminants database (245 sequences). Protein results were filtered for 1% FDR on the protein and peptide level and a minimum of 2 peptides identified.

File: JJoyce_NCSU_UNC_Peptide_ID_20251009.jpg

Image of Coomassie stained gel used for protein ID. Band designations: Band 01 (sample 1), Band 02 (sample 2), Band 03 (sample 3), Band 04 (sample 4), Band 05 (sample 5). 

File: JJoyce_NCSU_UNC_NIR_Peptide_ID_20251009.jpg

NIR image of gel used for protein ID. Band designations: Band 01 (sample 1), Band 02 (sample 2), Band 03 (sample 3), Band 04 (sample 4), Band 05 (sample 5). 

 
description: Isobutylene (2-methylpropene; IB) is the simplest branched alkene and is used industrially to produce a wide range of products ranging from fuel oxygenates to automobile tires. Only members of the Mycobacteriaceae family, including Mycolicibacterium sp. ELW1, are known to aerobically catabolize IB as a sole carbon and energy source. A multi-omics approach established two gene clusters on a large catabolic plasmid, pELW1-1, carried by Mycolicibacterium sp. ELW1. These two clusters separately contain the predicted genes necessary for the catabolism of IB to 2-hydroxyisobutyrate (2-HIBA) (cluster 1) and the biosynthesis of cobalamin and the further cobalamin-dependent transformation of 2-HIBA to acetyl-CoA (cluster 2). Curing strain ELW1 of pELW1-1 resulted in the loss of the ability of ELW1 to grow on IB and downstream metabolites including isobutylene oxide (IBO), 2-methyl-1,2-propanediol (MPD), and 2-hydroxyisobutyric acid (2-HIBA). Activity-based labeling (ABL), growth, and activity assays, combined with transcriptomic and proteomic analyses, confirmed the large catabolic plasmid is necessary for IB catabolism and that the plasmid-borne ibc genes are strongly induced during growth on IB. Diethyl ether (DEE) catabolism by ELW1 appears to induced the same plasmid-borne genes to initially oxidize DEE.