From accurate genome sequence to biotechnological application: The thermophile Mycolicibacterium hassiacum as experimental model

• Published in: Microbial biotechnology Vol. 17; no. 1; pp. e14290 - n/a
• Main Authors: Sánchez‐Costa, Mercedes, Gola, Susanne, Rodríguez‐Sáiz, Marta, Barredo, José‐Luis, Hidalgo, Aurelio, Berenguer, José
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01.01.2024; John Wiley and Sons Inc; Wiley
• Subjects: Antibiotics; Bacteria - genetics; Biotechnology; Carbon; Chemical synthesis; CRISPR; Gene transfer; Genetic transformation; Genomes; Glycerol; High temperature; Insertion sequences; Microorganisms; Microscopy; Mycobacteriaceae; Nucleotide sequence; Pathogenicity; Pathogens; Phytosterols; Plasmids; Steroids; Sterols; Tuberculosis
• ISSN: 1751-7915; 1751-7915
• DOI: 10.1111/1751-7915.14290

Mycobacteria constitute a large group of microorganisms belonging to the phylum Actinobacteria encompassing some of the most relevant pathogenic bacteria and many saprophytic isolates that share a unique and complex cell envelope. Also unique to this group is the extensive capability to use and synthesize sterols, a class of molecules that include active signalling compounds of pharmaceutical use. However, few mycobacterial species and strains have been established as laboratory models to date, Mycolicibacterium smegmatis mc2155 being the most common one. In this work, we focus on the use of a thermophilic mycobacterium, Mycolicibacterium hassiacum, which grows optimally above 50°C, as an emerging experimental model valid to extend our general knowledge of mycobacterial biology as well as for application purposes. To that end, accurate genomic sequences are key for gene mining, the study of pathogenicity or lack thereof and the potential for gene transfer. The combination of long‐ and short‐massive sequencing technologies is strictly necessary to remove biases caused by errors specific to long‐reads technology. By doing so in M. hassiacum, we obtained from the curated genome clues regarding the genetic manipulation potential of this microorganism from the presence of insertion sequences, CRISPR‐Cas, type VII ESX secretion systems, as well as lack of plasmids. Finally, as a proof of concept of the applicability of M. hassiacum as a laboratory and industrial model, we used this high‐quality genome of M. hassiacum to successfully knockout a gene involved in the use of phytosterols as source of carbon and energy, using an improved gene cassette for thermostable selection and a transformation protocol at high temperature. Mycolicibacterium hassiacum, the most thermophilic mycobacterium so far studied, is able to use phytosterols as carbon source. A curated version of the genome combining long and short read sequencing technologies was used to identify genes involved in sterols metabolism. The first marker for thermostable selection in thermophilic mycobacteria has been developed and used for the directed isolation of mutants defective in phytosterols degradation.