Woychik, N. A. | bioRxiv Science

bioRxiv2026

Application of the ORBIT Protocol for Targeted Gene Deletion to Mycobacterium abscessus

Mycobacterium abscessus (Mab) is an emerging opportunistic pathogen characterized by extensive intrinsic drug resistance, underscoring the need for precise genetic tools to define mechanisms of pathogenicity and persistence. The ORBIT (oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting) system developed by the Sassetti group enables precise gene deletions but has primarily been optimized for Mycobacterium tuberculosis and Mycobacterium smegmatis. Although ORBIT has been used in Mab, here we describe an optimized ORBIT workflow for efficient gene deletion in Mab. We tested key parameters, including plasmid selection, oligonucleotide design, and recombineering conditions. We evaluated oligonucleotide length and purification to improve recovery of targeted mutants and established conditions that enabled reproducible deletion of multiple target genes. Our findings establish a robust and adaptable ORBIT protocol tailored to the genetic characteristics of Mab. This optimized workflow provides a practical framework for precise genetic manipulation and can facilitate functional studies of genes that contribute to Mab physiology, antimicrobial resistance, and pathogenesis.

microbiology↗

bioRxiv2026

Two potent, functionally redundant MazF toxins inactivate the 16S rRNA anti-Shine-Dalgarno sequence and are differentially expressed in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) remains a major global health threat, sustained by lengthy, multidrug treatment regimens that promote poor compliance and antibiotic resistance. The pathogen's ability to evade clearance is facilitated in part by a transition to a nonreplicating, persistent state, a process thought to involve the abundant type II toxin-antitoxin (TA) systems. The Mtb chromosome encodes 11 MazEF TA systems whose MazF toxins are single-strand, sequence-specific endoribonucleases yet the precise RNA targets and physiological consequences of most remain unknown. Here we applied a specialized 5'-end RNA-seq approach to pinpoint the Mtb MazF-mt1 toxin recognition sequence and RNA target. MazF-mt1 specifically cleaves at U/CCUU within the anti-Shine-Dalgarno (aSD) sequence, releasing it from the 3' end of the 16S rRNA. Notably, the recently described MazF-mt11 toxin also targets the aSD for removal, cleaving at a different sequence 4 nts upstream of the MazF-mt1 site. Consistent with the essential role of the aSD in translation, both toxins rapidly inhibit new protein synthesis. This functional redundancy prompted us to mine Mtb transcriptomic datasets to compare how the two toxins respond to infection-relevant stresses. MazF-mt1 and MazF-mt11 are differentially regulated following exposure to diverse antibiotic and host-imposed stresses, with distinct temporal expression patterns. Thus, Mtb may coordinate transcription of these functionally redundant toxins to more precisely control the establishment and maintenance of nonreplicating persistence, thereby confounding pathogen clearance.

microbiology↗