Friend or Foe: Exploring the Biological Function of the Mycobacterium Tuberculosis RecA Intein

Author

Ryan Schneider, University at Albany, State University of New YorkFollow

ORCID

https://orcid.org/0009-0004-1169-3348

Date of Award

Winter 2025

Language

English

Embargo Period

1-14-2027

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Program

Biomedical Sciences

First Advisor

Kathleen McDonough

Committee Members

Joseph Wade, Guangchun Bai, Paul Masters, Janice Pata

Keywords

intein, exaptation, tuberculosis, RecA, gene regulation, SOS response

Subject Categories

Microbial Physiology | Microbiology | Molecular Biology | Pathogenic Microbiology

Abstract

Mycobacterium tuberculosis (Mtb) is the leading source of death due to a single infectious agent, and increasingly drug resistant strains threaten to quash progress in battling tuberculosis (TB) disease. Three of Mtb’s genes, the iron-sulfur cluster assembly protein sufB, the DNA gyrase dnaB and the recombinase recA, have been invaded by an intervening protein, or intein. Inteins are translated in frame with their host gene. The resulting precursor consists of the intein flanked by each half of the invaded protein, termed the N- and C-exteins (NE and CE). This hybrid protein is non-functional until the intein splices itself out, with concomitant ligation of the NE and CE to produce mature host protein, also called ligated exteins (LE).

Splicing is coordinated by two catalytic residues: the first residue of the intein, which can be a cysteine or serine, and the first residue of the CE, which can be a cysteine, serine or threonine. In addition to productive splicing, off-pathway cleavage products can occur when the NE or CE are liberated (also called N-terminal or C-terminal cleavage: NTC and CTC respectively). Inteins were thought to be mobile genetic parasites that provided no benefit to the host, but recent evidence suggest that this paradigm is untrue for some inteins. Rather, they act as important post-translational regulators of their host proteins, generally by splicing in response to specific environments such as pH, temperature, or DNA damage.

The RecA protein is an important hub for two DNA repair pathways: 1) as a regulator for the SOS DNA repair response, which has been implicated in generation of drug resistance, and 2) as the driver of homologous recombination, the high-fidelity double-strand break repair pathway. Mtb RecA is provided with a unique opportunity for post-translational regulation through the intein, which is specific to Mtb complex mycobacteria. Despite being the first bacterial intein discovered and well-studied in vitro using non-native expression systems, the role the Mtb RecA intein plays in Mtb biology is unknown.

I leveraged the non-essentiality of Mtb’s recA to study the impact of the recA intein on regulation and production of RecA protein in Mtb. I followed RecA species production in various conditions (LE, intein and NE) via western blot analysis, and recA transcription via a green fluorescent protein-based recA transcriptional reporter. DNA damaging therapeutics mitomycin C and ofloxacin increased the amount of recA transcription, RecA splicing and NE production. Non-DNA damaging therapeutics isoniazid and rifampicin did not increase transcription, splicing or NTC.

Treatment of cells grown in hypoxia impacted RecA splicing and NTC. Copper, a heavy metal used as an antimicrobial by macrophages, had no effect on RecA in ambient air conditions, but increased splicing and decreased NTC in hypoxia. Cisplatin, a chemotherapeutic that has been suggested as a potential splicing inhibitor and anti-Mtb therapeutic, had no effect on RecA in ambient air, but increased splicing in hypoxic conditions.

This is the first report of NTC of a WT intein in its native environment and notably, NTC was affected by various conditions. I explored if NTC was biologically important. The NE retains all features needed to activate the SOS response: the ssDNA binding motifs, ATP binding motifs, interfaces for multimerization with other RecA monomers, and the key residues for activating cleavage of the SOS transcriptional repressor, LexA. By using western blot analysis, I found that the NE retained LexA-activating capability when heterologously expressed in E. coli.

Interestingly, unspliced RecA precursor was not detected in Mtb under any environment tested, including those, such as copper and cisplatin, that inhibit RecA splicing in vitro. This runs counter to the initial paradigm that post-translational regulation by an intein would involve producing a pool of precursor protein, which could then have splicing triggered by specific environmental signals. RecA precursor was only detectable in Mtb when one or both catalytic cysteine residues were mutated, suggesting rapid splicing of the WT RecA intein. Rapid splicing was specific to the Mtb RecA intein, as splicing of a second Mycobacterial RecA intein, intein B, did not occur when expressed in Mtb. Rapid splicing of intein B could be induced by moving intein B to the position of the Mtb RecA intein.

I also provide preliminary data on recA regulation in hypoxic air conditions and non-replicating persister populations, and how RecA may interact with the NucS mismatch repair system. These results provide the first evidence of how the Mtb RecA intein impacts regulation of RecA, and that regulation of the Mtb RecA intein in Mtb proper differs from previous in vitro results.

License

This work is licensed under the University at Albany Standard Author Agreement.

Recommended Citation

Schneider, Ryan, "Friend or Foe: Exploring the Biological Function of the Mycobacterium Tuberculosis RecA Intein" (2025). Electronic Theses & Dissertations (2024 - present). 104.
https://scholarsarchive.library.albany.edu/etd/104