Biology subjects

Batko, I. Z.

Publications and source records attributed to Batko, I. Z..

3 recordsLinked to original sources

bioRxiv2025

Genetic entanglement enables ultra-stable biocontainment in the mammalian gut

Imbalances in the mammalian gut are associated with acute and chronic conditions, and using engineered probiotic strains to deliver synthetic constructs to treat them is a promising strategy. However, high rates of mutational escape and genetic instability in vivo limit the effectiveness of biocontainment circuits needed for safe and effective use. Here, we describe STALEMATE (Sequence enTAngLEd Multi lAyered geneTic buffEring), a dual-layered failsafe biocontainment strategy that entangles genetic sequences to create pseudo-essentiality and buffer against mutations. We entangled the colicin E9 immunity protein (Im9) with a thermoregulated meganu-clease (TSM) by overlapping the reading frames. Mutations that disrupted this entanglement simultaneously inactivated both biocontainment layers, leading to cell death by the ColE9 nuclease and the elimination of escape mutants. By lengthening the entangled region, refining ColE9 expression, and optimizing the TSM sequence against IS911 insertion, we achieved escape rates below 10-10 as compared to rates of 10-5 with the non-entangled TSM. The STALEMATE system contained plasmids in E. coli Nissle 1917 for over a week in the mouse gastrointestinal tract with nearly undetectable escape rates upon excretion. STALEMATE offers a modular and simple biocontainment approach to buffer against mutational inactivation in the mammalian gut without a requirement for engineered bacteria or exogenous signaling ligands.

synthetic biology↗

bioRxiv2023

Intern-based thermoregulated meganucleases for biocontainment of genetic material

Limiting the spread of synthetic genetic information outside of the intended use is essential for applications where biocontainment is critical. In particular, biocontainment of engineered probiotics and plasmids that are excreted from the mammalian gastrointestinal tract is needed to prevent escape and acquisition of genetic material that could confer a selective advantage to microbial communities. Here, we built a simple and lightweight biocontainment system that post-translationally activates a site-specific DNA endonuclease to degrade DNA at 18{degrees}C and not at higher temperatures. We constructed an orthogonal set of temperature sensitive-meganucleases, or TSMs, by inserting the yeast VMA1 L212P temperature-sensitive intein into the coding regions of LAGLIDADG homing endonucleases. We showed that the TSMs eliminated plasmids carrying the cognate TSM target site from laboratory strains of Escherichia coli at the permissive 18{degrees}C but not at higher restrictive temperatures. Plasmid elimination is dependent on both TSM endonuclease activity and intein splicing. We demonstrated that TSMs eliminated plasmids from the E. coli Nissle 1917 strain after passage through the mouse gut when fecal resuspensions were incubated at 18{degrees}C but not at 37{degrees}C. Collectively, our data demonstrates the potential of thermoregulated meganucleases as a means of restricting engineered plasmids and probiotics to the mammalian gut.

synthetic biology↗

bioRxiv2021

Heme-dependent siderophore utilization promotes iron-restricted growth of the Staphylococcus aureus hemB small colony variant

The ability to acquire iron is essential for Staphylococcus aureus to cause infection. Respiration deficient S. aureus small colony variants (SCVs) frequently cause persistent infections, which necessitates they too acquire iron. How SCVs obtain iron remains unknown and so here we addressed this outstanding question by creating a stable hemB mutant in S. aureus USA300 strain LAC. The mutant, auxotrophic for hemin, was assessed for its ability to grow under iron- restriction and with various iron sources. The hemB SCV utilizes exogenously supplied heme but was attenuated for growth under conditions of iron starvation. RNA-seq analyses showed that both WT S. aureus and the hemB mutant sense and respond to iron starvation, however, growth assays show that the hemB mutant is defective for siderophore-mediated iron acquisition. Indeed, the hemB SCV demonstrates limited utilization of endogenous staphyloferrin B or exogenously provided staphyloferrin A, Desferal, and epinephrine, which enabled the SCV to sustain only minimal growth in iron deplete media. Direct measurement of intracellular ATP in hemB and WT S. aureus revealed that both strains can generate comparable levels of ATP during exponential growth suggesting defects in ATP production cannot account for the inability to efficiently utilize siderophores. Defective siderophore utilization by hemB bacteria was also evident in vivo. Indeed, the administration of Desferal failed to promote hemB bacterial growth in vivo, in contrast to WT, in every organ analyzed except for the murine kidney where growth was enhanced. In support of the hypothesis that S. aureus accesses heme in kidney abscesses, in vitro analyses revealed that increased heme availability enables hemB bacteria to utilize siderophores for growth when iron availability is restricted. Taken together, our data support the conclusion that heme is not only used as an iron source itself, but as a nutrient that promotes utilization of siderophore-iron complexes. Author summaryS. aureus small colony variants (SCVs) represent a difficult to treat subpopulation of bacteria that are associated with chronic recurrent infection and worsened clinical outcome. Indeed, SCVs persist within the host despite appropriate administration of antibiotics. This study yields insight into the mechanisms by which S. aureus SCVs acquire iron which, during infection of a host, is a difficult-to-acquire metal nutrient. Under heme limited conditions, hemB S. aureus is severely impaired for siderophore-dependent growth and, in agreement, murine infection indicates that hemin-deficient SCVs meet their nutritional requirement for iron through utilization of heme in vivo. Importantly, we demonstrate that hemB SCVs rely upon heme as a nutrient to promote siderophore utilization. Therefore, perturbation of heme biosynthesis and/or utilization represents a viable to strategy to mitigate the ability of SCV bacteria to acquire siderophore- bound iron during infection.

microbiology↗