Biology subjects

Kiley, P. J.

Publications and source records attributed to Kiley, P. J..

4 recordsLinked to original sources

bioRxiv2025

IscR-mediated sensing of iron-sulfur cluster demand coordinates virulence gene expression in Yersinia pseudotuberculosis

The type III secretion system (T3SS) is a needle-like appendage that translocates effector proteins into host cells to disrupt host defenses. Strict control of T3SS expression is critical for facultative pathogens such as Yersinia pseudotuberculosis, as the T3SS is indispensable for virulence but is metabolically costly. We previously showed that the iron-sulfur (Fe-S) cluster-coordinating transcription factor IscR controls expression of LcrF, the master regulator of the Yersinia T3SS and the YadA adhesin. Clusterless apo-IscR, the predominant IscR form during high cellular Fe-S cluster demand (aerobic, low iron conditions), promotes LcrF, T3SS, and YadA expression. Importantly, binding of apo-IscR to the lcrF promoter at a type II IscR binding site facilitates Yersinia disseminated infection. Here, we show that mutating the lcrF promoter to allow only [2Fe-2S]-IscR binding (lcrFpTypeI) results in hyperexpression of LcrF, the T3SS, and YadA during low Fe-S cluster demand (anaerobic, iron replete conditions). These data suggest that switching the form of IscR that can bind to the lcrF promoter reversed how iron and oxygen regulate Yersinia virulence factors. We used barcoded Y. pseudotuberculosis to probe how control of the lcrF promoter in response to iron and oxygen modifies infection dynamics. We found that the lcrFpTypeI mutant experiences a tighter bottleneck in the cecum, where Yersinia is expected to experience a low oxygen, iron replete environment. Taken together, these findings suggest that by tying T3SS and YadA expression to cellular Fe-S cluster demand, Yersinia can fine-tune its virulence repertoire to the host tissue microenvironment. ImportanceIron and oxygen availability fluctuate spatially across mammalian tissues as well as temporally during the course of bacterial infection. The [2Fe-2S] cluster coordinating transcription factor IscR senses changes in iron and oxygen levels, and plays a pivotal role in enabling pathogens like Yersinia, Salmonella, and Vibrio to express critical virulence genes. While prior research has established that iron availability and oxygen tension influence IscR abundance and DNA-binding specificity, it is unclear how these changes control the timing and location of virulence factor expression during infection. In this study, we engineered a bacterial strain to reverse the way in which iron and oxygen drive expression of two critical virulence factors through IscR. This mutant displayed altered host infection dynamics, revealing that uncoupling virulence gene expression from host tissue cues decreases bacterial fitness.

microbiology↗

bioRxiv2025

Orthogonal chemical genomics approaches reveal genomic targets for increasing anaerobic chemical tolerance in Zymomonas mobilis

Genetically-engineered microbes have the potential to increase efficiency in the bioeconomy by overcoming growth-limiting production stress. Screens of gene perturbation libraries against production stressors can identify high-value engineering targets, but follow-up experiments needed to guard against false positives are slow and resource-intensive. In principle, the use of orthogonal gene perturbation approaches could increase recovery of true positives over false positives because the strengths of one technique compensate for the weaknesses of the other, but, in practice, two parallel screens are rarely performed at the genome-scale. Here, we screen genome-scale CRISPRi (CRISPR interference) knockdown and TnSeq (transposon insertion sequencing) libraries of the bioenergy-relevant Alphaproteobacterium, Zymomonas mobilis, against growth inhibitors commonly found in deconstructed plant material. Integrating data from the two gene perturbation techniques, we established an approach for defining engineering targets with high specificity. This allowed us to identify all known genes in the cytochrome bc1 and cytochrome c synthesis pathway as potential targets for engineering resistance to phenolic acids under anaerobic conditions, a subset of which we validated using precise gene deletions. Strikingly, this finding is specific to the cytochrome bc1 and cytochrome c pathway and does not extend to other branches of the electron transport chain. We further show that exposure of Z. mobilis to ferulic acid causes substantial remodeling of the cell envelope proteome, as well as the downregulation of TonB-dependent transporters. Our work provides a generalizable strategy for identifying high-value engineering targets from gene perturbation screens that is broadly applicable. IMPORTANCEEngineering microorganisms to tolerate harsh production conditions stands to increase bioproduct yields of engineered microbes. In this study, we systematically identified Z. mobilis genes that confer resistance or susceptibility to chemical stressors found in deconstructed plant material. We used complementary genetic techniques to cross-validate these genes at scale, providing a widely applicable method for precisely identifying genetic alterations that increase chemical resilience. We discovered genetic modifications that improve anaerobic growth of Z. mobilis in the presence of inhibitory chemicals found in renewable plant-based feedstocks. These results have implications in engineering robust production strains to support efficient and resilient bioproduction. Our methodologies can be broadly applied to understand microbial responses to chemicals across systems, paving the way for developments in biomanufacturing, therapeutics, and agriculture.

genomics↗

bioRxiv2021

The transcription factor IscR promotes Yersinia type III secretion system activity by antagonizing the repressive H-NS-YmoA histone-like protein complex

The type III secretion system (T3SS) is a appendage used by many bacterial pathogens, such as pathogenic Yersinia, to subvert host defenses. However, because the T3SS is energetically costly and immunogenic, it must be tightly regulated in response to environmental cues to enable survival in the host. Here we show that expression of the Yersinia Ysc T3SS master regulator, LcrF, is orchestrated by the opposing activities of the repressive YmoA/H-NS histone-like protein complex and induction by the iron and oxygen-regulated IscR transcription factor. Although IscR has been shown to bind the lcrF promoter and is required for in vivo expression of lcrF, in this study we show IscR alone fails to enhance lcrF transcription in vitro. Rather, we find that in a ymoA mutant, IscR is no longer required for LcrF expression or T3SS activity. Additionally, a mutation in YmoA that prevents H-NS binding (ymoAD43N) rescues the T3SS defect of a {Delta}iscR mutant, suggesting that a YmoA/H-NS complex is needed for this repressive activity. Furthermore, chromatin immunoprecipitation analysis revealed that H-NS is enriched at the lcrF promoter at environmental temperatures, while IscR is enriched at this promoter at mammalian body temperature under aerobic conditions. Importantly, CRISPRi knockdown of H-NS leads to increased lcrF transcription. Collectively, our data suggest that as IscR levels rise with iron limitation and oxidative stress, conditions Yersinia experiences during extraintestinal infection, IscR antagonizes YmoA/H-NS-mediated repression of lcrF transcription to drive T3SS activity and manipulate host defense mechanisms. Author SummaryFacultative pathogens must silence virulence gene expression during growth in the environment, while retaining the ability to upregulate these genes upon infection of a host. H-NS is an architectural DNA binding protein proposed to silence horizontally acquired genes, regulating virulence genes in a number of pathogens. Indeed, H-NS was predicted to regulate plasmid-encoded virulence genes in pathogenic Yersinia. However, Yersinia H-NS is reported to be essential, complicating testing of this model. We used chromatin immunoprecipitation and inducible CRISPRi knockdown to show that H-NS binds to the promoter of a critical plasmid-encoded virulence gene, silencing its expression. Importantly, under conditions that mimic Yersinia infection of a mammalian host, the transcriptional regulator IscR displaces H-NS to drive virulence factor expression.

microbiology↗

bioRxiv2019

Elevated Expression of a Functional Suf Pathway in the E. coli BL21(DE3) Cell Line Enhances Recombinant Production of an Iron-Sulfur Cluster Containing Protein.

Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overexpress [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an inframe fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is - inactive in [Fe-S] biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (~3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxido-reductase complex. These results show that this engineered SufFeScient derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein.\n\nIMPORTANCELarge quantities of recombinantly overexpressed iron-sulfur cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that increase Suf expression can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

biochemistry↗