Biology subjects

Fondell, J. D.

Publications and source records attributed to Fondell, J. D..

4 recordsLinked to original sources

bioRxiv2025

A lineage-specific heat-induced feedback loop controls HrcA to promote chlamydial fitness under stress

Bacterial stress responses rely on transcriptional regulation by sigma factors and repressors. In Chlamydia, which lacks a dedicated heat shock sigma factor, repressor HrcA limits chaperone gene expression under non-stressed conditions. While HrcA function may be enhanced by the chaperone GroESL in a positive feedback loop, here we identify a heat-induced negative feedback loop mediated by HagF, a protein unique to Chlamydiota and conserved across Chlamydia species. We show that HrcA represses hagF, but heat shock induces hagF expression, leading to HagF accumulation and binding to HrcA. Structural modeling and pulldown assays indicate that HagF blocks HrcA dimerization, impairing DNA binding. This relieves repression of the hrcA-grpE-dnaK operon and promotes secondary differentiation in the developmental cycle, enabling infectious elementary bodies to form under heat stress. Our findings reveal dual feedback regulation that tunes chaperone gene expression and illustrate how transcriptional repression can be modulated in a minimal-genome pathogen.

microbiology↗

bioRxiv2023

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance

Chlamydia, an obligate intracellular bacterial pathogen, has a unique developmental cycle involving the differentiation of invading elementary bodies (EBs) to noninfectious reticulate bodies (RBs), replication of RBs, and redifferentiation of RBs into progeny EBs. Progression of this cycle is regulated by three sigma factors, which direct the RNA polymerase to their respective target gene promoters. We hypothesized that the Chlamydia-specific transcriptional regulator GrgA, previously shown to activate {sigma}66 and {sigma}28, plays an essential role in chlamydial development and growth. To test this hypothesis, we applied a novel genetic tool known as dependence on plasmid-mediated expression (DOPE) to create Chlamydia trachomatis with conditional GrgA-deficiency. We show that GrgA-deficient C. trachomatis RBs have a growth rate that is approximately half of the normal rate and fail to transition into progeny EBs. In addition, GrgA-deficient C. trachomatis fail to maintain its virulence plasmid. Results of RNA-seq analysis indicate that GrgA promotes RB growth by optimizing tRNA synthesis and expression of nutrient-acquisition genes, while it enables RB-to-EB conversion by facilitating the expression of a histone and outer membrane proteins required for EB morphogenesis. GrgA also regulates numerous other late genes required for host cell exit and subsequent EB invasion into host cells. Importantly, GrgA stimulates the expression of {sigma}54, the third and last sigma factor, and its activator AtoC, and thereby indirectly upregulating the expression of {sigma}54-dependent genes. In conclusion, our work demonstrates that GrgA is a master transcriptional regulator in Chlamydia and plays multiple essential roles in chlamydial pathogenicity. IMPORTANCEHallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed DOPE that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

microbiology↗

bioRxiv2022

Identification and structural modeling of the chlamydial RNA polymerase omega subunit

Gene transcription in bacteria is carried out by the multisubunit RNA polymerase (RNAP), which is composed of a catalytic core enzyme and a promoter-recognizing {sigma} factor. RNAP core enzyme comprises two subunits, one {beta} subunit, one {beta} s subunit, and one {omega} (omega) subunit. Across multiple bacterial taxa, the RNAP {omega} subunit plays critical roles in the assembly of RNAP core enzyme and in other cellular functions, including regulation of bacterial growth, stress response, and biofilm formation. However, for several intracellular bacterium, including the obligate intracellular bacterium Chlamydia, no RNAP {omega} subunit previously has been identified. Here, we report the identification of Chlamydia trachomatis hypothetical protein CTL0286 as the chlamydial RNAP {omega} ortholog, based on sequence, synteny, and AlphaFold and AlphaFold-Multimer three-dimensional-structure predictions. We conclude that CTL0286 functions as the previously missing chlamydial {omega} ortholog. Extensions of our analysis indicate that all obligate intracellular bacteria have {omega} orthologs. IMPORTANCEChlamydiae are common mammalian pathogens. Chlamydiae have a unique developmental cycle characterized with an infectious but nondividing elementary body (EB), which can temporarily survive outside host cells, and a noninfectious reticulate body (RB), which replicates only intracellularly. Chlamydial development inside host cells can be arrested during persistence in response to adverse environmental conditions. Transcription plays a central role in the progression of the chlamydial developmental cycle as well as entry into and recovery from persistence. The identification of the elusive {omega} subunit of chlamydial RNAP makes possible future study of its regulatory roles in gene expression during chlamydial growth, development, and stress responses. This discovery also paves the way to prepare and study the intact chlamydial RNAP and its interactions with inhibitors in vitro.

molecular biology↗

bioRxiv2018

A role for GrgA in regulation of σ28-dependent transcription in the obligate intracellular bacterial pathogen Chlamydia trachomatis

The sexually transmitted obligate intracellular bacterial pathogen Chlamydia trachomatis has a unique developmental cycle consisting of two contrasting cellular forms. Whereas the primary Chlamydia sigma factor, {sigma}66, is involved in the expression of the majority of chlamydial genes throughout the developmental cycle, expression of several late genes requires the alternative sigma factor {sigma}28. In prior work we identified GrgA as a Chlamydia-specific transcription factor that activates {sigma}66-dependent transcription by binding DNA and interacting with a non-conserved region (NCR) of {sigma}66. Here, we extend these findings by showing GrgA can also activate {sigma}28-dependent transcription through direct interaction with {sigma}28. We measure the binding affinity of GrgA for both {sigma}66and {sigma}28, and we identify regions of GrgA important for {sigma}28-dependent transcription. Similar to results obtained with {sigma}66, we find that GrgAs interaction with {sigma}28 involves a NCR located upstream of conserved region 2 of {sigma}28. Our findings suggest GrgA is an important regulator of both {sigma}66- and {sigma}28-dependent transcription in C. trachomatis and further highlight NCRs of bacterial RNA polymerase as targets for regulatory factors unique to particular organisms.

microbiology↗