Biology subjects

Joseph, A. M.

Publications and source records attributed to Joseph, A. M..

7 recordsLinked to original sources

bioRxiv2025

Variability in intrinsic promoter strength underlies the temporal hierarchy of the Caulobacter SOS response induction

Bacteria encode for gene regulatory networks crucial for sensing and repairing DNA damage. Upon exposure to genotoxic stress, these transcriptional networks are induced in a temporally structured manner. A case in point is of the highly conserved SOS response thwat is regulated by the LexA repressor. Studies have proposed that affinity of LexA towards promoters of SOS response genes is the primary determinant of its expression dynamics. Here, we describe an additional level of regulation beyond LexA properties that modulates the SOS response gene expression pattern. Using transcriptomic analyses, we reveal a distinct temporal hierarchy in the induction of SOS-regulated genes in Caulobacter crescentus. We observe that LexA properties are insufficient in predicting the temporal hierarchy of these genes. Instead, we find that intrinsic promoter strength underlies the order of gene activation, with differential sigma factor association as a key factor in modulating gene expression timing. Our findings highlight a novel regulatory layer in SOS dynamics and underscore the importance of promoter properties in shaping bacterial stress responses.

microbiology↗

bioRxiv2024

Live-tracking of replisomes reveals nutrient-dependent regulation of replication elongation rates in Caulobacter crescentus

In bacteria, commitment to genome replication (initiation) is intricately linked to nutrient availability. Whether growth conditions affect other stages of replication beyond initiation, remains to be systematically studied. To address this, we assess the replication dynamics of Caulobacter crescentus, a bacterium that undergoes only single round of replication per cell cycle, by tracking the replisome across various growth phases and nutrient conditions. We find that the replication elongation rates slowdown as cells transition from exponential (high-nutrient) to stationary (low-nutrient) phase, and this contributes significantly to the overall cell cycle delay. While elongation rates are correlated with growth rates, both properties appear to be differentially influenced by nutrient status. This slowdown in replication progression is not associated with increased mutagenesis or upregulation of the DNA damage responses. We conclude that the growth conditions not only dictate the commitment to replication but also the rates of genome duplication. Such regulation appears to be distinct from stress-induced replication slowdown and likely serves as an adaptive mechanism to cope with fluctuations in nutrient availability in the environment.

microbiology↗

bioRxiv2024

Chlamydia muridarum Causes Persistent Subclinical Infection and Elicits Innate and Adaptive Immune Responses in C57BL/6J, BALB/cJ and J:ARC(S) Mice Following Exposure to Shedding Mice

Chlamydia muridarum (Cm) has reemerged as a moderately prevalent infectious agent in research mouse colonies. Despite its experimental use, few studies evaluate Cms effects on immunocompetent mice following its natural route of infection. A Cm field isolate was administered (orogastric gavage) to 8-week-old female BALB/cJ (C) mice. After confirming shedding (through 95d), these mice were cohoused with naive C57BL/6J (B6), C, and Swiss (J:ARC[S]) mice (n=28/strain) for 30 days. Cohoused mice (n=3-6 exposed and 1-6 control/strain) were evaluated 7, 14, 21, 63, 120, and 180 days post-cohousing (DPC) via hemograms, serum biochemistry analysis, fecal qPCR, histopathology, and Cm MOMP immunohistochemistry. Immunophenotyping was performed on spleen (B6, C, S; n=6/strain) and intestines (B6; n=6) at 14 and 63 DPC. Serum cytokine concentrations were measured (B6; n=6 exposed and 2 control) at 14 and 63 DPC. All B6 mice were shedding Cm by 3 through 180 DPI. One of 3 C and 1 of 6 S mice began shedding Cm at 3 and 14 DPC, respectively, with the remaining shedding thereafter. Clinical pathology was nonremarkable. Minimal-to-moderate enterotyphlocolitis and gastrointestinal associated lymphoid tissue (GALT) hyperplasia was observed in 15 and 47 of 76 Cm-infected mice, respectively. Cm antigen was frequently detected in GALT-associated surface intestinal epithelial cells. Splenic immunophenotyping revealed increased monocytes and shifts in T cell population subsets in all strains/timepoints. Gastrointestinal immunophenotyping (B6) revealed sustained increases in total inflammatory cells and elevated cytokine production in innate lymphoid cells and effector T cells (large intestine). Elevated concentrations of pro-inflammatory cytokines were detected in the serum (B6). Results demonstrate that while clinical disease was not appreciated, 3 commonly utilized strains of mice are susceptible to chronic enteric Cm infection which may alter various immune responses. Considering the widespread use of mice to model GI disease, institutions should consider excluding Cm from their colonies.

microbiology↗

bioRxiv2023

Chromosome organization shapes replisome dynamics in Caulobacter crescentus

DNA replication in bacteria takes place on highly compacted chromosomes, where segregation, transcription, and repair must occur simultaneously. Within this dynamic environment, colocalization of sister replisomes has been observed in many bacterial species, driving the hypothesis that a physical linker may tether them together. However, replisome splitting has also been reported in many of the same species, leaving the principles behind replisome organization a long-standing puzzle. Here, by tracking the replisome {beta}-clamp subunit in live Caulobacter crescentus, we find that rapid DNA segregation can give rise to a second focus which resembles a replisome, but does not replicate DNA. Sister replisomes can remain colocalized, or split apart to travel along DNA separately upon disruption of chromosome inter-arm alignment. Furthermore, chromosome arm-specific replication-transcription conflicts differentially modify replication speed on the two arms, facilitate the decoupling of the two replisomes. With these observations, we conclude that the dynamic chromosome organization flexibly shapes the organization of sister replisomes, and we outline principles which can help to reconcile previously conflicting models of replisome architecture.

microbiology↗

bioRxiv2022

Intrabacterial regulation of a cytotoxic effector by its cognate metaeffector promotes Legionella pneumophila virulence

Legionella pneumophila is a natural pathogen of unicellular protozoa that can opportunistically infect macrophages and cause Legionnaires Disease. Intracellular replication is driven by hundreds of bacterial effector proteins that are translocated into infected host cells by a Dot/Icm type IV secretion system. L. pneumophila effectors are temporally regulated in part by a unique family of translocated regulatory effectors, termed metaeffectors, which bind and modulate the function of a cognate effector in host cells. We discovered that regulation of the cytotoxic effector SidI by its metaeffector, MesI, is critical for L. pneumophila virulence in natural and opportunistic hosts. MesI binds and negatively regulates SidI activity in vitro but how dysregulation of SidI impairs L. pneumophila intracellular replication is unclear. Using a chromosomally-encoded inducible expression system, we discovered that dysregulation of SidI, via loss of MesI, was toxic to L. pneumophila. SidI enzymatic activity was required for toxicity since L. pneumophila growth was unaffected by induced expression of a catalytically inactive sidI allele. We found MesI translocation into host cells was dispensable for intracellular replication and that MesI-deficient bacteria were rapidly degraded within host cells. Together, our data suggest a unique role for intrabacterial effector regulation by a translocated metaeffector in L. pneumophila virulence. ImportanceLegionella pneumophila replicates within phagocytic host cells using hundreds of effector protein virulence factors, which canonically subvert the function of host proteins and pathways. L. pneumophila encodes a unique family of translocated effectors called metaeffectors, which bind and regulate the function of a cognate effector in host cells. The metaeffector MesI promotes L. pneumophila virulence by regulating the cytotoxic effector SidI; however, the MesI regulatory mechanism is poorly understood. We discovered a unique intrabacterial role for MesI in L. pneumophila virulence. When uncoupled from MesI, SidI was toxic to L. pneumophila in vitro and triggered robust bacterial degradation in host cells. Importantly, translocation of MesI was dispensable for intracellular replication, demonstrating that intrabacterial regulation of SidI contributes to L. pneumophila virulence. These data show a unique and important role for MesI regulation of SidI within the bacterium, which challenges the dogma that effectors function exclusively within host cells.

microbiology↗

bioRxiv2021

Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria

Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of the polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair, as replisome components fail to localize in its absence. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.

microbiology↗

bioRxiv2019

The Legionella pneumophila metaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity.

Legionella pneumophila, the etiological agent of Legionnaires Disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and SusF is not mutually exclusive and these proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyltransferase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505, SusF (suppressor of SidI function). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

microbiology↗