Biology subjects

Sadhir, I.

Publications and source records attributed to Sadhir, I..

4 recordsLinked to original sources

bioRxiv2025

Activation of the Yersinia type III secretion system induces large-scale chromosomal and virulence plasmid DNA rearrangements

The type III secretion system (T3SS) is used by Gram-negative bacteria, including important pathogens, to manipulate eukaryotic target cells by injecting effector proteins. Type III secretion and bacterial physiology are known to be tightly interconnected and influence each other. Most notably, secreting cells undergo growth arrest in the T3SS model organisms Yersinia, Salmonella and Shigella. The molecular basis of this phenotype, referred to as secretion-associated growth inhibition, is debated. In Yersinia, T3SS genes are encoded extra-chromosomally in the plasmid of Yersinia virulence (pYV), whose copy number increases upon induction of T3SS secretion. In this study, we characterize the link between T3SS activity and subcellular organization by localizing and quantifying the pYV and chromosomal DNA in Yersinia enterocolitica. We find that activation of secretion not only increases the number of pYV plasmids per bacterium, but that the plasmids also move towards the membrane and poles. This relocalization is not caused by transertion (coupled transcription, translation and translocation) of effectors, but part of a broader DNA rearrangement, leading to a distinct relocalization of chromosomal DNA to mid-cell. We hypothesize that these striking DNA rearrangements occurring during secretion are a main factor in the secretion-associated growth inhibition of pathogenic bacteria.

microbiology↗

bioRxiv2023

Mid-cell migration of the chromosomal terminus is coupled to origin segregation in Escherichia coli

Bacterial chromosomes are dynamically and spatially organised within cells. In slow-growing Escherichia coli, the chromosomal terminus is initially located at the new pole and must therefore migrate to midcell during replication to reproduce the same pattern in the daughter cells. Here, we use high-throughput time-lapse microscopy to quantify this transition, its timing and its relationship to chromosome segregation. We find that terminus centralisation is a rapid discrete event that occurs ~25 min after initial separation of duplicated origins and ~50 min before the onset of bulk nucleoid segregation but with substantial variation between cells. Despite this variation, its movement is tightly coincident with the completion of origin segregation, even in the absence of its linkage to the divisome, suggesting a coupling between these two events. Indeed, we find that terminus centralisation does not occur if origin segregation away from mid-cell is disrupted, which results in daughter cells having an inverted chromosome organisation. Overall, our study quantifies the choreography of origin-terminus positioning and identifies an unexplored connection between these loci, furthering our understanding of chromosome segregation in this bacterium.

microbiology↗

bioRxiv2022

Star Track: Inferred counting and tracking of replicating DNA loci

Fluorescent microscopy is the primary method to study DNA organization within cells. However the variability and low signal-to-noise commonly associated with live-cell time lapse imaging challenges quantitative measurements. In particular, obtaining quantitative or mechanistic insight often depends on the accurate tracking of fluorescent particles. Here, we present [*]Track, an inference method that determines the most likely temporal tracking of replicating intracellular particles such DNA loci while accounting for missing, merged and spurious detections. It allows the accurate prediction of particle copy numbers as well as the timing of replication events. We demonstrate [*]Tracks abilities and gain new insight into plasmid copy number control and the volume dependence of bacterial chromosome replication initiation. By enabling the accurate tracking of DNA loci, [*]Track can help to uncover the mechanistic principles of chromosome organisation and dynamics across a range of systems. SignificanceMicroscopy is one of the main tools for studying the intracellular organisation of cells. In particular, fluorescent proteins allow us to study the dynamics of many cellular processes. However, this requires the accurate tracking of fluorescent foci. Here, we present [*]Track a tool tailored to the tracking of replicating persistent subcellular particles such as DNA loci. [*]Track provides accurate predictions of particle copy number and replication timing even in the presence of substantial noise. The knowledge of these quantities are critical for uncovering the mechanisms behind many cell-cycle dependent processes, such the control of chromosome and plasmid replication initiation.

biophysics↗

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↗