Biology subjects

Pambos, O. J.

Publications and source records attributed to Pambos, O. J..

5 recordsLinked to original sources

bioRxiv2025

DeepTRACE: Flexible Machine Learning for Analysis and Discovery in Single Molecule Tracks

DeepTRACE is a machine learning tool for analysing long, complex single-molecule tracks in living cells; learning from sequences of molecular events using spatial, temporal, and photometric context. It traces how molecular processes unfold over time and space, incorporating past interactions, subcellular location, and photometric properties. DeepTRACE requires only a few hundred annotated tracks and minutes of CPU training, yet outperforms traditional methods and supports extensive downstream analysis, including the discovery of relationships absent from the training data.

biophysics↗

bioRxiv2023

In vivo single-molecule imaging of RecB reveals efficient repair of DNA damage in Escherichia coli

Efficient DNA repair is crucial for maintaining genome integrity and ensuring cell survival. In Escherichia coli, RecBCD plays a crucial role in processing DNA ends following a DNA double-strand break (DSB) to initiate repair by homologous recombination. While RecBCD has been extensively studied in vitro, less is known about how it contributes to rapid and efficient repair in living bacteria. Here, we perform single-molecule microscopy to investigate DNA repair in real-time in E. coli. We quantify RecB single-molecule mobility and monitor the induction of the DNA damage response (SOS response) in individual cells. We show that RecB binding to broken DNA ends leads to efficient repair without SOS induction. In contrast, in a RecB mutant with modified activities leading to the activation of an alternative repair pathway, repair is less efficient and leads to high SOS induction. Our findings reveal how subtle alterations in RecB activity profoundly impact the efficiency of DNA repair in E. coli.

molecular biology↗

bioRxiv2022

Single-molecule tracking reveals the functional allocation, in vivo interactions, and spatial organization of universal transcription factor NusG

Bacterial gene expression is highly regulated to allow cells to grow and adapt. Much regulation occurs during transcription elongation, where RNA polymerase (RNAP) extends nascent RNA transcripts aided by global and universally-conserved elongation factor NusG. NusG modulates transcription by inhibiting pausing and backtracking; promoting anti-termination on ribosomal RNA (rrn) operons; coupling transcription with translation on mRNA genes; and stimulating Rho-dependent termination on toxic genes. Despite extensive work on NusG, its functional allocation and spatial distribution in vivo is unknown. Here, we addressed these long-standing questions using single-molecule tracking and super-resolution imaging of NusG in live E. coli cells. We found that, under conditions of moderate growth, NusG is mainly present as a population that associates indirectly with the chromosome via RNAP in transcription elongation complexes, and a slowly diffusing population we identified as a NusG complex with the 30S ribosomal subunit; this complex offers a "30S-guided" path for NusG to enter transcription elongation. Only ~10% of total NusG was fast-diffusing, with the mobility of this population suggesting that free NusG interacts non-specifically with DNA for >50% of the time. Using antibiotics and deletion mutants, we showed that most chromosome-associated NusG is involved in rrn anti-termination and in transcriptiontranslation coupling. NusG involvement in rrn anti-termination was mediated via its participation in phase-separated transcriptional condensates. Our work illuminates the diverse activities of a central regulator while offering a guide on how to dissect the roles of multi-functional machines using in vivo imaging.

molecular biology↗

bioRxiv2022

RNA polymerase redistribution and increased gene dosage support growth in E. coli strains with a minimal number of ribosomal RNA operons

Bacterial transcription by RNA polymerase (RNAP) is spatially organised. RNAPs transcribing highly expressed genes locate in the nucleoid periphery, and form clusters in rich media, with several studies linking RNAP clustering and transcription of ribosomal RNA (rrn). However, the nature of RNAP clusters and their association with rrn transcription remains unclear. Here we address these questions by using single-molecule tracking to monitor the subcellular distribution of mobile and immobile RNAP in strains with a heavily reduced number of chromosomal rrn operons ({Delta}rrn strains). Strikingly, we find that the fraction of chromosome-associated RNAP (which is mainly engaged in transcription) is robust to deleting 5 or 6 of the 7 chromosomal rrn operons. Spatial analysis in{Delta} rrn strains showed substantial RNAP redistribution during moderate growth, with clustering increasing at the cell end-caps, where the remaining rrn operons reside. These results support a model where RNAPs in{Delta} rrn strains relocate to copies of the remaining rrn operons. We also show that{Delta} rrn strains experience increased rrn gene dosage in rich media, minimising growth defects due to rrn deletions. Our study further links RNAP clusters and rrn transcription, and offers insight on how bacteria maintain growth in the presence of only 1-2 rrn operons.

microbiology↗

bioRxiv2019

Rapid functionalisation and detection of viruses via a novel Ca2+-mediated virus-DNA interaction

Current virus detection methods often take significant time or can be limited in sensitivity and specificity. The increasing frequency and magnitude of viral outbreaks in recent decades has resulted in an urgent need for diagnostic methods that are facile, sensitive, rapid and inexpensive. Here, we describe and characterise a novel, calcium-mediated interaction of the surface of enveloped viruses with DNA, that can be used for the functionalisation of intact virus particles via chemical groups attached to the DNA. Using DNA modified with fluorophores, we have demonstrated the rapid and sensitive labelling and detection of influenza and other viruses using single-particle tracking and particle-size determination. With this method, we have detected clinical isolates of influenza in just one minute, significantly faster than existing rapid diagnostic tests. This powerful technique is easily extendable to a wide range of other enveloped pathogenic viruses and holds significant promise as a future diagnostic tool.

microbiology↗