Biology subjects

Harwood, C.

Publications and source records attributed to Harwood, C..

4 recordsLinked to original sources

bioRxiv2025

Analysis of (p)ppGpp metabolism and signaling using a dynamic luminescent reporter

As rapidly growing bacteria begin to exhaust nutrients, their growth rate slows, ultimately leading to the non-replicative state of quiescence. Adaptation to nutrient limitation requires widespread metabolic remodeling that is in part mediated by the phosphorylated nucleotides guanosine tetra- and penta-phosphate, collectively (p)ppGpp. We have developed a novel reporter of (p)ppGpp abundance in the Gram-positive bacterium Bacillus subtilis based on the recent identification of a riboswitch that binds (p)ppGpp and modulates transcription via regulation of a transcriptional terminator. Placement of an unstable reporter, firefly luciferase, downstream of the riboswitch allows for sensitive and dynamic assessment of (p)ppGpp. We first confirm that the reporter accurately reflects (p)ppGpp abundance in a variety of well-established conditions. We then proceed to use it to demonstrate the physiological importance of several mechanisms of regulation of (p)ppGpp metabolism previously observed only in vitro including allosteric interactions between (p)ppGpp synthesis enzymes and the hydrolytic activity of a (p)ppGpp synthase. (p)ppGpp signaling has been implicated in the regulation of gene expression, and we demonstrate a close temporal association between gene expression and (p)ppGpp abundance, indicating a rapid, and therefore likely direct mechanism of (p)ppGpp dependent gene activation. Thus, this reporter provides a new, comprehensive analysis of (p)ppGpp signaling in vivo and offers the potential ability to sensitively monitor the temporal dynamics of (p)ppGpp abundance under diverse environmental conditions. Author SummaryMost bacteria adapt to stressful conditions such as nutrient limitation by synthesizing a signaling molecule, known as ppGpp, that consists of a hyper-phosphorylated GTP. Synthesis of ppGpp affects most aspects of cellular physiology including replication, transcription and translation. We present here a method that for the first time allows measurement of ppGpp abundance in living cells, greatly facilitating investigation into ppGpp metabolism.

microbiology↗

bioRxiv2022

Phosphoproteomic analysis of the AKT signalling axis in cutaneous squamous carcinoma progression reveals novel therapeutic targets

Cutaneous Squamous Cell Carcinoma (cSCC) represents about 20% of all non-melanoma skin cancers. Whilst generally low risk to patients, metastases are associated with a poor prognosis. cSCC incidence is increasing, owing to an ageing population, greater exposure to UV radiation, and more patients receiving immunosuppressive treatments associated with organ transplants. Therefore, there is interest in identifying new biomarkers that may be to track progression of the disease and to exploit as therapeutic vulnerabilities. We show dynamic changes in AKT expression in precursor lesions and in SCC tumour tissue, with initial loss of AKT activity followed by progressive and widespread increase in AKT activity in SCC. Phosphoproteomic analysis and kinase substrate enrichment analysis on a panel of isogenic cSCC cell lines representing different stages of the disease from premalignancy to metastasis revealed several up-regulated kinases and AKT-targets. From this analysis we chose DNA dependent protein kinase (DNA-PK), a key kinase upstream of AKT phoshorlyation, and N-Myc downstream-regulated gene 2 (NDRG2) a downstream AKT phosphorylation target, to investigate in further detail. Both proteins were up-regulated and mis-expressed in a panel of SCC tissue from different patients. We therefore explored the potential of inhibiting DNA-PK and NDRG2 as cSCC treatments. Treatment with the iron chelator Dp44mT decreased levels of phosphorylated NDRG2 and led to significant losses to viability and reduced migration in our cSCC cell lines, while DNA-PK inhibition promoted the differentiation of premalignant and early-stage SCC cell lines. Our results suggest that NDRG2 and DNA-PK may be viable targets in cSCC treatment, with effectiveness at different stages of SCC progression.

cancer biology↗

bioRxiv2019

Heterogeneity in surface sensing produces a division of labor in Pseudomonas aeruginosa populations

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition from planktonic to biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. The current thinking in the field is that once cells attach to a surface, they uniformly respond with elevated c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration.

microbiology↗

bioRxiv2016

Tunable Extracellular Self-Assembly of Multi-Protein Conjugates from Bacillus subtilis

The ability to stably and specifically conjugate recombinant proteins to one another is a powerful in vitro technique for engineering multifunctional enzymes, protein therapeutics and novel biological materials. However, for many applications spontaneous in vivo protein conjugation would be preferable to in vitro methods. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically-encoded to covalently conjugate in culture media following programmable secretion by Bacillus subtilis. Using this novel approach, we demonstrate how self-conjugation of a secreted industrial enzyme, XynA, dramatically increases its resilience to boiling and we show that cellular consortia can be engineered to self-assemble functional multi-protein complexes with tunable composition. This genetically-encoded modular system provides a new, flexible strategy for protein conjugation harnessing the substantial advantages of extracellular self-assembly.

synthetic biology↗