Biology subjects

Franklin, M. J.

Publications and source records attributed to Franklin, M. J..

3 recordsLinked to original sources

bioRxiv2025

A novel non-catalytic function of PA2803-encoded PcrP contributes to polymyxin B resistance in P. aeruginosa and redefines the functional role of the PA2803 subfamily.

The opportunistic human pathogen Pseudomonas aeruginosa (Pa), a leading cause of severe infections, becomes increasingly resistant to antibiotics, including the last resort antibiotic, polymyxin-B (PMB). Previous studies have shown that calcium (Ca2+) at the levels encountered during infections increases Pa resistance to PMB. However, the mechanisms of this Ca2+ regulation are not known. Here, we identified three novel genes (PA2803, PA3237 and PA5317) that contribute to the Ca2+-dependent PMB resistance in Pa. PA2803, the focus of this work, encodes a putative phosphonatase and is a founding member of the PA2803 subfamily from the Haloacid Dehalogenase Superfamily. Since the transcription of this gene is regulated by both Ca2+ and inorganic phosphate (Pi), we named it "Pi and Ca2+ regulated protein, PcrP". Congruent with sequence-based predictions, we showed that PcrP lacks catalytic activity and instead binds protein partners, revealing a novel non-catalytic function for PA2803 subfamily proteins. By using pull-down assays and bacterial two-hybrid system, we identified and validated two protein partners of PcrP: Acp3 and PA3518. We show that PcrP is involved in oxidative stress responses in Pa, which are likely mediated by its interactions with Acp3, and may support its role in PMB resistance. In addition, PcrP imparts a Ca2+-dependent growth advantage to Pa during Pi starvation and plays a role in polyphosphate accumulation in a Ca2+-dependent manner. Overall, this study identified a novel protein-binding function for the PA2803 subfamily representative that mediates Pa responses to elevated Ca2+ and Pi starvation and enhances PMB resistance. IMPORTANCEPseudomonas aeruginosa (Pa) is a critical human pathogen that presents significant clinical challenges, underscoring the urgent need for understanding its resistance mechanisms. Previous studies have shown that calcium (Ca2+) at the levels detected during infections increase Pa resistance to the last resort antibiotic polymyxin-B (PMB). For the first time, we identified three novel genes, whose products are required for the Ca2+-dependent PMB resistance in Pa. One of them, PA2803, regulated by Ca2+ and phosphate, was named phosphate and Ca2+ regulated protein, PcrP. This study discovered a novel protein-binding function of PcrP and identified two protein partners. The protein-binding function is likely shared by the entire PA2803 subfamily of proteins, which redefines their previous functional assignment as enzymes.

microbiology↗

bioRxiv2025

Single-cell bacterial culturing and antibiotic susceptibility testing using permeable hydrogel-shelled microcapsules

Microbial communities, such as biofilms, consist of bacteria that exhibit cell-to-cell heterogeneity in their physiological properties, including enzyme activity and gene expression. This single-cell heterogeneity influences the communitys overall metabolic activity and contributes to stress tolerance, such as antimicrobial resistance. To study the impact of single-cell heterogeneity on population-level behaviors, methods have been developed to isolate and characterize bacteria at the single-cell level. One such method includes water-in-oil drop-based microfluidics. However, a limitation of this approach is that the droplet contents cannot be exchanged during experiments, making it difficult to investigate how cells respond to changing environmental conditions. To address this limitation, we developed a drop-based microfluidic technique called Bioflex, which creates permeable hydrogel-shell microcapsules that act as growth chambers for individual bacterial cells. The microcapsules are formed by crosslinking a shell made from a PEG-based hydrogel (4-arm PEG-maleimide) around a dextran core. After cross-linking, the dextran core diffuses out of the capsules and is replaced with buffer or growth medium. By adjusting fluid flow rates during the process, we can control the size and shell thickness of the microcapsules, allowing the production of different capsule architectures. The Bioflex capsules are biocompatible, supporting the encapsulation and growth of Pseudomonas aeruginosa by allowing nutrient transport across the capsule shell. Moreover, the capsules remained permeable to antimicrobial treatments introduced during P. aeruginosa incubation. For example, P. aeruginosa cells in Bioflex capsules responded to externally delivered ciprofloxacin treatments, with their responses varying depending on the timing of the antibiotic introduction. In summary, Bioflex capsules provide a novel, high-throughput platform for isolating single-cells and testing how single-cell derived communities react to time-dependent stresses, such as antibiotic treatments. Graphical AbstractBacterial cultivation using permeable microscale Bioflex capsules. (A) P. aeruginosa PAO1 expressing eGFP cultured in a Bioflex capsule starting from a single cell. (B) Bioflex capsules are permeable to nutrient and waste transport. (C) The capsules allow for exchange of media to study the responses of single cells or small populations of cells within the drops using time-lapse imaging. O_FIG O_LINKSMALLFIG WIDTH=101 HEIGHT=200 SRC="FIGDIR/small/647129v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@69d8bforg.highwire.dtl.DTLVardef@11507b6org.highwire.dtl.DTLVardef@1ad6649org.highwire.dtl.DTLVardef@1167ae0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2017

A Fluorogenic Array Tag for Temporally Unlimited Single Molecule Tracking

Cellular processes take place over many timescales, prompting the development of precision measurement technologies that cover milliseconds to hours. Here we describe ArrayG, a bipartite fluorogenic system composed of a GFP-nanobody array and monomeric wtGFP binders. The free binders are initially dim but brighten 15 fold upon binding the array, suppressing background fluorescence. By balancing rates of intracellular binder production, photo-bleaching, and stochastic binder exchange on the array, we achieved temporally unlimited tracking of single molecules. Fast (20-180Hz) tracking of ArrayG tagged kinesins and integrins, for thousands of frames, revealed repeated state-switching and molecular heterogeneity. Slow (0.5 Hz) tracking of single histones for as long as 1 hour showed fractal dynamics of chromatin. We also report ArrayD, a DHFR-nanobody-array tag for dual color imaging. The arrays are aggregation resistant and combine high brightness, background suppression, fluorescence replenishment, and extended choice of fluorophores, opening new avenues for seeing and tracking single molecules in living cells.

biophysics↗