Biology subjects

Kamp, D.

Publications and source records attributed to Kamp, D..

4 recordsLinked to original sources

bioRxiv2025

A versatile dual-color bacterial reporter system highlights two distinct Pseudomonas aeruginosa Type 3 secretion system intracellular populations.

Since their discovery, fluorescent reporters have revolutionized our ability to track gene and protein expression in real time. Ideally, two reporters are used, one constitutive signal for tracking viable bacteria and the other for measuring the expression of the gene/protein of interest. Unfortunately, these valuable tools are not available for most bacterial species, and if available are often not optimized for fluorophore protein folding rates and fluorescence intensity. Here we present a versatile dual reporter system, pCG-VmS, optimized for both transcriptional and translation fusions in Gram-negative bacteria. Using the important pathogen Pseudomonas aeruginosa for proof of concept, we demonstrate pCG-VmS utility in tracking transcriptional expression with flow cytometry and within a complex biofilm, and using a translational reporter fusion, monitor protein expression and visualize subcellular protein localization. We then analyzed T3SS-associated exoS toxin expression in infected host cells, which highlighted two distinct T3SS-dependent intracellular populations, one where the exoS promoter is turned on and the other where it is turned off in a smaller sub-population of bacterial cells (hereon referred to as T3SS-on and T3SS-off, respectively). Finally, we demonstrate the feasibility of spatiotemporal imaging in whole animals by using our system to monitor expression of an alternative sigma factor during Vibrio fischeri colonization of its squid host. Our findings demonstrate the versatile uses for the pCG-VmS vectors in microbiology, and that this vector can be used to visualize and separate distinct populations with precision for both in vitro and in vivo applications.

microbiology↗

bioRxiv2024

Organ structure and bacterial microbiogeography in a reproductive organ of the Hawaiian bobtail squid reveal dimensions of a defensive symbiosis

Many plants and animals house symbiotic microorganisms in specialized tissues or organs. Here, we used multidimensional in-situ imaging techniques to illuminate how host organ structure and bacterial microbiogeography contribute to the symbiotic function of an organ in the Hawaiian bobtail squid, Euprymna scolopes. Along with the well-studied light organ, female E. scolopes harbor a community of bacteria in the accessory nidamental gland (ANG). The ANG is a dense network of epithelium-lined tubules, some of which are dominated by a single bacterial taxon. These bacteria are deposited into squid eggs, where they defend the developing embryos from harmful biofouling. This study used a combination of imaging techniques to visualize different dimensions of the ANG and its bacterial communities. Imaging entire organs with light sheet microscopy revealed that the ANG is a composite tissue of individual, non-intersecting tubules that each harbor their own bacterial population. The organ is bisected, with tubules converging towards two points in the organ. At these points, tubules empty in a space where bacteria can mix with squid jelly to be deposited onto eggs. Observations of bacterial populations correlated bacterial taxa with cell morphology and show that tubule populations varied: some contained populations of mixed phyla while some tubules contained only one genus of bacteria. Together, these data shed light on how bacterial populations interact within the ANG and how the host uses physical structure to maintain and employ a symbiotic bacterial population in a defensive context. IMPORTANCESequence-based microbiome studies have revealed much about how hosts interact with communities of symbiotic microbiota, but often lack a spatial understanding of how microbes relate with each other and the host in which they reside. This study used a combination of microscopy techniques to reveal how the structure of a symbiotic organ in the female bobtail squid, Euprymna scolopes houses diverse, beneficial bacterial populations and deploys them for egg defense. These findings suggest that spatial partitioning may be key to harboring a diverse population of antimicrobial-producing bacteria and establish a foundation for further understanding how host structures mediate symbiotic interactions.

microbiology↗

bioRxiv2023

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. We investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWIs mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWIs diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a monkey-bar model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

biochemistry↗

bioRxiv2021

The loader complex Scc2/4 forms co-condensates with DNA as loading sites for cohesin

The genome is organized by diverse packaging mechanisms like nucleosome formation, loop extrusion and phase separation, which all compact DNA in a dynamic manner. Phase separation additionally drives protein recruitment to condensed DNA sites and thus regulates gene transcription. The cohesin complex is a key player in chromosomal organization that extrudes loops to connect distant regions of the genome and ensures sister chromatid cohesion after S-phase. For stable loading onto the DNA and for activation, cohesin requires the loading complex Scc2/4. As the precise loading mechanism remains unclear, we investigated whether phase separation might be the initializer of the cohesin recruitment process. We found that, in absence of cohesin, budding yeast Scc2/4 forms phase separated co-condensates with DNA, which comprise liquid-like properties shown by droplet shape, fusion ability and reversibility. We reveal in DNA curtain and optical tweezer experiments that these condensates are built by DNA bridging and bending through Scc2/4. Importantly, Scc2/4-mediated condensates recruit cohesin efficiently and increase the stability of the cohesin complex. We conclude that phase separation properties of Scc2/4 enhance cohesin loading by molecular crowding, which might then provide a starting point for the recruitment of additional factors and proteins.

biophysics↗