Biology subjects

Tseng, B. S.

Publications and source records attributed to Tseng, B. S..

5 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

The structure of the Tad pilus alignment complex reveals a periplasmic conduit for pilus extension

The Tad (Tight adherence) pilus is a bacterial appendage implicated in bacterial virulence, cell-cell aggregation, and biofilm formation. Despite its homology to the well-characterised Type IV pilus, the structure and assembly mechanism of the Tad pilus are poorly understood. Here, we investigate the role of the protein RcpC from Pseudomonas aeruginosa. Our analyses reveal that RcpC forms a dodecameric periplasmic complex, anchored to the inner membrane by a transmembrane helix, and interacting with the outer membrane secretin RcpA. We use single-particle Cryo-EM to elucidate the structure of this RcpC dodecamer, and cell-based assays to demonstrate that the RcpC-RcpA complex is essential for Tad-mediated cell-cell aggregation. Collectively, these data demonstrate that RcpC forms the Tad pilus alignment complex, which provides a conduit across the periplasm for the Tad pilus filament to access the extracellular milieu. Our experimental data and structure-based models allow us to propose a mechanism for Tad plus assembly.

microbiology↗

bioRxiv2023

OprF impacts Pseudomonas aeruginosa biofilm matrix eDNA levels in a nutrient-dependent manner

The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In the Pseudomonas aeruginosa biofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin of P. aeruginosa cells. However, current data describing the effects of OprF in the P. aeruginosa biofilm is limited. Here we identify a nutrient-dependent effect of OprF in static biofilms, whereby {Delta}oprF cells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate that P. aeruginosa {Delta}oprF biofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of mature P. aeruginosa biofilms by retaining eDNA in the matrix. IMPORTANCEMany pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized. However, the effects of P. aeruginosa matrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here we describe a conditional effect of the abundant matrix protein OprF on late-stage P. aeruginosa biofilms. A {Delta}oprF strain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective {Delta}oprF biofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in mature biofilms.

microbiology↗

bioRxiv2020

Inhibition of AlgU by MucA is required for viability in Pseudomonas aeruginosa

During decades-long infections in the cystic fibrosis (CF) airway, Pseudomonas aeruginosa undergoes selection. One bacterial genetic adaptation often observed in CF isolates is mucA mutations. MucA inhibits the sigma factor AlgU. Mutations in mucA lead to AlgU misregulation, resulting in a mucoid phenotype that is associated with poor CF disease outcomes. Due to its ability to be mutated, mucA is assumed to be dispensable for bacterial viability. Here we show that, paradoxically, a portion of mucA is essential in P. aeruginosa. We demonstrate that mucA is no longer required in a strain lacking algU, that mucA alleles encoding for proteins that do not bind to AlgU are insufficient for viability, and that mucA is no longer essential in mutant strains containing AlgU variants with reduced sigma factor activity. Furthermore, we found that overexpression of algU prevents cell growth in the absence of MucA, and that this phenotype can be rescued by overproduction of RpoD, the housekeeping sigma factor. Together, these results suggest that in the absence of MucA, the inability to regulate AlgU activity results in the loss of bacterial viability. Finally, we speculate that essentiality of anti-sigma factors that regulate envelope function may be a widespread phenomenon in bacteria.

microbiology↗

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↗