Biology subjects

Hallinen, K. M.

Publications and source records attributed to Hallinen, K. M..

5 recordsLinked to original sources

bioRxiv2026

Release of the Staphylococcus aureus autoinducing peptide is enabled by active export of the biosynthetic intermediate

Staphylococcus aureus is a species of Gram-positive bacteria that uses the accessory gene regulator (agr) quorum sensing system to regulate many aspects of cell behavior such as virulence and toxin production. Quorum sensing is activated in dense S. aureus populations by the extracellular accumulation of a signaling molecule called the autoinducing peptide (AIP). While the biosynthetic enzymes that produce AIP have been elucidated in certain subgroups of S. aureus, the mechanism of AIP secretion is unknown. Here, we show that the biosynthetic intermediate known as AgrD (1-32) thiolactone is actively exported into the surrounding environment in certain S. aureus subgroups. Using a high-throughput transposon mutagenesis screen, we identify the integral membrane transporters, AbcA and PmtCD, as being involved in the biosynthesis of certain AIPs. Subsequent genetic complementation and biochemical experiments reveal that these proteins actively transport AgrD (1-32) thiolactone out of the cell. This enables the production of AIP directly on the cell surface following the action of MroQ, an integral membrane protease demonstrated here to possess an active site facing the extracellular milieu. Furthermore, we show that media supplementation with catalytically-active MroQ nanodiscs rescues AIP production in an mroQ knockout strain. This observation suggests the possibility that some proportion of AgrD (1-32) thiolactone exported by one cell may travel to neighboring cell surfaces for further processing into the diffusive AIP molecule. Collectively, our studies shed light on a poorly understood step in the agr quorum sensing pathway with clear implications for the design of novel therapeutic strategies.

biochemistry↗

bioRxiv2026

Cooperative antibiotic response in coupled biofilm and planktonic E. faecalis communities

Bacterial communities grow as dynamic populations that respond to their environments. A clinically relevant example is the inactivation of beta-lactam antibiotics by intracellular beta-lactamase in E. faecalis resistant strains. In these populations, resistant bacteria act as antibiotic sinks, detoxifying the environment and allowing sensitive bacteria to survive treatment through a cooperative interaction. In this work, we study strongly coupled planktonic and biofilm populations of mixed sensitive-resistant E. faecalis bacteria under antibiotic stress using fluorescent microscopy. The presence of resistant bacteria in the system benefits both resistant and sensitive cells, leading to mixed planktonic and biofilm populations at super-inhibitory drug concentrations. We show that a beta-lactam antibiotic with or without the addition of a beta-lactam inhibitor can lead to a population inversion effect, characterized by a non-monotonic relation between initial and final fractions of resistant bacteria. The effect is observed in both the planktonic and biofilm populations and is modulated by the total initial cell density. A well-mixed model with competition mediated by resource sharing and cooperation from global degradation of toxins predicts the experimentally observed behavior. These observations suggest underlying population-level mechanisms that are largely independent of biofilm spatial structure.

biophysics↗

bioRxiv2023

Community behaviors of bacteria in flow: Divergent roles for transport and collective morphology

Fluid flows can impact bacterial behaviors in unexpected ways (1-3). The high shear rate in heart valves should reduce colonization, but in endocarditis, valves are often counter-intuitively colonized by Staphylococcus aureus and Enterococcus faecalis (4, 5). Here we discover bacteria-specific mechanisms for preferential surface colonization in higher shear rate environments. This behavior enables bacteria that are outcompeted in low flow to dominate in high flow. Flow-dependent colonization by S. aureus and E. faecalis are mediated by distinct mechanisms that depend on each species microcolony morphologies: transport of a dispersal signaling molecule for clustered S. aureus and mechanical forces for linear chains of E. faecalis. These results suggest that microcolony morphologies have previously unappreciated costs and benefits in different environments, like those introduced by flow. One-Sentence SummaryBacterial surface colonization in high fluid flow depends upon the species clustered or chained microcolony morphologies.

microbiology↗

bioRxiv2019

Fluorescent reporter plasmids for single-cell and bulk-level composition assays in E. faecalis

Fluorescent reporters are an important tool for monitoring dynamics of bacterial populations at the single cell and community level. While there are a large range of reporter constructs available-particularly for common model organisms like E. coli-fewer options exist for other species, including E. faecalis, a gram-positive opportunistic pathogen. To expand the potential toolkit available for E. faecalis, we modified a previously developed reporter plasmid (pBSU101) to express one of nine different fluorescent reporters and confirmed that all constructs exhibited detectable fluorescence in single E. faecalis cells and mixed biofilm communities. To identify promising constructs for bulk-level experiments, we then measured the fluorescence spectra from E. faecalis populations in microwell plate (liquid) cultures during different growth phases. Cultures showed density- and reporter-specific variations in fluorescent signal, though spectral signatures of all reporters become clear in late-exponential and stationary-phase populations. Based on these results, we identified six pairs of reporters that can be combined with simple spectral unmixing to accurately estimate population composition in 2-strain mixtures at or near stationary phase. This approach offers a simple and scalable method for selection and competition experiments in simple two-species populations. Finally, we modified the construct to express codon-optimized variants of blue (BFP) and red (RFP) reporters and show that they lead to increased fluorescence in exponentially growing cells. As a whole, the results inform the scope of application of different reporters and identify both single reporters and reporter pairs that are promising for fluorescence-based assays at bulk and single-cell levels in E. faecalis.

microbiology↗

bioRxiv2019

Delayed antibiotic exposure induces population collapse in enterococcal communities with drug-resistant subpopulations

Bacteria exploit a diverse set of defenses to survive exposure to antibiotics. While the molecular and genetic underpinnings of antibiotic resistance are increasingly understood, less is known about how these molecular events influence microbial dynamics on the population scale. In this work, we show that the dynamics of E. faecalis communities exposed to antibiotics can be surprisingly rich, revealing scenarios where-for example-increasing population size or delaying drug exposure can promote population collapse. Specifically, we combine experiments in computer-controlled bioreactors with simple mathematical models to reveal density-dependent feedback loops that couple population growth and antibiotic efficacy when communities include drug-resistant ({beta}-lactamase producing) subpopulations. The resulting communities exhibit a wide range of behavior, including population survival, population collapse, or one of two qualitatively distinct bistable behaviors where survival is favored in either small or large populations. These dynamics reflect competing density-dependent effects of different subpopulations, with growth of drug-sensitive cells increasing but growth of drug-resistant cells decreasing effective drug inhibition. Guided by these results, we experimentally demonstrate how populations receiving immediate drug influx may sometimes thrive, while identical populations exposed to delayed drug influx (and lower average drug concentrations) collapse. These results illustrate that the spread of drug resistant determinants--even in a simplified single-species communities--may be governed by potentially counterintuitive dynamics driven by population-level interactions.

microbiology↗