Fink, M. M. | Search bioRxiv

bioRxiv2025

Real-time simultaneous monitoring of multiple analytes in bacterial cultures

Bacterial metabolites are essential for biological processes, influencing human health, ecosystems, and industrial applications. Simultaneous real-time monitoring of these metabolites is critical in understanding microbial dynamics, particularly in bioreactors and food or drug manufacturing. Current approaches often rely on offline methods, which are labor-intensive and susceptible to contamination, or genetic engineering techniques limited to single-analyte monitoring. Here, we present a novel method utilizing engineered periplasmic binding proteins (PBPs) conjugated with fluorophores to track multiple metabolites simultaneously in Escherichia coli cultures. This system continuously monitors the levels of multiple analytes such as glucose, arabinose, ribose, glutamate, and arginine, providing high temporal resolution while maintaining sensor stability over 24 hours. Our findings confirm hierarchical substrate utilization in E. coli and demonstrate the versatility of PBP-based multi-sensor arrays. This approach offers a non-invasive, modular, and scalable tool for bacterial metabolite analysis, paving the way for advances in both fundamental discoveries and practical applications in microbiology. ImportanceReal-time monitoring of metabolites in bacterial cultures is crucial for advancing our understanding of microbial physiology, metabolic fluxes, and dynamic responses to environmental changes. This capability enables researchers to capture transient metabolic states that are often missed in endpoint measurements. The use of engineered periplasmic binding proteins as biosensors for this real-time metabolite monitoring represents a groundbreaking approach. By leveraging the natural specificity and high affinity of PBPs for small molecules, these biosensors can be engineered to detect a wide range of metabolites with exceptional sensitivity and temporal resolution. The integration of PBP-based biosensors into microbial research not only enhances our ability to study real-time metabolism but also provides a versatile tool for optimizing industrial bioprocesses and exploring bacterial infections and complex microbial ecosystems

bioengineering↗

bioRxiv2024

Pseudomonas aeruginosa Alkyl Quinolone Response is dampened by Enterococcus faecalis

The bacterium Pseudomonas aeruginosa is an opportunistic pathogen that can cause lung, skin, wound, joint, urinary tract, and eye infections. While P. aeruginosa is known to exhibit a robust competitive response towards other bacterial species, this bacterium is frequently identified in polymicrobial infections where multiple species survive. For example, in prosthetic joint infections (PJIs), P. aeruginosa can be identified along with other pathogenic bacteria including Staphylococcus aureus, Enterococcus faecalis, and Corynebacterium striatum. Here we have explored the survival and behavior of such microbes and find that E. faecalis readily survives culturing with P. aeruginosa while other tested species do not. In each of the tested conditions, E. faecalis growth remained unchanged by the presence of P. aeruginosa, indicating a unique mutualistic interaction between the two species. We find that E. faecalis proximity leads P. aeruginosa to attenuate competitive behaviors as exemplified by reduced production of Pseudomonas quinolone signal (PQS) and pyocyanin. Reduced alkyl quinolones is important to E. faecalis as it will grow in supernatant from a quinolone mutant but not P. aeruginosa wildtype in planktonic culture. The reduced pyocyanin production of P. aeruginosa is attributable to production of ornithine by E. faecalis, which we recapitulate by adding exogenous ornithine to P. aeruginosa mono-cultures. Similarly, co-culture with an ornithine-deficient strain of E. faecalis leads P. aeruginosa to yield near mono-culture amounts of pyocyanin. Here, we directly demonstrate how notorious pathogens such as P. aeruginosa might persist in polymicrobial infections under the influence of metabolites produced by other bacterial species. ImportanceWhile we now appreciate that many infections are polymicrobial, we understand little of the specific actions between a given set of microbes to enable combinatorial survival and pathogenesis. The bacteria Pseudomonas aeruginosa and Enterococcus faecalis are both prevalent pathogens in wound, urinary tract, and bacteremic infections. While P. aeruginosa often kills other species in standard laboratory culture conditions, we present here that E. faecalis can be reliably co-cultured with P. aeruginosa. We specifically detail that ornithine produced by E. faecalis reduces the Pseudomonas Quinolone Signal response of P. aeruginosa. This reduction of the Pseudomonas Quinolone Signal response aids E. faecalis growth.

microbiology↗