Biology subjects

VanDieren, A. J.

Publications and source records attributed to VanDieren, A. J..

3 recordsLinked to original sources

bioRxiv2026

Refactored genetic parts for modular assembly of the E. coli MccV type I secretion system used to screen class II microcin candidates from plant-associated bacteria

BackgroundMicrocins are small antibacterial proteins secreted by gram-negative bacteria. The activities of new microcins discovered using bioinformatic searches need to be validated and characterized to understand how they mediate competition in microbiomes and to evaluate their potential as new therapeutics for combating antibiotic resistance. Engineered plasmids containing the type I secretion system associated with Escherichia coli Microcin V (MccV) can secrete heterologous proteins, including other class II microcins, and this system functions in other bacterial hosts. However, existing microcin secretion constructs are not designed for easily swapping components -- such as origins of replication, resistance genes, promoters, and signal peptides -- that may need to be changed for compatibility with other chassis. ResultsWe refactored the E. coli MccV type I secretion system into genetic parts compatible with a modular Golden Gate assembly scheme and used these parts to construct two-plasmid microcin secretion systems. In our design, one plasmid encodes the type I secretion system proteins, and the other encodes a signal peptide fused to the cargo protein that will be secreted. We tested two versions of a system with inducible promoters separately controlling expression of the components on each plasmid. One used plasmids that replicate in E. coli and its close relatives. The other used broad-host-range plasmids. When induced to secrete MccV, both versions produced similar zones of inhibition against a susceptible strain of E. coli. Next, we identified putative class II microcins in genomes of bacteria from plant-associated genera (Pantoea, Erwinia, and Xanthomonas) using an existing bioinformatics pipeline. We screened 23 of these putative microcins for E. coli self-inhibition. Seven exhibited some inhibition, mostly later in growth curves, but none had effects that were comparable in strength to MccV. ConclusionsThe genetic parts we created can be assembled in various combinations into tailored systems for secreting small proteins from diverse bacterial chassis. These systems can be used to further characterize the targets of novel microcins and to secrete these or other small proteins for various applications. For example, beneficial bacteria used in crop protection could be engineered to secrete microcins that kill or inhibit plant pathogens to increase their efficacy.

synthetic biology↗

bioRxiv2026

Genetic biosensor for optimizing double-stranded RNA production by bacterial symbionts

There is growing interest in engineering animal and plant microbiomes to deliver double-stranded RNA (dsRNA) for RNA interference (RNAi) applications. We developed a genetically encoded biosensor that uses bimolecular fluorescence complementation to monitor dsRNA levels within bacterial cells to accelerate the symbiont-mediated RNAi design-build-test cycle. We validated performance of the sensor in Escherichia coli and demonstrated enhanced dsRNA accumulation in engineered strains of the aphid symbiont Serratia symbiotica.

synthetic biology↗

bioRxiv2024

Evolution in response to prophage activation attenuates the virulence of culturable Serratia symbiotica relatives of aphid endosymbionts

Serratia symbiotica bacteria exhibit a range of relationships with aphids. They may be co-obligate mutualists, commensals, or even pathogens depending on the strain, aphid host species, and environment. Serratia symbiotica CWBI-2.3T (CWBI), a culturable member of this group, is transmitted to embryos transovarially when it is injected into pea aphids (Acyrthosiphon pisum), the same route used by S. symbiotica strains that are vertically inherited endosymbionts. Yet, aphids colonized with CWBI die before they give birth to infected offspring. We evolved laboratory populations of CWBI through 15-30 serial passages at two different temperatures in rich media. These nutrient-replete conditions mimic aspects of the environment within aphid hosts that lead to the evolution of reduced endosymbiont genomes. Unexpectedly, all S. symbiotica populations propagated at one temperature appeared to evolve slower growth after only a few days due to reactivation of a lytic prophage from the CWBI genome. Though these populations continued to reach saturating cell densities slower than cultures of the ancestor throughout the experiment, representative clones isolated from them had mutations affecting lipopolysaccharide biosynthesis and were resistant to the phage. Some evolved strains exhibited less virulence when injected into aphids, and we observed instances of gene inactivation and loss mediated by insertion elements. Our results illustrate how transposons and prophages can dominate laboratory evolution of newly cultured bacteria, particularly those that are host-associated in nature and have genomes rife with selfish DNA elements. They also suggest that bacteria-phage coevolution can catalyze evolutionary paths that contribute to converting pathogens into stably inherited endosymbionts. IMPORTANCELaboratory experiments can be used to explore evolutionary innovations in how microbes associate with animal hosts. Serratia symbiotica bacteria exhibit a variety of interactions with aphids. Some strains are obligate endosymbionts. Others have facultative associations with benefits or costs depending on the environmental context. S. symbiotica CWBI-2.3T (CWBI) resembles aphid endosymbionts in how it can be transovarially transmitted to aphid embryos. However, adults injected with CWBI do not survive long enough to give birth to infected offspring. We evolved this aphid protosymbiont in rich media to see if this would attenuate its virulence and recapitulate genome reduction observed in endosymbionts. We observed large deletions and gene inactivation, but reactivation of a prophage from the CWBI genome and then evolution of phage resistance dominated. Some evolved strains became less virulent to aphids, suggesting that evolution driven by selfish DNA elements can contribute to the emergence of new endosymbionts from pathogen ancestors.

microbiology↗