Biology subjects

Mihajlovic, L.

Publications and source records attributed to Mihajlovic, L..

3 recordsLinked to original sources

bioRxiv2025

Modulating gene expression and protein secretion in the bacterial predator Bdellovibrio bacteriovorus

The predatory bacterium Bdellovibrio bacteriovorus kills and consumes other bacteria, thrives in diverse environments and holds great potential to address major challenges in medicine, agriculture, and biotechnology. As a bacterial predator it represents an alternative to traditional antimicrobial strategies to combat multidrug-resistant bacterial pathogens and prevent food waste, while the multitude of predatory enzymes it produces hold potential for biotechnological applications. However, the limited availability of versatile genetic tools and secretion assays constrain both fundamental studies and bioengineering of B. bacteriovorus. Here, we developed a molecular toolbox for B. bacteriovorus by systematically tuning gene expression and secretion of a reporter protein. We investigated functional native and synthetic promoters from the Anderson library with varying expression levels and demonstrated their efficacy in driving expression of the fluorescent reporter protein mScarletI3 at both the population and single-cell level. Additionally, we evaluated different ribosomal binding sites (RBS) to fine-tune gene expression. To examine secretion, we established a novel protocol to quantify extracellular release of a Nanoluc luciferase reporter protein in B. bacteriovorus using different native Sec-dependent signal sequences. We anticipate that the newly developed genetic toolkit and techniques will advance research on this fundamental predator-prey system, laying the foundation for its broader application and future bioengineering efforts. This work will pave the way for tailored applications of B. bacteriovorus in microbial ecology, agriculture, biotechnology, and medicine.

microbiology↗

bioRxiv2023

A direct experimental test of Ohno's hypothesis

Gene duplication drives evolution by providing raw material for proteins with novel functions. The oldest and historically most influential hypothesis about the evolutionary fate and potential of duplicated genes has been proposed by Susumu Ohno in 1970. This hypothesis essentially posits that gene duplication can help genes tolerate new mutations and thus facilitates the evolution of new phenotypes. Competing hypotheses argue that deleterious mutations will usually inactivate gene duplicates too rapidly for Ohnos hypothesis to work. Here, we provide a first direct experimental test of Ohnos hypothesis. Specifically, we evolved one or exactly two copies of a gene encoding a fluorescent protein in Escherichia coli through multiple rounds of mutagenesis and selection. We then analyzed the genotypic and phenotypic evolutionary dynamics of the evolving populations through high-throughput DNA sequencing, biochemical assays, and engineering of selected variants. In support of Ohnos hypothesis, populations carrying two gene copies displayed higher mutational robustness than those carrying a single gene copy. As a consequence, the double-copy populations experienced relaxed purifying selection, evolved higher phenotypic and genetic diversity, carried more mutations and accumulated combinations of key beneficial mutations earlier. However, their phenotypic evolution was not accelerated, possibly because one gene copy rapidly became inactivated by deleterious mutations. Our work provides an experimental platform to test models of evolution by gene duplication, and it supports alternatives to Ohnos hypothesis that point to the importance of gene dosage.

evolutionary biology↗

bioRxiv2017

Mechanistic causes of constrained phenotypic variation revealed by synthetic gene regulatory networks

Phenotypic variation is the raw material of adaptive Darwinian evolution. The phenotypic variation found in organismal development is biased towards certain phenotypes, but the molecular mechanisms behind such restrictions are still poorly understood. Gene regulatory networks have been proposed as one cause of constrained phenotypic variation. However, most of the evidence for this is theoretical rather than experimental. Here, we study evolutionary biases in two synthetic gene regulatory circuits expressed in E. coli that produce a gene expression stripe - a pivotal pattern in embryonic development. The two parental circuits produce the same phenotype, but create it through different regulatory mechanisms. We show that mutations cause distinct novel phenotypes in the two networks and use a combination of experimental measurements, mathematical modelling and DNA sequencing to understand why mutations bring forth only some but not other novel gene expression phenotypes. Our results reveal that the regulatory mechanisms of networks restrict the possible phenotypic variation upon mutation. Consequently, seemingly equivalent networks can indeed be distinct in how they constrain the outcome of further evolution.

systems biology↗