# A molecular toolbox to modulate gene expression and protein secretion in the bacterial predator Bdellovibrio bacteriovorus

## TL;DR

Researchers developed a genetic toolkit to control gene expression and protein secretion in Bdellovibrio bacteriovorus, a predatory bacterium with potential in medicine and biotechnology.

## Contribution

The study introduces a molecular toolbox with promoters and ribosomal binding sites to modulate gene expression and a novel secretion assay for B. bacteriovorus.

## Key findings

- Functional promoters and ribosomal binding sites were tested to fine-tune gene expression in B. bacteriovorus.
- A new protocol was established to quantify extracellular protein secretion using a luciferase reporter.
- The toolkit enhances the potential of B. bacteriovorus for bioengineering and practical applications.

## Abstract

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 have potential for biotechnological applications. However, while a limited set of genetic tools exist, the lack of secretion assays and fine-tuning of secretion constrain both fundamental studies and bioengineering of B. bacteriovorus. Here, we present a molecular toolbox for B. bacteriovorus by systematically tuning gene expression and secretion of a reporter protein. Building on functional native and synthetic promoters from the Anderson library with varying expression levels of fluorescent reporter protein mScarletI3, 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.

The predatory bacterium Bdellovibrio bacteriovorus kills and consumes other bacteria, including dangerous pathogens. It lives in a variety of environments and has great potential in medicine, agriculture, and biotechnology. Furthermore, the multitude of secreted predatory enzymes of B. bacteriovorus possess biotechnological potential. However, research and engineering efforts have been limited by a lack of secretion assay and fine-tuning of protein secretion tailored to this organism. Here, we present a molecular toolbox to modulate gene expression and protein secretion in B. bacteriovorus. We tested a range of native and synthetic gene promoters at population level and evaluated the effect of different ribosomal binding sites. Further, we established a protocol to quantify extracellular release of a reporter protein. By enabling more precise secretion control, our work brings B. bacteriovorus a step closer to practical use as a biological tool to address antibiotic resistance and other microbial challenges.