Toolbox of Characterized Genetic Parts for Staphylococcus aureus

Abstract

Staphylococcus aureus is an important clinical bacterium prevalent in human-associated microbiomes and the cause of many diseases. However, S. aureus has been intractable to synthetic biology approaches due to limited characterized genetic parts for this nonmodel Gram-positive bacterium. Moreover, genetic manipulation of S. aureus has relied on cumbersome and inefficient cloning strategies. Here, we report the first standardized genetic parts toolbox for S. aureus, which includes characterized promoters, ribosome binding sites, terminators, and plasmid replicons from a variety of bacteria for precise control of gene expression. We established a standard relative expression unit (REU) for S. aureus using a plasmid reference and characterized genetic parts in standardized REUs using S. aureus ATCC 12600. We constructed promoter and terminator part plasmids that are compatible with an efficient Type IIS DNA assembly strategy to effectively build multipart DNA constructs. A library of 24 constitutive promoters was built and characterized in S. aureus, which showed a 380-fold activity range. This promoter library was also assayed in Bacillus subtilis (122-fold activity range) to demonstrate the transferability of the constitutive promoters between these Gram-positive bacteria. By applying an iterative design-build-test-learn cycle, we demonstrated the use of our toolbox for the rational design and engineering of a tetracycline sensor in S. aureus using the P Xyl-TetO aTc-inducible promoter that achieved 25.8-fold induction. This toolbox greatly expands the growing number of genetic parts for Gram-positive bacteria and will allow researchers to leverage synthetic biology approaches to study and engineer cellular processes in S. aureus.

SubtiToolKit - a bioengineering kit forBacillus subtilisand Gram-positive bacteria

“…In the same way, the STK syntax is not compatible with other tool kits, but parts can be easily domesticated by PCR. Despite new boxes being expected to contain specific parts from the target organisms, Gram-positive bacteria show a high transferability of parts between the groups of bacteria(Rondthaler et al, 2024). However, these parts need to be characterized in the new host as it is not rare that parts behave differently in different species, even from the same genus as we show in our results on different species of Geobacillus and Parageobacillus (Figure 6A).…”

“…Large libraries of native promoters (Liu et al, 2018) and RBSs (Guiziou et al, 2016) have been already characterized for B. subtilis and other Gram-positive species (Rondthaler et al, 2024). The STK contains a collection of 4 inducible and 9 constitutive promoters, 11 RBSs, and 12 terminators (Figure 4).…”

Utilizing 5′ UTR Engineering Enables Fine-Tuning of Multiple Genes within Operons to Balance Metabolic Flux in Bacillus subtilis

“…Promoters and ribosome binding sites (RBSs) are frequently utilized to enhance gene expression to achieve the maximum formation of desired products. In Gram-positive bacteria, a library comprising 24 constitutive promoters was established, exhibiting an activity range of 380-fold in Staphylococcus aureus and 122fold in B. subtilis [26]. In the production of bacteriocins, incorporating a robust ribosomal binding sequence into the 5 ′ -UTR of the B. subtilis bac operon led to a 2.87-fold increase in bacteriocin production [27].…”