Biology and engineering of integrative and conjugative elements: Construction and analyses of hybrid ICEs reveal element functions that affect species-specific efficiencies

Abstract

Integrative and conjugative elements (ICEs) are mobile genetic elements that reside in a bacterial host chromosome and are prominent drivers of bacterial evolution. They are also powerful tools for genetic analyses and engineering. Transfer of an ICE to a new host involves many steps, including excision from the chromosome, DNA processing and replication, transfer across the envelope of the donor and recipient, processing of the DNA, and eventual integration into the chromosome of the new host (now a stable transconjugant). Interactions between an ICE and its hosts throughout the life cycle likely influence the efficiencies of acquisition by new hosts. Here, we investigated how different functional modules of two ICEs, Tn916 and ICEBs1, affect the transfer efficiencies into different host bacteria. We constructed hybrid elements that utilize the high-efficiency regulatory and excision modules of ICEBs1 and the conjugation genes of Tn916. These elements produced more transconjugants than Tn916, likely due to increased excision frequencies. We also found that several Tn916 and ICEBs1 components can substitute for one other. Using B. subtilis donors and three Enterococcus species as recipients, we found that different hybrid elements were more readily acquired by some species than others, demonstrating species-specific interactions in steps of the ICE life cycle. This work demonstrates that hybrid elements utilizing the efficient regulatory functions of ICEBs1 can be built to enable efficient transfer into and engineering of a variety of other species.Author summary (non-technical)Horizontal gene transfer helps drive microbial evolution, enabling bacteria to rapidly acquire new genes and traits. Integrative and conjugative elements (ICEs) are mobile genetic elements that reside in a bacterial host chromosome and are prominent drivers of horizontal gene transfer. They are also powerful tools for genetic analyses and engineering. Some ICEs carry genes that confer obvious properties to host bacteria, including antibiotic resistances, symbiosis, and pathogenesis. When activated, an ICE-encoded machine is made that can transfer the element to other cells, where it then integrates into the chromosome of the new host. Specific ICEs transfer more effectively into some bacterial species compared to others, yet little is known about the determinants of the efficiencies and specificity of acquisition by different bacterial species. We made and utilized hybrid ICEs, composed of parts of two different elements, to investigate determinants of transfer efficiencies. Our findings demonstrate that there are species-specific interactions that help determine efficiencies of stable acquisition, and that this explains, in part, the efficiencies of different ICEs. These hybrid elements are also useful in genetic engineering and synthetic biology to move genes and pathways into different bacterial species with greater efficiencies than can be achieved with naturally occurring ICEs.

Activation of the integrative and conjugative element Tn916causes growth arrest and death of host bacteria

“…Tn 916 , like many conjugative elements, only becomes active and excises from the genome in a small portion (∼0.1-3%) of the cells in a population (Celli and Trieu-Cuot, 1998; Celli et al, 1997; Marra and Scott, 1999; Scott et al, 1994; Wright and Grossman, 2016). In previous work, we created hybrid ICEs that contained the DNA processing and conjugation functions of Tn 916 and the efficient regulatory and recombination (integration and excision) systems encoded by ICE Bs1 (Bean et al, 2021). We refer to this hybrid element as (ICE Bs1 -Tn 916 )-H1, or H1 for short (Figure 1).…”

“…We refer to this hybrid element as (ICE Bs1 -Tn 916 )-H1, or H1 for short (Figure 1). ICE Bs1 and H1 can be activated in ∼25-90% of the cells in a population by overproduction of its activator protein RapI (Auchtung et al, 2005, 2007; Bean et al, 2021; Lee et al, 2007). The ability to activate Tn 916 genes needed for DNA processing and conjugation in a large proportion of cells enables population-level analyses of effects of these genes on host cells.…”

“…Excision assays. qPCR was used to monitor excision (and therefore activation) of Tn916, H1, and ICEBs1, as previously described (Bean et al, 2021;Wright and Grossman, 2016).…”

“…The construction of (ICE Bs1 -Tn 916 )-H1, a hybrid integrative and conjugative element, was previously described (Bean et al, 2021). Essentially, we replaced the DNA processing and conjugation genes of ICE Bs1 ( helP - cwlT ) with those of Tn 916 ( orf23-orf13 ) (Figure 1).…”

“…In doing so, the Tn 916 genes are controlled by the P xis promoter from ICE Bs1 (regulated by ImmR, ImmA, and RapI), and use the integration/excision components of ICE Bs1 (Int, Xis). Therefore, we can activate Tn 916 gene expression in a greater proportion of cells than in wild type Tn 916 through overproduction of the ICE Bs1 -encoded activator protein RapI (Bean et al, 2021).…”