Integrative and Conjugative Elements (ICEs): What They Do and How They Work

Abstract

Horizontal gene transfer plays a major role in microbial evolution, allowing microbes to acquire new genes and phenotypes. Integrative and conjugative elements (ICEs, a.k.a. conjugative transposons) are modular mobile genetic elements integrated into a host genome and are passively propagated during chromosomal replication and cell division. Induction of ICE gene expression leads to excision, production of the conserved conjugation machinery (a type IV secretion system), and the potential to transfer DNA to appropriate recipients. ICEs typically contain cargo genes that are not usually related to the ICE life cycle and that confer phenotypes to host cells. We summarize the life cycle and discovery of ICEs, some of the regulatory mechanisms, and how the types of cargo have influenced our view of ICEs. We discuss how ICEs can acquire new cargo genes and describe challenges to the field and various perspectives on ICE biology.

Keywords: ICE; ICEBs1; Tn916; bacteria; conjugation; gene transfer.

Figure 1

The ICE (integrative and conjugative element) life cycle. A model of ICE conjugation is shown. The bacterium bearing the ICE (the donor) is shown in gray, and the bacterium acquiring the ICE (the recipient) is shown in green. The ICE DNA is shown in blue. (a) The ICE is found integrated into the host chromosome. Most ICE genes are not expressed, because of either repression or lack of activation. (b)When ICE gene expression is induced, the ICE excises from the host chromosome and forms a dsDNA circular plasmid. ICE-encoded proteins are produced, some of which assemble into the mating pore (cylinders spanning the donor cell envelope). (c) The ICE-encoded relaxase nicks one strand of the ICE dsDNA and covalently attaches to the 5′ end of the nicked DNA, forming the transfer DNA (T-DNA). (d) If an appropriate recipient is available, the conjugation machinery transports the T-DNA into the recipient cell. (e) In the recipient cell, the relaxase ligates the 5′ and 3′ ends of the DNA to form a covalently closed ssDNA circle. The complementary DNA strand is synthesized to generate a dsDNA circle that is the substrate for integration into the host chromosome. In the donor, the remaining DNA strand likely serves as the template for rolling-circle replication, generating a dsDNA circle that can then reintegrate into the host chromosome. Without this synthesis and reintegration, the ICE would be lost from cells in which it had excised. (f) In both the donor and recipient, the circular dsDNA ICE integrates into the host chromosome.

Figure 2

Mechanisms that generate diversity between ICEs (integrative and conjugative elements). Double black lines represent the host chromosome and double red, orange, and blue lines represent mobile genetic elements as indicated. Double green lines indicate regions of homology. Rectangles behind double lines indicate att (attachment) sites or insertion sites. (a) An ICE can insert in an att site (purple boxes) next to a heterologous mobile element, such as a CIME (cis-mobilizable element), generating a construct with a total of three att sites. During excision the ICE might utilize the two att sites farthest apart, thereby generating a single dsDNA circle that contains both elements (CIME and ICE) that then serves as the substrate for conjugation. (b) An ICE can insert into an att site next to another ICE that is occupying the preferred att site, forming a tandem insertion. The tandem ICEs can recombine at regions of homology, removing the intervening sequence and forming a single, chimeric ICE. (c,d) Other mobile genetic elements can insert into an ICE. (c) Elements can transpose from another chromosomal location (Ci), or extrachromosomal elements can recombine into an ICE (Cii) by either site-specific recombination or homologous recombination. These elements then become part of the ICE and are transferred with the ICE in cis during conjugation. (d) T-DNA from an external element can recombine into a cognate oriT site on the ICE. Insertion is more efficient when the oriT is located on the lagging strand, likely indicating that the target is ssDNA, as shown. The relaxase is bound to the transferred DNA at one catalytic tyrosine residue, and nicks and binds the oriT of the resident element with a second catalytic tyrosine residue [both catalytic tyrosine residues are required for this activity (1)]. The relaxase also joins the free 3′-OH end of the chromosomal nick to the 5′ end of the T-DNA and the 3′-OH end of the T-DNA to the 5′ end of the chromosomal nick. The insertion is then replicated by the host machinery and becomes established in the chromosome.

Figure 3

Conjugation resembles rolling-circle replication. Both processes require similar initial steps that generate a substrate that may be suitable for transfer during conjugation. The ICE (integrative and conjugative element) is shown as double blue lines. In both processes, a relaxase recognizes and nicks a cognate ori, binding to the free 5′ end. Helicase activity (purple triangle) and a single-strand binding protein (turquoise circle) are required to unwind the single-stranded DNA. Replacement synthesis of the unwound strand can occur but is not required for unwinding. If replacement synthesis occurs, a second nicking event at the reconstituted ori is likely to be required to generate a free 3′-OH group for recircularization of the unwound strand.

Figure 4

Cell-cell signaling induces ICEBs1. The pathway by which cell-cell signaling regulates ICEBs1 gene expression is shown. Arrows indicate positive regulatory effects. Lines with cross bars indicate negative regulatory effects. Proteins and peptides are shown with a brief explanation of their role or activity. The PhrI signaling peptide is likely either a pentapeptide (5) or hexapeptide (104) with sequence (A)DRVGA.

Figure 5

Excision of Tn916 allows expression of conjugation genes. Both linear and circular maps of Tn916 are shown. Genes are shown as arrows on the map. Some known promoters are shown as bent arrows. While the element is in the chromosome, there is a low level of transcription (red dashed arrows) of genes in the regulatory region (white arrows), including xis and int. The genes upstream of tetM, including the relaxase and conjugation genes, are not expressed. Excision and circularization of Tn916 make orf20 (relaxase) and the conjugation operon contiguous and codirectional with the regulatory region, allowing all these genes to be transcribed. The dashed red arrows are used to illustrate this phenomenon and do not depict the known variety or relative abundance of transcripts produced by Tn916.