Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow

Key Points

  • Integrative and conjugative elements (ICEs) are found in a diverse array of Gram-negative and Gram-positive bacteria. ICEs are integrated into host chromosomes but can excise, circularize and transfer (through conjugation) to neighbouring cells.

  • The genes encoding key components of the ICE life cycle are often grouped into functional modules. Modules may be exchanged among ICEs as well as with other mobile elements that comprise the mobilome.

  • In addition to the core modules that mediate ICE integration, excision, conjugation and regulation, ICEs routinely encode a range of accessory functions, including virulence factors and resistance proteins for antibiotic and heavy metal resistance.

  • ICEs integrate with varying degrees of site specificity. Integrases, which mediate integration, are typically tyrosine recombinases, although there are a few cases of ICEs using a DDE transposase or a serine recombinase for this function. Integrases are also required for excision, although other factors are usually required in addition.

  • Conjugal transfer requires DNA processing, which is accomplished by a relaxase. Rolling circle replication is thought to be the primary process that liberates a single-stranded DNA molecule for transfer. The type IV secretion system seems to be the most common mechanism used by ICEs for horizontal DNA transfer.

  • There are varied and complex mechanisms that govern ICE transfer. Many ICEs encode unique factors that influence their excision and transfer frequencies.

Abstract

Integrative and conjugative elements (ICEs) are a diverse group of mobile genetic elements found in both Gram-positive and Gram-negative bacteria. These elements primarily reside in a host chromosome but retain the ability to excise and to transfer by conjugation. Although ICEs use a range of mechanisms to promote their core functions of integration, excision, transfer and regulation, there are common features that unify the group. This Review compares and contrasts the core functions for some of the well-studied ICEs and discusses them in the broader context of mobile-element and genome evolution.

Main

New genetic material can arise in bacteria either de novo, by internal genetic mutation, or from external sources, through horizontal gene transfer (HGT). HGT allows bacteria to rapidly acquire complex new traits, and it has been and remains a key driving force in bacterial evolution1,2,3,4,5,6,7. Conjugation, transduction and transformation are the main mechanisms of HGT. Conjugation requires direct contact between donor and recipient cells — the donor cell synthesizes a multiprotein apparatus that connects the two cells (called the 'mating pair') and enables the transfer of DNA. The DNA to be transferred is often processed in the donor cell to become single stranded, and it is then converted to double-stranded DNA by the replication machinery of the recipient cell. Most of the identified conjugative systems are encoded by plasmids, and plasmid-derived DNA is the typical substrate for these systems. However, in recent years it has become evident that conjugation systems are also widespread in chromosome-borne mobile genetic elements (MGEs). Such elements are often referred to as integrative and conjugative elements (ICEs). In this Review we discuss the steps of the ICE life cycle, including integration, excision, conjugation and regulation, in several experimentally characterized ICEs.

Integrative and conjugative elements

ICEs are self-transmissible MGEs that encode the machinery for conjugation as well as intricate regulatory systems to control their excision from the chromosome and their conjugative transfer8,9,10. They integrate into and replicate as part of the host chromosome. Certain conditions induce the excision of ICEs from the chromosome, after which they circularize and are replicated and then transferred to new hosts by the conjugation machinery encoded in the elements. The ICE in the recipient cell integrates into the chromosome, and the copy of the ICE that remains in the donor cell reintegrates into the donor cell chromosome (Fig. 1). Thus, these elements combine features of other classes of MGEs, such as phages (which often integrate into and excise from the host chromosome but are not transmitted by conjugation), transposons (which integrate into and excise from the chromosome but are not transferred horizontally) and plasmids (which sometimes transfer from cell to cell by conjugation but replicate autonomously). ICEs, unlike plasmids, cannot be maintained in an extrachromosomal state, as they seem to be incapable of autonomous replication, although this is still under investigation (discussed further below).

Figure 1: Schematic of a typical integrative and conjugative element life cycle.
figure1

An integrative and conjugative element (ICE) is integrated into one site in the host chromosome and is bounded by specific sequences on the right (attR) and left (attL). Excision yields a covalently closed circular molecule as a result of recombination between attL and attR to yield attP (in the ICE) and attB (in the host chromosome). An ICE-free cell can serve as a potential recipient. During conjugation, the donor and recipient are brought in close contact, and a single DNA strand is transferred to the new host through the action of rolling circle replication. Following transfer, DNA polymerase in the recipient synthesizes the complementary strand to regenerate the double-stranded, circular form. A recombination event between attP and attB results in integration into the host chromosome.

The ICEs encompass all self-transmissible integrative and conjugative mobile elements regardless of their mechanisms of integration or conjugation10,11,12, including elements that are commonly characterized as conjugative transposons, which often integrate into the host chromosome with minimal sequence specificity and, consequently, are capable of both intracellular and intercellular transfer (for example, the first known MGEs with ICE-like properties: Tn916 in Enterococcus faecalis and CTnDOT in Bacteriodes thetaiotaomicron )13,14. In addition, ICEs include elements for which the integration sites are far more restricted, such as the Vibrio cholerae -derived ICE SXT, which was the first MGE with ICE-like properties to be described in the Gammaproteobacteria15,16. Certain chromosomal elements that have been classified as genomic islands, such as ICEclcB13 and ICEMlSymR7A (Refs 17, 18), also fall within the ICE rubric, and many genomic islands that are not mobile may be defective ICEs. ICEs have been difficult to identify experimentally, as they are usually physically linked to the bacterial chromosome. However, in silico analyses of numerous complete bacterial genomes have revealed that ICEs are probably found in many bacterial subdivisions10.

ICEs and related elements such as integrative and mobilizable elements (IMES) and cis-mobilizable elements (CIMES) (Box 1) can constitute a notable proportion of the variable DNA found in bacterial chromosomes. For example, a comparative analysis of the genomes of different isolates of Streptococcus agalactiae (also known as group B Streptococcus) revealed that nearly two-thirds of the regions of diversity in the isolates are comprised of ICEs or ICE-like elements19. Even more striking was the finding that over one-third of the entire 2 Mb genome of Orientia tsutsugamushi , an obligate intracellular bacterium belonging to the family Rickettsiaceae, is derived from an ICE known as OtAGE20. This 33 kb ICE underwent massive amplification and then subsequent degradation; it is not known how many of the copies, if any, are now self-transmissible.

ICEs typically have modular structures, with genes clustered according to the process to which they contribute. In particular, all ICEs contain three distinct modules that mediate their integration and excision, conjugation, and regulation (Box 2; Fig. 2). However, these essential functions are not carried out in the same way by all ICEs; instead, they proceed using a diverse range of genes and mechanisms. Below, we discuss in more detail several of the different functional ICE modules that have been characterized.

Figure 2: Schematic of the genetic organization of SXT–R391 family integrative and conjugative elements.
figure2

a | All SXT–R391 family integrative and conjugative elements (ICEs) have 52 nearly identical core genes. These ICEs contain different DNA insertions at five hotspots (labelled HS1–HS5). b | The proposed minimal gene set required for SXT function. These can be divided into four modules: intxis is the integration and excision module; mob is the DNA mobilization and processing module; mpf is the mating-pair formation module and reg is the regulation module. Orange genes are conjugation related, purple genes are involved in regulation, red genes are involved in integration and excision, and grey genes have accessory or unknown functions. int, integrase; oriT, origin of transfer; tra, conjugal transfer; xis, excisionase.

Despite having a common life cycle (Fig. 1) and modular structure, ICEs can usually be distinguished by element-specific properties (Table 1). Individual ICEs have been found to bestow a wide range of phenotypes on their hosts, including resistance to antibiotics15,21,22,23 and heavy metals21,24 and the capacity to degrade aromatic compounds17. In addition, complex traits such as the ability to colonize a eukaryotic host25, fix nitrogen25 or promote virulence and biofilm formation have been documented24,26,27. Finally, the link between ICEs and the dissemination of antibiotic resistance genes in several pathogens means that these mobile elements have substantial clinical relevance23,28,29.

Table 1 Key characteristics of experimentally described integrative and conjugative elements (ICEs)

Integration

All ICEs encode an integrase (Int) that enables their integration into the host chromosome. These integrases are necessary and sufficient to mediate integration and are also required for excision, although this process requires additional factors in most cases. Integrases determine the site (or sites) of ICE insertion and, often, the frequency of ICE excision. Moreover, regulation of int expression is one of the key means of controlling ICE transmission.

Most of the known ICE integrases are members of the tyrosine recombinase family containing a signature R..H..R..Y tetrad in the carboxy-terminal catalytic core of the protein30,31,32. The best studied recombinase of this family is the Int encoded by phage λ. This Int uses a topoisomerase I-like mechanism to promote site-specific recombination between identical or near-identical sequences in the host chromosome (referred to as attB sites) and the phage chromosome (the attP site)33. The strand exchange reactions catalysed by Int do not require a high-energy cofactor such as ATP, and no sequence duplication or deletion results from recombination. Many ICEs, including members of the SXT–R391 family, integrate into the host chromosome by a similar mechanism to phage λ15,16,34. The Int encoded by this ICE family is 46% similar to Int from the lambdoid phage φ80. SXT Int mediates site-specific recombination between identical attP and attB sites; the attB site is located in the peptide chain release factor 3 gene (prfC), which encodes a non-essential release factor that is important for translation termination16.

Integrases of numerous other ICEs, including the phage P4-like integrases of ICEclcB13 (Ref. 35) and ICEMlSymR7A(Refs 25, 36), the XerC/XerD-like integrases of PAPI-1 and ICEHin1056 (Refs 28, 37, 38), and integrases that are not currently classified in a subfamily, such as the pSAM2 and ICEBs1 recombinases, probably function in a similar manner to SXT Int. Notably, although these ICEs are present in diverse host backgrounds, including Mesorhizobium loti (ICEMlSymR7A), Haemophilus influenzae (ICEHin1056), Bacillus subtilis (ICEBs1) and Pseudomonas spp. (PAPI-1 and ICEclcB13), they all mediate integration into the 3′ ends of tRNA loci. Most bacteria contain multiple alleles encoding a particular target tRNA, but ICEs integrate into only one of these loci, probably owing to optimal sequence conservation between the attB and the attP sites. Two exceptions are ICEclcB13, found in Pseudomonas knackmussii str. B13 (Refs 17, 39), which can integrate in either of two proximally located tRNAGly genes, and PAPI-1, found in Pseudomonas aeruginosa , which uses either of two tRNALys genes38. Recently, it was shown that ICEclcB13 often reintegrates into the other tRNAGly alleles following excision40. Insertion mediated by Int into a particular site (or sites) reflects a preference for the typical attB sequence rather than an absolute dependence on it, as several ICEs integrate with varying efficiencies into secondary insertion sites when primary sites are not available41,42.

By contrast, some Int proteins mediate ICE integration with lower sequence specificity. For example, the Int of Tn916, an 18 kb ICE originally described in E. faecalis str. DS16 (Ref. 14), has a very broad site preference, inserting in sequences that are AT-rich or bent43,44. The Tn916 Int belongs to the transposase subfamily of tyrosine recombinases31,32, members of which have a distinct evolutionary origin from the phage λ Int but are thought to guide strand exchanges similarly45. During recombination, attP and attB are cleaved to yield 6 bp overhangs (known as 'coupling sequences') at both sites46,47. As these overhangs do not base pair, either replication or mismatch repair must resolve the heteroduplex generated during integration. The Int of CTnDOT, a 65 kb ICE found in Bacteroides species13, functions in a similar manner as the Int of Tn916, even though the two enzymes do not share primary-sequence similarity48,49. CTnDOT favours sites that are characterized by a consensus sequence of GTANNTTTTGC48,50, thus inserting into fewer sites than Tn916.

Not all ICEs rely on tyrosine recombinases for their integration and excision. TnGBS2, an ICE detected in S. agalactiae, uses a DDE transposase that contains a catalytically active triad of acidic amino acids (D..D..E)51. This ICE integrates primarily in intergenic regions upstream of promoters regulated by RNA polymerase factor σA (also known as RpoD)52.

Finally, Tn5397, a 21 kb ICE identified in Clostridium difficile that shares large regions of sequence similarity with Tn916 but has distinct 5′ and 3′ ends, uses a serine recombinase, TndX, which mediates integration using a catalytic serine residue in its amino terminus. All sites of Tn5397 insertion contain a central GA dinucleotide, but no other consensus insertion sequence has been identified53. Tn916 and Tn5397 encode similar conjugation machineries54,55 but integrate and excise using different mechanisms owing to the difference in their recombinases, underscoring the idea that ICEs can be thought of as amalgams of functional modules. The exchange of integration modules among ICEs or between ICEs and other integrating MGEs (such as phages) can potentially create new ICEs with altered insertion site specificities and, perhaps, altered host ranges.

Excision

To transfer to a new host, an integrated ICE must give rise to an extrachromosomal form of the element. For those ICEs that have been characterized, excision also requires Int. In many cases, the insertion site is left perfectly intact after excision owing to the conservative mechanism of Int-mediated recombination. As excision proceeds in the reverse direction to insertion through recombination of the sequences generated by integration (attR and attL), similarly to Int-mediated excision of the λ prophage, additional 'recombination directionality factors' (RDFs) are often used to bias the process towards excision rather than integration. RDFs are small, positively charged DNA-binding proteins that influence DNA architecture or DNA–protein interactions to promote Int-mediated excision and inhibit Int-mediated integration56. For several characterized ICEs no RDFs have been detected to date, which may reflect the fact that they are difficult to detect using bioinformatic approaches. Thus, additional RDFs are likely to be discovered even in characterized elements.

Excisionase (Xis), the RDF of Tn916, is required for excision in Gram-positive hosts and substantially increases the frequency of excision in Gram-negative hosts57,58. It binds to the ends of the integrated element adjacent to Int binding sites59,60; mutations that prevent Xis from binding to the left end of Tn916 reduce the excision frequency, whereas mutations that block Xis binding to the right end of Tn916 increase the excision frequency58,60. Excision of Tn916 occurs at a low frequency (although this varies markedly depending on host background61) and requires both Int and Xis; therefore, Xis must outcompete Int for binding of the right end of the element to keep the element integrated in the chromosome58.

CTnDOT and Tn916 each form a heteroduplex DNA produced by pairing of nonhomologous coupling sequences during excision. However, these elements use nonhomologous integrases and therefore rely on distinct auxiliary factors for excision. Excision of CTnDOT requires Exc, which has in vitro topoisomerase activity62,63 and does not seem to function as a traditional RDF, and two other proteins. The roles of these three proteins and the complete mechanism of CTnDOT excision are not fully understood.

The dynamics of ICE integration and excision are a characteristic feature of individual ICEs that can constrain the rate of ICE transfer. For example, the excision frequency of SXT is far higher than its transfer frequency, and factors that increase excision do not necessarily increase transfer, indicating that excision is not always the limiting step for transfer of ICEs42. Interestingly, excision of SXT does not require the encoded Xis, but excision is reduced 1,000-fold in the absence of Xis42. Moreover, SXT Xis and Int are not co-regulated, suggesting that the relative levels of these proteins probably vary according to whether the element needs to integrate (for example, after conjugation) or whether excision is preferable42.

Environmental factors can also influence the frequency of ICE excision. For example, excision of ICEclcB13 increases as the host cells enter stationary phase35. ICEclcB13 encodes genes for the utilization of 3-chlorobenzoate (CBA) as a sole carbon and energy source17; supplementing the growth media with CBA induces Int expression and ICEclcB13 excision and transfer during stationary phase. Thus, CBA may modulate the balance between a positive regulator and a putative repressor to promote ICE excision and transfer64. Excision of ICEMlSymR7A shows a similar growth-phase dependence, although the process by which it is induced is unknown36.

ICE maintenance

Excision of an ICE from the chromosome could lead to loss of the element from the host cell. If chromosome replication or cell division occurs after ICE excision, the element may not be transmitted to progeny. The frequency with which ICE-free cells arise is thought to be low, in part because replication of the excised ICE increases the number of elements available for reintegration, as proposed for ICEMlSymR7A and ICEBs136,65. Reintegration after excision, rather than loss, has been observed for at least one element40, and other elements have been found to encode factors that minimize the emergence of ICE-free cells.

Two distinct processes that prevent ICE loss are known. In P. aeruginosa, maintenance of PAPI-1 requires an ICE-encoded homologue of Soj, a protein that has been implicated in chromosome partitioning in B. subtilis66,67 and in plasmid maintenance68. Although wild-type PAPI-1 is excised from the chromosome in only 0.16% of host cells, PAPI-1 lacking Soj is lost from all cells38. Expression of the protein occurs only when PAPI-1 is excised and circularized, when Soj activity is required. It has been proposed that Soj stabilizes the excised PAPI-1, although its precise mechanism of action remains to be determined.

By contrast, SXT maintenance is promoted by the presence of a functional toxin–antitoxin pair, MosT and MosA69,70,71,72. Deletion of mosA and mosT increases the frequency of ICE loss by several orders of magnitude72. As with PAPI-1 soj, expression of mosA and mosT is upregulated when the ICE is excised, thus promoting SXT maintenance when the element is vulnerable to loss. However, the low frequency of SXT loss even in the absence of mosA and mosT suggests that this element may encode additional factors to promote its maintenance.

Conjugative transfer

DNA processing. Conjugal DNA transfer requires processing of extrachromosomal ICE DNA. Most models of ICE DNA processing are based on those of plasmid processing, as the proteins involved in plasmid and ICE transfer are similar. For plasmid transfer, a relaxase covalently binds to and nicks the plasmid DNA at the origin of transfer (oriT) to enable initiation of rolling circle replication73. The relaxase remains bound to the displaced single-stranded DNA and interacts with a coupling protein (typically conjugal transfer protein G (TraG)) to target the nucleoprotein complex to the mating pore for transfer of the DNA to the recipient cell74,75.

Putative and experimentally proven oriT sequences and their cognate relaxases have been identified in several ICEs. The ICEBs1 oriT was discovered through a genetic screen and by the identification of an inverted repeat, which is a common feature of oriT sequences in conjugative plasmids76. The repeat is located directly upstream of the gene encoding the relaxase, NicK, which cleaves oriT in the inverted repeat76. ICEBs1 oriT and NicK are highly similar to the oriT and relaxase (TecH) of Tn916 (Refs 77, 78). Interestingly, the SXT relaxase, traI, is separated from its cognate oriT79 by 40 kb. Basic local alignment search tool (BLAST) analyses suggest that sequences similar to the SXT oriT are also present in various chromosomes independently of ICE sequences. These oriT-like sequences seem to be parts of genetic islands that can be mobilized in trans by SXT (Burrus, V., personal communication).

Replication. After the single-stranded DNA is transferred to a recipient cell, it becomes a template for DNA polymerase to reconstitute the double-stranded circular molecule (Fig. 1). Thus, according to this model, ICEs are not truly replicative, as the copy number of the element does not increase in the donor following rolling-circle replication. However, some ICEs may have replicative forms. In E. coli, the number of SXT attP sites (which are on the unintegrated element) is slightly higher than the number of attB sites (which are on the chromosome)42, suggesting that more than one copy of extrachromosomal SXT can exist in a cell. Furthermore, the copy number of the excised form of ICEBs1 can increase twofold to fivefold under inducing conditions65, in a process that requires NicK65. Interestingly, pSAM2 undergoes genuine rolling circle replication in the donor cell, producing multiple copies of this ICE. However, this element is transferred as a double-stranded DNA molecule, unlike the transfer of single-stranded DNA that has been described for other ICEs and plasmids80,81. Finally, ICEHin1056 may be capable of autonomous replication82,83.

DNA secretion systems. There have been few experimental studies of ICE-encoded conjugation apparatuses. However, bioinformatic analyses suggest that systems similar to type IV secretion systems (T4SSs) are most commonly used to transfer ICE DNA in Gram-negative bacteria. T4SSs consist of a protein complex that forms a membrane-spanning secretion channel and often include an extracellular component such as a pilus84; these systems have been adapted for the transport of proteins and for DNA exchange with the environment85.

Nearly all ICEs found in Gram-negative bacteria contain at least one gene with a homologue in a type IVA secretion system, and these genes have been designated tra, vir or trb to indicate their similarity to genes found in the F, pTi or RP4 plasmids, respectively.

Several ICEs encode entire transfer systems that are similar to plasmid-borne T4SSs. For example, the proteins that mediate SXT conjugation have 34–78% identity with those that mediate conjugation of the IncA–IncC plasmids, and the genes encoding these proteins are organized similarly86,87. This suggests that they share a common ancestor and further underscores the concept that ICEs are composed of potentially interchangeable functional modules and such modules can be exchanged between different types of MGEs.

Little is known about the conjugative systems in the ICEs of Gram-positive bacteria. However, ICEBs1 ConE is similar to VirB4, an ATPase that may provide energy for substrate transfer or machinery assembly84. ConE localizes to the cell poles and is required for ICEBs1 conjugation88. However, none of the other transfer genes of Gram-positive ICEs is clearly related to a gene of a T4SS.

In addition, some newly discovered ICE-encoded T4SSs are not obviously related to known T4SS proteins. Thus, a category of 'other T4SSs' has been proposed to encompass these distinct systems84,89. For example, ICEHin1056 and ICEclcB13 encode evolutionarily distinct T4SSs that have thus far been identified only in ICEs or genomic islands90,91.

Some ICE T4SSs are adapted for purposes other than conjugative transfer. For example, M. loti uses ICEMlSymR7A to mediate its symbiotic relationship with the plant Lotus corniculatus. This ICE encodes two separate T4SSs. The first T4SS, encoded by the trbB operon, mediates conjugal transfer between bacteria, whereas the second T4SS is a VirB/D4-like T4SS that mediates the secretion of at least two effector proteins into plants and thereby induces the formation of nitrogen-fixing nodules on the plant roots92,93.

Other ICEs rely on secretion systems that are not T4SSs. One of these ICEs, pSAM2, found in Streptomyces ambofaciens , uses a DNA transfer system similar to those found in Streptomyces plasmids. When hyphal tips of S. ambofaciens grow in close proximity, their intertwining can create sufficient cell-to-cell contact to enable conjugal DNA transfer. Remarkably, only one gene, traSA, is required for the inter-mycelial transfer of pSAM2 (Ref. 4). TraSA is similar to B. subtilis SpoIIIE and E. coli FtsK, which are DNA translocases that act as pumps for double-stranded DNA during sporulation and chromosome segregation, respectively80,95. TraB, a TraSA homologue encoded on a Streptomyces plasmid, has sequence-specific DNA-binding activity that facilitates plasmid transfer96. In contrast to other ICE conjugation systems, TraSA probably transfers double-stranded DNA80. After transfer, pSAM2 spreads among adjacent mycelial hyphae in a process mediated by additional pSAM2 genes such as spdABCD, which encode small hydrophobic proteins94.

ICE exclusion. Several mobile elements can prevent host cells from acquiring other closely related elements. The members of the SXT–R391 family of ICEs can be divided into two exclusion groups, denoted S and R97. Hosts that harbour an S group ICE limit the acquisition of another S group ICE but not the acquisition of an R group ICE. This exclusion activity is mediated by TraG and entry exclusion protein (Eex), which are inner-membrane proteins expressed in donor and recipient cells, respectively98. Surprisingly, it is the cytoplasmic domains of these proteins that mediate the exclusion specificity, suggesting that TraG may be transferred to the recipient cell during conjugation99. Exclusion is only partial and redundant transfer (that is, transfer into a cell that already contains an ICE) can occur. This results in the formation of tandem arrays of identical ICEs100; however, owing to the instability of these arrays100,101 only a single SXT–R391 family ICE remains in the host chromosome.

By contrast, the exclusion system of ICEBs1 is similar to bacteriophage immunity systems. This ICE encodes the transcriptional regulator ImmR, which functions as an immunity factor in addition to its function as master regulator, similarly to the phage λ repressor protein, CI (discussed below)102. Overexpression of ImmR in a recipient renders a cell resistant to transfer of ICEBs1 by preventing Int expression; expression of Int in trans can overcome ImmR-mediated exclusion103. Another method is seen in pSAM2, which uses pSAM2 immunity factor (Pif) to prevent transfer initiation in a donor104. Exclusion requires the hydrolase nudix motif in Pif, but the exact mechanism remains unclear104.

Host range. Many ICEs can transfer to a wide range of species. For example, Tn916 and Tn916-like elements have been detected in Proteobacteria (including Pseudomonas spp., Aeromonas spp., E. coli and Haemophilus spp.), Actinobacteria and Firmicutes105. ICEclcB13 can transfer between several species of Gammaproteobacteria and Betaproteobacteria, and can do so in a 'real world' environment of activated sludge and waste water106,107. ICEBs1 can transfer to Bacillus anthracis as well as Listeria monocytogenes 108, and ICEMlSymR7A can transfer among several Rhizobium species; thus, these ICEs can confer the ability to nodulate L. corniculatus on bacteria that are not typically symbionts18. SXT can be transferred from V. cholerae and E. coli to a range of Gram-negative species in the laboratory15. However, the factors that determine the host range of most ICEs have not been extensively explored. Antirestriction genes that might augment the establishment of ICEs in new host genomes have been described in Tn916 (Ref. 109), and other factors that regulate the transfer of ICEs between unrelated species will undoubtedly be uncovered in the future.

Regulation

ICE transmission is governed by complex regulatory networks that can be activated and repressed by environmental stimuli. These signals influence the expression and activity of ICE-derived and host-derived factors to modulate ICE gene expression and ICE transfer (Fig. 3).

Figure 3: Examples of integrative and conjugative element regulation.
figure3

Integrative and conjugative elements (ICEs) have complex regulatory networks to control integration, excision and transfer. Conjugation machinery is encoded by multiple genes, with the exception of the single pSAM2 gene traSA. a | In SXT, the SOS response mediates autoproteolysis of SetR through activated recombinase A (RecA), relieving SetR-mediated repression of an operon including the activator genes setCD. SetC and SetD increase expression of the integrase gene (int) as well as the genes encoding the conjugation machinery. b | In ICEBs1, the SOS response as well as response regulator aspartate phosphatase I (RapI) positively regulate the ImmR–ImmA operon. Proteolytic inactivation of ImmR by ImmA derepresses an operon encoding excisionase (Xis) and the conjugation machinery. When phosphatase RapI inhibitor (PhrI) levels are high or in the presence of AbrB, rapI is repressed. c | In CTnDOT, tetracycline enables elongation of an mRNA (dashed blue line) that originates from PtetQ and extends through rteA and rteB via a translation attenuation mechanism. RteB upregulates the expression of rteC. RteC positively regulates the excision genes, which in turn lead to upregulation of the genes encoding the conjugation machinery. However, a small non-coding RNA, rteR (dashed turquoise line), which is controlled by the same promoter as the excision genes, can independently repress the expression of the conjugation machinery. d | In Tn916, a transcript originating at tetM and continuing through to orf7 (dashed blue line) extends through to orf8 (dashed red line) in the presence of tetracycline. The products of orf7 and orf8 directly upregulate expression from a promoter upstream of orf7. Transcription through orf9 results in the production of an antisense RNA (dashed purple line) that represses the translation of the sense orf9 mRNA, which is a repressor of Porf7. Transcripts originating from orf7 (dashed grey line) are then elongated through xis, int and the conjugation genes if Tn916 is excised and in its circular form. e | In ICEclcB13, integrase regulator (InrR) upregulates expression from Pint to promote excision. Following excision, a strong constitutive promoter, Pcirc, is brought in proximity to int, thereby driving reintegration into the original host chromosome or into the chromosome of a new host following conjugation. f | In pSAM2, putative replication activator (Pra) is thought to influence transfer rates by directly upregulating the expression of the replication initiator gene (repSA), xis and int. KorSA represses expression of pra. CBA, 3-chlorobenzoate.

For all SXT–R391 family ICEs, SetR, a homologue of the phage λ repressor CI, is the main regulator of transfer (Fig. 3a). This protein binds to operators located immediately upstream of setR110 to inhibit the expression of an operon that includes setD and setC, which encode the master transcriptional activators of the SXT genes required for SXT transfer, including the int and tra operons42. Overexpression of setC and setD is toxic to host cells, indicating that unfettered expression of the SXT T4SS damages the host cell. SetC and SetD also upregulate the expression of various genes that are not required for transfer of SXT, including the genes of the toxin–antitoxin pair MosA and MosT72.

DNA-damaging agents that induce the host SOS response, such as mitomycin C or quinolone antibiotics, can increase the transfer of SXT by several hundred times. As SetR is similar to phage λ CI, the SetR-mediated repression of transfer genes may be relieved by recombinase A (RecA)-assisted autocleavage of SetR111,112. This model is supported by the finding that the transfer frequency of an SXT element encoding a SetR variant that is resistant to RecA cleavage is not increased by mitomycin C111. Hence, the widespread use of quinolone antibiotics such as ciprofloxacin may promote the spread of SXT and related ICEs.

Induction of the SOS response also promotes the transfer of ICEBs1 (Fig. 3b). Mitomycin C treatment induces a notable increase in RecA-dependent excision and transfer of ICEBs1 (Ref. 108), but the molecular events underlying this activation differ from those observed for SXT. The repressor ImmR normally inhibits expression of xis and additional downstream genes but is cleaved following induction of the SOS response. This cleavage is mediated by the antirepressor metallopeptidase (ImmA), encoded downstream of ImmR113. Homologues of ImmA and ImmR have been identified in a range of prophages and putative MGEs in Gram-positive bacteria, suggesting that antirepressor-mediated cleavage of repressors may be a common regulatory mechanism of transfer for diverse MGEs113. Quorum sensing can also play an important part in the regulation of ICE transfer, as shown for ICEMlSymR7A and ICEBs1 (Refs 36, 114) (Fig. 3b). In ICEBs1, the activity of response regulator aspartate phosphatase I (RapI), the ICE-encoded transcriptional activator that controls the expression of many ICEBs1 genes — including xis — is blocked by the quorum-signalling peptide phosphatase RapI inhibitor (PhrI)108. PhrI accumulates at high densities of ICEBs1-bearing cells, blocking RapI activity and inhibiting excision and transfer of ICEBs1. By contrast, in stationary phase cultures containing a minority of ICEBs1 donors, PhrI levels are low and ICEBs1 transfer can occur108. However, little ICEBs1 excision occurs in exponential cultures regardless of PhrI levels, possibly owing to negative regulation of rapI by a chromosome-encoded transition state regulator, AbrB. As a result of this regulation, ICEBs1 transfer is inhibited when the number of potential recipients is low108.

Inhibition of protein synthesis by tetracycline can promote the transfer of Tn916 and is required for the excision of CTnDOT115. For CTnDOT, tetracycline activates expression of tetQ, a tetracycline resistance gene, and 2 downstream genes, rteA and rteB, ultimately leading to increased transcription of rteC and several genes required for CTnDOT excision, including exc116,117. Tetracycline stimulation of CTnDOT excision and subsequent transfer occurs through a translation attenuation mechanism involving the leader region 5′ of tetQ118,119,120. In contrast to its effect on CTnDOT, tetracycline does not increase the excision of Tn916 (Ref. 121) but, instead, enables transcription of the tra genes required for transfer of the ICE (Fig. 3d). Transcription of two important regulatory genes, orf7 and or f 8, is driven from an upstream promoter, PtetM, located 5′ of the ribosomal tetracycline resistance gene tetM, as well as from a promoter upstream of orf7 (Ref. 122). In the absence of tetracycline, transcripts initiating from PtetM are short, as the polymerase stalls on a leader sequence that encodes a peptide of rare amino acids, thereby allowing the formation of an RNA stem loop that acts as a transcription terminator105,122,123. However, in the presence of tetracycline, transcription extends through orf7 and orf8, which encode proteins that promote transcription from Porf7122. This extended transcript also includes RNA that is complementary to transcripts from orf9, a gene that is upstream of and in the opposite orientation to orf7 and orf8, and which encodes a putative repressor of Porf7. Thus, transcription from PtetM in the presence of tetracycline both reduces the Orf9-mediated repression of Porf7 and promotes Porf7 autoactivation, which in turn leads to transcription of int and xis (which are downstream of orf8). In addition, once the ICE has circularized, Porf7 also drives expression of the tra genes, which lie at the opposite end of the integrated element.

Expression of int in ICEclcB13 is also regulated by circularization (Fig. 3e). In the integrated state, int is transcribed from Pint, a weak promoter under the control of a positive integrase regulator (InrR) and various environmental factors, including CBA (a compound that can be degraded by ICEclcB13-encoded proteins)124,125. However, circularization places a strong constitutive promoter, Pcirc, upstream of int, increasing int expression independently of factors that regulate Pint124. Thus, circularization of ICEclcB13 augments int expression, aiding the integration of the element back into the host genome or, following conjugation, into the genome of a new host.

Less is known about the factors that regulate transfer of the ICEs of actinomycete species. In the case of pSAM2 (Fig. 3f), the regulator protein putative replication activator (Pra) promotes expression of an operon containing the replication initiator gene (repSA) as well as int and xis126,130. Expression of pra is repressed by KorSA94,127, but how this repression is alleviated remains to be defined.

Future areas for enquiry

ICE distribution and diversity. With the availability of an increasing number of fully sequenced bacterial genomes, continued efforts to annotate and understand the diversity and distribution of ICEs are warranted. As experimental characterization of ICEs has been carried out on only a few elements, it is unclear if these characterized ICEs are representative of all ICEs. However, many ICEs may not be recognizable by bioinformatic approaches, and devising experimental methods to identify new ICEs is an important challenge for future studies.

ICE effects on host gene expression and fitness. On the basis of studies with plasmids and transposons, it is thought that the acquisition of foreign DNA temporarily decreases host fitness128,129,130,131,132. ICEs challenge the host both with their gene contents and owing to the potential disruption of the integration site. Experimental evidence shows different responses to the introduction of ICEs. For example, the presence of CTnDOT in Bacteroides thetaiotaomicron VPI-5482 alters the expression of various genes, including genes of unknown function and genes that are encoded by other MGEs133. By contrast, the acquisition of ICEclcB13 by P. aeruginosa str. PAO1, a strain that does not normally harbour this ICE, resulted in few observable phenotypes or changes in gene expression and did not affect fitness as tested in competition experiments134. This lack of negative consequences may facilitate transmission of ICEclcB13 to new hosts. However, owing to the diverse insertion sites of ICEs and their extremely variable genetic content, it may be that not all ICEs have such minimal effects on their hosts. Finally, it is possible that ICEs will be found to encode factors that can minimize the fitness cost of acquiring new DNA, similarly to the recently discovered H-NS-like protein encoded on the Shigella flexneri plasmid pSf-R27, which minimizes changes in host transcription levels following acquisition of the plasmid132.

ICE cell biology. Despite recent progress in understanding the life cycles of various ICEs, many questions remain. We need a better understanding of the factors that govern the expression and activities of Int and Xis, as these proteins determine the balance between ICE integration and excision. Furthermore, the mechanisms that mediate and regulate mating-pair formation remain poorly understood, even though bacterial conjugation was first described over 50 years ago135. This is particularly true for Gram-positive conjugative elements. Applications of recent advances in fluorescence microscopy will help to address some of these questions88,136.

As most studies of ICEs have used cell populations to draw inferences regarding ICE regulatory processes, little is known about ICE biology at the single-cell level. It is clear that the state (for example, integrated versus excised) of an ICE in a population is not homogeneous. Recently, fluorescence microscopy techniques have been adapted to study ICE behaviour at the single-cell level, and such studies might reveal subtleties that are obscured when considering the entire population. For example, studies might determine whether elements generally reintegrate after excision, as suggested by analyses of ICEclcB13 integration site switching, or are instead lost and the now-ICE-free cells are therefore selected against using factors such as the SXT-encoded MosT toxin. Single-cell studies might also reveal the extent to which ICE gene expression is heterogeneous in a population. Indeed, by examining ICEclcB13 excision on the single-cell level, it was found that upregulation of inrR, a positive regulator of int, occurs in a bistable manner64. Thus, despite the recent progress in our understanding of ICE biology, much remains to be discovered.

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Acknowledgements

Our work on ICEs is supported by the US National Institute of Allergy and Infectious Diseases (grant R37 AI-42347) and the Howard Hughes Medical Institute. We thank B. Davis and A. Mandlik for comments on the manuscript and previous laboratory members B. Hochhut, J. Beaber, V. Burrus and J. Marrero, who all contributed to our studies of ICEs.

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DATABASES

Entrez Genome Project

Bacillus anthracis

Bacillus subtilis

Bacteriodes thetaiotaomicron

Clostridium difficile

Enterococcus faecalis

Haemophilus influenzae

Listeria monocytogenes

Orientia tsutsugamushi

Pseudomonas aeruginosa

Shigella flexneri

Streptococcus agalactiae

Streptomyces ambofaciens

Vibrio cholerae

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Glossary

Conjugation

Direct transfer of genetic material between two bacteria.

Transduction

Bacteriophage-mediated DNA transfer.

Transformation

The uptake of exogenous DNA from the environment.

Integrative and conjugative element

A chromosomal element that can be excised and transferred to another cell.

Hfr-like transfer

Transfer of adjacent chromosomal DNA by an integrated plasmid

Toxin–antitoxin pair

A system in which an unstable antitoxin prevents the action of a stable toxin. When the genes encoding this system are lost, the antitoxin is lost and the cell is killed by the toxin.

Bistable

Regulated in a manner that can result in one of two distinct states.

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Wozniak, R., Waldor, M. Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow. Nat Rev Microbiol 8, 552–563 (2010). https://doi.org/10.1038/nrmicro2382

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