Unbridled Integrons: A Matter of Host Factors

Abstract

Integrons are powerful recombination systems found in bacteria, which act as platforms capable of capturing, stockpiling, excising and reordering mobile elements called cassettes. These dynamic genetic machineries confer a very high potential of adaptation to their host and have quickly found themselves at the forefront of antibiotic resistance, allowing for the quick emergence of multi-resistant phenotypes in a wide range of bacterial species. Part of the success of the integron is explained by its ability to integrate various environmental and biological signals in order to allow the host to respond to these optimally. In this review, we highlight the substantial interconnectivity that exists between integrons and their hosts and its importance to face changing environments. We list the factors influencing the expression of the cassettes, the expression of the integrase, and the various recombination reactions catalyzed by the integrase. The combination of all these host factors allows for a very tight regulation of the system at the cost of a limited ability to spread by horizontal gene transfer and function in remotely related hosts. Hence, we underline the important consequences these factors have on the evolution of integrons. Indeed, we propose that sedentary chromosomal integrons that were less connected or connected via more universal factors are those that have been more successful upon mobilization in mobile genetic structures, in contrast to those that were connected to species-specific host factors. Thus, the level of specificity of the involved host factors network may have been decisive for the transition from chromosomal integrons to the mobile integrons, which are now widespread. As such, integrons represent a perfect example of the conflicting relationship between the ability to control a biological system and its potential for transferability.

Keywords: antibiotics resistances; bacterial evolution; bacterial genetics; horizontal gene transfer; host factors; integron; mobile elements; site specific recombination.

Figure 1

Organization of the integron system. Integrons include a stable platform and a variable cassette array. The platform is composed of the integrase gene (intI) under the control of the integrase promoter (P_int) and encoding the integron integrase (IntI). The platform also contains the cassette promoter (P_C), which directs transcription of proximal cassettes and the attI recombination site in which cassettes are inserted. The cassettes in the variable cassette array are represented by successive arrows. Their color intensities reflect their level of expression. Integrase catalyzes both excision (attC × attC) and insertion (attI × attC) of cassettes, leading to cassette shuffling.

Figure 2

att recombination sites. (A) Sequence of the double-stranded attI1 site. Direct repeats (DR1 and DR2), and both left and right (L and R) imperfect inverted repeats are indicated by grey arrows. Putative integrase binding sites are represented by green boxes. The cleavage site (CS) is indicated by a red arrow. (B) Schematic representation of the double-stranded (ds) and single-stranded (ss) bottom strand (bs) attC sites. Only the conserved nucleotides are indicated. Inverted repeats (R″, L″, L′, and R′) are indicated by grey arrows. Green boxes show putative IntI binding sites, and the cleavage site (CS) is indicated by a red arrow. R, purine; Y, pyrimidine; N, any bases; bs, bottom strand; and ts, top strand. For the attCbs, the structural features, namely, the unpaired central spacer (UCS), the extrahelical bases (EHBs), and the variable terminal structure (VTS), are indicated. The dotted lines represent the VTS length variability among attC sites.

Figure 3

Regulation of the Vibrio cholerae SCI promoters. The intIA and cassette promoters are regulated by several processes. The main triggering signal for integrase expression is the bacterial SOS response. Indeed, the integrase expression is repressed in normal conditions by the binding of the LexA repressor on the P_int LexA box. In stress conditions (genotoxic stress) or in conditions of horizontal transfer of single-stranded DNA (ssDNA), the RecA- ss nucleofilament is formed and induces the autoproteolysis of the LexA repressor, and the integrase is expressed. Both integrase (P_int) and cassette (P_C) promoters are regulated by the catabolic repression since both share a CRP box. For instance, in presence of glucose, the P_int and P_C promoters do not efficiently function.

Figure 4

attC site folding pathways. The different possible cellular pathways allowing proper folding of the attC sites are shown. The attC sites can be folded during the single-stranded DNA pathway (1) when delivered during replication, conjugation, phage infection, and transformation. They can also be folded from supercoiled double-stranded DNA (2) as cruciform structures. IntI and SSB monomers are, respectively, represented as grey ovals and grey circles. The origin of replication is represented by a purple circle and the newly synthesized leading and lagging strands by dashed purple lines.

Figure 5

Model of the SSB protein flattening the folded attC sites. The model shows how SSB opens the attC hairpin via the attC-6–SSB state, followed by the SSB-65 and SSB-35 modes, ensuring the integrity of the attC sites in absence of integrase (IntI). The integrase acts by capturing the attC sites at the moment of their extrusion, preventing the melting effect of SSB. Thus, SSB is released and the attC folded structure is stabilized.

Figure 6

Replicative resolution pathway during cassette insertion in attI site. Both substrates (attI-containing replicon and the cassette) are presented by purple lines, and the single-stranded attC bottom strand (ss attC_bs) and double-stranded attI (ds attI) sites are represented, respectively, by green and red lines. Note that the top strand of the attC site is represented as a dotted line because we do not know the nature of the cassettes (ss or ds). Synaptic complexes during the first strand cleavage and transfer are shown. Only one strand from each duplex is cleaved and transferred forming an atypical Holliday junction (aHJ) due to the single-stranded nature of the attC site. Each time, the four integrase protomers are shown. The two activated protomers are represented by dark gray ovals and the inactive ones by light gray ovals. The aHJ resolution implies a replication step. The origin of replication is represented by a purple circle and the newly synthesized leading and lagging strands by dashed purple lines. Both products are represented: the inserted cassette results from the bottom strand replication and the initial attI-containing replicon from the top strand replication. Hybrid attC and attI sites are indicated.

Figure 7

Model of the RecA mechanism of action in Vibrio cholerae during cassette insertion in attIA site mediated by IntIA. The first steps are the same as in Figure 3, up to the formation of the atypical Holliday junction (aHJ). After that, the model suggests an arrest of the replication fork due to the structure of the aHJ. In order to ensure the replication fork restart, we propose the intervention of both SSB (tetramers of SSB, grey circles) and RecA (orange circles) proteins. SSB-assisted RecA filament growth on hairpin DNA would allow for the replication fork to restart and resolve the aHJ. The SSB protein can bind the attC site in one of two binding modes, SSB-35 or SSB-65, but here we represented the SSB-35 mode.

Figure 8

Evolution of integrons toward a limited host factor dependence. The figure shows the evolution of integrases followed by the capture of sedentary chromosomal integrons (SCI) by transposons and their mobilization on conjugative plasmids. The captured SCI are transferred by conjugation in bacteria and now called Mobile Integrons (MI). Their maintenance and dissemination in new bacteria reside in their capacity to have evolved limiting their specific host factor (HF) dependence.