A λ Cro-Like Repressor Is Essential for the Induction of Conjugative Transfer of SXT/R391 Elements in Response to DNA Damage

Abstract

Integrative and conjugative elements (ICEs) of the SXT/R391 family are the main contributors to acquired multidrug resistance in the seventh pandemic lineage of Vibrio cholerae, the etiological agent of the diarrheal disease cholera. Conjugative transfer of SXT/R391 ICEs is triggered by antibiotics and agents promoting DNA damage through RecA-dependent autoproteolysis of SetR, an ICE-encoded CI-like repressor. Here, we describe the role of CroS, a distant Cro homolog, as a key component contributing to the regulation of expression of the activator SetCD that orchestrates the expression of the conjugative transfer genes. We show that deletion of croS abolishes the SOS response-dependent induction of SXT despite the presence of a functional setR gene. Using quantitative reverse transcription-PCR and lacZ reporter assays, we also show that CroS represses setR and setCD expression by binding to operator sites shared with SetR. Furthermore, we provide evidence of an additional operator site bound by SetR and CroS. Finally, we show that SetCD expression generates a positive feedback loop due to SXT excision and replication in a fraction of the cell population. Together, these results refine our understanding of the genetic regulation governing the propagation of major vectors of multidrug resistance. IMPORTANCEHealthcare systems worldwide are challenged by an alarming drug resistance crisis caused by the massive and rapid propagation of antibiotic resistance genes and the associated emergence of multidrug-resistant pathogenic bacteria. SXT/R391 ICEs contribute to this phenomenon not only in clinical and environmental vibrios but also in several members of the family Enterobacteriaceae. We have identified and characterized here the regulator CroS as a key factor in the stimulation of conjugative transfer of these ICEs in response to DNA-damaging agents. We have also untangled conflicting evidence regarding autoactivation of transfer by the master activator of SXT/R391 ICEs, SetCD. Discovery of CroS provides a clearer and more complete understanding of the regulatory network that governs the dissemination of SXT/R391 ICEs in bacterial populations. Cholera is a severe infectious disease caused by the ingestion of food or water contaminated by Vibrio cholerae. This disease remains widespread in regions with limited access to clean water and where poor sanitation allows easy dissemination of the bacterium in drinking water sources. Cholera is characterized by a profuse watery diarrhea that rapidly induces massive fluid loss, causing severe dehydration of the patient, which may lead to death within 24 h of symptom onset. Although epidemic cholera is usually caused by V. cholerae O1, the unusual serogroup O139 emerged in the early 1990s as the cause of a cholera outbreak in India (1). O139 clinical isolates were found to be resistant to sulfamethoxazole and trimethoprim, two antibiotics commonly used for the treatment of severe cases of cholera (2). This resistance was found to be transmissible and linked to an integ...

Genome analysis of a clinical isolate of Shewanella sp. uncovered an active hybrid integrative and conjugative element carrying an integron platform inserted in a novel genomic locus

“…1, blue arrows; Table 1). It also contained a regulatory region (eex, setCD, croS and setR), which can be activated upon DNA damage (Beaber et al, 2002;Garriss et al, 2013;Poulin-Laprade & Burrus, 2015) and a recombination system (bet/exo) responsible for generating hybrid forms . Moreover, sequence analysis of the entry exclusion gene, eex, confirmed that ICESh95 belongs to ICE exclusion group S (99 % nucleotide identity to eexS from ICE SXT MO10 ) (Marrero & Waldor, 2007).…”

Characterization of Three Novel SXT/R391 Integrating Conjugative Elements ICEMfuInd1a and ICEMfuInd1b, and ICEMprChn1 Identified in the Genomes of Marinomonas fungiae JCM 18476T and Marinomonas profundimaris Strain D104

“…Moreover, G+C content was 44.8, 47.2, and 46.9% for ICE Mfu Ind1a, ICE Mfu Ind1b, and ICE Mpr Chn1, respectively. Most of the core genes of SXT/R391 ICEs (Beaber et al, 2002; Wozniak et al, 2009; Spagnoletti et al, 2014; Carraro et al, 2015; Poulin-Laprade and Burrus, 2015; Poulin-Laprade et al, 2015) were found to be preserved and were arranged in the same syntenic order in the three elements (Figure 1A, Supplementary Table 1) and showed 60–99% sequence identity at the level of amino acid to the corresponding proteins encoded by SXT/R391 ICEs (Supplementary Table 2). …”

“…All the SXT/R391 ICEs are chromosomal MGEs sharing a conserved integrase that mediates site-specific integration into the 5′ end of prfC or t-RNA-ser in the absence of a prfC site (Hochhut and Waldor, 1999; Hochhut et al, 2001; Burrus and Waldor, 2003; Burrus et al, 2006; Taviani et al, 2012; Carraro and Burrus, 2014; Luo et al, 2016). Members of this ICE family contain 52 conserved core genes, many of which are involved in integration/excision, conjugative transfer and regulation of the ICEs (Beaber et al, 2002; Burrus et al, 2006; Wozniak et al, 2009; Bi et al, 2012; Spagnoletti et al, 2014; Carraro et al, 2015; Poulin-Laprade and Burrus, 2015; Poulin-Laprade et al, 2015). In addition, five hotspots (HS1—5) and five variable (VRI—V) regions have also been identified (Lei et al, 2016), which contain variable genes conferring element-specific properties and providing beneficial phenotypes to their hosts (Osorio et al, 2008; Wozniak et al, 2009; Rodríguez-Blanco et al, 2012; Balado et al, 2013; Poulin-Laprade et al, 2015).…”

A four-step regulatory cascade controls bistable transfer competence development of the integrative and conjugative element ICEclcinPseudomonas

“…That switch entails essentially a balance of the counteracting transcription factors CI, CII and Cro [46,47]. Interestingly, other ICEs of the SXT/R391 family carry this typical double negative feedback loop architecture, which may therefore control their (bistable) activation [20,22,32].…”

“…In terms of its genetic makeup, ICE clc is very distinct from the well-known SXT/R391 family of ICEs and from ICE St1 /ICE St3 of Streptococcus thermophilus [11,20,21]. These carry analogous genetic regulatory circuitry to the lambda prophage, which is characterized by a typical double-negative feedback control [20–22]. Transcriptomic studies indicated that about half of the ICE clc coding capacity (~50 kb) is higher expressed in stationary phase cultures grown on 3-chlorobenzoate (3-CBA), and organized in at least half a dozen transcriptional units [23].…”