A Conserved Helicase Processivity Factor Is Needed for Conjugation and Replication of an Integrative and Conjugative Element

Integrative and conjugative elements (ICEs) are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT) by the ICE–encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP), encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.     

Autonomous plasmid‐like replication of Bacillus ICEBs1: a general feature of integrative conjugative elements?

Integrative conjugative elements (ICEs) occur frequently in Gram‐positive and Gram‐negative bacteria. In contrast to plasmids, they are stably integrated in the bacterial genome, often inserted in a tRNA gene. They are excised from the host chromosome upon induction in order to be transferred to a recipient cell. When conjugative transfer is completed, they stably reintegrate in the chromosome. It is generally thought that ICEs are incapable of autonomous replication, instead relying on replication and segregation along with the host chromosome. In this issue of Molecular Microbiology Lee and co‐workers demonstrate that ICEBs1 from Bacillus subtilis is capable of autonomous plasmid‐like replication in its circular form after excision. The authors show that ICEBs1 replication is unidirectional; it initiates at oriT_ICEBs1 and requires the ICEBs1‐encoded conjugative relaxase NicK. Replication also requires the catalytic subunit of the host DNA polymerase PolC, the host processivity clamp DnaN and the host‐encoded alternative helicase PcrA. Autonomous replication of ICEBs1 appears to be important for its stable maintenance, but not for horizontal transfer of the element. Lee and co‐workers argue that plasmid‐like replication is likely a common property of ICEs, probably contributing to stability and maintenance of ICEs in bacterial populations. I discuss these findings in context with data on other ICEs from Gram‐positive and Gram‐negative bacteria and with respect to possible consequences of the findings for basic research on mobile genetic elements from Gram‐positive bacteria and their applications in biotechnology.     

Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso''s in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.     

Identification,characterization and benefits of an exclusion system in an integrative and conjugative element of Bacillus subtilis

Integrative and conjugative elements (ICEs) are mobile genetic elements that transfer from cell to cell by conjugation (like plasmids) and integrate into the chromosomes of bacterial hosts (like lysogenic phages or transposons). ICEs are prevalent in bacterial chromosomes and play a major role in bacterial evolution by promoting horizontal gene transfer. Exclusion prevents the redundant transfer of conjugative elements into host cells that already contain a copy of the element. Exclusion has been characterized mostly for conjugative elements of Gram‐negative bacteria. Here, we report the identification and characterization of an exclusion mechanism in ICEBs1 from the Gram‐positive bacterium Bacillus subtilis. We found that cells containing ICEBs1 inhibit the activity of the ICEBs1‐encoded conjugation machinery in other cells. This inhibition (exclusion) was specific to the cognate conjugation machinery and the ICEBs1 gene yddJ was both necessary and sufficient to mediate exclusion by recipient cells. Through a mutagenesis and enrichment screen, we identified exclusion‐resistant mutations in the ICEBs1 gene conG. Using genes from a heterologous but related ICE, we found that the exclusion specificity was determined by ConG and YddJ. Finally, we found that under conditions that support conjugation, exclusion provides a selective advantage to the element and its host cells.     

Selective Pressures to Maintain Attachment Site Specificity of Integrative and Conjugative Elements

Integrative and conjugative elements (ICEs) are widespread mobile genetic elements that are usually found integrated in bacterial chromosomes. They are important agents of evolution and contribute to the acquisition of new traits, including antibiotic resistances. ICEs can excise from the chromosome and transfer to recipients by conjugation. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial genome. Site-specific ICEs can also integrate into secondary locations, particularly if the primary site is absent. However, little is known about the consequences of integration of ICEs into alternative attachment sites or what drives the apparent maintenance and prevalence of the many ICEs that use a single attachment site. Using ICEBs1, a site-specific ICE from Bacillus subtilis that integrates into a tRNA gene, we found that integration into secondary sites was detrimental to both ICEBs1 and the host cell. Excision of ICEBs1 from secondary sites was impaired either partially or completely, limiting the spread of ICEBs1. Furthermore, induction of ICEBs1 gene expression caused a substantial drop in proliferation and cell viability within three hours. This drop was dependent on rolling circle replication of ICEBs1 that was unable to excise from the chromosome. Together, these detrimental effects provide selective pressure against the survival and dissemination of ICEs that have integrated into alternative sites and may explain the maintenance of site-specific integration for many ICEs.     

Identification of host genes that affect acquisition of an integrative and conjugative element in Bacillus subtilis

Conjugation, a major type of horizontal gene transfer in bacteria, involves transfer of DNA from a donor to a recipient using donor‐encoded conjugation machinery. Using a high‐throughput screen (Tn‐seq), we identified genes in recipients that contribute to acquisition of the integrative and conjugative element ICEBs1 by Bacillus subtilis. We found that null mutations in some genes caused an increase, and others a decrease in conjugation efficiency. Some mutations affected conjugation only when present in recipients. Other mutations affected conjugation when present in donors or recipients. Most of the genes identified are known or predicted to affect the cell envelope. Several encode enzymes involved in phospholipid biosynthesis and one encodes a homologue of penicillin‐binding proteins. Two of the genes identified also affected conjugation of Tn916, indicating that their roles in conjugation may be general. We did not identify any genes in recipients that were essential for ICEBs1 conjugation, indicating that if there are such genes, then these are either essential for cell growth or redundant. Our results indicate that acquisition of ICEBs1, and perhaps other conjugative elements, is robust and not easily avoided by mutation and that several membrane‐related functions affect the efficiency of conjugation.     

Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage

Most bacterial genomes contain horizontally acquired and transmissible mobile genetic elements, including temperate bacteriophages and integrative and conjugative elements. Little is known about how these elements interact and co-evolved as parts of their host genomes. In many cases, it is not known what advantages, if any, these elements provide to their bacterial hosts. Most strains of Bacillus subtilis contain the temperate phage SPß and the integrative and conjugative element ICEBs1. Here we show that the presence of ICEBs1 in cells protects populations of B. subtilis from predation by SPß, likely providing selective pressure for the maintenance of ICEBs1 in B. subtilis. A single gene in ICEBs1 (yddK, now called spbK for SPß killing) was both necessary and sufficient for this protection. spbK inhibited production of SPß, during both activation of a lysogen and following de novo infection. We found that expression spbK, together with the SPß gene yonE constitutes an abortive infection system that leads to cell death. spbK encodes a TIR (Toll-interleukin-1 receptor)-domain protein with similarity to some plant antiviral proteins and animal innate immune signaling proteins. We postulate that many uncharacterized cargo genes in ICEs may confer selective advantage to cells by protecting against other mobile elements.     

The Bifunctional Cell Wall Hydrolase CwlT Is Needed for Conjugation of the Integrative and Conjugative Element ICEBs1 in Bacillus subtilis and B. anthracis

The mobile genetic element ICEBs1 is an integrative and conjugative element (ICE) found in Bacillus subtilis. One of the ICEBs1 genes, cwlT, encodes a cell wall hydrolase with two catalytic domains, a muramidase and a peptidase. We found that cwlT is required for ICEBs1 conjugation. We examined the role of each of the two catalytic domains and found that the muramidase is essential, whereas the peptidase is partially dispensable for transfer of ICEBs1. We also found that the putative signal peptide in CwlT is required for CwlT to function in conjugation, consistent with the notion that CwlT is normally secreted from the cytoplasm. We found that alteration of the putative lipid attachment site on CwlT had no effect on its role in conjugation, indicating that if CwlT is a lipoprotein, the lipid attachment is not required for conjugation. Finally, we found conditions supporting efficient transfer of ICEBs1 into and out of Bacillus anthracis and that cwlT was needed for ICEBs1 to function in B. anthracis. The mature cell wall of B. anthracis is resistant to digestion by CwlT, indicating that CwlT might act during cell wall synthesis, before modifications of the peptidoglycan are complete.     

Genomic analysis of a novel integrative conjugative element in Vibrio cholerae

Integrative conjugative elements (ICEs) are a class of self-transmissible mobile elements that mediate horizontal gene transfer in bacteria, and play an important role in bacterial evolution. Since 1992, ICEs of the SXT/R391 family have been found to be widely distributed among Vibrio cholerae strains isolated in Asian countries. Here we describe ICEVchB33, an ICE found in the genomes of two V. cholerae O1 Eltor strains, one isolated in India, 1994, and the other from Mozambique, 2004. ICEVchB33 revealed a new genetic organization, different from other ICEs of the SXT/R391 family, demonstrating the genomic plasticity of these elements.     

Site-specific accretion of an integrative conjugative element together with a related genomic island leads to cis mobilization and gene capture

Genomic islands, flanked by attachment sites, devoid of conjugation and recombination modules and related to the integrative and conjugative element (ICE) ICESt3, were previously found in Streptococcus thermophilus. Here, we show that ICESt3 transfers to a recipient harbouring a similar engineered genomic island, CIMEL₃catR₃, and integrates by site‐specific recombination into its attachment sites, leading to their accretion. The resulting composite island can excise, showing that ICESt3 mobilizes CIMEL₃catR₃ in cis. ICESt3, CIMEL₃catR₃ and the whole composite element can transfer from the strain harbouring the composite structure. The ICESt3 transfer to a recipient bearing CIMEL₃catR₃ can also lead to retromobilization, i.e. its capture by the donor. This is the first demonstration of specific conjugative mobilization of a genomic island in cis and the first report of ICE‐mediated retromobilization. CIMEL₃catR₃ would be the prototype of a novel class of non‐autonomous mobile elements (CIMEs: CIs mobilizable elements), which hijack the recombination and conjugation machinery of related ICEs to excise, transfer and integrate. Few genome analyses have shown that CIMEs could be widespread and have revealed internal repeats that could result from accretions in numerous genomic islands, suggesting that accretion and cis mobilization have a key role in evolution of genomic islands.     

Polar Positioning of a Conjugation Protein from the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

ICEBs1 is an integrative and conjugative element found in the chromosome of Bacillus subtilis. ICEBs1 encodes functions needed for its excision and transfer to recipient cells. We found that the ICEBs1 gene conE (formerly yddE) is required for conjugation and that conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE. ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4. We found that a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells. At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles, as ConE-GFP was dispersed throughout the cytoplasm in a strain lacking ICEBs1. We also visualized the subcellular location of ICEBs1. When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell, a position characteristic of that chromosomal region. Following excision, ICEBs1 was more frequently found near a cell pole. Excision of ICEBs1 also caused altered positioning of at least one component of the replisome. Taken together, our findings indicate that ConE is a critical component of the ICEBs1 conjugation machinery, that conjugative transfer of ICEBs1 from B. subtilis likely initiates at a donor cell pole, and that ICEBs1 affects the subcellular position of the replisome.Integrative and conjugative elements (also known as conjugative transposons) and conjugative plasmids are key elements in horizontal gene transfer and are capable of mediating their own transfer from donor to recipient cells. ICEBs1 is an integrative and conjugative element found in some Bacillus subtilis strains. Where found, ICEBs1 is integrated into the leucine tRNA gene trnS-leu2 (Fig. ​(Fig.1)1) (7, 14, 21).Open in a separate windowFIG. 1.Genetic map of ICEBs1. conE (formerly yddE), regulatory genes (gray arrows), and genes required for integration, excision, and nicking (hatched arrows) are indicated. The number of transmembrane (TM) segments for each protein predicted by cPSORTdb (46) is indicated below each gene. Other topology programs yield similar but not identical predictions.ICEBs1 gene expression, excision, and potential mating are induced by activation of RecA during the SOS response following DNA damage (7). In addition, ICEBs1 is induced by increased production or activation of the ICEBs1-encoded regulatory protein RapI. Production and activity of RapI are indicative of the presence of potential mating partners that do not contain a copy of ICEBs1 (7). Under inducing conditions, the ICEBs1 repressor ImmR (6) is inactivated by proteolytic cleavage mediated by the antirepressor and protease ImmA (12). Most ICEBs1 genes then become highly expressed (7). One of these genes (xis) encodes an excisionase, which in combination with the element''s integrase causes efficient excision and formation of a double-stranded circle (7, 38). The circular form is nicked at the origin of transfer, oriT, by a DNA relaxase, the product of nicK (39). Under appropriate conditions, ICEBs1 can then be transferred by mating into B. subtilis and other species, including the pathogens Listeria monocytogenes and Bacillus anthracis (7). Once transferred to a recipient, ICEBs1 can be stably integrated into the genome at its attachment site in trnS-leu2 by the ICEBs1-encoded integrase (38).In contrast to what is known about ICEBs1 genes and proteins involved in excision, integration, and gene regulation, less is known about the components that make up gram-positive organisms'' mating machinery, defined as the conjugation proteins involved in DNA transfer (18, 24). The well-characterized mating machinery of gram-negative organisms can serve as a preliminary model (15, 16, 37, 48). Gram-negative organisms'' mating machinery is a type IV secretion system composed of at least eight conserved proteins that span the cell envelope. For example, the conjugation apparatus of the Agrobacterium tumefaciens Ti plasmid (pTi) is composed of 11 proteins (VirB1 through VirB11), including the ATPase VirB4 (16). VirB4 family members interact with several components of their cognate secretion systems and may energize machine assembly and/or substrate transfer (16, 48). The secretion substrate is targeted to the conjugation machinery by a coupling protein. Coupling proteins, such as VirD4 of pTi, interact with a protein attached to the end of the DNA substrate and couple the substrate to other components of the conjugation machinery. Coupling proteins might also energize the translocation of DNA through the machinery. Both VirB4 and VirD4 belong to the large HerA/FtsK superfamily of ATPases (29). Two other characterized members of this superfamily are the chromosome-partitioning proteins FtsK and SpoIIIE (29), which are ATP-dependent DNA pumps (reviewed in reference 2).Some of the proteins encoded by the conjugative elements of gram-positive organisms are homologous to components of the conjugation machinery from gram-negative organisms (1, 9, 14, 29), indicating that some aspects of conjugative DNA transfer may be similar in gram-positive and gram-negative organisms. For example, ConE (formerly YddE) of ICEBs1 has sequence similarities to VirB4 (29). YdcQ may be the ICEBs1-encoded coupling protein, as it is phylogenetically related to other coupling proteins (29, 44). Despite some similarities, the cell envelopes and many of the genes encoding the conjugation machinery are different between gram-positive and gram-negative organisms, indicating that there are likely to be significant structural and mechanistic differences as well.To begin to define the conjugation machinery of ICEBs1 and to understand spatial aspects of conjugation, we examined the function and subcellular location of ConE of ICEBs1. Our results indicate that ConE is likely a crucial ATPase component of the ICEBs1 conjugation machinery. We found that ConE and excised ICEBs1 DNA were located at or near the cell poles. We propose that the conjugation machinery is likely located at the cell poles and that mating might occur from a donor cell pole.     

Dissemination and loss of a biofilm‐related genomic island in marine Pseudoalteromonas mediated by integrative and conjugative elements

Acquisition of genomic islands (GIs) plays a central role in the diversification and adaptation of bacteria. Some GIs can be mobilized in trans by integrative and conjugative elements (ICEs) or conjugative plasmids if the GIs carry specific transfer‐related sequences. However, the transfer mechanism of GIs lacking such elements remains largely unexplored. Here, we investigated the transmissibility of a GI found in a coral‐associated marine bacterium. This GI does not carry genes with transfer functions, but it carries four genes required for robust biofilm formation. Notably, this GI is inserted in the integration site for SXT/R391 ICEs. We demonstrated that acquisition of an SXT/R391 ICE results in either a tandem GI/ICE arrangement or the complete displacement of the GI. The GI displacement by the ICE greatly reduces biofilm formation. In contrast, the tandem integration of the ICE with the GI in cis allows the GI to hijack the transfer machinery of the ICE to excise, transfer and re‐integrate into a new host. Collectively, our findings reveal that the integration of an ICE into a GI integration site enables rapid genome dynamics and a new mechanism by which SXT/R391 ICEs can augment genome plasticity.     

Uncovering the prevalence and diversity of integrating conjugative elements in actinobacteria

Horizontal gene transfer greatly facilitates rapid genetic adaptation of bacteria to shifts in environmental conditions and colonization of new niches by allowing one-step acquisition of novel functions. Conjugation is a major mechanism of horizontal gene transfer mediated by conjugative plasmids and integrating conjugative elements (ICEs). While in most bacterial conjugative systems DNA translocation requires the assembly of a complex type IV secretion system (T4SS), in Actinobacteria a single DNA FtsK/SpoIIIE-like translocation protein is required. To date, the role and diversity of ICEs in Actinobacteria have received little attention. Putative ICEs were searched for in 275 genomes of Actinobacteria using HMM-profiles of proteins involved in ICE maintenance and transfer. These exhaustive analyses revealed 144 putative FtsK/SpoIIIE-type ICEs and 17 putative T4SS-type ICEs. Grouping of the ICEs based on the phylogenetic analyses of maintenance and transfer proteins revealed extensive exchanges between different sub-families of ICEs. 17 ICEs were found in Actinobacteria from the genus Frankia, globally important nitrogen-fixing microorganisms that establish root nodule symbioses with actinorhizal plants. Structural analysis of ICEs from Frankia revealed their unexpected diversity and a vast array of predicted adaptive functions. Frankia ICEs were found to excise by site-specific recombination from their host's chromosome in vitro and in planta suggesting that they are functional mobile elements whether Frankiae live as soil saprophytes or plant endosymbionts. Phylogenetic analyses of proteins involved in ICEs maintenance and transfer suggests that active exchange between ICEs cargo-borne and chromosomal genes took place within the Actinomycetales order. Functionality of Frankia ICEs in vitro as well as in planta lets us anticipate that conjugation and ICEs could allow the development of genetic manipulation tools for this challenging microorganism and for many other Actinobacteria.     

An ICEBs1-like element may be associated with the extreme radiation and desiccation resistance of Bacillus pumilus SAFR-032 spores

Comparisons of the genomes of Bacillus pumilus SAFR-032 and the closely related type strain, B. pumilus ATCC7061^T, exposed an extended region of non-homologous genes. A detailed examination of this region revealed the presence of an ICEBs1-like integrative conjugative element in SAFR-032. A similar element was subsequently located elsewhere in the ATCC7061^T genome. A detailed comparison of these elements and the ICEBs1 of B. subtilis revealed extremely rapid flux in gene content, genome organization and sequence similarity. It is not clear if the B. pumilus elements as they are currently structured are functional. However, it is clear that the past involvement of these elements has brought multiple genes of unknown function to the SAFR-032 genome and these genes may be responsible for the rapid evolution that led to the extreme radiation and desiccation resistance of this organism’s spores.     

The repertoire of ICE in prokaryotes underscores the unity, diversity, and ubiquity of conjugation

Horizontal gene transfer shapes the genomes of prokaryotes by allowing rapid acquisition of novel adaptive functions. Conjugation allows the broadest range and the highest gene transfer input per transfer event. While conjugative plasmids have been studied for decades, the number and diversity of integrative conjugative elements (ICE) in prokaryotes remained unknown. We defined a large set of protein profiles of the conjugation machinery to scan over 1,000 genomes of prokaryotes. We found 682 putative conjugative systems among all major phylogenetic clades and showed that ICEs are the most abundant conjugative elements in prokaryotes. Nearly half of the genomes contain a type IV secretion system (T4SS), with larger genomes encoding more conjugative systems. Surprisingly, almost half of the chromosomal T4SS lack co-localized relaxases and, consequently, might be devoted to protein transport instead of conjugation. This class of elements is preponderant among small genomes, is less commonly associated with integrases, and is rarer in plasmids. ICEs and conjugative plasmids in proteobacteria have different preferences for each type of T4SS, but all types exist in both chromosomes and plasmids. Mobilizable elements outnumber self-conjugative elements in both ICEs and plasmids, which suggests an extensive use of T4SS in trans. Our evolutionary analysis indicates that switch of plasmids to and from ICEs were frequent and that extant elements began to differentiate only relatively recently. According to the present results, ICEs are the most abundant conjugative elements in practically all prokaryotic clades and might be far more frequently domesticated into non-conjugative protein transport systems than previously thought. While conjugative plasmids and ICEs have different means of genomic stabilization, their mechanisms of mobility by conjugation show strikingly conserved patterns, arguing for a unitary view of conjugation in shaping the genomes of prokaryotes by horizontal gene transfer.     

Transcriptome analysis of the mobile genome ICE<Emphasis Type="Italic">clc</Emphasis> in <Emphasis Type="Italic">Pseudomonas knackmussii</Emphasis> B13

Background  

Integrative and conjugative elements (ICE) form a diverse group of DNA elements that are integrated in the chromosome of the bacterial host, but can occasionally excise and horizontally transfer to a new host cell. ICE come in different families, typically with a conserved core for functions controlling the element's behavior and a variable region providing auxiliary functions to the host. The ICEclc element of Pseudomonas knackmussii strain B13 is representative for a large family of chromosomal islands detected by genome sequencing approaches. It provides the host with the capacity to degrade chloroaromatics and 2-aminophenol.     

Worldwide Occurrence of Integrative Conjugative Element Encoding Multidrug Resistance Determinants in Epidemic Vibrio cholerae O1

In the last decades, there has been an increase of cholera epidemics caused by multidrug resistant strains. Particularly, the integrative and conjugative element (ICE) seems to play a major role in the emergence of multidrug resistant Vibrio cholerae. This study fully characterized, by whole genome sequencing, new ICEs carried by multidrug resistant V. cholerae O1 strains from Nigeria (2010) (ICEVchNig1) and Nepal (1994) (ICEVchNep1). The gene content and gene order of these two ICEs are the same, and identical to ICEVchInd5, ICEVchBan5 and ICEVchHai1 previously identified in multidrug resistant V. cholerae O1. This ICE is characterized by dfrA1, sul2, strAB and floR antimicrobial resistance genes, and by unique gene content in HS4 and HS5 ICE regions. Screening for ICEs, in publicly available V. cholerae genomes, revealed the occurrence and widespread distribution of this ICE among V. cholerae O1. Metagenomic analysis found segments of this ICE in marine environments far from the direct influence of the cholera epidemic. Therefore, this study revealed the epidemiology of a spatio-temporal prevalent ICE in V. cholerae O1. Its occurrence and dispersion in V. cholerae O1 strains from different continents throughout more than two decades can be indicative of its role in the fitness of the current pandemic lineage.     

A new flavor of entry exclusion in ICE elements provides a selective advantage for the element and its host

Entry exclusion has been described in many bacterial conjugation systems, but their molecular mechanisms are not well understood. In the current issue, Avello et al. describe a new exclusion system in the conjugative element ICEBs1. They identify the yddJ gene as the functional exclusion gene and its target as the protein product of the conG gene. They provide evidence for a possible mechanism and for the contribution of the system to reduce fitness costs of ICE expression.     

Evolution of copper resistance in the kiwifruit pathogen Pseudomonas syringae pv. actinidiae through acquisition of integrative conjugative elements and plasmids

Horizontal gene transfer can precipitate rapid evolutionary change. In 2010 the global pandemic of kiwifruit canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) reached New Zealand. At the time of introduction, the single clone responsible for the outbreak was sensitive to copper, however, analysis of a sample of isolates taken in 2015 and 2016 showed that a quarter were copper resistant. Genome sequences of seven strains showed that copper resistance – comprising czc/cusABC and copABCD systems – along with resistance to arsenic and cadmium, was acquired via uptake of integrative conjugative elements (ICEs), but also plasmids. Comparative analysis showed ICEs to have a mosaic structure, with one being a tripartite arrangement of two different ICEs and a plasmid that were isolated in 1921 (USA), 1968 (NZ) and 1988 (Japan), from P. syringae pathogens of millet, wheat and kiwifruit respectively. Two of the Psa ICEs were nearly identical to two ICEs isolated from kiwifruit leaf colonists prior to the introduction of Psa into NZ. Additionally, we show ICE transfer in vitro and in planta, analyze fitness consequences of ICE carriage, capture the de novo formation of novel recombinant ICEs, and explore ICE host‐range.