Nicolas Carraro

Integrative and conjugative elements (ICEs) are widespread autonomous mobile DNA within bacterial chromosomes. ICEs contain the genes necessary for excision from the chromosome, conjugative transfer to a new recipient cell, and chromosomal reintegration. They can also carry accessory genes that, while not essential for transfer, confer adaptive phenotypes to the host, contributing to host survival under stressful or changing conditions. Genome studies have indicated that Pseudomonas aeruginosa clinical isolates carry a wide range of related ICEs with adaptive genes enriched for heavy metal resistance and efflux systems; however, their mobility has remained understudied. Here, we studied the activation and transfer mechanisms of a representative subset of ICE clc -type elements. We found that ICE excision could be induced in P. aeruginosa by ectopic expression of BisDC, the known master regulator of ICE clc activation, pointing to a similar regulatory cascade. A number of elements could be transferred to P. putida , where they conferred increased tolerance to specific heavy metals. We also assessed ICE excision rates in response to different classes of stressors using qPCR-based quantification. Sub-lethal copper exposure significantly increased ICE excision rates in several P. aeruginosa strains, although this response was strongly strain-dependent and absent in isolates with enhanced copper tolerance, highlighting the importance of host background. Despite elevated excision, copper did not stimulate ICE transfer or induce conjugation gene expression, indicating that ICE excision and conjugation can be uncoupled processes. Transcriptomic analyses revealed strain-specific regulatory responses to copper stress, including differential activation of metal-responsive regulators, oxidative stress pathways, and virulence-associated systems. IMPORTANCE: Integrative and conjugative elements (ICEs) play a major role in bacterial adaptation by mediating horizontal gene transfer; however, the environmental cues governing their activation remain poorly understood. Here, we demonstrate that ICE clc -type elements in Pseudomonas aeruginosa are transferable at low frequencies and that their excision rates can be selectively increased by specific stress conditions, notably copper exposure and hypoosmotic stress. Our findings reveal that ICE excision and conjugative transfer can be uncoupled and are strongly influenced by host genetic background, underscoring the complexity of ICE regulation. This work aimed to explore whether clinical conditions or antimicrobial treatment could inadvertently promote ICE-mediated gene transfer, with implications for understanding the evolution of antibiotic resistance and virulence.

Conjugation, the exchange of genetic material between different bacterial species, is an important process underlying adaptation. The reason for this is that conjugative elements carry additional genes potentially providing adaptive benefit to the host, such as antibiotic resistance or pathways for degradation of environmental contaminants. However, there is increasing evidence that the conjugative process itself is also responsive to cellular or environmental cues and may, thus, be under rate-dependent selection. Here, we investigate the integrative and conjugative element ICE clc as a model for both metabolic pathway “engrafting” (i.e., how a Pseudomonas putida -encoded benzoate pathway is expanded to 3-chlorobenzoate; 3-CBA) and understanding how metabolic cues or states may lead to ICE activation. We compared differences in gene expression of P. putida with or without ICE clc grown on 3-CBA, benzoate, and succinate, both in exponential and stationary phase conditions. Mutants were constructed in the 3-CBA metabolic pathway, and effects on ICE-transfer were examined. We further deleted parts of a seemingly redundant methyltetrahydrofolate (mTHF) biosynthesis operon that was highly expressed during 3-CBA growth and studied its effects on ICE-activation. Our results demonstrate that 3-CBA metabolism is necessary for ICE activation and reveal an unexpected connection between mTHF pathway induction and ICE clc activation and transfer. These findings show how metabolic states may arise that are perceived by the ICE and lead to an increase in its transfer frequency, thereby mobilizing the very genes that partly caused this state to appear. IMPORTANCE: Bacterial conjugation is widely appreciated for its diversity, its molecular details, and evolutionary consequences. However, the regulation of the onset of the conjugative process in or by the host cell is frequently taken for granted, whereas any influence of environmental or cellular cues on the rates of conjugation can have drastic consequences on both positive and negative outcomes of conjugation-dependent adaptation (e.g., the distribution of genes for antibiotic resistance). We study here a particular case of an integrative and conjugative element (ICEclc), representative of a widely distributed family of elements pervasive in pseudomonads, which has a modulable conjugation rate in donor populations that is dependent on the proportion of transfer-competent cells arising in stationary phase. Transfer-competent cell appearance permits to quantify and understand conditions favoring ICE activation and transfer, and we show here how the ICE reacts to differences in the physiological states of the host cell under influence of its metabolism of aromatic compounds.

Les éléments intégratifs conjugatifs (ICE) sont des îlots génomiques qui codent leur excision du chromosome, leur transfert par conjugaison et leur intégration. Ils présentent une organisation modulaire, chaque module incluant tous les gènes nécessaires pour conférer une fonction biologique. Ce travail a porté sur l'étude de la régulation ainsi que les modalités de transfert et de maintien d'ICESt1 et ICESt3, deux ICE de Streptococcus thermophilus présentant une région core étroitement apparentée et une région variable non apparentée. Les résultats obtenus ont montré que, bien qu'ICESt3 s'excise et se transfère à beaucoup plus haute fréquence qu'ICESt1, l'excision des deux éléments est activée par des stimuli identiques et est dépendante de la souche hôte. Chacun de ces ICE code des homologues de deux types de régulateurs différents, cI et ImmR, ce qui implique un mécanisme de régulation complexe et original qui pourrait être conservée chez de nombreux ICE apparentés identifiés lors de ce travail. Selon la définition initiale, les ICE se maintiendraient uniquement sous forme intégrée et ne se répliqueraient pas de façon intracellulaire. Cependant, les dommages à l'ADN induisent non seulement l'excision et le transfert d'ICESt3, mais aussi sa présence en copies multiples extrachromosomiques. Les résultats obtenus impliquent une réplication sous forme extrachromosomique, réplication codée par la région core et qui serait impliquée dans le maintien de l'élément. Une telle réplication pourrait être impliquée dans le maintien de nombreux ICE en plus de leur intégration.

Integrative and conjugative elements (ICEs) are widespread autonomous mobile DNA within bacterial chromosomes. ICEs contain the genes necessary for excision from the chromosome, conjugative transfer to a new recipient cell, and chromosomal reintegration. They can also carry accessory genes that, while not essential for transfer, confer adaptive phenotypes to the host, contributing to host survival under stressful or changing conditions. Genome studies have indicated that Pseudomonas aeruginosa clinical isolates carry a wide range of related ICEs with adaptive genes enriched for heavy metal resistance and efflux systems, however, their mobility has remained understudied. Here, we studied the activation and transfer mechanisms of a representative subset of ICEclc-type elements. We found that ICE excision could be induced in P. aeruginosa by ectopic expression of BisDC, the known master regulator of ICEclc activation, pointing to a similar regulatory cascade. A number of elements could be transferred to P. putida, where they conferred increased tolerance to specific heavy metals. We also assessed ICE excision rates in response to different classes of stressors using qPCR-based quantification. Sub-lethal copper exposure significantly increased ICE excision rates in several P. aeruginosa strains, although this response was strongly strain-dependent and absent in isolates with enhanced copper tolerance, highlighting the importance of host background. Despite elevated excision, copper did not stimulate ICE transfer or induce conjugation gene expression, indicating that ICE excision and conjugation can be uncoupled processes. Transcriptomic analyses revealed strain-specific regulatory responses to copper stress, including differential activation of metal-responsive regulators, oxidative stress pathways, and virulence-associated systems.

Manganese (Mn) oxides are commonly precipitated by bacteria and fungi. Recent studies have shown that Mn-oxidizing bacteria can harbor multiple Mn oxidases, but environmental controls on the regulation of these enzymes are unknown. Here, we examine the activation of the genes encoding for MnxG and McoA, two Mn oxidases in Pseudomonas putida GB-1, in response to varying Mn(II) concentrations. Using reporter gene fusion strains, we found that mnxG and mcoA are activated only by a fraction of the cells in the population and at different threshold Mn concentrations, with an increasing proportion of cells expressing mnxG (10 to 500 μM). Kinetic modeling showed that coexpression of mnxG and mcoA not only alleviates substrate saturation and inhibition of MnxG at high Mn(II) concentrations but also results in an 8-fold increase in the initial rate of Mn oxidation in the wild type compared to strains lacking mnxG or mcoA. The population-level control of gene activation and, ultimately, Mn oxide precipitation, in response to the initial Mn(II) concentration, shows that P. putida fine-tunes the regulation of multiple Mn oxidases to operate under varying environmental conditions.

Summary: Integrative conjugative elements (ICEs) are mobile DNA that remain integrated within the host bacterial genome until they activate their excision and transfer to recipient cells via conjugation. ICE transfer is initiated in a small subpopulation of cells that undergo a hierarchical gene expression cascade leading to transfer competence formation. In this study, we demonstrate that transfer competence formation of the ICE clc element in stationary phase Pseudomonas putida cells is regulated by the coordinated activity of a two-component transcriptional activator, BisC and BisD. Chromatin immunoprecipitation of tagged BisC or BisD followed by high throughput sequencing showed that both proteins accumulate at similar sites around ICE clc transfer competence promoters in P. putida cells. Genetic dissection and single cell microscopy showed that BisD is a dual domain protein, with a C-terminal gene activator domain and an N-terminal ParB-like domain, forming dimeric DNA clamps that self-load at distant sites and reach target promoters after extensive one-dimensional DNA sliding. Expressed mCherry-BisD in P. putida ICE clc cells form discrete fluorescent foci, dependent on parS- like sequences on the ICE. This focus formation is similar as what is seen with canonical ParB proteins accumulating on chromosomal DNA, but in case of BisD corresponds to both chromosomal and excised ICE-molecules. Sliding of BisD over ∼50 kb of ICE-DNA is asymmetric and follows the direction of ICE gene transcription. This may help to establish a temporal order of activation of transfer competence formation, optimizing ICE transfer. Given that ICE clc is activated in stationary phase cells, we hypothesize that BisD is not involved in segregating excised ICE-DNA among daughter cells, but rather in actively directing ICE-DNA molecules towards the (multiple) conjugative systems that are produced in transfer competent cells. BisD thus serves as a twin function protein, integrating gene activation and DNA segregation functions.

Microbiome engineering – the targeted manipulation of microbial communities – is considered a promising strategy to restore ecosystems, but experimental support and mechanistic understanding are required. Here, we show that bacterial inoculants for soil microbiome engineering may fail to establish because they inadvertently facilitate growth of native resident microbiomes. By generating soil microcosms in presence or absence of standardized soil resident communities, we show how different nutrient availabilities limit outgrowth of focal bacterial inoculants (three Pseudomonads), and how this might be improved by adding an artificial, inoculant-selective nutrient niche. Through random paired interaction assays in agarose micro-beads, we demonstrate that, in addition to direct competition, inoculants lose competitiveness by facilitating growth of resident soil bacteria. Metatranscriptomics experiments with toluene as selective nutrient niche for the inoculant Pseudomonas veronii indicate that this facilitation is due to loss and uptake of excreted metabolites by resident taxa. Generation of selective nutrient niches for inoculants may help to favor their proliferation for the duration of their intended action while limiting their competitive loss.

Integrative Conjugative Elements (ICEs) are mosaics containing functional modules allowing maintenance by site-specific integration and excision into and from the host genome and conjugative transfer to a specific host range. Many ICEs encode a range of adaptive functions that aid bacterial survival and evolution in a range of niches. ICEs from the SXT/R391 family are found in γ-Proteobacteria. Over 100 members have undergone epidemiological and molecular characterization allowing insight into their diversity and function. Comparative analysis of SXT/R391 elements from a wide geographic distribution has revealed conservation of key functions, and the accumulation and evolution of adaptive genes. This evolution is associated with gene acquisition in conserved hotspots and variable regions within the SXT/R391 ICEs catalysed via element-encoded recombinases. The elements can carry IS elements and transposons, and a mutagenic DNA polymerase, PolV, which are associated with their evolution. SXT/R391 ICEs isolated from different niches appear to have retained adaptive functions related to that specific niche; phage resistance determinants in ICEs carried by wastewater bacteria, antibiotic resistance determinants in clinical isolates and metal resistance determinants in bacteria recovered from polluted environments/ocean sediments. Many genes found in the element hotspots are undetermined and have few homologs in the nucleotide databases.

Bacterial genomes are prone to rapidly evolve in content and size as a result of horizontal gene transfer (HGT), i.e., the exchange and acquisition of genetic material. Mobile genetic elements (MGEs) have a major impact on this genome plasticity, driving rapid bacterial adaptation and evolution. During the past fifty years, extensive work has been done by the scientific community to identify and classify MGEs, characterize their mechanisms of transfer, lifestyle aspects, or uncover interactions with the bacterial host and/or other MGEs in the same cell. This detailed mini-review published by Haudiquet and collaborators covers basic and advanced aspects of various classes of MGEs, and their impact on microbial populations. The review focuses on well-known conjugative elements and phages, but also mentions newly discovered parasitic non-autonomously transferring elements in the same cell. It summarizes key characteristics of MGEs, notably, the different steps of their lifecycle, adaptive functions they can confer, or the burden they can impose on their host. It gives a taste of the interactions between different classes of MGEs and potential evolutionary consequences. It also highlights the complex interplay between MGEs, environmental cues, and host characteristics that can favor and hinder the spread of genetic material among and between microbial communities. This is a nice stimulatory mini-review for those who want a rapid entry to the field, and emphasizes key challenges for future research.

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that is a model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. Previous work showed bimodal ICE activation, but by using single cell time-lapse microscopy coupled to combinations of chromosomally integrated single copy ICE promoter-driven fluorescence reporters, RNA sequencing and mutant analysis, we now describe the complete regulon leading to the arisal of differentiated dedicated transfer competent cells. The regulon encompasses at least three regulatory nodes and five (possibly six) further conserved gene clusters on the ICE that all become expressed under stationary phase conditions. Time-lapse microscopy indicated expression of two regulatory nodes (i.e., bisR and alpA-bisDC) to precede that of the other clusters. Notably, expression of all clusters except of bisR was confined to the same cell subpopulation, and was dependent on the same key ICE regulatory factors. The ICE thus only transfers from a small fraction of cells in a population, with an estimated proportion of between 1.7–4%, which express various components of a dedicated transfer competence program imposed by the ICE, and form the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements.