Biology subjects

Carraro, N.

Publications and source records attributed to Carraro, N..

11 recordsLinked to original sources

bioRxiv2026

Horizontal transfer of ICEclc-like elements in Pseudomonas aeruginosa clinical isolates

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. IMPORTANCEIntegrative and conjugative elements (ICEs) play a major role in bacterial adaptation by mediating horizontal gene transfer, yet the environmental cues governing their activation remain poorly understood. Here, we demonstrate that ICEclc-type elements in Pseudomonas aeruginosa are transferable at low frequencies and that their excision can be selectively induced 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.

microbiology↗

bioRxiv2024

Population-level control of two manganese oxidases expands the niche for bacterial manganese biomineralization

The enzymatic oxidation of aqueous divalent manganese (Mn) is a widespread microbial trait that produces reactive Mn(III, IV) oxide minerals. These biominerals drive carbon, nutrient, and trace metal cycles, thus playing important environmental and ecological roles. However, the regulatory mechanisms and physiological functions of Mn biomineralization are unknown. This challenge arises from the common occurrence of multiple Mn oxidases within the same organism and the use of Mn oxides as indicators of combined gene activity. Through detection of gene activation in individual cells, we discover that expression of mnxG and mcoA, two Mn oxidase-encoding genes in Pseudomonas putida GB-1, is confined to subsets of cells within the population, with each gene showing distinct spatiotemporal patterns that reflect local microenvironments. These coordinated intra-population dynamics control Mn biomineralization and illuminate the strategies used by microbial communities to dictate the extent, location and timing of biogeochemical transformations.

microbiology↗

bioRxiv2024

Orchestrated long-distance gene activation by a ParB-like BisD-CTP DNA clamp in low-frequency transfer competence development in Pseudomonas putida

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 ICEclc 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 ICEclc 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 ICEclc 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 ICEclc 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.

microbiology↗

bioRxiv2023

Niche Availability and Competitive Facilitation Control Proliferation of Bacterial Strains Intended for Soil Microbiome Interventions

Microbiome engineering, the rational manipulation of microbial communities and their habitats, is considered a crucial strategy to revert dysbiosis. However, the concept is in its infancy and lacks experimental support. Here we study the ecological factors controlling the proliferation of focal bacterial inoculants into taxa-complex soil communities and their impact on resident microbiota. We demonstrate using standardized soil microbiomes with different growth phases that the proliferation of typical soil inoculants depends on niche competition. By adding an artificial, inoculant selective niche to soil we improve inoculant proliferation and show by metatranscriptomics to give rise to a conjoint metabolic network in the soil microbiome. Furthermore, using random paired growth assays we demonstrate that, in addition to direct competition, inoculants lose competitiveness with soil bacteria because of metabolite sharing. Thus, the fate of inoculants in soil is controlled by niche availability and competitive facilitation, which may be manipulated by selective niche generation. TeaserTypical bacterial inoculants for soil microbiome engineering suffer from facilitating growth of native resident microorganisms

ecology↗

bioRxiv2023

Diversity and Evolution of an Abundant ICEclc-Family of Integrative and Conjugative Elements in Pseudomonas aeruginosa

Integrative and conjugative elements (ICEs) are widespread autonomous mobile DNA, containing the genes necessary for their excision, conjugative transfer, and insertion into a new host cell. ICEs can carry additional genes that are non-essential for their transfer, but that can confer adaptive phenotypes to the host. Our aim here was to better characterize the presence, distribution and evolution of ICEs related to the well-described ICEclc among Pseudomonas aeruginosa clinical isolates, and to understand their potential role in spreading genes with adaptive benefit. We examined a total of 181 P. aeruginosa genome sequences obtained from patient or hospital environment isolates. More than 90% of the isolates carried one or more ICEclc-like elements, with different degrees of conservation to the known ICEclc-lifestyle and transfer genes. ICE clones closely matched their host clonal phylogeny, but not exclusively, indicating that both clonal evolution and ICE-horizontal transfer are occurring in the hospital environment. Variable gene regions among the clinical P. aeruginosa ICEclc-type elements were notably enriched for heavy metal resistance genes, toxin-antitoxin systems, potential efflux systems and multidrug resistance proteins, a metalloprotease and for a variety of regulatory systems, but not for specific recognizable antibiotic resistance cassettes. Clonal persistence suggests adaptive benefits of these functional categories; and micro-patterns of gene gain and loss indicate ongoing ICE evolution within the P. aeruginosa hosts.

microbiology↗

bioRxiv2021

A bistable orthogonal prokaryotic differentiation system underlying development of conjugative transfer competence

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 stands model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. The ICE only transfers from a small fraction of cells in a population, which we uncover here, results from a dedicated transfer competence program imposed by the ICE. Transfer competence is orthogonally maintained in individual cells in which it is activated, making them 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.

microbiology↗

bioRxiv2020

Crucial Role of Salmonella Genomic Island 1 Master Activator in Parasitism of IncC plasmids

IncC conjugative plasmids and the multiple variants of Salmonella Genomic Island 1 (SGI1) are two functionally interacting families of mobile genetic elements commonly associated with multidrug resistance in Gammaproteobacteria. SGI1 and its siblings are specifically mobilised in trans by IncC conjugative plasmids. Conjugative transfer of IncC plasmids is activated by the plasmid-encoded master activator AcaCD. SGI1 carries five AcaCD-responsive promoters that drive the expression of genes involved in its excision, replication, and mobilisation. SGI1 encodes an AcaCD homologue, the transcriptional activator complex SgaCD (also known as FlhDCSGI1) that seems to recognise and activate the same SGI1 promoters. Here, we investigated the relevance of SgaCD in SGI1s lifecycle. Mating assays revealed the requirement for SgaCD and its IncC-encoded counterpart AcaCD in the mobilisation of SGI1. An integrative approach combining ChIP-exo, Cappable-seq, and RNA-seq confirmed that SgaCD activates each of the 18 AcaCD-responsive promoters driving the expression of the plasmid transfer functions. A comprehensive analysis of the activity of the complete set of AcaCD-responsive promoters in both SGI1 and IncC plasmid was performed through reporter assays. qPCR and flow cytometry assays revealed that SgaCD is essential for the excision and replication of SGI1, and the destabilisation of the helper IncC plasmid.

molecular biology↗

bioRxiv2020

An analog to digital converter controls bistable transfer competence of a widespread integrative and conjugative element

Conjugative transfer of the integrative and conjugative element ICEclc in Pseudomonas requires development of a transfer competence state in stationary phase, which arises only in 3-5% of individual cells. The mechanisms controlling this bistable switch between non-active and transfer competent cells have long remained enigmatic. Using a variety of genetic tools and epistasis experiments in P. putida, we uncovered an upstream cascade of three consecutive transcription factor-nodes, which controls transfer competence initiation. Initiation activates a feedback loop, which stochastic modeling and experimental data demonstrated acts as a scalable converter of unimodal input to bistable output. The feedback loop further enables prolonged production of a transcription factor that ensures downstream transfer competence formation in activated cells. Phylogenetic analyses showed that the ICEclc regulatory factors are widespread among Gamma- and Beta-proteobacteria, highlighting its evolutionary conservation and prime importance to control the behaviour of this wide family of conjugative elements.

genomics↗

bioRxiv2019

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

Genetic bistability controls different phenotypic programs in defined subpopulations of genetically identical bacteria. Conjugative transfer of the integrative and conjugative element ICEclc in Pseudomonas requires development of a transfer competence state in stationary phase, but this state arises only in 3-5% of individual cells. The mechanisms controlling and underlying the bistable switch between non-active and transfer competence cells have long remained enigmatic. Using a variety of genetic tools combined with stochastic modeling, we characterize here the factors and overall network architecture controlling bistable ICEclc activation of transfer competence. Two new key regulators (BisR and BisDC) were uncovered, that link the hierarchical cascade of ICEclc transfer competence activation to in total four regulatory nodes. The final activator complex named BisDC drives a positive feedback on its own transcription, and directly controls the "late" ICE promoters for excision and transfer. Stochastic mathematical modeling conceptually explained the arisal and maintenance of bistability by the feedback loop, and demonstrated its importance to guarantee consistent prolonged downstream output in activated cells. A minimized gene set allowing controllable bistable output in a Pseudomonas putida in absence of the ICEclc largely confirmed model predictions. Phylogenetic analyses further showed that the two new ICEclc regulatory factors are widespread among putative ICEs found in Gamma- and Beta- proteobacteria, highlighting the conceptual importance of our findings for the behaviour of this wide family of conjugative elements. Author summaryIntegrative and conjugative elements (ICEs) are mobile genetic elements present in virtually every bacterial species, which can confer adaptive functions to their host, such as antibiotic resistance or xenometabolic pathways. Integrated ICEs maintain by replication along with the genome of their bacterial host, but in order to transfer, the ICE excises and conjugates into a new recipient cell. Single-cell studies on a unique but widely representative ICE model from Pseudomonas (ICEclc) showed that transfer only occurs from a small dedicated subpopulation of cells that arises during stationary phase conditions. This bistable subpopulation differentiation is highly significant for ICE behaviour and fitness, but how it is regulated has remained largely unknown. The present work unveiled the architecture of the ICEclc transfer competence regulation, and showed its widespread occurrence among ICEs of the same family. Stochastic mathematical modeling explained how bistability is generated and maintained, prolonging the capacity of stationary phase cells to complete all stages of ICE activation.

microbiology↗

bioRxiv2018

Heterologous Expression of Pseudomonas putida Methyl-Accepting Chemotaxis Proteins Yields Escherichia coli Chemotactic to Aromatic Compounds

Escherichia coli, commonly used in chemotaxis studies, is attracted mostly by amino acids, sugars and peptides. We envisioned modifying chemotaxis specificity of E. coli by expressing heterologous chemoreceptors from Pseudomonas putida enabling attraction either to toluene or benzoate. The mcpT gene encoding the type 40H methyl-accepting chemoreceptor for toluene from Pseudomonas putida MT53 and the pcaY gene for the type 40H receptor for benzoate and related molecules from P. putida F1 were expressed from the trg promoter on a plasmid in motile wild-type E. coli MG1655. E. coli cells expressing McpT accumulated in chemoattraction assays to sources with 60-200 M toluene; less strongly than the response to 100 M serine, but statistically significantly stronger than to sources without any added attractant. An McpT-mCherry fusion protein was detectably expressed in E. coli and yielding weak but distinguishable membrane and polar foci in 1% of cells. E. coli expressing PcaY showed weak attraction to 0.1-1 mM benzoate but 50-70% of cells localized the PcaY-mCherry fusion to their membrane. We conclude that implementing heterologous receptors in the E. coli chemotaxis network is possible and, upon improvement of the compatibility of the type 40H chemoreceptors, may bear interest for biosensing.\n\nIMPORTANCEBacterial chemotaxis might be harnessed for the development of rapid biosensors, in which chemical availability is deduced from cell accumulation to chemoattractants over time. Chemotaxis of Escherichia coli has been well-studied, but the bacterium is not attracted to chemicals of environmental concern, such as aromatic solvents. We show here that heterologous chemoreceptors for aromatic compounds from Pseudomonas putida at least partly functionally complement the E. coli chemotaxis network, yielding cells attracted to toluene or benzoate. Complementation was still inferior to native chemoattractants like serine, but our study demonstrates the potential for obtaining selective sensing for aromatic compounds in E. coli.

microbiology↗

bioRxiv2016

Whole genome sequence accuracy is improved by replication in a population of mutagenized sorghum.

The accurate detection of induced mutations is critical for both forward and reverse genetics studies. Experimental chemical mutagenesis induces relatively few single base changes per individual. In a complex eukaryotic genome, false positive detection of mutations can occur at or above this mutagenesis rate. We demonstrate here, using a population of ethyl methanesulfonate (EMS) treated Sorghum bicolor BTx623 individuals, that using replication to detect false positive induced variants in next-generation sequencing data permits higher throughput variant detection with greater accuracy. We used a lower sequence coverage depth (average of 7X) from 586 independently mutagenized individuals and detected 5,399,493 homozygous SNPs. Of these, 76% originated from only 57,872 genomic positions prone to false positive variant calling. These positions are characterized by high copy number paralogs where the error-prone SNP positions are at copies containing a variant at the SNP position. The ability of short stretches of homology to generate these error prone positions suggests that incompletely assembled or poorly mapped repeated sequences are one driver of these error prone positions. Removal of these false positives left 1,275,872 homozygous and 477,531 heterozygous EMS-induced SNPs which, congruent with the mutagenic mechanism of EMS, were greater than 98% G:C to A:T transitions. Through this analysis we generated a database of sequence indexed mutants of Sorghum. This collection contains 4,035 high impact homozygous mutations in 3,637 genes and 56,514 homozygous missense mutations in 23,227 genes. Each line contains, on average, 2,177 annotated homozygous SNPs per genome, including seven likely gene knockouts and 96 missense mutations. The number of mutations in a transcript was linearly correlated with the transcript length and also the G+C count, but not with the GC/AT ratio. Analysis of the detected mutagenized positions identified CG-rich patches, and flanking sequences strongly influenced EMS-induced mutation rates. Our method for detecting false-positive induced mutations is generally applicable to any organism, is independent of the choice of in silico variant-calling algorithm, and is most valuable when the true mutation rate is likely to be low, such as in laboratory induced mutations or somatic mutation detection in medicine.

genetics↗