Biology subjects

Charpentier, X.

Publications and source records attributed to Charpentier, X..

17 recordsLinked to original sources

bioRxiv2026

Widespread immune systems protect bacteria against conjugative plasmids

Conjugative plasmids are a class of mobile genetic elements capable of efficient transfer between bacterial cells. Although they can introduce beneficial traits such as antibiotic resistance to recipients, they may also behave as genetic parasites. Bacteria would thus be expected to have evolved barriers to plasmid conjugation. However, the distribution of these barriers and their underlying mechanisms remain poorly understood. Here, we performed a large-scale analysis of 364 diverse strains of the opportunistic pathogen Acinetobacter baumannii as recipients of the broad-host-range conjugative plasmids R388 and RP4. Major variations in host susceptibilities to conjugation, with limited phylogenetic association, suggested multiple and fast-evolving plasmid-specific barriers. Functional genetic analyses revealed a role for core genes, pointing to epistasis or genetic background effects. This is illustrated by the previously unrecognized role of H-NS expression in alleviating conjugation barriers in a strain-dependent manner. Most importantly, we identified three novel immune systems protecting bacteria against conjugation by R388 and RP4. Their patchy distribution within the species, and that of their homologs across bacteria, indicate that they are part of a dynamic repertoire of immune systems against conjugation. While the Ishtar system promotes plasmid loss through putative HEPN nuclease domains, Namtar and Attar sense distinct components of the R388 type IV secretion system (T4SS) to trigger a non-proliferative, energetically depleted state, analogously to the abortive infection response of anti-phage defenses. Live imaging of conjugation showed Namtar halting cell division in Escherichia coli recipients, conferring population-level immunity against plasmid spread via horizontal and vertical transmission. The existence of immune systems specifically targeting T4SS components suggests that conjugative plasmids impose a selective disadvantage greater than previously thought. This work reveals an additional layer of bacterial immunity directed at a class of genetic elements driving dissemination of antibiotic resistance.

microbiology↗

bioRxiv2026

Heterogeneous dynamics of mobile genetic elements encode and spread antibiotic persistence in bacteria

Bacterial persisters are nonreplicating cells that transiently evade antibiotic killing within an otherwise susceptible, replicating population. While persister formation has traditionally been attributed to phenotypic heterogeneity, reversible functional variations among genetically identical cells, most studies lack direct genomic evidence to exclude the contribution of cryptic and reversible genetic variations. Here, we demonstrate that the dynamics of the Legionella pneumophila integrative and conjugative element (ICEs) pP36 encode and disseminate antibiotic persistence-like traits. During biofilm formation, pP36 undergoes heterogeneous chromosomal excision generating a subpopulation of nonreplicating persisters-like bacteria with a distinct molecular profile. pP36-mediated L. pneumophila growth rate heterogeneity significantly enhances bacterial survival under fluoroquinolone treatment. Our findings reveal that pP36 dynamics not only orchestrate physiological diversity but also serve as a vehicle for horizontal transfer of antibiotic persistence mechanisms among clinical Legionella isolates. While ICEs are recognized as key drivers of antibiotic resistance dissemination, this study establishes that ICEs-mediated phenotypic variations is a critical determinant of persister-like cell formation, reshaping our understanding of bacterial adaptability to antibiotic stress.

microbiology↗

bioRxiv2026

Calcium signals natural transformation in Acinetobacter baumannii

The acquisition of resistance to antibiotics in the opportunistic pathogen Acinetobacter baumannii may be linked to its capacity to undergo natural transformation. This mode of horizontal gene transfer relies on the import of extracellular DNA and its chromosomal integration by homologous recombination. Type IV pilus activity initiates the capture of extracellular DNA, which is then transported to the cytoplasm for recombination in the chromosome. While most Acinetobacter baumannii strains are transformable, the conditions allowing the expression of type IV pilus and other transformation genes remain largely unexplored. By investigating transformation-permissive conditions, we uncovered that calcium is a potent inducer of natural transformation. Type IV pilus genes and other transformation-specific genes (comEA, dprA) are upregulated by submillimolar concentrations of calcium ions, in a growth phase-dependent manner. In contrast, sodium chloride represses expression of pilA, counteracting the calcium-dependent induction, explaining the reported absence of transformation in NaCl-containing medium (such as LB). Independently of transcriptional induction, calcium ions also directly bind the type IV pilus through the calcium-dependent adhesin PilY1. Our data support a model in which calcium strengthens the interaction of PilY1 with the minor pilin complex, increasing pilus dynamics and subsequent pilus-dependent DNA capture. Hence, calcium signals natural transformation through both transcriptional and structural activation of type IV pilus. In addition to providing new insights into the regulation of natural transformation in A. baumannii this work led us to establish a protocol for genetic engineering of A. baumannii by natural transformation. ImportanceAcinetobacter baumannii is a nosocomial pathogen considered a critical research priority due to its resistance to last resort antibiotics. Understanding how A. baumannii evolves and acquires resistance to antibiotics is thus of prime importance. This species is capable of natural transformation, a means to acquire and spread genetic information, including antibiotic resistance genes. However, the conditions under which this process is active in this species remain elusive. We identify calcium ions as potent inducers of natural transformation and propose a model of the signaling of natural transformation by calcium ions. This opens the way for further investigations into the contribution of natural transformation to acquisition of antibiotic resistance. In addition, it provides an efficient way to genetically manipulate most A. baumannii strains.

microbiology↗

bioRxiv2025

Impact of natural transformation on the acquisition of novel genes in bacteria

Natural transformation is the only process of gene exchange under the exclusive control of the recipient bacteria. It has often been considered as a source of novel genes but quantitative assessments of this claim are lacking. To investigate the potential role of natural transformation in gene acquisition, we analysed a large collection of genomes of Acinetobacter baumannii (Ab) and Legionella pneumophila (Lp) for which transformation rates were experimentally determined. Natural transformation rates are weakly correlated with genome size. But they are negatively associated with gene flow in both species. This might result from a negative balance between transformations ability to cure the chromosome from mobile genetic elements (MGEs), resulting in gene loss, and its facilitation of gene acquisition. By focusing on the latter, we found that transformation was significantly associated with small gene acquisition events while MGEs-driven gene acquisition tend to be associated with larger ones. Events of gene gain by transformation were spread more evenly in the chromosome than MGEs encoding the ability to integrate autonomously. We estimated the contribution of natural transformation to gene gains by comparing recombination-driven gene acquisition rates between transformable and non-transformable strains. Natural transformation may have caused the acquisition of up to 6.4% (Ab) and 1.1% (Lp) of the novel genes. This low contribution of natural transformation to the acquisition of novel genes implies that most novel genes must have been acquired by other means. Interestingly, the ones potentially acquired by transformation include almost 15% of the recently acquired antibiotic resistance genes in A. baumannii. Hence, natural transformation may drive the acquisition of relatively few novel genes but these may have a high fitness impact.

genomics↗

bioRxiv2024

Host shapes microevolution of MDR Acinetobacter baumannii during long-term infection

Acinetobacter baumannii is a nosocomial pathogen associated with various infections, including urinary tract infections (UTIs). In the course of an infection, A. baumannii is known to rapidly become resistant to antibiotic therapy, but much less is known about possible adaptation without antibiotic pressure. Through a retrospective study, we investigated within-host genetic diversity during a subclinical five-year UTI in an animal-patient after withdrawal of colistin treatment. We conducted whole-genome sequencing and phenotypic assays on seventeen clonally related isolates from the Sequence Type 25 lineage. Phylogenomic analysis revealed their proximity with animal and human strains from the same country suggesting zoonotic transmission (France). In this case study, the clonally related strains presented variations in genome sizes and nucleotide sequences. Over the course of the infection, A. baumannii underwent genome reduction through insertion sequence (IS) recombination, phage excision, or plasmid curing. Alongside this global genome reduction, we observed an expansion of IS17, initially located on the endogenous large plasmid. Genetic variations were mainly located in biofilm formation and metabolism genes. We observed repeated variation affecting three biofilm genes and two adhesion operons associated with weak biofilm-forming capacity. Conversely, only two metabolic genes were recurrently affected and phenotypic assays indicated a rather stable metabolism profile between the isolates suggesting minor adaptations to its host. Lastly, an overall decreased antibiotic resistance - expected in the absence of antibiotic treatment - contrasted with a conserved colistin resistance due to a pmrB mutation among the isolates. Impact statementThis study brings a new insight on the genome evolution of the opportunistic pathogen Acinetobacter baumannii during a five-year urinary tract infection in the absence of antibiotic treatment. It relies on genomic and phenotypic analyses of multiple isolates from the same animal-patient highlighting the necessity of studying bacterial diversity as opposed to solely on single-isolate approaches. Over the course of the infection, we observed a reduction in genome size, resulting however in a limited loss of metabolism flexibility, but we observed recurrent loss in adhesion capacities and antibiotic resistance. However, this study stands out by the unexpected conserved colistin resistance of the study isolates in the absence of treatment. Data summaryThe newly sequenced genomes are listed in Table S1, these genomes have been submitted to the NCBI and their BioProject number is PRJNA1100485. The publicly available genomes of bacteria from human and animal origin used in this study are listed in Table S2 All the supplementary tables are available in the Supplementary Material File.

microbiology↗

bioRxiv2024

Interplay between the Xer recombination system and the dissemination of antibioresistance in Acinetobacter baumannii

Antibiotic-resistant infections pose a pressing challenge in clinical settings. Plasmids are widely recognized for hastening the emergence of resistance by facilitating horizontal gene transfer of antibiotic resistance genes among bacteria. We explore this inquiry in Acinetobacter baumannii, a globally emerging nosocomial pathogen responsible for a wide array of infections with worrying accumulation of resistances, notably involving plasmids. In this specie, plasmids of the Rep_3 family harbor adaptive genes within variable regions edged by potential site-specific recombination sites recognized by the XerCD recombinase. We first show that the Xer system of Acinetobacter baumannii functions as described in Escherichia coli, resolving chromosome dimers at the dif site as well as recombining plasmid-borne sites. The multiple Xer recombination sites found in Rep_3 plasmids do not, however, allow excising plasmid fragments. They rather recombine to co-integrate plasmids, which may then further evolve to exchange genes. Co-integrates represent a significative part of the plasmid population and their formation is controlled by the sequence of the recombination sites determining their compatibility between the recombining sites. We conclude that plasmids frequently exchange genes in Acinetobacter baumannii using Xer recombination, allowing a high level yet controlled plasticity involved in the acquisition and combination of resistance genes.

microbiology↗

bioRxiv2024

YraN is a helicase-associated nuclease fostering extended recombination events by natural transformation

Natural transformation is a widespread mechanism driving genetic exchanges in bacteria. It proceeds by the capture and internalization of exogenous DNA in linear single strands, ultimately integrated in the genome by homologous recombination. It is unknown how the RecA-directed D-loop intermediate of this dedicated recombination pathway is processed. We report that resolution of the transformation D-loop depends on two endonucleases of opposing phylogenetic distribution in bacteria. One is YraN, which has co-evolved and interacts with the ComM helicase, known to extend DNA recombination at the transformation D-loop. The other is CoiA, which is restricted to the Bacillota. CoiA is shown to be a resolvase of the transformation D-loop, extended by the RadA helicase in these species. We demonstrate that both YraN and CoiA act synergistically with their cognate helicases. These findings reveal that bacteria have evolved two helicase/nuclease pairs for the maturation and recombination extension of the transformation D-loop.

microbiology↗

bioRxiv2023

Intragenomic conflicts with plasmids and chromosomal mobile genetic elements drive the evolution of natural transformation within species

Natural transformation is the only mechanism of genetic exchange controlled by the recipient bacteria. We quantified its rates in 1282 strains of the human pathogens Legionella pneumophila (Lp) and Acinetobacter baumannii (Ab) and found that transformation rates evolve by large quick changes as a jump process across six orders of magnitude. Close to half of the strains are non-transformable in standard conditions. Transitions to non-transformability were frequent and recent, suggesting that they are deleterious and subsequently purged by natural selection. Accordingly, we find that transformation decreases genetic linkage in both species, which often accelerates adaptation. Intragenomic conflicts with chromosomal mobile genetic elements (MGEs) and plasmids could explain these transitions and a GWAS confirmed systematic negative associations between transformation and MGEs: plasmids and other conjugative elements in Lp, prophages in Ab, and transposable elements in both. In accordance with the modulation of transformation rates by genetic conflicts, transformable strains have fewer MGEs. Defense systems against the latter are associated with lower transformation except the adaptive CRISPR-Cas systems which show the inverse trend. The two species have different lifestyles and gene repertoires, but they exhibit very similar trends in terms of variation of transformation rates and its determinants, suggesting that genetic conflicts could drive the evolution of natural transformation in many bacteria.

genomics↗

bioRxiv2023

Manipulation of natural transformation by AbaR-type islands promotes fixation of antibiotic resistance in Acinetobacter baumannii populations

The opportunistic pathogen Acinetobacter baumannii, a major global public health concern, has developed multiple variants of AbaR-type genomic islands that confer multidrug resistance. The mechanisms facilitating the persistence of these potentially costly islands within A. baumannii populations have remained enigmatic. In this study, we employed a combination of investigative methods to shed light on the factors contributing to their selective advantage and long-term persistence. The dissemination of AbaR islands is intricately linked to their horizontal transfer via natural transformation, a process through which bacteria can import and recombine exogenous DNA, facilitating allelic recombination, genetic acquisition, and deletion. In experimental populations, we first quantified the rate at which natural transformation events occur between individuals. Our findings indicate that the rate of AbaR deletion events is marginally higher than the rate of their acquisition. When this result is integrated into a model of population dynamics not exposed to antibiotic selection pressure, it leads to the swift removal of AbaRs from the population, a pattern that stands in contrast to AbaR prevalence in genomes. Yet, genomic analyses show that nearly all AbaRs-carrying A. baumannii have at least one AbaR disrupting comM, a gene encoding a helicase critical for natural transformation. We discovered that such disruption differentially inhibits the rate of genetic acquisition and deletion. Specifically, they significantly impede the removal of AbaRs while still enabling the host cell to acquire and recombine short sequences, such as allelic variants. Through mathematical evolutionary modeling, we demonstrate that AbaRs inserted into comM gain a selective advantage over AbaRs inserted in sites that do not inhibit or completely inhibit transformation, in line with the genomic observations. The persistence of AbaRs within populations can be ascribed to their targeted integration into a gene, substantially diminishing the likelihood of their removal from the bacterial genome. In contrast, this integration enables the host cell to preserve the ability to acquire and eliminate various short heterologous sequences, enabling the host bacterium - and thus its AbaR - to adapt to the unpredictability of the environment and persist over the long term. This work underscores how AbaRs, and potentially other Mobile Genetic Elements (MGEs), can manipulate natural transformation to ensure their persistence in populations, ultimately leading to the high prevalence of multidrug resistance.

microbiology↗

bioRxiv2021

Interbacterial transfer of carbapenem resistance and large antibiotic resistance islands by natural transformation in pathogenic Acinetobacter

Acinetobacter baumannii infection poses a major health threat with recurrent treatment failure due to antibiotic resistance, notably to carbapenems. While genomic analyses of clinical strains indicate that homologous recombination plays a major role in the acquisition of antibiotic resistance genes, the underlying mechanisms of horizontal gene transfer often remain speculative. Our understanding of the acquisition of antibiotic resistance is hampered by the lack of experimental systems able to reproduce genomic observations. We here report the detection of recombination events occurring spontaneously in mixed bacterial populations and which can result in the acquisition of resistance to carbapenems. We show that natural transformation is the main driver of intra-, but also inter-strain recombination events between A. baumannii clinical isolates and pathogenic species of Acinetobacter. We observed that interbacterial natural transformation in mixed populations is more efficient at promoting the acquisition of large resistance islands (AbaR4, AbaR1) than when the same bacteria are supplied with large amounts of purified genomic DNA. Importantly, analysis of the genomes of the recombinant progeny revealed large recombination tracts (from 13 to 123 kb) similar to those observed in the genome of clinical isolates. Moreover, we highlight that transforming DNA availability is a key determinant of the rate of recombinants and results from both spontaneous release and interbacterial predatory behavior. In the light of our results, natural transformation should be considered as a leading mechanism of genome recombination and horizontal gene transfer of antibiotic resistance genes in Acinetobacter baumannii. ImportanceAcinetobacter baumannii is a multidrug resistant pathogen responsible for difficult-to-treat hospital-acquired infections. Understanding the mechanisms leading to the emergence of the multi-drug resistance in this pathogen is today crucial. Horizontal gene transfer is assumed to largely contribute to this multidrug resistance. However, in A. baumannii, the mechanisms leading to genome recombination and the horizontal transfer of resistance genes are poorly understood. We bring experimental evidence that natural transformation, a horizontal gene transfer mechanism recently highlighted in A. baumannii, allows the highly efficient interbacterial transfer of genetic elements carrying resistance to last line antibiotic carbapenems. Importantly, we demonstrated that natural transformation, occurring in mixed populations of Acinetobacter, enables the transfer of large resistance island mobilizing multiple resistance genes.

microbiology↗

bioRxiv2020

Transposon-insertion sequencing in a clinical isolate of Legionella pneumophila identifies essential genes and determinants of natural transformation

Legionella pneumophila is a Gram-negative bacterium ubiquitous in freshwater environments which, if inhaled, can cause a severe pneumonia in humans. The emergence of L. pneumophila is linked to several traits selected in the environment, the acquisition of some of which involved intra- and interkingdown horizontal gene transfer events. Transposon-insertion sequencing (TIS) is a powerful method to identify the genetic basis of selectable traits as well as to identify fitness determinants and essential genes, possible antibiotic targets. TIS has not yet been used to its full power in L. pneumophila, possibly because of difficulty to obtain a high-saturation transposon insertion library. Indeed, we found that ST1 isolates, to which belong the commonly used laboratory strains, are poorly permissive to saturating mutagenesis by conjugation-mediated transposon delivery. In contrast, we obtained high-saturation libraries in non-ST1 clinical isolates, offering the prospect of using TIS on unaltered L. pneumophila strains. Focusing on one of them, we therefore used TIS to identify essential genes in L. pneumophila. We also revealed that TIS could be used to identify genes controlling vertical transmission of mobile genetic elements. We then applied TIS to identify all the genes required for L. pneumophila to develop competence and undergo natural transformation, defining the set of major and minor Type IV pilins that are engaged in DNA uptake. This work paves the way for the functional exploration of the L. pneumophila genome by TIS and the identification of the genetic basis of other life traits of this species. ImportanceLegionella pneumophila is the etiologic agent of a severe form of nosocomial and community-acquired pneumonia in humans. L. pneumophila is found in man-made and freshwater environments which are the causing source of the infection. The environmental life traits of L. pneumophila, such as its abilities to form biofilms, resist biocides and unicellular predators, are essential to its ability to accidentally infect humans. A comprehensive identification of the genetic basis of these life traits could be obtained through the use of transposon-insertion sequencing. Yet, this powerful approach, had not been fully implemented in L. pneumophila. Here we described the successful implementation of the transposon-sequencing approach in a clinical isolate of L. pneumophila. We identify essential genes, potential drug targets, and genes required for horizontal gene transfer by natural transformation. This work represents an important step towards identifying the genetic basis of the many life traits of this environmental and pathogenic species.

microbiology↗

bioRxiv2020

Curing of large resistance island AbaR results in antibiotic susceptibility and increased natural transformability in Acinetobacter baumannii.

With a great diversity in gene composition including multiple putative antibiotic-resistance genes, AbaR islands are potential contributors to multi-drug resistance in Acinetobacter baumannii. However, the effective contribution of AbaR to antibiotic resistance and bacterial physiology remains elusive. To address this, we sought to accurately remove AbaR islands and restore the integrity of their insertion site. To this end, we devised a versatile scarless genome editing strategy. We performed this genetic modification in two recent A. baumannii clinical strains: the strain AB5075 and the nosocomial strain AYE which carry AbaR11 and AbaR1 islands of 19.7 kbp and 86.2 kbp, respectively. Antibiotic susceptibilities were then compared between the parental strains and their AbaR-cured derivatives. As anticipated by the predicted function of the ORF of this island, the antibiotic resistance profiles were identical between the wild type and the AbaR11-cured AB5075 strains. In contrast, AbaR1 carries 25 ORFs with a predicted resistance to several classes of antibiotics and the AYE AbaR1-cured derivative showed restored susceptibility to multiple classes of antibiotics. Moreover, curing of AbaRs restored high levels of natural transformability. Indeed, most AbaR islands are inserted into the comM gene involved in natural transformation. Our data indicate that AbaR insertion effectively inactivates comM and that the restored comM is functional. Curing of AbaR consistently resulted in highly transformable, and therefore, easily genetically tractable strains. Emendation of AbaR provides insight into the functional consequences of AbaR acquisition.

microbiology↗

bioRxiv2019

Orchestrated delivery of Legionella effectors by the Icm/Dot secretion system

Secretion of bacterial effector proteins into host cells plays a key role in bacterial virulence. Yet, the dynamics of the secretion systems activity remains poorly understood, especially when machineries deal with the export of numerous effectors. We address the question of multi-effector secretion by focusing on the Legionella pneumophila Icm/Dot T4SS that translocates a record number of 300 effectors. We set up a kinetic translocation assay, based on the {beta}-lactamase translocation reporter system combined with the effect of the protonophore CCCP. When used for translocation analysis of Icm/Dot substrates constitutively produced by L. pneumophila, this assay allows a fine monitoring of the secretion activity of the T4SS, independently of the expression control of the effectors. We observed that effectors are translocated with a specific timing, suggesting a control of their docking/translocation by the T4SS. Their delivery is accurately organized to allow effective manipulation of the host cell, as exemplified by the sequential translocation of effectors targeting Rab1, namely SidM/DrrA, LidA, LepB. Remarkably, the timed delivery of effectors does not depend only on their interaction with chaperone proteins but implies cyclic-di-GMP signaling, as the diguanylate cyclase Lpl0780/Lpp0809, contributes to the timing of translocation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/754762v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@17b7245org.highwire.dtl.DTLVardef@18b2443org.highwire.dtl.DTLVardef@1f9b7ceorg.highwire.dtl.DTLVardef@19275ff_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe dynamics of multi-effector secretion is addressed in the paradigm Legionella Icm/Dot T4SS C_LIO_LIA kinetic translocation assay allowing a fine monitoring of the T4SS activity is set up C_LIO_LISpecific secretion patterns consistent with sequential functions in the host are reported C_LIO_LIT4SS-dependent translocation is promoted by a diguanylate cyclase C_LIO_LIUnreported control of Type 4 secretion by the second messenger c-di-GMP signaling is revealed C_LI

microbiology↗

bioRxiv2019

Diverse conjugative elements silence natural transformation in Legionella species

Natural transformation, i.e. the uptake of DNA and its stable integration in the chromosome, is a major mechanism of horizontal gene transfer and is common in bacteria. The vast majority of bacterial genomes carry the specific genes involved in natural transformation, yet only a fraction of species are deemed naturally transformable. This is typically explained by the inability of standard laboratory conditions to induce this phenotypic trait. However, even when the inducing conditions are known, large intraspecific variations have been reported. In this study, we investigated the conservation and distribution of natural transformability in the human pathogen Legionella pneumophila. Using a panel of 113 clinical isolates, we found that natural transformability is relatively conserved but shows large variations inconsistent with the phylogeny. By conducting a genome-wide association study (GWAS) we identified the conjugative plasmid pLPL as a source of these intraspecific variations. We further show that the plasmid inhibits transformation by simultaneously silencing the genes required for DNA uptake and recombination, comEC, comEA, comF and comM. We identified a plasmid-encoded small RNA (sRNA), RocRp, as solely responsible for the silencing of natural transformation. RocRp is homologous to the highly conserved and chromosome-encoded RocR which controls the transient expression of the DNA uptake system. We show that RocRp can take over the function of RocR, by acting as a substitute, ensuring that the bacterial host of the conjugative plasmid does not become naturally transformable. Distinct homologs of this plasmid-encoded sRNA are found in diverse conjugative elements in other Legionella species, suggesting that silencing natural transformation is beneficial to these genetic elements. We propose that transformation-interfering factors are frequent genetic cargo of mobile genetic elements, accounting for intraspecific variations in natural transformation but also responsible for the apparent non-transformability of some species.

microbiology↗

bioRxiv2019

Bacterial transformation buffers environmental fluctuations through the reversible integration of mobile genetic elements

Horizontal gene transfer (HGT) is known to promote the spread of genes in bacterial communities, which is of primary importance to human health when these genes provide resistance to antibiotics. Among the main HGT mechanisms, natural transformation stands out as being widespread and encoded by the bacterial core genome. From an evolutionary perspective, transformation is often viewed as a mean to generate genetic diversity and mixing within bacterial populations. However, another recent paradigm proposes that its main evolutionary function would be to cure bacterial genomes from their parasitic mobile genetic elements (MGEs). Here, we propose to combine these two seemingly opposing points of view because MGEs, although costly for bacterial cells, can carry functions that are point-in-time beneficial to bacteria under stressful conditions (e.g. antibiotic resistance genes under antibiotic exposure). Using computational modeling, we show that, in stochastic environments (unpredictable stress exposure), an intermediate transformation rate maximizes bacterial fitness by allowing the reversible integration of MGEs carrying resistance genes but costly for the replication of host cells. By ensuring such reversible genetic diversification (acquisition then removal of MGEs), transformation would be a key mechanism for stabilizing the bacterial genome in the long term, which would explain its striking conservation.

microbiology↗

bioRxiv2018

Fluorescence-based detection of natural transformation in drug resistant Acinetobacter baumannii

Acinetobacter baumannii is a nosocomial agent with a high propensity for developing resistance to antibiotics. This ability relies on horizontal gene transfer mechanisms occurring in the Acinetobacter genus, including natural transformation. To study natural transformation in bacteria, the most prevalent method uses selection for the acquisition of an antibiotic resistance marker in a target chromosomal locus by the recipient cell. Most clinical isolates of A. baumannii are resistant to multiple antibiotics limiting the use of such selection-based method. Here we report the development of a phenotypic and selection-free method based on flow cytometry to detect transformation events in multidrug resistant (MDR) clinical A. baumannii isolates. To this end, we engineered a translational fusion between the abundant and conserved A. baumannii nucleoprotein (HU) and the superfolder green fluorescent protein (sfGFP). The new method was benchmarked against the conventional antibiotic selection-based method. Using this new method, we investigated several parameters affecting transformation efficiencies and identified conditions of transformability one hundred times higher than those previously reported. Using optimized transformation conditions, we probed natural transformation in a set of MDR clinical and non-clinical animal A. baumannii isolates. Regardless of their origin, the majority of the isolates displayed natural transformability, indicative of a conserved trait in the species. Overall, this new method and optimized protocol will greatly facilitate the study of natural transformation in the opportunistic pathogen A. baumannii.

microbiology↗

bioRxiv2017

KKL-35 Exhibits Potent Antibiotic Activity Against Legionella Species Independently Of Trans-Translation Inhibition

Trans-translation is a ribosome rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibit trans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogen Legionella pneumophila and other related species that can also cause Legionnaires disease (LD). Consistent with the essential nature of trans-translation in L. pneumophila, KKL-35 inhibits growth of all tested strains at sub-micromolar concentrations. KKL-35 is also active against other LD-causing Legionella species. KKL-35 remains equally active against L. pneumophila mutants that have evolved resistance to macrolides. KKL-35 inhibits multiplication of L. pneumophila in human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 is not synergistic with other ribosome-targeting antibiotics and does not induce the filamentation phenotype observed in cells defective for trans-translation. Importantly, KKL-35 remains active against L. pneumophila mutants expressing an alternate ribosome-rescue system and lacking tmRNA, the essential component of trans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition of trans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogen L. pneumophila and with no detectable resistance. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment.

microbiology↗