Zeynep Baharoglu

Conjugative DNA Transfer Induces the Bacterial SOS Response and Promotes Antibiotic Resistance Development through Integron Activation

Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response controls a series of genes responsible for DNA damage repair, which can lead to recombination and mutagenesis. In this manuscript, we show that conjugative transfer of ssDNA induces the bacterial SOS stress response, unless an anti-SOS factor is present to alleviate this response. We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements. Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell. Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria.

Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

ABSTRACT Antibiotic resistance development has been linked to the bacterial SOS stress response. In Escherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS in Vibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation with E. coli, all these antibiotics induce SOS in V. cholerae.

Folded DNA in Action: Hairpin Formation and Biological Functions in Prokaryotes

SUMMARY Structured forms of DNA with intrastrand pairing are generated in several cellular processes and are involved in biological functions. These structures may arise on single-stranded DNA (ssDNA) produced during replication, bacterial conjugation, natural transformation, or viral infections. Furthermore, negatively supercoiled DNA can extrude inverted repeats as hairpins in structures called cruciforms. Whether they are on ssDNA or as cruciforms, hairpins can modify the access of proteins to DNA, and in some cases, they can be directly recognized by proteins. Folded DNAs have been found to play an important role in replication, transcription regulation, and recognition of the origins of transfer in conjugative elements. More recently, they were shown to be used as recombination sites. Many of these functions are found on mobile genetic elements likely to be single stranded, including viruses, plasmids, transposons, and integrons, thus giving some clues as to the manner in which they might have evolved. We review here, with special focus on prokaryotes, the functions in which DNA secondary structures play a role and the cellular processes giving rise to them. Finally, we attempt to shed light on the selective pressures leading to the acquisition of functions for DNA secondary structures.

RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases

We observed that cells lacking Rep and UvrD, two replication accessory helicases, and the recombination protein RecF are cryo‐sensitive on rich medium. We isolated five mutations that suppress this Luria–Bertani (LB)‐cryo‐sensitivity and show that they map in the genes encoding the RNA polymerase subunits RpoB and RpoC. These rpoB (D444G, H447R and N518D) and rpoC mutants (H113R and P451L) were characterized. rpoBH447R and rpoBD444G prevent activation of the Prrn core promoter in rich medium, but only rpoBH447R also suppresses the auxotrophy of a relA spoT mutant (stringent‐like phenotype). rpoCH113R suppresses the thermo‐sensitivity of a greA greB mutant, suggesting that it destabilizes stalled elongation complexes. All mutations but rpoCP451L prevent R‐loop formation. We propose that these rpo mutations allow replication in the absence of Rep and UvrD by destabilizing RNA Pol upon replication–transcription collisions. In a RecF+ context, they improve growth of rep uvrD cells only if DinG is present, supporting the hypothesis that Rep, UvrD and DinG facilitate progression of the replication fork across transcribed sequences. They rescue rep uvrD dinG recF cells, indicating that in a recF mutant replication forks arrested by unstable transcription complexes can restart without any of the three known replication accessory helicases Rep, UvrD and DinG.

Connecting Environment and Genome Plasticity in the Characterization of Transformation-Induced SOS Regulation and Carbon Catabolite Control of the Vibrio cholerae Integron Integrase

The human pathogen Vibrio cholerae carries a chromosomal superintegron (SI). The SI contains an array of hundreds of gene cassettes organized in tandem which are stable under conditions when no particular stress is applied to bacteria (such as during laboratory growth). Rearrangements of these cassettes are catalyzed by the activity of the associated integron integrase. Understanding the regulation of integrase expression is pivotal to fully comprehending the role played by this genetic reservoir for bacterial adaptation and its connection with the development of antibiotic resistance. Our previous work established that the integrase is regulated by the bacterial SOS response and that it is induced during bacterial conjugation. Here, we show that transformation, another horizontal gene transfer (HGT) mechanism, also triggers integrase expression through SOS induction, underlining the importance of HGT in genome plasticity. Moreover, we report a new cyclic AMP (cAMP)-cAMP receptor protein (CRP)-dependent regulation mechanism of the integrase, highlighting the influence of the extracellular environment on chromosomal gene content. Altogether, our data suggest an interplay between different stress responses and regulatory pathways for the modulation of the recombinase expression, thus showing how the SI remodeling mechanism is merged into bacterial physiology.

RpoS Plays a Central Role in the SOS Induction by Sub-Lethal Aminoglycoside Concentrations in Vibrio cholerae

Bacteria encounter sub-inhibitory concentrations of antibiotics in various niches, where these low doses play a key role for antibiotic resistance selection. However, the physiological effects of these sub-lethal concentrations and their observed connection to the cellular mechanisms generating genetic diversification are still poorly understood. It is known that, unlike for the model bacterium Escherichia coli, sub-minimal inhibitory concentrations (sub-MIC) of aminoglycosides (AGs) induce the SOS response in Vibrio cholerae. SOS is induced upon DNA damage, and since AGs do not directly target DNA, we addressed two issues in this study: how sub-MIC AGs induce SOS in V. cholerae and why they do not do so in E. coli. We found that when bacteria are grown with tobramycin at a concentration 100-fold below the MIC, intracellular reactive oxygen species strongly increase in V. cholerae but not in E. coli. Using flow cytometry and gfp fusions with the SOS regulated promoter of intIA, we followed AG-dependent SOS induction. Testing the different mutation repair pathways, we found that over-expression of the base excision repair (BER) pathway protein MutY relieved this SOS induction in V. cholerae, suggesting a role for oxidized guanine in AG-mediated indirect DNA damage. As a corollary, we established that a BER pathway deficient E. coli strain induces SOS in response to sub-MIC AGs. We finally demonstrate that the RpoS general stress regulator prevents oxidative stress-mediated DNA damage formation in E. coli. We further show that AG-mediated SOS induction is conserved among the distantly related Gram negative pathogens Klebsiella pneumoniae and Photorhabdus luminescens, suggesting that E. coli is more of an exception than a paradigm for the physiological response to antibiotics sub-MIC.

Formation of a Stable RuvA Protein Double Tetramer Is Required for Efficient Branch Migration in Vitro and for Replication Fork Reversal in Vivo

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2KaP, specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2KaP is fully active for homologous recombination in vivo. RuvA2KaP is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2KaP for forks is decreased compared with wild type. Accordingly, RuvA2KaP is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2KaP is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2KaP is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.

Identification of genes involved in low aminoglycoside-induced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase

Sub-inhibitory concentrations (sub-MIC) of antibiotics play a very important role in selection and development of resistances. Unlike Escherichia coli, Vibrio cholerae induces its SOS response in presence of sub-MIC aminoglycosides. A role for oxidized guanine residues was observed, but the mechanisms of this induction remained unclear. To select for V. cholerae mutants that do not induce low aminoglycoside-mediated SOS induction, we developed a genetic screen that renders induction of SOS lethal. We identified genes involved in this pathway using two strategies, inactivation by transposition and gene overexpression. Interestingly, we obtained mutants inactivated for the expression of proteins known to destabilize the RNA polymerase complex. Reconstruction of the corresponding mutants confirmed their specific involvement in induction of SOS by low aminoglycoside concentrations. We propose that DNA lesions formed on aminoglycoside treatment are repaired through the formation of single-stranded DNA intermediates, inducing SOS. Inactivation of functions that dislodge RNA polymerase leads to prolonged stalling on these lesions, which hampers SOS induction and repair and reduces viability under antibiotic stress. The importance of these mechanisms is illustrated by a reduction of aminoglycoside sub-MIC. Our results point to a central role for transcription blocking at DNA lesions in SOS induction, so far underestimated.

Multiple Pathways of Genome Plasticity Leading to Development of Antibiotic Resistance

The emergence of multi-resistant bacterial strains is a major source of concern and has been correlated with the widespread use of antibiotics. The origins of resistance are intensively studied and many mechanisms involved in resistance have been identified, such as exogenous gene acquisition by horizontal gene transfer (HGT), mutations in the targeted functions, and more recently, antibiotic tolerance through persistence. In this review, we focus on factors leading to integron rearrangements and gene capture facilitating antibiotic resistance acquisition, maintenance and spread. The role of stress responses, such as the SOS response, is discussed.

Influence of very short patch mismatch repair on SOS inducing lesions after aminoglycoside treatment in Escherichia coli.

Low concentrations of aminoglycosides induce the SOS response in Vibrio cholerae but not in Escherichia coli. In order to determine whether a specific factor present in E. coli prevents this induction, we developed a genetic screen where only SOS inducing mutants are viable. We identified the vsr gene coding for the Vsr protein of the very short patch mismatch repair (VSPR) pathway. The effect of mismatch repair (MMR) mutants was also studied. We propose that lesions formed upon aminoglycoside treatment are preferentially repaired by VSPR without SOS induction in E. coli and by MMR when VSPR is impaired.