Bacillus subtilis as a host for natural product discovery and engineering of biosynthetic gene clusters.

Covering: up to October 2023Many bioactive natural products are synthesized by microorganisms that are either difficult or impossible to cultivate under laboratory conditions, or that produce only small amounts of the desired compound. By transferring biosynthetic gene clusters (BGCs) into alternative host organisms that are more easily cultured and engineered, larger quantities can be obtained and new analogues with potentially improved biological activity or other desirable properties can be generated. Moreover, expression of cryptic BGCs in a suitable host can facilitate the identification and characterization of novel natural products. Heterologous expression therefore represents a valuable tool for natural product discovery and engineering as it allows the study and manipulation of their biosynthetic pathways in a controlled setting, enabling innovative applications. Bacillus is a genus of Gram-positive bacteria that is widely used in industrial biotechnology as a host for the production of proteins from diverse origins, including enzymes and vaccines. However, despite numerous successful examples, Bacillus species remain underexploited as heterologous hosts for the expression of natural product BGCs. Here, we review important advantages that Bacillus species offer as expression hosts, such as high secretion capacity, natural competence for DNA uptake, and the increasing availability of a wide range of genetic tools for gene expression and strain engineering. We evaluate different strain optimization strategies and other critical factors that have improved the success and efficiency of heterologous natural product biosynthesis in B. subtilis. Finally, future perspectives for using B. subtilis as a heterologous host are discussed, identifying research gaps and promising areas that require further exploration.

Optimizing mRNA transfection on a high-definition electroporation microelectrode array results in 98% efficiency and multiplexed gene delivery.

Spatially resolved transfection, intracellular delivery of proteins and nucleic acids, has the potential to drastically speed up the discovery of biologically active cargos, for instance for the development of cell therapies or new genome engineering tools. We recently demonstrated the use of a high-density microelectrode array for the targeted electrotransfection of cells grown on its surface, a process called High-Definition Electroporation (HD-EP). We also developed a framework based on Design of Experiments to quickly establish optimized electroporation conditions across five different electrical pulse parameters. Here, we used this framework to optimize the transfection efficiency of primary fibroblasts with a mCherry-encoding mRNA, resulting in 98% of the cells expressing the desired fluorescent protein without any sign of cell death. That transfection yield is the highest reported so far for electroporation. Moreover, varying the pulse number was shown to modulate the fluorescence intensity of cells, indicating the dosage-controlled delivery of mRNA and protein expression. Finally, exploiting the single-electrode addressability of the microelectrode array, we demonstrated spatially resolved, high efficiency, sequential transfection of cells with three distinct mRNAs. Since the chip can be easily redesigned to feature a much large number of electrodes, we anticipate that this methodology will enable the development of dedicated screening platforms for analysis of mRNA variants at scale.

Amplification Efficiency and Template Accessibility as Distinct Causes of Rain in Digital PCR: Monte Carlo Modeling and Experimental Validation.

Partitions in digital PCR (dPCR) assays do not reach the detection threshold at the same time. This heterogeneity in amplification results in intermediate endpoint fluorescence values (i.e., rain) and misclassification of partitions, which has a major impact on the accuracy of nucleic acid quantification. Rain most often results from a reduced amplification efficiency or template inaccessibility; however, exactly how these contribute to rain has not been described. We developed and experimentally validated an analytical model that mechanistically explains the relationship between amplification efficiency, template accessibility, and rain. Using Monte Carlo simulations, we show that a reduced amplification efficiency leads to broader threshold cycle (Ct) distributions that can be fitted using a log-normal probability distribution. From the fit parameters, the amplification efficiency can be calculated. Template inaccessibility, on the other hand, leads to a different rain pattern, in which a distinct exponential tail in the Ct distribution can be observed. Using our model, it is possible to determine if the amplification efficiency, template accessibility, or another source is the main contributor of rain in dPCR assays. We envision that this model will facilitate and speed up dPCR assay optimization and provide an indication for the accuracy of the assay.

High-definition electroporation: Precise and efficient transfection on a microelectrode array.

Intracellular delivery is critical for a plethora of biomedical applications, including mRNA transfection and gene editing. High transfection efficiency and low cytotoxicity, however, are often beyond the capabilities of bulk techniques and synonymous with extensive empirical optimization. Moreover, bulk techniques are not amenable to large screening applications. Here, we propose an expeditious workflow for achieving optimal electroporation-based intracellular delivery. Using the multiplexing ability of a high-definition microelectrode array (MEA) chip, we performed a sequence of carefully designed experiments, multiple linear regression modelling and validation to obtain optimal conditions for on-chip electroporation of primary fibroblasts. Five electric pulse parameters were varied to generate 32 different electroporation conditions. The effect of the parameters on cytotoxicity and intracellular delivery could be evaluated with just two experiments. Most successful electroporation conditions resulted in no cell death, highlighting the low cytotoxicity of on-chip electroporation. The resulting delivery models were then used to achieve dosage-controlled delivery of small molecules, delivery of Cas9-GFP single-guide RNA complexes and transfection with an mCherry-encoding mRNA, resulting in previously unreported high-efficiency, single-cell transfection on MEAs: cells expressed mCherry on 81% of the actuated electrodes, underscoring the vast potential of CMOS MEA technology for the transfection of primary cells.

Molecular detection of SARS-COV-2 in exhaled breath at the point-of-need.

The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It assesses contagiousness directly, the sample is easy and comfortable to obtain, sampling can be standardized, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an RT-qPCR molecular assay to detect SARS-CoV-2 shedding. Our portable, silicon impactor allowed for the efficient capture (>85%) of respiratory particles down to 300 nm without the need for additional equipment. We demonstrate using both conventional off-chip and in-situ PCR directly on the silicon chip that sampling subjects' breath in less than a minute yields sufficient viral RNA to detect infections as early as standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test remains positive during the first week but is the first to consistently report a negative result, putatively signalling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections.

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis.

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters.

STRide probes: Single-labeled short tandem repeat identification probes.

The demand for forensic DNA profiling at the crime scene or at police stations is increasing. DNA profiling is currently performed in specialized laboratories by PCR amplification of Short Tandem Repeats (STR) followed by amplicon sizing using capillary electrophoresis. The need for bulky equipment to identify alleles after PCR presents a challenge for shifting to a decentralized workflow. We devised a novel hybridization-based STR-genotyping method, using Short Tandem Repeat Identification (STRide) probes, which could help tackle this issue. STRide probes are fluorescently labeled oligonucleotides that rely on the quenching properties of guanine on fluorescein derivatives. Mismatches between STRide probes and amplicons can be detected by melting curve analysis after asymmetric PCR. The functionality of the STRide probes was demonstrated by analyzing synthetic DNA samples for the D16S539 locus. Next, STRide probes were developed for five different CODIS core loci (D16S539, TH01, TPOX, FGA, and D7S820). These probes were validated by analyzing 13 human DNA samples. Successful genotyping was obtained using inputs as low as 31 pg of DNA, demonstrating high sensitivity. The STRide probes are ideally suited to be implemented in a microarray and present an important step towards a portable device for fast on-site forensic DNA fingerprinting.

Single-cell transfection technologies for cell therapies and gene editing.

Advances in gene editing and cell therapies have recently led to outstanding clinical successes. However, the lack of a cost-effective manufacturing process prevents the democratization of these innovative medical tools. Due to the common use of viral vectors, the step of transfection in which cells are engineered to gain new functions, is a major bottleneck in making safe and affordable cell products. A promising opportunity lies in Single-Cell Transfection Technologies (SCTTs). SCTTs have demonstrated higher efficiency, safety and scalability than conventional transfection methods. They can also feature unique abilities such as substantial dosage control over the cargo delivery, single-cell addressability and integration in microdevices comprising multiple monitoring modalities. Unfortunately, the potential of SCTTs is not fully appreciated: they are most often restricted to research settings with little adoption in clinical settings. To encourage their adoption, we review and compare recent developments in SCTTs, and how they can enable selected clinical applications. To help bridge the gap between fundamental research and its translation to the clinic, we also describe how Good Manufacturing Practices (GMP) can be integrated in the design of SCTTs.

Image-Based Dynamic Phenotyping Reveals Genetic Determinants of Filamentation-Mediated ß-Lactam Tolerance.

Antibiotic tolerance characterized by slow killing of bacteria in response to a drug can lead to treatment failure and promote the emergence of resistance. ß-lactam antibiotics inhibit cell wall growth in bacteria and many of them cause filamentation followed by cell lysis. Hence delayed cell lysis can lead to ß-lactam tolerance. Systematic discovery of genetic factors that affect ß-lactam killing kinetics has not been performed before due to challenges in high-throughput, dynamic analysis of viability of filamented cells during bactericidal action. We implemented a high-throughput time-resolved microscopy approach in a gene deletion library of Escherichia coli to monitor the response of mutants to the ß-lactam cephalexin. Changes in frequency of lysed and intact cells due to the antibiotic action uncovered several strains with atypical lysis kinetics. Filamentation confers tolerance because antibiotic removal before lysis leads to recovery through numerous concurrent divisions of filamented cells. Filamentation-mediated tolerance was not associated with resistance, and therefore this phenotype is not discernible through most antibiotic susceptibility methods. We find that deletion of Tol-Pal proteins TolQ, TolR, or Pal but not TolA, TolB, or CpoB leads to rapid killing by ß-lactams. We also show that the timing of cell wall degradation determines the lysis and killing kinetics after ß-lactam treatment. Altogether, this study uncovers numerous genetic determinants of hitherto unappreciated filamentation-mediated ß-lactam tolerance and support the growing call for considering antibiotic tolerance in clinical evaluation of pathogens. More generally, the microscopy screening methodology described here can easily be adapted to study lysis in large numbers of strains.

Model-Driven Controlled Alteration of Nanopillar Cap Architecture Reveals its Effects on Bactericidal Activity.

Nanostructured surfaces can be engineered to kill bacteria in a contact-dependent manner. The study of bacterial interactions with a nanoscale topology is thus crucial to developing antibacterial surfaces. Here, a systematic study of the effects of nanoscale topology on bactericidal activity is presented. We describe the antibacterial properties of highly ordered and uniformly arrayed cotton swab-shaped (or mushroom-shaped) nanopillars. These nanostructured surfaces show bactericidal activity against Staphylococcus aureus and Pseudomonas aeruginosa. A biophysical model of the cell envelope in contact with the surface, developed ab initio from the infinitesimal strain theory, suggests that bacterial adhesion and subsequent lysis are highly influenced by the bending rigidity of the cell envelope and the surface topography formed by the nanopillars. We used the biophysical model to analyse the influence of the nanopillar cap geometry on the bactericidal activity and made several geometrical alterations of the nanostructured surface. Measurement of the bactericidal activities of these surfaces confirms model predictions, highlights the non-trivial role of cell envelope bending rigidity, and sheds light on the effects of nanopillar cap architecture on the interactions with the bacterial envelope. More importantly, our results show that the surface nanotopology can be rationally designed to enhance the bactericidal efficiency.

GTP Binding is Necessary for the Activation of a Toxic Mutant Isoform of the Essential GTPase ObgE.

Even though the Obg protein is essential for bacterial viability, the cellular functions of this universally conserved GTPase remain enigmatic. Moreover, the influence of GTP and GDP binding on the activity of this protein is largely unknown. Previously, we identified a mutant isoform of ObgE (the Obg protein of Escherichia coli) that triggers cell death. In this research we explore the biochemical requirements for the toxic effect of this mutant ObgE* isoform, using cell death as a readily accessible read-out for protein activity. Both the absence of the N-terminal domain and a decreased GTP binding affinity neutralize ObgE*-mediated toxicity. Moreover, a deletion in the region that connects the N-terminal domain to the G domain likewise abolishes toxicity. Taken together, these data indicate that GTP binding by ObgE* triggers a conformational change that is transmitted to the N-terminal domain to confer toxicity. We therefore conclude that ObgE*-GTP, but not ObgE*-GDP, is the active form of ObgE* that is detrimental to cell viability. Based on these data, we speculate that also for wild-type ObgE, GTP binding triggers conformational changes that affect the N-terminal domain and thereby control ObgE function.

Bacterial Heterogeneity and Antibiotic Survival: Understanding and Combatting Persistence and Heteroresistance.

For decades, mankind has dominated the battle against bacteria, yet the tide is slowly turning. Our antibacterial strategies are becoming less effective, allowing bacteria to get the upper hand. The alarming rise in antibiotic resistance is an important cause of anti-infective therapy failure. However, other factors are at play as well. It is widely recognized that bacterial populations display high levels of heterogeneity. Population heterogeneity generates phenotypes specialized in surviving antibiotic attacks. Nonetheless, the presence of antibiotic-insensitive subpopulations is not considered when initiating treatment. It is therefore time to reevaluate how we combat bacterial infections. We here focus on antibiotic persistence and heteroresistance, phenomena in which small fractions of the population are tolerant (persisters) and resistant to antibiotics, respectively. We discuss molecular mechanisms involved, their clinical importance, and possible therapeutic strategies. Moving forward, we argue that these heterogeneous phenotypes should no longer be ignored in clinical practice and that better diagnostic and therapeutic approaches are urgently needed.

Antibiotics: Combatting Tolerance To Stop Resistance.

Antibiotic resistance poses an alarming and ever-increasing threat to modern health care. Although the current antibiotic crisis is widely acknowledged, actions taken so far have proved insufficient to slow down the rampant spread of resistant pathogens. Problematically, routine screening methods and strategies to restrict therapy failure almost exclusively focus on genetic resistance, while evidence for dangers posed by other bacterial survival strategies is mounting. Antibiotic tolerance, occurring either population-wide or in a subpopulation of cells, allows bacteria to transiently overcome antibiotic treatment and is overlooked in clinical practice. In addition to prolonging treatment and causing relapsing infections, recent studies have revealed that tolerance also accelerates the emergence of resistance. These critical findings emphasize the need for strategies to combat tolerance, not only to improve treatment of recurrent infections but also to effectively address the problem of antibiotic resistance at the root.

Biochemical determinants of ObgE-mediated persistence.

Obg is a versatile GTPase that plays a pivotal role in bacterial persistence. We previously showed that the Escherichia coli homolog ObgE exerts this activity through transcriptional activation of a toxin-antitoxin module and subsequent membrane depolarization. Here, we assessed the role of G-domain functionality in ObgE-mediated persistence. Through screening of a mutant library, we identified five obgE alleles (with substitutions G166V, D246G, S270I, N283I and I313N) that have lost their persistence function and no longer activate hokB expression. These alleles support viability of a strain otherwise deprived of ObgE, indicating that ObgE's persistence function can be uncoupled from its essential role. Based on the ObgE crystal structure, we designed two additional mutant proteins (T193A and D286Y), one of which (D286Y) no longer affects persistence. Using isothermal titration calorimetry, stopped-flow experiments and kinetics, we subsequently assessed nucleotide binding and GTPase activity in all mutants. With the exception of the S270I mutant that is possibly affected in protein-protein interactions, all mutants that have lost their persistence function display severely reduced binding to GDP or the alarmone ppGpp. However, we find no clear relation between persistence and GTP or pppGpp binding nor with GTP hydrolysis. Combined, our results signify an important step toward understanding biochemical determinants underlying persistence.

High-throughput time-resolved morphology screening in bacteria reveals phenotypic responses to antibiotics.

Image-based high-throughput screening strategies for quantifying morphological phenotypes have proven widely successful. Here we describe a combined experimental and multivariate image analysis approach for systematic large-scale phenotyping of morphological dynamics in bacteria. Using off-the-shelf components and software, we established a workflow for high-throughput time-resolved microscopy. We then screened the single-gene deletion collection of Escherichia coli for antibiotic-induced morphological changes. Using single-cell quantitative descriptors and supervised classification methods, we measured how different cell morphologies developed over time for all strains in response to the ß-lactam antibiotic cefsulodin. 191 strains exhibit significant variations under antibiotic treatment. Phenotypic clustering provided insights into processes that alter the antibiotic response. Mutants with stable bulges show delayed lysis, contributing to antibiotic tolerance. Lipopolysaccharides play a crucial role in bulge stability. This study demonstrates how multiparametric phenotyping by high-throughput time-resolved imaging and computer-aided cell classification can be used for comprehensively studying dynamic morphological transitions in bacteria.

Silicon µPCR Chip for Forensic STR Profiling with Hybeacon Probe Melting Curves.

The demand to perform forensic DNA profiling outside of centralized laboratories and on the crime scene is increasing. Several criminal investigations would benefit tremendously from having DNA based information available in the first hours rather than days or weeks. However, due to the complexity and time-consuming nature of standard DNA fingerprinting methods, rapid and automated analyses are hard to achieve. We here demonstrate the implementation of an alternative DNA fingerprinting method in a single microchip. By combining PCR amplification and HyBeacon melting assays in a silicon Lab-on-a-chip (LoC), a significant step towards rapid on-site DNA fingerprinting is taken. The small form factor of a LoC reduces reagent consumption and increases portability. Additional miniaturization is achieved through an integrated heating element covering 24 parallel micro-reactors with a reaction volume of 0.14 µl each. The high level of parallelization allows the simultaneous analysis of 4 short tandem repeat (STR) loci and the amelogenin gender marker commonly included in forensic DNA analysis. A reference and crime scene sample can be analyzed simultaneously for direct comparison. Importantly, by using industry-standard semiconductor manufacturing processes, mass manufacturability can be guaranteed. Following assay design and optimization, complete 5-loci profiles could be robustly generated on-chip that are on par with those obtained using conventional benchtop real-time PCR thermal cyclers. Together, our results are an important step towards the development of commercial, mass-produced, portable devices for on-site testing in forensic DNA analysis.

Bacterial persistence promotes the evolution of antibiotic resistance by increasing survival and mutation rates.

Persisters are transiently antibiotic-tolerant cells that complicate the treatment of bacterial infections. Both theory and experiments have suggested that persisters facilitate genetic resistance by constituting an evolutionary reservoir of viable cells. Here, we provide evidence for a strong positive correlation between persistence and the likelihood to become genetically resistant in natural and lab strains of E. coli. This correlation can be partly attributed to the increased availability of viable cells associated with persistence. However, our data additionally show that persistence is pleiotropically linked with mutation rates. Our theoretical model further demonstrates that increased survival and mutation rates jointly affect the likelihood of evolving clinical resistance. Overall, these results suggest that the battle against antibiotic resistance will benefit from incorporating anti-persister therapies.

Development and validation of a glass-silicon microdroplet-based system to measure sulfite concentrations in beverages.

Sulfite is often added to beverages as an antioxidant and antimicrobial agent. In fermented beverages, sulfite is also naturally produced by yeast cells. However, sulfite causes adverse health effects in asthmatic patients and accurate measurement of the sulfite concentration is therefore very important. Current sulfite analysis methods are time- and reagent-consuming and often require costly equipment. Here, we present a system allowing sensitive, ultralow-volume sulfite measurements based on a reusable glass-silicon microdroplet platform on which microdroplet generation, addition of enzymes through chemical-induced emulsion destabilization and pillar-induced droplet merging, emulsion restabilization, droplet incubation, and fluorescence measurements are integrated. In a first step, we developed and verified a fluorescence-based enzymatic assay for sulfite by measuring its analytical performance (LOD, LOQ, the dynamic working range, and the influence of salts, colorant, and sugars) and comparing fluorescent microplate readouts of fermentation samples with standard colorimetric measurements using the 5,5'-dithiobis-(2-nitrobenzoic acid) assay of the standard Gallery Plus Beermaster analysis platform. Next, samples were analyzed on the microdroplet platform, which also showed good correlation with the standard colorimetric analysis. Although the presented platform does not allow stable reinjection of droplets due to the presence of a tight array of micropillars at the fluidics entrances to prevent channel clogging by dust, removing the pillars, and integrating miniaturized pumps and optics in a future design would allow to use this platform for high-throughput, automated, and portable screening of microbes, plant, or mammalian cells. Graphical abstract ᅟ.

Ultra-fast, sensitive and quantitative on-chip detection of group B streptococci in clinical samples.

PCR enables sensitive and specific detection of infectious disease agents, but application in point-of-care diagnostic testing remains scarce. A compact tool that runs PCR assays in less than a few minutes and that relies on mass-producible, disposable reactors could revolutionize while-you-wait molecular testing. We here exploit well-established semiconductor manufacturing processes to produce silicon ultra-fast quantitative PCR (UF-qPCR) chips that can run PCR protocols with limited assay optimization. A total of 110 clinical samples were analyzed for the detection of group B streptococci using both a validated benchtop and an on-chip qPCR assay. For the on-chip assay, the total reaction time was reduced after optimization to less than 5 min. The standard curve, spanning a concentration range of 5 log units, yielded a PCR efficiency of 94%. The sensitivity obtained was 96% (96/100; CI: 90-98%) and the specificity 70% (7/10; CI: 40-90%). We show that if melting analyses would be integrated, the obtained sensitivity would drop slightly to 93% (CI: 86-96%), while the specificity would increase to 100% (CI: 72% - 100%). In comparison to the benchtop reference qPCR assay performed on a LightCycler©96, the on-chip assay demonstrated a highly significant qualitative (Spearman's rank correlation) and quantitative (linear regression) correlation. Using a mass-producible qPCR chip and limited assay optimization, we were able to develop a validated qPCR protocol that can be carried out in less than five minutes. The analytical performance of the microchip-based UF-qPCR system was shown to match that of a benchtop assay. This is the first report to provide UF-qPCR validation using clinical samples. We demonstrate that qPCR-based while-you-wait testing is feasible without jeopardizing assay performance.

The Putative De-N-acetylase DnpA Contributes to Intracellular and Biofilm-Associated Persistence of Pseudomonas aeruginosa Exposed to Fluoroquinolones.

Persisters are the fraction of antibiotic-exposed bacteria transiently refractory to killing and are recognized as a cause of antibiotic treatment failure. The putative de-N-acetylase DnpA increases persister levels in Pseudomonas aeruginosa upon exposure to fluoroquinolones in broth. In this study the wild-type PAO1 and its dnpA insertion mutant (dnpA::Tn) were used in parallel and compared for their capacity to generate persisters in broth (surviving fraction after exposure to high antibiotic concentrations) and their susceptibility to antibiotics in models of intracellular infection of THP-1 monocytes and of biofilms grown in microtiter plates. Multiplication in monocytes was evaluated by fluorescence dilution of GFP (expressed under the control of an inducible promoter) using flow cytometry. Gene expression was measured by quantitative RT-PCR. When exposed to fluoroquinolones (ciprofloxacin or levofloxacin) but not to meropenem or amikacin, the dnpA::Tn mutant showed a 3- to 10-fold lower persister fraction in broth. In infected monocytes, fluoroquinolones (but not the other antibiotics) were more effective (difference in Emax: 1.5 log cfu) against the dnpA::Tn mutant than against the wild-type PAO1. Dividing intracellular bacteria were more frequently seen (1.5 to 1.9-fold) with the fluoroquinolone-exposed dnpA::Tn mutant than with its parental strain. Fluoroquinolones (but not the other antibiotics) were also 3-fold more potent to prevent biofilm formation by the dnpA::Tn mutant than by PAO1 as well as to act upon biofilms (1-3 days of maturity) formed by the mutant than by the parental strain. Fluoroquinolones induced the expression of gyrA (4.5-7 fold) and mexX (3.6-5.4 fold) in the parental strain but to a lower extent (3-4-fold for gyrA and 1.8-2.8-fold for mexX, respectively) in the dnpA::Tn mutant. Thus, our data show that a dnpA insertion mutant of P. aeruginosa is more receptive to fluoroquinolone antibacterial effects than its parental strain in infected monocytes or in biofilms. The mechanism of this higher responsiveness could involve a reduced overexpression of the fluoroquinolone target.