Fluorescent reporters give new insights into antibiotics-induced nonsense and frameshift mistranslation.

We developed a reporter system based on simultaneous expression of two fluorescent proteins: GFP as a reporter of the capacity of protein synthesis and mutated mScarlet-I as a reporter of translational errors. Because of the unique stop codons or frameshift mutations introduced into the mScarlet-I gene, red fluorescence was produced only after a mistranslation event. These reporters allowed us to estimate mistranslation at a single cell level using either flow cytometry or fluorescence microscopy. We found that laboratory strains of Escherichia coli are more prone to mistranslation compared to the clinical isolates. As relevant for uropathogenic E. coli, growth in human urine elevated translational frameshifting compared to standard laboratory media, whereas different standard media had a small effect on translational fidelity. Antibiotic-induced mistranslation was studied by using amikacin (aminoglycoside family) and azithromycin (macrolide family). Bactericidal amikacin induced preferably stop-codon readthrough at a moderate level. Bacteriostatic azithromycin on the other hand induced both frameshifting and stop-codon readthrough at much higher level. Single cell analysis revealed that fluorescent reporter-protein signal can be lost due to leakage from a fraction of bacteria in the presence of antibiotics, demonstrating the complexity of the antimicrobial activity.

Fibrous matrices facilitate pleurocidin killing of wound associated bacterial pathogens.

Conventional wound infection treatments neither actively promote wound healing nor address the growing problem of antibacterial resistance. Antimicrobial peptides (AMPs) are natural defense molecules, released from host cells, which may be rapidly bactericidal, modulate host-immune responses, and/or act as endogenous mediators for wound healing. However, their routine clinical use has hitherto been hindered due to their instability in the wound environment. Here we describe an electrospun carrier system for topical application of pleurocidin, demonstrating sufficient AMP release from matrices to kill wound-associated pathogens including Acinetobacter baumannii and Pseudomonas aeruginosa. Pleurocidin can be incorporated into polyvinyl alcohol (PVA) fiber matrices, using coaxial electrospinning, without major drug loss with a peptide content of 0.7% w/w predicted sufficient to kill most wound associated species. Pleurocidin retains its activity on release from the electrospun fiber matrix and completely inhibits growth of two strains of A. baumannii (AYE; ATCC 17978) and other ESKAPE pathogens. Inhibition of P. aeruginosa strains (PAO1; NCTC 13437) is, however, matrix weight per volume dependent, with only larger/thicker matrices maintaining complete inhibition. The resulting estimation of pleurocidin release from the matrix reveals high efficiency, facilitating a greater AMP potency. Wound matrices are often applied in parallel or sequentially with the use of standard wound care with biocides, therefore the presence and effect of biocides on pleurocidin potency was tested. It was revealed that combinations displayed additive or modestly synergistic effects depending on the biocide and pathogens which should be considered during the therapy. Taken together, we show that electrospun, pleurocidin-loaded wound matrices have potential to be investigated for wound infection treatment.

Development of In Vitro and Ex Vivo Biofilm Models for the Assessment of Antibacterial Fibrous Electrospun Wound Dressings.

Increasing evidence suggests that the chronicity of wounds is associated with the presence of bacterial biofilms. Therefore, novel wound care products are being developed, which can inhibit biofilm formation and/or treat already formed biofilms. A lack of standardized assays for the analysis of such novel antibacterial drug delivery systems enhances the need for appropriate tools and models for their characterization. Herein, we demonstrate that optimized and biorelevant in vitro and ex vivo wound infection and biofilm models offer a convenient approach for the testing of novel antibacterial wound dressings for their antibacterial and antibiofilm properties, allowing one to obtain qualitative and quantitative results. The in vitro model was developed using an electrospun (ES) thermally crosslinked gelatin-glucose (GEL-Glu) matrix and an ex vivo wound infection model using pig ear skin. Wound pathogens were used for colonization and biofilm development on the GEL-Glu matrix or pig skin with superficial burn wounds. The in vitro model allowed us to obtain more reproducible results compared with the ex vivo model, whereas the ex vivo model had the advantage that several pathogens preferred to form a biofilm on pig skin compared with the GEL-Glu matrix. The in vitro model functioned poorly for Staphylococcus epidermidis biofilm formation, but it worked well for Escherichia coli and Staphylococcus aureus, which were able to use the GEL-Glu matrix as a nutrient source and not only as a surface for biofilm growth. On the other hand, all tested pathogens were equally able to produce a biofilm on the surface of pig skin. The developed biofilm models enabled us to compare different ES dressings [pristine and chloramphenicol-loaded polycaprolactone (PCL) and PCL-poly(ethylene oxide) (PEO) (PCL/PEO) dressings] and understand their biofilm inhibition and treatment properties on various pathogens. Furthermore, we show that biofilms were formed on the wound surface as well as on a wound dressing, indicating that the demonstrated methods mimic well the in vivo situation. Colony forming unit (CFU) counting and live biofilm matrix as well as bacterial DNA staining together with microscopic imaging were performed for biofilm quantification and visualization, respectively. The results showed that both wound biofilm models (in vitro and ex vivo) enabled the evaluation of the desired antibiofilm properties, thus facilitating the design and development of more effective wound care products and screening of various formulations and active substances.

Making Antimicrobial Susceptibility Testing More Physiologically Relevant with Bicarbonate?

Azithromycin is a clinically important drug for treating invasive salmonellosis despite poor activity in laboratory assays for MIC. Addition of the main buffer in blood, bicarbonate, has been proposed for more physiologically relevant and more predictive testing conditions. However, we show here that bicarbonate-triggered lowering of azithromycin MIC is entirely due to alkalization of insufficiently buffered media. In addition, bicarbonate is unlikely to be altering efflux pump activity.

Uropathogenic Escherichia coli Shows Antibiotic Tolerance and Growth Heterogeneity in an In Vitro Model of Intracellular Infection.

Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can invade different types of host cells. To compare the pharmacodynamic properties of antibiotics against intra- and extracellular UPEC, an in vitro model of intracellular infection was established in J774 mouse macrophages infected by the UPEC strain CFT073. We tested antibiotics commonly prescribed against urinary tract infections (gentamicin, ampicillin, nitrofurantoin, trimethoprim, sulfamethoxazole, and ciprofloxacin) and the investigational fluoroquinolone finafloxacin. The metabolic activity of individual bacteria was assessed by expressing the fluorescent reporter protein TIMERbac within CFT073. Concentration-response experiments revealed that all tested antibiotics were much less effective against intracellular bacteria than extracellular ones. Most antibiotics, except fluoroquinolones, were unable to reach a bactericidal effect intracellularly at clinically achievable concentrations. Ciprofloxacin and finafloxacin killed 99.9% of extracellular bacteria at concentrations around the MIC, while for intracellular bacteria, concentrations more than 100× over the MIC were required to achieve a bactericidal effect. Time-kill curves showed that finafloxacin was more rapidly bactericidal in acidic medium than at neutral pH, while the reverse observation was made for ciprofloxacin. Intracellularly, kill curves showed biphasic kinetics for both fluoroquinolones, suggesting the presence of drug-tolerant subpopulations. Flow cytometry analysis of TIMERbac fluorescence revealed a marked heterogeneity in intracellular growth of individual bacteria, suggesting that the presence of subpopulations reaching a state of metabolic dormancy was the main reason for increased antibiotic tolerance of intracellular UPEC.

In Vitro Studies of Persister Cells.

Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which survive treatment with bactericidal antibiotics in otherwise antibiotic-sensitive cultures and were extensively studied in a hope to discover the mechanisms that cause treatment failures in chronically infected patients; however, most of these studies were conducted in the test tube. Research into antibiotic persistence has uncovered large intrapopulation heterogeneity of bacterial growth and regrowth but has not identified essential, dedicated molecular mechanisms of antibiotic persistence. Diverse factors and stresses that inhibit bacterial growth reduce killing of the bulk population and may also increase the persister subpopulation, implying that an array of mechanisms are present. Hopefully, further studies under conditions that simulate the key aspects of persistent infections will lead to identifying target mechanisms for effective therapeutic solutions.

Antibacterial Porous Electrospun Fibers as Skin Scaffolds for Wound Healing Applications.

Electrospun fiber scaffolds have a huge potential for the successful treatment of infected wounds based on their unique properties. Although several studies report novel drug-loaded electrospun fiber-based biomaterials, many of these do not provide information on their interactions with eukaryotic and bacterial cells. The main aim of this study was to develop antibacterial drug-loaded porous biocompatible polycaprolactone (PCL) fiber scaffolds mimicking the native extracellular matrix for wound healing purposes. Mechanical property evaluation and different biorelevant tests were conducted in order to understand the structure-activity relationships and reveal how the surface porosity of fibers and the fiber diameter affect the scaffold interactions with the living bacterial and eukaryotic fibroblast cells. Cell migration and proliferation assays and antibiofilm assays enabled us to enlighten the biocompatibility and safety of fiber scaffolds and their suitability to be used as scaffolds for the treatment of infected wounds. Here, we report that porous PCL microfiber scaffolds obtained using electrospinning at high relative humidity served as the best surfaces for fibroblast attachment and growth compared to the nonporous microfiber or nonporous nanofiber PCL scaffolds. Porous chloramphenicol-loaded microfiber scaffolds were more elastic compared to nonporous scaffolds and had the highest antibiofilm activity. The results indicate that in addition to the fiber diameter and fiber scaffold porosity, the single-fiber surface porosity and its effect on drug release, mechanical properties, cell viability, and antibiofilm activity need to be understood when developing antibacterial biocompatible scaffolds for wound healing applications. We show that pores on single fibers within an electrospun scaffold, in addition to nano- and microscale diameter of the fibers, change the living cell-fiber interactions affecting the antibiofilm efficacy and biocompatibility of the scaffolds for the local treatment of wounds.

Monitoring of Antimicrobial Drug Chloramphenicol Release from Electrospun Nano- and Microfiber Mats Using UV Imaging and Bacterial Bioreporters.

New strategies are continuously sought for the treatment of skin and wound infections due to increased problems with non-healing wounds. Electrospun nanofiber mats with antibacterial agents as drug delivery systems provide opportunities for the eradication of bacterial infections as well as wound healing. Antibacterial activities of such mats are directly linked with their drug release behavior. Traditional pharmacopoeial drug release testing settings are not always suitable for analyzing the release behavior of fiber mats intended for the local drug delivery. We tested and compared different drug release model systems for the previously characterized electrospun chloramphenicol (CAM)-loaded nanofiber (polycaprolactone (PCL)) and microfiber (PCL in combination with polyethylene oxide) mats with different drug release profiles. Drug release into buffer solution and hydrogel was investigated and drug concentration was determined using either high-performance liquid chromatography, ultraviolet-visible spectrophotometry, or ultraviolet (UV) imaging. The CAM release and its antibacterial effects in disc diffusion assay were assessed by bacterial bioreporters. All tested model systems enabled to study the drug release from electrospun mats. It was found that the release into buffer solution showed larger differences in the drug release rate between differently designed mats compared to the hydrogel release tests. The UV imaging method provided an insight into the interactions with an agarose hydrogel mimicking wound tissue, thus giving us information about early drug release from the mat. Bacterial bioreporters showed clear correlations between the drug release into gel and antibacterial activity of the electrospun CAM-loaded mats.

Effects and efficacy of different sterilization and disinfection methods on electrospun drug delivery systems.

Microbiological quality of a pharmaceutical product is an essential requirement ensuring patient safety, thus effective sterilization/disinfection methods need to be found. The aim of this study was to evaluate the efficacy of different sterilization/disinfection methods on drug-loaded electrospun matrices and the impact of these treatments on the functionality related characteristics of these matrices. The sterilization efficacy of gamma-irradiation, ultraviolet-irradiation, in situ generated chlorine gas and low-pressure argon plasma treatment were evaluated on two different chloramphenicol-loaded electrospun matrices using pristine polycaprolactone (PCL) as a carrier polymer or PCL in combination with polyethylene oxide. Drug stability, solid state properties, morphology, mechanical properties, swelling, biodegradation and drug release kinetics were studied before and after the treatments. It was shown that all tested methods help to reduce bioburden and only plasma treated matrices were not sterile. At the same time drug degradation after the treatment can be considerable and depends not only on the susceptibility of the drug to degradation, but also on matrix properties (e.g. the nature of carrier polymers). Even though no morphological changes were observed, gamma sterilization increased the hardness and elasticity of PCL matrices as a result of increased crystallinity of the polymer. Plasma treatment was able to significantly enhance water absorption to otherwise hydrophobic PCL/CAM matrix and had tremendous impact on its drug release kinetics as the drug was instantly released from otherwise prolonged release formulation.

ABCF ATPases Involved in Protein Synthesis, Ribosome Assembly and Antibiotic Resistance: Structural and Functional Diversification across the Tree of Life.

Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterized VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase center and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance (ARE) factors from other ABCFs have not been explored. To address this, we analyzed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting that they were present in the last common ancestor of both. Surprisingly, currently known ARE ABCFs are not confined to a distinct lineage of the ABCF family tree, suggesting that ARE can readily evolve from other ABCF functions. Our data suggest that there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that the ribosome-binding resistance factor VmlR from Bacillus subtilis is localized to the cytoplasm, ruling out a role in antibiotic efflux.

Impact of PCL nanofiber mat structural properties on hydrophilic drug release and antibacterial activity on periodontal pathogens.

Electrospinning enables to design and manufacture novel drug delivery systems capable of advancing the local antibacterial therapy. In this study, two hydrophilic drugs - metronidazole and ciprofloxacin hydrochloride - were loaded both individually and in combination into hydrophobic poly(ε-caprolactone) (PCL) matrix using electrospinning. We aimed to develop prolonged release drug delivery systems suitable for the treatment of periodontal diseases and understand how different rarely studied structural features, such as nanofiber mat thickness, surface area, wettability, together with intrinsic properties, like solid state and localization of incorporated drugs in nanofibers, affect the drug release. Furthermore, the safety of nanofiber mats was assessed in vitro on fibroblasts, and their antibacterial activity was tested on selected strains of periodontopathogenic bacteria. The results showed that the structural properties of nanofiber mat are crucial in particular drug-polymer combinations, affecting the drug release and consequently the antibacterial activity. The hydrophobicity of a PCL nanofiber mat and its thickness are the key characteristics in prolonged hydrophilic drug release, but only when wetting is the rate-limiting step for the drug release. Combination of drugs showed beneficial effects by inhibiting the growth of all tested pathogenic bacterial strains important in periodontal diseases.

Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells.

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicillin. Moreover, microfluidic experiments performed with a strain reporting the induction of the yefM-yoeB TA system allowed the observation of a small number of type II persister cells that resume growth after removal of ampicillin. However, we were unable to establish a correlation between high fluorescence and persistence, since the fluorescence of persister cells was comparable to that of the bulk of the population and none of the cells showing high fluorescence were able to resume growth upon removal of the antibiotic. Altogether, these data show that there is no direct link between induction of TA systems and persistence to antibiotics.IMPORTANCE Within a growing bacterial population, a small subpopulation of cells is able to survive antibiotic treatment by entering a transient state of dormancy referred to as persistence. Persistence is thought to be the cause of relapsing bacterial infections and is a major public health concern. Type II toxin-antitoxin systems are small modules composed of a toxic protein and an antitoxin protein counteracting the toxin activity. These systems were thought to be pivotal players in persistence until recent developments in the field. Our results demonstrate that previous influential reports had technical flaws and that there is no direct link between induction of TA systems and persistence to antibiotics.

Interactions between Chloramphenicol, Carrier Polymers, and Bacteria-Implications for Designing Electrospun Drug Delivery Systems Countering Wound Infection.

Antibacterial drug-loaded electrospun nano- and microfibrous dressings are of major interest as novel topical drug delivery systems in wound care. In this study, chloramphenicol (CAM)-loaded polycaprolactone (PCL) and PCL/poly(ethylene oxide) (PEO) fiber mats were electrospun and characterized in terms of morphology, drug distribution, physicochemical properties, drug release, swelling, cytotoxicity, and antibacterial activity. Computational modeling together with physicochemical analysis helped to elucidate possible interactions between the drug and carrier polymers. Strong interactions between PCL and CAM together with hydrophobicity of the system resulted in much slower drug release compared to the hydrophilic ternary system of PCL/PEO/CAM. Cytotoxicity studies confirmed safety of the fiber mats to murine NIH 3T3 cells. Disc diffusion assay demonstrated that both fast and slow release fiber mats reached effective concentrations and had similar antibacterial activity. A biofilm formation assay revealed that both blank matrices are good substrates for the bacterial attachment and formation of biofilm. Importantly, prolonged release of CAM from drug-loaded fibers helps to avoid biofilm formation onto the dressing and hence avoids the treatment failure.

Single-cell level methods for studying the effect of antibiotics on bacteria during infection.

Considerable evidence about phenotypic heterogeneity among bacteria during infection has accumulated during recent years. This heterogeneity has to be considered if the mechanisms of infection and antibiotic action are to be understood, so we need to implement existing and find novel methods to monitor the effects of antibiotics on bacteria at the single-cell level. This review provides an overview of methods by which this aim can be achieved. Fluorescence label-based methods and Raman scattering as a label-free approach are discussed in particular detail. Other label-free methods that can provide single-cell level information, such as impedance spectroscopy and surface plasmon resonance, are briefly summarized. The advantages and disadvantages of these different methods are discussed in light of a challenging in vivo environment.

Phenotypic heterogeneity enables uropathogenic Escherichia coli to evade killing by antibiotics and serum complement.

Uropathogenic strains of Escherichia coli (UPEC) are the major cause of bacteremic urinary tract infections. Survival in the bloodstream is associated with different mechanisms that help to resist serum complement-mediated killing. While the phenotypic heterogeneity of bacteria has been shown to influence antibiotic tolerance, the possibility that it makes cells refractory to killing by the immune system has not been experimentally tested. In the present study we sought to determine whether the heterogeneity of bacterial cultures is relevant to bacterial targeting by the serum complement system. We monitored cell divisions in the UPEC strain CFT073 with fluorescent reporter protein. Stationary-phase cells were incubated in active or heat-inactivated human serum in the presence or absence of different antibiotics (ampicillin, norfloxacin, and amikacin), and cell division and complement protein C3 binding were measured by flow cytometry and immunofluorescence microscopy. Heterogeneity in the doubling times of CFT073 cells in serum enabled three phenotypically different subpopulations to be distinguished, all of them being recognized by the C3 component of the complement system. The population of rapidly growing cells resists serum complement-mediated lysis. The dominant subpopulation of cells with intermediate growth rate is susceptible to serum. The third population, which does not resume growth upon dilution from stationary phase, is simultaneously protected from serum complement and antibiotics.

The ColRS system is essential for the hunger response of glucose-growing Pseudomonas putida.

BACKGROUND: The survival of bacteria largely depends on signaling systems that coordinate cell responses to environmental cues. Previous studies on the two-component ColRS signal system in Pseudomonas putida revealed a peculiar subpopulation lysis phenotype of colR mutant that grows on solid glucose medium. Here, we aimed to clarify the reasons for the lysis of bacteria. RESULTS: We present evidence that the lysis defect of P. putida colR mutant is linked to hunger response. A subpopulation prone to lysis was located in the periphery of bacterial cultures growing on solid medium. Cell lysis was observed in glucose-limiting, but not in glucose-rich conditions. Furthermore, lysis was also alleviated by exhaustion of glucose from the medium which was evidenced by a lower lysis of central cells compared to peripheral ones. Thus, lysis takes place at a certain glucose concentration range that most probably provides bacteria a hunger signal. An analysis of membrane protein pattern revealed several hunger-induced changes in the bacterial outer membrane: at glucose limitation the amount of OprB1 channel protein was significantly increased whereas that of OprE was decreased. Hunger-induced up-regulation of OprB1 correlated in space and time with the lysis of the colR mutant, indicating that hunger response is detrimental to the colR-deficient bacteria. The amount of OprB1 is controlled post-transcriptionally and derepression of OprB1 in glucose-limiting medium depends at least partly on the carbon catabolite regulator protein Crc. The essentiality of ColR in hunger response can be bypassed by reducing the amount of certain outer membrane proteins. In addition to depletion of OprB1, the lysis defect of colR mutant can be suppressed by the down-regulation of OprF levels and the hindering of SecB-dependent protein secretion. CONCLUSIONS: We show that Pseudomonas putida growing on solid glucose medium adapts to glucose limitation through up-regulation of the sugar channel protein OprB1 that probably allows enhanced acquisition of a limiting nutrient. However, to survive such hunger response bacteria need signalling by the ColRS system. Hence, the ColRS system should be considered a safety factor in hunger response that ensures the welfare of the cell membrane during the increased expression of certain membrane proteins.

The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of Pseudomonas putida becomes evident only in growing bacteria.

BACKGROUND: We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida. RESULTS: By using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of colR-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the ttgABC operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the colR mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the colR-deficient P. putida are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type P. putida as well. Comparison of phenol tolerance of growing versus starving bacteria revealed that both ColRS and TtgABC systems affect phenol tolerance only under growth conditions and not under starvation. Flow cytometry analysis showed that phenol strongly inhibited cell division and to some extent also caused cell membrane permeabilization to propidium iodide. Single cell analysis of populations of the ttgC- and colRttgC-deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the colR mutant, respectively. However, cell division of P. putida with inactivated TtgABC pump seemed to be less sensitive to phenol than that of the parental strain. At the same time, cell division appeared to be more inhibited in the colR-mutant strain than in the wild-type P. putida. CONCLUSIONS: ColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of P. putida. However, as they affect phenol tolerance of growing bacteria only, this indicates that they participate in the regulation of processes which are active during the growth and/or cell division. Single cell analysis data indicated that the cell division step of cell cycle is particularly sensitive to the toxic effect of phenol and its inhibition can be considered as an adaptive response under conditions of phenol stress.

ColRS two-component system prevents lysis of subpopulation of glucose-grown Pseudomonas putida.

ColRS two-component system is well conserved in pseudomonads, but its exact role has remained obscure. Here, we report that Pseudomonas putida deficient in ColR experiences serious carbon source-specific stress that leads to the lysis of a subpopulation of bacteria growing on solid glucose medium. We observed that on glucose medium colR-deficient bacteria aggregated, produced a Congo Red-binding substance and had enhanced membrane permeability. Detection of a large amount of cytoplasmic beta-galactosidase and other proteins as well as chromosomal DNA in the growth medium of a colR mutant indicated that cell lysis took place if ColR was absent. Investigation of colony morphology revealed concavities in the centre of the colonies of colR mutant suggesting that cell lysis occurred mainly in the areas of the highest cell density. Analysis of bacteria at a single cell level by flow cytometry showed that population of glucose-grown colR-deficient cells was heterogeneous. In addition to the wild type-like population, we detected a subpopulation of cells with damaged membrane permeable to propidium iodide. Interestingly, inactivation of oprB1 encoding a glucose porin eliminated the cell lysis as well as autoaggregation and membrane leakiness of a colR mutant indicating that glucose influx could be responsible for membrane stress in the absence of ColRS system.

Study of factors which negatively affect expression of the phenol degradation operon pheBA in Pseudomonas putida.

Transcription of the plasmid-borne phenol catabolic operon pheBA in Pseudomonas putida is activated by the LysR-family regulator CatR in the presence of the effector molecule cis,cis-muconate (CCM), which is an intermediate of the phenol degradation pathway. In addition to the positive control of the operon, several factors negatively affect transcription initiation from the pheBA promoter. First, the activation of the pheBA operon depends on the extracellular concentration of phenol. The pheBA promoter is rapidly activated in the presence of micromolar concentrations of phenol in minimal growth medium, but the initiation of transcription from this promoter is severely delayed after sudden exposure of bacteria to 2.5 mM phenol. Second, the transcriptional activation from this promoter is impeded when the growth medium of bacteria contains amino acids. The negative effects of amino acids can be suppressed either by overproducing CatR or by increasing, the intracellular amount of CCM. However, the intracellular amount of CCM is a major limiting factor for the transcriptional activation of the pheBA operon, as accumulation of CCM in a P. putida catB-defective strain, unable to metabolize CCM (but expressing CatR at a natural level), almost completely relieves the negative effects of amino acids. The intracellular amount of CCM is negatively affected by the catabolite repression control protein via downregulating at the post-transcriptional level the expression of the pheBA-encoded catechol 1,2-dioxygenase and the phenol monooxygenase, the enzymes needed for CCM production.