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.

Impact of Bicarbonate-ß-Lactam Exposures on Methicillin-Resistant Staphylococcus aureus (MRSA) Gene Expression in Bicarbonate-ß-Lactam-Responsive vs. Non-Responsive Strains.

Methicillin-resistant Staphylococcus aureus (MRSA) infections represent a difficult clinical treatment issue. Recently, a novel phenotype was discovered amongst selected MRSA which exhibited enhanced ß-lactam susceptibility in vitro in the presence of NaHCO3 (termed 'NaHCO3-responsiveness'). This increased ß-lactam susceptibility phenotype has been verified in both ex vivo and in vivo models. Mechanistic studies to-date have implicated NaHCO3-mediated repression of genes involved in the production, as well as maturation, of the alternative penicillin-binding protein (PBP) 2a, a necessary component of MRSA ß-lactam resistance. Herein, we utilized RNA-sequencing (RNA-seq) to identify genes that were differentially expressed in NaHCO3-responsive (MRSA 11/11) vs. non-responsive (COL) strains, in the presence vs. absence of NaHCO3-ß-lactam co-exposures. These investigations revealed that NaHCO3 selectively repressed the expression of a cadre of genes in strain 11/11 known to be a part of the sigB-sarA-agr regulon, as well as a number of genes involved in the anchoring of cell wall proteins in MRSA. Moreover, several genes related to autolysis, cell division, and cell wall biosynthesis/remodeling, were also selectively impacted by NaHCO3-OXA exposure in the NaHCO3-responsive strain MRSA 11/11. These outcomes provide an important framework for further studies to mechanistically verify the functional relevance of these genetic perturbations to the NaHCO3-responsiveness phenotype in MRSA.

Structure Activity Relationship Study of the XIP Quorum Sensing Pheromone in Streptococcus mutans Reveal Inhibitors of the Competence Regulon.

The dental cariogenic pathogen Streptococcus mutans coordinates competence for genetic transformation via two peptide pheromones, competence stimulating peptide (CSP) and comX-inducing peptide (XIP). CSP is sensed by the comCDE system and induces competence indirectly, whereas XIP is sensed by the comRS system and induces competence directly. In chemically defined media (CDM), after uptake by oligopeptide permease, XIP interacts with the cytosolic receptor ComR to form the XIP::ComR complex that activates the expression of comX, an alternative sigma factor that initiates the transcription of late-competence genes. In this study, we set out to determine the molecular mechanism of XIP::ComR interaction. To this end, we performed systematic replacement of the amino acid residues in the XIP pheromone and assessed the ability of the mutated analogs to modulate the competence regulon in CDM. We were able to identify structural features that are important to ComR binding and activation. Our structure-activity relationship insights led us to construct multiple XIP-based inhibitors of the comRS pathway. Furthermore, when comCDE and comRS were both stimulated with CSP and XIP, respectively, a lead XIP-based inhibitor was able to maintain the inhibitory activity. Last, phenotypic assays were used to highlight the potential of XIP-based inhibitors to attenuate pathogenicity in S. mutans and to validate the specificity of these compounds to the comRS pathway within the competence regulon. The XIP-based inhibitors developed in this study can be used as lead scaffolds for the design and development of potential therapeutics against S. mutans infections.

Genotoxic effect of 2,2'-bis(bicyclo[2.2.1] heptane) on bacterial cells.

The toxic effect of strained hydrocarbon 2,2'-bis (bicyclo[2.2.1]heptane) (BBH) was studied using whole-cell bacterial lux-biosensors based on Escherichia coli cells in which luciferase genes are transcriptionally fused with stress-inducible promoters. It was shown that BBH has the genotoxic effect causing bacterial SOS response however no alkylating effect has been revealed. In addition to DNA damage, there is an oxidative effect causing the response of OxyR/S and SoxR/S regulons. The most sensitive to BBH lux-biosensor was E. coli pSoxS-lux which reacts to the appearance of superoxide anion radicals in the cell. It is assumed that the oxidation of BBH leads to the generation of reactive oxygen species, which provide the main contribution to the genotoxicity of this substance.

Methylene blue induces the soxRS regulon of Escherichia coli.

Extensive application of methylene blue (MB) for therapeutic and diagnostic purposes, and reports for unwanted side effects, demand better understanding of the mechanisms of biological action of this thiazine dye. Because MB is redox-active, its biological activities have been attributed to transfer of electrons, generation of reactive oxygen species, and antioxidant action. Results of this study show that MB is more toxic to a superoxide dismutase-deficient Escherichia coli mutant than to its SOD-proficient parent, which indicates that superoxide anion radical is involved. Incubation of E. coli with MB induced the enzymes fumarase C, SOD, nitroreductase A, and glucose-6-phosphate dehydrogenase, all controlled by the soxRS regulon. Induction of these enzymes was prevented by blocking protein synthesis with chloramphenicol and was not observed when soxRS-negative mutants were incubated with MB. These results show that MB is capable of inducing the soxRS regulon of E. coli, which plays a key role in protecting bacteria against oxidative stress and redox-cycling compounds. Irrespective of the abundance of heme-containing proteins in living cells, which are preferred acceptors of electrons from the reduced form of MB, reduction of oxygen to superoxide radical still takes place. Induction of the soxRS regulon suggests that in humans, beneficial effects of MB could be attributed to activation of redox-sensitive transcription factors like Nrf2 and FoxO. If defense systems are compromised or genes coding for protective proteins are not induced, MB would have deleterious effects.

A multi-omics analysis reveals the unfolded protein response regulon and stress-induced resistance to folate-based antimetabolites.

Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.

Transcriptional response of mar, sox and rob regulon against concentration gradient carbapenem stress within Escherichia coli isolated from hospital acquired infection.

OBJECTIVE: The present study was carried out to investigate the transcriptional response of marA (Multiple antibiotic resistance A gene), soxS (Superoxide S gene) and rob (Right-origin-binding gene) under carbapenem stress. RESULTS: 12 isolates were found over-expressing AcrAB-TolC efflux pump system and showed reduced expression of OmpF (Outer membrane porin) gene were selected for further study. Among them, over expression of marA and rob was observed in 7 isolates. Increasing pattern of expression of marA and rob against meropenem was observed. The clones of marA and rob showed reduced susceptibility towards carbapenems.

Designing cyclic competence-stimulating peptide (CSP) analogs with pan-group quorum-sensing inhibition activity in Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic human pathogen that utilizes the competence regulon, a quorum-sensing circuitry, to acquire antibiotic resistance genes and initiate its attack on the human host. Interception of the competence regulon can therefore be utilized to study S. pneumoniae cell-cell communication and behavioral changes, as well as attenuate S. pneumoniae infectivity. Herein we report the design and synthesis of cyclic dominant negative competence-stimulating peptide (dnCSP) analogs capable of intercepting the competence regulon in both S. pneumoniae specificity groups with activities at the low nanomolar range. Structural analysis of lead analogs provided important insights as to the molecular mechanism that drives CSP receptor binding and revealed that the pan-group cyclic CSPs exhibit a chimeric hydrophobic patch conformation that resembles the hydrophobic patches required for both ComD1 and ComD2 binding. Moreover, the lead cyclic dnCSP, CSP1-E1A-cyc(Dap6E10), was found to possess superior pharmacological properties, including improved resistance to enzymatic degradation, while remaining nontoxic. Lastly, CSP1-E1A-cyc(Dap6E10) was capable of attenuating mouse mortality during acute pneumonia caused by both group 1 and group 2 S. pneumoniae strains. This cyclic pan-group dnCSP is therefore a promising drug lead scaffold against S. pneumoniae infections that could be administered individually or utilized in combination therapy to augment the effects of current antimicrobial agents.

Turning off Bacillus cereus quorum sensing system with peptidic analogs.

We explored quenching of the PlcR-PapR quorum-sensing system in Bacillus cereus. We generated PapR7-peptidic derivatives that inhibit this system and thus the production of virulence factors, reflected by a loss in hemolytic activity, without affecting bacterial growth. To our knowledge, these peptides represent the first potent synthetic inhibitors of quorum-sensing in B. cereus.

Quantitative Proteomics Analysis Confirmed Oxidative Metabolism Predominates in Streptomyces coelicolor versus Glycolytic Metabolism in Streptomyces lividans.

Recent physiological studies indicated that S. lividans metabolism was mainly glycolytic, whereas S. coelicolor metabolism was mainly oxidative. To determine whether such metabolic characteristics were correlated with consistent proteomics features, a comparative label-free, shotgun proteomics analysis of these strains was carried out. Among 2024 proteins identified, 360 showed significant differences in abundance between the strains. This study revealed that S. coelicolor catabolized glucose less actively than S. lividans, whereas the amino acids present in the medium were catabolized less actively by S. lividans than by S. coelicolor. The abundance of glycolytic proteins in S. lividans was consistent with its high glycolytic activity, whereas the abundance of proteins involved in the catabolism of amino acids in S. coelicolor provided an explanatory basis for its predominantly oxidative metabolism. In this study, conducted under conditions of low O2 availability, proteins involved in resistance to oxidative stress and those belonging to a DosR-like dormancy regulon were abundant in S. coelicolor, whereas tellurium resistance proteins were abundant in S. lividans. This indicated that the strains reacted differently to O2 limitation. Proteins belonging to the CDA, RED, and ACT pathways, usually highly expressed in S. coelicolor, were not detected under these conditions, whereas proteins of siderophores, 5-hydroxyectoine, and terpenoid biosynthetic pathways were present.

Polypharmacology Approaches against the Pseudomonas aeruginosa MvfR Regulon and Their Application in Blocking Virulence and Antibiotic Tolerance.

Pseudomonas aeruginosa is an important nosocomial pathogen that is frequently recalcitrant to available antibiotics, underlining the urgent need for alternative therapeutic options against this pathogen. Targeting virulence functions is a promising alternative strategy as it is expected to generate less-selective resistance to treatment compared to antibiotics. Capitalizing on our nonligand-based benzamide-benzimidazole (BB) core structure compounds reported to efficiently block the activity of the P. aeruginosa multiple virulence factor regulator MvfR, here we report the first class of inhibitors shown to interfere with PqsBC enzyme activity, responsible for the synthesis of the MvfR activating ligands HHQ and PQS, and the first to target simultaneously MvfR and PqsBC activity. The use of these compounds reveals that inhibiting PqsBC is sufficient to block P. aeruginosa's acute virulence functions, as the synthesis of MvfR ligands is inhibited. Our results show that MvfR remains the best target of this QS pathway, as we show that antagonists of this target block both acute and persistence-related functions. The structural properties of the compounds reported in this study provide several insights that are instrumental for the design of improved MvfR regulon inhibitors against both acute and persistent P. aeruginosa infections. Moreover, the data presented offer the possibility of a polypharmacology approach of simultaneous silencing two targets in the same pathway. Such a combined antivirulence strategy holds promise in increasing therapeutic efficacy and providing alternatives in the event of a single target's resistance development.

The Proteasome Stress Regulon Is Controlled by a Pair of NAC Transcription Factors in Arabidopsis.

Proteotoxic stress, which is generated by the accumulation of unfolded or aberrant proteins due to environmental or cellular perturbations, can be mitigated by several mechanisms, including activation of the unfolded protein response and coordinated increases in protein chaperones and activities that direct proteolysis, such as the 26S proteasome. Using RNA-seq analyses combined with chemical inhibitors or mutants that induce proteotoxic stress by impairing 26S proteasome capacity, we defined the transcriptional network that responds to this stress in Arabidopsis thaliana This network includes genes encoding core and assembly factors needed to build the complete 26S particle, alternative proteasome capping factors, enzymes involved in protein ubiquitylation/deubiquitylation and cellular detoxification, protein chaperones, autophagy components, and various transcriptional regulators. Many loci in this proteasome-stress regulon contain a consensus cis-element upstream of the transcription start site, which was previously identified as a binding site for the NAM/ATAF1/CUC2 78 (NAC78) transcription factor. Double mutants disrupting NAC78 and its closest relative NAC53 are compromised in the activation of this regulon and notably are strongly hypersensitive to the proteasome inhibitors MG132 and bortezomib. Given that NAC53 and NAC78 homo- and heterodimerize, we propose that they work as a pair in activating the expression of numerous factors that help plants survive proteotoxic stress and thus play a central regulatory role in maintaining protein homeostasis.

Cth2 Protein Mediates Early Adaptation of Yeast Cells to Oxidative Stress Conditions.

Cth2 is an mRNA-binding protein that participates in remodeling yeast cell metabolism in iron starvation conditions by promoting decay of the targeted molecules, in order to avoid excess iron consumption. This study shows that in the absence of Cth2 immediate upregulation of expression of several of the iron regulon genes (involved in high affinity iron uptake and intracellular iron redistribution) upon oxidative stress by hydroperoxide is more intense than in wild type conditions where Cth2 is present. The oxidative stress provokes a temporary increase in the levels of Cth2 (itself a member of the iron regulon). In such conditions Cth2 molecules accumulate at P bodies-like structures when the constitutive mRNA decay machinery is compromised. In addition, a null Δcth2 mutant shows defects, in comparison to CTH2 wild type cells, in exit from α factor-induced arrest at the G1 stage of the cell cycle when hydroperoxide treatment is applied. The cell cycle defects are rescued in conditions that compromise uptake of external iron into the cytosol. The observations support a role of Cth2 in modulating expression of diverse iron regulon genes, excluding those specifically involved in the reductive branch of the high-affinity transport. This would result in immediate adaptation of the yeast cells to an oxidative stress, by controlling uptake of oxidant-promoting iron cations.

Expression signature based on TP53 target genes doesn't predict response to TP53-MDM2 inhibitor in wild type TP53 tumors.

A number of TP53-MDM2 inhibitors are currently under investigation as therapeutic agents in a variety of clinical trials in patients with TP53 wild type tumors. Not all wild type TP53 tumors are sensitive to such inhibitors. In an attempt to improve selection of patients with TP53 wild type tumors, an mRNA expression signature based on 13 TP53 transcriptional target genes was recently developed (Jeay et al. 2015). Careful reanalysis of TP53 status in the study validation data set of cancer cell lines considered to be TP53 wild type detected TP53 inactivating alterations in 23% of cell lines. The subsequent reanalysis of the remaining TP53 wild type cell lines clearly demonstrated that unfortunately the 13-gene signature cannot predict response to TP53-MDM2 inhibitor in TP53 wild type tumors.

The Carbonic Anhydrase Inhibitor Ethoxzolamide Inhibits the Mycobacterium tuberculosis PhoPR Regulon and Esx-1 Secretion and Attenuates Virulence.

Mycobacterium tuberculosis must sense and adapt to host environmental cues to establish and maintain an infection. The two-component regulatory system PhoPR plays a central role in sensing and responding to acidic pH within the macrophage and is required for M. tuberculosis intracellular replication and growth in vivo. Therefore, the isolation of compounds that inhibit PhoPR-dependent adaptation may identify new antivirulence therapies to treat tuberculosis. Here, we report that the carbonic anhydrase inhibitor ethoxzolamide inhibits the PhoPR regulon and reduces pathogen virulence. We show that treatment of M. tuberculosis with ethoxzolamide recapitulates phoPR mutant phenotypes, including downregulation of the core PhoPR regulon, altered accumulation of virulence-associated lipids, and inhibition of Esx-1 protein secretion. Quantitative single-cell imaging of a PhoPR-dependent fluorescent reporter strain demonstrates that ethoxzolamide inhibits PhoPR-regulated genes in infected macrophages and mouse lungs. Moreover, ethoxzolamide reduces M. tuberculosis growth in both macrophages and infected mice. Ethoxzolamide inhibits M. tuberculosis carbonic anhydrase activity, supporting a previously unrecognized link between carbonic anhydrase activity and PhoPR signaling. We propose that ethoxzolamide may be pursued as a new class of antivirulence therapy that functions by modulating expression of the PhoPR regulon and Esx-1-dependent virulence.

Mapping and manipulating the Mycobacterium tuberculosis transcriptome using a transcription factor overexpression-derived regulatory network.

BACKGROUND: Mycobacterium tuberculosis senses and responds to the shifting and hostile landscape of the host. To characterize the underlying intertwined gene regulatory network governed by approximately 200 transcription factors of M. tuberculosis, we have assayed the global transcriptional consequences of overexpressing each transcription factor from an inducible promoter. RESULTS: We cloned and overexpressed 206 transcription factors in M. tuberculosis to identify the regulatory signature of each. We identified 9,335 regulatory consequences of overexpressing each of 183 transcription factors, providing evidence of regulation for 70% of the M. tuberculosis genome. These transcriptional signatures agree well with previously described M. tuberculosis regulons. The number of genes differentially regulated by transcription factor overexpression varied from hundreds of genes to none, with the majority of expression changes repressing basal transcription. Exploring the global transcriptional maps of transcription factor overexpressing (TFOE) strains, we predicted and validated the phenotype of a regulator that reduces susceptibility to a first line anti-tubercular drug, isoniazid. We also combined the TFOE data with an existing model of M. tuberculosis metabolism to predict the growth rates of individual TFOE strains with high fidelity. CONCLUSION: This work has led to a systems-level framework describing the transcriptome of a devastating bacterial pathogen, characterized the transcriptional influence of nearly all individual transcription factors in M. tuberculosis, and demonstrated the utility of this resource. These results will stimulate additional systems-level and hypothesis-driven efforts to understand M. tuberculosis adaptations that promote disease.

Bacillus pumilus reveals a remarkably high resistance to hydrogen peroxide provoked oxidative stress.

Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus.

Transcription factors and genetic circuits orchestrating the complex, multilayered response of Clostridium acetobutylicum to butanol and butyrate stress.

BACKGROUND: Organisms of the genus Clostridium are Gram-positive endospore formers of great importance to the carbon cycle, human normo- and pathophysiology, but also in biofuel and biorefinery applications. Exposure of Clostridium organisms to chemical and in particular toxic metabolite stress is ubiquitous in both natural (such as in the human microbiome) and engineered environments, engaging both the general stress response as well as specialized programs. Yet, despite its fundamental and applied significance, it remains largely unexplored at the systems level. RESULTS: We generated a total of 96 individual sets of microarray data examining the transcriptional changes in C. acetobutylicum, a model Clostridium organism, in response to three levels of chemical stress from the native metabolites, butanol and butyrate. We identified 164 significantly differentially expressed transcriptional regulators and detailed the cellular programs associated with general and stressor-specific responses, many previously unexplored. Pattern-based, comparative genomic analyses enabled us, for the first time, to construct a detailed picture of the genetic circuitry underlying the stress response. Notably, a list of the regulons and DNA binding motifs of the stress-related transcription factors were identified: two heat-shock response regulators, HrcA and CtsR; the SOS response regulator LexA; the redox sensor Rex; and the peroxide sensor PerR. Moreover, several transcriptional regulators controlling stress-responsive amino acid and purine metabolism and their regulons were also identified, including ArgR (arginine biosynthesis and catabolism regulator), HisR (histidine biosynthesis regulator), CymR (cysteine metabolism repressor) and PurR (purine metabolism repressor). CONCLUSIONS: Using an exceptionally large set of temporal transcriptional data and regulon analyses, we successfully built a STRING-based stress response network model integrating important players for the general and specialized metabolite stress response in C. acetobutylicum. Since the majority of the transcription factors and their target genes are highly conserved in other organisms of the Clostridium genus, this network would be largely applicable to other Clostridium organisms. The network informs the molecular basis of Clostridium responses to toxic metabolites in natural ecosystems and the microbiome, and will facilitate the construction of genome-scale models with added regulatory-network dimensions to guide the development of tolerant strains.

KpnEF, a new member of the Klebsiella pneumoniae cell envelope stress response regulon, is an SMR-type efflux pump involved in broad-spectrum antimicrobial resistance.

Klebsiella pneumoniae has been frequently associated with nosocomial infections. Efflux systems are ubiquitous transporters that also function in drug resistance. Genome analysis of K. pneumoniae strain NTUH-K2044 revealed the presence of ∼15 putative drug efflux systems. We discuss here for the first time the characterization of a putative SMR-type efflux pump, an ebrAB homolog (denoted here as kpnEF) with respect to Klebsiella physiology and the multidrug-resistant phenotype. Analysis of hypermucoviscosity revealed direct involvement of kpnEF in capsule synthesis. The ΔkpnEF mutant displayed higher sensitivity to hyperosmotic (∼2.8-fold) and high bile (∼4.0-fold) concentrations. Mutation in kpnEF resulted in increased susceptibility to cefepime, ceftriaxone, colistin, erythromycin, rifampin, tetracycline, and streptomycin; mutated strains changed from being resistant to being susceptible, and the resistance was restored upon complementation. The ΔkpnEF mutant displayed enhanced sensitivity toward structurally related compounds such as sodium dodecyl sulfate, deoxycholate, and dyes, including clinically relevant disinfectants such as benzalkonium chloride, chlorhexidine, and triclosan. The prevalence of kpnEF in clinical strains broadens the diversity of antibiotic resistance in K. pneumoniae. Experimental evidence of CpxR binding to the efflux pump promoter and quantification of its expression in a cpxAR mutant background demonstrated kpnEF to be a member of the Cpx regulon. This study helps to elucidate the unprecedented biological functions of the SMR-type efflux pump in Klebsiella spp.

Nitrofurantoin, phenazopyridine, and the superoxide-response regulon soxRS of Escherichia coli.

Nitrofurantoin and phenazopyridine are two drugs commonly used against urinary tract infections. Both compounds exert oxidative damage in patients deficient in glucose-6-phosphate dehydrogenase. This study was done to assess the interactions of these drugs with the soxRS regulon of Escherichia coli, a superoxide-defense system (that includes a nitroreductase that yields the active metabolite of nitrofurantoin) involved in antibiotic multi-resistance. The effects of either nitrofurantoin or phenazopyridine, upon strains with different soxRS genotypes, were measured as minimum inhibitory concentrations (MICs) and growth curves. Also, the ability of these drugs to induce the expression of a soxS'::lacZ gene fusion was assessed. The effect of antibiotics in the presence of phenazopyridine, paraquat (a known soxRS inducer), or an efflux inhibitor, was measured using the disk diffusion method. A strain constitutively expressing the soxRS regulon was slightly more susceptible to nitrofurantoin, and more resistant to phenazopyridine, compared to wild-type and soxRS-deleted strains, during early treatment, but 24-h MICs were the same (8 mg/l nitrofurantoin, 1,000 mg/l phenazopyridine) for all strains. Both compounds were capable of inducing the expression of a soxS'::lacZ fusion, but less than paraquat. Subinhibitory concentrations of phenazopyridine increased the antimicrobial effect of ampicillin, chloramphenicol, tetracycline, and nitrofurantoin. The induction or constitutive expression of the soxRS regulon seems to be a disadvantage for E. coli during nitrofurantoin exposure; but might be an advantage during phenazopyridine exposure, indicating that the latter compound could act as a selective pressure for mutations related to virulence and antibiotic multi-resistance.