oxyR Mutant Research Articles

OxyR is required for oxidative stress resistance of the entomopathogenic bacterium Xenorhabdus nematophila and has a minor role during the bacterial interaction with its hosts.

Xenorhabdus nematophila is a Gram-negative bacterium, mutualistically associated with the soil nematode Steinernema carpocapsae, and this nemato-bacterial complex is parasitic for a broad spectrum of insects. The transcriptional regulator OxyR is widely conserved in bacteria and activates the transcription of a set of genes that influence cellular defence against oxidative stress. It is also involved in the virulence of several bacterial pathogens. The aim of this study was to identify the X. nematophila OxyR regulon and investigate its role in the bacterial life cycle. An oxyR mutant was constructed in X. nematophila and phenotypically characterized in vitro and in vivo after reassociation with its nematode partner. OxyR plays a major role during the X. nematophila resistance to oxidative stress in vitro. Transcriptome analysis allowed the identification of 59 genes differentially regulated in the oxyR mutant compared to the parental strain. In vivo, the oxyR mutant was able to reassociate with the nematode as efficiently as the control strain. These nemato-bacterial complexes harbouring the oxyR mutant symbiont were able to rapidly kill the insect larvae in less than 48 h after infestation, suggesting that factors other than OxyR could also allow X. nematophila to cope with oxidative stress encountered during this phase of infection in insect. The significantly increased number of offspring of the nemato-bacterial complex when reassociated with the X. nematophila oxyR mutant compared to the control strain revealed a potential role of OxyR during this symbiotic stage of the bacterial life cycle.

Bioactivity of Myrtus communis from the Montenegro coastline

Although numerous studies have demonstrated the biomedical potential of Myrtus communisL., (Myrtaceae) data on myrtle from Montenegro are scarce. To evaluate antioxidant, antimutagenic and antibacterial activity of myrtle methanolic extracts. Antioxidant activity was evaluated by measuring free radicals scavenging activity, reducing power and enzyme inhibition. The strongest scavenging activity was towards DPPH radical (2,2-diphenyl-1-picrylhydrazyl) (IC501.69-2.25 mg/mL) and superoxide anion (IC500.56 to 0.88 mg/mL), followed by high reducing power (428-472 mgAA/g.DE) and inhibition of XOD (IC500.308-0.6261mg/mL). Antimutagenic activity was evaluated in reverse mutation assays with Escherichia coliWP2 oxyR mutant IC202 and deficient in the induction of antioxidant enzymes. The myrtle extracts strongly inhibited mutagenesis induced by t-BOOH, reaching 70% at the highest concentration applied. Antimicrobial activity was examined on eight different bacterial strains. Gram-positive bacteria, S. epidermis, S. aureusand M. flavus demonstrated the highest sensitivity towards extracts (MICs 4.5-9 mg/mL), but significantly lower towards essential oil (MIC 0.42-3.32 mg/mL).

Manganese transporters regulate the resumption of replication in hydrogen peroxide-stressed Escherichia coli.

Following hydrogen peroxide treatment, ferrous iron (Fe2+) is oxidized to its ferric form (Fe3+), stripping it from and inactivating iron-containing proteins. Many mononuclear iron enzymes can be remetallated by manganese to restore function, while other enzymes specifically utilize manganese as a cofactor, having redundant activities that compensate for iron-depleted counterparts. DNA replication relies on one or more iron-dependent protein(s) as synthesis abates in the presence of hydrogen peroxide and requires manganese in the medium to resume. Here, we show that manganese transporters regulate the ability to resume replication following oxidative challenge in Escherichia coli. The absence of the primary manganese importer, MntH, impairs the ability to resume replication; whereas deleting the manganese exporter, MntP, or transporter regulator, MntR, dramatically increases the rate of recovery. Unregulated manganese import promoted recovery even in the absence of Fur, which maintains iron homeostasis. Similarly, replication was not restored in oxyR mutants, which cannot upregulate manganese import following hydrogen peroxide stress. Taken together, the results define a central role for manganese transport in restoring replication following oxidative stress.

Genotoxic Effect of Dicyclopropanated 5-Vinyl-2-Norbornene.

Dicyclopropanated 5-vinyl-2-norbornene (dcpVNB) is a strained polycyclic hydrocarbon compound with a high energy content, which makes it promising for the development of propellant components based on it. In this work, the genotoxic properties of dcpVNB were studied using whole-cell lux-biosensors based on Escherichia coli and Bacillus subtilis. It was shown that the addition of dcpVNB to bacterial cells leads to the appearance of DNA damage inducing the SOS response and Dps expression with slight activation of the OxyR-mediated response to oxidative stress. The highest toxic effect of dcpVNB is detected by the following lux-biosensors: E. coli pColD-lux, E. coli pDps, B. subtilis pNK-DinC, and B. subtilis pNK-MrgA, in which the genes of bacterial luciferases are transcriptionally fused to the corresponding promoters: Pcda, Pdps, PdinC, and PmrgA. It was shown that lux-biosensors based on B. subtilis, and E. coli are almost equally sensitive to dcpVNB, which indicates the same permeability to this compound of cell wall of Gram-positive and Gram-negative bacteria. The activation of Pdps after dcpVNB addition maintains even in oxyR mutant E. coli strains, which means that the Pdps induction is only partially determined by the OxyR/S regulon. Comparison of specific stress effects caused by dcpVNB and 2-ethyl(bicyclo[2.2.1]heptane) (EBH), characterized by the absence of cyclopropanated groups, shows that structural changes in hydrocarbons could significantly change the mode of toxicity.

Characterization of the oxidative stress response regulatory network in Bacteroides fragilis: An interaction between BmoR and OxyR regulons promotes abscess formation in a model of intra-abdominal infection

ObjectivesBacteroides fragilis is an anaerobic bacterium that is commonly found in the human gut microbiota and an opportunistic pathogen in extra-intestinal infections. B. fragilis displays a robust response to oxidative stress which allows for survival in oxygenated tissues such as the peritoneal cavity and lead to the formation of abscesses. In this study, we investigated the synergy of the oxidative stress response regulators OxyR and BmoR in the ability of B. fragilis to resist oxidative damage and to survive in extra-intestinal infection. MethodsA ΔbmoR ΔoxyR double mutant B. fragilis strain was constructed, and its oxidative stress response was compared to parental and single mutant strains in phenotypical assays and gene expression analysis. The pathogenic potential in an in vivo mouse model of abscess formation was also evaluated. ResultsExpression analysis showed a coordinated control of thioredoxin C (trxC) gene expression by BmoR and OxyR during oxygen exposure, with upregulation of trxC in the bmoR mutant strain (4.9-fold increase), downregulation in the oxyR mutant (2.5-fold decrease), and an intermediate level of deregulation (2-fold increase) in the double mutant strain compared to the parent strain. Expression analysis during oxidative stress conditions also showed that BmoR is a major repressor of the CoA-disulfide reductase gene (upregulated 47-fold in the bmoR mutant) while OxyR plays a minor repression role in this gene (upregulated 2.5-fold in the oxyR mutant). Exposure to atmospheric oxygen for up to 72 h revealed that the deletion of bmoR alone had no significant effect in in vitro survival phenotype assays, though it partially abolishes the OxyR sensitivity phenotype in the bmoR/oxyR double mutant strain compared to oxyR mutant. In vivo assays showed that bmoR and oxyR mutants were significantly impaired in the formation and development of abscesses compared to the parent strain in an experimental intra-abdominal infection mouse model. ConclusionAlthough the full extent of genes whose expression are modulated by BmoR and OxyR is yet to be defined, we present evidence that these regulators have overlapping functions in B. fragilis response to oxidative stress and ability to form abscess in extra-intestinal tissues.

OxyR contributes to virulence of Acidovorax citrulli by regulating anti-oxidative stress and expression of flagellin FliC and type IV pili PilA.

In many bacteria, OxyR acts as a transcriptional regulator that facilitates infection via degrading hydrogen peroxide (H2O2) generated by the host defense response. Previous studies showed that OxyR also plays an important role in regulating biofilm formation, cell motility, pili relate-genes expression, and surface polysaccharide production. However, the role of OxyR has not been determined in Acidovorax citrulli strain xjl12. In the current study, the qRT-PCR and western blot assays revealed that the expression level of oxyR was significantly induced by H2O2. The oxyR deletion mutant of A. citrulli was significantly impaired bacterial tolerance to oxidative stress and reduced catalase (CAT) activity. In addition, oxyR mutant resulted in reduced swimming motility, twitching motility, biofilm formation, virulence, and bacterial growth in planta by significantly affecting flagellin and type IV pili-related gene (fliC and pilA) expression. The qRT-PCR assays and western blot revealed that OxyR positively regulated the expression of fliC and pilA. Furthermore, bacterial one-hybrid assay demonstrated that OxyR directly affected pilA and fliC promoter. Through bacterial two-hybrid assay, it was found that OxyR can directly interact with PilA and FliC. These results suggest that OxyR plays a major role in the regulating of a variety of virulence traits, and provide a foundation for future research on the global effects of OxyR in A. citrulli.

OxyR inactivation reduces the growth rate and oxidative stress defense in Capnocytophaga ochracea

ObjectiveThe human oral cavity harbors several bacteria. Among them, Capnocytophaga ochracea, a facultative anaerobe, is responsible for the early phase of dental plaque formation. In this phase, the tooth surface or tissue is exposed to various oxidative stresses. For colonization in the dental plaque phase, a response by hydrogen peroxide (H2O2)-sensing transcriptional regulators, such as OxyR, may be necessary. However, to date, no study has elucidated the role of OxyR protein in C. ochracea. MethodsInsertional mutagenesis was used to create an oxyR mutant, and gene expression was evaluated by reverse transcription-polymerase chain reaction and quantitative real-time reverse transcription-polymerase chain reaction. Bacterial growth curves were generated by turbidity measurement, and the sensitivity of the oxyR mutant to H2O2 was assessed using the disc diffusion assay. Finally, a two-compartment system was used to assess biofilm formation. ResultsThe oxyR mutant grew slower than the wild-type under anaerobic conditions. The agar diffusion assay revealed that the oxyR mutant had increased sensitivity to H2O2. The transcript levels of oxidative stress defense genes, sod, ahpC, and trx, were lower in the oxyR mutant than in the wild-type strain. The turbidity of C. ochracea, simultaneously co-cultured with Streptococcus gordonii, was lower than that observed under conditions of homotypic growth. Moreover, the percentage decrease in growth of the oxyR mutant was significantly higher than that of the wild-type. ConclusionsThese results show that OxyR in C. ochracea regulates adequate in vitro growth and escapes oxidative stress.

OxyR-controlled surface polysaccharide production and biofilm formation in Acinetobacter oleivorans DR1.

The genomes of several Acinetobacter species possess three distinct polysaccharide-producing operons [two poly-N-acetyl glucosamine (PNAG) and one K-locus]. Using a microfluidic device, an increased amount of polysaccharides and enhanced biofilm formation were observed following continuous exposure to H2O2 and removal of the H2O2-sensing key regulator, OxyR, in Acinetobacter oleivorans DR1 cells. Gene expression analysis revealed that genes located in PNAG1, but not those in PNAG2, were induced and that genes in the K-locus were expressed in the presence of H2O2. Interestingly, the expression of the K-locus gene was enhanced in the PNAG1 mutant and vice versa. The absence of either OxyR or PNAG1 resulted in enhanced biofilm formation, higher surface hydrophobicity, and increased motility, implying that K-locus-driven polysaccharide production in both the oxyR and PNAG1 deletion mutants may be related to these phenotypes. Both the oxyR and K-locus deletion mutants were more sensitive to H2O2 compared with the wildtype and PNAG1 mutant strains. Purified OxyR binds to the promoter regions of both polysaccharide operons with a higher affinity toward the K-locus promoter. Although oxidized OxyR could bind to both promoter regions, the addition of dithiothreitol further enhanced the binding efficiency of OxyR, suggesting that OxyR might function as a repressor for controlling these polysaccharide operons.

Oxidative Disinfectants Activate Different Responses in Vibrio parahaemolyticus

Vibrio parahaemolyticus is a prevalent seafoodborne enteropathogen that has become a global concern since the spread of its pandemic strain in 1996. This study investigates the responses of this pathogen to the oxidative disinfectants hydrogen peroxide, chlorine dioxide, and peracetic acid. Expression of the regulator genes oxyR and rpoS, determined by reverse transcription PCR, in V. parahaemolyticus wild-type, oxyR mutant, and rpoS mutant strains exhibited similar patterns in response to the tested oxidative disinfectants. The transcription of the rpoS gene was markedly enhanced in the oxyR mutant strain in the exponential phase. The expression of catalase KatE1 was tracked by using a LacZ fusion reporter in these strains. The experimental results revealed that KatE1 was a significant scavenger of hydrogen peroxide and peracetic acid in V. parahaemolyticus, and RpoS may partially compensate for the regulatory role of OxyR in the oxyR mutant strain. In contrast to its responses to hydrogen peroxide and paracetic acid, KatE1 was not the primary scavenger of chlorine dioxide in these V. parahaemolyticus strains. This study shows that these disinfectants activated a basic oxidative response in this pathogen with different features.

Oxidized OxyR Up-Regulates ahpCF Expression to Suppress Plating Defects of oxyR- and Catalase-Deficient Strains.

It is well established that in bacteria, such as Escherichia coli, OxyR is a transcriptional regulator that mediates the response to H2O2 by activating the OxyR regulon, which consists of many genes that play vital roles in oxidative stress resistance. In Shewanella, OxyR regulates, however, in both reduced and oxidized states, the production of H2O2 scavengers, including major catalase KatB and NADH peroxidase AhpCF. Here we showed that the oxyR mutant carried a plating defect manifested as division arresting, a phenotype that can be completely suppressed by an OxyR variant constitutively existing in oxidized form (OxyRL197P). This effect of OxyRL197P could not be solely attributed to the increment in KatB production, since the suppression was also observed in the absence of KatB. Although expression of peroxidase CcpA was greatly activated by OxyRL197P, the contribution of the protein in alleviating plating defect was negligible. We eventually identified AhpCF as the critical factor, when produced at substantially elevated levels by OxyRL197P, to protect the cell from H2O2 attack. Our data indicate that AhpCF is a particularly important peroxidase in oxidative stress resistance in Shewanella, not only playing a compensatory role for catalase, but also by itself providing sufficient protection from killing of H2O2 generated abiotically.

Quorum-sensing systems trigger catalase expression to reverse the oxyR deletion-mediated VBNC state in Salmonella typhimurium

OxyR is an important regulatory protein that plays a key role in anti-oxygenation, and its deletion causes a special VBNC (Viable But Non-Culturable) state in many bacteria including Salmonella typhimurium. The S. typhimurium in the VBNC state can grow in LB broth but cannot grow on an LB plate unless its concentration is sufficiently high. However, the mechanism that reverses this state is not clear. In this study, conditioned media containing autoinducer collected from the wild type strain can restore the growth of low concentrations of the oxyR mutant strain on LB plates, and S. typhimurium collected from the plate has higher catalase activity than that from the broth, suggesting that a quorum-sensing system can trigger catalase expression to resuscitate the organism from the VBNC state independent of the OxyR regulon. We discovered a novel catalase (STM14_2049, Cat) whose expression is strictly concentration-dependent. The purified Cat protein has obvious catalase activity in vitro and in vivo and can restore the growth of the low concentration oxyR mutant strain. Thus, we believed Cat plays a role in VBNC resuscitation process. By understanding this mechanism, we can further understand the antioxidation and quorum-sensing systems in Salmonella typhimurium.

OxyR positively and directly regulates Vi polysaccharide capsular antigen in Salmonella enterica serovar Typhi

Salmonella enterica serovar Typhi (S. Typhi) is a human enteropathogen that can overcome oxidative stress and survive in macrophages. OxyR is an important part of the antioxidant defense system. S. Typhi expresses a virulence (Vi) polysaccharide capsular antigen, which provides the bacterium with the ability to avoid host defenses and suppress detection by the innate immune system. This study investigated the effect of OxyR on Vi antigen in S. Typhi. In the oxyR mutant strain, microarray analysis, quantitative real time PCR and β-galactosidase assay confirmed that the viaB operon was positively regulated by OxyR. The Vi enzyme-linked immunosorbent assay and flow cytometry results showed that Vi capsule level was decreased in the oxyR mutant strain. Also, the EMSA revealed that OxyR directly binds to the promoter region of tviA. Thus, we propose that S. Typhi OxyR positively regulates expression of Vi capsule antigen in a direct manner.

Molecular response of Vibrio parahaemolyticus to the sanitizer peracetic acid

Peracetic acid (PAA) is a common oxidative sanitizer that is used in the food industry against various microorganisms. Limited information on the response of bacteria to this biocide is available. This study investigates the molecular response of the prevalent seafood-borne pathogenic Vibrio parahaemolyticus to PAA using mutants of peroxide scavenging genes. Among katE1, katE2, katG1, katG2, ahpC1 and ahpC2, and their regulator oxyR gene mutants, oxyR and katE mutants were highly susceptible to PAA. The growth and lethality of V. parahaemolyticus were harmed by 15 ppm of PAA in the △katE1E2 double mutant, and were significantly ameliorated in the presence of the katE1 gene in the wild-type strain and the gene-complementary strains that were pre-adapted in 2 ppm of PAA or 100 μM hydrogen peroxide. The application of PAA to these strains induced the accumulation of reactive oxygen species. The reduction of the level of hydrogen peroxide and gene expression during this treatment was influenced by the presence of katE genes. This investigation confirmed the major role of katE1 and a compensatory role of katE2 in the resistance of V. parahaemolyticus to PAA, and demonstrated some minor differences in the responses of this bacterium against PAA and hydrogen peroxide.

OxyR of Haemophilus parasuis is a global transcriptional regulator important in oxidative stress resistance and growth

Haemophilus parasuis is an opportunistic pathogen and the causative agent of Glässer's disease in swine. This disease has high morbidity and mortality rates in swine populations, and is responsible for major economic losses worldwide. Survival of H. parasuis within the host requires mechanisms for coping with oxidative stress conditions. In many bacteria, OxyR is known to mediate protection against oxidative stress; however, little is known about the role of OxyR in H. parasuis. In the current study, an oxyR mutant strain was constructed in H. parasuis strain SC1401 and designated H. parasuis SC1401∆oxyR. The oxyR mutant strain had a slower growth rate and impaired biofilm formation compared to the wild type strain. Complementation restored the growth-associated phenotypes to wild type levels. Oxidative stress susceptibility testing, using a range of concentrations of H2O2, indicated that H. parasuis SC1401∆oxyR was more sensitive to oxidative stress than the wild type strain. RNA sequencing transcriptome analysis comparing H. parasuis SC1401 with H. parasuis SC1401∆oxyR identified 466 differentially expressed genes. These genes were involved in a wide range of biological processes, including: oxidative stress, transcriptional regulation, and DNA replication, recombination, and repair. These findings provide a foundation for future research to examine the role of OxyR as a global transcriptional regulator and to better define its role in oxidative stress resistance in H. parasuis.

The OxyR-regulated phnW gene encoding 2-aminoethylphosphonate:pyruvate aminotransferase helps protect Pseudomonas aeruginosa from tert-butyl hydroperoxide

The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H2O2 and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, Pseudomonas aeruginosa. We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H2O2 in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire P. aeruginosa PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N7-CTAT-N7-ATAG-N7-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were not predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an oxyR mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, phnW, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the oxyR mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the phnW promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a phnW mutant was more susceptible to t-butyl-hydroperoxide (t-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade t-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the phnW mutant.

PprM, a Cold Shock Domain-Containing Protein from Deinococcus radiodurans, Confers Oxidative Stress Tolerance to Escherichia coli.

Escherichia coli is a representative microorganism that is frequently used for industrial biotechnology; thus its cellular robustness should be enhanced for the widespread application of E. coli in biotechnology. Stress response genes from the extremely radioresistant bacterium Deinococcus radiodurans have been used to enhance the stress tolerance of E. coli. In the present study, we introduced the cold shock domain-containing protein PprM from D. radiodurans into E. coli and observed that the tolerance to hydrogen peroxide (H2O2) was significantly increased in recombinant strains (Ec-PprM). The overexpression of PprM in E. coli elevated the expression of some OxyR-dependent genes, which play important roles in oxidative stress tolerance. Particularly, mntH (manganese transporter) was activated by 9-fold in Ec-PprM, even in the absence of H2O2 stress, which induced a more than 2-fold increase in the Mn/Fe ratio compared with wild type. The reduced production of highly reactive hydroxyl radicals (·OH) and low protein carbonylation levels (a marker of oxidative damage) in Ec-PprM indicate that the increase in the Mn/Fe ratio contributes to the protection of cells from H2O2 stress. PprM also conferred H2O2 tolerance to E. coli in the absence of OxyR. We confirmed that the H2O2 tolerance of oxyR mutants reflected the activation of the ycgZ-ymgABC operon, whose expression is activated by H2O2 in an OxyR-independent manner. Thus, the results of the present study showed that PprM could be exploited to improve the robustness of E. coli.

Tolerance to oxidative stress is required for maximal xylem colonization by the xylem-limited bacterial phytopathogen, Xylella fastidiosa.

Bacterial plant pathogens often encounter reactive oxygen species (ROS) during host invasion. In foliar bacterial pathogens, multiple regulatory proteins are involved in the sensing of oxidative stress and the activation of the expression of antioxidant genes. However, it is unclear whether xylem-limited bacteria, such as Xylella fastidiosa, experience oxidative stress during the colonization of plants. Examination of the X. fastidiosa genome uncovered only one homologue of oxidative stress regulatory proteins, OxyR. Here, a knockout mutation in the X. fastidiosa oxyR gene was constructed; the resulting strain was significantly more sensitive to hydrogen peroxide (H2 O2 ) relative to the wild-type. In addition, during early stages of grapevine infection, the survival rate was 1000-fold lower for the oxyR mutant than for the wild-type. This supports the hypothesis that grapevine xylem represents an oxidative environment and that X. fastidiosa must overcome this challenge to achieve maximal xylem colonization. Finally, the oxyR mutant exhibited reduced surface attachment and cell-cell aggregation and was defective in biofilm maturation, suggesting that ROS could be a potential environmental cue stimulating biofilm development during the early stages of host colonization.

The role of oxidative stress genes and effect of pH on methylene blue sensitized photooxidation of Escherichia coli.

In this study, the survival time of wild type E. coli W3110 and 11 mutants was analysed with a plate count method in methylene blue added or control groups under daylight fluoroscence illumination (4950 lux) at different pH values (5.0, 6.0, 7.0, and 8.0) in phosphate buffer. As a result, while the number of bacteria did not decrease under photooxidative stress at pH 5.0 and 6.0 during a 6-hour incubation, the wild type and all mutants decreased more than 2 log. at pH 8.0, and approximately one log. at pH 7.0. It was determined that a 2 log decrease in wild type E. coli takes 3.7 h according to t99 value at pH 8, these values were 2.39 h in the katE mutant, 2.64 h in the soxR mutant, 2.67 h in the oxyR mutant, 2.71 h in the sodB mutant, 3 h in the btuE mutant, 3.38 h in the zwf mutant and 3.40 h in the soxS mutant, respectively (p < 0.05). The roles of these genes were proved with complement tests. Finally, it is found that the effectiveness of photooxidative stress is in direct relation with pH, and the katE, soxR, oxyR, sodB, btuE, zwf, and soxS genes are important for the protection against this stress.

Molecular mechanism involved in the response to hydrogen peroxide stress in Acinetobacter oleivorans DR1.

Two-dimensional gel electrophoresis was conducted to investigate the effect of H2O2 on whole protein expression in Acinetobacter oleivorans DR1. Functional classification of 13 upregulated proteins using MALDI-TOF mass spectrometry showed relationships with oxidative stress, energy production and conversion, nucleotide and amino acid metabolism, membrane-related, ion transport, and chaperone-related functions. Alignment of OxyR-binding regions from Pseudomonas aeruginosa and Escherichia coli with promoters of identified proteins revealed that only ahpC, ahpF, and trxB (thioredoxin-disulfide reductase) genes, along with a newly found oprC (putative outer membrane receptor protein) gene, have OxyR-binding sites. The oxyR and ahpC mutants were more sensitive to H2O2 and showed growth defects in both nutritional and n-hexadecane-amended media. Four catalases present in the genome of A. oleivorans DR1 were not detected, which led us to confirm the expression and activity of those catalases in the presence of H2O2. The expression patterns of the four catalase genes differed at different concentrations of H2O2. Interestingly, the promoters of both known OxyR-controlled katG gene (AOLE_17390) and putative small catalase gene (AOLE_09800) have OxyR-binding sites. Gel-shift assay confirmed OxyR binding to the promoter regions of newly identified OxyR-controlled genes encoding OprC and a putative catalase. Hierarchical expression and OxyR-binding of several OxyR-controlled genes suggested that concentration is an important factor in inducing the set of genes under H2O2 stress.

Essential oil of Eucalyptus gunnii hook. As a novel source of antioxidant, antimutagenic and antibacterial agents.

The present study describes radical scavenging capacity (RSC), antimutagenic and antibacterial properties of the essential oil (EO) of the leaves of Eucalyptus gunnii Hook. (Southern Montenegro). Chemical composition was evaluated by gas chromatography-mass spectrometry (GC-MS). In oil, 1,8-cineole (67.8%) and α-pinene (14.12%) were the major compounds comprising almost 82% of total EO. EO exhibited moderate DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity, with IC50 value of 7.19 µL/mL. The antimutagenic properties were assayed against the spontaneous and t-BOOH-induced mutagenesis in Escherichia coli IC202 oxyR mutant strain, deficient in removing radical oxygen species (ROS). Reduction of the spontaneous mutagenesis in the presence of E. gunnii EO was only slight, up to 12% at the highest concentration tested. However, when the oxidative mutagen was used, EO displayed more significant reduction of mutagenesis (maximum 23%) in a concentration dependent manner. Antibacterial activity was tested against the selected strains from ATTC and NCIB collections: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Micrococcus flavus, Klebsiella pneumoniae, and the two Escherichia coli strains from our laboratory collection (SY252 and IB112) using both the disk-diffusion and MIC assays. The greatest sensitivity was shown by M. flavus, K. pneumoniae and E. coli lpcA (MIC = 0.83 mg/mL), while the highest resistance was shown by E. coli (ATTC 25922) and S. epidermidis. This study represents the first report on chemical composition and biological activity of the Eucalyptus gunnii in the South Balkan region and beyond.