Most bacteria control oxidative stress through the H2O2-responsive transactivator OxyR, a member of the LTTR family (LysR Type Transcriptional Regulators), which activates the expression of defensive genes such as those encoding catalases, alkyl hydroperoxide reductases and superoxide dismutases. In the human opportunistic pathogen Pseudomonas aeruginosa, OxyR positively regulates expression of the oxidative stress response genes katA, katB, ahpB and ahpCF. To identify additional targets of OxyR in P. aeruginosa PAO1, we performed chromatin immunoprecipitation in combination with whole genome tiling array analyses (ChIP-chip). We detected 56 genes including all the previously identified defensive genes and a battery of novel direct targets of OxyR. Electrophoretic mobility shift assays (EMSAs) for selected newly identified targets indicated that ∼70% of those were bound by purified oxidized OxyR and their regulation was confirmed by quantitative real-time polymerase chain reaction. Furthermore, a thioredoxin system was identified to enzymatically reduce OxyR under oxidative stress. Functional classification analysis showed that OxyR controls a core regulon of oxidative stress defensive genes, and other genes involved in regulation of iron homeostasis (pvdS), quorum-sensing (rsaL), protein synthesis (rpsL) and oxidative phosphorylation (cyoA and snr1). Collectively, our results indicate that OxyR is involved in oxidative stress defense and regulates other aspects of cellular metabolism as well.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptorbinding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other Stype pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

Soluble (S‐type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa cells by gaining entry via a specific receptor, which, in the case of pyocin S2, is the siderophore pyoverdine receptor FpvAI, and in the case of pyocin S3, FpvAII. The nucleic acid sequence at the positions 4327697–4327359 of P. aeruginosa PAO1 genome was not annotated, but it was predicted to encode the immunity gene of the flanking pyocin S4 gene () based on our analysis of the genome sequence. Using RT‐PCR, the expression of the immunity gene was detected, confirming the existence of an immunity gene overlapping the S4 pyocin gene. The coding for pyocin S4 and the downstream gene coding for the immunity protein were cloned and expressed in Escherichia coli and the His‐tagged S4 pyocin was obtained in pure form. Forty‐three P. aeruginosa strains were typed via PCR to identify their ferripyoverdine receptor gene (fpvAI–III) and were tested for their sensitivity to pyocin S4. All S4‐sensitive strains had the type I ferripyoverdine receptor fpvA gene. Some S4‐resistant type I fpvA‐positive strains were detected, but all of them had the S4 immunity gene, and, following the deletion of the immunity gene, became S4‐sensitive. The fpvAI receptor gene was deleted in a S4‐sensitive strain, and, as expected, the mutant became resistant to S4. The N‐terminal receptor binding domain (RBD) of pyocin S2, which also uses the FpvAI receptor to enter the cell, was cloned in the pET‐15b vector, and expressed in E. coli. When the purified RBD was mixed with pyocin S4 at different ratios, an inhibition of killing was observed, indicating that S2 RBD competes with the pyocin S4 for the binding to the FpvAI receptor. The S2 RBD was also shown to enhance the expression of the pvdA pyoverdine gene, suggesting that it, like pyoverdine, works via the known siderophore‐mediated signalization pathway.

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNA in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (tA) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using tA-deficient yeast derivatives, it is shown that tA is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the tA synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE mutant was devoid of tA. However, we describe here a novel and a more sensitive hybridization-based tA detection method (compared to HPLC) that showed tA was still present in the S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans tA synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of tA modification ratios and of tA synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.

Objectives:This study evaluates the ability of a non-white rot fungus strain, HESHAM-1, to degrade a mixture of low (naphthalene and phenanthrene) and high (chrysene and benzo(a)pyrene) molecular weight polycyclic aromatic hydrocarbons (LMW and HMW PAHs). Methods:Strain HESHAM-1 was isolated from oil polluted soil by enrichment method using phenanthrene as the sole source of carbon and energy. The strain showed the ability to tolerate and degrade a mixture of both low and high molecular weight PAHs. In the presences of LMW-PAHs (naphthalene and phenanthrene) as co-substrate, chrysene and benzo(a)pyrene (HMW-PAHs) were, respectively degraded by the fungus strain HESHAM-1 which was confirmed by GC-MS analyses. Results:The degradation rate was found as 84.82% for naphthalene, 40.09% for phenanthrene, 57.84% for chrysene and 71.06% for benzo(a)pyrene at the end of 10 days. This is the first report describing the biodegradation of a mixture of four PAH compounds by non-white rot fungus strain HESHAM-1 isolated from Egyptian oil-polluted soil. The fungus strain HESHAM-1 was identified by morphological characteristics and molecular genetics technique based on PCR amplification and sequencing of the internal transcribed spacers (ITSs) of the rDNA region and intervening 5.8S rRNA gene. Blast result and phylogenetic analysis of gene sequencing suggested that strain HESHAM-1 was closely related to Fusarium solani with 100% sequence identity. Conclusion:The present study clearly demonstrates that, strain HESHAM-1 could be used to remove the crude oil from the environment.

Microorganisms have an effective capability to degrade synthetic dyestuff enzymatically. In this study, two 16S rRNA identified Pseudomonas aeruginosa bacterial isolates (ASU3 and ASU6) have been screened to enzymatically degrade both Disperse Blue 64 (DB64) and Acid Yellow 17 (AY17) which resembles 2 of the synthetic dyes that causes aquatic environmental pollution. The expression of the azoR1 gene has been detected in the strains, the degradation percentage of DB64 and AY17 by stain ASU6 was found to exceed the ASU3. This returned to the differences in the expression levels of azoR1 between the ASU6 and the ASU3 which was confirmed by RT-PCR. In addition, the specific activity (U/mg protein) of azoreductase in ASU6 was higher than ASU3 when MethylRed (MR), DB64 and AY17 were used as a substrate, separately. The azoreductase kinetics was studied to find the optimum condition for maintaining the stability of the enzyme. The degraded products separated by HPLC/MS were tested for their toxicity on two crop plants and they were nontoxic to the plants.

BackgroundThe infectious prion protein (PrPSc or prion) is derived from its cellular form (PrPC) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrPC to PrPSc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrPC (BVPrP) is highly susceptible to PrPSc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease.ResultsTo explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion.ConclusionsOur results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.

Background Plant-parasitic nematodes are extremely dangerous pests in a variety of economically important crops. The purpose of this study was a survey of all nematode species existing in banana from three sites in Assiut Governorate, Egypt and to characterize the most common species by morphological, morphometric and molecular techniques (PCR with species-specific primers). Then, study of resistance or sensitivity of some banana cultivars to root-knot nematodes.Methods and Results Four nematodes, Meloidogyne, Rotylenchulus reniformis, Helicotylenchus and Pratylenchus were isolated and identified from soil and root samples collected from banana plants. Most frequently occurring of plant parasitic nematode species in banana was Meloidogyne. Former research found differences in species and in resistance to root-knot nematodes among the examined plant cultivars. Identification of Root-knot nematodes by Characterize of morphometric, molecularly, morphological isolate of Meloidogyne related to banana plants. The results revealed that the identified nematode species, Meloidogyne javanica, is the most common plant-parasitic nematodes in all locations. Data on the susceptibility of the tested banana cultivars to M. javanica revealed that Grand Naine was highly susceptible (HS) however, Magraby was susceptible (S) but Williams and Hindi cultivars were moderately resistant (MR).Conclusions we concluded that a survey revealed the significant prevalence of Meloidogyne javanica, the most important nematodes on banana in Assiut. The morphometric, morphological, and molecular identification were harmonic with one another. In addition to the host response of certain banana cultivars, to M. javanica that resistance is of significance and can be helpful to incorporate through planning control measures for root- knot nematodes.