The role of thiol redox systems in the response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to far-UV irradiation

Escherichia coli mutants deficient in glutathione (gshA), glutaredoxin (grxA), thioredoxin (trxA), and thioredoxin reductase (trxB) synthesis were studied with respect to their resistance to far-UV (UV₂₅₄) exposure. The trxA, trxB, and grxA mutants subjected to a short-term UV exposure were found to be more resistant to UV irradiation than the parent cells. Under the same conditions, the trxA and trxB mutants demonstrated a high level of induction of the sulA gene, a component of the SOS regulon. The mutagenic effect of long-term UV exposure of all the mutants with redox deficiencies was more pronounced than in the case of the parent strain, and the trxA and trxB mutants were found to be the least viable microorganisms. Pretreatment of the cells with low concentrations of the thiol-oxidizing agent diamide enhanced the sulA gene expression; however, high concentrations of diamide inhibited sulA expression. The data obtained indicate that the thiol redox systems of E. coli are involved in its response to far-UV irradiation.     

Glutathione reductase gene expression depends on chloroplast signals in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Glutathione reductase (EC 1.6.4.2) is one of the main antioxidant enzymes of the plant cell. In Arabidopsis thaliana, glutathione reductase is encoded by two genes: the gr1 gene encodes the cytosolic-peroxisomal form, and the gr2 gene encodes the chloroplast-mitochondrial form. Little is known about the regulation of expression of plant glutathione reductase genes. In the present work, we have demonstrated that gr2 (but not gr1) gene expression in Arabidopsis leaves changes depending on changes in redox state of the photosynthetic electron transport chain. Expression of both the gr1 and gr2 genes was induced by reactive oxygen species. In heterotrophic suspension cell culture of Arabidopsis, expression of both studied genes did not depend on H₂O₂ level or on changes in the redox state of the mitochondrial electron transport chain. Our data indicate that chloroplasts are involved in the regulation of the glutathione reductase gene expression in Arabidopsis.     

The role of thiol redox systems in the peroxide stress response of Escherichia coli

The effect of mutations in the genes encoding glutathione, glutaredoxin, thioredoxin, and thioredoxin reductase on the response of growing Escherichia coli to oxidative stress was studied. The gshA mutants defective in glutathione synthesis had the lowest resistance to high doses of H2O2, whereas the trxB mutants defective in thioredoxin reductase synthesis had the highest resistance to this oxidant, exceeding that of the parent strain. Among the studied mutants, the trxB cells demonstrated the highest basic levels of catalase activity and intracellular glutathione; they were able to rapidly reach the normal GSH level after oxidative stress. At the same time, these bacteria showed high frequency of induced mutations. The expression of the katG and sulA genes suggests that, having different sensitivity to high oxidant concentrations, the studied mutants differ primarily in their ability to induce the antioxidant genes of the OxyR and SOS regulons.     

Revealing physiological and genetic properties of a dominant maize dwarf <Emphasis Type="Italic">Dwarf11</Emphasis> (<Emphasis Type="Italic">D11</Emphasis>) by integrative analysis

Plant height is an important agronomic trait involved in lodging resistance and harvest index. The identification and characterization of mutants that are defective in plant height have implications for trait improvement in breeding programs. Two dominant maize dwarf mutants D8 and D9 have been well-characterized. Here, we report the characterization of a dominant maize dwarf mutant Dwarf11 (D11). Dwarf stature of D11 was mainly attributed to the inhibition of longitudinal cell elongation. The levels of bioactive GA₃ were significantly lower in D11. Contrarily, D8 mutant accumulates markedly higher levels of GA₃. The expression of GA biosynthetic and catabolic genes was dramatically decreased in D11. Expression variations of d8 and d9 genes were not observed in D11 mutant. Moreover, genetic suppressors of D11 were identified in inbred line Chang 7-2. Integrated omics data indicated that D11 is a novel dominant maize dwarf. The ultimate D11 gene cloning and its regulatory network elucidation may strengthen our understanding of the genetic basis of plant architecture and provide cues for breeding of crops with plant height ideotypes.     

A <Emphasis Type="Italic">pqr2</Emphasis> mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 μM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na₂WO₄, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.     

Interaction between checkpoint genes <Emphasis Type="Italic">RAD9, RAD17, RAD24</Emphasis>, and <Emphasis Type="Italic">RAD53</Emphasis> involved in the determination of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> sensitivity to ionizing radiation

Mechanisms for genetic control of cell division cycle (checkpoint control) have been studied in most detail in yeast Saccharomyces cerevisiae. To clarify the role of checkpoint genes RAD9, RAD17, RAD24, and RAD53 in cell radioresistance, double mutants were analyzed for cell sensitivity to ionizing radiation. Double mutants carrying mutations in combination with mutation rad9Δ were shown to manifest the epistatic type of interaction. Our results suggest that checkpoint genes RAD9, RAD17, RAD24, and RAD53 belong to a single epistatic group designated RAD9 and govern the same pathway. Genes RAD9 and RAD53 have a positive effect on sensitivity to γ-radiation, whereas RAD17 and RAD24 have a negative effect. Interactions between mutations may differ when considering their sensitivity to γ-radiation and UV light; mutations rad9Δ and rad24Δ were shown to manifest the additive effect in the first case and epistatic effect in the second.     

Features of expression of the <Emphasis Type="Italic">PsSst1</Emphasis> and <Emphasis Type="Italic">PsIgn1</Emphasis> genes in nodules of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) symbiotic mutants

The sequences of the PsSst1 and PsIgn1 genes of pea (Pisum sativum L.) homologous to the symbiotic LjSST1 and LjIGN1 genes of Lotus japonicus (Regel.) K. Larsen are determined. The expression level of PsSst1 and PsIgn1 genes is determined by real-time PCR in nodules of several symbiotic mutants and original lines of pea. Lines with increased (Sprint-2Fix^– (Pssym31)) and decreased (P61 (Pssym25)) expression level of both genes are revealed along with the lines characterized by changes in the expression level of only one of these genes. The revealed features of the PsSst1 and PsIgn1 expression allow us to expand the phenotypic characterization of pea symbiotic mutants. In addition, PsSst1 and PsIgn1 cDNA is sequenced in selected mutant lines, characterized by a decreased expression level of these genes in nodules, but no mutations are found.     

IutB participates in the ferric-vulnibactin utilization system in <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> M2799

Vibrio vulnificus, an opportunistic pathogen that causes a serious, often fatal, infection in humans, requires iron for its growth. This bacterium utilizes iron from the environment via the vulnibactin-mediated iron uptake system. The mechanisms of vulnibactin biosynthesis, vulnibactin export, and ferric-vulnibactin uptake systems have been reported, whereas the ferric-vulnibactin reduction mechanism in the cell remains unclear. The results of our previous study showed that VuuB, a member of the flavin adenine dinucleotide-containing siderophore-interacting protein family, is a ferric-vulnibactin reductase, but there are other reductases that can complement for the defective vuuB. The aim of this study was to identify these proteins that can complement the loss of function of VuuB. We constructed mutants of genes encoding putative reductases in V. vulnificus M2799, and analyzed their growth under low-iron conditions. Complementation analyses confirmed that IutB, which functions as a ferric-aerobactin reductase, participates in ferric-vulnibactin reduction in the absence of VuuB. This is the first genetic evidence that ferric-vulnibactin is reduced by a member of the ferric-siderophore reductase protein family. In the aerobactin-utilization system, IutB plays a major role in ferric-aerobactin reduction in V. vulnificus M2799, and VuuB and DesB can compensate for the defect of IutB. Furthermore, the expression of iutB and desB was found to be regulated by iron and a ferric uptake regulator.     

Genetic Collection of Meiotic Mutants of Rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

Genetic collection of meiotic mutants of winter rye Secale cereale L. (2n = 14) was created. Mutations were detected in inbred F₂ generations after self-fertilization of the F₁ hybrids, obtained by individual crossing of rye plants (cultivar Vyatka) or weedy rye with plants from autofertile lines. The mutations cause partial or complete plant sterility and are maintained in collection in a heterozygous state. Genetic analysis accompanied by cytogenetic study of meiosis has revealed six mutation types. (1) Nonallelic asynaptic mutations sy1 and sy9 caused the formation of only axial chromosome elements in prophase and anaphase. The synaptonemal complexes (SCs) were absent, the formation of the chromosome “bouquet” was impaired, and all chromosomes were univalent in meiotic metaphase I in 96.8% (sy1) and 67% (sy2) of cells. (2) Weak asynaptic mutation sy3, which hindered complete termination of synapsis in prophase I. Subterminal asynaptic segments were always observed in the SC, and at least one pair of univalents was present in metaphase I, but the number of cells with 14 univalents did not exceed 2%. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19, which caused partially nonhomologous synapsis: change in pairing partners and fold-back chromosome synapsis in prophase I. In metaphase I, the number of univalents varied and multivalents were observed. (4) Mutation mei6, which causes the formation of ultrastructural protrusions on the lateral SC elements, gaps and branching of these elements. (5) Allelic mutations mei8 and mei8-10, which caused irregular chromatin condensation along chromosomes in prophase I, sticking and fragmentation of chromosomes in metaphase I. (6) Allelic mutations mei5 and mei10, which caused chromosome hypercondensation, defects of the division spindle formation, and random arrest of cells at different meiotic stages. However, these mutations did not affect the formation of microspore envelopes even around the cells, whose development was blocked at prophase I. Analysis of cytological pictures of meiosis in double rye mutants reveled epistatic interaction in the mutation series sy9 > sy1 > sy3 > sy19, which reflects the order of switching these genes in the course of meiosis. The expression of genes sy2 and sy19 was shown to be controlled by modifier genes. Most meiotic mutations found in rye have analogs in other plant species.     

Hydrogen sulfide inhibits the growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> through oxidative damage

Many studies have shown that hydrogen sulfide (H₂S) is both detrimental and beneficial to animals and plants, whereas its effect on bacteria is not fully understood. Here, we report that H₂S, released by sodium hydrosulfide (NaHS), significantly inhibits the growth of Escherichia coli in a dose-dependent manner. Further studies have shown that H₂S treatment stimulates the production of reactive oxygen species (ROS) and decreases glutathione (GSH) levels in E. coli, resulting in lipid peroxidation and DNA damage. H₂S also inhibits the antioxidative enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) and induces the response of the SoxRS and OxyR regulons in E. coli. Moreover, pretreatment with the antioxidant ascorbic acid (AsA) could effectively prevent H₂S-induced toxicity in E. coli. Taken together, our results indicate that H₂S exhibits an antibacterial effect on E. coli through oxidative damage and suggest a possible application for H₂S in water and food processing.     

Altered somatic mutation level and DNA repair gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exposed to ultraviolet C,salt, and cadmium stresses

It is known that somatic mutations arising during animal growth and ageing contribute to the development of neurodegenerative and other animal diseases. For plants, several studies showed that small-scale somatic DNA mutations accumulated during Arabidopsis life cycle. However, there is a lack of data on the influence of environmental stresses on somatic DNA mutagenesis in plants. In this study, we analyzed the effects of ultraviolet C (UV-C) irradiation, high soil salinity, and cadmium (CdI₃) stresses on the level of small-scale somatic DNA mutations in Arabidopsis thaliana. The number of DNA mutations was examined in the Actin2 3′UTR (Actin-U1), ITS1-5.8rRNA-ITS2 (ITS), and ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) DNA regions. We found that somatic mutation levels considerably increased in CdI₃-treated Arabidopsis plants, while the mutation levels declined in the UV-C- and NaCl-treated A. thaliana. Cadmium is a mutagen that is known to inhibit DNA repair processes. The detected stress-induced alterations in somatic DNA mutation levels were accompanied by markedly increased expression of base excision repair genes (AtARP, AtDME, AtDML2, AtDML3, AtMBD4, AtROS, AtUNG, and AtZDP), nucleotide excision repair genes (AtDDB1a, AtRad4, and AtRad23a), mismatch repair genes (AtMSH2, AtMSH3, and AtMSH7), and photoreactivation genes (AtUVR2, AtUVR3). Thus, the results demonstrated that UV-C, high soil salinity, and cadmium stresses influence both the level of DNA mutations and expression of DNA repair genes. Salt- and UV-induced activation of DNA repair genes could contribute to the stress-induced decrease in somatic mutation level.     

Jasmonic acid regulates the ascorbate–glutathione cycle in <Emphasis Type="Italic">Malus baccata</Emphasis> Borkh. roots under low root-zone temperature

Jasmonic acid (JA), which is an important phytohormone, plays a key role in plant growth, development and stress responses. Here, Malus baccata Borkh. seedlings were used to study the mechanism by which JA alleviates the oxidative damage induced by low root-zone temperature (5 °C) through regulating the ascorbate–glutathione (AsA–GSH) cycle. The roots of M. baccata Borkh. were subjected to three treatments [5 °C, 5 °C + JA, and 5 °C + ibuprofen (IBU)] for 0, 12, 24, and 48 h. The results showed that treatment with low root-zone temperature could modulate the non-enzymatic and enzymatic components of the AsA–GSH cycle, significantly inducing the accumulation of MDA and H₂O₂. Additionally, the endogenous JA content changed dramatically, and the expression levels of the related genes [lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC)] showed different trends. In plants pretreated with JA, the endogenous JA content increased at 24 h, and the gene expression levels of LOX, AOS, and AOC were upregulated. We also found a marked increase in the activities of antioxidant enzymes [ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR)], a decrease in oxidized glutathione (GSSG) and an increased GSH/GSSG ratio, which resulted in lower MDA and H₂O₂ contents. Thus, the oxidative stress was alleviated. Plants pretreated with IBU experienced an opposite effect on the function of the AsA–GSH cycle and the gene expression in the JA synthesis route relative to those subjected to exogenous JA treatment, indicating that endogenous JA can alleviate oxidative damage by regulating the function of the AsA–GSH cycle under low root-zone temperature.     

Participation of <Emphasis Type="Italic">SRM5/CDC28</Emphasis>, <Emphasis Type="Italic">SRM8/NET1</Emphasis>, and <Emphasis Type="Italic">SRM12/HFI1</Emphasis> genes in checkpoint control in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

About twenty genes participating in checkpoint control are known in yeast Saccharomyces cerevisiae. The involvement of SRM genes in the cell cycle arrest under the action of DNA damaging agents was studied in this work. These genes were earlier defined as genes affecting genetic stability and radiosensitivity. It was shown that mutations srm5/cdc28-srm, srm8/net1-srm, and srm12/hfi1-srm fail the cell cycle arrest in the presence of DNA damage and influence the checkpoint arrest in G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm5, srm12), and G₂/M (srm5). It seems likely that genes SRM5/CDC28, SRM12/HFI1/ADA1, and SRM8/NET1 are involved in a cell response to DNA damage, and in checkpoint regulation in particular.     

Hydrogen sulfide is involved in the regulation of ascorbate and glutathione metabolism by jasmonic acid in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

This study investigated the role of hydrogen sulfide (H₂S) in the regulation of the ascorbate (AsA) and glutathione (GSH) metabolism by jasmonic acid (JA) in the leaves of Arabidopsis thaliana by using H₂S scavenger hypotaurine (HT) and H₂S synthetic mutant (SALK_041918, designated Atl-cdes). The results showed that JA significantly increased the H₂S content, the activities of L-cysteine desulfhydrase (L-CDes), D-cysteine desulfhydrase (D-CDes), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), L-galactono-1,4-lactone dehydrogenase (GalLDH) and γ-glutamylcysteine synthetase (γ-ECS), the ratio of AsA to dehydroascorbate (DHA), and decreased the content of malondialdehyde (MDA) and H₂O₂ in the wild type of A. thaliana, compared to control. The above effects of JA except the increased activities of L-CDes and D-CDes were suppressed by addition of HT. However, JA and HT+JA had no significant effects on the ratio of reduced GSH to oxidized GSH (GSSG) in the wild type of A. thaliana. Application of HT to the control decreased H₂S content, AsA/DHA ratio, and activities of APX, GR, DHAR, MDHAR, γ-ECS, and GalLDH, but had no effects on MDA content, activities of L-CDes and D-CDes, and GSH/GSSG ratio. In the H₂S synthetic mutant, JA had no obvious effects on above mentioned parameters except the D-CDes activity compared with the control. Our results suggest that JA-induced H₂S, which is a signal that leads to the up-regulation of the AsA and GSH metabolism.     

A novel NADPH-dependent reductase of <Emphasis Type="Italic">Sulfobacillus acidophilus</Emphasis> TPY phenol hydroxylase: expression,characterization, and functional analysis

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X ₄ -Cys-X ₂ -Cys-X _29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.     

The pleiotropic nature of <Emphasis Type="Italic">rib80, hit1</Emphasis>, and <Emphasis Type="Italic">red6</Emphasis> mutations affecting riboflavin biosynthesis in the yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis>

The yeast Pichia guilliermondii is capable of riboflavin overproduction under iron deficiency. The rib80, hit1, and red6 mutants of this species, which exhibit impaired riboflavin regulation, are also distinguished by increased iron concentrations in the cells and mitochondria, morphological changes in the mitochondria, as well as decreased growth rates (except for red6) and respiratory activity. With sufficient iron supply, the rib80 and red6 mutations cause a 1.5–1.8-fold decrease in the activity of such Fe-S cluster proteins as aconitase and flavocytochrome b ₂, whereas the hit1 mutation causes a six-fold decrease. Under iron deficiency, the activity of these enzymes was equally low in all of the studied strains.     

New Approach to Identification of Genes Controlling Cell Wall Biogenesis in the Yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

MCD4 codes for a protein presumably adding the phosphoethanolamine moiety to the first mannose residue of glycosylphosphatidylinositol (GPI) precursors in the yeast Saccharomyces cerevisiae. The role of this modification is still unclear. The phenotypic effects of some MCD4 mutations are probably unrelated to defects in GPI synthesis, suggesting additional functions for Mcd4p. To study the Mcd4p functions in more detail, a search for the genes whose mutations are lethal or semilethal in combination with the ssu21 mutation of MCD4 was performed. Six such mutations were isolated, including some mutations causing sensitivity to SDS and/or calcofluor white. Genes complementing two out of the six mutations were cloned and identified as MNN9, which is involved in the formation of outer chains of N-linked glycans of secreted proteins, and GWT1, which codes for an endoplasmic reticulum protein involved in GPI biosynthesis. In both cases, growth inhibition was probably caused by defective biogenesis of the cell wall and a misfolding of secreted proteins. The proposed approach is suitable for seeking new genes controlling cell wall biogenesis.     

Identification of essential genes of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> for its growth in airway mucus

Pseudomonas aeruginosa has been identified as an important causative agent of airway infection, mainly in cystic fibrosis. This disease is characterized by defective mucociliary clearance induced in part by mucus hyper-production. Mucin is a major component of airway mucus and is heavily O-glycosylated, with a protein backbone. Airway infection is known to be established with bacterial adhesion to mucin. However, the genes involved in mucin degradation or utilization remain elusive. In this study, we sought to provide a genetic basis of P. aeruginosa airway growth by identifying those genes. First, using RNASeq analyses, we compared genome-wide expression profiles of PAO1, a prototype P. aeruginosa laboratory strain, grown in M9-mucin (M9M) and M9-glucose (M9G) media. Additionally, a PAO1 transposon (Tn) insertion mutants library was screened for mutants defective in growth in M9M medium. One mutant with a Tn insertion in the xcpU gene (PA3100) was determined to exhibit faulty growth in M9M medium. This gene contributes to the type II secretion system, suggesting that P. aeruginosa uses this secretion system to produce a number of proteins to break down and assimilate the mucin molecule. Furthermore, we screened the PAO1 genome for genes with protease activity. Of 13 mutants, one with mutation in PA3247 gene exhibited defective growth in M9M, suggesting that the PA3247-encoded protease plays a role in mucin utilization. Further mechanistic dissection of this particular process will reveal new drug targets, the inhibition of which could control recalcitrant P. aeruginosa infections.