Inhibiting sulfate-reducing bacteria in biofilms by expressing the antimicrobial peptides indolicidin and bactenecin

To identify novel, less-toxic compounds capable of inhibiting sulfate-reducing bacteria (SRB), Desulfovibrio vulgaris and Desulfovibrio gigas in suspension cultures were exposed to several antimicrobial peptides. The bacterial peptide antimicrobials gramicidin S, gramicidin D, and polymyxin B as well as the cationic peptides indolicidin and bactenecin from bovine neutrophils decreased the viability of both SRB by 90% after a 1-h exposure at concentrations of 25–100 μg ml⁻¹. To reduce corrosion by inhibiting SRB in biofilms, the genes for indolicidin and bactenecin were expressed in Bacillus subtilisBE1500 and B. subtilis WB600 under the control of the constitutive alkaline protease (apr) promoter, and the antimicrobials were secreted into the culture medium using the apr signal sequence. Bactenecin was also synthesized and expressed as a fusion to the pro-region of barnase from Bacillus amyloliquefaciens. Concentrated culture supernatants of B. subtilis BE1500 expressing bactenecin at 3 μg ml⁻¹ decreased the viability of Escherichia coli BK6 by 90% and the reference SRB D. vulgaris by 83% in suspension cultures. B. subtilis BE1500 and B. subtilis WB600 expressing bactenecin in biofilms also inhibited the SRB-induced corrosion of 304 stainless steel six to 12-fold in continuous reactors as evidenced by the lack of change in the impedance spectra (resistance polarization) upon addition of SRB and by the reduction in hydrogen sulfide and iron sulfide in batch fermentations with mild steel. A 36-fold decrease in the population of D. vulgaris in a B. subtilis BE1500 biofilm expressing bactenecin was also observed. This is the first report of an antimicrobial produced in a biofilm for in vivo applications and represents the first application of a beneficial, genetically-engineered biofilm for combating corrosion. Received 27 October 1998/ Accepted in revised form 21 February 1999     

Growth and biochemical responses of jojoba (Simmondsia chinensis (Link) Schneid) explants cultured under mannitol-simulated drought stress in vitro

Jojoba explants were cultured under four levels of mannitol (control plus 50, 100, 250 and 500 mM mannitol)-induced osmotic stress during the proliferation stage in vitro. Explants grown under control condition exhibited the highest growth, while the more severe the stress was, the lower was the growth of explants. Electrolyte leakage, lipid peroxidation, H₂O₂ content and proline concentration were highest and relative water content lowest under the highest level of osmotic stress. Concentration of phenolic compounds (total phenolic compounds, o-diphenols and flavanols, as well as protocatechuic, vanillic, p-coumaric and ferulic acids and rutin), putrescine and total polyamines decreased with increasing stress level. Mannitol, glucose and pinitol concentrations increased, whereas that of inositol decreased with increasing stress level. Explants were transferred to the rooting stage, separately per stress treatment. Explants grown under stress conditions during the proliferation stage exhibited lower rooting percentage than controls, as the stress became more severe, the lower was the rooting response. Jojoba tolerated osmotic stress to some extent (till 100–250 mM mannitol), exhibiting sufficient growth rate and good rooting response as well as low oxidative damage (based on electrolyte leakage and lipid peroxidation indices).     

Transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses

Despite the high isoform multiplicity of aquaporins in plants, with 35 homologues including 13 plasma membrane intrinsic proteins (PIPs) in Arabidosis thaliana, the individual and integrated functions of aquaporins under various physiological conditions remain unclear. To better understand aquaporin functions in plants under various stress conditions, we examined transgenic Arabidopsis and tobacco plants that constitutively overexpress Arabidopsis PIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates and water transport were found between the transgenic and wild-type plants when grown under favorable growth conditions. The transgenic plants overexpressing PIP1;4 or PIP2;5 displayed a rapid water loss under dehydration stress, which resulted in retarded germination and seedling growth under drought stress. In contrast, the transgenic plants overexpressing PIP1;4 or PIP2;5 showed enhanced water flow and facilitated germination under cold stress. The expression of several PIPs was noticeably affected by the overexpression of PIP1;4 or PIP2;5 in Arabidopsis under dehydration stress, suggesting that the expression of one aquaporin isoform influences the expression levels of other aquaporins under stress conditions. Taken together, our results demonstrate that overexpression of an aquaporin affects the expression of endogenous aquaporin genes and thereby impacts on seed germination, seedling growth, and stress responses of the plants under various stress conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of nitrogen-deficiency on midday photoinhibition in flag leaves of different rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars

Effects of nitrogen (N)-deficiency on midday photoinhibition in flag leaves were compared between two contrastive Japanese rice cultivars, a traditional japonica cultivar with low yield, cv. Shirobeniya (SRB), and a japonica-indica intermediate type with high yield, cv. Akenohoshi (AKN). Both cultivars were grown under high-N and low-N conditions. At midday, low-N supply resulted in more intensive reductions in net photosynthetic rate, stomatal conductance, maximal quantum yield of photosystem II (PSII) and quantum yield of PSII electron transport in SRB than in AKN, indicating that SRB was more strongly photoinhibited than AKN under low-N condition. At midday, the low-N plants of two cultivars showed higher superoxide dismutase (SOD) activities than the high-N plants. However, ascorbate peroxidase (APX) activity was maintained in AKN but significantly decreased in SRB under low-N condition (N-deficiency). In contrast, hydrogen peroxide (H₂O₂) content in SRB significantly increased under low-N condition, indicating that the susceptibility to midday photoinhibition in the low-N plants of SRB is related to the increased H₂O₂ accumulation. It is suggested that the midday depression in photosynthesis may be a result of oxidative stress occurring in the low-N plants in which antioxidant capacity is not enough to cope with the generation of H₂O₂. Therefore, H₂O₂-scavenging capacity could be an important factor in determining the cultivar difference of midday photoinhibition in flag leaves of rice under low-N condition.     

Heat Production Rate as an Indicator of the Ability of Plant Cell to Adapt to Environmental Conditions

The heat production rate in the suspension of unicellular alga (Chlorella vulgaris) was studied by the method of microcalorimetry during adaptation of the alga to stress conditions (NaCl and 45°C). The heat production rate slightly increased after the addition of 75 mM NaCl to cell suspension. A two-phase response was observed at 150 and 450 mM NaCl and at elevated temperature. The heat production rate was high during the first 3–4 h; then, it decreased and leveled off. This characteristic dramatically decreased at a high salt concentration (500 mM). Cell was unable to adapt to a high NaCl concentration (550 mM), which led to energy dissipation manifested in a high heat production rate. We conclude that the heat production rate, an integral indicator of plant cell activity can be used to assess plant cell adaptation to the impact of stress factors.     

Microalgae Improve the Photosynthetic Performance of Rice Seedlings (<i>Oryza sativa</i> L.) under Physiological Conditions and Cadmium Stress

The aim of this study was to assess the impact of the microalgae Chlorella vulgaris on the rice seedlings at physiological conditions and under cadmium (Cd) stress. We examined the effects of C. vulgaris in the nutrient solution on rice seedlings grown hydroponically in the presence and the absence of 150 μM CdCl₂, using the low (77 K) temperature and pulse amplitude modulated (PAM) chlorophyll fluorescence, P700 photooxidation measurements, photochemical activities of both photosystems, kinetic parameters of oxygen evolution, oxidative stress markers (MDA, H₂O₂ and proline), pigment content, growth parameters and Cd accumulation. Data revealed that the application C. vulgaris not only stimulates growth and improves the functions of photosynthetic apparatus under physiological conditions, but also reduces the toxic effect of Cd on rice seedlings. Furthermore, the presence of the green microalgae in the nutrient solution of the rice seedlings during Cd exposure, significantly improved the growth, photochemical activities of both photosystems, the kinetic parameters of the oxygen-evolving reactions, pigment content and decreased lipid peroxidation, H₂O₂ and proline content. Data showed that the alleviation of Cd-induced effects in rice seedlings is a result of the Cd sorption by microalgae, as well as the reduced Cd accumulation in the roots and its translocation from the roots to the shoots.     

Analysis of copper corrosion in compacted bentonite clay as a function of clay density and growth conditions for sulfate‐reducing bacteria

Aims: To investigate the relationships between sulfate‐reducing bacteria (SRB), growth conditions, bentonite densities and copper sulfide generation under circumstances relevant to underground, high‐level radioactive waste repositories. Methods and Results: Experiments took place 450 m underground, connected under in situ pressure to groundwater containing SRB. The microbial reduction of sulfate to sulfide and subsequent corrosion of copper test plates buried in compacted bentonite were analysed using radioactive sulfur (³⁵SO₄^2?) as tracer. Mass distribution of copper sulfide on the plates indicated a diffusive process. The relationship between average diffusion coefficients (D_s) and tested density (ρ) was linear. D_s (m² s^?1) = ?0·004 × ρ (kg m^?3) + 8·2, decreasing by 0·2 D_s units per 50 kg m^?3 increase in density, from 1·2 × 10^?11 m² s^?1 at 1750 kg m^?3 to 0·2 × 10^?11 m² s^?1 at 2000 kg m^?3. Conclusions: It is possible that sulfide corrosion of waste canisters in future radioactive waste repositories depends mainly on sulfide concentration at the boundary between groundwater and the buffer, which in turn depends on SRB growth conditions (e.g., sulfate accessibility, carbon availability and electron donors) and geochemical parameters (e.g., presence of ferrous iron, which immobilizes sulfide). Maintaining high bentonite density is also important in mitigating canister corrosion. Significance and Impact of the Study: The sulfide diffusion coefficients can be used in safety calculations regarding waste canister corrosion. The work supports findings that microbial activity in compacted bentonite will be restricted. The study emphasizes the importance of growth conditions for sulfate reduction at the groundwater boundary of the bentonite buffer and linked sulfide production.     

The use of magnesium peroxide for the inhibition of sulfate-reducing bacteria under anoxic conditions

Sulfate-reducing bacteria (SRB), which cause microbiologically influenced material corrosion under anoxic conditions, form one of the major groups of microorganisms responsible for the generation of hydrogen sulfide. In this study, which is aimed at reducing the presence of SRB, a novel alternative approach involving the addition of magnesium peroxide (MgO₂) compounds involving the use of reagent-grade MgO₂ and a commercial product (ORC™) was evaluated as a means of inhibiting SRB in laboratory batch columns. Different concentrations of MgO₂ were added in the columns when black sulfide sediment had appeared in the columns. The experimental results showed that MgO₂ is able to inhibit biogenic sulfide. The number of SRB, the sulfide concentration and the sulfate reducing rate (SRR) were decreased. ORC™ as an additive was able to decrease more effectively the concentration of sulfide in water and the SRB-control effect was maintained over a longer time period when ORC™ was used. The level of oxidation–reduction potential (ORP), which has a linear relationship to the sulfide/sulfate ratio, is a good indicator of SRB activity. As determined by fluorescence in-situ hybridization (FISH), most SRB growth was inhibited under increasing amounts of added MgO₂. The concentration of sulfide reflected the abundance of the SRB. Utilization of organic matter greater than the theoretical SRB utilization rate indicated that facultative heterotrophs became dominant after MgO₂ was added. The results of this study could supply the useful information for further study on evaluating the solution to biocorrosion problems in practical situations.     

Experimental evolution reveals nitrate tolerance mechanisms in Desulfovibrio vulgaris

Elevated nitrate in the environment inhibits sulfate reduction by important microorganisms of sulfate-reducing bacteria (SRB). Several SRB may respire nitrate to survive under elevated nitrate, but how SRB that lack nitrate reductase survive to elevated nitrate remains elusive. To understand nitrate adaptation mechanisms, we evolved 12 populations of a model SRB (i.e., Desulfovibrio vulgaris Hildenborough, DvH) under elevated NaNO₃ for 1000 generations, analyzed growth and acquired mutations, and linked their genotypes with phenotypes. Nitrate-evolved (EN) populations significantly (p < 0.05) increased nitrate tolerance, and whole-genome resequencing identified 119 new mutations in 44 genes of 12 EN populations, among which six functional gene groups were discovered with high mutation frequencies at the population level. We observed a high frequency of nonsense or frameshift mutations in nitrosative stress response genes (NSR: DVU2543, DVU2547, and DVU2548), nitrogen regulatory protein C family genes (NRC: DVU2394-2396, DVU2402, and DVU2405), and nitrate cluster (DVU0246-0249 and DVU0251). Mutagenesis analysis confirmed that loss-of-functions of NRC and NSR increased nitrate tolerance. Also, functional gene groups involved in fatty acid synthesis, iron regulation, and two-component system (LytR/LytS) known to be responsive to multiple stresses, had a high frequency of missense mutations. Mutations in those gene groups could increase nitrate tolerance through regulating energy metabolism, barring entry of nitrate into cells, altering cell membrane characteristics, or conferring growth advantages at the stationary phase. This study advances our understanding of nitrate tolerance mechanisms and has important implications for linking genotypes with phenotypes in DvH.Subject terms: Population genetics, Mutation     

The Effect of Silicon on Photosynthesis and Expression of Its Relevant Genes in Rice (Oryza sativa L.) under High-Zinc Stress

The main objectives of this study were to elucidate the roles of silicon (Si) in alleviating the effects of 2 mM zinc (high Zn) stress on photosynthesis and its related gene expression levels in leaves of rice (Oryza sativa L.) grown hydroponically with high-Zn stress. The results showed that photosynthetic parameters, including net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO₂ concentration, chlorophyll concentration and the chlorophyll fluorescence, were decreased in rice exposed to high-Zn treatment. The leaf chloroplast structure was disordered under high-Zn stress, including uneven swelling, disintegrated and missing thylakoid membranes, and decreased starch granule size and number, which, however, were all counteracted by the addition of 1.5 mM Si. Furthermore, the expression levels of Os08g02630 (PsbY), Os05g48630 (PsaH), Os07g37030 (PetC), Os03g57120 (PetH), Os09g26810 and Os04g38410 decreased in Si-deprived plants under high-Zn stress. Nevertheless, the addition of 1.5 mM Si increased the expression levels of these genes in plants under high-Zn stress at 72 h, and the expression levels were higher in Si-treated plants than in Si-deprived plants. Therefore, we conclude that Si alleviates the Zn-induced damage to photosynthesis in rice. The decline of photosynthesis in Zn-stressed rice was attributed to stomatal limitation, and Si activated and regulated some photosynthesis-related genes in response to high-Zn stress, consequently increasing photosynthesis.     

Fe2O3 nanoparticles induced biochemical responses and expression of genes involved in rosmarinic acid biosynthesis pathway in Moldavian balm under salinity stress

The effect of iron oxide nanoparticle (NP) at four concentrations (0, 30, 60 and 90 ppm) and salinity at three levels (0, 50 and 100 mM) were investigated on rosmarinic acid (RA) production in 5-week-old Moldavian balm (Dracocephalum moldavica L.) plants. Salinity and spraying iron oxide NPs significantly affected tyrosine (Tyr), phenylalanine (Phe) and proline (Pro) amino acids content, Phenylalanine Ammonia-Lyase (PAL), Tyrosine Aminotransferase (TAT) and Rosmarinic Acid Synthase (RAS) genes expression levels, RA content, Polyphenol Oxidase (PPO), PAL and Superoxide Dismutase (SOD) activities, malondialdehyde (MDA) content and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity. PAL, TAT and RAS genes expression rate and content of RA were enhanced in Moldavian balm plants exposed by NaCl + NPs. The results of high performance liquid chromatography (HPLC) revealed that simultaneous application of 50 mM NaCl and 90 ppm NPs increases the RA content in leaf by 81.15% as compared to control plants. The Tyr and Phe contents decreased in Moldavian balm plants exposed to salt stress. Application of NPs had a positive effect on the content of these amino acids. Proline content increased under salinity stress and application of iron NPs induced a significant increase in the Pro content of leaf. The results revealed that PAL, PPO and SOD enzymes activities increased under salinity conditions. The highest activity of PPO and SOD was observed in 100 mM NaCl + 60 ppm NPs treatment. Simultaneous application of 100 mM NaCl + 90 ppm NPs increased the MDA content and DPPH radical scavenging activity compared to control plants. It can be concluded that the application of appropriate levels of NPs moderates the effect of salinity stress in D. moldavica L. and results in an increased amount of RA compared to control plants.     

The cotton endocycle‐involved protein SPO11‐3 functions in salt stress via integrating leaf stomatal response,ROS scavenging and root growth

The SPORULATION 11 (SPO11) proteins are among eukaryotic the topoisomerase VIA (Topo VIA) homologs involved in modulating various important biological processes, such as growth, development and stress response via endoreduplication in plants, but the underlying mechanism response to stress remains largely unknown under salt treatment. Here, we attempted to characterize a homolog of TOP VIA in upland cotton (Gossypium hirsutum L.), designated as GhSPO11‐3. The silencing of GhSPO11‐3 in cotton plants resulted in a dwarf phenotype with a failure of cell endoreduplication and a phase shift in the ploidy levels. The GhSPO11‐3‐silenced plants also showed substantial changes including accumulated malondialdehyde, significantly reduced chlorophyll and proline contents and decreased antioxidative enzyme activity after salt treatment. In addition, transgenic Arabidopsis lines overexpressing GhSPO11‐3 accelerated both leaf and root growth with cell expansion and endopolyploidy. Both leaf stomatal density and aperture were markedly decreased, and the transgenic Arabidopsis lines were more tolerant with expression of stress‐responsive genes under salinity stress. Furthermore, consistent with the reduced reactive oxygen species (ROS), the expression of ROS scavenging‐related genes was largely reinforced, and antioxidant enzyme activities were accordingly significantly enhanced in transgenic Arabidopsis lines under salt stress. In general, these results indicated that GhSPO11‐3 likely respond to salt stress by positively regulating root growth, stomatal response, ROS production and the expression of stress‐related genes to cope with adverse conditions in plants.     

Expression of ferritin gene in <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> plants under different supply with iron and different intensity of oxidative stress

In plants of the facultative halophyte Mesembryanthemum crystallinum L. cultivated under climate-controlled conditions, expression of one of ferritin genes, McFer, the ortholog of arabidopsis AtFer1 gene was studied for the first time. The level of this gene expression occurring in response to oxidative stress and changes in the iron status was similar to that of AtFer1 gene. A dependence of McFer gene expression and ferritin content on the regime of plant supplying with Fe-EDTA on the background of medium salinity (300 mM NaCl), oxidative stress modeling by leaf treatment with paraquat (PQ, 100 μM), or in the presence of antioxidant spermidine (Spd, 1 mM) was analyzed. The level of gene expression was assessed by RT-PCR, whereas the content of ferritin by Western blotting, using the primary polyclonal antibody against pea ferritin. An enhanced production of superoxide radical and hydrogen peroxide at leaf treatment with PQ activated gene expression and ferritin content, whereas ROS scavenging with the antioxidant Spd suppressed gene expression. It is concluded that ferritin deposits in the halophyte M. crystallinum, which we have observed earlier in the chloroplasts of the mesophyll and parenchyma of the vascular system, fulfill not only storage but also protective role by binding the excessive Fe²⁺, a catalyzer of OH^·− production.     

Dinitrophenol influences the rate and yield of Acetobacter methanolicus during the growth on glucose

Acetobacter methanolicuswas grown on glucose in the presence of dinitrophenol (DNP) under carbon/energy-limited conditions. DNP affected both the growth yield and the growth rate (D_sh) at which the energy generation was shifted from a complete to an incomplete substrate oxidation by using the PQQ-linked glucose dehydrogenase. The more the growth yield was decreased, the higher both the DNP concentration and the growth rate became. At about 0.53 mM DNP, growth was completely stopped. D_sh decreased from 0.21h^?1in the absence of DNP to 0.175 h^?1and 0.075 h^?1in the presence of 0.2 mM and 0.4 mM DNP, respectively. The experimental data are discussed in terms of the limitations in the generation of energy and some stress situations which are exerted by the presence of the uncoupler.     

Microbial reduction of structural iron in interstratified illite-smectite minerals by a sulfate-reducing bacterium

Clay minerals are ubiquitous in soils, sediments, and sedimentary rocks and could coexist with sulfate‐reducing bacteria (SRB) in anoxic environments, however, the interactions of clay minerals and SRB are not well understood. The objective of this study was to understand the reduction rate and capacity of structural Fe(III) in dioctahedral clay minerals by a mesophilic SRB, Desulfovibrio vulgaris and the potential role in catalyzing smectite illitization. Bioreduction experiments were performed in batch systems, where four different clay minerals (nontronite NAu‐2, mixed‐layer illite‐smectite RAr‐1 and ISCz‐1, and illite IMt‐1) were exposed to D. vulgaris in a non‐growth medium with and without anthraquinone‐2,6‐disulfonate (AQDS) and sulfate. Our results demonstrated that D. vulgaris was able to reduce structural Fe(III) in these clay minerals, and AQDS enhanced the reduction rate and extent. In the presence of AQDS, sulfate had little effect on Fe(III) bioreduction. In the absence of AQDS, sulfate increased the reduction rate and capacity, suggesting that sulfide produced during sulfate reduction reacted with the phyllosilicate Fe(III). The extent of bioreduction of structural Fe(III) in the clay minerals was positively correlated with the percentage of smectite and mineral surface area of these minerals. X‐ray diffraction, and scanning and transmission electron microscopy results confirmed formation of illite after bioreduction. These data collectively showed that D. vulgaris could promote smectite illitization through reduction of structural Fe(III) in clay minerals.