Cloning and characterization of the new multiple stress responsible gene I (<Emphasis Type="Italic">MuSI</Emphasis>) from sweet potato

New multiple-stress related gene isolated from sweet potato and designated it as MusI (multiple stress responsible gene I). Sequence analysis revealed that its full length cDNA was 998 bp long and included a 717 bp open reading frame encoding for 238 amino acids. Comparison of its cDNA and genomic DNA sequence showed that 3 exons were divided by 2 introns in its ORF region. Its deduced amino acid sequence contained a conserved rubber elongation factor (REF) domain and showed high homology with many stress-related proteins. Therefore, it was named MuSI (multiple stress responsible gene I). Southern hybridization analysis indicated that the MuSI gene may belong to a multi-gene family. Expression pattern of the MuSI gene showed that it was differently expressed among roots, stems, leaves, and flowers of a sweet potato, and its expression level was especially high in flowers and white fibrous roots. Its expression was also highly induced by various stress signals including dehydration, high salt, heavy metal, oxidation, and plant hormones. Stress tolerance experiment using transgenic plants overexpressing the MuSI gene showed that all independent transgenic tobacco lines have enhanced tolerance to high temperature stress. Among them, transgenic line 6 particularly showed tolerance to salt, heavy metal, and osmotic stress as well. These results suggest that the MuSI gene functions as a positive regulator of various stress responses and may be useful in improving stress tolerance of transgenic plants.     

Heavy metal accumulation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells armed with metal binding hexapeptides targeted to the inner face of the plasma membrane

Accumulation of heavy metals without developing toxicity symptoms is a phenotype restricted to a small group of plants called hyperaccumulators, whose metal-related characteristics suggested the high potential in biotechnologies such as bioremediation and bioextraction. In an attempt to extrapolate the heavy metal hyperaccumulating phenotype to yeast, we obtained Saccharomyces cerevisiae cells armed with non-natural metal-binding hexapeptides targeted to the inner face of the plasma membrane, expected to sequester the metal ions once they penetrated the cell. We describe the construction of S. cerevisiae strains overexpressing metal-binding hexapeptides (MeBHxP) fused to the carboxy-terminus of a myristoylated green fluorescent protein (myrGFP). Three non-toxic myrGFP-MeBHxP (myrGFP-H6, myrGFP-C6, and myrGFP-(DE)3) were investigated against an array of heavy metals in terms of their effect on S. cerevisiae growth, heavy metal (hyper) accumulation, and capacity to remove heavy metal from contaminated environments.     

The <Emphasis Type="Italic">yajC</Emphasis> gene from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and its role in ethanol tolerance

The yajC gene (Lbuc_0921) from Lactobacillus buchneri NRRL B-30929 was identified from previous proteomics analyses in response to ethanol treatment. The YajC protein expression was increased by 15-fold in response to 10 % ethanol vs 0 % ethanol. The yajC gene encodes the smaller subunit of the preprotein translocase complex, which interacts with membrane protein SecD and SecF to coordinate protein transport and secretion across cytoplasmic membrane in Escherichia coli. The YajC protein was linked to sensitivity to growth temperatures in E. coli, involved in translocation of virulence factors during Listeria infection, and stimulating a T cell-mediated response of Brucella abortus. In this study, the L. buchneri yajC gene was over-expressed in E. coli. The strain carrying pET28byajC that produces YajC after isopropyl β-d-1-thiogalactopyranoside induction showed tolerance to 4 % ethanol in growth media, compared to the control carrying pET28b. This is the first report linking YajC to ethanol stress and tolerance.     

Toxic metals accumulation in <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> and <Emphasis Type="Italic">T. harzianum</Emphasis>

Heavy metal contamination represents an important environmental issue due to the toxic effects of metals on different organisms. Filamentous fungi play an important impact in the bioremediation of heavy metal-contaminated wastewater and soil. The purpose of this investigation was to observe fungal uptake behavior toward heavy metal. For this aim Trichoderma asperellum TS141 and T. harzianum TS103 at growth period were screened for their tolerance and uptake capability of cadmium (Cd), lead (Pb) and nickel (Ni) at different concentrations (0, 25, 50, 100, and 200 mg/L) in PDB media (potato dextrose broth as a complex medium). Results showed that both fungi were able to survive at the maximum concentration of 200 mg/L of the heavy metals, and remove them. T. asperellum had a better uptake capacity for Cd compared to Pb and Ni in the highest metal concentration in media. Maximum removal efficiency of Pb (68.4%) at 100 mg/L and Ni (78%) at 200 mg/L was performed by T. asperellum. For Cd, the highest removal efficiency (82.1%) was recorded by T. harzianum at 200 mg/L Cd in aqueous solution. The uptake of Cd was highly dependent on pH of solution than Pb and Ni so that the optimal pH of Cd uptake was 9 for T. asperellum and 4 for T. harzianum. Also, optimal temperature was 35°C for Cd and Pb uptake in both fungi, whereas for Ni uptake was 30 and 35°C in T. harzianum and T. asperellum, respectively. We propose that T. asperellum TS141 and T. harzianum TS103 can be used as a bioremediation agent for metal remediation from wastewater and heavy metal-contaminated soils.     

Molecular cloning and functional characterization of a Cu/Zn superoxide dismutase gene (<Emphasis Type="Italic">CsCSD1</Emphasis>) from <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Superoxide dismutase (SOD) proteins, which are widely present in the plant kingdom, play vital roles in response to abiotic stress. However, the functions of cucumber SOD genes in response to environmental stresses remain poorly understood. In this study, a SOD gene CsCSD1 was identified and functionally characterized from cucumber (Cucumis sativus). The CsCSD1 protein was successfully expressed in E. coli, and its overexpression significantly improved the tolerance of host E. coli cells to salinity stress. Besides, overexpression of CsCSD1 enhanced salinity tolerance during germination and seedling development in transgenic Arabidopsis plants. Further analyses showed that the SOD and CAT (catalase) activities of transgenic plants were significantly higher than those of wild-type (WT) plants under normal growth conditions as well as under NaCl treatment. In addition, the expression of stress-response genes RD22, RD29B and LEA4-5 was significantly elevated in transgenic plants. Our results demonstrate that the CsCSD1 gene functions in defense against salinity stress and may be important for molecular breeding of salt-tolerant plants.     

Overexpression of a Common Wheat Gene <Emphasis Type="Italic">GALACTINOL SYNTHASE3</Emphasis> Enhances Tolerance to Zinc in <Emphasis Type="Italic">Arabidopsis</Emphasis> and Rice Through the Modulation of Reactive Oxygen Species Production

Galactinol synthase (GolS, EC 2.4.1.123), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), plays roles in plant growth and developmental processes. The in vitro roles of GolS in plant responses against heavy metal stress are not well clarified. In the present study, a suppression-subtractive hybridization (SSH) cDNA library has been constructed using RNA extracted from wheat cultivar Jinan 18 treated with ZnCl₂ as the tester and RNA from untreated seedlings as the driver. Sixteen expressed sequence tags (ESTs) highly homologous with known proteins associated with stress tolerance have been obtained. Among these, a 1000-bp cDNA sequence encoding GolS protein has been isolated and designated as TaGolS3. Real-time quantitative PCR (qPCR) analysis revealed that TaGolS3 was mainly expressed in young roots and upregulated by exogenous ABA treatment and several abiotic stresses, such as ZnCl₂, CuCl₂, low temperature, and NaCl. Subcellular localization analysis showed that TaGolS3 protein is a nuclear-localized protein. A detailed analysis of Arabidopsis and rice transgenic plants overexpressing TaGolS3 gene displayed that transgenic plants exhibited increased lateral root number, primary root length, plant survival rate, and plant height. Moreover, in comparison with the wild-type (WT) plants, the TaGolS3-overexpressing lines showed a higher expression of ROS-scavenging genes, activities of antioxidative enzymes, proline contents, and a lower level of malondialdehyde (MDA) contents and electrolyte leakage under zinc stress. These results confirmed the positive roles of TaGolS3 in improving plant tolerance to heavy metal stress, indicating a potential resource in the transgenic breeding to enhance heavy metal stress tolerance in crop plants.     

Improved acid-stress tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000 and <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 by overexpression of the anti-acid component <Emphasis Type="Italic">recT</Emphasis>

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.     

Cloning of <Emphasis Type="Italic">rec</Emphasis>A gene of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and phenotypic complementation of <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM^®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm², while non-transformed cells tolerated only up to dose 0.08 J/cm². It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.     

<Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> RecX and <Emphasis Type="Italic">Escherichia coli</Emphasis> RecX proteins are capable to replace each other in vivo and in vitro

A plasmid carrying the Deinococcus radiodurans recX gene under the control of a lactose promoter decreases the Escherichia coli cell resistance to UV irradiation and γ irradiation and also influences the conjugational recombination process. The D. radiodurans RecX protein functions in the Escherichia coli cells similarly to the E. coli RecX protein. Isolated and purified D. radiodurans RecX and E. coli RecX proteins are able to replace each other interacting with the E. coli RecA and D. radiodurans RecA proteins in vitro. Data obtained demonstrated that regulatory interaction of RecA and RecX proteins preserves a high degree of conservatism despite all the differences in the recombination reparation system between E. coli and D. radiodurans.     

A novel heavy metal ATPase peptide from <Emphasis Type="Italic">Prosopis juliflora</Emphasis> is involved in metal uptake in yeast and tobacco

Heavy metal pollution of agricultural soils is one of the most severe ecological problems in the world. Prosopis juliflora, a phreatophytic tree species, grows well in heavy metal laden industrial sites and is known to accumulate heavy metals. Heavy Metal ATPases (HMAs) are ATP driven heavy metal pumps that translocate heavy metals across biological membranes thus helping the plant in heavy metal tolerance and phytoremediation. In the present study we have isolated and characterized a novel 28.9 kDa heavy metal ATPase peptide (PjHMT) from P. juliflora which shows high similarity to the C-terminal region of P_1B ATPase HMA1. It also shows the absence of the invariant signature sequence DKTGT, and the metal binding CPX motif but the presence of conserved regions like MVGEGINDAPAL (ATP binding consensus sequence), HEGGTLLVCLNS (metal binding domain) and MLTGD, GEGIND and HEGG motifs which play important roles in metal transport or ATP binding. PjHMT, was found to be upregulated under cadmium and zinc stress. Heterologous expression of PjHMT in yeast showed a higher accumulation and tolerance of heavy metals in yeast. Further, transgenic tobacco plants constitutively expressing PjHMT also showed increased accumulation and tolerance to cadmium. Thus, this study suggests that the transport peptide from P. juliflora may have an important role in Cd uptake and thus in phytoremediation.     

Improved Tolerance of <Emphasis Type="Italic">Escherichia coli</Emphasis> to Propionic Acid by Overexpression of Sigma Factor RpoS

Propionic acid (PA) is an economically important compound, but large-scale microbial production of PA confronts obstacle such as acid stress on microbial cells. Here, we show that overexpressing sigma factor RpoS improves the acid tolerance of Escherichia coli. Four genes including rpoS, fur, pgi and dnaK (encoding RNA polymerase sigma factor, ferric uptake regulator, phosphoglucoisomerase, and chaperone, respectively) were independently overexpressed in E. coli. The recombinant E. coli overexpressing rpoS showed the highest PA tolerance. This strain could grow in M9 medium at pH 4.62, whereas wild type E. coli survived only at pHs above 5.12. Moreover, in the shake-flask cultivation, the E. coli strain overexpressing rpoS grew faster than wild type. Notably, the minimum inhibitory concentration of PA for this recombinant strain was 7.81 mg/mL, which was 2-fold higher in comparison with wild type. Overall these results indicated that overexpression of sigma factor rpoS significantly enhanced E. coli tolerance to PA.     

Over-expression of tobacco <Emphasis Type="Italic">UBC1</Emphasis> encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca²⁺/H⁺ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.     

A single gene <Emphasis Type="Italic">all3940</Emphasis> (Dps) overexpression in <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120 confers multiple abiotic stress tolerance via proteomic alterations

DNA-binding proteins (Dps) induced during starvation play an important role in gene regulation and maintaining homeostasis in bacteria. The nitrogen-fixing cyanobacterium, Anabaena PCC7120, has four genes annotated as coding for Dps; however, the information on their physiological roles is limiting. One of the genes coding for Dps, ‘all3940’ was found to be induced under different abiotic stresses in Anabaena and upon overexpression enhanced the tolerance of Anabaena to a multitude of stresses, which included salinity, heat, heavy metals, pesticide, and nutrient starvation. On the other hand, mutation in the gene resulted in decreased growth of Anabaena. The modulation in the levels of All3940 in Anabaena, achieved either by overexpression of the protein or mutation of the gene, resulted in changes in the proteome, which correlated well with the physiological changes observed. Proteins required for varied physiological activities, such as photosynthesis, carbon-metabolism, oxidative stress alleviation, exhibited change in protein profile upon modulation of All3940 levels in Anabaena. This suggested a direct or an indirect effect of All3940 on the expression of the above stress-responsive proteins, thereby enhancing tolerance in Anabaena PCC7120. Thus, All3940, though categorized as a Dps, is possibly a general stress protein having a global role in regulating tolerance to multitude of stresses in Anabaena.     

Gene <Emphasis Type="Italic">yddG</Emphasis> of <Emphasis Type="Italic">Escherichia coli</Emphasis> encoding the putative exporter of aromatic amino acids: Constitutive transcription and dependence of the expression on the cell growth rate

Gene yddG of Escherichia coli encodes a protein of the inner membrane. Data obtained earlier demonstrated that under conditions of aromatic amino acids overproduction YddG promotes their export from E. coli cells. In this work, a method of primer extension was used to localize the P _yddG promoter, which corresponds to E. coli promoters recognized by RNA polymerase in complex with σ⁷⁰ or σ^S subunits. By constructing a gene of the hybrid protein YddG’-LacZ at the intrinsic site of gene yddG location in the E. coli chromosome and analyzing the activity of β-galactosidase in cells growing on laboratory media LB and M9, the constitutive type of yddG expression at a low level was demonstrated (the activity was about 3 to 4% of the LacZ level under induction of the lac operon in E. coli wild-type cells). The expression of yddG had a twofold increase under conditions of retarded cell growth upon the stress caused by the high NaCl content (0.6 M) or by the presence of phenylalanine excess quantities (>1 mM) in the culture medium.