Exploiting the <Emphasis Type="Italic">ZIP4</Emphasis> homologue within the wheat <Emphasis Type="Italic">Ph1</Emphasis> locus has identified two lines exhibiting homoeologous crossover in wheat-wild relative hybrids

Despite possessing related ancestral genomes, hexaploid wheat behaves as a diploid during meiosis. The wheat Ph1 locus promotes accurate synapsis and crossover of homologous chromosomes. Interspecific hybrids between wheat and wild relatives are exploited by breeders to introgress important traits from wild relatives into wheat, although in hybrids between hexaploid wheat and wild relatives, which possess only homoeologues, crossovers do not take place during meiosis at metaphase I. However, in hybrids between Ph1 deletion mutants and wild relatives, crossovers do take place. A single Ph1 deletion (ph1b) mutant has been exploited for the last 40 years for this activity. We show here that chemically induced mutant lines, selected for a mutation in TaZIP4-B2 within the Ph1 locus, exhibit high levels of homoeologous crossovers when crossed with wild relatives. Tazip4-B2 mutant lines may be more stable over multiple generations, as multivalents causing accumulation of chromosome translocations are less frequent. Exploitation of such Tazip4-B2 mutants, rather than mutants with whole Ph1 locus deletions, may therefore improve introgression of wild relative chromosome segments into wheat.     

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.     

Repair of cisplatin-DNA adducts in mutants for genes controlling spontaneous and induced mutagenesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeast

Sensitivity to the lethal action of the anticancer substance cisplatin was studied in the yeast mutants him1, hsm2, hsm3, and hsm6, deficient for repair of spontaneous and induced mutations. The him1 and hsm3 mutants were as resistant to the agent under study as the wild-type strain. The survival of the double mutant rad2 hsm3 was higher than that of the single mutant rad2. The hsm2 and hsm6 mutants were more cisplatin-sensitive than the wild type. Cisplatin was shown to have high mutagenic and recombinogenic effects on yeast cells.     

Proteomics analysis of global regulatory cascades involved in clavulanic acid production and morphological development in <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>

The genus Streptomyces comprises bacteria that undergo a complex developmental life cycle and produce many metabolites of importance to industry and medicine. Streptomyces clavuligerus produces the β-lactamase inhibitor clavulanic acid, which is used in combination with β-lactam antibiotics to treat certain β-lactam resistant bacterial infections. Many aspects of how clavulanic acid production is globally regulated in S. clavuligerus still remains unknown. We conducted comparative proteomics analysis using the wild type strain of S. clavuligerus and two mutants (ΔbldA and ΔbldG), which are defective in global regulators and vary in their ability to produce clavulanic acid. Approximately 33.5 % of the predicted S. clavuligerus proteome was detected and 192 known or putative regulatory proteins showed statistically differential expression levels in pairwise comparisons. Interestingly, the expression of many proteins whose corresponding genes contain TTA codons (predicted to require the bldA tRNA for translation) was unaffected in the bldA mutant.     

Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.     

Dot1 and Set2 histone methylases control the spontaneous and UV-induced mutagenesis levels in the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeasts

In the Saccharomyces cerevisiae yeasts, the DOT1 gene product provides methylation of lysine 79 (K79) of histone H3 and the SET2 gene product provides the methylation of lysine 36 (K36) of the same histone. We determined that the dot1 and set2 mutants suppress the UV-induced mutagenesis to an equally high degree. The dot1 mutation demonstrated statistically higher sensitivity to the low doses of MMC than the wild type strain. The analysis of the interaction between the dot1 and rad52 mutations revealed a considerable level of spontaneous cell death in the double dot1 rad52 mutant. We observed strong suppression of the gamma-induced mutagenesis in the set2 mutant. We determined that the dot1 and set2 mutations decrease the spontaneous mutagenesis rate in both single and double mutants. The epistatic interaction between the dot1 and set2 mutations and almost similar sensitivity of the corresponding mutants to the different types of DNA damage allow one to conclude that both genes are involved in the control of the same DNA repair pathways, the homologous-recombination-based and the postreplicative DNA repair.     

Non-capsulated mutants of a chemical-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> strain

Objectives

To investigate the outcomes of capsule lost on cell transformation efficiency and chemicals (1,3-propanediol, 2,3-butanediol, and 2-ketogluconic acid) production by Klebsiella pneumoniae.

Results

The cps gene cluster showed low sequence homology with pathogenic strains. The wza is a highly conserved gene in the cps cluster that encodes an outer membrane protein. A non-capsulated mutant was constructed by deletion of wza. Phenotype studies demonstrated that non-capsulated cells were less buoyant and easy to sediment. The transformation efficiency of the non-capsulated mutant reached 6.4 × 10⁵ CFU μg^?1 DNA, which is 10 times higher than that of the wild strain. 52.2 g 1,3-propanediol L^?1, 30.7 g 2,3-butanediol L^?1, and 175.9 g 2-ketogluconic acid L^?1 were produced by non-capsulated mutants, which were 10–40% lower compared to wild strain. Furthermore, viscosities of the three fermentation broths decreased to approximately 1.3 cP from the range of 1.8–2.2 cP.

Conclusions

Non-capsulated K. pneumoniae mutants should allay concerns regarding biological safety, improve transformation efficiency, lower viscosity, and subsequently ameliorate the financial burden of the downstream process of chemicals production.

     

A rice <Emphasis Type="Italic">White-stripe leaf3</Emphasis> (<Emphasis Type="Italic">wsl3</Emphasis>) mutant lacking an HD domain-containing protein affects chlorophyll biosynthesis and chloroplast development

Leaf-color mutants are ideal genetic materials for understanding the mechanism of chloroplast development and chlorophyll (Chl) biosynthesis. Here we isolated and identified a new leaf-color mutant of rice, named white-stripe leaf3 (wsl3), from a ⁶⁰Co-irradiated mutant pool. The wsl3 mutant displayed a visible white-stripe leaf in both young seedlings and flag leaves of mature plant. Chl content in homozygous wsl3 mutant was approximately 47% of that in the wild type. Besides, chloroplast development in the mutant was severely arrested. By a map-based cloning strategy, the wsl3 gene was finely confined to a 50.8 kb region on chromosome 1. Moreover, a 9-bp deletion was identified in the genomic region of LOC_Os01g01920, which encodes an HD (histidine and aspartic acid) domaincontaining protein. Genetic complementation confirmed that LOC_Os01g01920 could recover the lesion of wsl3 mutation. Real-time PCR analyses showed that the expression levels of WSL3 were the highest in young and flag leaves among various tissues, and most of the genes associated with Chl biosynthesis were significantly down-regulated in the wsl3 mutant. Meanwhile, in contrast to many nuclear gene-encoded phage-type RNA polymerase(s) (NEP) transcribed genes were up-regulated, most of plastid-encoded bacterialtype RNA polymerase (PEP) transcribed genes were downregulated. These results demonstrated that the WSL3 gene, as an HD domain-containing protein, is involved in chl biosynthesis and chloroplast development in rice.     

Mutational analysis of substrate specificity in a <Emphasis Type="Italic">Citrus paradisi</Emphasis> flavonol 3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

Citrus paradisi 3-O-glucosyltransferase (Cp3GT, Genbank Protein ID: ACS15351) and Citrus sinensis 3-O-glucosyltransferase (Cs3GT, Genbank Protein ID: AAS00612.2) share 95% amino acid sequence identity. Cp3GT was previously established as a flavonol-specific 3-O-glucosyltransferase by direct enzymatic analysis. Cs3GT is annotated as a flavonoid-3-O-glucosyltransferase and predicted to use anthocyanidins as substrates based on gene expression analysis correlated with the accumulation of anthocyanins in C. sinensis cv. Tarocco, a blood orange variety. Mutant enzymes in which amino acids found in Cs3GT were substituted for position equivalent residues in Cp3GT were generated, heterologously expressed in yeast, and characterized for substrate specificity. Structure–function relationships were investigated for wild type and mutant glucosyltransferases by homology modelling using a crystallized Vitis vinifera anthocyanidin/flavonol 3-O-GT (PDB: 2C9Z) as template and subsequent substrate docking. All enzymes showed similar patterns for optimal temperature, pH, and UDP/metal ion inhibition with differences observed in kinetic parameters. Although changes in the activity of the mutant proteins as compared to wild type were observed, cyanidin was never efficiently accepted as a substrate.     

Co-expression of Beta-Glucosidase and Laccase in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> by Random Insertion with Enhanced Filter Paper Activity

Trichoderma reesei strain Rut-C30 was modified with enhanced beta-glycosidase (BGL) activity to balance the cellulase system and generated laccase (LAC) protein for lignin degradation. Initially, the binary plasmid p1300-w1 was constructed to express T. reesei bgl2 under the control of promoter P _pki and T-nos terminator. Random insertion was performed via Agrobacterium tumefaciens-mediated transformation. A total of 353 mutants were obtained, and 34PTrb2 was exceptionally stable with increased FPA and BGL activity after screening for extracellular enzyme activity. Subsequently, 34PTrb2 was used as parent strain via the same method to insert the lac gene from Fomes lignosus, with promoter P _gpd , followed by cbh1 signal peptide trss and T-nos as terminator. Several mutants successfully expressed enzyme LAC with stable activity of approximately 0.13 U/mL. The mutant 15Gsslac increased activity by 40.4% FPA compared with that of the host Rut-C30.     

Analysis the role of arabidopsis <Emphasis Type="Italic">CKRC6/ASA1</Emphasis> in auxin and cytokinin biosynthesis

The crosstalk between auxin and cytokinin (CK) is important for plant growth and development, although the underlying molecular mechanisms remain unclear. Here, we describe the isolation and characterization of a mutant of Arabidopsis Cytokinin-induced Root Curling 6 (CKRC6), an allele of ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) that encodes the á-subunit of AS in tryptophan (Trp) biosynthesis. The ckrc6 mutant exhibits root gravitropic defects and insensitivity to both CK and the ethylene precursor 1-aminocyclopropane-1-carboxylicacid (ACC) in primary root growth. These defects can be rescued by exogenous indole-3-acetic acid (IAA) or tryptophan (Trp) supplementation. Furthermore, our results suggest that the ckrc6 mutant has decreased IAA content, differential expression patterns of auxin biosynthesis genes and CK biosynthesis isopentenyl transferase (IPT) genes in comparison to wild type. Collectively, our study shows that auxin controls CK biosynthesis based on that CK sensitivity is altered in most auxin-resistant mutants and that CKs promote auxin biosynthesis but inhibit auxin transport and response. Our results also suggest that CKRC6/ASA1 may be located at an intersection of auxin, CK and ethylene metabolism and/or signaling.     

The <Emphasis Type="Italic">APX4</Emphasis> locus regulates seed vigor and seedling growth in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The amino acid sequence of APX4 is similar to other ascorbate peroxidases (APXs), a group of proteins that protect plants from oxidative damage by transferring electrons from ascorbate to detoxify peroxides. In this study, we characterized two apx4 mutant alleles. Translational fusions with GFP indicated APX4 localizes to chloroplasts. Both apx4 mutant alleles formed chlorotic cotyledons with significantly reduced chlorophyll a, chlorophyll b and lutein. Given the homology of APX to ROS-scavenging proteins, this result is consistent with APX4 protecting seedling photosystems from oxidation. The growth of apx4 seedlings was stunted early in seedling development. In addition, APX4 altered seed quality by affecting seed coat formation. While apx4 seed development appeared normal, the seed coat was darker and more permeable than the wild type. In addition, accelerated aging tests showed that apx4 seeds were more sensitive to environmental stress than the wild-type seeds. If APX4 affects seed pigment biosynthesis or reduction, the seed coat color and permeability phenotypes are explained. apx4 mutants had cotyledon chlorosis, increased H₂O₂ accumulation, and reduced soluble APX activity in seedlings. These results indicate that APX4 is involved in the ROS-scavenging process in chloroplasts.     

Mutant tRNA<Superscript>His</Superscript> Ineffective in Repression and Lacking Two Pseudouridine Modifications

TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons^1–4. Histidyl-tRNA^His has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium^5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNA^His as the wild type⁴ and in the one example sequenced, contains tRNA^HIS with a structure identical to that of the wild type⁸. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNA^HISin vitro². The amounts of charged and uncharged tRNA^His present in vivo during physiological derepression of the wild type and in the six classes of regulatory mutants, have been determined⁹. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNA^Hisin vivo and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNA^His. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNA^His) and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNA^His which these mutants contain.     

Interaction of gibberellic acid and 24-epibrassinolide in the regulation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seedling scotomorphogenesis

The effects of GA₃, 24-epibrassinolide (EBL), and their combination on morphogenesis of Arabidopsis thaliana (L.) Heynh seven-day-old seedlings were studied. Four plant lines were analyzed: wild type Ler and ga4-1 mutant, belonging to the Landsberg erecta ecotype and wild type Col and det2 mutant, both of the Columbia ecotype. In ga4-1 and det2, GA_4/1-and brassinosteroid-deficient mutants, the highest hypocotyl growth response to the lack of hormones was noted. The cotyledon shape and size were dependent on EBL, and the root length was both GA₃-and EBL-regulated, indicating organ specificities in the responses to these hormones. Simultaneous treatment of dark-grown plants with GA₃ and EBL exerted an additive stimulatory effect on the root growth of det2, reduced the inhibitory effect of EBL on hypocotyl elongation of ga4-1, and enhanced the effect of EBL on hypocotyl and cotyledon elongation of det2.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

Functional Characterization of Potential Probiotic Lactic Acid Bacteria Isolated from <Emphasis Type="Italic">Kalarei</Emphasis> and Development of Probiotic Fermented Oat Flour

Considerable variations among probiotics with respect to their health benefitting attributes fuel the research on bioprospecting of proficient probiotic strains from various ecological niches especially the poorly unexplored ones. In the current study, kalarei, an indigenous cheese-like fermented milk product, and other dairy-based sources like curd and raw milk were used for isolation of lactic acid bacteria (LAB). Among 34 LAB isolates, 7 that could withstand simulated gastrointestinal (GI) conditions were characterized for functional probiotic attributes, viz. adhesion ability, aggregation and coaggregation, extracellular enzyme producing capability, antibacterial activity against pathogens and antibiotic resistance. The isolate M-13 (from kalarei) which exhibited most of the desirable probiotic functional properties was identified as Lactobacillus plantarum based on 16S ribosomal DNA sequence analysis and designated as L. plantarum M-13. The sequence was submitted to GenBank (accession number KT592509). The study presents the first ever report of isolation of potential probiotic LAB, i.e. L. plantarum M-13 from indigenous food kalarei, and its application for development of potential probiotic fermented oat flour (PFOF). PFOF was analysed for parameters like viability of L. plantarum M-13, acidity and pH. Results show that PFOF serves as a good matrix for potential probiotic L. plantarum M-13 as it supported adequate growth of the organism (14.4 log cfu/ml after 72 h of fermentation). In addition, appreciable acid production by L. plantarum M-13 and consequential pH reduction indicates the vigorous and active metabolic status of the potential probiotic organism in the food matrix. Thus, study shows that fermented oat flour may possibly be developed as a potential probiotic carrier especially in view of the problems associated with dairy products as probiotic vehicles.     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

Isocitrate dehydrogenase mutation in <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> results in virulence attenuation and immunoprotection in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss)</Emphasis>

Background

Vibrio anguillarum is an extracellular bacterial pathogen that is a causative agent of vibriosis in finfish and crustaceans with mortality rates ranging from 30% to 100%. Mutations in central metabolism (glycolysis and the TCA cycle) of intracellular pathogens often result in attenuated virulence due to depletion of required metabolic intermediates; however, it was not known whether mutations in central metabolism would affect virulence in an extracellular pathogen such as V. anguillarum.

Results

Seven central metabolism mutants were created and characterized with regard to growth in minimal and complex media, expression of virulence genes, and virulence in juvenile rainbow trout (Oncorhynchus mykiss). Only the isocitrate dehydrogenase (icd) mutant was attenuated in virulence against rainbow trout challenged by either intraperitoneal injection or immersion. Further, the icd mutant was shown to be immunoprotective against wild type V. anguillarum infection. There was no significant decrease in the expression of the three hemolysin genes detected by qRT-PCR. Additionally, only the icd mutant exhibited a significantly decreased growth yield in complex media. Growth yield was directly related to the abundance of glutamate. A strain with a restored wild type icd gene was created and shown to restore growth to a wild type cell density in complex media and pathogenicity in rainbow trout.

Conclusions

The data strongly suggest that a decreased growth yield, resulting from the inability to synthesize α-ketoglutarate, caused the attenuation despite normal levels of expression of virulence genes. Therefore, the ability of an extracellular pathogen to cause disease is dependent upon the availability of host-supplied nutrients for growth. Additionally, a live vaccine strain could be created from an icd deletion strain.