<Emphasis Type="Italic">bvg</Emphasis>-Negative Regulation of Repeated Sequence Transposition in <Emphasis Type="Italic">Bordetella pertussis</Emphasis> Cells

A method of monitoring the sequential events of IS481 transposition into the ctag site of bvg operon of Bordetella pertussis has been developed. Reproduction of virulent B. pertussis cells in vitro is accompanied by intrachromosomal site-specific IS481 transposition, which, in turn, results in inactivation of bvg operon of the causative agent and cell avirulent state. Avirulent bvg mutants of B. pertussis are incapable of intramolecular IS481 transposition. The frequency of the transposition increases when MgSO₄ and nicotinic acid are present the culture medium. In the absence of these modulating factors, IS481 transposition along B. pertussis chromosome is inhibited but not arrested completely. Negative regulation of the bvg-repressed genes of B. pertussis seems to be a mechanism that controls bvg-dependent IS481 transposition.     

Accumulation of large non-circular forms of the chromosome in recombination-defective mutants of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

Double-strand breakage of chromosomal DNA is obviously a serious threat to cells because various activities of the chromosome depend on its integrity. However, recent experiments suggest that such breakage may occur frequently during "normal" growth in various organisms – from bacteria through vertebrates, possibly through arrest of a replication fork at some endogenous DNA damage.

Results

In order to learn how the recombination processes contribute to generation and processing of the breakage, large (> 2000 kb) linear forms of Escherichia coli chromosome were detected by pulsed-field gel electrophoresis in various recombination-defective mutants. The mutants were analyzed in a rich medium, in which the wild-type strain showed fewer of these huge broken chromosomes than in a synthetic medium, and the following results were obtained: (i) Several recB and recC null mutants (in an otherwise rec⁺ background) accumulated these huge linear forms, but several non-null recBCD mutants (recD, recC1001, recC1002, recC1003, recC1004, recC2145, recB2154, and recB2155) did not. (ii) In a recBC sbcA background, in which RecE-mediated recombination is active, recA, recJ, recQ, recE, recT, recF, recO, and recR mutations led to their accumulation. The recJ mutant accumulated many linear forms, but this effect was suppressed by a recQ mutation. (iii) The recA, recJ, recQ, recF and recR mutations led to their accumulation in a recBC sbcBC background. The recJ mutation showed the largest amount of these forms. (iv) No accumulation was detected in mutants affecting resolution of Holliday intermediates, recG, ruvAB and ruvC, in any of these backgrounds.

Conclusion

These results are discussed in terms of stepwise processing of chromosomal double-strand breaks.

     

Mycorrhization between <Emphasis Type="Italic">Cistus ladanifer</Emphasis> L. and <Emphasis Type="Italic">Boletus edulis</Emphasis> Bull is enhanced by the mycorrhiza helper bacteria <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Migula

Boletus edulis Bull. is one of the most economically and gastronomically valuable fungi worldwide. Sporocarp production normally occurs when symbiotically associated with a number of tree species in stands over 40 years old, but it has also been reported in 3-year-old Cistus ladanifer L. shrubs. Efforts toward the domestication of B. edulis have thus focused on successfully generating C. ladanifer seedlings associated with B. edulis under controlled conditions. Microorganisms have an important role mediating mycorrhizal symbiosis, such as some bacteria species which enhance mycorrhiza formation (mycorrhiza helper bacteria). Thus, in this study, we explored the effect that mycorrhiza helper bacteria have on the efficiency and intensity of the ectomycorrhizal symbiosis between C. ladanifer and B. edulis. The aim of this work was to optimize an in vitro protocol for the mycorrhizal synthesis of B. edulis with C. ladanifer by testing the effects of fungal culture time and coinoculation with the helper bacteria Pseudomonas fluorescens Migula. The results confirmed successful mycorrhizal synthesis between C. ladanifer and B. edulis. Coinoculation of B. edulis with P. fluorescens doubled within-plant mycorrhization levels although it did not result in an increased number of seedlings colonized with B. edulis mycorrhizae. B. edulis mycelium culture time also increased mycorrhization levels but not the presence of mycorrhizae. These findings bring us closer to controlled B. edulis sporocarp production in plantations.     

<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 %.     

Regulation of growth and development in phytochrome mutants of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by solar UV

Phytochrome mutants (phyA, phyB and phyAB) of Arabidopsis thaliana were grown under ambient and UV-excluded sunlight to understand their influence on growth and development by mutual exclusion. Phytochrome A and B played a complementary role in the regulation of germination. Suppression of hypocotyl length was predominantly under the control of phytochrome B; UV photoreceptors were active in suppression of hypocotyl growth only in phyB and phyAB mutants. Exclusion of UV promoted the number and the area of rosette leaves only in presence of phytochrome A and B. Phytochrome mutation reduced petiole length, whereas UV exclusion led to an increase. Requirement of long-day period for flowering was removed in the mutants. Under short-day conditions, flowering was predominantly under the control of phytochrome B, since phyB mutants flowered earlier than phyA mutants. Solar UV regulates the number of boltings and number of siliques per plant. Overall biomass of the plants is enhanced by the exclusion of UV only in the wild type. The interaction of phytochromes with UV photoreceptors is discussed in the paper.     

Update on <Emphasis Type="Italic">transparent testa</Emphasis> mutants from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: characterisation of new alleles from an isogenic collection

Main conclusion

We present a comprehensive overview on flavonoid-related phenotypes of A. thaliana tt and tds mutants, provide tools for their characterisation, increase the number of available alleles and demonstrate that tds3 is allelic to tt12 and tds5 to aha10.Flavonoid biosynthesis is one of the best-studied secondary metabolite pathways in plants. In the model system Arabidopsis thaliana it leads to the synthesis of three phenolic compound classes: flavonol glycosides, anthocyanins and proanthocyanidins (PAs). PAs appear brown in their oxidised polymeric forms, and most A. thaliana mutants impaired in flavonoid accumulation were identified through screens for lack of this seed coat pigmentation. These mutants are referred to as transparent testa (tt) or tannin-deficient seed (tds). More than 20 mutants of these types have been published, probably representing most of the genes relevant for PA accumulation in A. thaliana. However, data about the genes involved in PA deposition or oxidation are still rather scarce. Also, for some of the known mutants it is unclear if they represent additional loci or if they are allelic to known genes. For the present study, we have performed a systematic phenotypic characterisation of almost all available tt and tds mutants and built a collection of mutants in the genetic background of the accession Columbia to minimise effects arising from ecotype variation. We have identified a novel tt6 allele from a forward genetic screen and demonstrated that tds3 is allelic to tt12 and tds5 to aha10.

     

A glucuronokinase gene in<Emphasis Type="Italic"> Arabidopsis</Emphasis>, <Emphasis Type="Italic">AtGlcAK</Emphasis>, is involved in drought tolerance by modulating sugar metabolism

Arabidopsis glucuronokinase (AtGlcAK), as a member of the GHMP kinases family, is implicated in the de novo synthesis of UDP-glucuronic acid (UDP-GlcA) by the myo-inositol oxygenation pathway. In this study, two T-DNA insertion homozygous mutants of AtGlcAK, atglcak-1 and atglcak-2, were identified. AtGlcAK was highly expressed in roots and flowers. There was reduced primary root elongation and lateral root formation in atglcak mutants under osmotic stress. The atglcak mutants displayed enhanced stomatal opening in response to abscisic acid (ABA), elevated water loss and impaired drought tolerance. Under water stress, the accumulation of reducing and soluble sugars was reduced in atglcak mutants, and the metabolism of glucose and sucrose was affected by the synthetic pathway of UDP-GlcA. Furthermore, a reduced level of starch in atglcak mutants was observed under normal conditions. The phylogenetic analysis suggested that GlcAK was conserved in numerous dicots and monocots plants. In short, AtGlcAK mutants displayed hypersensitivity to ABA and reduced root development under water stress, rendering the plants more susceptible to drought stress.     

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.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

<Emphasis Type="Italic">CKB1</Emphasis> is involved in abscisic acid and gibberellic acid signaling to regulate stress responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Casein kinase II (CK2), an evolutionarily well-conserved Ser/Thr kinase, plays critical roles in all higher organisms including plants. CKB1 is a regulatory subunit beta of CK2. In this study, homozygous T-DNA mutants (ckb1-1 and ckb1-2) and over-expression plants (35S:CKB1-1, 35S:CKB1-2) of Arabidopsis thaliana were studied to understand the role of CKB1 in abiotic stress and gibberellic acid (GA) signaling. Histochemical staining showed that although CKB1 was expressed in all organs, it had a relatively higher expression in conducting tissues. The ckb1 mutants showed reduced sensitivity to abscisic acid (ABA) during seed germination and seedling growth. The increased stomatal aperture, leaf water loss and proline accumulation were observed in ckb1 mutants. In contrast, the ckb1 mutant had increased sensitivity to polyaluminum chloride during seed germination and hypocotyl elongation. We obtained opposite results in over-expression plants. The expression levels of a number of genes in the ABA and GA regulatory network had changed. This study demonstrates that CKB1 is an ABA signaling-related gene, which subsequently influences GA metabolism, and may play a positive role in ABA signaling.     

The role of thiol redox systems in the peroxide stress response of <Emphasis Type="Italic">Escherichia coli</Emphasis>

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.     

Inactivation of <Emphasis Type="Italic">RAD52</Emphasis> and <Emphasis Type="Italic">HDF1</Emphasis> DNA repair genes leads to premature chronological aging and cellular instability

The present study aims to investigate the role of radiation sensitive 52 (RAD52) and high-affinity DNA binding factor 1 (HDF1) DNA repair genes on the life span of budding yeasts during chronological aging. Wild type (wt) and rad52, hdf1, and rad52 hdf1 mutant Saccharomyces cerevisiae strains were used. Chronological aging and survival assays were studied by clonogenic assay and drop test. DNA damage was analyzed by electrophoresis after phenol extraction. Mutant analysis, colony forming units and the index of respiratory competence were studied by growing on dextrose and glycerol plates as a carbon source. Rad52 and rad52 hdf1 mutants showed a gradual decrease in surviving fraction in relation to wt and hdf1 mutant during aging. Genomic DNA was spontaneously more degraded during aging, mainly in rad52 mutants. This strain showed an increased percentage of revertant colonies. Moreover, all mutants showed a decrease in the index of respiratory competence during aging. The inactivation of RAD52 leads to premature chronological aging with an increase in DNA degradation and mutation frequency. In addition, RAD52 and HDF1 contribute to maintain the metabolic state, in a different way, during chronological aging. The results obtained could have important implications in the chronobiology of aging.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Reduced gene expression at the branch point of chlorophyll and heme biosynthesis in Arctic <Emphasis Type="Italic">Chlorella</Emphasis> ArM0029B

The gene expression at the branch point of chlorophyll and heme synthesis in the model microalga, Chlamydomonas reinhardtii, is different from that of higher plants. Another green alga, Arctic Chlorella, was recently isolated from Arctic sea ice and may be a promising candidate for a biofuel. To understand the chlorophyll metabolic pathway and relevant nuclear gene expression in Chlorella sp., we characterized chlorophyll-deficient mutants of the Arctic Chlorella sp. ArM0029B. First, we characterized the chlorophyll and heme biosynthetic pathways based on genes identified by bioinformatics analysis of the genome of Arctic Chlorella sp. ArM0029B. Then, we isolated and analyzed nine chlorophyll-deficient mutants that showed reduced expression of the ChlM gene, which encodes Mg-protoporphyrin methyltransferase. Expression of 5-amino levulinic acid dehydratase (encoded by ALAD) and glutamyl-tRNA reductase (encoded by HemA) was reduced in all nine independent mutants compared to wild type. These results indicated that Arctic Chlorella ArM0029B may have a regulatory mechanism of gene expression at earlier steps of the Mg-porphyrin branch that is more similar to higher plants than to the microalga C. reinhardtii. This study provides useful insight into the regulation of porphyrin precursor formation in Chlorella and related microalgae.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Botryosphaeria dothidea</Emphasis>

Botryosphaeria dothidea is a severe causal agent of die-back and cankers of many woody plants and causes great losses in many regions. The pathogenic mechanism of this pathogen has not been well explored due to lack of mutants and genetic information. In this study, we developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for B. dothidea protoplasts using vector pBHt2 containing the hph gene as a selection marker under the control of trp C promoter. Using this protocol we successfully generated the B. dothidea transformants with efficiency about 23 transformants per 10⁵ protoplasts. This is the first report of genetic transformation of B. dothidea via ATMT and this protocol provides an effective tool for B. dothidea genome manipulation, gene identification and functional analysis.     

Diversity of endophytic fungal and bacterial communities in <Emphasis Type="Italic">Ilex paraguariensis</Emphasis> grown under field conditions

The composition and diversity of the endophytic community associated with yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. Fungal and bacterial diversity did not show significant differences between organ age. The highest fungal diversity was registered during fall season and the lowest in winter. Bacterial diversity was higher in stems and increased from summer to winter, in contrast with leaves, which decreased. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Actinobacteria represented 57.5 % of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. Other bacteria frequently found were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. Nitrogen fixation and phosphate solubilization activity, ACC deaminase production and antagonism against plant fungal pathogens were assayed in endophytic bacterial strains. In the case of fungi, strains of Trichoderma, Penicillium and Aspergillus were assayed for antagonism against pathogenic Fusarium sp. All microbial isolates assayed showed at least one growth promoting activity. Strains of Bacillus, Pantoea, Curtobacterium, Methylobacterium, Brevundimonas and Paenibacillus had at least two growth-promoting activities, and Bacillus, Paenibacillus and the three endophytic fungi showed high antagonistic activity against Fusarium sp. In this work we have made a wide study of the culturable endophytic community within yerba mate plants and found that several microbial isolates could be considered as potential inoculants useful for improving yerba mate production.     

A reexamination on the deficiency of riboflavin accumulation in Malpighian tubules in larval translucent mutants of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A variety of insects accumulate high contents of riboflavin (vitamin B₂) in their Malpighian tubules (MTs). Although this process is known to be genetically controlled, the mechanism is not known. In the 1940s and the 1950s, several studies showed that riboflavin contents were low in the MTs of some Bombyx mori (silkworm) mutants with translucent larval skin mutations (e.g., w-3, od, oa, and otm) and that genes responsible for these translucent mutations also affected riboflavin accumulation in the MTs. Since the 2000s, it has been shown that the w-3 gene encodes an ABC transporter, whereas genes responsible for od, oa, and otm mutations encode for the biogenesis of lysosome-related organelles. These findings suggest that some genes of ABC transporters and biogenesis of lysosome-related organelles may control the accumulation of riboflavin in MTs. Therefore, we reexamined the effects that translucent mutations have on the accumulation of riboflavin in MTs by using the translucent and wild-type segregants in mutant strains to measure the specific effect that each gene has on riboflavin accumulation (independent of genomic background). We used nine translucent mutations (w-3^oe, oa, od, otm, Obs, oy, or, oh, and obt) even though the genes responsible for some of these mutations (Obs, oy, or, oh, and obt) have not yet been isolated. Through observation of larval MTs and measurements of riboflavin content using high-performance liquid chromatography, we found that the oa, od, otm, and or mutations were responsible for low contents of riboflavin in MTs, whereas the Obs and oy mutations did not affect riboflavin accumulation. This indicates that the molecular mechanism for riboflavin accumulation is similar but somewhat different than the mechanism responsible for uric acid accumulation in epidermal cells. We found that the genes responsible for oa, od, and otm mutations were consistent with those already established for uric acid accumulation in larval epidermis. This suggests that these three genes control riboflavin accumulation in MTs through a mechanism similar to that of uric acid accumulation, although we do not yet know why the or mutation also controls riboflavin accumulation.     

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.