Changes in the microbial community during repeated anaerobic microbial dechlorination of pentachlorophenol

Pentachlorophenol (PCP) has been widely used as a pesticide in paddy fields and has imposed negative ecological effect on agricultural soil systems, which are in typically anaerobic conditions. In this study, we investigated the effect of repeated additions of PCP to paddy soil on the microbial communities under anoxic conditions. Acetate was added as the carbon source to induce and accelerate cycles of the PCP degradation. A maximum degradation rate occurred at the 11th cycle, which completely transformed 32.3 μM (8.6 mg L^?1) PCP in 5 days. Illumina high throughput sequencing of 16S rRNA gene was used to profile the diversity and abundance of microbial communities at each interval and the results showed that the phyla of Bacteroidates, Firmicutes, Proteobacteria, and Euryarchaeota had a dominant presence in the PCP-dechlorinating cultures. Methanosarcina, Syntrophobotulus, Anaeromusa, Zoogloea, Treponema, W22 (family of Cloacamonaceae), and unclassified Cloacamonales were found to be the dominant genera during PCP dechlorination with acetate. The microbial community structure became relatively stable as cycles increased. Treponema, W22, and unclassified Cloacamonales were firstly observed to be associated with PCP dechlorination in the present study. Methanosarcina that have been isolated or identified in PCP dechlorination cultures previously was apparently enriched in the PCP dechlorination cultures. Additionally, the iron-cycling bacteria Syntrophobotulus, Anaeromusa, and Zoogloea were enriched in the PCP dechlorination cultures indicated they were likely to play an important role in PCP dechlorination. These findings increase our understanding for the microbial and geochemical interactions inherent in the transformation of organic contaminants from iron rich soil, and further extend our knowledge of the PCP-transforming microbial communities in anaerobic soil conditions.     

The <Emphasis Type="Italic">angora</Emphasis> gene weakens the effect of interaction of the mutant genes <Emphasis Type="Italic">wellhaarig</Emphasis> and <Emphasis Type="Italic">waved alopecia</Emphasis> in mice

The interaction of the mutant genes wellhaarig (we) and waved alopecia (wal) in mice was earlier demonstrated in our laboratory. The we gene significantly accelerates the appearance of alopecia in double we/wewal/wal homozygotes as compared to that in single +/+wal/wal homozygotes. It has been found in this work that the mutant gene angora-Y (Fgf5 ^go-Y ) weakens the effect of interaction of the we and wal genes. The first signs of alopecia appear in mice of the we/wewal/wal genotype at the age of 14 days, in triple Fgf5 ^go-Y /Fgf5 ^go-Y we/wewal/wal homozygotes alopecia is observed seven days later, i. e., in 21-day-old animals. The progression of alopecia in triple homozygotes is expressed to a lesser degree than in double +/+we/wewal/wal homozygotes. A single dose of the Fgf5 ^go-Y gene also decreases the effect of interaction of the we and wal genes, but less than a double dose of this gene. The first signs of alopecia in mice of the +/Fgf5 ^go-Y we/wewal/wal genotype appear only three days later than in double +/+we/wewal/wal homozygotes. The data obtained demonstrate that the Fgf5 ^go-Y gene is a powerful modifier of mutant genes determining the process of alopecia.     

Cloning and Functional Analysis of <Emphasis Type="Italic">SaCLCc1</Emphasis>, a Gene Belonging to the Chloride Channel Family (<Emphasis Type="Italic">CLC</Emphasis>), from the Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis> (L.) Pall.

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe²⁺-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn²⁺. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl^– ions.     

Cell wall glycopolymers of type strains from three species of the genus <Emphasis Type="Italic">Actinoplanes</Emphasis>

The structures of cell wall glycopolymers from the type strains of three Actinoplanes species were investigated using chemical methods, NMR spectroscopy, and mass spectrometry. Actinoplanes digitatis VKM Ac-649^T contains two phosphate-containing glycopolymers: poly(diglycosyl-1-phosphate) →6)-α-D-GlcpNAc-(1-P-6)-α-D-GlcpN-(1→ and teichoic acid →1)-sn-Gro-(3-P-3)-β-[β-D-GlcpNAc-(1→2]-D-Galp-(1→. Two glycopolymers were identified in A. auranticolor VKM Ac-648^T and A. cyaneus VKM Ac-1095^T: minor polymer–unsubstituted 2,3-poly(glycerol phosphate), widely abundant in actinobacteria (Ac-648^T), and mannan with trisaccharide repeating unit →2)-α-D-Manp-(1→2)-α-D-Manp(1→6)-α-D-Manp-(1→(Ac-1095^T). In addition, both microorganisms contain a teichuronic acid of unique structure containing a pentasaccharide repeating unit with two residues of glucopyranose and three residues of diaminouronic acids in D-manno- and/or D-gluco-configuration. Each of the strains demonstrates peculiarities in the structure of teichuronic acid with respect to the ratio of diaminouronic acids and availability and location of O-methyl groups in glucopyranose residues. All investigated strains contain a unique set of glycopolymers in their cell walls with structures not described earlier for prokaryotes.     

Crystal structure of wild-type centrin 1 from <Emphasis Type="Italic">Mus musculus</Emphasis> occupied by Ca<Superscript>2+</Superscript>

Mus musculus centrin 1 (MmCen1) is located at the cilium of photoreceptor cells connecting the outer segment through signal transduction components to the metabolically active inner segment. In the cilium, MmCen1 is involved in the translocation of transducin between compartments as a result of photoreceptor activation. In this study, we report the crystal structure of wild-type MmCen1 and its Ca²⁺-binding properties using structure-based mutagenesis. The crystal structure exhibits three structural features, i.e. four Ca²⁺ equally occupied at each EF-hand motif, structural changes accompanying helix motion at the N- and C-lobes, and adoption of N–C type dimerization when Ca²⁺ binds to EF-hand I and II in the N-lobe. The presence of MmCen1 dimers was confirmed in solution by native PAGE. Isothermal titration calorimetry data showed sequential binding of Ca²⁺ at four independent sites. Mutations S45A and D49A in EF-hand I alone disrupted the Ca²⁺-binding property of the wild-type protein. Based on the crystal structure of MmCen1, we suggest that a dimerization mode between the N- and C-lobes may be required by Ca²⁺ binding at the N-lobe.     

A saponin isolated from <Emphasis Type="Italic">Agapanthus africanus</Emphasis> differentially induces apoplastic peroxidase activity in wheat and displays fungicidal properties

A spirostane with an attached trisaccharide, (25R)-5α-spirostane-2α,3β,5α-triol 3-O-(O-α-l-rhamnopyranosyl-(1 → 2)-O-(β-d-galactopyranosyl-(1 → 3))-β-d-glucopyranoside), was isolated and identified from the aerial parts of Agapanthus africanus by activity-guided fractionation. Fungicidal properties of the crude extract, semi-purified fractions as well as the purified active saponin from A. africanus were screened in vitro against Fusarium oxysporum. At a concentration of 1 mg mL^?1, the crude extract and semi-purified ethyl acetate and dichloromethane fractions showed significant antifungal activity. The purified saponin inhibited the in vitro mycelial growth of F. oxysporum completely (100 %) at a concentration of 125 µg mL^?1. Furthermore, to verify previously observed induced resistance by crude extracts of A. africanus towards leaf rust, intercellular PR-protein activity was determined in wheat seedlings following foliar application of the purified saponin at 100 µg mL^?1. In vitro peroxidase enzyme activity increased significantly (60 %) in wheat seedlings 48 h after treatment with the purified saponin, demonstrating its role as an elicitor to activate a defence reaction in wheat.     

Salt oversensitivity derived from mutation breeding improves salinity tolerance in barley <Emphasis Type="Italic">via</Emphasis> ion homeostasis

Salt stress imposes a major environmental threat to agriculture, therefore, understanding the basic physiology and genetics of cell under salt stress is crucial for developing any breeding strategy. In the present study, the expression profile of genes involved in ion homeostasis including salt overly sensitive (HvSOS1, HvSOS2, HvSOS3), vacuolar Na⁺/H⁺ antiporter (HvNHX1), and H⁺-ATPase (HVA) along with ion content measurement were investigated in two genotypes of Hordeum vulgare under 300 mM NaCl. The gene expressions were measured in the roots and shoots of a salt-tolerant mutant genotype M4-73-30 and in its wild-type cv. Zarjou by real-time qPCR technique. The critical differences between the salt-tolerant mutant and its wild-type were observed in the expressions of HvSOS1 (105-fold), HvSOS2 (24-fold), HvSOS3 (31-fold), and HVA (202-fold) genes in roots after 6-h exposure to NaCl. The parallel early up-regulation of these genes in root samples of the salt-tolerant mutant genotype indicated induction of Na⁺/H⁺ antiporters activity and Na+ exclusion into apoplast and vacuole. The earlier up-regulation of HvSOS1, HVA, and HvNHX1 genes in shoot of the wild-type genotype corresponded to the relative accumulation of Na⁺ which was not observed in salt-tolerant mutant genotype because of efficient inhibitory role of the root in Na+ transport to the shoot. In conclusion, the lack of similarity in gene expression patterns between the two genotypes with similar genetic background may confirm the hypothesis that mutation breeding could change the ability of salt-tolerant mutant genotype for efficient ion homeostasis via salinity oversensitivity response.     

Loss or Mislocalization of Aquaporin-4 Affects Diffusion Properties and Intermediary Metabolism in Gray Matter of Mice

The first aim of this study was to determine how complete or perivascular loss of aquaporin-4 (AQP4) water channels affects membrane permeability for water in the mouse brain grey matter in the steady state. Time-dependent diffusion magnetic resonance imaging was performed on global Aqp4 knock out (KO) and α-syntrophin (α-syn) KO mice, in the latter perivascular AQP4 are mislocalized, but still functioning. Control animals were corresponding wild type (WT) mice. By combining in vivo diffusion measurements with the effective medium theory and previously measured extra-cellular volume fractions, the effects of membrane permeability and extracellular volume fraction were uncoupled for Aqp4 and α-syn KO. The second aim was to assess the effect of α-syn KO on cortical intermediary metabolism combining in vivo [1-¹³C]glucose and [1,2-¹³C]acetate injection with ex vivo ¹³C MR spectroscopy. Aqp4 KO increased the effective diffusion coefficient at long diffusion times by 5%, and a 14% decrease in membrane water permeability was estimated for Aqp4 KO compared with WT mice. α-syn KO did not affect the measured diffusion parameters. In the metabolic analyses, significantly lower amounts of [4-¹³C]glutamate and [4-¹³C]glutamine, and percent enrichment in [4-¹³C]glutamate were detected in the α-syn KO mice. [1,2-¹³C]acetate metabolism was unaffected in α-syn KO, but the contribution of astrocyte derived metabolites to GABA synthesis was significantly increased. Taken together, α-syn KO mice appeared to have decreased neuronal glucose metabolism, partly compensated for by utilization of astrocyte derived metabolites.     

Molecular characterization of the <Emphasis Type="Italic">Aspergillus fumigatus NCS-1</Emphasis> homologue,NcsA

Here, we characterize the Aspergillus fumigatus homologue ncsA Neuronal Calcium Sensor. We showed that ncsA is not an essential gene and ?ncsA growth was decreased in the presence of EGTA and SDS. Furthermore, the ?ncsA mutant is more resistant to calcium chloride. NcsA:mRFP localizes to the cytoplasm and its cellular localization is not affected by the cellular response to either calcium chloride or EGTA. The ?ncsA mutant strain is more sensitive to voriconazole, itraconazole, and amphotericin. Polar growth in the ΔncsA mutant was also considerably more affected by lovastatin than in the wild type strain. The Spitzenkörper can be visualized in both strains and although the vacuolar system does not seem to be very different, there is an increase in the staining intensity on the germling surface of the ?ncsA strain. NcsA promotes pmcA and pmcB expression and therefore there is a reduced expression of these ion pumps in the ΔncsA mutant background, and also of other genes involved in the response to calcium in A. fumigatus. The ncsA inactivation mutation is not causing loss of virulence in a low dose murine infection when compared to the corresponding wild type strain.     

Rhamnose-Containing Cell Wall Glycopolymers from <Emphasis Type="Italic">Rathayibacter toxicus</Emphasis> VKM Ac-1600 and “<Emphasis Type="Italic">Rathayibacter tanaceti</Emphasis>” VKM Ac-2596

Structures of the cell wall glycopolymers from two representatives of the genus Rathayibacter were investigated using chemical, NMR spectroscopy, and optical methods. The R. toxicus VKM Ac-1600 strain contains two neutral glycopolymers–a linear rhamnomannan →2)-α-D-Rhap-(1→3)-α-D-Manp-(1→ and a branched polysaccharide containing in the repeating unit the residues of D-Manp, D-Glcp, and L-Rhap in the ratios of 2: 4: 1, respectively (the structure is presented in the text). The “Rathayibacter tanaceti” VKM Ac-2596 contains a rhamnomannan that is different from the above-described one by localization of glycosidic bonds on the residues of α-Rhap and α-Manp, i.e. →3)-α-D-Rhap (1→2)-α-D-Manp-(1→. The structures of all identified glycopolymers are described for the first time in actinobacteria. The data obtained make it possible to characterize representatives of the studied actinobacteria more fully and can be used to differentiate Rathayibacter species at the phenotype level.     

The crystal structure of methanol dehydrogenase,a quinoprotein from the marine methylotrophic bacterium <Emphasis Type="Italic">Methylophaga aminisulfidivorans</Emphasis> MP<Superscript>T</Superscript>

The first crystal structure of a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) from a marine methylotrophic bacterium, Methylophaga aminisulfidivorans MP^T (MDH_Mas), was determined at 1.7 Å resolution. The active form of MDH_Mas (or MDHI_Mas) is a heterotetrameric α₂β₂, where each β-subunit assembles on one side of each of the α-subunits, in a symmetrical fashion, so that two β-subunits surround the two PQQ-binding pockets on the α-subunits. The active site consists of a PQQ molecule surrounded by a β-propeller fold for each α-subunit. Interestingly, the PQQ molecules are coordinated by a Mg²⁺ ion, instead of the Ca²⁺ ion that is commonly found in the terrestrial MDHI, indicating the efficiency of osmotic balance regulation in the high salt environment. The overall interaction of the β-subunits with the α-subunits appears tighter than that of terrestrial homologues, suggesting the efficient maintenance of MDHI_Mas integrity in the sea water environment to provide a firm basis for complex formation with MxaJ_Mas or Cyt c_L. With the help of the features mentioned above, our research may enable the elucidation of the full molecular mechanism of methanol oxidation by taking advantage of marine bacterium-originated proteins in the methanol oxidizing system (mox), including MxaJ, as the attainment of these proteins from terrestrial bacteria for structural studies has not been successful.     

Defects in meiotic recombination delay progression through pachytene in <Emphasis Type="Italic">Tex19.1</Emphasis><Superscript><Emphasis Type="Italic">−/−</Emphasis></Superscript> mouse spermatocytes

Recombination, synapsis, chromosome segregation and gene expression are co-ordinately regulated during meiosis to ensure successful execution of this specialised cell division. Studies with multiple mutant mouse lines have shown that mouse spermatocytes possess quality control checkpoints that eliminate cells with persistent defects in chromosome synapsis. In addition, studies on Trip13^mod/mod mice suggest that pachytene spermatocytes that successfully complete chromosome synapsis can undergo meiotic arrest in response to defects in recombination. Here, we present additional support for a meiotic recombination-dependent checkpoint using a different mutant mouse line, Tex19.1^?/?. The appearance of early recombination foci is delayed in Tex19.1^?/? spermatocytes during leptotene/zygotene, but some Tex19.1^?/? spermatocytes still successfully synapse their chromosomes and we show that these spermatocytes are enriched for early recombination foci. Furthermore, we show that patterns of axis elongation, chromatin modifications and histone H1t expression are also all co-ordinately skewed towards earlier substages of pachytene in these autosomally synapsed Tex19.1^?/? spermatocytes. We also show that this skew towards earlier pachytene substages occurs in the absence of elevated spermatocyte death in the population, that spermatocytes with features of early pachytene are present in late stage Tex19.1^?/? testis tubules and that the delay in histone H1t expression in response to loss of Tex19.1 does not occur in a Spo11 mutant background. Taken together, these data suggest that a recombination-dependent checkpoint may be able to modulate pachytene progression in mouse spermatocytes to accommodate some types of recombination defect.     

Effects of Disruption of <Emphasis Type="Italic">PMC1</Emphasis> in the <Emphasis Type="Italic">tfp1</Emphasis>∆/∆ Mutant on Calcium Homeostasis,Oxidative and Osmotic Stress Resistance in <Emphasis Type="Italic">Candida albicans</Emphasis>

The vacuolar-type H⁺-ATPase (V-ATPase) is essential for many cell processes. Our previous study has demonstrated that Tfp1 is a putative subunit of V-ATPase, loss of which causes disorders in calcium homeostasis and decreased resistance to oxidative stress. In this study, we found that further deletion of PMC1, a vacuolar calcium pump, in tfp1?/? mutant led to more severe dysregulation of calcium homeostasis. Besides, the tfp1?/?pmc1?/? mutant was more sensitive to H₂O₂ and had a higher ROS level. As is known, V-ATPase mutants are sensitive to NaCl, and PMC1 mutant is resistant against NaCl. However, the tfp1?/?pmc1?/? mutant exhibited sensitivity to NaCl. Mechanism study demonstrated that their sensitivity was associated with reduced osmotic resistance caused by relatively low expression of GPD1. In addition, we first found that NaCl addition significantly declined ROS levels in tfp1?/? and tfp1?/?pmc1?/? mutants. In tfp1?/? mutant, decreased ROS levels were relevant to enhanced antioxidant activities. However, in tfp1?/?pmc1?/? mutant, reduced ROS resulted from decreased total calcium content, revealing that NaCl affected ROS levels in the two mutants through different mechanisms. Taken together, our data indicated that loss of both TFP1 and PMC1 further affected calcium homeostasis and other cellular processes in Candida albicans and provides a potential antifungal target.     

Gene <Emphasis Type="Italic">angora</Emphasis> as a modifier of the <Emphasis Type="Italic">hairless</Emphasis> gene in mouse

The interaction between mouse angora-Y (Fgf5 ^go-Y) and hairless (hr) genes have been studied. Homozygous mutant gene Fgf5 ^go-Y increases length of all hair types, while homozygotes for the h gene lose hair completely starting on day 14 after birth. We obtained mice with genotypes +/+ hr/hr F₂, +/Fgf5 ^go-Y hr/hr and Fgf5 ^go-Y/Fgf5 ^go-Y hr/hr. Both +/Fgf5 ^go-Y hr/hr and +/+ hr/hr mice began to loose hair from their heads on day 14. This further extended on the whole body. On day 21 the mice were completely deprived of hair. Therefore a single dose of gene Fgf5 ^go-Y does not modify alopecia in mice homozygous for hr. However in double homozygotes Fgf5 ^go-Y/Fgf5 ^go-Y hr/hr alopecia started 4 days later, namely on day 18. It usually finished 10–12 days after detection of first bald patches. On days 28–30 double homozygotes lose coat completely. Hair loss in double homozygous mice was 1.5-fold slower than in +/+ hr/hr mice. This resulted from a significant extension of anagen phase induced by a mutant homozygous gene Fgf5 ^go-Y in morphogenesis of the hair follicle. The hr gene was expressed at the transmission phase from anagen to catagen. Our data shows that the angora gene is a modifier of the hairless gene and this results in a strong repression of alopecia progression in double homozygous mice compared to +/+ hr/hr animals.     

Steroid Compounds from Far Eastern Starfishes <Emphasis Type="Italic">Henricia aspera</Emphasis> and <Emphasis Type="Italic">H. tumida</Emphasis>

Six new natural compounds were isolated from two Far Eastern starfish species, Henricia aspera and H. tumida, collected in the Sea of Okhotsk. Two new glycosylated steroid polyols were obtained from H. aspera: asperoside A and asperoside B, which were shown to be (20R,24R, 25S)-3-O-(2,3-di-O-methyl-β -D-xylopyranosyl)-24-methyl-5α-cholest-4-ene-3β, 6β,8,15α,16β,26-hexaol and (20R, 24R,25S,22E)-3-O-(2,4-di-O-methyl-β-D-xylopyranosyl)-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,26-hexaol, respectively. Two other glycosylated polyols, tumidoside A, with the structure elucidated as (20R, 22E)-3-O-(2,4-di-O-methyl-β -D-xylopyranosyl)-26,27-dinor-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,25-hexaol, and tumidoside B, whose structure was elucidated as (20R,24S)-3-O-(2,3-di-O-methyl-β-D-xylopyranosyl)-5α-cholestan-3β,4β,6β,8,15α,24-hexaol, were isolated from the two starfish species. (20R, 24S)-5α-Cholestan-3β,6β,15α,24-tetraol and (20R, 24S)-5α-cholestan-3β,6β,8,15α,24-pentaol were identified only in H. tumida. The known monoglycosides henricioside H1 and laeviuscolosides H and G were also identified in both species.     

Isolation and functional analysis of apple MdHMGR1 and MdHMGR4 gene promoters in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Cereal grains offer great potential as a storage system for production of highly valuable proteins using biotechnological approaches, but such applications require tight temporal and spatial control of transgene expression. Towards this aim, we have undertaken a detailed analysis of α-kafirin (α-kaf) promoter and α-kaf signal peptide (sp) in transgenic sorghum plants, using green fluorescent protein gene (gfp) as a reporter. Constructs containing either the α-kaf promoter or the constitutive maize ubiquitin-1 (ubi) promoter driving either gfp or sp-gfp translational fusion were introduced into Sorghum bicolor inbred line Tx430 by particle bombardment. We show for the first time that the α-kaf promoter directs endosperm-specific transgene expression, with activity first detected at 10 days post-anthesis (dpa), peaking at 20 dpa, and remaining active through to physiological maturity. Furthermore, we demonstrate for the first time that the α-kafirin sp is sufficient to direct foreign protein to protein bodies in the endosperm. The evidence is also provided for possible mis-targeting by α-kaf sp in vegetative tissues of transgenic lines with ubi-sp-gfp, resulting in loss of reporter gene translational activity that no GFP signal was observed. These results demonstrate that α-kaf promoter and α-kaf sp are well suited for seed bioengineering to produce recombinant proteins in sorghum endosperm or deposit foreign proteins into sorghum protein bodies.