Yeast communities of <Emphasis Type="Italic">Formica aquilonia</Emphasis> colonies

Yeast abundance and species diversity in the colonies of Formica aquilonia ants in birch–pine grass forest near Novosibirsk, Russia, were studied. The average yeast number in the anthill material was 10³–10⁴ CFU/g, reaching 10⁵ CFU/g in the hatching chambers. Typical litter species (Trichosporon moniliiforme and Cystofilobasidium capitatum) were predominant in soil and litter around the anthills. Apart from these species, ascomycete species of the family Debaryomycetaceae, Debaryomyces hansenii, and Schwanniomyces vanrijiae were predominant in the anthill material. Yeast population of the ant’ bodies consisted exclusively of the members of the last two species. Thus, highly specific yeast communities formed in the colonies of Formica aquilonia ants differ from the communities of surrounding soil. These differences are caused by environment-forming activity of the ants.     

Adaptive mutation related to cellulose producibility in <Emphasis Type="Italic">Komagataeibacter medellinensis</Emphasis> (<Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>) NBRC 3288

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.     

In vitro investigation of <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> strains for potential probiotic properties

In this study, 23 Debaryomyces hansenii strains, isolated from cheese and fish gut, were investigated in vitro for potential probiotic properties i.e. (1) survival under in vitro GI (gastrointestinal) conditions with different oxygen levels, (2) adhesion to Caco-2 intestinal epithelial cells and mucin, and (3) modulation of pro- and anti-inflammatory cytokine secretion by human monocyte-derived dendritic cells. As references two commercially available probiotic Saccharomyces cerevisiae var. boulardii (S. boulardii) strains were included in the study. Our results demonstrate that the different D. hansenii yeast strains had very diverse properties which could potentially lead to different probiotic effects. One strain of D. hansenii (DI 09) was capable of surviving GI stress conditions, although not to the same degree as the S. boulardii strains. This DI 09 strain, however, adhered more strongly to Caco-2 cells and mucin than the S. boulardii strains. Additionally, two D. hansenii strains (DI 10 and DI 15) elicited a higher IL-10/IL-12 ratio than the S. boulardii strains, indicating a higher anti-inflammatory effects on human dendritic cells. Finally, one strain of D. hansenii (DI 02) was evaluated as the best probiotic candidate because of its outstanding ability to survive the GI stresses, to adhere to Caco-2 cells and mucin and to induce a high IL-10/IL-12 ratio. In conclusion, this study shows that strains of D. hansenii may offer promising probiotic traits relevant for further study.     

Association of polymorphic markers of chemokine genes,their receptors,and <Emphasis Type="Italic">CD14</Emphasis> gene with coronary atherosclerosis

Atherosclerosis represents an inflammatory response to the disturbance of the endothelial layer in the arterial bloodstream. In the present study, an analysis of associations of polymorphic markers for the genes controlling synthesis of proteins involved in atherosclerosis pathogenesis in coronary atherosclerosis (CA) patients (217 subjects) and in a control group (250 subjects) was conducted. The following genes were examined: rs991804 (CCL2 gene), rs1126579 (CXCR2 gene), rs4074 (CXCL1 gene), rs4073 (CXCL8 gene), rs333 (CCR5 gene), rs2471859 (CXCR4 gene), rs1801157 (CXCL12 gene), and rs2569190 (CD14 gene). Using the Monte Carlo and Markov chain (APSampler) method, allele/genotype combinations associated with both low and high CA risk were revealed. The most important findings included the following: CXCR4*T/T + CCL2*C + CCR5*I/I (P_perm = 1 × 10^–6, OR = 0.44, 95% CI 0.3–0.63), CXCR2*C + CD14*C + CXCL12*G + CCL2*C + CCR5*D (P_perm = 4 × 10^–6, OR = 5.78, 95% CI 2.34–14.28), CD14*C + CCL2*C/C + CCR5*D (P_perm = 6.3 × 10^–6, OR = 5.81, 95% CI 2.17–15.56), CXCL8*A + CXCR2*C + CD14*T + CXCR4*C (P_perm = 0.01, OR = 3.21, 95% CI 1.63–6.31).     

<Emphasis Type="Italic">Limnobacter humi</Emphasis> sp. nov., a thiosulfate-oxidizing,heterotrophic bacterium isolated from humus soil,and emended description of the genus <Emphasis Type="Italic">Limnobacter</Emphasis> Spring <Emphasis Type="Italic">et al.</Emphasis> 2001

Three Gram-negative, strictly aerobic, chemolithoheterotrophic bacterial strains, designated UCM-30, UCM-33, and UCM-39^T, were isolated in South Korea. Based on their 16S rRNA gene sequences, the three isolated strains were found to be similar to Limnobacter thiooxidans CS-K2^T (97.41–97.68%), Limnobacter litoralis KP1-19^T (95.55–95.76%), and various genera belonging to the class Betaproteobacteria (90.34–93.34%). DNA-DNA hybridization showed 79.3–83.9% similarity between the genomic DNA of UCM-39^T, UCM-30, and UCM-33, while the sequence similarity between UCM-39^T and L. thiooxidans KACC 13837T or L. litoralis LMG 24869T was 23.7% and 18.6%, respectively. The DNA G+C content of UCM 39T was 59.7 mol%, the major ubiquinone was Q-8, and the optimal oxidation rate was observed at 10 mM thiosulfate. The major fatty acids (≥ 10%) were summed features 3 (C_16:1 ω7c and/or C_16:1 ω6c) and 8 (C_18:1 ω7c and/or C_18:1 ω6c), and C_16:0. The major polar lipids (diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol) were found in all members of genus Limnobacter. Based on phenotypic, physiological, and phylogenetic analyses, the UCM-39T strain was found to be significantly distinct to represent a novel species affiliated to the genus Limnobacter. We propose to name it Limnobacter humi sp. nov. with the type strain UCM-39^T (=KACC 18574^T =NBRC 111650^T).     

Alginate biosynthesis by <Emphasis Type="Italic">Azotobacter</Emphasis> bacteria

The ability of representatives of various species of the bacterial genus Azotobacter (A. chroococcum 7B, A. chroococcum 12B, A. chroococcum 12BS, A. agile 12, A. indicum 8, A. vinelandii 17, and A. vinelandii 5B) to alginate synthesis has been studied. It has been shown that all tested bacterial strains have this ability to different extents. Capsular alginate comprises from 2.6 to 32% of the total amount of synthesized alginate in various bacterial species. Strains that are able to active synthesis of alginate have been selected; the effect of the medium composition on their biosynthesis has been studied. The optimal conditions for alginate synthesis by the A. chroococcum 12BS producer strain include the presence of mannitol (40 g/L), yeast extract (1%), and low concentration of phosphates (KH₂PO₄—0.008 g/L, K₂HPO₄—0.032 g/L) in the medium; alginate production under these conditions is 4.5 g/L. The effect of aeration on polymer biosynthesis has been revealed: an increase in aeration causes an increase in alginate synthesis, while its decrease promotes the synthesis of poly-3-hydroxybutirate. It has been shown by IR spectroscopy that alginates obtained under various conditions of cultivation contain different ratios of residues of mannuronic and guluronic acids (M/G from 70/30 to 80/20) in the polymer chain and also differ in the amount of acetyl groups (from 10 to 25%) in the polyme structure.     

<Emphasis Type="Italic">Nibribacter flagellatus</Emphasis> sp. nov., isolated from rhizosphere of <Emphasis Type="Italic">Hibiscus syriacus</Emphasis> and emended description of the genus <Emphasis Type="Italic">Nibribacter</Emphasis>

A Gram-stain negative, aerobic, motile by flagella, rod-shaped strain (THG-T16^T) was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10–40 °C (optimum 28–30 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–1.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-T16^T were identified as Nibribacter koreensis KACC 16450^T (98.6%), Rufibacter roseus KCTC 42217^T (94.7%), Rufibacter immobilis CCTCC AB 2013351^T (94.5%) and Rufibacter tibetensis CCTCC AB 208084^T (94.4%). The DNA G+C content of strain THG-T16^T was determined to be 46.7 mol%. DNA–DNA hybridization values between strain THG-T16^T and N. koreensis KACC 16450^T, R. roseus KCTC 42217^T, R. immobilis CCTCC AB 2013351^T, R.tibetensis CCTCC AB 208084^T were 33.5?±?0.5% (31.7?±?0.7% reciprocal analysis), 28.1?±?0.2% (25.2?±?0.2%), 17.1?±?0.9% (10.2?±?0.6%) and 8.1?±?0.3% (5.2?±?0.1%). The polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified aminolipid and three unidentified lipids. The quinone was identified as MK-7 and the polyamine as sym-homospermidine. The major fatty acids were identified as C_16:1 ω5c, C_17:1 ω6c, iso-C_15:0, summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) and summed feature 4 (iso-C_17:1 I and/or anteiso-C_17:1 B). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA–DNA hybridization data, strain THG-T16^T represents a novel species of the genus Nibribacter, for which the name Nibribacter flagellatus sp. nov. is proposed. The type strain is THG-T16^T(=?KACC 19188^T?=?CCTCC AB 2016246^T).     

Intertidal benthic communities associated with the macroalgae <Emphasis Type="Italic">Iridaea cordata</Emphasis> and <Emphasis Type="Italic">Adenocystis utricularis</Emphasis> in King George Island,Antarctica

Antarctic benthos has been a main target in Antarctic research, but very few quantitative studies have been carried out in the littoral zone, which may be seasonally covered by macroalgae. In this work, we studied (1) cover and biomass of the macroalgae Iridaea cordata and Adenocystis utricularis, and (2) composition of macrobenthic assemblage associated with these macroalgal species at three locations at King George Island: Mareograph Beach (1 M), Tank’s Bay (2R) and Ardley Bay (3R). Iridaea cordata was collected by completely detaching the algae from the substrate, while A. utricularis was scraped. Adenocystis utricularis covered more than 80 % of the substrate at all locations, while coverage of Iridaea cordata was below 53 % or absent (3R). Fresh biomass of I. cordata was 0.8–61.4 g/individual and 4.7–93.0 g/100 cm² for A. utricularis. The assemblage associated with both macroalgae differed significantly between sites. The studied fauna was composed mainly of amphipods, gastropods and bivalves. Species diversity was higher in the community associated with A. utricularis. A total of ~27 ind/g DW were found associated with I. cordata, while ~112 ind/g DW were found associated with A. utricularis. The most abundant groups associated with I. cordata were amphipods at 1 M (57 %) and gastropods at 2R (46 %). Both groups were responsible for the dissimilarity between localities (62.50 %). The most abundant groups associated with A. utricularis were the gastropods at all localities reaching up to 82 % at 1 M. This study provides a first baseline on the diversity and abundance of benthic assemblages associated with intertidal macroalgae in the southwest of King George Island.     

Enhancement of crystallinity of cellulose produced by <Emphasis Type="Italic">Escherichia coli</Emphasis> through heterologous expression of <Emphasis Type="Italic">bcsD</Emphasis> gene from <Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>

Objectives

To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001.

Results

The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD.

Conclusion

The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

     

Dielectric Dispersion in Ga<Subscript>2</Subscript>S<Subscript>3</Subscript> Thin Films

In this work, the structural, compositional, optical, and dielectric properties of Ga₂S₃ thin films are investigated by means of X-ray diffraction, scanning electron microscopy, energy dispersion X-ray analysis, and ultraviolet—visible light spectrophotometry. The Ga₂S₃ thin films which exhibited amorphous nature in its as grown form are observed to be generally composed of 40.7 % Ga and 59.3 % S atomic content. The direct allowed transitions optical energy bandgap is found to be 2.96 eV. On the other hand, the modeling of the dielectric spectra in the frequency range of 270–1,000 THz, using the modified Drude-Lorentz model for electron-plasmon interactions revealed the electrons scattering time as 1.8 (fs), the electron bounded plasma frequency as ~0.76–0.94 (GHz) and the reduced resonant frequency as 2.20–4.60 ×10¹⁵ (Hz) in the range of 270–753 THz. The corresponding drift mobility of electrons to the terahertz oscillating incident electric field is found to be 7.91 (cm ²/Vs). The values are promising as they nominate the Ga₂S₃ thin films as effective candidates in thin-film transistor and gas sensing technologies.     

Association analysis of alcohol metabolizing enzymes <Emphasis Type="Italic">ADH1B,ADH7, CYP2E1</Emphasis> gene polymorphism with risk for coronary atherosclerosis

The allele and genotype distribution of two alcohol dehydrogenase genes ADH1B (exon 3 polymorphism A/G (47His)), ADH7 (intron 5 polymorphism G/C) and cytochrome P450 2E1 gene (CYP2E1; 5′-flanking region G/C and intron 6 T/A polymorphisms) were examined in Russian (Tomsk, n = 125) healthy population and in coronary atherosclerosis patients (CA, n = 92). The genotype frequencies followed the Hardy-Weinberg equilibrium and the alleles were in linkage equilibrium or gametic equilibrium in the control sample. Only two CYP2E1 gene polymorphisms were in linkage disequilibrium. The frequencies of the derived alleles at ADH1B ^* G (+MslI) allele, CYP2E1 ^* C2 (+PstI) allele and CYP2E1 ^* C (-DraI) allele were 8.48 ± 1.86, 1.20 ± 0.69, and 10.00 ± 1.90%, respectively. The ADH7 gene polymorphism showed a high level of heterozygosity; the frequency of the ADH7 ^* C (-StyI) allele was 44.58 ± 3.21%. A significantly higher frequency of CYP2E1 PstI C2 allele has been revealed in the CA group (P = 0.043; OR = 4.23; 95% CI 1.03–20.01). The tendency to significant effect of A1A2 genotype in ADH1B MslI polymorphism was observed for systolic blood pressure in the control group (P = 0.068). The statistically significant two-way interaction effects of ADH7 StyI and CYP2E1 DraI on diastolic blood pressure (P = 0.029) and on the serum high density lipoprotein level (P = 0.042) were also revealed. Association of A1A2 genotype in ADH1B MslI polymorphism with reduced amount in a serum of a very low density lipoprotein level (P = 0.045) have also been shown. This may result from multifunctional activity of alcohol metabolizing enzymes and their involvement in many metabolic and free radical reactions in the body.     

<Emphasis Type="Italic">Roseomonas aeriglobus</Emphasis> sp. nov., isolated from an air-conditioning system

A novel pale pink-coloured, strictly aerobic, Gram-stain negative bacterial strain, designated strain KER25-12^T, was isolated from a laboratory air-conditioning system in South Korea. Cells were observed to be non-motile cocci showing positive catalase and oxidase reactions. Strain KER25-12^T was found to grow at 10–30 °C (optimum, 25–30 °C), at pH 4.0–9.0 (optimum, pH 6.0–7.0) and in the presence of 0–2% (w/v) NaCl (optimum, 0%). Ubiquinone-10 and spermidine were detected as the sole respiratory quinone and the predominant polyamine, respectively. The major fatty acids were identified as summed feature 8 (comprising C_18:1 ω7c and/or C_18:1 ω6c), summed feature 3 (comprising C_16:1 ω7c and/or C_16:1 ω6c), C_16:0 and C_18:0. The genomic DNA G+C content of strain KER25-12^T was determined to be 70.0 mol%. The major polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unidentified aminolipid. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain KER25-12^T belongs to the genus Roseomonas and shows high sequence similarity to Roseomonas aerilata 5420S-30^T (98.57%), Roseomonas pecuniae N75^T (97.44%) and Roseomonas vinacea CPCC 100056^T (97.40%). Based on the morphological, physiological, chemotaxonomic and phylogenetic features, strain KER25-12^T is concluded to represent a novel species of the genus Roseomonas, for which the name Roseomonas aeriglobus sp. nov. is proposed. The type strain is KER25-12^T (= KACC 19282^T = JCM 32049^T).     

Simultaneous removal of hexavalent chromium and <Emphasis Type="Italic">o</Emphasis>-dichlorobenzene by isolated <Emphasis Type="Italic">Serratia marcescens</Emphasis> ZD-9

Heavy metals–organics mixture pollution is increasingly concerned and simultaneous removal of organic pollutants and heavy metals is becoming significant. In this study, a strain was isolated from the sediment of a tannery effluent outfalls, which can remove o-dichlorobenzene (o-DCB) and Cr(VI) simultaneously. The bacterial isolate was identified as Serratia marcescens by the 16S rRNA gene sequences. The strain removed about 90% of o-DCB and more than 80% of Cr(VI) at the concentration of 1.29 g L^?1 o-DCB and 20 mg L^?1 Cr(VI). In the presence of concomitant pollutant o-DCB, the optimal pH (8.0) and temperature (30 °C) were determined for Cr(VI) removal. Changes of the bacterial cells and intracellular black Cr(III) sediments were observed by the TEM auxiliary analysis. The results of the FTIR spectroscopy analysis indicated that hydroxyl, amide and polysaccharides were involved in the process of Cr(VI) removal.     

<Emphasis Type="Italic">Paradevosia shaoguanensis</Emphasis> gen. nov., sp. nov., Isolated from a Coking Wastewater

A Gram staining negative, rod-shaped, aerobic bacterial strain J5-3^T with a single polar flagellum was isolated from coking wastewater collected from Shaoguan, Guangdong, China. It was motile and capable of optimal growth at pH 6–8, 30 °C, and 0–2 % (w/v) NaCl. Its predominant fatty acids were 11-methyl C_18:1 ω7c (29.2 %), C_16:0 (20.6 %), C_19:0 cyclo ω8c (18.2 %), C_18:0 (11.0 %), and C_18:1 ω7c/C_18:1 ω6c (10.9 %) when grown on trypticase soy agar. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two unknown glycolipids (GL1, GL2), and two unknown phospholipid (PL1, PL2). The predominant ubiquinone was Q-10, and the genome DNA G+C content was 61.7 mol %. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain J5-3^T belonged to the family Hyphomicrobiaceae in Alphaproteobacteria. It shared the 16S rRNA gene sequence similarities of 93.8–96.1 % with the genus Devosia, 94.5–94.8 % with the genus Pelagibacterium, and <92.0 % with all the other type strains in family Hyphomicrobiaceae. It can be distinguished from the closest phylogenetic neighbors based on several phenotypic and genotypic features, including α-galactosidase activity, tetracycline susceptibility, major fatty acid composition, polar lipid profile, DNA gyrase B subunit (gyrB) gene sequence, and random-amplified polymorphic DNA profile. Therefore, we consider strain J5-3^T to represent a novel species of a novel genus within the family Hyphomicrobiaceae, for which the name Paradevosia shaoguanensis gen. nov., sp. nov. is proposed. The type strain of Paradevosia shaoguanensis is J5-3^T (=CGMCC 1.12430^T =LMG 27409^T).     

<Emphasis Type="Italic">Ponticoccus alexandrii</Emphasis> sp. nov., a novel bacterium isolated from the marine toxigenic dinoflagellate <Emphasis Type="Italic">Alexandrium minutum</Emphasis>

During an investigation of the biodiversity of the cultivable bacterial community associated with paralytic shellfish poisoning toxin-producing marine dinoflagellate, Alexandrium minutum a novel algal-associated bacterium, designated strain AT2-A^T was isolated. 16S rRNA gene sequence similarity analysis showed that the strain is a member of the genus Ponticoccus, with high sequence similarity to Ponticoccus litoralis DSM 18986^T (97.9%) and Ponticoccus lacteus JCM 30379^T (96.0%). However, based on the data obtained for the physiological and biochemical characteristics, and low level of DNA–DNA relatedness analysis, the strain could be genotypically and phenotypically differentiated from two type strains of the genus Ponticoccus. Therefore, this algal-associated bacterial strain is concluded to represent a novel species of the genus Ponticoccus, for which the name Ponticoccus alexandrii sp. nov. is proposed. The type strain is AT2-A^T (CCTCC AB 2017228 ^T = KCTC 52626 ^T ).     

<Emphasis Type="Italic">Spirosoma metallicus</Emphasis> sp. nov., isolated from an automobile air conditioning system

A Gram-stain-negative and orangish yellow-pigmented bacterial strain, designated PR1014K^T, was isolated from an automobile evaporator core collected in Korea. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain PR1014K^T was related with the members of the genus Spirosoma (94.7–90.2%) and closely related with Spirosoma lacussanchae CPCC 100624^T (94.7%), Spirosoma knui 15J8-12^T (94.3%), and Spirosoma soli MIMBbqt12^T (93.3%). The strain grew at 15–40°C (optimum, 25°C), pH 6.5–7.0 (optimum, 6.5) and 0–1% (w/v) NaCl (optimum, 0%). The predominant fatty acids were summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c), C_16:0, iso-C_15:0, C_16:1 ω5c, and iso-C_17:0 3-OH. The major menaquinone was MK-7. The polar lipid profile of the strain indicated that the presence of one phosphatidylethanolamine, one unidentified aminolipid, two unidentified aminophospholipids, and three unidentified lipids. The DNA G+C content of the strain was 47.4 mol%. On the basis of the phenotypic, genotypic and chemotaxonomic characteristics, strain PR1014K^T represents a novel species in the genus Spirosoma, for which the name Spirosoma metallicus sp. nov. (=KACC 17940^T =NBRC 110792^T) is proposed.     

Expressing accessory proteins in cellulolytic <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to improve the conversion yield of recalcitrant cellulose

Background

A recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate.

Results

The production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against β-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively.

Conclusions

This study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.

     

<Emphasis Type="Italic">Hoeflea prorocentri</Emphasis> sp. nov., isolated from a culture of the marine dinoflagellate <Emphasis Type="Italic">Prorocentrum mexicanum</Emphasis> PM01

A Gram-stain negative, aerobic, rod-shaped, non-motile, yellow-pigmented and non-spore-forming bacterial strain, designated PM5-8^T, was isolated from a culture of a marine toxigenic dinoflagellate Prorocentrum mexicanum PM01. Strain PM5-8^T grew at 15–35 °C (optimum, 25–30 °C) and pH 6–11 (optimum, 7.5–8). Cells required at least 1.5% (w/v) NaCl for growth, and can tolerate up to 7.0% with the optimum of 4%. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the strain PM5-8^T is closely related to members of the genus Hoeflea, with high sequence similarities with Hoeflea halophila JG120-1^T (97.06%) and Hoeflea alexandrii AM1V30^T (97.01%). DNA–DNA hybridization values between the isolate and other type strains of recognized species of the genus Hoeflea were between 11.8 and 25.2%, which is far below the value of 70% threshold for species delineation. The DNA G?+?C content was 50.3 mol%. The predominant cellular fatty acids of the strain were identified as summed feature 8 (C_16:1 ω7c and/or C_16:1 ω6c; 51.5%), C_18:1 ω7c 11-methyl (20.7%), C_16:0 (17.2%) and C_18:0 (5.7%). The major respiratory quinone was Q-10. Polar lipids profiles contained phosphatidylcholine, phosphatidylglycerol, sulfoquinovosyl diacylglycerol, phosphatidylmono- methylethanolamine, phosphatidylethanolamine and four unidentified lipids. On the basis of the polyphasic taxonomic data presented, strain PM5-8^T (=?CCTCC AB 2016294^T?=?KCTC 62490^T) represents a novel species of the genus Hoeflea, for which the name Hoeflea prorocentri sp. nov. is proposed.     

Irregular xylem 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis

Large quantities of mucilage are synthesized in seed coat epidermis cells during seed coat differentiation. This process is an ideal model system for the study of plant cell wall biosynthesis and modifications. In this study, we show that mutation in Irregular Xylem 7 (IRX7) results in a defect in mucilage adherence due to reduced xylan biosynthesis. IRX7 was expressed in the seeds from 4 days post-anthesis (DPA) to 13 DPA, with the peak of expression at 13 DPA. The seed coat epidermis cells of irx7 displayed no aberrant morphology during differentiation, and these cells synthesized and deposited the same amount of mucilage as did wild type (WT) cells. However, the distribution of the water-soluble vs. adherent mucilage layers was significantly altered in irx7 compared to the WT. Both the amount of xylose and the extent of glycosyl linkages of xylan was dramatically decreased in irx7 water-soluble and adherent mucilage compared to the WT. The polymeric structure of water-soluble mucilage was altered in irx7, with a total loss of the higher molecular weight polymer components present in the WT. Correspondingly, whole-seed immunolabeling assays and dot-immunoassays of extracted mucilage indicated dramatic changes in rhamnogalacturonan I (RG I) and xylan epitopes in irx7 mucilage. Furthermore, the crystalline cellulose content was significantly reduced in irx7 mucilage. Taken together, these results indicate that xylan synthesized by IRX7 plays an essential role in maintaining the adhesive property of seed coat mucilage, and its structural role is potentially implemented through its interaction with cellulose.