Overexpression of <Emphasis Type="Italic">flv3</Emphasis> improves photosynthesis in the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803 by enhancement of alternative electron flow

Background

To ensure reliable sources of energy and raw materials, the utilization of sustainable biomass has considerable advantages over petroleum-based energy sources. Photosynthetic algae have attracted attention as a third-generation feedstock for biofuel production, because algae cultivation does not directly compete with agricultural resources, including the requirement for productive land and fresh water. In particular, cyanobacteria are a promising biomass feedstock because of their high photosynthetic capability.

Results

In the present study, the expression of the flv3 gene, which encodes a flavodiiron protein involved in alternative electron flow (AEF) associated with NADPH-coupled O₂ photoreduction in photosystem I, was enhanced in Synechocystis sp. PCC6803. Overexpression of flv3 improved cell growth with corresponding increases in O₂ evolution, intracellular ATP level, and turnover of the Calvin cycle. The combination of in vivo¹³C-labeling of metabolites and metabolomic analysis confirmed that the photosynthetic carbon flow was enhanced in the flv3-overexpressing strain.

Conclusions

Overexpression of flv3 improved cell growth and glycogen production in the recombinant Synechocystis sp. PCC6803. Direct measurement of metabolic turnover provided conclusive evidence that CO₂ incorporation is enhanced by the flv3 overexpression. Increase in O₂ evolution and ATP accumulation indicates enhancement of the AEF. Overexpression of flv3 improves photosynthesis in the Synechocystis sp. PCC6803 by enhancement of the AEF.

     

Inactivation of virginiamycin by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Objective

To test the inactivation of the antibiotic, virginiamycin, by laccase-induced culture supernatants of Aureobasidium pullulans.

Results

Fourteen strains of A. pullulans from phylogenetic clade 7 were tested for laccase production. Three laccase-producing strains from this group and three previously identified strains from clade 5 were compared for inactivation of virginiamycin. Laccase-induced culture supernatants from clade 7 strains were more effective at inactivation of virginiamycin, particularly at 50 °C. Clade 7 strain NRRL Y-2567 inactivated 6 µg virginiamycin/ml within 24 h. HPLC analyses indicated that virginiamycin was degraded by A. pullulans.

Conclusions

A. pullulans has the potential for the bioremediation of virginiamycin-contaminated materials, such as distiller’s dry grains with solubles (DDGS) animal feed produced from corn-based fuel ethanol production.

     

Characterization of an aryl-alcohol oxidase from the plant saprophytic basidiomycete <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis> with broad substrate specificity against aromatic alcohols

Objectives

The aim of the study was to obtain information about the enzymatic properties of aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea (rCcAAO), which is classified into the auxiliary activities family 3 subfamily 2 (AA3_2).

Results

The gene encoding AAO from the plant saprophytic basidiomycete Coprinopsis cinerea (CcAAO) was cloned, and the recombinant CcAAO (rCcAAO) was heterologously expressed in the methylotrophic yeast Pichia pastoris. The purified rCcAAO showed significant activity not only against trans,trans-2,4-hexadien-1-ol but also against a broad range of aromatic alcohols including aromatic compounds that were reported to be poor substrates for known AAOs. Moreover, site-directed mutagenesis analysis demonstrated that mutants with substitutions from leucine to phenylalanine and tryptophan at position 416 exhibited decreases of activity for aromatic alcohols but still maintained the activity for trans,trans-2,4-hexadien-1-ol.

Conclusions

Leucine 416 in CcAAO contributes to the broad substrate specificity against various aromatic alcohols, which is useful for the production of hydrogen peroxide using this enzyme.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Niches and routes of transmission of <Emphasis Type="Italic">Xanthomonas citri</Emphasis> pv. <Emphasis Type="Italic">fuscans</Emphasis> to bean seeds

Aims

Seeds are vectors of a diversified microbiota including plant pathogens. To better understand transmission of common bacterial blight (CBB) agents to bean seeds, we analyzed the role of non-pathogenic xanthomonads on seed transmission efficiency and investigated the location of Xanthomonas citri pv. fuscans (Xcf) into seeds and plantlets.

Methods

Competition between CBB and NP strains was initially assessed in vitro and then extended in planta to monitor the impact of co-inoculation on Xcf seed transmission. Moreover, location of Xcf strains in seeds and seedlings was visualized using a combination of gfp-tagged strain and DOPE-FISH/CSLM.

Results

Whereas CBB agent growth was inhibited in vitro by some seed-borne non-pathogenic xanthomonads strains, these strains did not transmit efficiently to seed through floral pathway and did not affect Xcf seed transmission. Xcf cells were observed entering seed through vascular elements and parenchyma of funiculus, but also micropyle and testa. Xcf cells were observed, moreover, among other bacteria on radicle surfaces, especially tip, in cotyledons, and plumules.

Conclusions

CBB agents are more efficient than non-pathogenic xanthomonads in using the floral route to colonize seeds. CBB agents are located within different niches in the seed tissues up to the embryonic axis.

     

<Emphasis Type="Italic">massPix</Emphasis>: an R package for annotation and interpretation of mass spectrometry imaging data for lipidomics

Introduction

Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools.

Objectives

We have developed massPix—an R package for analysing and interpreting data from MSI of lipids in tissue.

Methods

massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries.

Results

Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering.

Conclusion

massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications.

     

<Emphasis Type="Italic">Rhizobium</Emphasis> strains in the biological control of the phytopathogenic fungi <Emphasis Type="Italic">Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>) <Emphasis Type="Italic">rolfsii</Emphasis> on the common bean

Aims

To identify Rhizobium strains’ ability to biocontrol Sclerotium rolfsii, a fungus that causes serious damage to the common bean and other important crops, 78 previously isolated rhizobia from common bean were assessed.

Methods

Dual cultures, volatiles, indole-acetic acid (IAA), siderophore production and 16S rRNA sequencing were employed to select strains for pot and field experiments.

Results

Thirty-three antagonistic strains were detected in dual cultures, 16 of which were able to inhibit ≥84% fungus mycelial growth. Antagonistic strains produced up to 36.5 μg mL^?1 of IAA, and a direct correlation was verified between IAA production and mycelium inhibition. SEMIA 460 inhibited 45% of mycelial growth through volatile compounds. 16S rRNA sequences confirmed strains as Rhizobium species. In pot condition, common bean plants grown on S. rolfsii-infested soil and inoculated with SEMIA 4032, 4077, 4088, 4080, 4085, or 439 presented less or no disease symptoms. The most efficient strains under field conditions, SEMIA 439 and 4088, decreased disease incidence by 18.3 and 14.5% of the S. rolfsii-infested control.

Conclusions

Rhizobium strains could be strong antagonists towards S. rolfsii growth. SEMIA 4032, 4077, 4088, 4080, 4085, and 439 are effective in the biological control of the collar rot of the common bean.

     

Emergence of Resistance to Fluconazole in <Emphasis Type="Italic">Candida albicans</Emphasis> Isolated From Vaginal Discharge

Purpose of Review

To provide information about the emergence of fluconazole resistance in Candida albicans isolated from vaginal discharge, in a global context, and to update the in vitro susceptibility profile of this species from Argentina.

Recent Findings

Vulvovaginal candidiasis is the second most common vaginal infection after vaginal bacteriosis. C. albicans remains the prevalent etiological yeast species, and despite antifungal treatment, the rate of recurrence remains high, which may be associated to antifungal resistance.

Summary

Data here presented were obtained from the study of C. albicans strains isolated from patients with clinical signs of vulvovaginal candidiasis from 1996 to 2017. Data obtained could represent the susceptibility profile of C. albicans strains circulating in Argentina and could be of potential usefulness to monitor and guide therapy, and also suggests the need for greater surveillance programs to detect fluconazole resistance over time.

     

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Objective

To develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential.

Results

Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain.

Conclusion

A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.

     

Diverse bacterial taxa inhabit root nodules of lucerne (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) in New Zealand pastoral soils

Background and aims

Pseudomonas spp. have previously been isolated from lucerne nodules. The aims of this study were to: 1) investigate the microbiome within a lucerne nodule; and 2) assess the ability of two Pseudomonas spp. isolated from lucerne nodules to form nodules.

Methods

The microbial community within 27 lucerne nodules, collected from plants inoculated with Sinorhizobium meliloti as a seed coat or peat slurry and an uninoculated control, was identified using 16S rRNA based Illumina sequencing. Lucerne seedlings were inoculated with the two Pseudomonas spp. strains. The plants were grown in sterile conditions for 6 weeks and nodulation was assessed. 16S rRNA, nodC, nodA and nifH genes were amplified.

Results

Sinorhizobium was the dominant genus in nodules, comprising 90–99% of all sequences regardless of inoculation treatment. Overall, 9 other genera were identified, with each represented by <3% of the total sequences. Both Pseudomonas strains were able to form nodules with lucerne. From one of these strains, a nodC gene was detected.

Conclusion

Lucerne nodules contained a diverse assemblage of bacterial species, some of which were capable of forming nodules in the absence of rhizobia.

     

Effects of temperature on competition and relative dominance of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">Bradyrhizobium elkanii</Emphasis> in the process of soybean nodulation

Background and aims

Bradyrhizobium japonicum and Bradyrhizobium elkanii dominated soybean nodules in temperate and subtropical regions in Nepal, respectively, in our previous study. The aims of this study were to reveal the effects of temperature on the nodulation dominancy of B. japonicum and B. elkanii and to clarify the relationship between the effects of temperature and the climate-dependent distribution of Bradyrhizobium species.

Methods

A laboratory competition experiment was conducted between B. japonicum and B. elkanii strains isolated from the same temperate location in Nepal. A mixture of each strain was inoculated into sterilized vermiculite with or without soybean seeds, and inoculated samples were incubated at 33/27 (day/night) and 23/17 °C. Relative populations in the non-rhizosphere, rhizosphere, and nodules were determined by competitive PCR using specific primers for each strain at 0, 1, 2, and 4 weeks after inoculation.

Results

Both separately inoculated B. japonicum and B. elkanii strains formed nodules at both temperatures. Under competitive conditions, B. japonicum strains dominated at low temperature; however, at high temperature, both strains achieved co-nodulation in 1 week, with B. elkanii dominating after 2 weeks. The relative populations of both strains were similar in the non-rhizosphere and rhizosphere at low temperature, but B. elkanii strains dominated in these regions at high temperature.

Conclusions

The domination of B. japonicum strains in nodules at the low temperature appeared to be due to preferential infection, while the domination of B. elkanii strains at high temperature appeared to be due to the higher population of B. elkanii in the non-rhizosphere and rhizosphere, in addition to its domination in nodules after co-nodulation. The effects of temperature on the competition between B. japonicum and B. elkanii strains were remarkable and corresponded with the distribution of bradyrhizobial species in Nepal.

     

Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803

Background

Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria.

Results

Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent.

Conclusions

Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.     

Study on the Clinical Features and Prognosis of <Emphasis Type="Italic">Penicilliosis marneffei</Emphasis> Without Human Immunodeficiency Virus Infection

Objective

To improve the diagnosis and treatment of Penicilliosis marneffei without human immunodeficiency virus infection.

Methods

Analyze and review the clinical features, diagnosis and treatment of six cases of P. marneffei without human immunodeficiency virus infection at The First Affiliated Hospital of Fujian Medical University.

Results

Two cases were diagnosed in the ENT Department, three cases in the respiratory department and one case in the dermatological department. Penicillium marneffei infection was confirmed by sputum culture, blood culture and tissue biopsy. After definite diagnosis, one refused further treatment, and others showed significant improvement.

Conclusion

Penicilliosis marneffei is insidious onset and easy to be escaped and misdiagnosed. To achieve early diagnosis and appropriate treatment, doubtful cases should be alerted for the diagnoses as P. marneffei.

     

A versatile <Emphasis Type="Italic">Trichoderma reesei</Emphasis> expression system for the production of heterologous proteins

Objectives

To develop a versatile Trichoderma reesei (teleomorph Hypocrea jecorina) expression system for the high-purity production of heterologous proteins.

Results

The versatile T. reesei expression system is based on xyn1 and xyn2 promoters, A824V transition in XYRI, and a bicomponent carbon source strategy. Red fluorescent protein gene rfp and alkaline endoglucanase EGV gene egv3 from Humicola insolens were used as reporter genes to test our versatile expression system

Conclusions

The versatile T. reesei expression system can be applied to produce heterologous proteins with high purity and high yield.

     

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.