Barcoded Asaia bacteria enable mosquito in vivo screens and identify novel systemic insecticides and inhibitors of malaria transmission.

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control.

Production of high crystallinity type-I cellulose from Komagataeibacter hansenii JR-02 isolated from Kombucha tea.

In this study, a bacterial cellulose (BC) producing strain was isolated from Kombucha tea and identified as Komagataeibacter hansenii JR-02 by morphological, physiological, and biochemical characterization and 16S rRNA sequence. Then, the media components and culture conditions for BC production were optimized. Result showed that the highest BC yield was 3.14 ± 0.22 and 8.36 ± 0.19 g/L after fermentation for 7 days under shaking and static cultivation, respectively. Moreover, it was interesting that JR-02 could produce BC in nitrogen-free medium with the highest yield of 0.76 ± 0.06 g/L/7days, and the possible nitrogen fixation gene nifH was cloned from its genomic DNA. The BC produced by JR-02 was type-I cellulose with high crystallinity and thermodynamic stability, which was revealed from Fourier transform infrared spectroscopy, X-ray diffraction, and thermogravimetric analysis methods. The crystallinity of static and shaking cultured BC were 91.76% and 90.69%, respectively. The maximum rate of weight loss of static and shaking BC occurred at temperature of approximately 373.1 °C and 369.1 °C, respectively. Overall, these results indicated that K. hansenii JR-02 had great potential to produce high crystallinity type-I BC in manufacture.

Endobacter medicaginis gen. nov., sp. nov., isolated from alfalfa nodules in an acidic soil.

A bacterial strain designated M1MS02(T) was isolated from a surface-sterilized nodule of Medicago sativa in Zamora (Spain). The 16S rRNA gene sequence of this strain showed 96.5 and 96.2 % similarity, respectively, with respect to Gluconacetobacter liquefaciens IFO 12388(T) and Granulibacter bethesdensis CGDNIH1(T) from the family Acetobacteraceae. The novel isolate was a Gram-stain-negative, non-sporulating, aerobic coccoid to rod-shaped bacterium that was motile by a subpolar flagellum. The major fatty acid was C18 : 1ω7c and the major ubiquinone was Q-10. The lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, two aminophospholipids, three aminolipids, four glycolipids, two phospholipids and one lipid. Strain M1MS02(T) was catalase-positive and oxidase- and urease-negative. Acetate and lactate were not oxidized. Acetic acid was produced from ethanol in culture media supplemented with 2 % CaCO3. Ammonium sulphate was assimilated in glucose medium. The strain produced dihydroxyacetone from glycerol. Phylogenetic and phenotypic analyses commonly used to differentiate genera within the family Acetobacteraceae showed that strain M1MS02(T) should be classified as representing a novel species of a new genus within this family, for which the name Endobacter medicaginis gen. nov., sp. nov. is proposed. The type strain of the type species is M1MS02(T) ( = LMG 26838(T) = CECT 8088(T)). To our knowledge, this is the first report of a member of the Acetobacteraceae occurring as a legume nodule endophyte.

Novel nitrogen-fixing acetic acid bacteria, Gluconacetobacter johannae sp. nov. and Gluconacetobacter azotocaptans sp. nov., associated with coffee plants.

Diazotrophic bacteria were isolated, in two different years, from the rhizosphere and rhizoplane of coffee (Coffea arabica L.) plants cultivated in Mexico; they were designated as type DOR and type SAd isolates. They showed characteristics of the family Acetobacteraceae, having some features in common with Gluconacetobacter (formerly Acetobacter) diazotrophicus, the only known N2-fixing species of the acetic acid bacteria, but they differed from this species with regard to several characteristics. Type DOR isolates can be differentiated phenotypically from type SAd isolates. Type DOR isolates and type SAd isolates can both be differentiated from Gluconacetobacter diazotrophicus by their growth features on culture media, their use of amino acids as nitrogen sources and their carbon-source usage. These results, together with the electrophoretic mobility patterns of metabolic enzymes and amplified rDNA restriction analysis, suggested that the type DOR and type SAd isolates represent two novel N2-fixing species. Comparative analysis of the 16S rRNA sequences revealed that strains CFN-Cf55T (type DOR isolate) and CFN-Ca54T (type SAd isolate) were closer to Gluconacetobacter diazotrophicus (both strains had sequence similarities of 98.3%) than to Gluconacetobacter liquefaciens, Gluconacetobacter sacchari (similarities < 98%) or any other acetobacteria. Strain CFN-Cf55T exhibited low levels of DNA-DNA reassociation with type SAd isolates (mean 42%) and strain CFN-Ca54T exhibited mean DNA-DNA reassociation of 39.5% with type DOR isolates. Strains CFN-Cf55T and CFN-Ca54T exhibited very low DNA reassociation levels, 7-21%, with other closely related acetobacterial species. On the basis of these results, two novel N2-fixing species are proposed for the family Acetobacteraceae, Gluconacetobacter johannae sp. nov. (for the type DOR isolates), with strain CFN-Cf55T (= ATCC 700987T = DSM 13595T) as the type strain, and Gluconacetobacter azotocaptans sp. nov. (for the type SAd isolates), with strain CFN-Ca54T (= ATCC 70098ST = DSM 13594T) as the type strain.

Microbial reduction of Fe(III) in acidic sediments: isolation of Acidiphilium cryptum JF-5 capable of coupling the reduction of Fe(III) to the oxidation of glucose.

To evaluate the microbial populations involved in the reduction of Fe(III) in an acidic, iron-rich sediment, the anaerobic flow of supplemental carbon and reductant was evaluated in sediment microcosms at the in situ temperature of 12 degrees C. Supplemental glucose and cellobiose stimulated the formation of Fe(II); 42 and 21% of the reducing equivalents that were theoretically obtained from glucose and cellobiose, respectively, were recovered in Fe(II). Likewise, supplemental H(2) was consumed by acidic sediments and yielded additional amounts of Fe(II) in a ratio of approximately 1:2. In contrast, supplemental lactate did not stimulate the formation of Fe(II). Supplemental acetate was not consumed and inhibited the formation of Fe(II). Most-probable-number estimates demonstrated that glucose-utilizing acidophilic Fe(III)-reducing bacteria approximated to 1% of the total direct counts of 4', 6-diamidino-2-phenylindole-stained bacteria. From the highest growth-positive dilution of the most-probable-number series at pH 2. 3 supplemented with glucose, an isolate, JF-5, that could dissimilate Fe(III) was obtained. JF-5 was an acidophilic, gram-negative, facultative anaerobe that completely oxidized the following substrates via the dissimilation of Fe(III): glucose, fructose, xylose, ethanol, glycerol, malate, glutamate, fumarate, citrate, succinate, and H(2). Growth and the reduction of Fe(III) did not occur in the presence of acetate. Cells of JF-5 grown under Fe(III)-reducing conditions formed blebs, i.e., protrusions that were still in contact with the cytoplasmic membrane. Analysis of the 16S rRNA gene sequence of JF-5 demonstrated that it was closely related to an Australian isolate of Acidiphilium cryptum (99.6% sequence similarity), an organism not previously shown to couple the complete oxidation of sugars to the reduction of Fe(III). These collective results indicate that the in situ reduction of Fe(III) in acidic sediments can be mediated by heterotrophic Acidiphilium species that are capable of coupling the reduction of Fe(III) to the complete oxidation of a large variety of substrates including glucose and H(2).