Characterization of the cellulase-secretome produced by the Antarctic bacterium Flavobacterium sp. AUG42

Flavobacterium sp. AUG42 is a cellulase-producing bacterium isolated from the Antarctic oligochaete Grania sp. (Annelida). In this work, we report that AUG42 produces a glycoside hydrolase cocktail with CMCase, PASCase and cellobiase activities (optimum pHs and temperatures ranging from 5.5 to 6.5 and 40 to 50 °C, respectively). The time-course analyses of the bacterial growth and cellulase production showed that the cocktail has maximal activity at the stationary phase when growing at 16 °C with filter paper as a cellulosic carbon source, among the tested substrates. The analyses of the CAZome and the identification of secreted proteins by shotgun Mass Spectrometry analysis showed that five glycoside hydrolyses are present in the bacterial secretome, which probably cooperate in the degradation of the cellulosic substrates. Two of these glycoside hydrolyses may harbor putative carbohydrate binding modules, both with a cleft-like active site. The cellulolytic cocktail was assayed in saccharification experiments using carboxymethylcellulose as a substrate and results showed the release of glucose (a fermentable sugar) and other reducing-sugars, after 24 h incubation. The ecological relevance of producing cellulases in the Antarctic environment, as well as their potential use in the bio-refinery industry, are discussed

Identification, recombinant production and partial biochemical characterization of an extracellular cold-active serine-metalloprotease from an Antarctic Pseudomonas isolate

Cold-adapted enzymes are generally derived from psychrophilic microorganisms and have features that make them very attractive for industrial and biotechnological purposes. In this work, we identified a 50 kDa extracellular protease (MP10) from the Antarctic isolate Pseudomonas sp. AU10. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and partially characterized. MP10 is an alkaline thermosensitive serine-metallo protease with optimal activity at pH 8.0 and 40 ℃, in the presence of 1.5 mM Ca2+. MP10 showed 100% residual activity and stability (up to 60 min) when incubated with 7% of non-ionic surfactants (Triton X-100, Tween-80 and Tween-20) and 1.5% of the oxidizing agent hydrogen peroxide. The 3D MP10 structure was predicted and compared with the crystal structure of mesophilic homologous protease produced by Pseudomonas aeruginosa PA01 (reference strain) and other proteases, showing similarity in surface area and volume of proteins, but a significantly higher surface pocket area and volume of MP10. The observed differences presumably may explain the enhanced activity of MP10 for substrate binding at low temperatures. These results give insight to the potential use of MP10 in developing new biotechnologically processes active at low to moderate temperatures, probably with focus in the detergent industry

Five-year bio-monitoring of aquatic ecosystems near Artigas Antarctic Scientific Base, King George Island

Fildes Peninsula, in King George Island, Antarctica, has a great concentration of international facilities, and it has clearly been affected by human activities. The objective of this 5-year study was to assess the impact of anthropogenic activities on the bacterial abundance in water bodies close to Artigas Antarctic Scientific Base (BCAA, in Spanish Base Científica Antártica Artigas). Water samples from areas under different human influence (Uruguay Lake, nearby ponds, and meltwater from Collins Glacier) were aseptically collected and refrigerated until processed. The number of heterotrophic bacteria and Pseudomonas spp. was analyzed using a culture-dependent approach. Physico-chemical properties of the water samples (temperature, pH, and conductivity) were also determined. Results showed that water from the highly affected area, Uruguay Lake, where the pump that provides water to the BCAA is located, did not suffer significant fluctuations in heterotrophic bacterial abundance (104–105 CFU∙mL−1); however, Pseudomonas abundance increased until becoming the predominant population. In other water samples, the number of heterotrophic bacteria and Pseudomonas gradually increased during this 5-year study, by 2014 reaching similar values to those observed for Uruguay Lake. The implications of human activities on Antarctic bacterial abundance are discussed

Lobophorin K, a new natural product with cytotoxic activity produced by Streptomyces sp. M-207 associated with the deep-sea coral Lophelia pertusa

The present article describes the isolation of a new natural product of the lobophorin family, designated as lobophorin K (1), from cultures of the marine actinobacteria Streptomyces sp. M-207, previously isolated from the cold-water coral Lophelia pertusa collected at 1800 m depth during an expedition to the submarine Avilés Canyon. Its structure was determined using a combination of spectroscopic techniques, mainly ESI-TOF MS and 1D and 2D NMR. This new natural product displayed cytotoxic activity against two human tumor cell lines, such as pancreatic carcinoma (MiaPaca-2) and breast adenocarcinoma (MCF-7). Lobophorin K also displayed moderate and selective antibiotic activity against pathogenic Gram-positive bacteria such as Staphylococcus aureus

Searching for glycosylated natural products in actinomycetes and identification of novel macrolactams and angucyclines

Many bioactive natural products are glycosylated compounds in which the sugar components usually participate in interaction and molecular recognition of the cellular target. Therefore, the presence of sugar moieties is important, in some cases essential, for bioactivity. Searching for novel glycosylated bioactive compounds is an important aim in the field of the research for natural products from actinomycetes. A great majority of these sugar moieties belong to the 6-deoxyhexoses and share two common biosynthetic steps catalyzed by a NDP-D-glucose synthase (GS) and a NDP-D-glucose 4,6-dehydratase (DH). Based on this fact, seventy one Streptomyces strains isolated from the integument of ants of the Tribe Attini were screened for the presence of biosynthetic gene clusters (BGCs) for glycosylated compounds. Total DNAs were analyzed by PCR amplification using oligo primers for GSs and DHs and also for a NDP-D-glucose-2,3-dehydratases. Amplicons were used in gene disruption experiments to generate non-producing mutants in the corresponding clusters. Eleven mutants were obtained and comparative dereplication analyses between the wild type strains and the corresponding mutants allowed in some cases the identification of the compound coded by the corresponding cluster (lobophorins, vicenistatin, chromomycins and benzanthrins) and that of two novel macrolactams (sipanmycin A and B). Several strains did not show UPLC differential peaks between the wild type strain and mutant profiles. However, after genome sequencing of these strains, the activation of the expression of two clusters was achieved by using nutritional and genetic approaches leading to the identification of compounds of the cervimycins family and two novel members of the warkmycins family. Our work defines a useful strategy for the identification new glycosylated compounds by a combination of genome mining, gene inactivation experiments and the activation of silent biosynthetic clusters in Streptomyces strains

Vitrescent dark spot of peach: a new fruit calcium-related disorder?

Comunicación oral (S10-O3) presentada en la Reunión de la Sociedad Española de Fisiología Vegetal (SEFV) (18ª. Zaragoza. 8-11 septiembre 2009).Late-season peach cultivars are a specialty of the Calanda area, (Zaragoza, Spain) and the only horticultural commodity protected under a designation of origin in Spain. Such peaches are bagged shortly after thinning and are sold as agrochemical-free due to the limited exposure to sprays and also to pest and diseases. Due to their outstanding quality, Calanda peaches a have great market potential and Spanish consumers already show a clear preference for such commodities. However, the occurrence of a physiological disorder called “vitrescent dark spot” has been reported by growers in the area already more than 15 years ago[1]. The major decrease in quality and associated loss of marketability of peaches affected by such disorder, claim for the development of scientific efforts to characterise it and prevent it in the future. Work over the last 3 years enabled a preliminary characterisation of such unknown peach disorder, which will be herein contrasted with a well-know calcium (Ca)-related disorder of apple, i.e., bitter pit[2][3]. Results regarding tissue structure, protein patterns, induction of damaged tissues and mineral element composition of peach and apple affected areas such that “vitrescent dark spot” of peach may be related to Ca metabolism alike bitter pit in apple. A comprehensive comparison of sound, affected and adjacent tissues relating to such parameters (i.e., tissue structure, protein pattern, induction of damaged tissues and mineral element composition) will be provided, which supports the hypothesis that vitrescent dark spot may be a Ca-related disorder.Peer reviewe

Improving the performance of calcium-containing spray formulations to limit the incidence of bitter pit in apple (Malus x domestica Borkh.)

17 Pag., 3 Tabl., 3 Fig.Laboratory and field experiments were carried out with apples (Malus x domestica Borkh.) cv. ‘Golden Reinders’, to assess the efficacy of sodium salt of carboxymethyl ether of cellulose (0.5%, CMC) as an adjuvant for Ca spray formulations containing either Ca-chloride or Ca-propionate as active ingredient (120 or 250 mM Ca). This additive significantly increased the retention of Ca-containing solutions by the apple skin and prolonged the process of drying of the solution at room temperature. Four days after immersion of apples in 0.5% CMC plus CaCl2 or Ca-propionate solutions (120 and 250 mM Ca) significant Ca increases were recorded in the peel and cortex of treated fruits. Application to apple trees of in-season sprays containing 250 mM CaCl2 plus 0.05% Tween 20, Ca-propionate (120 and 250 mM Ca) plus 0.5% CMC or 250 mM CaCl2 plus 0.5% CMC had no impact on fruit yield and quality, but significantly limited the rate of bitter pit incidence during the following 3-month cold-storage period. Evidence is provided that addition of appropriate adjuvants to Ca sprays can favour the distribution of Ca into the apple fruit and helps to reduce the incidence of Ca-related disorders over the postharvest cold-storage period.This work was financed by MICINN (Spanish Ministry of Science and Innovation) and FEDER funds under the projects INIA, PET2007-09-C5 and AGL2009-08501. Work of Victoria Fernández was supported by a “Juan de la Cierva” MEC post-doctoral contract, co-financed by the European Social Fund. Currently, she is supported by a “Torres Quevedo contract” (MICINN-PTQ), also co-financed by the European Social Fund.Peer reviewe