Publications

Publications

Publications between 2011-2015

60. "TITLE"
AUTHORS
Genomics, doi: (YEAR). Request copy
Abstract:TEXT

67. " The Kolumbo submarine volcano of Santorini island is a large pool of bacterial strains with antimicrobial activity"
M. Bourbouli, E.A. Katsifas, E. Papathanassiou, A.D. Karagouni
Archives of Microbiology, doi:10.1007/s00203-015-1086-3 (2015). Request copy
Abstract: Microbes in hydrothermal vents with their unique secondary metabolism may represent an untapped potential source of new natural products. In this study, samples were collected from the hydrothermal field of Kolumbo submarine volcano in the Aegean Sea, in order to isolate bacteria with antimicrobial activity. Eight hundred and thirty-two aerobic heterotrophic bacteria were isolated and then differentiated through BOX-PCR analysis at the strain level into 230 genomic fingerprints, which were screened against 13 different type strains (pathogenic and nonpathogenic) of Gram-positive, Gram-negative bacteria and fungi. Forty-two out of 176 bioactive-producing genotypes (76 %) exhibited antimicrobial activity against at least four different type strains and were selected for 16S rDNA sequencing and screening for nonribosomal peptide (NRPS) and polyketide (PKS) synthases genes. The isolates were assigned to genus Bacillus and Proteobacteria, and 20 strains harbored either NRPS, PKS type I or both genes. This is the first report on the diversity of culturable mesophilic bacteria associated with antimicrobial activity from Kolumbo area; the extremely high proportion of antimicrobial-producing strains suggested that this unique environment may represent a potential reservoir of novel bioactive compounds.

66. "Insights into the functionality and stability of designer cellulosomes at elevated temperatures"
A.P. Galanopoulou, S. Morais, A. Georgoulis, E. Morag, E.A. Bayer, D.G. Hatzinikolaou
Applied Microbiology and Biotechnology, doi: 1007/s00253-016-7594-5 (2016). Request copy
Abstract:Enzymatic breakdown of lignocellulose is a major limiting step in second generation biorefineries. Assembly of the necessary activities into designer cellulosomes increases the productivity of this step by enhancing enzyme synergy through the proximity effect. However, most cellulosomal components are obtained from mesophilic microorganisms, limiting the applications to temperatures up to 50 degrees C. We hypothesized that a scaffoldin, comprising modular components of mainly mesophilic origin, can function at higher temperatures when combined with thermophilic enzymes, and the resulting designer cellulosomes could be employed in higher temperature reactions. For this purpose, we used a tetravalent scaffoldin constituted of three cohesins of mesophilic origin as well as a cohesin and cellulose-binding module derived from the thermophilic bacterium Clostridium thermocellum. The scaffoldin was combined with four thermophilic enzymes from Geobacillus and Caldicellulosiruptor species, each fused with a dockerin whose specificity matched one of the cohesins. We initially verified that the biochemical properties and thermal stability of the resulting chimeric enzymes were not affected by the presence of the mesophilic dockerins. Then we examined the stability of the individual single-enzyme-scaffoldin complexes and the full tetravalent cellulosome showing that all complexes are stable and functional for at least 6 h at 60 degrees C. Finally, within this time frame and conditions, the full complex appeared over 50 % more efficient in the hydrolysis of corn stover compared to the free enzymes. Overall, the results support the utilization of scaffoldin components of mesophilic origin at relatively high temperatures and provide a framework for the production of designer cellulosomes suitable for high temperature biorefinery applications.

65. Purine utilization proteins in the Eurotiales: Cellular compartmentalization, phylogenetic conservation and divergence K. Galanopoulou, C. Scazzocchio, M.E. Galinou, W.W. Liu, F. Borbolis, M.Karachaliou, N. Oestreicher, D.G. Hatzinikolaou, G. Diallinas, S. Amillis Fungal Genetics and Biology, doi:10.1016/j.fgb.2014.06.005 (2014) Abstract:The purine utilization pathway has been thoroughly characterized in Aspergillus nidulans. We establish here the subcellular distribution of seven key intracellular enzymes, xanthine dehydrogenase (HxA), urate oxidase (UaZ), 5-hydroxy-isourate hydrolase (UaX), 2-oxo-4-hydroxy-4-carboxy ureido imidazoline decarboxylase (UaW), allantoinase (AlX), allantoicase (AaX), ureidoglycolate lyase (UglA), and the fungal-specific alpha-ketoglutarate Fe(II)-dependent dioxygenase (XanA). HxA, AlX, AaX, UaW and XanA are cytosolic, while UaZ, UaX and UglA are peroxisomal. Peroxisomal localization was confirmed by using appropriate pex mutants. The pathway is largely, but not completely conserved in the Eurotiomycetes, noticeably in some species AaX is substituted by an alternative enzyme of probable bacterial origin. UaZ and the urate-xanthine UapA and UapC transporters, are also localized in specific cells of the conidiophore. We show that metabolic accumulation of uric acid occurring in uaZ null mutations is associated with an increased frequency of appearance of morphologically distinct colony sectors, diminished conidiospore production, UV resistance and an altered response to oxidation stress, which may provide a rationale for the conidiophore-specific localization. The pathway-specific transcription factor UaY is localized in both the cytoplasm and nuclei under non-inducing conditions, but it rapidly accumulates exclusively to the nuclei upon induction by uric acid.

64. Evaluation of Paecilomyces variotii potential in bioethanol production from lignocellulose through consolidated bioprocessing A. Zerva, A.L. Savvides, E.A. Katsifas, A.D. Karagouni, D.G. Hatzinikolaou Bioresource Technology, doi:10.1016/j.biortech.2014.03.137 (2014) Abstract:The ascomycete Paecillomyces variotii was evaluated for the first time as a candidate species for the production of bioethanol from lignocellulose through consolidated bioprocessing (CBP) approaches. The examined strain (ATHUM 8891) revealed all the necessary phenotypic characteristics required for 2nd generation biofuel production. The fungus is able to efficiently ferment glucose and xylose to ethanol, with yields close to the theoretical maximum. Nitrogen supplementation greatly affected ethanol production with nitrate-nitrogen presenting the best results. Notably, ethanol yield on xylose fermentation was higher than that of glucose, while in co-fermentation of glucose-xylose mixtures no distinguished diauxic behavior was observed. Furthermore, the fungus seems to possess the necessary enzyme factory for the degradation of lignocellulosic biomass, as it was able to grow and produce ethanol on common agro-industrial derivatives. Overall, the results of our study indicate that P. variotii is a new and possibly powerful candidate for CBP applications.

63. “Bacterial Deterioration of Marble Monuments: A Case Study of the Conservation Project of Acropolis Monuments” A.L. Savvides, T.L. Nikolakopoulou, N. Kyratsous, E.A. Katsifas, G. Kanini, A.D. Karagouni Geomicrobiology Journal, doi:10.1080/01490451.2014.885618, (2014) Abstract:The deterioration of the Acropolis monuments' surface can be attributed to a combination of mechanical, physical, chemical and biological factors. The aim of this work was to investigate the potential use of biocides suitable for marble surface in order to confront the biodeterioration phenomenon due to heterotrophic bacteria. Surface bacterial populations were studied in respect to the application of three biocide agents. A significant decrease of the bacterial counts was occurred after the biocides' application and remained less compared with the counts occurred on the untreated surfaces for more than 14 months. A gradual diminution of bacterial diversity was also observed leading to the survival of specific Gram positive strains.

62. Constitutive homologous expression of phosphoglucomutase and transaldolase increases the metabolic flux of Fusarium oxysporum G.E. Anasontzis, E. Kourtoglou, D. Mamma, S.G. Villas-Boas, D.G. Hatzinikolaou, P. Christakopoulos Microbial Cell Factories, DOI10.1186/1475-2859-13-43 (2014) Abstract:Background: Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism. Results: Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher biomass to xylose yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth on glucose revealed higher abundance of most determined metabolites between 1.5-and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source. Conclusions: Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics.

61. Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum E. Kourtoglou, G.E. Anasontzis, D. Mamma, E. Topakas, D.G. Hatzinikolaou, P. Christakopoulos Enzyme And Microbial Technology, doi:10.1016/j.enzmictec.2010.10.007 (2011) Abstract:The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45 degrees C but the enzyme became unstable at temperatures above 40 degrees C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co2+. The apparent K-m for Co2+ was found to be 10 mu M. The enzyme was also active with other divalent metal ions such as Mn2+, Mg2+, Ni2+ and Ca2+ but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 mu mol mg(protein)(-1) min(-1); k(cat), 44.2 min(-1); K-m(G1P), 0.10 mM: K-m(G1,6diP), 1.03 mu M; k(cat)/K-m(G1P), 443 mM(-1) min(-1) and k(cat)/K-m(G1,6diP), 42,860 mM(-1) min(-1). The enzyme was considered to follow a Ping Pang substituted enzyme or enzyme isomerization mechanism.