Lipid production from indigenous Greek microalgae: a possible biodiesel source.

OBJECTIVE: Microalgae gained interest for potential use as biodiesel producers, since they synthesize and accumulate significant quantities of lipids. The aim of this work was to isolate indigenous microalgae strains from Greek habitats, study their physicochemical growth conditions and finally select the best ones with respect to overall lipid production and profile. RESULTS: Two sampling sites of marine aquatic ecosystems were selected in Attica prefecture, Greece in order to screen for novel wild type strains with lipid production capacity. Microalgae isolates (59) were obtained from the selected areas and were morphologically and molecularly characterized. Fatty acids were estimated through Flow Cytometry combined with BODIPY staining method. Four isolates were selected for their lipid production properties and were cultivated in 15 L tank cultures. The four isolates were also identified by 18S rDNA gene sequencing. Two of them, Chlorella sp. ΑCΑ9 and ACA17, exhibited both maximum biomass and lipid productivity. Optimization of growth conditions with respect to pH and initial NaNO3 concentration was performed for the two microalgae in 15 L cultures. Finally, 20 L fed batch cultures were set up using the optimum culture conditions. Lipid profiles were stabilized for both strains at dry biomass levels over 1 g L-1 and lipid content of 25% (w/w). CONCLUSIONS: Two Chlorella strains (ACA9 and ACA17) were promising candidates for biodiesel production as they were easily grown in sea water in fed batch systems and produce lipids suitable for biodiesel-especially Chlorella sp. ACA9.

The Kolumbo submarine volcano of Santorini island is a large pool of bacterial strains with antimicrobial activity.

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.

Frequent gene fissions associated with human pathogenic bacteria.

Gene fusion and fission events are important for evolutionary studies and for predicting protein-protein interactions. Previous studies have shown that fusion events always predominate over fission events and, in their majority, they represent singular events throughout evolution. In this project, the role of fusion and fission events in the genome evolution of 104 human bacterial pathogens was studied. 141 protein pairs were identified to be involved in gene fusion or fission events. Surprisingly, we find that, in the species analyzed, gene fissions prevail over fusions. Moreover, while most events appear to have occurred only once in evolution, 23% of the gene fusion and fission events identified are deduced to have occurred independently multiple times. Comparison of the analyzed bacteria with non-pathogenic close relatives indicates that this impressive result is associated with the recent evolutionary history of the human bacterial pathogens, and thus is probably caused by their pathogenic lifestyle.

Carbapenemase-producing Pseudomonas aeruginosa from central Greece: molecular epidemiology and genetic analysis of class I integrons.

BACKGROUND: Multidrug-resistant Pseudomonas aeruginosa is a serious challenge for antimicrobial therapy of nosocomial infections, as it possesses several mechanisms of antimicrobial resistance. In Central Greece, a sudden increase of infections caused by carbapenem-resistant P. aeruginosa was observed during 2011, indicating the need for further analysis. METHODS: Five-hundred and sixty-eight P. aeruginosa isolates were collected consecutively during an 8-month period in 2011 from inpatients treated in three hospitals in the Thessaly region (1,000,000 habitants) of Greece. Carbapenem-resistant P. aeruginosa (n = 284) were characterized by antimicrobial susceptibility testing and ß-lactamase content, and the genetic relatedness of carbapenemase-producing isolates was assessed by BOX-PCR, multilocus sequence typing, and eBURST analysis. Mapping of the class I integrons of Verona integron-encoded metallo-ß-lactamase (VIM)-carrying isolates was also performed, and clinical data of the VIM producers were reviewed. RESULTS: Eighty (14.1%) out of the 568 P. aeruginosa isolates recovered from clinical specimens were VIM producers. Multilocus sequence typing revealed high prevalence of the international clones ST111 and ST235 among blaVIM-2- and blaVIM-4-positive isolates, respectively. blaVIM-17 was identified in an isolate of a novel sequence type (ST1457). blaVIM gene cassettes were carried by five distinct class I integrons, including two novel ones. CONCLUSIONS: Since the first report of VIM-producing P. aeruginosa in 2000, this microorganism still remains among the most prevalent multidrug resistant pathogens in Greece. The spread of VIM-producers belonging to the most common international clones (ST111 and ST235), the spread of integrons of divergent structures, and the emergence of novel integrons underscore their ongoing evolution.

Assessment of the biomass hydrolysis potential in bacterial isolates from a volcanic environment: biosynthesis of the corresponding activities.

The biomass degrading enzymatic potential of 101 thermophilic bacterial strains isolated from a volcanic environment (Santorini, Aegean Sea, Greece) was assessed. 80 % of the strains showed xylanolytic activity in Congo Red plates, while only eight could simultaneously hydrolyze cellulose. Fifteen isolates were selected on the basis of their increased enzyme production, the majority of which was identified as Geobacilli through 16S rDNA analysis. In addition, the enzymatic profile was evaluated in liquid cultures using various carbon sources, a procedure that revealed lack of correlation on xylanase levels between the two cultivation modes and the inability of solid CMC cultures to fully unravel the cellulose degrading potential of the isolates. Strain SP24, showing more than 99 % 16S DNA similarity with Geobacillus sp. was further studied for its unique ability to simultaneously exhibit cellulase, xylanase, ß-glucosidase and ß-xylosidase activities. The first two enzymes were produced mainly extracellularly, while the ß-glycosidic activities were primarily detected in the cytosol. Maximum enzyme production by this strain was attained using a combination of wheat bran and xylan in the growth medium. Bioreactor cultures showed that aeration was necessary for both enhanced growth and enzyme production. Aeration had a strong positive effect on cellulase production while it negatively affected expression of ß-glucosidase. Xylanase and ß-xylosidase production was practically unaffected by aeration levels.

Diversity of cyanobacterial phylotypes in a Mediterranean drinking water reservoir (Marathonas, Greece).

The structure of the cyanobacterial community in a large drinking water reservoir (Marathonas, Greece) was investigated in October 2007 and September 2008. Cyanobacteria-specific primers were used for the PCR amplification of cyanobacterial 16S rDNAs from three water column sites and the water collection tank. In total, 199 clones were sequenced representing 52 unique cyanobacterial, including chloroplast-related, and 11 non-cyanobacterial phylotypes. All cyanobacterial phylotypes belonged to the order Chroococcales. Cluster analysis showed that the cyanobacterial communities in 2007 in the three water column sites showed high similarity between the stations and low diversity (H=1.17-1.44), due to the occurring common phylotypes, while all sites in 2008 had very low similarities between them and higher diversity (H=1.56-2.40). Some of the most abundant phylotypes were closely related (>98%) to members of the genus Gloeocapsa and a potentially toxin-producing strain of Microcystis aeruginosa. The non-cyanobacterial phylotypes were either unaffiliated or belonged to the Verrucomicrobia, and were related with sequences originating from lake water habitats.

Τhe Nematicidal Potential of Bioactive Streptomyces Strains Isolated from Greek Rhizosphere Soils Tested on Arabidopsis Plants of Varying Susceptibility to Meloidogyne spp.

A total of 461 indigenous Streptomycetes strains recovered from various Greek rhizosphere habitats were tested for their bioactivity. All isolates were examined for their ability to suppress the growth of 12 specific target microorganisms. Twenty-six were found to exert antimicrobial activity and were screened for potential nematicidal action. S. monomycini ATHUBA 220, S. colombiensis ATHUBA 438, S. colombiensis ATHUBA 431, and S. youssoufensis ATHUBA 546 were proved to have a nematicidal effect and thus were further sequenced. Batch culture supernatants and solvent extracts were assessed for paralysis on Meloidogyne javanica and Meloidogyne incognita second-stage juveniles (J2). The solvent extracts of S. monomycini ATHUBA 220 and S. colombiensis ATHUBA 438 had the highest paralysis rates, so these Streptomycetes strains were further on tested for nematodes' biological cycle arrest on two Arabidopsis thaliana plants; the wild type (Col-0) and the katanin mutant fra2, which is susceptible to M. incognita. Interestingly, S. monomycini ATHUBA 220 and S. colombiensis ATHUBA 438 were able to negatively affect the M. incognita biological cycle in Col-0 and fra2 respectively, and increased growth in Col-0 upon M. incognita infection. However, they were ineffective against M. javanica. Fra2 plants were also proved susceptible to M. javanica infestation, with a reduced growth upon treatments with the Streptomyces strains. The nematicidal action and the plant-growth modulating abilities of the selected Streptomycetes strains are discussed.

Purification and partial characterization of the xanthine-uric acid transporter (UapA) of Aspergillus nidulans.

UapA, the uric acid-xanthine permease from the filamentous ascomycete Aspergillus nidulans, is one of the most thoroughly characterized purine/H(+) transporters in eukaryotes. Detailed studies have addressed its regulation of expression, at both the transcriptional and post-translational levels, in response to physiological and developmental signals. An extensive kinetic profile towards a plethora of purines and mutational analyses have established models on how UapA recognizes the purine ring and revealed specific amino acid residues involved in proper folding, topogenesis, function and specificity. The present work describes for the first time the purification of the UapA transporter of A. nidulans through overexpression via the strong, ethanol-inducible, glucose-repressible, alcA promoter. Purification, almost to homogeneity, was achieved by Ni(2+) affinity chromatography using a functional His-tagged UapA protein version. It is subsequently shown, by Circular Dichroism (CD) spectroscopy, that the purified protein is structured with a high alpha-helical content, as expected from the in silico predictions. The result of this work opens the way for further, analytical and biochemical studies on UapA at the protein level.

Prevalence of tetracycline resistance genes in Greek seawater habitats.

The presence of selected tetracycline resistance (TcR) genes was studied in different Greek seawater habitats, originated from wastewater treatment facilities, fishfarm, and coastal environments. The methods employed included assessment of the presence of twelve gene clusters by PCR, followed by hybridization with specific probes, in habitat extracted DNA, Tc(R) bacteria, and exogenous isolated plasmids conferring TcR. The direct DNA-based analysis showed that tet(A) and tet(K) genes were detected in all habitats, whilst tet(C) and tet(E) were present in fishfarm and wastewater effluent samples and tet(M) was detected in fish-farm and coastal samples. Resistance genes tet(h), tet(C), tet(K), and tet(M) were detected in 60 of the 89 isolates screened. These isolates were identified by fatty acid methyl ester analysis (FAME) as Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Staphylococcus strains. The presence of the TcR genes in 15% of the bacterial isolates coincided with the presence of IncP plasmids. A habitat-specific dissemination of IncP alpha plasmids in wastewater effluent isolates and of IncP beta plasmids in fishfarm isolates was observed. Exogenous isolation demonstrated the presence of plasmids harbouring Tc(R) genes in all the habitats tested. Plasmids were shown to carry tet(h), tet(C), tet(E), and tet(K) genes. It is concluded that TcR genes are widespread in the seawater habitats studied and often occur on broad host range plasmids that seem to be well disseminated in the bacterial communities.

A refinery sludge deposition site: presence of nahH and alkJ genes and crude oil biodegradation ability of bacterial isolates.

204 bacterial isolates from four Greek refinery sludge deposition sites were investigated for the presence of nahH and alkJ genes encoding key enzymes of both aromatic and aliphatic hydrocarbon degradation pathways by PCR and DNA hybridisation. Members of Pseudomonas, Acinetobacter, Bacillus, Rhodococcus and Arthrobacter play important role in bioremediation processes in sandy/loam soil contaminated with oil and nahH and alkJ genes were present in the 73% of the isolates. Consortia of bacterial isolates that were used for biodegradation of aliphatic and aromatic hydrocarbons in crude oil using liquid cultures exhibited rates from 35% to 48% within 10 days of incubation.

Fluostatins C-E, novel members of the fluostatin family produced by Streptomyces strain Acta 1383.

Three new members of the fluostatin family, fluostatins C-E, were discovered in a culture filtrate extract of strain Acta 1383 during an HPLC screening program. The producing strain belongs to the genus Streptomyces and is closely related to type strains classified in the Streptomyces lavendulae 16S rRNA subclade. Fluostatins are named by their characteristic fluorenone chromophore. Fluostatin C shows moderate activity against selected human tumor cell lines.

Evaluation of Paecilomyces variotii potential in bioethanol production from lignocellulose through consolidated bioprocessing.

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.

Streptomyces rochei ACTA1551, an indigenous Greek isolate studied as a potential biocontrol agent against Fusarium oxysporum f.sp. lycopersici.

Many studies have shown that several Greek ecosystems inhabit very interesting bacteria with biotechnological properties. Therefore Streptomyces isolates from diverse Greek habitats were selected for their antifungal activity against the common phytopathogenic fungus Fusarium oxysporum. The isolate encoded ACTA1551, member of Streptomyces genus, could strongly suppress the fungal growth when examined in antagonistic bioassays in vitro. The isolate was found phylogenetically relative to Streptomyces rochei after analyzing its 16S rDNA sequence. The influence of different environmental conditions, such as medium composition, temperature, and pH on the expression of the antifungal activity was thoroughly examined. Streptomyces rochei ACTA1551 was able to protect tomato seeds from F. oxysporum infection in vivo while it was shown to promote the growth of tomato plants when the pathogen was absent. In an initial effort towards the elucidation of the biochemical and physiological nature of ACTA1551 antifungal activity, extracts from solid streptomycete cultures under antagonistic or/and not antagonistic conditions were concentrated and fractionated. The metabolites involved in the antagonistic action of the isolate showed to be more than one and produced independently of the presence of the pathogen. The above observations could support the application of Streptomyces rochei ACTA1551 as biocontrol agent against F. oxysporum.

Unraveling the lipolytic activity of thermophilic bacteria isolated from a volcanic environment.

In a bioprospecting effort towards novel thermostable lipases, we assessed the lipolytic profile of 101 bacterial strains isolated from the volcanic area of Santorini, Aegean Sea, Greece. Screening of lipase activity was performed both in agar plates and liquid cultures using olive oil as carbon source. Significant differences were observed between the two screening methods with no clear correlation between them. While the percentage of lipase producing strains identified in agar plates was only 17%, lipolytic activity in liquid culture supernatants was detected for 74% of them. Nine strains exhibiting elevated extracellular lipase activities were selected for lipase production and biochemical characterization. The majority of lipase producers revealed high phylogenetic similarity with Geobacillus species and related genera, whilst one of them was identified as Aneurinibacillus sp. Lipase biosynthesis strongly depended on the carbon source that supplemented the culture medium. Olive oil induced lipase production in all strains, but maximum enzyme yields for some of the strains were also obtained with Tween-80, mineral oil, and glycerol. Partially purified lipases revealed optimal activity at 70-80°C and pH 8-9. Extensive thermal stability studies revealed marked thermostability for the majority of the lipases as well as a two-step thermal deactivation pattern.

Granulomatous infection of the hand and wrist due to Azospirillum spp.

We report a case of Azospirillum infection manifestating as granulomatous tenosynovitis of the right hand, in an immunocompetent middle-aged female. We highlight the unusual source of the infection, the diagnostic workup, as well as the treatment approach.

Variability of prokaryotic community structure in a drinking water reservoir (Marathonas, Greece).

The structure of the Bacteria and Archaea community in a large drinking water reservoir (Marathonas, Greece; MR) was investigated in October 2007 and September 2008, using 16S rRNA gene clone libraries. The bacterial communities were more diverse than archaeal communities (Shannon diversity index H' 0.81-3.28 and 1.36-1.77, respectively). The overall bacterial community composition was comparable to bacterioplankton community described in other freshwater habitats. Within the Bacteria, Betaproteobacteria dominated, while representatives of Alpha-, Gamma- and Deltaproteobacteria also occurred. Other important phyla were Actinobacteria and Bacteroidetes, while representatives of Acidobacteria, Cyanobacteria, Chloroflexi, Planctomycetes and Verrucomicrobia were also retrieved. Several phylotypes in Alpha- and Betaproteobacteria and Bacteroidetes were related to bacteria capable of cyanotoxin degradation and with aromatic compounds/iron oxidizers or polymer degraders. Euryarchaeota dominated (60.5%) the Archaea community mostly with phylotypes related to Methanobacteriales and Methanosarcinales. Among the Thaumarchaeota, the two most abundant phylotypes were affiliated (97% similarity) with the only cultivated mesophilic thaumarchaeote of marine origin, Nitrosopumilus maritimus. Temporal and spatial comparison of the prokaryotic community structure revealed that three of the most abundant prokaryotic phylotypes, belonging to Actinobacteria, were recovered from all sites both years, suggesting that these Actinobacteria could be important key players in MR ecosystem functioning.

SAFE Software and FED Database to Uncover Protein-Protein Interactions using Gene Fusion Analysis.

Domain Fusion Analysis takes advantage of the fact that certain proteins in a given proteome A, are found to have statistically significant similarity with two separate proteins in another proteome B. In other words, the result of a fusion event between two separate proteins in proteome B is a specific full-length protein in proteome A. In such a case, it can be safely concluded that the protein pair has a common biological function or even interacts physically. In this paper, we present the Fusion Events Database (FED), a database for the maintenance and retrieval of fusion data both in prokaryotic and eukaryotic organisms and the Software for the Analysis of Fusion Events (SAFE), a computational platform implemented for the automated detection, filtering and visualization of fusion events (both available at: http://www.bioacademy.gr/bioinformatics/projects/ProteinFusion/index.htm). Finally, we analyze the proteomes of three microorganisms using these tools in order to demonstrate their functionality.

Homologous overexpression of xylanase in Fusarium oxysporum increases ethanol productivity during consolidated bioprocessing (CBP) of lignocellulosics.

In an effort to increase ethanol productivity during the consolidated bioprocessing (CBP) of lignocellulosics by Fusarium oxysporum, we attempted the constitutive homologous overexpression of one of the key process enzymes, namely an endo-xylanase. The endo-ß-1,4-xylanase 2 gene was incorporated into the F. oxysporum genome under the regulation of the gpdA promoter of Aspergillus nidulans. The transformation was effected through Agrobacterium tumefaciens and resulted in 12 transformants, two of which were selected for further study due to their high extracellular xylanase activities under normally repressing conditions (glucose as sole carbon source). During natural induction conditions (growth on xylan) though, the extracellular enzyme levels of the transformants were only marginally higher (5-10%) compared to the wild type despite the significantly stronger xylanase 2 mRNA signals. SDS-PAGE verified enzyme assay results that there was no intracellular xylanase 2 accumulation in the transformants, suggesting the potential regulation in a post transcriptional or translational level. The fermentative performance of the transformants was evaluated and compared to that of the wild type in simple CBP systems using either corn cob or wheat bran as sole carbon sources. Both transformants produced approximately 60% more ethanol compared to the wild type on corn cob, while for wheat bran this picture was repeated for only one of them. This result is attributed to the high extracellular xylanase activities in the transformants' fermentation broths that were maintained 2-2.5-fold higher compared to the wild type.

Biodegradation of crude oil by thermophilic bacteria isolated from a volcano island.

One-hundred and fifty different thermophilic bacteria isolated from a volcanic island were screened for detection of an alkane hydroxylase gene using degenerated primers developed to amplify genes related to the Pseudomonas putida and Pseudomonas oleovorans alkane hydroxylases. Ten isolates carrying the alkJ gene were further characterized by 16s rDNA gene sequencing. Nine out of ten isolates were phylogenetically affiliated with Geobacillus species and one isolate with Bacillus species. These isolates were able to grow in liquid cultures with crude oil as the sole carbon source and were found to degrade long chain crude oil alkanes in a range between 46.64% and 87.68%. Results indicated that indigenous thermophilic hydrocarbon degraders of Bacillus and Geobacillus species are of special significance as they could be efficiently used for bioremediation of oil-polluted soil and composting processes.

PCR detection of oxytetracycline resistance genes otr(A) and otr(B) in tetracycline-resistant streptomycete isolates from diverse habitats.

A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (Tc(R)) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of Tc(R) streptomycetes originated from bulk and rhizosphere soil. Fewer Tc(R) streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene-carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which Tc(R) streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common.