Stefano Ventura

Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

ABSTRACT The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.

Exopolysaccharide production by a unicellular cyanobacterium isolated from a hypersaline habitat

The unicellular cyanobacterial strain 16Som2, isolated from a Somaliland saltpan and identified asCyanothece sp., is characterized by cells surrounded by a thick polysaccharidic capsule, the external part of which dissolves into the medium during growth, causing a progressive increase in culture viscosity. In spite of this, the thickness of the capsule remained almost constant under all the culture conditions tested, demonstrating that the processes of its synthesis and solubilization occurred at a similar rate. The synthesis of carbohydrates was neither enhanced by increasing salinity (sea-water enriched with NaCl in the range 0 to 2.0 M) nor by Mg2+, K+ or Ca2+ deficiencies. In contrast, N-limitation and, to a lesser extent, P-limitation induced a significant enhancement of carbohydrate synthesis; in particular, N-deficiency stimulated the synthesis of all the carbohydrate fractions (intracellular, capsular and soluble). The soluble polysaccharide, separated from the culture medium and hydrolyzed with 2N trifluoroacetic acid, showed a sugar composition consisting of glucuronic acid: galacturonic acid: galactose: glucose: mannose: xylose: fucose in a molar ratio of 1: 2: 2.4: 6.8: 4.8: 2.9: 1.6.Cyanothece sp. culture subjected to nitrogen starvation synthesized polysaccharide with a mean productivity of 115 mg (EPS) l−1d−1, for the polymer solubilized into the medium, and of 15 mg (CPS) l−1d−1 for the capsular polysaccharide.

DNA restriction fingerprint analysis of the soil bacterium Azospirillum

Total DNAs of 18 strains of Azospirillum from different sources and geographical areas were compared by restriction endonuclease pattern analysis. Fragments obtained with HindIII or BglII were separated by PAGE and stained with silver nitrate. Each strain possessed a unique and reproducible fingerprint with each enzyme, thereby facilitating strain recognition. UPGMA analysis recovered clusters of band patterns that were compared to the distribution of species within the genus Azospirillum.

Biodiversity of Phototrophic Biofilms Dwelling on Monumental Fountains

Among the stone monumental assets, artistic fountains are particularly affected by microbial colonization due to constant contact with water, giving rise to biodegradation processes related with physical–chemical and aesthetical alterations. In this paper, we make an overview of reported biodiversity of the phototrophic patina developed in various fountains of Italy and Spain. The microbial composition of four fountains (two from Florence, Italy and two from Granada, Spain) was investigated using traditional and/or molecular techniques. The results indicated many common similarities with regard the phototrophic biodiversity for all the investigated fountains. Automated ribosomal RNA intergenic spacer analysis (ARISA), a molecular fingerprint tool, was used to examine the eubacterial and cyanobacterial community for two of the investigated fountains. The principal component analysis of ARISA profiles strengthens the results obtained by traditional methods and revealed separate clusters, as a consequence of the differences of micro-environmental conditions for each fountain.

Species attribution and strain typing of Oenococcus oeni (formerly Leuconostoc oenos) with restriction endonuclease fingerprints

In several wines, malolactic fermentation is required to improve the organoleptic characters and to stabilize the final product. In order to establish a controlled malolactic fermentation in wine, easy identification and sensitive typing of strains of Oenococcus oeni (new name of the malolactic bacterium Leuconostoc oenos) used as starter cultures are necessary. To accomplish these tasks, several strains of Oenococcus oeni isolated from wines of the Chianti region (Italy), along with reference strains and strains of L. mesenteroides subsp. mesenteroides, L. carnosum, L. fallax, L. pseudomesenteroides, L. lactis and Weisella paramesenteroides, were studied with RFLP of ribosomal genes and ultrasensitive total DNA restriction pattern analysis performed on polyacrylamide gel. With each of four restriction endonucleases used, identical restriction profiles of ribosomal genes were obtained for all strains of O. oeni. These ribopatterns, being strongly dissimilar to profiles of the other lactic acid bacteria tested, appear to be well suited for the attribution of wine lactic acid bacteria to the species O. oeni. Cluster analysis performed on two total DNA restriction profile data sets showed that the species O. oeni possesses a good degree of genomic homogeneity. Very sensitive typing of strains of O. oeni was obtained with total DNA restriction profiles. The potential of an integrated approach using restriction profiles for species assignment and typing of selected malolactic bacteria is demonstrated.

The Primitive Thylakoid-Less Cyanobacterium Gloeobacter Is a Common Rock-Dwelling Organism.

Cyanobacteria are an ancient group of photosynthetic prokaryotes, which are significant in biogeochemical cycles. The most primitive among living cyanobacteria, Gloeobacter violaceus, shows a unique ancestral cell organization with a complete absence of inner membranes (thylakoids) and an uncommon structure of the photosynthetic apparatus. Numerous phylogenetic papers proved its basal position among all of the organisms and organelles capable of plant-like photosynthesis (i.e., cyanobacteria, chloroplasts of algae and plants). Hence, G. violaceus has become one of the key species in evolutionary study of photosynthetic life. It also numbers among the most widely used organisms in experimental photosynthesis research. Except for a few related culture isolates, there has been little data on the actual biology of Gloeobacter, being relegated to an “evolutionary curiosity” with an enigmatic identity. Here we show that members of the genus Gloeobacter probably are common rock-dwelling cyanobacteria. On the basis of morphological, ultrastructural, pigment, and phylogenetic comparisons of available Gloeobacter strains, as well as on the basis of three new independent isolates and historical type specimen, we have produced strong evidence as to the close relationship of Gloeobacter to a long known rock-dwelling cyanobacterial morphospecies Aphanothece caldariorum. Our results bring new clues to solving the 40 year old puzzle of the true biological identity of Gloeobacter violaceus, a model organism with a high value in several biological disciplines. A probable broader distribution of Gloeobacter in common wet-rock habitats worldwide is suggested by our data, and its ecological meaning is discussed taking into consideration the background of cyanobacterial evolution. We provide observations of previously unknown genetic variability and phenotypic plasticity, which we expect to be utilized by experimental and evolutionary researchers worldwide.

The cyanobaterial genus Phormidesmis.

Characteristics and detailed morphology of the genus Characterization and detailed morphology of the genus Phormidesmis TURICCHIA et al. 2009 are presented. This genus is more related to thin cyanobacteria of the family Pseudanabaenaceae than to the genus Phormidium sensu stricto, which is confirmed by ultrastructural analysis, showing mainly parietal arrangement of thylakoids. Other potential and similar morphotypes from the genus Phormidium are discussed.

The taxonomic validation of the cyanobacterial genus Halothece

M.C. Margheri, S. Ventura, J. Kaštovský and J. Komárek. 2008. The taxonomic validation of the cyanobacterial genus Halothece. Phycologia 47: 477–486. DOI: 10.2216/07-87.1. In 1998, Garcia-Pichel and coworkers studied 13 strains of extremely halotolerant, unicellular cyanobacteria. One strain, which was isolated from a coastal salty habitat in Baja California, Mexico, was found to represent a genetically distinct subcluster, different from another related coherent group of 12 morphologically similar types. This subcluster was designated as ‘true Halothece’, in comparison with ‘Euhalothece’ strains. The genetic characters clearly indicated their separation of Halothece at the generic level (less than 91% of genetic similarity), but the formal description, corresponding with bacteriological or botanical nomenclatoric rules, was never published. The present article comprises the valid description of the genus Halothece based on the type strain MPI 96P605, supported by new original studies of the morphological, ecophysiological and ultrastructural variability of the type strain.

Genotypic diversity of oscillatoriacean strains belonging to the genera Geitlerinema and Spirulina determined by 16S rDNA restriction analysis

Genotypic diversity of several cyanobacterial strains mostly isolated from marine or brackish waters, belonging to the genera Geitlerinema and Spirulina, was investigated by amplified 16S ribosomal DNA restriction analysis and compared with morphological features and response to salinity. Cluster analysis was performed on amplified 16S rDNA restriction profiles of these strains along with profiles obtained from sequence data of five Spirulina-like strains, including three representatives of the new genus Halospirulina. Our strains with tightly coiled trichomes from hypersaline waters could be assigned to the Halospirulina genus. Among the uncoiled strains, the two strains of hypersaline origin clustered together and were found to be distant from their counterparts of marine and freshwater habitat. Moreover, another cluster, formed by alkali-tolerant strains with tightly coiled trichomes, was well delineated.

Revision of species delineation in the genus Ectothiorhodospira.

When the type strains and other strains of the six currently defined species of the genus Ectothiorhodospira were examined by DNA-DNA reassociation and RFLP of 16S/23S rDNA (ribotype), only four genospecies could be found. The possibility of defining taxonomically meaningful species corresponding to these four genospecies was investigated by combining DNA relatedness and ribotype data with other genotypic and phenotypic characters already described in the literature, an approach known as polyphasic taxonomy. Following this comparison, the type strain and another strain of Ectothiorhodospira vacuolata were found to be very similar to the type strain of Ectothiorhodospira shaposhnikovii and have been transferred to this latter species. Also, the type strain of Ectothiorhodospira marismortui and another previously unidentified strain were found to be very similar to the type strain of Ectothiorhodospira mobilis and have been transferred to this latter species. Due to the limited degree of reciprocal DNA relatedness, strains belonging either to Ectothiorhodospira marina or to Ectothiorhodospira haloalkaliphila are still considered as belonging to separate species, even though they show a remarkable phenotypic similarity. This revision has led to the delineation of only four species in the genus Ectothiorhodospira, namely E. mobilis, E. shaposhnikovii, E. marina and E. haloalkaliphila. E. vacuolata is recognized as a junior synonym of E. shaposhnikovii and E. marismortui as a junior synonym of E. mobilis.