Mario De Stefano

Marine diatoms as optical chemical sensors

Complex micro- and nanostructured materials for optical sensing purposes are designed and fabricated using top technologies. A completely different approach to engineering systems at the nanoscale consists in recognizing the nanostructures and morphologies that nature has optimized during life’s history on earth. We have found that the photoluminescence emission from silica skeleton of marine diatoms Thalassiosira rotula Meunier is strongly dependent on the surrounding environment. Both the optical intensity and the peaks positions are affected by gases and organic vapors. Depending on the electronegativity and polarizing ability, some substances quench the luminescence, while others effectively enhance it. These phenomena allow the discrimination between different substances. These naturally occurring organisms are thus good candidates as optical sensing materials.

Lensless light focusing with the centric marine diatom Coscinodiscus walesii.

In this work, we report on the light focusing ability exploited by the microshell of a marine organism: the Coscinodiscus wailesii diatom. A 100 microm spot size of a red laser beam is narrowed up to less than 10 microm at a distance of 104 microm after the transmission through the regular geometry of the diatom structure, which thus acts as a microlens. Numerical simulations of the electromagnetic field propagation show a good qualitative agreement with the experimental results. The focusing effect is due to the superposition of the waves scattered by the holes present on the surface of the diatom valve. Very interesting applications in micro-optic devices are feasible due to the morphological and biological characteristic of these unicellular organisms.

Clonothrix fusca Roze 1896, a Filamentous, Sheathed, Methanotrophic γ-Proteobacterium

ABSTRACT Crenothrix polyspora Cohn 1870 and Clonothrix fusca Roze 1896 are two filamentous, sheathed microorganisms exhibiting complex morphological differentiation, whose phylogeny and physiology have been obscure for a long time due to the inability to cultivate them. Very recently, DNA sequencing data from uncultured C. polyspora-enriched material have suggested that Crenothrix is a methane-oxidizing γ-proteobacterium (39). In contrast, the possible ecological function of C. fusca, originally considered a developmental stage of C. polyspora, is unknown. In this study, temporal succession of two filamentous, sheathed microorganisms resembling Cohns Crenothrix and Rozes Clonothrix was observed by analyzing the microbial community of an artesian well by optical microscopy. Combined culture-based and culture-independent approaches enabled us to assign C. fusca to a novel subgroup of methane-oxidizing γ-proteobacteria distinct from that of C. polyspora. This assignment was supported by (i) methane uptake and assimilation experiments, (ii) ultrastructural data showing the presence in C. fusca cytoplasm of an elaborate membrane system resembling that of methanotrophic γ-proteobacteria, and (iii) sequencing data demonstrating the presence in its genome of a methanol dehydrogenase α subunit-encoding gene (mxaF) and a conventional particulate methane mono-oxygenase α subunit-encoding gene (pmoA) that is different from the unusual pmoA (u-pmoA) of C. polyspora.

The microbial community of Vetiver root and its involvement into essential oil biogenesis.

Vetiver is the only grass cultivated worldwide for the root essential oil, which is a mixture of sesquiterpene alcohols and hydrocarbons, used extensively in perfumery and cosmetics. Light and transmission electron microscopy demonstrated the presence of bacteria in the cortical parenchymatous essential oil-producing cells and in the lysigen lacunae in close association with the essential oil. This finding and the evidence that axenic Vetiver produces in vitro only trace amounts of oil with a strikingly different composition compared with the oils from in vivo Vetiver plants stimulated the hypothesis of an involvement of these bacteria in the oil metabolism. We used culture-based and culture-independent approaches to analyse the microbial community of the Vetiver root. Results demonstrate a broad phylogenetic spectrum of bacteria, including alpha-, beta- and gamma-Proteobacteria, high-G+C-content Gram-positive bacteria, and microbes belonging to the Fibrobacteres/Acidobacteria group. We isolated root-associated bacteria and showed that most of them are able to grow by using oil sesquiterpenes as a carbon source and to metabolize them releasing into the medium a large number of compounds typically found in commercial Vetiver oils. Several bacteria were also able to induce gene expression of a Vetiver sesquiterpene synthase. These results support the intriguing hypothesis that bacteria may have a role in essential oil biosynthesis opening the possibility to use them to manoeuvre the Vetiver oil molecular structure.

Optical properties of diatom nanostructured biosilica in Arachnoidiscus sp: micro-optics from mother nature.

Some natural structures show three-dimensional morphologies on the micro- and nano- scale, characterized by levels of symmetry and complexity well far beyond those fabricated by best technologies available. This is the case of diatoms, unicellular microalgae, whose protoplasm is enclosed in a nanoporous microshell, made of hydrogenated amorphous silica, called frustule. We have studied the optical properties of Arachnoidiscus sp. single valves both in visible and ultraviolet range. We found photonic effects due to diffraction by ordered pattern of pores and slits, accordingly to an elaborated theoretical model. For the first time, we experimentally revealed spatial separation of focused light in different spots, which could be the basis of a micro-bio-spectrometer. Characterization of such intricate structures can be of great inspiration for photonic devices of next generation.

The HrpB–HrpA two‐partner secretion system is essential for intracellular survival of Neisseria meningitidis

In this study we used HeLa cells to investigate the role of the HrpB–HrpA two‐partner secretion (TPS) system in the meningococcal infection cycle. Although there is evidence that several pathogenic microorganisms may use TPS systems to colonize epithelial surfaces, the meningococcal HrpB–HrpA TPS system was not primarily involved in adhesion to or invasion of HeLa cells. Instead, this system was essential for intracellular survival and escape from infected cells. Gentamicin protection assays, immunofluorescence and transmission electron microscopy analyses demonstrated that, in contrast to the wild‐type strain, HrpB–HrpA‐deficient mutants were primarily confined to late endocytic vacuoles and trapped in HeLa cells. Haemolytic tests using human erythrocytes suggested that the secreted HrpA proteins could act as manganese‐dependent lysins directly involved in mediating vacuole escape. In addition, we demonstrated that escape of wild‐type meningococci from infected cells required the use of an intact tubulin cytoskeleton and that the hrpB–hrpA genes, which are absent in other Neisseria spp., were upregulated during infection.

Seasonal variations of epilithic diatoms on different hard substrates, in the northern Adriatic Sea

The colonization of epilithic diatoms on artificial hard substrates (marble, quartzite and slate) was investigated on a seasonal basis in a subtidal site of the northern Adriatic Sea to determine if substrate-dependent differences in colonization occurs and to define the seasonal variations of micro-epilithic communities in terms of abundance, biomass and community structure. Artificial substrates (smoothed small discs mounted on a Plexiglas sheet) were placed at a depth of 8 m in April 2003, July 2003, January 2004 and February 2004. The discs were collected after 6-7 weeks for counting and taxonomic identification of the diatoms. The community structure of epilithic diatoms showed a dominance of motile species over the entire study period, followed by erect, adnate and tube-dwelling diatoms. Diatom density showed a marked seasonal variability, ranging from 365 ±407 (winter 2004) to 557156 ±82602 cells cm -2 (spring 2004). Biomass ranged from 0.02 ±0.01 to 17.53 ±3.20 μg Ccm -2 . Abundance and biomass values did not present any significant differences for the three substrates examined.

Shedding light on diatom photonics by means of digital holography

Diatoms are among the dominant phytoplankters in the worlds oceans, and their external silica investments, resembling artificial photonic crystals, are expected to play an active role in light manipulation. Digital holography allowed studying the interaction with light of Coscinodiscus wailesii cell wall reconstructing the light confinement inside the cell cytoplasm, condition that is hardly accessible via standard microscopy. The full characterization of the propagated beam, in terms of quantitative phase and intensity, removed a long-standing ambiguity about the origin of the light confinement. The data were discussed in the light of living cell behavior in response to their environment.

A comparative study of Cocconeis scutellum Ehrenberg and its varieties (Bacillariophyta)

We present an ultrastructure-based revision (using light and electron microscopy) of the type species of the genus Cocconeis, C. scutellum var. scutellum, and four often-mentioned but poorly described, morphologically related varieties: C. scutellum var. baldjikiana, C. scutellum var. clinoraphis, C. scutellum var. parva, C. scutellum var. posidoniae. In addition, we introduce three new taxa, C. scutellum var. gorensis var. nov., C. scutellum var. posidoniae f. decussata f. nov. and C. scutellum var. sullivanensis var. nov. The taxonomic relationships between these varieties and the remaining validly described C. scutellum varieties are analyzed on the basis of their ultrastructural differences. We also provide additional information on the geographical distribution of all analyzed C. scutellum taxa.

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of priA and its role in intracellular replication

BackgroundIn vitro studies with cell line infection models are beginning to disclose the strategies that Neisseria meningitidis uses to survive and multiply inside the environment of the infected host cell. The goal of this study was to identify novel virulence determinants that are involved in this process using an in vitro infection system.ResultsBy using reverse transcriptase-PCR differential display we have identified a set of meningococcal genes significantly up-regulated during residence of the bacteria in infected HeLa cells including genes involved in L-glutamate transport (gltT operon), citrate metabolism (gltA), disulfide bond formation (dsbC), two-partner secretion (hrpA-hrpB), capsulation (lipA), and DNA replication/repair (priA). The role of PriA, a protein that in Escherichia coli plays a central role in replication restart of collapsed or arrested DNA replication forks, has been investigated. priA inactivation resulted in a number of growth phenotypes that were fully complemented by supplying a functional copy of priA. The priA-defective mutant exhibited reduced viability during late logarithmic growth phase. This defect was more severe when it was incubated under oxygen-limiting conditions using nitrite as terminal electron acceptors in anaerobic respiration. When compared to wild type it was more sensitive to hydrogen peroxide and the nitric oxide generator sodium nitroprusside. The priA-defective strain was not affected in its ability to invade HeLa cells, but, noticeably, exhibited severely impaired intracellular replication and, at variance with wild type and complemented strains, it co-localized with lysosomal associated membrane protein 1.ConclusionIn conclusion, our study i.) demonstrates the efficacy of the experimental strategy that we describe for discovering novel virulence determinants of N. meningitidis and ii.) provides evidence for a role of priA in preventing both oxidative and nitrosative injury, and in intracellular meningococcal replication.