Genotypic and phenotypic analysis of strains assigned to the widespread cyanobacterial morphospecies Phormidium autumnale (Oscillatoriales).

In this study, ten cyanobacterial strains assigned to the oscillatorian species Phormidium autumnale have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, and pigment content. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The strains were quite homogenous in their morphologic features. Their thylakoids showed a stacked or fascicular pattern. Some, but not all strains contained phycoerythrin. Only one strain (P. autumnale UTCC 476) deviated significantly in its phenotype by lacking a calyptra. In neighbour-joining and maximum Parsimony trees most 16S rRNA sequences were located on a single well-defined branch, which, however, also harboured sequences assigned to other cyanobacterial genera. Two strains (P. autumnale UTCC 476 and P. autumnale UTEX 1580) were found on distant branches. The presence of phycoerythrin was not correlated with the strains' position in the phylogenetic trees. Our results reconfirm that the morphospecies P. autumnale and the Phormidium group in general are not phylogenetically coherent and require revision. However, as indicated by sequence similarities most of the strains assigned to P. autumnale except P. autumnale UTCC 476 and P. autumnale UTEX 1580 are phylogenetically related and might belong to a single genus.

Characterization of aerobic bacterial and fungal microbiota on surfaces of historic Scottish monuments.

Twenty samples were taken from the inner or outer surfaces of stone monuments of six historic Scottish buildings and ruins. Biofilms developing on mineral substrates were analysed by in situ scanning electron microscopy and cultivation. Various methods were used to characterize the isolates including automated ribotyping, RAPD and sequencing of the 16S rRNA gene for bacteria, and stereomicroscopy and sequencing of the Internal Transcribed Spacers (ITS) for fungi. Most samples contained microbes between 10(5) and 10(7)cfug(-1) substrate. Actinobacteria belonging to the genus Streptomyces (17 samples/5 monuments) or Arthrobacter (12/3) and Pseudomonas (9/3) were frequently detected. Most streptomycetes were in terms of their 16S rRNA gene sequence most closely related to S. microflavus (10/3) or to the undescribed species S. "vulgaris" (8/3). Indoor and outdoor biofilms exhibited significant differences in their microbiota, as shown by both microscopy and isolation studies. Pigmented coccoid Arthrobacter species were typical for the outdoor samples, whereas Pseudomonas species were common in the indoor samples. Based on the low phylogenetic relationship to a known species (type strain), potential novel pigmented bacterial species belonging to the genera Arthrobacter, Brevundimonas, Cryseobacterium, Deinococcus and Dyadobacter were detected from the outdoor samples and to Pseudomonas from the indoor samples. Hyaline fungal species of Acremonium (10/4) mainly occurred in indoor samples, whereas pigmented species of Cladosporium (8/3), Penicillium (6/3) and Phialophora (6/2) were found outdoors. Using in situ microscopy diatom algae were also detected.

Genotypic and phenotypic diversity of cyanobacteria assigned to the genus Phormidium (Oscillatoriales) from different habitats and geographical sites.

In this study, 30 strains of filamentous, non-heterocystous cyanobacteria from different habitats and different geographical regions assigned to diverse oscillatorian genera but here collectively referred to as members of the Phormidium group have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, phycoerythrin content, and complementary chromatic adaptation. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The sequences were located on multiple branches of the inferred cyanobacterial 16S rRNA tree. For some, but not all, strains with identical 16S rDNA sequences, a higher level of discrimination was achieved by analyses of the less conserved ITS sequences. As shown for other cyanobacteria, no correlation was found between position of the strains in the phylogenetic tree and their geographic origin. Genetically similar strains originated from distant sites while other strains isolated from the same sampling site were in different phylogenetic clusters. Also the presence of phycoerythrin was not correlated with the strains' position in the phylogenetic trees. In contrast, there was some correlation among inferred phylogenetic relationship, original environmental habitat, and morphology. Closely related strains came from similar ecosystems and shared the same morphological and ultrastructural features. Nevertheless, structural properties are insufficient in themselves for identification at the genus or species level since some phylogenetically distant members also showed similar morphological traits. Our results reconfirm that the Phormidium group is not phylogenetically coherent and requires revision.

Phylogenetic evaluation of cyanobacteria preserved as historic herbarium exsiccata.

Dried herbarium specimens of cyanobacteria (exsiccata) deposited over 100 years ago were analysed and characterized using combined morphological and molecular approaches. Six representative coccoid and filamentous cyanobacteria from two historic collections and a 15-year-old air-dried environmental sample were studied. Morphological features observed by light and electron microscopy were correlated with the results of 16S rRNA gene sequencing. Historic identifications achieved by means of classical morphology could thus be confirmed by extracted, amplified and sequenced 16S rRNA gene fragments. The results of this study open the possibility of providing genotypic characterizations to botanical type specimens, thus reconciling the botanical and bacteriological approaches to the taxonomic treatment of these micro-organisms.

Association of fucoxanthin chlorophyll a/c-binding polypeptides with photosystems and phosphorylation in the centric diatom Cyclotella cryptica.

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.

Differential circadian expression of genes fcp2 and fcp6 in Cyclotella cryptica.

The steady-state mRNA concentrations of two fcp genes encoding fucoxanthin chlorophyll a/c light-harvesting polypeptides of the centric diatom Cyclotella cryptica were investigated over a 4-day period by RNA dot-blotting experiments. Before and during the first day of the experiment, the cultures were grown under a 12-h light/12-h dark regime. On the following 3 days, the algae were kept in darkness. On the first day, the steady-state mRNA concentration of fcp2 followed a diurnal pattern, with a maximum occurring around noon, approximately 6 h after the onset of light. The gene fcp6 also had a diurnal pattern on the first day. Its maximum, however, occurred immediately after the onset of light. During the subsequent incubation period in darkness, the diurnal pattern of expression of both fcp genes continued, thus demonstrating that their steady-state mRNA concentrations oscillated in a circadian manner.

A methodological approach to investigate steady state fucoxanthin chlorophyll a/c binding protein mRNA levels in Wadden Sea sediments.

A method was established to investigate the steady state levels of mRNAs from genes encoding fucoxanthin chlorophyll a/c binding proteins (Fcp) of diatoms in situ. During the study, which was performed with Wadden Sea sediments from the German North Sea shore near Dangast, oxygenic photosynthesis was carried out mainly by pennate diatoms. Field samples were taken after tidal exposure from dawn up to late afternoon at 2-hourly intervals, and frozen in liquid nitrogen. In the laboratory, total RNA was isolated by isopycnic ultracentrifugation in caesium chloride gradients. Yields of approximately 10-300 micro g RNA per gram wet sediment were obtained. Defined amounts of total RNA were blotted onto nylon membranes and hybridised with probes against the fcp2 and 18S rDNA genes of Cyclotella cryptica. To estimate the steady state amount of fcp mRNAs, fcp signal intensities were normalized to the signal intensities obtained from hybridisation to an 18S rDNA gene probe. In the two time-course studies performed to demonstrate the applicability of the method, the steady state levels of fcp mRNA increased up to 12-fold with the onset of light, reaching a maximum 6-8 h after sunrise before they decreased again. Possible reasons for this time-course are discussed.

Lysineurethanedimethacrylate--a novel generation of amino acid based monomers for bone cements and tissue repair.

A novel amino acid based dimethacrylate monomer (lysineurethanedimethacrylate, LUDM) was prepared by the addition of hydroxyethylmethacrylate (HEMA) to lysinediisocyanate (LDI). The structure was confirmed by FT-IR and 1H and 13C NMR spectroscopy as well as FAB-MS. Photopolymerized LUDM exhibited low volume shrinkage upon polymerization, good mechanical properties (Young's modulus: 3740 MPa) and high thermal stability. Osteoblast adhesion and growth on polymerized LUDM samples evidenced the biocompatibility. Further improvement of the mechanical properties was obtained by using Ca-hydroxyapatite as inorganic filler varying between 10 and 30 wt%. The Young's and flexural moduli increased with increasing filler content ranging from 3740 to 5250 MPa and from 2020 to 3690 MPa, respectively. The mechanical properties and the good biocompatibility of the lysine-based methacrylate networks make them interesting materials for medical applications, e.g. bone cements, and tissue engineering.