Erhard Rhiel

Phenotype variability of identical genotypes: the need for a combined approach in cyanobacterial taxonomy demonstrated on Merismopedia-like isolates

Five Merismopedia-like cyanobacterial strains were collected from microbial mats at Norderney Island, subcultured in the laboratory, and finally grown as unicyanobacterial cultures. As a sixth strain, Merismopediaglauca from the „Sammlung von Algenkulturen“ at Göttingen (SAG) was used for comparisons. According to morphological and physiological characteristics initially observed in the field and during initial subculturing, the five strains were assigned to the species Merismopedia glauca, Merismopedia punctata, or Merismopedia elegans. However, after prolonged maintenance under laboratory conditions, the formation of platelet-like colonies stopped, whereas cell sizes, production of extracellular polymeric substances, and division patterns were stably maintained. These physiological and morphological parameters allowed us to divide the six strains into two clusters. This division was further supported by the profiling of total cell protein and phycobilisomes using SDS-PAGE. The nearly complete 16S rDNA sequence of three of the six isolates was determined. The comparative sequencing analysis revealed an almost 100% identity of these three Merismopedia-like strains. The evolutionary distance dendrogram constructed placed this Merismopedia cluster into a common line of descent with Synechocystis sp. strain PCC6906. Based on the analysis of common stretches of 1,050 nucleotides, the overall similarity between the sequence types of „Merismopedia“ and „Synechocystis“ is 96–97%. The values of the different methods for taxonomic classification of unicyanobacterial strains, the relationship of the cyanobacterial genera Merismopedia, Synechococcus, Synechocystis, and Eucapsis sp., and the functional role of different Merismopedia morphologies within microbial mats are discussed. It is suggested that all analyzed Merismopedia strains be combined into one species, namely Merismopediapunctata Meyen (1839).

Morphological, physiological, and molecular characterization of actinomycetes isolated from dry soil, rocks, and monument surfaces.

Abstract In an extended study on the biodiversity of rock-dwelling bacteria, the colony and cell morphology, physiology, protein patterns, and 16S rDNA sequences of 17 bacterial strains isolated from different surfaces of rocks, stones, and monuments and from various geographical locations were characterized. All except one strain, which was found to be a Bacillus, were members of the order Actinomycetales. The majority of the strains either were closely related to Geodermatophilus obscurus, which was also analyzed in this study, or formed a closely related sister taxon. All of these strains were isolated from the surface of marble in Namibia and Greece and from limestone from the Negev desert, Israel. One strain, G10, of Namibia origin was equidistantly related to Geodermatophilus obscurus, Frankia alni, Sporichthya polymorpha, and Acidothermus cellulolyticus. Three strains from rock varnish in the Mojave desert, California, were found to be highly related to Arthrobacter (formerly Micrococcus) agilis. All clusters could be confirmed from results of studies on morphological and physiological properties and from banding patterns of whole cell proteins. Based on the results of tests, four additional strains were assigned to the lineage defined by strain G10.

The membrane-intrinsic light-harvesting complex of the red alga Galdieria sulphuraria (formerly Cyanidium caldarium): biochemical and immunochemical characterization

Abstract The membrane-intrinsic light-harvesting complex of the red alga Galdieria sulphuraria (formerly Cyanidium caldarium ) could be isolated by gel-electrophoresis as a green band with an apparent molecular mass of about 20 kDa. The band had a long-wavelength absorption maximum at 672 nm and a fluorescence maximum (77 K) at 680 nm and reacted with an antibody against light-harvesting proteins of higher plant Photosystem I. Screening of thylakoid membranes with antisera directed against various chlorophyll a/b and chlorophyll a/c light-harvesting proteins indicated the existence of at least 4 distinct light-harvesting polypeptides with apparent molecular masses between 17 and 20 kDa. Isolation of Photosystem I and of a fraction enriched in Photosystem II showed that these polypeptides are exclusively bound to Photosystem I, thus forming a holocomplex which binds at least 205 molecules of chlorophyll a , and 33 and 37 molecules of zeaxanthin and β-carotene, respectively. Additionally, there is some evidence for the existence of a second Photosystem I pool without light-harvesting complexes. In-vitro translation experiments showed that at least two of the five polypeptides which constitute the membrane-intrinsic light-harvesting complex of Galdieria sulphuraria are translated from the poly(A)-enriched RNA fraction. They could be immunoprecipitated as preproteins being 3 to 4 kDa larger in size than the mature polypeptides.

Dilution cultivation of marine heterotrophic bacteria abundant after a spring phytoplankton bloom in the North Sea

The roles of individual bacterioplankton species in the re-mineralization of algal biomass are poorly understood. Evidence from molecular data had indicated that a spring diatom bloom in the German Bight of the North Sea in 2009 was followed by a rapid succession of uncultivated bacterioplankton species, including members of the genera Ulvibacter, Formosa, Polaribacter (class Flavobacteria) and Reinekea (class Gammaproteobacteria). We isolated strains from the same site during the diatom bloom in spring 2010 using dilution cultivation in an artificial seawater medium with micromolar substrate and nutrient concentrations. Flow cytometry demonstrated growth from single cells to densities of 10(4) -10(6) cells ml(-1) and a culturability of 35%. Novel Formosa, Polaribacter and Reinekea strains were isolated and had 16S rRNA gene sequence identities of > 99.8% with bacterioplankton in spring or summer 2009. Genomes of selected isolates were draft sequenced and used for read recruitment of metagenomes from bacterioplankton in 2009. Metagenome reads covered 93% of a Formosa clade B, 91% of a Reinekea and 74% of a Formosa clade A genome, applying a ≥ 94.5% nucleotide identity threshold. These novel strains represent abundant bacterioplankton species thriving on coastal phytoplankton blooms in the North Sea.

EFFECTS OF SALINITY, LIGHT AND TIME ON THE VERTICAL MIGRATION OF DIATOM ASSEMBLAGES

The vertical migration behaviour of diatom assemblages as affected by changes of salinity, light intensity and time of day after (re-)mixing has been studied in the laboratory using Low temperature scanning electron microscopy and epifluorescence light microscopy. Further, the response in migratory speed towards changing salinities was investigated. The migration of diatoms to the sediment surface was partially inhibited by darkness but strongly enhanced by light. Inhibition and enhancement were reversible. Increasing light intensities led to increases in cell numbers on the sediment surface and resulted in variations in species composition migrating to the sediment surface. Diatoms were most responsive to light for upward migration in the morning; remixing or mixing around noon or afternoon resulted in fewer cells migrating to the surface. Maximum upward migratory behaviour was found at 35 ppm salinity. Fewer cells surfaced when the salinity was either lowered or increased. The migratory speed which was approx. 1 μm/s averaged over the entire diatom population remained similar during experiments in which salinity was either lowered to 5 ppm or increased to 60 ppm.

Investigations on Gene Copy Number, Introns and Chromosomal Arrangement of Genes Encoding the Fucoxanthin Chlorophyll a/c-Binding Proteins of the Centric Diatom Cyclotella cryptica

The gene arrangement, existence of introns and the number of gene copies of genes (fcps) encoding fucoxanthin chlorophyll a/c-binding proteins (Fcps) of the centric diatom Cyclotella cryptica were investigated by polymerase chain reaction (PCR), Southern blotting and denaturing gradient gel electrophoresis (DGGE) experiments. PCR-mediated amplification of the fcp genes using chromosomal DNA as template demonstrated the absence of introns within the amplified regions. Clustering of genes could not be demonstrated in these experiments. Digestion of chromosomal DNA of Cy. cryptica followed by Southern blotting and hybridization with specific fcp probes revealed minimum and maximum values of 12 and 20, respectively, for the gene copies. In addition, the DGGE technique confirmed and strengthened the results obtained from Southern blotting experiments as amplification of gene fragments from genomic DNA with different sets of specific primers revealed values of 21 and 23, for the minimum and maximum gene copy number, respectively.

Structural Analysis and Electrochemical Properties of Bimetallic Palladium–Platinum Aerogels Prepared by a Two‐Step Gelation Process

Multimetallic aerogels have emerged as promising unsupported and high‐surface‐area metal materials for different applications in heterogeneous catalysis and electrochemistry, which are fabricated using a gelation process characterized by the controlled aggregation of metallic nanoparticles to form a macroscopic network structure in aqueous solution. However, the achievement of the structural homogeneity of the multimetallic aerogels in terms of the diameter of the nanochains and the chemical composition at the nano‐ and the macro‐scale is still a great challenge. In this paper, we investigated two Pd‐Pt aerogels prepared by the two‐step gelation method. The structural homogeneity and chemical distribution of both metals inside the aerogels were analyzed by using high‐resolution (scanning) transmission microscopy, energy‐dispersive X‐ray spectroscopy, extended X‐ray absorption fine structure spectroscopy and cyclic voltammetry. The Pd‐Pt aerogels show the presence of Pd/Pt‐rich domains inside the long‐range framework. It is evident that the initial monometallic features dominate over alloying during the gelation process. Although the same synthetic approach for Pd‐Pt aerogels with different atomic ratios was used, we observed that the sizes of these monometallic domains varied strongly between the Pd‐rich and Pt‐rich aerogels. The presence of such metal clusters influenced the electrochemical robustness of the Pd‐Pt aerogels dramatically. Electrochemical durability investigations revealed that the aerogels with a high content of Pd are less stable because of the gradual dissolution of the less noble metal particularly inside the Pd‐rich domains. A chemical and structural homogeneity might improve the lifetime of the Pd‐Pt aerogels under electrochemical conditions. In this work, we provide a better understanding of the structure and chemical distribution of the bimetallic aerogel framework prepared by the two‐step gelation process.

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF PUTATIVE REGULATORY DNA SEQUENCES OF FCP GENES IN THE CENTRIC DIATOM CYCLOTELLA CRYPTICA

In order to investigate the transcriptional regulation of light harvesting proteins in diatoms we cloned and sequenced the regions encoding the N-termini together with the respective upstream DNA stretches of 11 different fucoxanthin chlorophyll a/c binding polypeptides (FCP) of Cyclotella cryptica. The deduced N-terminal amino acid sequences almost completely matched those deduced earlier from cDNA clones. No introns were found in the regions coding for the N-terminal plastid targeting sequences or shortly downstream of them. A PLACE search for cis-acting DNA elements which are known from higher plants to be involved in the light-dependent or circadian expression of Lhc genes encoding chlorophyll a/b binding light-harvesting polypeptides in green plants resulted in the detection of several motifs. A comparative analysis with DNA stretches, upstream of the Lhc, LhcaR and Fcp genes of the green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana, however, did not show any conservation of these elements. This indicates that green plants, red algae and diatoms most probably do not share the same conserved elements for light-regulated gene expression. Furthermore a 924 bp DNA fragment habouring a stretch of 292 bp upstream DNA and the entire Fcp6 gene was amplified from genomic DNA of Cyclotella cryptica, cloned into a modified derivative of the transformation vector pPha-T1 and expressed in the pennate diatom Phaeodactylum tricornutum. Reverse transcriptase polymerase chain reaction using RNA of the transformants as template showed that the introduced Fcp6 gene indeed was transcribed, indicating that the regulatory sequence may be generally functional in diatoms.

An immunochemical in situ approach to detect adaptation processes in the photosynthetic apparatus of diatoms of the Wadden Sea sediment surface layers.

Intertidal Wadden Sea sediment surface layers located near the North Sea shore at Dangast (Germany) were subjected to quantitative chlorophyll and protein extractions followed by SDS-PAGE and Western immunoblotting. During the study, benthic diatoms were almost exclusively the only group of microphytobenthos in this area performing oxygenic photosynthesis. Three successive extractions with 90% acetone yielded more than 98% of the extractable pigments. The absorption spectra of the extracts of sediment samples were nearly identical to those obtained from the diatom Phaeodactylum tricornutum. Ten repetitive extractions with SDS containing loading buffer used for SDS-PAGE ensured that more than 98% of the extractable protein was recovered. Subsequent Western immunoblotting with an antiserum directed against the subunits of the main light harvesting complex of the diatom Cyclotella cryptica showed that the antiserum immunodecorated selectively subunits of diatomaceous light harvesting complexes. This finding demonstrated that a taxon specific class of polypeptides could be visualized and quantified directly in sediment samples. In shading experiments, shaded sedimient areas generally revealed higher amounts of light harvesting subunits which could be immunodecorized. The improved mnethodological approach and the results are discussed in the context of the current development of direct molecular methods for the investigation of activities and adaptation processes of specific groups of microorganisms in their natural habitats.

Swarm-like migratory behaviour in the laboratory of a pennate diatom isolated from North Sea sediments

An unidentified Navicula species was isolated from the surface sediments in two different localities of the German North Sea shore in 1997. Cells were maintained in culture on agar plates under various environmental conditions. The diatom cells showed a previously undescribed migratory behaviour, moving synchronically from the starting point towards the periphery of the Petri dish, resulting in a swarm-like migration and giving rise to ring-like banding patterns. This mode of movement takes place horizontally on a rather small scale (cm) and usually within days; it might represent, in addition to dispersal by water, another way of colonizing new areas of its natural environment. Ring formation occurred only when diatom cells were cultured on agar plates either in continuous white light or in a 14:10 h light:dark regime at an irradiance between 5 and 10 μmol photons m−2 s−1, and not when the agar plates were kept in the dark. Salinity above or beyond 34 psu, as well as a lack of nitrate or phosphate had a negative impact on this migratory behaviour and resulted in reduced numbers of rings or lack of migration. This migratory pattern was recorded repeatedly from October to the end of March, but ceased to occur afterwards.