Host and symbiont intraspecific variability: The case of Paramecium calkinsi and "Candidatus Trichorickettsia mobilis".

Newly isolated strains of the ciliate Paramecium calkinsi and their cytoplasmic bacterial endosymbionts were characterized by a multidisciplinary approach, including live observation, ultrastructural investigation, and molecular analysis. Despite morphological resemblance, the characterized P. calkinsi strains showed a significant molecular divergence compared to conspecifics, possibly hinting for a cryptic speciation. The endosymbionts were clearly found to be affiliated to the species "Candidatus Trichorickettsia mobilis" (Rickettsiales, Rickettsiaceae), currently encompassing only bacteria retrieved in an obligate intracellular association with other ciliates. However, a relatively high degree of intraspecific divergence was observed as well, thus it was possible to split "Candidatus Trichorickettsia" into three subspecies, one of which represented so far only by the newly characterized endosymbionts of P. calkinsi. Other features distinguished the members of each different subspecies. In particular, the endosymbionts of P. calkinsi resided in the cytoplasm and possessed numerous peritrichous flagella, although no motility was evidenced, whereas their conspecifics in other hosts were either cytoplasmic and devoid of flagella, or macronuclear, displaying flagellar-driven motility. Moreover, contrarily to previously analyzed "Candidatus Trichorickettsia" hosts, infected P. calkinsi cells frequently became amicronucleate and demonstrated abnormal cell division, eventually leading to decline of the laboratory culture.

Covalent incorporation of tobacco mosaic virus increases the stiffness of poly(ethylene glycol) diacrylate hydrogels.

Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA (M n = 700 g mol-1) hydrogels are combined with a thiol-group presenting TMV mutant (TMVCys). By covalent coupling of TMVCys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMVCys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies.

Magnetic spin imaging under ambient conditions with sub-cellular resolution.

The detection of small numbers of magnetic spins is a significant challenge in the life, physical and chemical sciences, especially when room temperature operation is required. Here we show that a proximal nitrogen-vacancy spin ensemble serves as a high precision sensing and imaging array. Monitoring its longitudinal relaxation enables sensing of freely diffusing, unperturbed magnetic ions and molecules in a microfluidic device without applying external magnetic fields. Multiplexed charge-coupled device acquisition and an optimized detection scheme permits direct spin noise imaging of magnetically labelled cellular structures under ambient conditions. Within 20 s we achieve spatial resolutions below 500 nm and experimental sensitivities down to 1,000 statistically polarized spins, of which only 32 ions contribute to a net magnetization. The results mark a major step towards versatile sub-cellular magnetic imaging and real-time spin sensing under physiological conditions providing a minimally invasive tool to monitor ion channels or haemoglobin trafficking inside live cells.

Spirostomum spp. (Ciliophora, Protista), a suitable system for endocytobiosis research.

Among ciliate genera, only Paramecium and Euplotes species have been studied extensively as host organisms of bacterial endocytobionts. In this article, we show that members of the genus Spirostomum may also serve as a suitable system for endocytobiosis research. Two strains of Spirostomum minus (Heterotrichea, Ciliophora) collected in Germany and Italy, respectively, were found to harbor different types of bacterial infections. Bacteria of various sizes and shapes were observed in the cytoplasm or in the nuclei of the ciliates. The bacteria in the cytoplasm were either surrounded by a peribacterial membrane or lay naked. One of the bacterial species was found in the vicinity of the contractile fibrillar system (myonemes) of the ciliates. In rare cases, another type of bacteria was observed associated with mitochondria. The macronuclei of both the Italian and the German strains were crowded with endocytobionts. The endonuclear bacteria in the two S. minus strains differed with respect to their cytoplasmic structures but they were of similar size and both were rod shaped. According to the results of in situ hybridization, the endonuclear bacteria of the Italian strain belong to the subgroup of alphaproteobacteria, whereas the bacteria associated with the fibrillar system appeared to be gram-positive bacteria with high G+C content. While both the German and the Italian strains were found to permanently maintain their endocytobionts, they were at least partly colonized by different bacteria. This is taken as an indication that geographically separated populations of ciliates may be stably infected by different endocytobionts, possibly due to different ecological conditions. For S. minus and S. ambiguum a total of 7 different bacterial endocytobionts have now been recorded. We recommend the members of the genus Spirostomum as a suitable system for endocytobiosis research.

Characterization of intracellular bacteria in the freshwater dinoflagellate Peridinium cinctum.

Intracellular bacteria belonging to two phylogenetically different groups of eubacteria were found in cultures of the freshwater dinoflagellate Peridinium cinctum (O.F. Müller) Ehrenberg isolated from the eutrophic lake Plusssee (Federal Republic of Germany). The phylogenetic relationships of the bacteria were studied with fluorochrome-conjugated oligonucleotides specific for archaebacteria, eubacteria, alpha-, beta- and gamma-proteobacteria, complementary to 16S rRNA and 23S rRNA sequences, respectively. The bacteria are members of the eubacterial alpha- and gamma-subgroups of proteobacteria.

The Toxic Symbiont Caedibacter caryophila in the Cytoplasm of Paramecium novaurelia.

Endosymbiotic bacteria were observed to inhabit the cytoplasm of the freshwater ciliate Paramecium novaurelia. Transmission electron microscopy and toxicity tests with sensitive paramecia showed that the endosymbionts belong to the genus Caedibacter. The bacteria conferred a killer trait to their host paramecia. The production of a proteinaceous inclusion body ("R-body") in the bacterial cell makes them toxic to other paramecia after they become enclosed in food vacuoles. R-bodies of Caedibacter sp were associated with phages, which are known in most other Caedibacter species to code for the R-body proteins. The killer-effect of P. novaurelia on sensitive P. caudatum strains was of the "paralysis" type, which is a characteristic of the symbiont species Caedibacter caryophila. Until now C. caryophila was known to inhabit the macronucleus of Paramecium caudatum only. Sequencing of the 16S rRNA-gene proved that Caedibacter sp from the cytoplasm of P. novaurelia belongs to the species C. caryophila as well. The rDNA-sequence of 1695 bp length differed in a total of only 1 bp from the corresponding gene in C. caryophila from the macronucleus of P. caudatum. The results indicate that the infection of specific host cell compartments may depend on host genes, but not on different traits of the infecting symbiont species. The occurrence of killer and sensitive paramecia strains together in one pond is discussed with respect to the competitive advantage of the killer trait.

Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors.

COPI-coated vesicle budding from lipid bilayers whose composition resembles mammalian Golgi membranes requires coatomer, ARF, GTP, and cytoplasmic tails of putative cargo receptors (p24 family proteins) or membrane cargo proteins (containing the KKXX retrieval signal) emanating from the bilayer surface. Liposome-derived COPI-coated vesicles are similar to their native counterparts with respect to diameter, buoyant density, morphology, and the requirement for an elevated temperature for budding. These results suggest that a bivalent interaction of coatomer with membrane-bound ARF[GTP] and with the cytoplasmic tails of cargo or putative cargo receptors is the molecular basis of COPI coat assembly and provide a simple mechanism to couple uptake of cargo to transport vesicle formation.

Coincidence Counting in Time-of-Flight Mass Spectrometry: A Test for Chemical Microhomogeneity.

Coincidence counting techniques have been combined with time-of-flight mass spectrometry in the examination of surfaces for chemical microhomogeneity. A mathematical formalism was developed to describe the principles underlying this coincidence counting technique and was used to produce a quantitative method for handling the data obtained. This technique of testing for chemical homogeneity has been demonstrated with a sample that consists of a physical mixture of polystyrene and crystals of NaF which were tenths of micrometers in diameter. Ultimately this approach is expected to be useful for the routine testing of surfaces for chemical homogeneity at the level of tens of nanometers.