Fusobacterium nucleatum ATCC 10953 Requires Actinomyces naeslundii ATCC 43146 for Growth on Saliva in a Three-Species Community That Includes Streptococcus oralis 34

Formation of dental plaque is a developmental process involving initial and late colonizing species that form polymicrobial communities. Fusobacteria are the most numerous gram-negative bacteria in dental plaque, but they become prevalent after the initial commensal colonizers, such as streptococci and actinomyces, have established communities. The unusual ability of these bacteria to coaggregate with commensals, as well as pathogenic late colonizers, has been proposed to facilitate colonization by the latter organisms. We investigated the integration of Fusobacterium nucleatum into multispecies communities by employing two in vitro models with saliva as the sole nutritional source. In flow cell biofilms, numbers of cells were quantified using fluorescently conjugated antibodies against each species, and static biofilms were analyzed by quantitative real-time PCR (q-PCR) using species-specific primers. Unable to grow as single-species biofilms, F. nucleatum grew in two-species biofilms with Actinomyces naeslundii but not with Streptococcus oralis. However, enhanced growth of fusobacteria was observed in three-species biofilms, indicating that there was multispecies cooperation. Importantly, these community dynamics yielded an 18-fold increase in the F. nucleatum biomass between 4 h and 18 h in the flow cell inoculated with three species. q-PCR analysis of static biofilms revealed that maximum growth of the three species occurred at 24 h to 36 h. Lower numbers of cells were observed at 48 h, suggesting that saliva could not support higher cell densities as the sole nutrient. Integration of F. nucleatum into multispecies commensal communities was evident from the interdigitation of fusobacteria in coaggregates with A. naeslundii and S. oralis and from the improved growth of fusobacteria, which was dependent on the presence of A. naeslundii.The human mouth contains microbiologically diverse communities. While collectively humans harbor more than 700 bacterial phylotypes, each individual is estimated to have fewer than 100 such phylotypes (1), and approximately 50% of human oral bacteria have yet to be cultivated. Although biofilm communities on tooth enamel are polymicrobial (3, 20), more than 60 to 90% of the bacteria found in initial plaque on saliva-coated tooth enamel are streptococci (6, 19). Other bacterial genera that are among the initial commensal colonizers include Actinomyces, Veillonella, and Neisseria (6, 16, 19), and these organisms contribute to the polymicrobial nature of initial plaque.The structure of a community is dependent upon the nature of the foundation. An integral feature of an oral bacterial biofilm foundation is the ability to coaggregate, which is defined as cell-cell recognition and binding between genetically distinct bacteria. After routine oral hygiene treatment, freshly cleaned tooth enamel is quickly coated with a salivary pellicle, which provides a set of receptor molecules recognized by primary colonizing bacteria, such as streptococci and actinomyces. Besides recognizing salivary receptors, these bacteria coaggregate and provide a foundation for the subsequent attachment and growth of other bacteria, such as veillonellae, that form close metabolic relationships with streptococci (12, 15). As initial colonizers develop into biofilm communities with anaerobic microenvironments, incorporation of the obligate anaerobic fusobacteria into these communities becomes possible. Fusobacteria as a group coaggregate with all other oral bacteria and have been suggested, therefore, to be a crucial link between primary colonizing species and later colonizing pathogens (13, 14). Thus, a foundation consisting of coaggregating streptococci, actinomyces, and veillonellae populates the tooth surface, and these organisms are recognized by fusobacteria, which colonize and become the dominant gram-negative bacterial species. The new foundation is a substratum containing fusobacterial surface receptors available for recognition by late colonizing pathogens. Supporting the crucial link is clinical evidence that fusobacteria appear in dental plaque after commensal species and before the pathogenic “red” complex consisting of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia (22, 23).Coaggregation partnerships are highly specific. A significant role for coaggregation in the formation of dental plaque biofilms and particularly in accretion of secondary colonizers to the pioneer species in plaque has been proposed (14) and has been demonstrated for the development of a spatially organized community (20). However, coaggregation may also provide some metabolic advantages (e.g., cross feeding and enzyme complementation) to neighboring cells by facilitating physical juxtaposition of partner cells, as has been shown for glucose metabolism of coaggregates of actinomyces and streptococci (7, 8). One aim of the present study was to examine the structures of two- and three-species communities composed of Actinomyces naeslundii, Streptococcus oralis, and Fusobacterium nucleatum in model biofilm systems. The first two species are initial colonizers and are considered commensals, whereas fusobacteria are secondary colonizers and are postulated to be a coaggregation bridge between initial and late colonizers (14). Our second aim was to investigate the integration and growth of fusobacteria in polymicrobial communities.A variety of experimental methods have been developed to study the formation of biofilms. Model systems often rely on the flow of nutrients over a surface on which bacteria are able to attach and grow. In the present study we used two distinct in vitro models, a saliva-fed flow cell and a polystyrene peg immersed in static saliva. Biofilm communities form naturally and are undisturbed (3, 20, 21). The spatial organization of a multispecies community resulting from colonization and growth is preserved and can be examined noninvasively by confocal laser scanning microscopy (CLSM). In the static system, the amount of each species in multispecies biofilms formed on polystyrene pegs can be measured by real-time quantitative PCR (q-PCR). We show here with both models that fusobacteria are unable to grow as single species, but they integrate into commensal streptococcus-actinomyces communities and grow. Integration and growth are required for fusobacteria to become crucial links between commensal communities and later colonizing pathogenic communities. In the three-species community studied here, A. naeslundii is required for F. nucleatum to integrate and grow.     

The effects of temperature, pressure and peptide incorporation on ternary model raft mixtures--a Laurdan fluorescence spectroscopy study

Recently, an increasing evidence accumulated for the existence of lipid microdomains, called lipid rafts, in cell membranes, which may play an important role in many important membrane-associated biological processes. Suitable model systems for studying biophysical properties of lipid rafts are lipid vesicles composed of three-component lipid mixtures, such as POPC/SM/cholesterol, which exhibit a rich phase diagram, including raft-like liquid-ordered/liquid-disordered phase coexistence regions. We explored the temperature, pressure and concentration-dependent phase behavior of such canonical model raft mixtures using the Laurdan fluorescence spectroscopic technique. Hydrostatic pressure has not only been used as a physical parameter for studying the stability and energetics of these systems, but also because high pressure is an important feature of certain natural membrane environments. We show that the liquid-disordered/liquid-ordered phase coexistence regions of POPC/SM/cholesterol model raft mixtures extends over a very wide temperature range of about 50 degrees C. Upon pressurization, an overall ordered membrane state is reached at pressures of approximately 1,000 bar at 20 degrees C, and of approximately 2,000 bar at 40 degrees C. Incorporation of 5 mol% gramicidin as a model ion channel slightly increases the overall order parameter profile in the l(o)+l(d) two-phase coexistence region, probably by selectively partitioning into l(d) domains, does not change the overall phase behavior, however. This behavior is in contrast to the effect of the peptide incorporation into simple, one-component phospholipid bilayer systems.     

The Calsequestrin Mutation CASQ2D307H Does Not Affect Protein Stability and Targeting to the Junctional Sarcoplasmic Reticulum but Compromises Its Dynamic Regulation of Calcium Buffering

Mutations in cardiac ryanodine receptor (RYR2) and cardiac calsequestrin (CASQ2) genes are linked to catecholaminergic polymorphic ventricular tachycardia, a life-threatening genetic disease. They predispose young individuals to cardiac arrhythmia in the absence of structural abnormalities. One such mutation that changes an aspartic residue to histidine at position 307 in CASQ2 has been linked to catecholaminergic polymorphic ventricular tachycardia. In this study we made a transgenic mouse model expressing the mutant CASQ2^D307H protein in a CASQ2 null background and investigated if the disease is caused by accelerated degradation of the mutant protein. Our data suggest that the mutant protein can be expressed, is relatively stable, and targets appropriately to the junctional sarcoplasmic reticulum. Moreover, it partially normalizes the ultrastructure of the sarcoplasmic reticulum, which was altered in the CASQ2 null background. In addition, overexpression of the mutant protein does not cause any pathology and/or structural changes in the myocardium. We further demonstrate, using purified protein, that the mutant protein is very stable under chemical and thermal denaturation but shows abnormal Ca²⁺ buffering characteristics at high calcium concentrations. In addition, trypsin digestion studies reveal that the mutant protein is more susceptible to protease activity only in the presence of high Ca²⁺. These studies collectively suggest that the D307H mutation can compromise the dynamic behavior of CASQ2 including supramolecular rearrangement upon Ca²⁺ activation.     

On the origin of heterogeneity of fluorescence decay kinetics of reduced nicotinamide adenine dinucleotide

The fluorescence lifetimes of reduced nicotinamide adenine dinucleotide and other dihydronicotinamide derivatives were measured by picosecond laser excited time correlated single photon counting technique. All the dihydronicotinamide derivatives (including the simple model compound N-methyl-nicotinamide) had fluorescence decay profiles which could be fitted to double and triple exponentials in neutral aqueous solutions and in dimethyl sulfoxide respectively. It was concluded that the heterogeneity in the measured lifetimes arises from the inherent photoprocess of the dihydronicotinamide chromophore and not due to any intramolecular interaction as assumed in earlier studies. Some of the possible schemes for the fluorescence decay are discussed.     

Cloning and characterization of the crystal protein-encoding gene of Bacillus thuringiensis subsp. yunnanensis.

Molecular cloning and characterization of a novel cry gene, cry32Aa, of Bacillus thuringiensis subsp. yunnanensis was carried out. The Cry32Aa protein was predicted to have a molecular mass of 139.2 kDa and was found to have an unusual 42-amino-acid-long tail at the C terminus. The cry32Aa gene was localized on the 103-MDa plasmid of the organism. Bioassays showed no toxicity against several moths and mosquitoes. However, it exhibited weak toxicity against larvae of the diamondback moth, Plutella xylostella.