A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

The Family Acholeplasmataceae (Including Phytoplasmas)The Prokaryotes

The family Acholeplasmataceae was originally established to accommodate the genus Acholeplasma, comprising the mollicutes that could be cultivated without the supplement of cholesterol and that use UGA as a stop codon instead of coding for tryptophan. It was later shown that the phytoplasmas, a large group of uncultivable, wall-less, non-helical mollicutes that are associated with plants and insects, shared taxonomically relevant properties with members of the genus Acholeplasma. Being not cultivable in vitro in axenic culture, the phytoplasmas could not be classified using the standards used for other mollicutes and are named using the category of Candidatus, as “Ca. Phytoplasma.” Although phytoplasmas are associated with habitats and ecology different from acholeplasmas, the two genera Acholeplasma and “Candidatus Phytoplasma” are phylogenetically related and form a distinct clade within the Mollicutes. The persisting inability to grow the phytoplasmas in vitro hinders the identification of their distinctive phenotypic traits, important criteria for mollicute classification. Until supplemental phenotypic traits become available, the genus “Candidatus Phytoplasma” is designated, on the basis of phylogeny, as a tentative member in the family Acholeplasmataceae. Phylogenetic analysis based on gene sequences, in particular, ribosomal sequences, has provided the major supporting evidence for the composition and taxonomic subdivision of this group of organisms with diverse habitats and ecology and has become the mainstream for the Acholeplasmataceae systematics. However, without the ability to determine phenotypic properties, the circumscription of related species among the non culturable members of the family remains a major issue. The genus Acholeplasma comprises 14 species predominantly associated with animals and isolated from mammalian fluids but regarded as not normally pathogenic. Conversely, the genus “Ca. Phytoplasma” includes plant pathogens of major economic relevance worldwide. To date, 36 “Ca. Phytoplasma species” have been described