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

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field