Die Bildung und die Reversion von Sphäroplasten einer Diaminopimelinsäure-auxotrophen Mutante von Escherichia coli

1. The formation and reversion of spheroplasts of the diaminopimelic acid-auxotrophic mutant Escherichia coli K 12, 335, dap ⁻, R⁺TEM in a medium lacking diaminopimelic acid have been investigated by microphotography: During their development from rod form cells to spheroplasts cells on slide-surface-agar preparations underwent two successive cell divisions in the course of which the cells retained their rod form. The cells formed by these divisions partitioned into a varying number of spheroplasts of different size. The reversion of spheroplasts to rod form cells, started by the addition of diaminopimelic acid showed two characteristic steps: Each spheroplast partitioned again into several spheroplast-like cell bodies; most of them reverted directly to rod form cells.
2. The release of the R-factor mediated periplasmic TEM-β-lactamase, E. C. 3.4.2.6., into the growth medium during the development of spheroplasts attained more than 50% of the entire TEM-β-lactamase activity.

The spheroplasts showed a multiple enhancement of TEM-β-lactamase activity per mg cell protein compared with rod form cells.     

Phenetic relationships among varanid lizards based upon comparative electrophoretic data and karyotypic analyses

Comparative electrophoretic phenotypes of 18 of the 32 species of the lizard genus Varanus have been determined for four proteins. The animals studied were representative of species from Africa, Israel, Southeast Asia and Australia. Malate dehydrogenase (A₂) exhibited a single phenotype throughout. Lactate dehydrogenase (B₄) showed four distinctive electrophoretic forms which grouped the various subgenera as follows: (1) Polydaedalus, Empagusia (African); (2) Psammosaurus (Israel); (3) three species of Varanus, V. gouldii, V. spenceri, V. mertensi (Australian); (4) Dendrovaranus, Indovaranus (Southeast Asian), other Varanus species, Odatria (Australian). Electrophoretic and previously reported karyotypic data were used to interpret the phylogenetic relationships as well as the mode and direction of evolution of these animals. In particular, the results questioned the reality of the subgenus Varanus as a taxonomic unit, since four distinct karyotypic forms and two LDH-B₄ phenotypes were observed for these animals, of which one belongs to another subgenus. Serum albumin and carbonic anhydrase phenotypes were of little use in deciding phenotypic groupings.     

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.     

Genetic and Anatomical Basis of the Barrier Separating Wakefulness and Anesthetic-Induced Unresponsiveness

A robust, bistable switch regulates the fluctuations between wakefulness and natural sleep as well as those between wakefulness and anesthetic-induced unresponsiveness. We previously provided experimental evidence for the existence of a behavioral barrier to transitions between these states of arousal, which we call neural inertia. Here we show that neural inertia is controlled by processes that contribute to sleep homeostasis and requires four genes involved in electrical excitability: Sh, sss, na and unc79. Although loss of function mutations in these genes can increase or decrease sensitivity to anesthesia induction, surprisingly, they all collapse neural inertia. These effects are genetically selective: neural inertia is not perturbed by loss-of-function mutations in all genes required for the sleep/wake cycle. These effects are also anatomically selective: sss acts in different neurons to influence arousal-promoting and arousal-suppressing processes underlying neural inertia. Supporting the idea that anesthesia and sleep share some, but not all, genetic and anatomical arousal-regulating pathways, we demonstrate that increasing homeostatic sleep drive widens the neural inertial barrier. We propose that processes selectively contributing to sleep homeostasis and neural inertia may be impaired in pathophysiological conditions such as coma and persistent vegetative states.