Genetic analysis of Bacillus subtilis stable L-forms obtained via long-term cultivation.

Various bacteria can change to a spherical cell-wall-deficient state, called L-from, in the presence of antibiotics that inhibit cell wall synthesis. L-forms are classified into two types: unstable and stable L-forms. Unstable L-forms revert to a normal walled state in the absence of antibiotics, while stable L-forms remain in their wall-deficient state. The conversion from unstable to stable L-forms has been often observed during long-term cultivation. However, the genetic cause for this conversion is not yet fully understood. Here, we obtained stable Bacillus subtilis L-form strains from unstable L-form strains via three independent long-term culturing experiments. The whole genome sequencing of the long-cultured strains identified many mutations, and some mutations were commonly found in all three long-cultured strains. The knockout strain of one of the commonly mutated genes, tagF, in the ancestral strain lost the ability to revert to walled state (rod shape), supporting that eliminating the function of tagF gene is one of the possible methods to convert unstable L forms to a stable state.

Viridiuvalis adhaerens gen. et sp. nov., a novel colony-forming chlorarachniophyte.

A new chlorarachniophyte, Viridiuvalis adhaerens gen. et sp. nov. was isolated from the mucus on a coral reef from Zanpa Beach, Okinawa, Japan. The main vegetative stage of V. adhaerens consisted of unicellular coccoid cells with cell walls, although sarcinoid colonies and uniflagellate zoospores were also observed. V. adhaerens had chloroplasts with nucleomorphs and pyrenoids that were completely embedded in the chloroplast. A deep plate-like invagination of the periplastidal compartment (PPC) almost partitioned the pyrenoid and chloroplast components, which were surrounded by two membranes. The nucleomorph was positioned in the base of the invagination of the PPC. Molecular phylogenetic analyses using rRNA genes showed that V. adhaerens branched as a sister lineage of the Amorphochlora clade. The sarcinoid colony, pyrenoid embedded in the chloroplast, and nucleomorph located at the base of the deep invagination of the PPC have not been reported in other chlorarachniophytes. Based on these morphological and ultrastructural characteristics and the results of the molecular phylogenetic analyses, we propose V. adhaerens as a new genus and species of chlorarachniophyte.