Protein Synthesis in the Isolated Forespores from Sporulating Cells of Bacillus subtilis

Abstract

Developing forespores were isolated from Bacillus subtilis at different stages of sporulation and protein synthesis in the forespore compartment was examined. Pulse-labeling experiments indicated that [14C]phenylalanine was continuously incorporated into the sporangium throughout sporulation, and at t5 (early stage V of sporulation) 58% of the radioactivity was located in the forespore compartment. Significantly high incorporation of [14C]phenylalanine was observed when the isolated forespores at ts were incubated with the corresponding mother-cell cytoplasmic fraction or an amino acid mixture. About 73% of the radioactivity incorporated into the isolated forespore at t5 was found in the cytoplasmic fraction and 26% in the membranous fraction. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the 14C-labeled cytoplasmic protein had a-molecular weight of about 20,000, and that a protein having the same molecular weight was present in the ts forespore as a slight protein band and also in the mature spore as a clear protein band. Gel electrophoresis also revealed that the 14C-labeled membranous-soluble protein (prepared by solubilization with detergents) had broad peaks with molecular weights of about 74,000, 33,000, 20,000, and 12,000.After a final round of chromosomal replication, bacterial sporulation proceeds through a sequence of biochemical and morphological changes in the cell (20). Stationary phase cells are asymmetrically partitioned into two compartments by the rapid growth of a folded mother-cell double-membrane septum. The smaller of these compartments (forespore) is then engulfed as a discrete cell within the larger compartment (mother cell). Many important questions about the biochemical events and gene expression taking place in both compartments throughout the development of sporulation have remained unanswered because of the absence of reliable methods for isolating the forespore from the mother cell. Extensive protein turnover has been shown to occur during sporulation (4,12,13,22); at least 70% of the spore protein in Bacillus thuringiensis and B. subtilis has been shown to be newly synthesized during sporulation. These results were, however, obtained from the whole cells of an earlier stage of sporulation, which did not contain a discrete cell (forespore) within the mother-cell cytoplasm.

Prevalence and Duration of Asymptomatic Clostridium difficile Carriage among Healthy Subjects in Pittsburgh, Pennsylvania

“…This finding should provide a note of caution to investigators seeking to screen healthy donors for fecal microbiota therapy (FMT) as a treatment for CDIs without using toxigenic anaerobic cultures. This finding probably reflects the limit of detection for the illumigene assay, which is not optimized for organism densities lower than those typically encountered in CDI patients (ϳ10 6 CFU/g stool). Furthermore, nucleic acid-based assays may be insufficiently sensitive to identify asymptomatic, C. difficile-colonized, hospital inpatients, the active surveillance of whom may have a role in strategies for the reduction of hospital-acquired CDIs (17).…”

“…The source of C. difficile in community-acquired cases is unclear. Prevalence estimates for asymptomatic C. difficile colonization range from 7 to 15% among healthy, non-health care workers outside the United States to 1 to 4% among health care workers in the United States and abroad (5)(6)(7)(8)(9). Individuals colonized with C. difficile could represent a potential reservoir of strains imported into hospitals, as well as a source of false-positive clinical test results for CDIs among patients with community-acquired diarrheal illness, especially norovirus and Clostridium perfringens.…”

Purification and Characterization of Cytoplasmic Proteins Synthesized in the Developing Forespores of Bacillus subtilis during Sporulation

“…Procedures for isolation of intact forespores have been developed in many species of Bacillus (Andreoli et al, 1973;Ellar & Posgate, 1974;Fujita et al, 1977;Watabe et al, 1980;Nakayama et al, 1980) and some of the biochemical properties closely related to the developing forespores have been investigated (Eaton & Ellar, 1974;Ellar & Posgate, 1974;Andreoli et af., 1975Andreoli et af., , 1978Ellar et al, 1975;Fujita et al, 1977;Nakayama et al, 1980;Watabe et al, 1980Watabe et al, , 1981Setlow, 198 1). We have developed a [ Clphenylalanine-incorporation system using isolated forespores of Bacillus subtilis 60015, and found continuous protein synthesis in the young forespore throughout sporulation.…”

“…Preparation of forespores from sporulating cells at t 5 and confirmation of their purity were carried out as described previously (Watabe et al, 1981). The labelling experiment with the isolated forespores was performed by incubation with 0.1 pCi ~-[U-l~C]phenylalanine ml-1 (3-7 kBq m1-I) and10 p~ of each of 20 unlabelled amino acids (Watabe et al, 1981).…”

“…We have developed a [ Clphenylalanine-incorporation system using isolated forespores of Bacillus subtilis 60015, and found continuous protein synthesis in the young forespore throughout sporulation. About 75% of the radioactive amino acid incorporated into the forespore at t5 was located in the cytoplasmic fraction, and a major protein labelled with [ 14C]phenylalanine was found to have a molecular weight of about 20000 and lower molecular weight polypeptides also contained radioactivity (Watabe et al, 1981). In the present paper, l4 C-labelled proteins in the forespore cytoplasmic fraction were purified to homogeneity, and their amino acid composition and molecular weights were analysed.…”