Biochemical characterization of a cortexless mutant of a variant of Bacillus cereus

Abstract

Previous studies on this cortexless mutant of Bacillus cereus var. alesti indicated that the forespore membrane was the site of the biochemical lesion. This hypothesis is supported by the results presented here: fatty acid composition of sporulating cells of themutant is altered, while in vegetative cells it is comparable to the parent; soluble precursors of peptidoglycan synthesis are accumulated in the mutant, at the time of cortex formation; homogenates of the mutant prepared at the time of cortex formation are unable to incorporate tritiated diaminopimelic acid into peptidoglycan, while homogenates of cells forming germ cell wall do so to an extent comparable to that of the parent; lipid-linked intermediates are formed by the mutant as in the parent. Apparently the mutant is unable either to transfer disaccharide penta-peptide units from the carrier lipid to the growing peptidoglycan acceptor, or to transport lipid-linked intermediates across the forespore membrane.

Accessibility of the [ 14 C]Benzylpenicillin Binding Proteins in Membranes of Sporulating Bacilli

“…The fluctuations in specific binding capacity of sporulating cultures, in contrast with the 38 constant binding activity of nonsporulating organisms during post-logarithmic growth, agrees with other observations (2,3,4,15,(21)(22)(23) which suggest that functional peptidoglycan biosynthesis is required during two distinct phases of sporulation (septation or primodial cell wall synthesis and cortex formation). The binding patterns of a series of genetically mapped asporogenous mutants of Bacillus subtilis supports this hypothesis (17).…”

“…Sporulating cells were treated with Triton X-100 as previously described and incubated with various unlabeled semisynthetic penicillins for 15 min at 4 C. The specific ["4C]benzylpenicillin-binding capacity of these cells was then determined (Table 3). In all cases, the semisynthetic penicillin prevented binding of the [4C ]benzylpenicillin to the detergenttreated sporulating cells.…”

“…This polymer is believed to be synthesized by the outer forespore membrane (OFSM) (21). Penicillins and other antibiotics which inhibit cell division by interfering with functional peptidoglycan synthesis can prevent spore septum formation (4,10) or cortex biosynthesis (15) if added to a sporulating culture during the appropriate time intervals. The binding of radioactive benzylpenicillin to sporulating bacilli has been examined and compared to that of Staphylococcus aureus (a nonsporulating organism) during the stationary phase of growth (11).…”

“…Another aspect of the penicillin-binding properties of vegetative, and especially sporulating bacteria, which has been inadequately studied is the accessibility of all of the membranous penicillin-binding components to the antibiotic. This question assumes particular significance in sporulating cells since, in addition to the outermost vegetative membrane, they contain two additional membranes, the IFSM and OFSM, both apparently capable of peptidoglycan synthesis (4,15,21 10,000 x g, and the supematants were assayed for protein content by the method of Lowry et al (14). Corrections were made for the effect of the small amounts of Triton X-100 on the protein assay.…”

The cortical peptidoglycan from spores of Bacillus megaterium and Bacillus subtilis is not highly cross-linked

“…Spores which contain defective coat layers or which have been chemically stripped of their coats maintain full, or nearly full, heat resistance (4). However, changes in the structure or volume of the spore cortex result in a corresponding loss of heat resistance (1,5,10). The involvement of the cortex in spore heat resistance has generally been attributed to its role in maintaining the dehydrated state of the spore protoplast (4).…”