Cell Wall Polymers of Bacillus sphaericus 9602 II. Synthesis of the First Enzyme Unique to Cortex Synthesis During Sporulation

Abstract

The cell wall peptidoglycan of vegetative cells of Bacillus sphaericus 9602 contains l -lysine and d -isoasparagine and is devoid of diaminopimelic acid (Dap), whereas the peptidoglycan of its spore cortex is devoid of l -lysine and d -isoasparagine and contains meso -Dap. These two structures have a common biosynthetic precursor, uridine-diphospho- N -acetylmuramyl- l - alanyl- d -glutamic acid, which accepts either l -lysine or meso -Dap, the latter reaction being the first unique to the synthesis of the spore cortex peptidoglycan. l -lysine-adding activity decays at the end of vegetative growth to a level which is maintained until Dap-adding activity appears, when it declines rapidly again. Dap-adding activity is not detectable in refractile spores, in vegetative cells, or in sporulating cells until about 4 hr after the end of vegetative growth, when it increases rapidly for about 1.5 hr in a process dependent on continued protein and ribonucleic acid (RNA) synthesis. This process apparently involves transcription and translation during this period of a “sporulation-specific” gene whose product is essential for and unique to sporulation. It is closely followed by the acquirement of refractility. Another sporulation-specific gene, that for dipicolinate synthase, is apparently transcribed and translated in an overlapping period commencing about 0.5 hr later, although dipicolinate does not accumulate rapidly until 1.5 hr later, when about 75% of the cells are already refractile. Inhibition of protein synthesis with chloramphenicol or of RNA synthesis with streptolydigin inhibited accumulation of these enzymes in sporulating cells; this inhibition could be reversed by washing out the antibiotics after 1.5 hr. Sporulation recommenced with an unaltered sequence of events but with poorer synchrony. There was no evidence for a messenger RNA for either enzyme of lifetime greater than a small fraction of the period of enzyme accumulation, although dilution with 10 volumes of fresh medium failed to prevent synthesis of Dap-adding enzyme in cells which had become terminally swollen, a process preceding enzyme synthesis by about 1.5 hr. The synthesis of this enzyme in B. sphaericus is apparently dependent on programmed transcription of the appropriate gene.