Presence of Cysteic Acid in the Sporangium and its Metabolic Pathway during Sporulation of Bacillus subtilis NRRL B558

Abstract

Cells of Bacillus subtilis NRRL B558 at early stages of sporulation contained considerable amounts of cysteic acid. The cell cysteic acid content began to increase at the onset of sporulation and was maximal when synthesis of dipicolinic acid commenced; it then decreased as sporulation advanced. C ysteic acid was synthesized from cysteine and metabolized to sulpholactate in the mother-cell cytoplasm, and the sulpholactate was then incorporated into the forespore. This metabolic change is considered to be a general phenomenon in spore-formers that contain sulpholactate in their spores. I N T R O D U C T I O NIdentification of spore-specific materials and knowledge of their role during sporulation has been considered essential for studies on sporulation mechanisms. Studies of the chemical constituents of mature spores have revealed the existence of many spore-specific materials, such as dipicolinic acid (Powell, 1953) (Warth & Strominger, 1968). It has been generally accepted that these spore-specific materials are newly synthesized in sporangia from substances derived from vegetative cell constituents, in which case precursors of spore constituents are presumably present in the cells at an early stage of sporulation. No detailed information has yet been obtained on the chemical nature or biological roles of such precursors. Chemical and biochemical studies on precursors of spore-specific materials may contribute to our understanding of sporulation mechanisms. The present paper describes the identification of cysteic acid as a precursor of sulpholactate and the metabolic pathway of cysteine to sulpholactate via cysteic acid. M E T H O D SStrain and culture conditions. Bacillus subtilis NRRL B558 was used unless otherwise indicated. Bacilli from an overnight nutrient agar slope were suspended in Schaeffer's medium (Schaeffer & Ionesco, 1960) and incubated for 5 h at 37 OC. A sufficient volume of this culture was inoculated into fresh Schaeffer's medium to give an initial of 0.03 to 0.05, and the fresh culture was incubated with shaking (120 strokes min-', 3 cm amplitude) at 37 OC. Cells were harvested at various stages of sporulation and washed with chilled saline by centrifuging at 10000 rev. min-I for 10 min.Replacement sporulation was carried out in a medium containing (per litre) 50mmol acetic acid, 10 mmol glycine, 10 mmol serine, 1.5 g NaCl, 0.45 g KH,P04, 0.58 g K2HP04, 200 mg MgS04.7H20, 7 mg MnS04. 7H,O, 15 mg FeSO,. 7H,O and 73.5 mg CaCl,. 2H,O (pH 6.8).Trichloroacetic acid (TCA) extraction andpreparation offractions. Cells (100 mg dry wt) were treated in 15 ml 10% (w/v) TCA with stirring for 2 h at 0 OC, and the extract was centrifuged at 10000 rev. min-' for 20 min at 0 OC. The supernatant was extracted with 45 ml diethyl ether to remove the TCA and the water layer (TCA extract) was retained.

Microbial desulfonation

“…[161]), while the importance of methanesulfonate in the sulfur cycle has only now been recognized [69]. Cysteate was first observed in biological systems as a weathering product from cysteine in wool [162], but it is now better known as an intermediate in one pathway of taurine formation [1]or as a precursor [163]of the major spore component, sulfolactate [164]. Isethionate, initially found as a major anion in squid nerve [22], is now known to have a wider distribution [165].…”

Microbial desulfonation

“…[161]), while the importance of methanesulfonate in the sulfur cycle has only now been recognized [69]. Cysteate was ¢rst observed in biological systems as a weathering product from cysteine in wool [162], but it is now better known as an intermediate in one pathway of taurine formation [1] or as a precursor [163] of the major spore component, sulfolactate [164]. Isethionate, initially found as a major anion in squid nerve [22], is now known to have a wider distribution [165].…”

Identification of Coenzyme M Biosynthetic Phosphosulfolactate Synthase

“…These enzymes are probably used to synthesize sulfolactate from sulfate and Penolpyruvate during B. subtilis sporulation (14,48). This organism may also produce sulfolactate from cysteic acid (49) Phosphosulfolactate Synthase from M. jannaschiifrom L-cysteate, but not from L-cysteine (50,51). Rather, Com-ABC proteins in Cytophaga spp.…”