Isolation and characterization of a Clostridium sp with cinnamoyl esteraseactivity and unusual cell envelope ultrastructure

Microorganisms that hydrolyse the ester linkages between phenolic acids and polysaccharides in plant cell walls are potential sources of enzymes for t he degradation of lignocellulosic waste. An anaerobic, mesophilic, spore-fo rming, xylanolytic bacterium with high hydroxy cinnamic acid esterase activ ity was isolated from the gut of the grass-eating termite Tumilitermes past inator. The bacterium was motile and rod-shaped, stained gram-positive, had an eight-layered cell envelope, and.formed endospores. Phylogenetic analys is based on 16S rRNA indicated that the bacterium is closely related to Clo stridium xylanolyticum and is grouped with polysaccharolytic strains of clo stridia. A wide range of carbohydrates were fermented, and growth was stimu lated by either xylan or cellobiose as substrates. The bacterium hydrolysed and then hydrogenated the hydroxy cinnamic acids (ferulic and p-coumaric a cids), which are esterified to arabinoxylan in plant cell walls. Three cyto plasmic enzymes with hydroxy cinnamic acid esterase activity were identifie d using non-denaturing gel electrophoresis. This bacterium possesses an unu sual multilayered cell envelope in which both leaflets of the cytoplasmic m embrane, the peptidoglycan layer and the S layer are clearly discernible. T he fate of all these components was easily followed throughout the endospor e formation process. The peptidoglycan component persisted during the entir e morphogenesis. It was seen to enter the septum and to pass with the engul fing membranes to surround the prespore. It eventually expanded to form the cortex, verification for the peptidoglycan origin of the cortex. Sporogeni c vesicles, which are derived from the cell wall peptidoglycan, were associ ated with the engulfment process. Spore coat fragments appeared early, in s tage II, though spore coat formation was not complete until after cortex fo rmation.