Cell Wall-Associated Proteases of Streptococcus cremoris Wg2

Abstract

, have been studied in Streptococcus cremoris Wg2 by immunological methods. The components could not be separated by standard chromatography techniques because both proteins had almost identical molecular weights (about 140,000) and isoelectric points (pH 4.5). Specific antibodies were raised against proteins A and B by excision of the different immunoprecipitates from crossed immunoelectrophoresis gels. With these antibodies, protein A or B was removed from solutions containing both proteins. The purified proteins A and B possessed proteolytic activity and were inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. Each of these proteins accounted for approximately 50% of the total proteolytic activity isolated from S. cremoris Wg2. The specific antibodies against the proteases were also used for immuno-gold labeling studies. The proteases were clearly seen to be located at the outside of the cell wall. The proteases had the same location when the genetic information coding for the proteases was cloned in Streptococcus lactis and Bacillus subtilis.

Proteolytic enzymes of dairy starter cultures

“…The published photograph ( Fig. 1) shows the presence of gold-labelled antibody in the cell wall region but the rather high intracellular background and the scattered distribution of particles leave open to question the authors' conclusion [34] that the proteinases are 'clearly seen to be located at the outside of the cell wall'. In view of the possibility that polyclonal antibodies may bind to a variety of bacterial cell wall antigens, the inclusion of proteinase-negative (Prt-; see Section 7) controls treated in the same way would have improved this very useful and direct approach to the study of proteinase localization.…”

“…Ca ~ + is also involved in stabilizing the wall-bound enzyme [32,33]. In a recent study [34] the localization of proteinase in S. cremoris Wg2 has been investigated more directly using immunogold labelling and electron microscopy to detect the proteinase molecules in cell sections after electrophoretic purification of proteinases from this strain and the subsequent isolation of antibodies to the purified enzymes (see below). The published photograph ( Fig.…”

“…All four components were absent when cultures were grown in casein-free medium. In a subsequent study [34], attempts to separate proteins A and B from S. cremoris Wg2 using gel filtration, ion exchange chromatography and isoelectric focusing were unsuccessful indicating that the two proteins had very similar Mrs (140 kDa) and isoelectric points (pH 4.5). Separation was ultimately achieved by immunoprecipitation with antibodies raised to the individual proteins excised from the CIE gels.…”

“…However, in the case of the cell wall proteinase system of S. cremoris HP the presence of several distinct proteolytically active components has been attributed to autoproteolytic modification of a single original proteinase [33]. The two components differed slightly in molecular weight by an amount that may not have been detectable in the CIE system [34]. The lower M r component (126 kDa) was believed to be a more stable derivative of the larger protein (130 kDa).…”

“…Further lower Mr, proteolytically active, components originated during diafiltration of solutions of these proteins with a corresponding decline in the amount of the 130 kDa form. Amino acid sequence data on the different proteolytic components isolated in the above studies [33,34] are needed to establish whether autoproteolysis is a sufficient explanation for the existence of apparently distinct proteinases produced by a single strain.…”