Differential localization of the Streptococcus mutans GS-5 fructan hydrolase enzyme, FruA.
Streptococcus mutans GS-5 synthesizes an exo-beta-D-fructosidase, FruA, capable of degrading levans, inulins, sucrose and raffinose, with the greatest activity on levans. A previous analysis of the deduced amino acid sequence of the FruA protein revealed the presence of a C-terminus with an LPXTGX membrane sorting sequence and membrane spanning domain, characteristic of many Gram-positive cocci surface proteins. Here it is demonstrated that FruA, which had been previously shown to exist almost exclusively as an extracellular enzyme, can be detected in significant proportions at the surface of S. mutans cells. Moreover, growth of S. mutans GS-5 at steady state in continuous culture at pH values of 7.0, 6.0, or 5.0 revealed that the amount of cell-associated enzyme increased with decreasing pH values, such that roughly 50% of the total fructanase activity of pH 5.0-grown organisms was cell-associated. This result was confirmed using anti-recombinant-FruA antisera in Western blotting of culture supernate and cell-associated enzyme preparations from chemostat-grown cells. Incubation of S. mutans at pH values of 5.0, 6.0 or 7.0 in buffered media yielded results similar to those observed in the chemostat experiments. The release of FruA from S. mutans was also shown to be inhibitable by copper, which is known to interfere with the release of the surface adhesin, P1, from intact cells and protoplasts of S. mutans. These data provide evidence for a unique post-translational mechanism for the regulation of the catabolism of polysaccharides by bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)
Burne
R A
RA
Department of Dental Research, University of Rochester School of Medicine and Dentistry, New York 14642.
Penders
J E
JE
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins; D015153:Blotting, Western; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C021512:levanase