Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells.
Bacterial cells degrade intracellular proteins at elevated rates during starvation and can selectively degrade proteins by energy-dependent processes. Sporulating bacteria can degrade protein with apparent first-order rate constants of over 0.20 h-1. We have shown, with an optimized [14C]leucine-labeling and chasing procedure, in a chemically defined sporulation medium, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent. Sodium arsenate, sodium azide, carbonyl cyanide m-chlorophenylhydrozone, and N,N'-dicyclohexylcarbodiimide, at levels which did not induce appreciable lysis (less than or equal to 10%) over 10-h periods of sporulation, inhibited intracellular proteolysis by 13 to 93%. Exponentially growing cells acquired arsenate resistance. In contrast to earlier reports, we found that chloramphenicol (100 micrograms/ml) strongly inhibited proteolysis (68%) even when added 6 h into the sporulation process. Restricting the calcium ion concentration (less than 2 microM) in the medium had no effect on rates or extent of vegetative growth, strongly inhibited sporulation (98%), and inhibited rates of proteolysis by 60% or more. Inhibitors of energy metabolism, at the same levels which inhibited proteolysis, did not affect the rate or degree of uptake of Ca2+ by cells, which suggested that the Ca2+ and metabolic energy requirements of proteolysis were independent. Restricting the Ca2+ concentration in the medium reduced by threefold the specific activity in cells of the major intracellular serine proteinase after 12 h of sporulation. Finally, cells of a mutant of B. subtilis bearing an insertionally inactivated gene for the Ca2(+)-dependent intracellular proteinase-1 degraded protein in chemically defined sporulation medium at a rate indistinguishable from that of the wild-type cells for periods of 8 h.
O'Hara
M B
MB
Department of Chemistry, New Mexico State University, Las Cruces 88003.
Hageman
J H
JH
D001149:Arsenates / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D001426:Bacterial Proteins / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002701:Chloramphenicol / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004734:Energy Metabolism / Q000187:drug effects; D007700:Kinetics; D007930:Leucine / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D019810:Sodium Azide; D013171:Spores, Bacterial / Q000187:drug effects / Q000502:physiology
D001149:Arsenates; D001386:Azides; D001426:Bacterial Proteins; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D002701:Chloramphenicol; D019810:Sodium Azide; D010447:Peptide Hydrolases; D007930:Leucine; C025657:arsenic acid; D002118:Calcium