Further studies on an asporogenous mutant of Bacillus subtilis lacking in vivo proteolytic activity.

Cells of Bacillus subtilis 168 (trpC2) growing and sporulating in a single chemically defined medium carried out intracellular protein degradation and increased their levels of intracellular serine protease-1 in a manner very similar to what had previously been reported for cells sporulating in nutrient broth. The results were interpreted to mean that these processes are intrinsic to sporulation rather than medium dependent. To determine the cause of these increases in specific activity of proteinases, we purified the protease, prepared rabbit immunoglobulins directed against it, and monitored changes in protease antigen levels by performing rocket immunoelectrophoresis. In cells sporulating in nutrient broth, the protease antigen levels increased about 7-fold, whereas the specific activity increased about 150-fold, for an activation of about 20-fold. In cells sporulating in the single chemically defined sporulation medium, the protease antigen increased about 10-fold, whereas the specific activity increased at least 400-fold, for an activation of about 40-fold. These results were interpreted to mean that a posttranslational event activated the protease in vivo; a previously described endogenous proteinase inhibitor was confirmed to be present in the strain used. Chloramphenicol added to the cultures inhibited both the increases in antigen levels and in the specific activity of the proteinase.Bacillus subtilis cells produce at least two intracellular serine proteinase activities which are referred to as ISP-1 (15, 31, 38) and ISP-2 (37). Both ISP-1 and ISP-2 (7, 31, 37) increase in specific activity during sporulation, but ISP-1 appears to account for at least 80% of the intracellular azocasein or azocollagen hydrolyzing activity (11, 36; S. A. Smith and J. H. Hageman, unpublished data). These two proteinases are distinct from the three proteinases excreted by B. subtilis cells during sporulation (18, 30) and from other cytoplasmic or membrane-associated proteinases (20,34).Possible roles of proteinase activity in protein degradation in B. subtilis cells have been considered for some time (19,36), and evidence for such roles was reviewed most recently by Maurizi and Switzer (23). Genetic and physiological evidence has been presented for a role of ISP-1 in bulk protein degradation during sporulation (15,27), and evidence has been reported that mutations affecting ISP-2 (12, 32) also result in impaired ability of cells to degrade intracellular proteins during sporulation. In most previous studies (21, 29), workers have attempted to correlate intracellular proteinase activity and protein degradation in cells sporulating in complex or semidefined media. We show in this study that cells sporulating in a chemically defined sporulation medium (CDSM) (9) also elaborate intracellular proteinase and degrade intracellular proteins efficiently.Two to three hours after the onset of sporulation of B. subtilis cells in nutrient broth, ISP-1 undergoes a rapid increase in specific activity (7,29,31) 15,500) that disappears from ...

mentioning

confidence: 99%

Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation

Burnett¹,

Shankweiler²,

Hageman³

1986

“…4). Evidently, calcium ions must be present in the growth medium in order to produce normal synthesis and/or activation of ISP-1, which accounts for about 80% of the total proteinase activity in the soluble fraction (5,29). The data in Table 2 confirm that in Ca2"-containing CDSM medium, ISP-1 accounted for about 74% of the total azocaseinase activity in the cells at t1o.…”

Section: Effect Of L-leucine Concentrations On Protein Degradationmentioning

confidence: 63%

“…The results of these experiments are summarized in Table TABLE 1 (29,36), we studied the effect of Ca2" on sporulation, proteinase activity, and proteolysis in B. subtilis cells. In CDSM, cells were strongly dependent on the addition of calcium ions to the medium to achieve maximal sporulation ( Table 2).…”

Section: Effect Of L-leucine Concentrations On Protein Degradationmentioning

confidence: 99%

Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells

Ohara

Hageman

1990

(12,27,35). Degradation of bulk protein during starvation is apparently a first-order process and yields a first-order rate constant of about 0.04 h-1 (22; our calculation).In growing Bacillus subtilis cells (4), rates of bulk protein degradation (1 to 3% h-1, depending on nutritional conditions) are similar to those seen in the enteric bacteria. In contrast, during the developmental process of spore formation, cells of the species Bacillus have been measured to degrade intracellular protein with first-order rate constants of 0.070 to 0.27 h-1 (2, 40), depending on the experimental conditions. Not only is protein degradation relatively rapid during this period; it is also very extensive (40). Bernlohr (2) Rates of proteolysis and total intracellular proteinase activity were both reduced by limiting calcium ion concentrations in the medium. Since the metabolic poisons used did not restrict the uptake of calcium ions, we believe that the requirements for calcium ions and ATP or energized membranes are independent requirements for optimal proteolysis. Cells of a strain containing an insertionally inactivated intracellular serine proteinase-1 (ISP-1) degraded intracellular protein at the same rates as those of the parental strain over an extended period.

“…These data indicate that ISP-1 is synthesized beginning at to but is not enzymatically active until t4. The mechanism for maintaining ISP-1 in the inactive form or the nature of the inactive protein is unknown, although several forms of regulation have been proposed (12,13,19). A zymogen-like precursor of another intracellular protease, the small-acid-solubleprotein-degrading enzyme, has been observed in sporulating cells (7).…”

Section: Discussionmentioning

confidence: 99%

“…The exact role of this enzyme in sporulation has not been established, and little is known about the regulation of ISP-1 expression and control of protease activity. It has been suggested that ISP-1 activity might be regulated by interaction with a proteinaceous inhibitor, calcium availability, or activation by membrane-bound proteases (12,13,19). A recent report presented immunological evidence that ISP-1 was initially synthesized as an enzymatically inactive protein that was subsequently converted to an active protease (3).…”

mentioning

confidence: 99%

Control of intracellular serine protease expression in Bacillus subtilis

Ruppen¹,

Alstine²,

Band³

1988

Expression of the major intracellular serine protease (ISP-1) gene of Bacillus subtilis was studied by using a translational fusion plasmid in which the isp promoter regon was fused to the lacZ gene. ,-Galactosidase activity, used to measure transcription from the isp promoter, was produced immediately after the end of exponential growth, whereas intracellular protease activity was not detected until 4 h later. These results are consistent with a previous suggestion that ISP-1 initially accumulates in the cell in an enzymatically inactive form. ISP-1 activity was detected in all of the sporulation-deficient strains examined, and the amount of protease activity always corresponded to the amount of ,B-galactosidase activity. These results indicate that the activation of ISP-1 is not dependent on a sporulation-specific gene product. Expression of ISP-1 is regulated by a number of mutations known to affect the expression of extracellular enzymes. In sacU(h) and sacQ(h) mutants, the expression of ISP-1 was 10-fold higher than in the wild-type strain. In catA, hpr, and scoC strains, expression of ISP was stimulated two-to threefold, whereas in sacU mutants the expression of ISP-1 was reduced to less than 10% of the wild-type level. The temporal expression and activation of ISP-1 was not affected by any of these mutations. This is the first evidence that the expression of a native intracellular protein is affected by these hyperproduction mutations.Soon after the initiation of sporulation, Bacillus subtilis synthesizes an intracellular serine protease (ISP-1) (3,16). The isp gene has been cloned, and studies have shown that ISP-1 is not essential for normal sporulation (2, 10). The exact role of this enzyme in sporulation has not been established, and little is known about the regulation of ISP-1 expression and control of protease activity. It has been suggested that ISP-1 activity might be regulated by interaction with a proteinaceous inhibitor, calcium availability, or activation by membrane-bound proteases (12,13,19). A recent report presented immunological evidence that ISP-1 was initially synthesized as an enzymatically inactive protein that was subsequently converted to an active protease (3).Several mutations affecting the production of extracellular enzymes in B. subtilis have been described. Mutations in the sacU and sacQ genes are pleiotropic in nature, affecting the prpduction of many extracellular enzymes and some sporulation-related events (11,22). Other mutations (catA, hpr, ScoC), although from independently isolated mutants, may be mutations of the same gene. These mutations affect production of the extracellular proteases and catabolite repression of sporulation eyents (5,8,9). The only common feature of the target genes of these hyperproduction mutations seems to be their participation in adapting to starvation conditions requiring the utilization of a complex carbon or nitrogen source. Since one of the postulated roles of ISP-1 is to provide amino acids by degrading proteins during sporulation, it wa...