ATP-promoted interaction between Clp A and Clp P in activation of Clp protease from Escherichia coli.

Clp protease is a high relative molecular mass, ATP-dependent protease found in the cytoplasm of Escherichia coli. Clp protease is composed of two protein components, Clp A, which has ATPase activity, and Clp P, which has the proteolytic active site and is activated by Clp A in the presence of ATP. Clp P subunits (Mr = 21,500) are arranged in two hexagonal rings directly superimposed on each other, and under low salt conditions two dodecamers associate to form a particle with Mr approximately 440,000. Clp A (subunit Mr = 83,000) and Clp P do not associate in the absence of nucleotide, but Clp A with ATP bound associates with Clp P to form an active proteolytic complex with Mr approximately 700,000. Although adenosine 5'-[beta gamma-imido]triphosphate (AMPPNP) weakly promotes association between Clp A and Clp P, non-hydrolysable analogues of ATP do not activate proteolysis, indicating that association between the components is not sufficient to allow proteolysis. Association between Clp A and Clp P does not alter the basal ATPase activity of Clp A, but addition of protein substrates is accompanied by an increase in ATP hydrolysis by Clp A. Chemically-inactivated Clp P or inactive mutants of Clp P also associate with Clp A, but no increase in the ATPase activity of Clp A is observed, either in the presence or absence of protein substrates, when Clp P is inactive. Thus the increased ATP hydrolysis is dependent on active proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein degradation in extracts of exponential and stationary phase Vibrio cells

The degradation of the foreign protein [14C]methyl apohaemoglobin ([14C-me]globin) was stimulated by ATP in cell-free extracts from exponential phase and shaken and standing stationary phase Vibrio cells. A marked stimulation by ATP of the degradation of [14C-me]globin was observed with exponential phase cell extracts which were preincubated for 30 min at 30 degrees C. Maximum stimulation was obtained with 3 mM-ATP and optimum degradation was at pH 8.0-8.5. Preincubation of extracts from both types of stationary phase cells did not affect the degree of ATP stimulation. The amount of ATP stimulation of [14C-me]globin degradation by exponential phase extracts decreased markedly when the cells were starved in a growth limiting minimal medium before preparation of the cell extracts. In the exponential and both types of stationary phase extracts most of the activity was located in the cytoplasmic fractions. Although the periplasmic preparations contained a minor portion of the total activity, this activity showed a greater percentage stimulation by ATP. In the absence of ATP the specific proteolytic activities of the extracts from exponential and both types of stationary phase cells were similar. The proteolytic activities in all the cell extracts were inhibited to the same extent by phenylmethylsulphonyl fluoride, but the exponential and both types of stationary phase cell extracts were inhibited to different extents by EDTA and p-hydroxymercuribenzoate. The results suggest that the proteolytic systems responsible for the degradation of abnormal proteins are different in exponential and stationary phase Vibrio cells.     

ATP-dependent association between subunits of Clp protease in pea chloroplasts

The chloroplast ATP-dependent Clp protease (EC 3.4.21.92) is composed of the proteolytic subunit ClpP and the regulatory ATPase, ClpC. Although both subunits are found in the stroma, the interaction between the two is dynamic. When immunoprecipitation with antibodies against ClpC was performed on stroma from dark-adapted pea (Pisum sativum L. cv. Alaska) chloroplasts, ClpC but not ClpP was precipitated. However, when stroma was supplemented with ATP, both ClpC and ClpP were precipitated. Co-immunoprecipitation was even more efficient in the presence of ATP-gamma-S, suggesting that the association between regulatory and proteolytic subunits is dependent on binding of ATP to ClpC, but not its hydrolysis. To further test this association, stroma was fractionated by column chromatography, and the presence of Clp subunits in the different fractions was monitored immunologically. When stroma depleted of ATP was fractionated on an ion-exchange column, ClpP and ClpC migrated separately, whereas in the presence of ATP-gamma-S both subunits co-migrated. Similar results were observed in size-exclusion chromatography. To further characterize the precipitated enzyme, its proteolytic activity was assayed by testing its ability to degrade beta-casein. No degradation was observed in the absence of ATP, and degradation was inhibited in the presence of phenylmethylsulfonyl fluoride, consistent with Clp being an ATP-dependent serine protease. The activity of the isolated enzyme was further tested using chimeric OE33 as a model substrate. This protein was also degraded in an ATP-dependent manner, supporting the suggested role of Clp protease as a major housekeeping protease in the stroma.     

ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum

Bioluminescence activity and ATP pool were investigated in the cells of psychrophilic bacteria Photobacterium phosphoreum collected from the exponential and stationary growth phases and immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilized cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h). Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation from the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.     

Analysis of growth phase regulated KatA and CatE and their physiological roles in determining hydrogen peroxide resistance in Agrobacterium tumefaciens

Agrobacterium tumefaciens possesses two catalases, a bifunctional catalase-peroxidase, KatA and a homologue of a growth phase regulated monofunctional catalase, CatE. In stationary phase cultures and in cultures entering stationary phase, total catalase activity increased 2-fold while peroxidase activity declined. katA and catE were found to be independently regulated in a growth phase dependent manner. KatA levels were highest during exponential phase and declined as cells entered stationary phase, while CatE was detectable at early exponential phase and increased during stationary phase. Only small increases in H2O2 resistance levels were detected as cells entering stationary phase. The katA mutant was more sensitive to H2O2 than the parental strain during both exponential and stationary phase. Inactivation of catE alone did not significantly change the level of H2O2 resistance. However, the katA catE double mutant was more sensitive to H2O2 during both exponential and stationary phase than either of the single catalase mutants. The data indicated that KatA plays the primary role and CatE acts synergistically in protecting A. tumefaciens from H2O2 toxicity during all phases of growth. Catalase-peroxidase activity (KatA) was required for full H2O2 resistance. The expression patterns of the two catalases in A. tumefaciens reflect their physiological roles in the protection against H2O2 toxicity, which are different from other bacteria.     

Inhibition of 5′-nucleotidase activity after growth of Dictyostelium discoideum

The specific activity of 5′-nucleotidase activity in cell-free extracts of Dictyostelium discoideum at both exponential and stationary growth phases was determined. The 5′-nucleotidase activity of both membrane and soluble fractions was determined. The results show that at exponential growth more activity is found in the soluble fraction. Furthermore, the results show that stationary phase cells contain about 10-fold less activity than cells at exponential growth. To determine if stationary phase cells contained an inhibitor of 5′-nucleotidase, purified membranes were incubated with a high speed supernatant (S-100) prepared from cells at this stage. The results showed not only a time and concentration dependent loss of membrane bound activity, but also that most of the lost activity could be recovered in a soluble form. This result suggested that the 5′-nucleotidase was being released by a factor in the S-100. Additional studies showed inactivation of the releasing factor by a protease and further, that this inactivation could be prevented by serine protease inhibitors. The specificity of releasing factor with respect to two other membrane bound activities was determined. The results indicated no loss of either 3′5′-cyclic phosphodiesterase or adenylate cyclase. In addition, the results of a comparison of the activity of the releasing factor at two stages of growth showed similar values at both exponential and stationary growth phase. This latter finding suggests that the loss of 5′-nucleotidase activity at stationary phase is not due to modulation of the releasing factor activity. An alternative mechanism is proposed.     

Changes in bacterial cells induced by high pressure at subzero temperature

The aim of this study was to examine the effect of pressure treatment at 193 MPa and −20 °C on membrane damage, changes in activity of membrane-bound ATPases and degradation of nucleic acids. The experiments were carried out with three Escherichia coli strains, in the exponential and stationary phases of growth, and differing in sensitivity to pressure. All E. coli strains subjected to pressure in the exponential phase of growth were inactivated by 6 log cycles, independently of the strain, which was accompanied by a total loss of ability to plasmolyse, an increase in irreversible membrane permeability to PI, and a reduction of cellular ATP by more than 80%. After pressure treatment of stationary phase cells, the relationship between the inactivation level and the ability to plasmolyse was not as evident as in the case of exponential phase cells. Pressure treatment of two strains of E. coli K-12 and Ec160/59 in the stationary phase that decreased viability by no more than one log cycle led only to reversible permeabilization of bacterial membranes, while irreversible permeabilization was observed in the pressure sensitive E. coli IBA72 strain phase that was inactivated by 4.6 log cycles. The reduction of ATP and changes in ATPase activity after pressure treatment of tested E. coli strains in the stationary phase of growth depended on the stage of inactivation of the particular strain. Electrophoretic analysis showed degradation of RNA isolated after pressure treatment from cells of all E. coli strains tested in the exponential phase of growth. The changes of RNA induced by pressure were not visible in the case of cells in the stationary phase. The degradation of DNA isolated from pressure treated E. coli strains from the exponential as well as from the stationary phase of growth was not observed.     

Ribonuclease U2: cloning, production in Pichia pastoris and affinity chromatography purification of the active recombinant protein

A recombinant lipase, CWB-LipB, localized on the Bacillus subtilis cell surface and retaining lipase activity was unstable and not accumulated in a high yield. To improve the accumulation, we examined cell wall binding protease (wprA)- and/or sigma D (sigD)-deficient mutants, and also a NprE and AprA protease-deficient mutant as host strains. The nprE aprA mutation did not lead to a significant increase in the CWB-LipB accumulation. The wprA mutant accumulated a greater amount than the wild-type only in the stationary phase, but the sigD mutant accumulated a greater amount in both the exponential and stationary phases. The double mutant exhibited great accumulation of CWB-LipB, the amount being 36% of the total proteins extracted from the cell surface.     

Detection of an Intracellular Protease Inhibitor in Archaea

Proteolytic activity and a subtilisin inhibitor (NSI) were detected in Natrialba magadii cells. The proteolytic activity was due to two different proteases: a ∼90-kDa metallo protease (NMP) produced during exponential growth and a 246-kDa serine protease (NSP) detected in the stationary phase. Both proteases were detected in the cytosolic fraction. NSI activity was maximal during early stages of growth and decreased in the stationary phase. NSI is a 35-kDa thermosensitive protein; it inhibits NSP activity but has no effect on NMP, and it was detected as a soluble or membrane-bound protein depending on the growth phase. Our results suggest that NSI may regulate NSP activity in vivo and that this protease may have a role in stationary phase cells. To our knowledge, this is the first report on the occurrence of protease inhibitors in Archaea. Received: 4 May 2002 / Accepted: 10 July 2002     

Analysis of nucleotide pools during protease production with Bacillus licheniformis

Summary During development of a fed-batch procedure for protease production with Bacillus licheniformis the nucleotide pools of the culture were assayed. Transitions between different growth phases or different nutrient limitations could easily be discerned by alterations in the nucleotide pool. As already described for B. subtilis, induction of sporulation was marked by a drop in the guanosine triphosphate (GTP) pool of the cells, which always preceded protease production. This was also valid for closed-loop controlled fed-batch processes in which the concentration of ammonium, which repressed protease biosynthesis, was kept constant at low levels. A marked decrease in the GTP content of the cells, e.g. after addition of mycophenolic acid during the exponential phase, increased protease formation during the stationary phase. During protease production the energy charge was lower (0.6–0.8) than during the exponential and early stationary phases (0.8–0.95) although very low energy charges (<0.5) did not support protease formation. Offprint requests to: G. Bierbaum     

Metabolic stability of the two forms of initiation factor IF-3 in Escherichia coli during the growth cycle.

Possible alteration in the ratio of the long and short forms of initiation factor IF-3 (FEBS Lett. 79, 264-275, 1977) during the growth cycle of Escherichia coli was examined. The ratio was found to remain unchanged between the exponential and stationary growth phases. Contrary to an earlier report (Eur. J. Biochem. 29, 319-325, 1972), the total amount of IF-3 relative to the ribosome content in stationary phase cells was essentially the same as in midlogarithmic phase cells. The activity of IF-3, assayed after its separation from other initiation factors by chromatography, was also the same in extracts from midlogarithmic and stationary phase cells. The data show that in Escherichia coli the ratio of IF-3/ribosome is maintained constant. The ribosomes themselves have been shown to retain virtually full activity in vitro during this transition indicating that growth-cycle-dependent biochemical modifications of the ribosome do not affect its protein synthetic capacity per se.     

Clp P represents a unique family of serine proteases

The amino acid sequence of Clp P, the proteolytic subunit of the ATP-dependent Clp protease of Escherichia coli, closely resembles a protein encoded by chloroplast DNA, which is well conserved between chloroplasts of different plant species. The homology extends over almost the full length of the sequences of both proteins and consists of approximately 46% identical and approximately 70% similar amino acids. Antibodies against E. coli Clp P cross-reacted with proteins with Mr of 20,000-30,000 in bacteria, lower eukaryotes, plants, and animal cells. Since the regulatory subunit of Clp protease, Clp A, also has a homolog in plants, as well as in other bacteria and in lower eukaryotes, it is likely that ATP-dependent proteolysis in chloroplasts is catalyzed in part by a Clp-like protease and that both components of Clp-like proteases are widespread in living cells. We have identified Ser-111 as the active site serine in E. coli Clp P modified by diisopropyl fluorophosphate. Mutational alteration of Ser-111 or His-136 eliminates proteolytic activity of Clp P. Both residues are found in highly conserved regions of the protein. The sequences around the active site residues suggest that Clp P represents a unique class of serine protease. Amino-terminal processing of cloned Clp P mutated at either Ser-111 or His-136 occurs efficiently when wild-type clpP is present in the chromosome but is blocked in clpP- hosts. Processing of Clp P appears, therefore, to involve an intermolecular autocatalytic cleavage reaction. Since processing of Clp P occurs in clpA- cells, the autoprocessing activity of Clp P is independent of Clp A.     

Dynamics of the formation of extracellular protease and the ATP pool in a thermophilic strain of Bacillus brevis]

The dynamics of varying ATP concentration was studied in the cells of the thermophilic strain of Bacillus brevis 224 synthesizing a neutral exoprotease. Some irregularities during the primary and secondary growth of the culture corresponded to the changes in the amount of ATP. A sharp decrease (by a factor of ca. 100) in the level of ATP pool was detected in the bacterial cells after 2.5 hours of their exponential growth; their growth ceased and the synthesis of protease was depressed. A decrease in the rate of the enzyme synthesis coincides with an increase in the concentration of ATP in the cells.     

Effect of age on the membrane lipid composition of Streptococcus sanguis

The cell membrane of Streptococcus sanguis contains three classes of lipid: neutral lipid, glycolipid and phospholipid. A striking difference in membrane lipid composition between cells in the exponential and in the stationary phases of growth was observed. During the exponential phase, approx. 37–45%, 14–19% and 37–45% of the lipids synthesized were found to be neutral lipid, glycolipid and phospholipid, respectively. The amount of lipid synthesized reached a maximum at the early stationary phase. The amount of phospholipid drastically declined thereafter and that of neutral lipid slightly declined. In contrast, the amount of glycolipid markedly increased and exceeded the amount of phospholipid. The phospholipid present during the exponential phase was found to be mainly phosphatidylglycerol (82–88%) and a small amount of cardiolipin (12–18%). At the stationary phase, the amount of phosphatidylglycerol greatly decreased and reached approx. 16% of that in the early stationary phase, while cardiolipin steadily increased and became the major phospholipid in the late stationary phase. The glycolipid was found to be composed of mainly mono- and diglucosyldiglycerides. At the end of the experiment (after 8 h incubation), the distribution of lipids was found to be: neutral lipid, 46%; glycolipid (monoglucosyldiglyceride, 28%; diglucosyldiglyceride, 13%) 41%; and phospholipid (phosphatidylglycerol, 3%, cardiolipin, 8%) 13%.     

Concentration and metabolism of cyclic AMP during the early stages of hepatoma 22a growth]

Intracellular levels of cyclic AMP (cAMP), adenylate cyclase, and cAMP-phosphodiesterase activities at lag-period, exponential and stationary growth phases of hepatoma 22a were determined. It was shown that the transition of tumour cells from the lag-period to the exponential phase of growth was accompanied by the two-fold decrease of intracellular cAMP level on account of drastic activation of cAMP phosphodiesterase. Subsequently the cAMP level lowered more slowly until the cells entered the stationary phase of growth. In view of the fact that the adenylate cyclase activity failed to change at different growth phases of hepatoma 22a, it seems very proballe that the rise of cAMP phosphodiesterase activity could be a signal for the exit of tumour cells from the lag-period and their entrance into the mitotic cycle.     

Triaglycerol metabolism in Saccharomyces cerevisiae. Relation to phospholipid synthesis.

The acylglycerol content of Saccharomyces cerevisiae has been examined during cellular growth. The cells maintained a constant amount of phospholipid and diacylglycerol throughout growth. Triacylglycerol content fell in the early exponential phase of growth and then increased sharply upon entry of the culture into the stationary growth phase. Pulse-chase experiments with [1-14C]oleic acid and [2-3H]- and [1-14C]glycerol indicated that the triacylglycerol molecule was utilized for phospholipid synthesis in early exponential phase probably through a diacylglycerol intermediate. A substantial turnover of phospholipid during growth was also apparent. No role for the triacylglycerol could be found in regulating the fatty acid species of the phospholipid nor in the storage of fatty acid for energy metabolism.     

Quantitative Analysis of the Chloroplast Molecular Chaperone ClpC/Hsp93 in Arabidopsis Reveals New Insights into Its Localization,Interaction with the Clp Proteolytic Core,and Functional Importance

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.