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.     

Effects of starvation for potassium and other inorganic ions on protein degradation and ribonucleic acid synthesis in Escherichia coli.

Starvation of Escherichia coli for potassium, phosphate, or magnesium ions leads to a reversible increase in the rate of protein degradation and an inhibition of ribonucleic acid (RNA) synthesis. In cells deprived of potassium, the breakdown of the more stable cell proteins increased two- to threefold, whereas the hydrolysis of short-lived proteins, both normal ones and analog-containing polypeptides, did not change. The mechanisms initiating the enhancement of proteolysis during starvation for these ions were examined. Upon starvation for amino acids or amino acyl-transfer RNA (tRNA), protein breakdown increases in relA+ (but not relA) cells as a result of the rapid synthesis of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). However, a lack of amino acyl-tRNA does not appear to be responsible for the increased protein breakdown in cells starved for inorganic ions, since protein breakdown increased in the absence of these ions in both relA+ and relA cultures, and since a large excess of amino acids did not affect this response. In bacteria in which energy production is restricted, ppGpp levels also rise, and protein breakdown increases. The ion-deprived cultures did show a 40 to 75% reduction in adenosine-5'-triphosphate levels,l similar to that seen upon glucose starvation. However, this decrease in ATP content does not appear to cause the increase in protein breakdown or lead to an accumulation of ppGpp. No consistent change in intracellular ppGpp levels was found in relA+ or relA cells starved for these ions. In addition, in relX mutants, removal of these ions led to accelerated protein degradation even though relX cells are unable to increase ppGpp levels or proteolysis when deprived of a carbon source. In the potassium-, phosphate-, and magnesium-deprived cultures, the addition of choramphenicol or tetracycline caused a reduction in protein breakdown toward basal levels. Such findings, however, do not indicate that protein synthesis is essential for the enhancement of protein degradation, since blockage of protein synthesis by inactivation of a temperature-sensitive valyl-tRNA synthetase did not restore protein catabolism to basal levels. These various results and related studies suggest that the mechanism for increased protein catabolism on starvation for inorganic ions differs from that occurring upon amino acid or arbon deprivation and probably involves an enhanced susceptibility of various cell proteins (especially ribosomal proteins) to proteolysis.     

Study of the sensitivity of the L-forms of group A Streptococcus to antibiotics in artificial nutrient media and in human cell cultures

Sensitivity of L-forms of group A streptococci to 5 antibiotics such as erythromycin, lincomycin, tetracycline, gentamicin and chloramphenicol was studied in an artificial nutrient medium and cell cultures i.e. human fibroblast diploid cells and transplantable human heart cells (Girardi). In vitro investigation of the antibiotic effect on the streptococcal L-forms revealed their sensitivity to erythromycin (MIC, 0.4 micrograms/ml), lincomycin (MIC, 0.08 microgram/ml) and tetracycline (MIC, 2 micrograms/ml). The streptococcal L-forms were slightly sensitive to gentamicin (MIC, 6 micrograms/ml) and chloramphenicol (MIC, 30 micrograms/ml). Complete inhibition of the growth of the L-forms in the Girardi cells on the 1st day of the experiment after the antibiotics administration in single doses was induced by lincomycin, 5 micrograms/ml, erythromycin, 10 micrograms/ml, and tetracycline, 100 micrograms/ml. In the diploid cells, the respective figures were 50, 100 and 200 micrograms/ml. Chloramphenicol and gentamicin had an inhibitory effect on the growth of the L-forms but produced no sanative effect.     

Induction of cat-86 by chloramphenicol and amino acid starvation in relaxed mutants of Bacillus subtilis

The chloramphenicol acetyltransferase gene cat-86 is induced through a mechanism that is a variation of classical attenuation. Induction results from the destabilization of an RNA stem-loop that normally sequesters the cat-86 ribosome-binding site. Destabilization of the stem-loop is due to the stalling of a ribosome in the leader region of cat-86 mRNA at a position that places the A site of the stalled ribosome at leader codon 6. Two events can stall ribosomes at the correct location to induce cat-86 translation: addition of chloramphenicol to cells and starvation of cells for the amino acid specified by leader codon 6. Induction by amino acid starvation is an anomaly because translation of the cat-86 coding sequence requires all 20 amino acids. To explain this apparent contradiction we postulated that amino acid starvation triggers intracellular proteolysis, thereby providing levels of the deprived amino acid sufficient for cat-86 translation. Here we show that a mutation in relA, the structural gene for stringent factor, blocks intracellular proteolysis that is normally triggered by amino acid starvation. The relA mutation also blocks induction of cat-86 by amino acid starvation, but the mutation does not interfere with chloramphenicol induction. Induction by amino acid starvation can be demonstrated in relA mutant cells if the depleted amino acid is restored at very low levels (e.g., 2 micrograms/ml). A mutation in relC, which may be the gene for ribosomal protein L11, blocks induction of cat-86 by either chloramphenicol or amino acid starvation. We believe this effect is due to a structural alteration of the ribosome resulting from the relC mutation and not to the relaxed phenotype of the cells.     

Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12.

In a typical Escherichia coli K-12 culture starved for glucose, 50% of the cells lose viability in ca. 6 days (Reeve et al., J. Bacteriol. 157:758-763, 1984). Inhibition of protein synthesis by chloramphenicol resulted in a more rapid loss of viability in glucose-starved E. coli K-12 cultures. The more chloramphenicol added (i.e., the more protein synthesis was inhibited) and the earlier during starvation it was added, the greater was its effect on culture viability. Chloramphenicol was found to have the same effect on a relA strain as on an isogenic relA+ strain of E. coli. Addition of the amino acid analogs S-2-aminoethylcysteine, 7-azatryptophan, and p-fluorophenylalanine to carbon-starved cultures to induce synthesis of abnormal proteins had an effect on viability similar to that observed when 50 micrograms of chloramphenicol per ml was added at zero time for starvation. Both chloramphenicol and the amino acid analogs had delayed effects on viability, compared with their effects on synthesis of normal proteins. The need for protein synthesis did not arise from cryptic growth, since no cryptic growth of the starving cells was observed under the conditions used. From these and previous results obtained from work with peptidase-deficient mutants of E. coli K-12 and Salmonella typhimurium LT2 (Reeve et al., J. Bacteriol. 157:758-763, 1984), we concluded that a number of survival-related proteins are synthesized by E. coli K-12 cells as a response to carbon starvation. These proteins are largely synthesized during the early hours of starvation, but their continued activity is required for long-term survival.     

The mechanism of action of DuP 721, a new antibacterial agent: effects on macromolecular synthesis

Pulse labeling studies with Bacillus subtilis showed that DuP 721 inhibited protein synthesis. The IC50 of DuP 721 for protein synthesis was 0.25 micrograms/ml but it was greater than 32 micrograms/ml for RNA and DNA synthesis. In cell-free systems, DuP 721 concentrations up to 100 microM did not inhibit peptide chain elongation reactions under conditions where chloramphenicol, tetracycline and hygromycin B inhibited these reactions. Furthermore, Dup 721 did not cause phenotypic suppression of nonsense mutations suggesting that DuP 721 did not inhibit peptide chain termination. Thus, the mechanism of action of DuP 721 is at a target preceeding chain elongation.     

Degradation of L-glutamate dehydrogenase from Escherichia coli: allosteric regulation of enzyme stability.

L-glutamate dehydrogenase (GDH) is stable in exponentially growing Escherichia coli cells but is degraded at a rate of 20-30% per hour in cells starved for either nitrogen or carbon. GDH degradation is energy-dependent, and mutations in ATP-dependent proteases, ClpAP or Lon lead to partial stabilization. Degradation is inhibited by chloramphenicol and is completely blocked in relA mutant cells, suggesting that ribosome-mediated signaling may facilitate GDH degradation. Purified GDH has a single tight site for NADPH binding. Binding of NADPH in the absence of other ligands leads to destabilization of the enzyme. NADPH-induced instability and sensitivity to proteolysis is reversed by tri- and dicarboxylic acids or nucleoside di- and triphosphates. GTP and ppGpp bind to GDH at an allosteric site and reverse the destabilizing effects of NADPH. Native GDH is resistant to degradation by several purified ATP-dependent proteases: ClpAP, ClpXP, Lon, and ClpYQ, but denatured GDH is degraded by ClpAP. Our results suggest that, in vivo, GDH is sensitized to proteases by loss of a stabilizing ligand or interaction with an destabilizing metabolite that accumulates in starving cells, and that any of several ATP-dependent proteases degrade the sensitized protein.     

The Anton blood group antigen is the erythrocyte receptor for Haemophilus influenzae

Abstract The protein synthesis pattern was investigated in Bacillus subtilis relA ⁺ and relA after heat shock using the highly sensitive 2-dimensional O'Farrell technique [1]. The synthesis of several proteins is markedly enhanced upon temperature shift-up in both strains. At 52°C the growth rate is drastically diminished because the synthesis of cellular proteins is inhibited. However, the production of heat-shock proteins is maintained. The synthesis of some of these presumptive heat-shock proteins is stimulated at 37°C in cells treated with H₂O₂ as well as with norvaline, which induces a guanosine tetraphosphate (ppGpp)-dependent stringent response.     

Inhibition of uridine 5'-diphosphate-N-acetylmuramyl-L-alanine synthetase by beta-chloro-L-alanine in Escherichia coli

The synthesis of the nucleotide precursors for peptidoglycan is regulated by the relA gene in Escherichia coli. Thus, nucleotide precursors labeled with [3H]diaminopimelic acid accumulated in a relA strain but not in an isogenic relA+ strain during amino acid deprivation. Furthermore, nucleotide precursor synthesis was relaxed in the amino acid deprived relA+ strain by treatment with chloramphenicol. Uridine diphosphate-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) was the major component accumulated during the relaxed synthesis of nucleotide precursors in both relA+ and relA strains. The effect of beta-chloro-L-alanine (CLA) on the relaxed synthesis of nucleotide precursors for peptidoglycan was determined. At a low concentration (0.0625 mM) CLA inhibited the synthesis of UDP-MurNAc-pentapeptide and caused the accumulation of UDP-MurNAc-tripeptide. Thus, low concentrations of CLA probably inhibited alanine racemase, as reported previously. Higher concentrations of CLA also inhibited an earlier step in nucleotide precursor synthesis. This was shown to be due to the inhibition of UDP-MurNAc-L-alanine synthetase by CLA. CLA inhibited the activity of this enzyme in cell-free extracts as well as in intact cells.     

Control of lipopolysaccharide biosynthesis and release by Escherichia coli and Salmonella typhimurium.

The influence of the relA gene on lipopolysaccharide (LPS) biosynthesis and release by Escherichia coli and Salmonella typhimurium was investigated. Similar results were obtained with both species. The incorporation of [3H]galactose into LPS by galE mutants was inhibited by at least 50% (as compared with normal growing controls) during amino acid deprivation of relA+ strains. This inhibition could be prevented by the treatment of the amino acid-deprived relA+ bacteria with chloramphenicol, a known antagonist of the stringent control mechanism. Furthermore, LPS biosynthesis was not inhibited during amino acid deprivation of isogenic relA mutant strains. These results indicate that LPS synthesis is regulated by the stringent control mechanism. Normal growing cells of both relA+ and relA strains released LPS into the culture fluid at low rates. Amino acid deprivation stimulated the rate of LPS release by relA mutants but not by relA+ bacteria. Chloramphenicol treatment markedly stimulated the release of cell-bound LPS by amino acid-deprived relA+ cells. Thus, a low rate of LPS release was characteristic of normal growth and could be increased in nongrowing cells by relaxing the control of LPS synthesis.     

Inhibition of intracellular proteolysis by insulin in isolated rat hepatocytes. Possible role of internalized hormone

The mechanism of insulin's action upon intracellular proteolysis in isolated hepatocytes was studied. At 37 degrees C insulin inhibited intracellular degradation of intracellular proteins in a dose-dependent manner. A maximal 40% inhibition of intracellular proteolysis was achieved at an insulin concentration of 500 ng/ml with a half-maximal inhibition observed at 2.5 ng/ml of insulin. Insulin inhibited intracellular proteolysis both in the presence and in the absence of amino acids in the incubation mixture. Low concentrations of trypsin (10 micrograms/ml) mimicked insulin's effect upon glucose incorporation into glycogen, but not on intracellular proteolysis. Four protease inhibitors (phenylmethylsulfonyl fluoride (0.5 mM), p-nitrophenyl-p-guanidinobenzoate (0.25 mM), p-tosyl-L-arginine methyl ester (1 mM), and N alpha-p-tosyl-L-lysine chloromethyl ketone (1 mM) blocked the stimulatory effect of insulin upon [14C]glucose incorporation into glycogen, but did not affect the inhibitory action of insulin upon intracellular proteolysis. These results suggest that the mechanism of insulin's action upon intracellular proteolysis differs from that involved in stimulation of glycogenesis. Low temperature (15 degrees C) and short time exposure (10 min) of the hepatocytes to insulin eliminated the inhibitory effect of insulin on intracellular proteolysis. Similarly, insulin's effect on intracellular proteolysis was eliminated by dansylcadaverine, a transglutaminase inhibitor that blocked insulin internalization. In contrast, dansylcadaverine had no effect on insulin's ability to stimulate [14C]glucose incorporation into glycogen. These experiments strongly suggest the necessity of insulin internalization for its inhibitory effect on endogenous protein degradation.     

Binding and internalization of VIP in rat intestinal epithelial cells.

We have prepared villous cells from the jejunum of the rat small intestine and studied the effects of divalent cations and bacitracin on the binding and internalization of VIP. Villous epithelial cells (4 x 10(6) cells/ml) were suspended in a Hepes-NaCl buffer with 1.0% BSA, (pH 7.4) and the cells were incubated for varying periods of time with 125I-VIP at 24 degrees C. Specific binding of radiolabeled VIP was maximal within 10 min (10%) and slowly declined to 9.0 percent after 30 min. In the presence of 1.0 mg/ml bacitracin, however, maximal specific binding of VIP was only 2.7 percent (P less than or equal to 0.001). The addition of CA2+ or Mg2+ to the buffer significantly decreased binding of VIP in a concentration dependent manner. At 8.0, 4.0, 2.0 and 1.0 mM Ca2+, binding of 125I-VIP decreased by 70, 60, 40 and 25 percent, whereas in the presence of the same concentrations of Mg2+ binding was decreased to 50, 38, 25 and 10 percent (P less than or equal to 0.01). To determine if epithelial cells internalize VIP, we bound 125I-VIP to villous cells and then differentiated surface-bound and internalized radioactivity by treating with trypsin (150 micrograms/ml). Surface bound radioligand was the same at both 24 and 4 degrees C (5.3%), while internalized 125I-VIP was 4.0% at 24 degrees C compared to only 1.0% at 4 degrees C (P less than or equal to 0.001). At 24 and 4 degrees C, both Ca2+ (4.0 mM) and Mg2+ (8.0 mM) decreased surface bound radioligand by 60 percent (P less than or equal to 0.01) and lowered internalized radioactivity. These data demonstrate that (1) bacitracin decreases the binding of VIP to small intestinal epithelial cells, (2) both Ca2+ and Mg2+ affect the binding of VIP to its surface receptor and (3) VIP is internalized into epithelial cells.     

Antibiotic resistance of Shigella in different regions of the USSR]

Shigella were most sensitive to polymyxin ceporin, ampicillin, neomycin and furazolidone and resistant to chloramphenicol, tetracycline and streptomycin. Shigella resistant simultaneously to two or three drugs mainly to tetracycline + chloramphenicol, tetracycline + streptomycin and tetracycline + chloramphenicol + streptomycin were most frequent. The frequency of the Shigella strains carrying R-plasmids increased from 28 per cent in 1969--1970 to 72.6 per cent in 1977. The Shigella strains isolated during the dysentery outbreak in 1973--1977 carried the R-factor controlling resistance to tetracycline + chloramphenicol, tetracycline + chloramphenicol + streptomycin, tetracycline + chloramphenicol + streptomycin + neomycin. Interaction between separate biochemical types, colicinogenicity and drug resistance classes was found in the Shigella isolates. The data on the effect of antibiotic (tetracyclines) intensive use in stock-raising defining wide spread of the R-plasmids controlling resistance to these drugs were obtained.     

Calcium-stressed erythrocyte membrane structure and function for assessing glipizide effects on transglutaminase activation.

A proposed mechanism of action of hypoglycemic sulfonylureas is the prevention of transglutaminase-mediated endocytosis of insulin receptors. When activated by high levels of intracellular calcium, transglutaminase (TG) catalyzes the cross-linking of intracellular proteins to membrane proteins and modifies membrane structure and function. This study examined the effects of the sulfonylurea glipizide on TG activity in an erythrocyte model by assessing various membrane ATPase activities and high molecular weight protein polymer formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To activate TG, red blood cells were exposed to 1 mM intracellular Ca2+ using 10(-5) M Ca2(+)-ionophore A23187. In Ca2(+)-stressed cells, calmodulin stimulation (0.1 micrograms/ml) of (Ca2+ + Mg2+)-ATPase was decreased to 21.2% of control activity. Increasing concentrations of calmodulin (0.1-3.0 micrograms/ml) could not overcome the inhibitory effects of TG on the (Ca2+ + Mg2+)-ATPase in Ca2(+)-stressed cells with or without glipizide. An increased Ca2+ sensitivity of calmodulin-independent (Ca2+ + Mg2+)-ATPase due to Ca2+ stress was seen in all Ca2(+)-stressed cells even in the presence of 1 mM glipizide. Structural changes were observed in the form of high molecular weight polymer formation. Cells exposed to high Ca2+ and glipizide (3 x 10(-5)-10(-3) M) showed no improvement in ATPase activity or protection from protein cross-linking compared with cells without the drug. We conclude that in this model glipizide fails to inhibit TG induced protein cross-linking and does not prevent the decrease in (Ca2+ + Mg2+)-ATPase activation in Ca2(+)-stressed red blood cells. This finding considerably weakens the proposal that sulfonylureas act by inhibiting TG activity.     

Role of the stringent response in the expression and mechanism of near-ultraviolet induced growth delay

The near-ultraviolet (300-400 nm) induced growth delay of Escherichia coli cells was compared in isogenic relA+ and relA- cells illuminated either in the stationary or the exponential phase. In the latter case: (a) the relA- strains of K12 and B/r exhibited similar maximal growth lags (65 min and 55 min respectively); (b) the maximal lags were 1.5-fold and 4-fold longer, respectively, in the isogenic relA+ strains; (c) the rate of the relA- -dependent guanosine 3',5'-bis(diphosphate) (ppGpp) accumulation was three-times lower in the K12 relA+ strain as compared to the B/r relA- strain: (d) a K12 spoT mutant having an impaired rate of ppGpp degradation had a 2-fold longer lag. On the other hand, when illumination is performed in the stationary phase, isogenic relA+ and relA- cells (B/r or K12) exhibited similar growth lags at any fluences, indicating little if any involvement of the stringent response. These data extend previous observations of T.V. Ramabhadran an J. Jagger [(1976) Proc. Natl Acad. Sci. USA, 73, 59-63] but do not support their conclusion that the stringent response is the main factor responsible for growth delay. By monitoring the intracellular level of ppGpp in relA+ spoT- and relA+ spoT+ growing cells during illumination and the subsequent growth lag we observed that the initial burst of ppGpp decreases slowly all along the lag; in all relA+ strains checked the return of ppGpp to its basal level coincides with the recovery of normal growth. We conclude that it is the accumulation of ppGpp over the basal level due either to the stringent response or to prevention of ppGpp degradation that is responsible for an amplification of the growth lag.     

Molecular cloning of a tetracycline-resistance determinant from Bacillus subtilis chromosomal DNA and its expression in Escherichia coli and B. subtilis

Bacillus subtilis GSY908 DNA fragments (5.1 and 4.4 kilobase pairs (kb)) containing a tetracycline-resistance determinant were cloned in Escherichia coli using a shuttle plasmid vector pLS353. Restriction endonucelase analysis showed that the 4.4 kb fragment is a spontaneous deletion derivative of the 5.1 kb fragment. E. coli tetracycline-resistance transformants carrying pLS353 with the 5.1 kb fragment (named pTBS1) and that with 4.4 kb fragment (pTBS1.1) could grow at tetracycline concentrations up to 80 and 50 micrograms per ml, respectively. B. subtilis MI112 and RM125 were transformed by pTBS1, resulting in isolation of transformants of MI112 maintaining pTBS1 and RM125 maintaining either pTBS1 or pTBS1.1. Maximum tetracycline concentrations permitting growth of plasmidless MI112 and MI112 with pTBS1 were 4 and 10 micrograms per ml, respectively, while those of plasmidless RM125, RM125 with pTBS1 and RM125 with pTBS1.1 were 7, 50 and 80 micrograms per ml, respectively. It was interesting to note that the tetracycline-resistance level in E. coli conferred by the 5.1 kb fragment is higher than that conferred by the 4.4 kb fragment, but in B. subtilis the 4.4 kb fragment, in contrast, confers a higher level of tetracycline resistance. The level of tetracycline resistance in B. subtilis conferred by the cloned determinant clearly depends on the host strain. The tetracycline resistance conferred by the cloned determinant was associated with decreased accumulation of the drug into the cells. However, it was constitutive in E. coli, but inducible in B. subtilis. The cloned tetracycline-resistance determinant was detected specifically on the chromosome of B. subtilis Marburg 168 derivatives.     

Pressure dependence of equilibria and kinetics of Escherichia coli ribosomal subunit association

Equilibria and rates were observed over the ranges 1-1600 atm, 3-10 mM Mg2+, at 60 mM NH4Cl, pH 7.5, 20 degrees C, by light scattering. The main reaction is accurately represented at all conditions by the following phenomenological equations. 30 S + 50 S = 70 S, KA70 = ka/kd = [70 S]/[30 S][50 S] The equilibrium constants obey simple rules: the volume of association, delta VA0, has the constant value 242 +/- 9 ml/mol, independent of pressure, at all Mg2+ concentrations; the derived values of log KA70 at 1 atm increase linearly with log [Mg2+] at a slope of 7.5. In contrast, the rate constants show a clear break at 6 mM Mg2+: below 6 mM, log ka decreases with pressure with a delta Va of 105 +/- 9 ml/mol and increases with log [Mg2+] at a slope of 4.9; above 6 mM, these values are halved; a split can actually be seen at 6 mM Mg2+, near 500 atm. The usual two-step mechanism for second order reactions in solution, which would insert a 70 S' species, either an encounter complex or a true low concentration steady state intermediate, into the above equation can accommodate these results: as [Mg2+] increases, the rate of transformation of 70 S' into 70 S finally predominates over the rate of dissociation of 70 S' into subunits. The bulk of the pressure effects and all of the [Mg2+] dependence arise from the progressive increase in delta GA0 (electrostatic) that occurs when 30, 50, and 70 S particles all lose equivalent fractions of their internal Mg2+ in response to increases in pressure or decreases in [Mg2+].     

Susceptibility of sixty-five non-oral clinical isolates of the Streptococcus milleri group to seven antimicrobial agents.

Antimicrobial susceptibilities of sixty-five non-oral Streptococcus milleri group clinical isolates to penicillin, gentamicin, lincomycin, ampicillin, chloramphenicol, tetracycline and erythromycin were determined by an agar dilution method. All strains were penicillin-sensitive (MIC < or = 0.031 microgram/ml) and the majority (64/65) were susceptible to erythromycin (MIC < or = 0.125 microgram/ml). Low-level resistance to gentamicin was observed, and the majority of strains possessed an MIC of 8 micrograms/ml. Lincomycin and ampicillin at 0.5 microgram/ml inhibited 52/65 and 61/65 strains, respectively. Of the isolates 92% were inhibited by chloramphenicol at < or = 2 micrograms/ml. Twenty-two S. milleri group strains (of which thirteen were vaginal isolates) were resistant to tetracycline (MIC > or = 8 micrograms/ml).     

Disaggregation-induced changes of developmentally regulated proteins in Dictyostelium discoideum

By means of two-dimensional gel electrophoresis, we analyzed proteins present in a slug-shaped tissue mass of D. discoideum and examined the changes in their amounts after disaggregation of the slugs. Of approximately one hundred polypeptides, six were found to decrease in amount after disaggregation. The decreases of four polypeptides were inhibited by the presence of 1 mM cAMP or 250 micrograms/ml cycloheximide. The decreases of the two other proteins were not suppressed by cAMP or cycloheximide. The patterns of proteins present in vegetative and aggregative cells were also examined. None of the six proteins which showed a decrease after slug disaggregation was found in vegetative or preaggregative cells. These results indicate that both synthesis and degradation of these proteins are controlled by cell-cell contact.