Regulation of Staphylococcal Penicillinase Synthesis

5-Methyl tryptophan was found to be an efficient inducer of penicillinase synthesis in Staphylococcus aureus. Addition of actinomycin D or tryptophan to the culture medium shuts off the 5-methyl tryptophan-induced synthesis of penicillinase with an apparent half-life of approximately 1 to 2 min, respectively. Hence, in the induction of penicillinase synthesis, 5-methyl tryptophan seems to function as a structural analogue of penicillin rather than by becoming incorporated in proteins and thereby creating faulty penicillinase repressor or antirepressor. This conclusion is supported by similarities in the structures of the two compounds as revealed by solid atomic models. The fact that S. aureus exposed to (14)C-penicillin in the absence of protein synthesis failed to synthesize penicillinase at an increased level when cell growth was resumed strongly suggests that a protein involved in the regulation of penicillinase synthesis must be synthesized in the presence of the penicillinase inducer. In turn, this observation suggests that the penicillinase inducer promotes penicillinase synthesis by directing the penicillinase regulatory protein (i.e., the penicillinase antirepressor) to acquire a different conformation when it is synthesized in the presence of the penicillinase inducer. A working model for the regulation of penicillinase synthesis based on these and other data has been constructed and is presented.     

Characteristics of Secretion of Penicillinase, Alkaline Phosphatase, and Nuclease by Bacillus Species

The distribution of alkaline phosphatase and nuclease activity between cells and medium was examined in one strain of Bacillus licheniformis and four strains of B. subtilis. Over 95% of both activities was found in the medium of the B. licheniformis culture, but in the B. subtilis cultures the amount of enzyme activity found in the medium varied with the strain and the enzyme considered. B. licheniformis 749 and its penicillinase magnoconstitutive mutant 749/C were grown in continuous culture with phosphorous as the growth-limiting factor, and the kinetics of penicillinase formation and secretion were examined. Nutrient arrest halted secretion (usually after a lag of about 30 min) in both the inducible and constitutive strains. Chloramphenicol did not eliminate secretion, but under certain circumstances reduced its rate. In the inducible strain treated with a low level of inducer, the rate of secretion was more affected by the rate of synthesis than by the level of cell-bound enzyme. During induction, the onset of accretion of cell-bound penicillinase and secretion of the exoenzyme were nearly simultaneous. It seems unlikely that a long-lived, membrane- or cell-bound intermediate is mandatory in the secretion of the three enzymes by Bacillus species. In the case of penicillinase secretion, there are at least two different phases. When penicillinase synthesis is proceeding rapidly, the rate of secretion is five to six times greater at equivalent concentrations of membrane-bound penicillinase than it is when penicillinase synthesis is reduced. The data require that any membrane-bound intermediate in the formation of exoenzyme be much shorter-lived in cells with a high rate of synthesis than in cells with a low rate. Either there are two separate routes for the secretion of penicillinase or the characteristics of the process vary substantially between the early stages and the declining phase of induction.     

Kinetics of Penicillinase Induction and Variation of Penicillinase Translation in Staphylococcus aureus

At neutral pH, the rate of penicillinase synthesis by staphylococci declines gradually after removal of free inducer, while at pH 5.4 enzyme formation is generally linear for an extended period. Linear synthesis of penicillinase was observed at neutral pH in nonsaturating concentrations (1 μg/ml) of actinomycin D. The rate of enzyme synthesis, corrected for inhibition of growth caused by the antibiotic, was relatively independent of the time of actinomycin addition. The lag preceding linear enzyme formation increased with the interval between induction and the addition of actinomycin. The findings are consistent with the concept that, at neutral pH, “operons” activated by induction are rapidly repressed, while at pH 5.4, this process is delayed.

At a concentration of 4 μg/ml, actinomycin D blocked penicillinase messenger synthesis and also elicited a short-lived acceleration of the increase of penicillinase activity in uninduced and, late after induction, in induced cultures. This effect did not require a functional genomic repressor mechanism since it occurred also in a penicillinase-constitutive strain. It required protein synthesis and could not be attributed to a greater enzyme stability in the presence of actinomycin. The results suggest enhanced penicillinase translation after addition of actinomycin D.

     

Characteristics of Penicillinase Secretion by Growing Cells and Protoplasts of Bacillus licheniformis

Cultures of the inducible penicillinase-producing strain 749 of Bacillus licheniformis, induced with small amounts of benzylpenicillin, synthesized penicillinase at a high rate for a short period, after which the rate of synthesis slowly declined. During the period of active synthesis, the rate of secretion, as a fraction of the level of cell-bound penicillinase (which is originally high), gradually decreased to a constant level. Chloramphenicol, at a concentration (40 mug/ml) which completely inhibited synthesis of penicillinase, partially inhibited secretion if added during the period of active synthesis. During the phase of reduced synthesis, chloramphenicol was without effect on secretion. Penicillinase secretion, by actively growing cultures of the constitutive penicillinase-producing mutant 749/C, was inhibited by 75% immediately after addition of chloramphenicol. The secretion of part of the penicillinase released during active growth is probably dependent on synthesis of penicillinase, but part of the secreted penicillinase can be released in the absence of synthesis. Protoplasts were obtained from which periplasmic penicillinase has been removed, and these protoplasts were capable of substantial growth and penicillinase synthesis without lysis. At pH 7.5, there was no net incorporation of penicillinase into the cell membrane; the enzyme released was almost entirely of the exo form and was roughly equivalent to the amount of new enzyme formed. At pH 6.0, there was some incorporation of penicillinase into the plasma membrane, and approximately half of the extracellular penicillinase was in the exo form; the remainder perhaps represented membrane fragments. In the presence of chloramphenicol, a small amount of penicillinase was released at pH 7.5 as the exo form; at pH 6.0, practically none was released. We suggest that, with the removal from protoplasts of the periplasmic penicillinase-containing particles, a restriction on secretion has been lifted.     

Characterization of a protein synthesis system from rat liver. Translation of endogenous and exogenous messenger RNA

An in vitro system prepared from rat liver postmitochondrial supernatant exhibits a high rate of protein synthesis for an extended period of time. This system initiates translation of either endogenous or exogenous mRNA, incorporates Met at a rate of 13 pmol/mg of postmitochondrial supernatant protein/min, maintains this rate for at least 90 min, and performs several rounds of translation/mRNA molecule. Up to 50% of the activity is due to reinitiation of protein synthesis using endogenous mRNA. In addition, 60-70% of the protein synthesized was released from ribosomes into the medium. Addition of globin mRNA stimulates protein synthesis and results in the synthesis of a protein that comigrates with authentic rabbit globin. Black beetle virus mRNA 2 also stimulates protein synthesis and results in synthesis of a protein with molecular weight corresponding to that of the mature viral protein. With endogenous rat liver mRNA this system synthesizes a large number of proteins.     

Characteristics of penicillinase release by washed cells of Bacillus licheniformis

Saline-washed cells of Bacillus licheniformis strain 749/C (constitutive for penicillinase) were able to release exopenicillinase in the presence of concentrations of chloramphenicol that prevented protein synthesis completely. The release reaction was strongly pH-dependent, occurring at a faster rate at alkaline pH in anionic or cationic buffers than at neutral pH. A strongly pH-dependent release reaction was noted in growing cells also. The reaction in washed cells can be stopped completely by changing the pH to 6.0. Within 30 min at pH 9.0, about 55% of the cell-bound penicillinase was released; thereafter, release continued at a greatly reduced rate. Suspensions of washed cells retained their capacity to release penicillinase at pH 9.0 for 90 min. Penicillinase released at pH 9.0 from either cells or protoplasts was not readsorbed over a 60-min period after changing the pH to 6.0. The release reaction was strongly temperature-dependent. We examined the effect of a large number of metabolic inhibitors and other compounds on the pH-dependent release phenomenon. Quinacrine hydrochloride, chloroquine diphosphate, and chlorpromazine hydrochloride reduced secretion substantially at 10(-4)m. Deoxycholate and Triton X-100 were active at 10(-3)m, but tungstate, arsenate, and molybdate had small effects at 10(-1)m. The rate of exopenicillinase release at pH 9.0 from fully stabilized protoplasts was one-half that of intact cells. Protoplasts lysed in hypotonic media or detergents showed even greater reduction in releasing activity. Penicillinase released from washed cells at pH 7.5 or 9.0 appeared to be derived from the periplasmic tubule and vesicle fraction that was released by protoplast formation.     

Interleukin-3 inhibits cycloheximide of C-Jun mRNA in human monocytes: Possible role for a serine/threonine phosphatase

Cycloheximide is a strong inducer of the c-jun protooncogene mRNA at concentrations (≤50 ng/ml) that do not inhibit protein synthesis in human monocytes. This induction is transient lasting 30–60 min in contrast to the sustained induction obtained with concentrations that inhibit protein synthesis. The pluripotent colony stimulating factor interleukin-3 (IL-3) (10 ng/ml) is also a modest inducer of the c-jun gene in these cells; however, in combination with cycloheximide, IL-3 dramatically reduces the c-jun induction below levels induced by cycloheximide alone. This is a true inhibition and is not due to a change in temporal kinetics of induction because the suppression in the presence of IL-3 is observed at both 30 and 60 min after simultaneous addition of both IL-3 and cycloheximide. Preincubation of monocytes with 12.5 nM okadaic acid (a potent inhibitor of protein phosphatases 1 and 2A) and cycloheximide prior to addition of IL-3 restored the level of c-jun induction to that mediated by cycloheximide alone. This concentration of okadaic acid inhibited almost 70% of the phosphorylase phosphatase activity in monocyte lysates. These observations suggest that activation of protein serine/threonine phosphatase(s) underlies the ability of IL-3 to inhibit cycloheximide induction of c-jun in monocytes. © 1993 Wiley-Liss, Inc.     

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate

This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli. Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH. When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically. The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S-transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPARalpha LBD. Total yield of GST-PPARalpha LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPARalpha LBD, the only active form, increased with the length of induction period. Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 +/- 0.4 cell divisions. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific growth rate was observed when inducer was added in the lag-phase, and no soluble protein was produced. These results suggest that solely soluble GST-PPARalpha LBD acts as a growth inhibitor and that GST-PPARalpha LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction. Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth.     

Control of synthesis of mRNA''s for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase.

A 30 degrees C, functional messengers for dCMP hydroxymethylase first appeared 3 to 6 min postinfection and reached their maximum levels at 12 min. Chloramphenicol, added before the phage, reduced the rate of mRNA accumulation. When the antibiotic was added 6 min postinfection, mRNA levels increased at their normal rate but there was no obvious repression of messenger accumulation. Delaying the addition of drug until 8 or 12 min had progressively less effect on the pattern of hydroxymethylase mRNA metabolism. When chloramphenicol was present from preinfection times or from 6 min postinfection, all hydroxymethylase mRNA's synthesized were stable; at later times, however, the ability of the drug to stabilize mRNA decreased with its ability to delay the turnoff of mRNA production. An overaccumulation of hydroxymethylase mRNA was also seen when phage-specific DNA synthesis was inhibited either by mutational lesion in an essential viral gene or by 5-fluorodeoxyuridine. By min 20 of a DNA-negative program, hydroxymethylase mRNA synthesis was repressed to the point where it no longer compensated for decay. However, a finite level of hydroxymethylase mRNA synthesis was maintained at later times of a DNA-negative infection. Such results indicate that replication of the phage chromosome is necessary but not sufficient for a complete turnoff of hydroxymethylase mRNA production. Functions controlled by the maturation-defective proteins (the products of genes 55 and 33) played only a minor role in the regulation of hydroxymethylase mRNA, metabolism. Thus, we favor the hypothesis that a complete turnoff of hydroxymethylase messenger production requires one or more new proteins as well as an interval of DNA replication. The absence of DNA synthesis had no particular effect upon dihydrofolate reductase messenger production. The preinfection addition of chloramphenicol likewise had little effect on dihydrofolate reductase messenger metabolism. These latter data imply that prior synthesis of a phage-coded protein synthesis may not be required for the turnoff of reductase messenger production.     

Inducible synthesis of β-galactosidase inKluyveromyces fragilis

Lactose,d-galactose, andl-arabinose induce the synthesis of β-galactosidase inKluyveromyces fragilis. Lactose is the best inducer with a maximum effect at 1.4 mm. The induced synthesis of the enzyme in glycerol grown stationary phase cells is triggered within 30 min of inducer addition, the full induction being achieved within subsequent 30–40 min.     

Thionein gene expression in Cd++-variants of the CHO cell: correlation of thionein synthesis rates with translatable mRNA levels during induction, deinduction, and superinduction.

The relationship of thionein synthesis rates to translatable cytoplasmic thionein mRNA levels was investigated for the first time in a cultured cell system. Thionein synthesis was induced in Cdr, a cadmium-resistant variant of CHO, by exposure to 2 microM CdCl2. Following a short (1.5 hr) lag, thionein synthesis increases to a rate that is at least 30 times the uninduced rate 7-8 hr after addition of Cd++. This increase is blocked by the coincident addition of a actinomycin D. Cytoplasmic thionein mRNA levels, measured by translation in a modified wheat germ system, increase rapidly following induction to values approximately 25 times uninduced levels within 6-8 hr. The increase in thionein mRNA precede proportionate increases in thionein synthesis by 0.5-1.0 hr. Continued exposure to Cd++ results in a decreased thionein synthesis rate after 8 hr. By 30 hr, the rate is one-half that seen 6-8 hr after induction. Removal of Cd++ after 8 hr results in a rapid decrease in thionein synthesis (t 1/2 approximately 4 hr). Both decreases are inhibited by the addition of actinomycin. In all instances--induction, deinduction, and actinomycin-mediated "super-induction"--translatable thionein mRNA levels and thionein synthesis rates increase, decrease, or are maintained coordinately. The results suggest that thionein synthesis in Cdr is controlled primarily by the level of translatable cytoplasmic thionein mRNA.     

Dual regulation by arginine of the expression of the Escherichia coli argECBH operon.

The correlation between the level of messenger ribonucleic acid (mRNA) specific for the argECBH gene cluster (argECBH mRNA) measured by ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization and the rates of synthesis of N-acetylornithine deacetylase (argE enzyme) and of argininosuccinate lyase (argH enzyme) of Escherichia coli strain K-12 were determined for steady-state growth with and without added L-arginine and during the transition periods between these two states. During the transient period after arginine removal (transient derepression), the synthesis of enzymes argE and argH was initially three to five times greater than the steady-state derepressed rate finally reached 50 min later. The level of argECHB mRNA correlated well both quantitatively and temporally with the rates of enzyme synthesis during this transition. The level of in vivo charged arginyl-transfer RNA (tRNAarg), monitored simultaneously, was initially only 5 to 10% and gradually increased to a final level of 80% after 45 min. During the transient period after arginine addition (transient repression), the rates of synthesis of enzymes argE and argH decreased to almost zero and gradually reached steady-state repressed rates after about 180 min. The argECBH mRNA level remained constant at the steady-state repressed level throughout transient repression, revealing a discontinuity between the level of this mRNA and rates of enzyme synthesis. A similar discrepancy was noted during the transition after ornithine addition. In vivo charged tRNAarg remained constant at 80% during this transition. After removal of arginine, the zero-level transient enzyme synthesis developed after only 7.5 min of arginine deprivation and was maximum after 30 min. The results suggest an accumulation of a molecule regulated by arginine that plays a role in transient repression. Our data indicate that arginyl-tRNA synthetase is not this molecule since its synthesis was unaffected by arginine. The ratios of steady-state argE and argH enzyme synthesis without arginine to that with arginine were 12 and 20, respectively, whereas the similar ratio for argECBH mRNA was 2 to 3. The repressed level of argECBH mRNA was not affected by attempts to repress or derepress the ppc+ gene (carried on the DNA used for hybridization), and the repressed level of argECBH mRNA was lowered about 50% in cells carrying an internal argBH deletion. These data taken together indicate the presence of an excess of untranslated argECBH mRNA during both transient and steady-state repression by arginine. Thus, a second regulatory mechanism, not yet defined, appears to play an important role in arginine regulation of enzyme synthesis.     

Correlation of Carbohydrate Catabolism and Synthesis of Macromolecules During Enzyme Synthesis in Pseudomonas fluorescens.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Correlation of carbohydrate catabolism and synthesis of macromolecules during enzyme synthesis in Pseudomonas fluorescens. J. Bacteriol. 90: 23-28. 1965.-Glucose, ribose, and fructose shorten the lag period required for synthesis of protocatechuate oxygenase. Radioactivity from uracil-2-C(14) is incorporated into the hot trichloroacetic acid-soluble fraction after a lag period of approximately 20 min after addition of protocatechuic acid. Addition of glucose or ribose simultaneously with the inducer shortens the lag period to approximately 5 min and increases the rate of uracil incorporation. The inducer must be present to initiate incorporation of radioactivity, and the exogenous carbon source accelerates incorporation but is not sufficient to initiate synthesis by itself. The addition of protocatechuic acid increases the rate and total incorporation of radioactivity from uniformly labeled glucose or ribose-1-C(14) into the hot trichloroacetic acid-soluble fraction. Ribose decreases the incorporation of radioactivity from uniformly labeled glucose into the hot trichloroacetic acid-soluble fraction, and glucose shows a similar effect on incorporation of radioactivity from ribose-1-C(14), indicating the two sugars are serving in the same capacity to enhance enzyme synthesis. Radioactivity from glucose-1-C(14) is not incorporated into the hot trichloroacetic acid-soluble fraction. The results suggest that glucose and ribose shorten the lag period for inducible enzyme formation by serving as a "specific" carbon source for synthesis of macromolecules such as ribonucleic acid.     

Penicillinase (β-Lactamase) Induction in Bacillus licheniformis Under Pseudogratuitous Conditions by 2-(2′-Carboxyphenyl)-Benzoyl-6-Aminopenicillanic Acid

Induction of penicillinase (beta-lactamase) in Bacillus licheniformis 749 by 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid (CBAP) was examined, since this compound was reported to be a gratuitous inducer of penicillinase in Staphylococcus aureus. The specific activity of enzyme optimally induced by CBAP is slightly more than that formed in response to cephalosporin C and threefold the level induced by benzylpenicillin. The optimal inducer concentration of CBAP was not inhibitory toward the growth of penicillinase-deficient mutants, unlike benzylpenicillin or cephalosporin C which showed marked toxicities. CBAP is hydrolyzed by the Bacillus penicillinase, but as indicated by its "physiological efficiency" (V(max)/K(m)), CBAP is a poor substrate at low concentrations. At very high concentrations, CBAP inhibited benzylpenicillin hydrolysis. The overall effectiveness of CBAP as an inducer can be attributed to its low "physiological efficiency" which enables the use of nontoxic levels of CBAP for induction without its rapid hydrolysis. Although CBAP is not a true gratuitous inducer, operationally it approaches gratuity for induction of B. licheniformis penicillinase better than other known inducers.     

Resistance of Escherichia coli to penicillins. V. Physiological comparison of two isogenic strains, one with chromosomally and one with episomally mediated ampicillin resistance

Two essentially isogenic strains of Escherichia coli K-12 were compared: D31 had chromosomally and D1-R1 episomally mediated resistance to ampicillin. The two strains had the same ability to form colonies on ampicillin plates, but in other tests they were quite different. In serial dilution tests as well as in exponentially growing cultures, D1-R1 was far more resistant to ampicillin than was D31. The inoculum effect with D1-R1 was large and with D31 was rather small. On plates, D31 was more resistant to penicillin G than was D1-R1. The penicillinase activity of buffer suspended cells against dl-ampicillin was 15 times higher for D1-R1 than for D31, but the two strains showed about the same rate of hydrolysis of penicillin G. With dl-ampicillin as substrate, for D1-R1 the apparent K(m) was 1.7 x 10(-4)m, whereas D31 gave a slightly sigmoid curve with a half-saturation concentration of about 5 x 10(-3)m. No induction of penicillinase activity was found. When the growth rate was varied by a factor of four, the amount of penicillinase per cell mass was constant in both D1-R1 and D31, whereas in two wild-type strains the amounts of penicillinase increased with increasing growth rates. With exponentially growing D1-R1, ampicillin disappearance started within 3 min, but at low ampicillin concentrations the rate was less than 10% of the rate of hydrolysis by buffer-suspended cells. Before D31 started hydrolysis, there was a lag period that lasted at least one generation and depended on the concentration of ampicillin. After this lag period, the rate of hydrolysis was 10 times higher than that observed with buffer-suspended cells. These differences between growing and nongrowing cells indicate that both the chromosomally and the episomally mediated penicillinases are controlled by some products present in growing cells.