Gratuitous Synthesis of β-Lactamase in Staphylococcus aureus

The synthesis of beta-lactamase in response to 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid as inducer was studied in Staphylococcus aureus. The inducer was not detectably hydrolyzed by beta-lactamase and had minimal antibacterial activity. The kinetics of induction showed a lag of 4 to 6 min in a nutrient broth medium and 8 to 12 min in a defined medium, followed by constant differential rates of synthesis of beta-lactamase. The differential rate of beta-lactamase synthesis in nutrient broth was unaltered by supplementing the medium with glucose, galactose, lactose, arabinose, glycerol, or sucrose. Variations in the partial pressure of oxygen did not alter the differential rate of synthesis of beta-lactamase over the range 18 to 50% oxygen in nitrogen. Even when the rate of growth was considerably reduced by high-oxygen tension, the differential rate of synthesis of the enzyme remained the same. The differential rate of beta-lactamase synthesis at low inducer concentration increased after a shift down in growth rate. The effect was observed with several inducers and under different nutritional conditions, but was always preceded by a change in growth rate. It is suggested that the change in growth rate itself causes the increase in differential rate of beta-lactamase synthesis.     

The primary alkylsulfatase of Pseudomonas aeruginosa: inducer specificity and induction kinetics

The ability of primary alkylsulfate esters and alkanesulfonates to induce alkylsulfatase formation in Pseudomonas aeruginosa was compared on the basis of maximum enzyme levels, induction rate, and levels of induction as a function of inducer concentration. Apparent K inducer values for these effectors were calculated from linear relationships between reciprocals of induction rate and inducer concentration. Maximum enzyme levels estimated from linear progress relationships for each effector indicated that little major distinction could be made between effectors. Excepting carbon chain lengths of C8 which induced about the same level of enzyme, sulfate esters were generally better inducers than sulfonates with little or no apparent induction occurring with effectors of chain length less than or equal to C6. These observations also held true when rates were compared, except that the rate for the C8 ester was approximately ninefold greater than that for the analogous sulfonate. Apparent K inducer constants decreased with increasing alkyl chain length for the esters (C6-C12) and the sulfonates (C8-C14). Values for the esters were approximately sixfold greater than those for sulfonates of equivalent chain length. Plots of log apparent K inducer values against carbon chain length for each series of esters and sulfonates yielded straight-line relationships characteristic of an homologous series in each instance.     

Kinetics and regulation of beta-lactamase formation in methylotrophic bacterium

The regulation and kinetics of formation of an inducible, cell-bound oxacillin-hydrolyzing beta-lactamase (M-OXA) by a methanol-grown Pseudomonas strain were investigated in batch, chemostat, and two-stage continuous cultures. The extent to which enzyme production occurs declines at increased growth rates, and the rate of M-OXA beta-lactamase production follows a kinetic pattern that is partially growth independent and partially inversely growth linked. Growth and enzyme formation are regulated differently by medium constituents. The initial steps of M-OXA beta-lactamase synthesis takes place during the exponential growth phase, and active enzyme is produced by induced nonproliferating cells, probably through a turnover mechanism.     

Pullulanase synthesis in Klebsiella (Aerobacter) aerogenes strains growing in continuous culture

1. Pullulanase synthesis was studied in 16 classified (N.C.I.B.) strains and in an industrial strain (R) of Klebsiella aerogenes grown in chemostats containing maltose as inducer and sole carbon source. 2. Maximum synthesis was associated with carbon-limited growth at a low dilution rate (about 0.2h(-1)). The enzyme remained firmly cell-bound and seemed to be located on the cell surface. 3. Three strains had high activity (R, N.C.I.B. 5938, 8017), twelve were intermediate, and two (N.C.I.B. 8153, 9146) had negligible activity but were inducible with pullulan. 4. Pullulan similarly induced low, but adequate, activity in the other strains in conditions (nutrient limitation other than carbon-limitation) in which pullulanase was otherwise very seriously repressed. Nevertheless, in carbon limitation pullulan induced no more enzyme than did maltose, maltotriose or oligosaccharide mixtures, and ;hyperactivity' never developed on protracted culture. 5. Cyclic AMP relieved the transient repression produced by adding glucose to maltose-limited cultures and a further change to glucose-limited conditions led to constitutive pullulanase synthesis. 6. Amylomaltase and alpha-glucosidase activities were also examined but in less detail. 7. The presence of pullulanase in maltose-limited growth is discussed, but no clear function can be assigned to it at present. The molar growth yields for all the strains were very similar, and no correlation was found between the overgrowth of one strain by another and pullulanase activity. Further, any function as a general branching enzyme in polysaccharide synthesis seems unlikely.     

Beta-lactamase activity of bacteria of the genus Proteus]

beta-Lactamases of Proteus and their role in the mechanism of the microbe resistance to penicillins and ceporin were studied. It was found that the beta-lactamase of Proteus had low activity and were produced by both beta-lactamide resistant and sensitive clinical strains of Proteus. The resistant cultures of Proteus produced enzymes more frequently (3.4--5 times) than the sensitive ones. The synthesis of beta-lactamase in the clinical Proteus strains was inducable. The high induction coefficient was achieved only in the presence of high concentrations of the inductor. No significant dependence of the culture sensitivity level of ampicillin and ceporin on the induction level was observed. The most significant part of the constitutive enzyme in Proteus was intracellular, while that of the inducable enzyme was extracellular. No correlative dependence between the culture resistance levels to penicillins and ceporin and the enzyme activity was noted. The beta-lactamase activity was not found in the transconjugants with the in vitro acquired R-factor controlling the ampicillin and ceporin resistance, as well as in the resistant mutants selected on the media with increasing concentrations of the above antibiotics. Induction of beta-lactamase synthesis was not found in these strains either. The ability of Proteus to synthesize beta-lactamase can be lost on the strain storage under laboratory conditions which was not always accompanied by reduction of the culture sensitivity to ampicillin and ceporin. The enzymatic destruction of beta-lactamides was not the main mechanism of Proteus resistance to the above antibiotics.     

Induction of xylan-degrading enzymes inButyrivibrio fibrisolvens

The formation of xylanolytic enzymes byButyrivibrio fibrisolvens NCFB 2249 was induced by xylan, xylo-oligosaccharides, and xylobiose. Inhibition of RNA or protein synthesis prevented inducetion, and enzyme formation occured only when anaerobiosis was maintained. The rate of enzyme inducetion by xylan was affected by pH and inducer concentration, and highest levels of activity occurred when the initial pH and xylan concentration were pH 6.5–7 and 2 mg/ml respectively. The ability of the cells to respond to the inducer was reduced in slowly growing cells, although cells that were grown at dilution rates that appertain in the rumen ecosystem responded rapidly to the inducer.Butyrivibrio fibrisolvens also exhibited diauxic characteristics of carbohydrate utilization, and in consequence enzyme induction and xylanolysis were delayed until readily metabolized sugars (e. g., glucose, arabinose) had been consumed.     

Induction of lytic enzymes by the interaction of Ustilago maydis with Zea mays tissues

The nonpathogenic (FB-2) and pathogenic (FB-D12) strains of Ustilago maydis were grown in medium supplemented with different carbon sources including monosaccharides, polysaccharides, and plant tissues. Both strains were able to grow on all substrates, with doubling times varying from 2 to 25 h depending on the carbon source. Plant tissues supplied as carbon source induced lytic enzymes differentially; pectate lyase and cellulase activities were induced preferentially by apical stem meristem in strain FB-D12, whereas leaves preferentially induced xylanase and cellulase activities in strain FB2. Stems induced polygalacturonase activity in both strains. All enzyme activities, except cellulase in the FB-D12 strain, were detected at a low level when U. maydis was grown on glucose. In planta, chlorosis and production of teliospores were paralleled by an increase in pectate lyase activity. Anthocyanin production and formation of galls and teliospores correlated with polygalacturonase expression whereas cellulase activity increased only during the stage of anthocyanin production and gall formation. Expression of xylanase activity coincided with the last stage of teliospore formation.     

Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

AIMS: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the beta-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. METHODS AND RESULTS: Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and beta-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 microg ml(-1) and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 microg ml(-1). The highest concentration that gave permissible growth rate was 50 microg ml(-1). The isolated survivals were screened on the bases of PGA and beta-lactamase activities. Among the retained mutants, the occurrence of beta-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). CONCLUSIONS: In seven of the mutants, PGA activity was enhanced with considerable decrease in beta-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of beta-lactamase activity and twofold increase in PGA activity [12.7 mg 6-amino-penicillanic acid (6-APA) h(-1) mg(-1) wet cells] compared with that in the wild-type strain (6.3 mg 6-APA h(-1) mg(-1) wet cells). SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of beta-lactamase. These mutants could be used for industrial production of PGA.     

Physiological studies of the regulation of beta-lactamase expression in Pseudomonas maltophilia.

The kinetics of beta-lactamase induction in Pseudomonas maltophilia IID1275/873 were investigated. Upon induction with beta-lactam antibiotics, a correlation was seen between the increase in specific beta-lactamase activity and the generation time, as well as the concentration of inducer in the medium. The specific beta-lactamase activity increased slowly within the first 0.5 generation and then more rapidly; it decreased regularly after about 2 generations of growth in the presence of inducer. This decrease could presumably be attributed to the continuous breakdown of inducer by beta-lactamases in the culture medium. In a chemostat culture with continuous supply of fresh inducer-containing medium, the specific beta-lactamase activity could be stabilized at a high level over several generations. Removal of the beta-lactam after a certain induction time showed that a short exposure of the bacteria to inducer caused induction kinetics comparable to those resulting from continuous exposure of the cells to inducer. The two beta-lactamases of P. maltophilia, L1 and L2, were induced simultaneously under various experimental conditions.     

Half-of-the sites reactivity in the catalytic mechanism of yeast glyceraldehyde 3-phosphate dehydrogenase

Novick &; Weiner (1957) proposed a model in which induction of the lac operon with suboptimal concentrations of inducer generates a population containing both uninduced and fully induced cells. The latter arise as cells acquire the galactoside transport system, thus initiating an autocatalytic cycle of induction since this permease can transport an inducer for its own synthesis. Evidence in favor of this model has been obtained from direct measurements of the enzyme content of individual cells, using a fluorogenic assay sensitive to one molecule of β-d-galactosidase. Fully induced cells, at the predicted frequency, were found in suboptimally induced populations of wild type strains, and of a strain lacking thiogalactoside transacetylase, but not of a strain lacking galactoside permease. In the wild type, the frequency of cells with an enzyme content intermediate between uninduced and fully induced levels was greater than the frequency predicted for cells within the autocatalytic cycle of induction. According to the model, then, in some of these cells, induction of β-d-galactosidase has occurred without formation of the permease necessary to initiate accumulation of inducer.     

Salicylate decreases production of AmpC type beta-lactamases and increases susceptibility to beta-lactams in a Morganella morganii clinical isolate

The effect of salicylate, a marRAB inducer, on the resistance to beta-lactams was characterized in an AmpC beta-lactamase hyperproducer Morganella morganii clinical isolate (the M1 strain). Results were compared with those of the effect of salicylate in a wild-type M. morganii strain. Salicylate induced a decreased susceptibility to nalidixic acid, norfloxacin and tetracycline and simultaneously increased the susceptibility to beta-lactams apparently due to the repression of AmpC beta-lactamase synthesis in the M1 strain. Likewise, salicylate only repressed 46 kDa outer membrane protein expression in the wild-type strain, since the clinical isolate M1 did not express it.     

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.     

Continuous-culture studies of synthesis and regulation of extracellular beta(1-3) glucanase and protease enzymes from Oerskovia xanthineolytica

This article describes the synthesis and regulation of beta(1-3)glucanase and protease enzymes from the cell lytic system of Oerskovia xanthineolytica LL-G109 in continuous culture using different concentrations of carbon source (glucose) and inducer (glucan). These two enzyme activities are the main components of a lytic system capable of lysing and disrupting whole yeast cells; it is subject to catabolite repression by glucose and is induced by yeast glucan. Peaks of beta(1-3)glucanase and protease activity are obtained at dilution rates of between 0.05 and 0.15 h(-1). The glucanase-protease ratio is very high compared to other strains. At dilution rates above 0.15 h(-1) all activities are similar to those obtained in batch culture. The lytic enzyme system appears to contain several beta(1-3)glucanase enzymes. In continuous culture both productivity and enzyme concentrations are greatly in creased when compared to batch culture, 11- and 4.4-fold, respectively.     

Purification and properties of beta-lactamase from Proteus morganii.

The cephalosporin beta-lactamase was purified from a strain of Proteus morganii that showed resistance to beta-lactam antibiotics and produced the enzyme constitutively. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and consisted of a single polypeptide of molecular weight 38,000 to 40,000 from gel filtration of Sephadex G-100 and sodium dodecyl sulfate-acrylamide gel electrophoresis, its isoelectric point being pH 7.2 No cysteine residue was found in its amino acid composition. The specific activity was 190 mumol/min per mg of the purified enzyme protein for the hydrolysis of cephaloridine, the optimal pH was about 8.5 and the optimal temperature was 50 degrees C. Antibodies against the purified beta-lactamase inhibited not only the enzyme activity of the purified preparation, but also the enzyme activity of all of the other strains of P. morganii so far tested, regardless of whether the modes of their production were inducible or constitutive. None of the beta-lactamases produced by beta-lactam antibiotic-resistant strains of other species of Proteus was affected at all by the antibodies, thus showing that the purified cephalosporin beta-lactamase was of the species-specific type. The enzymological properties of the preparation have been compared with those of beta-lactamases derived from other gram-negative enteric bacteria.     

Expression of beta-lactamase by recombinant Escherichia coli strains containing plasmids of different sizes--effects of pH, phosphate, and dissolved oxygen

The characteristics of growth and synthesis of plasmid-encoded protein were studied for strains of recombinant E. coli JM103 which carried the beta-lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying-sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0-8.0, the bulk beta-lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7-8.7 kb), at which beta-lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for beta-lactamase synthesis. The bulk beta-lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and beta-lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid-encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.     

Possible Involvement of β-Lactamase in Sporulation in Bacillus cereus

Nonreverting beta-lactamase-negative strains were isolated from the beta-lactamase-constitutive strain, Bacillus cereus 569 H. These strains differed from both beta-lactamase-inducible and -constitutive strains not only in failure to produce beta-lactamase but also in failure to autolyze on aging, delayed sporulation, and failure to release free spores from sporangia when produced. The addition of B. cereus beta-lactamase of 15% purity to a final concentration of 10 IU/ml stimulates sporulation and particularly the release of free spores in culture from sporangia of strain 569 (inducible wild-type), 569/H (constitutive mutant of 569), and HPen(-), a nonreverting beta-lactamase strain isolated from 569/H in this laboratory. Cultures of HPen(-) did not release free spores without this treatment. Similar stimulation of sporulation and spore release by beta-lactamase from B. cereus were observed in another beta-lactamase-negative strain derived from 569/H as well as in certain sporogeny mutants of B. subtilis. The beta-lactamase preparation used in these experiments was free of peptidases, proteases, and autolysins capable of solubilizing wall from vegetative cells. These results, taken with our previous finding that a soluble peptidoglycan inducer becomes available in cultures of B. cereus only at sporulation and that normal derepression of beta-lactamase accompanies normal sporulation, suggest that beta-lactamase in B. cereus may be involved in peptidoglycan metabolism during sporulation and possibly the breakdown of sporangial wall with the concomitant release of mature spores.     

Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 1. Readjustment of metabolic enzyme synthesis

The metabolic burden and the stress load resulting from temperature-induced production of human basic fibroblast growth factor is connected to an increase in the respiratory activity of recombinant Escherichia coli, thereby reducing the biomass yield. To study the underlying changes in metabolic enzyme synthesis rates, the radiolabeled proteom was subjected to two-dimen- sional gel electrophoresis. After temperature-induction, the cAMP-CRP controlled dehydrogenases of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle (LpdA and SdhA) were induced four times, reaching a maximum 1 h after the temperature upshift. The more abundant tricarboxylic acid cycle dehydrogenases (Icd and Mdh) were initially produced at reduced rates but regained preshift rates within 30 min. The adenylate energy charge dropped immediately after the temperature upshift but recovered within 1 h. Similar profiles in dehydrogenase synthesis rates and adenylate energy charge were found in a control cultivation of a strain carrying the "empty" parental expression vector. Although both strains exhibited significant differences in growth pattern and respiration rates after the temperature upshift, the adaptation of the energetic state of the cells and the synthesis of enzymes from the energy-generating catabolic pathway did not seem to be affected by the strong overproduction of the recombinant growth factor. In contrast, the synthesis rates of enzymes belonging to the biosynthetic machinery, e.g., translational elongation factors, decreased more strongly in the culture synthesizing the recombinant protein. In control and producing culture, synthesis rates of elongation factors paralleled the respective growth rate profiles. Thus, cells seem to readjust their metabolic activities according to their energetic requirements and, if necessary, at the cost of their biosynthetic capabilities.     

Induced formation of ribulose 1,5-diphosphate carboxylase in Rhodopseudomonas spheroides with particular concern to its relation with chromatophore formation

A comparative study was made on features of the induced synthesisof RuDP carboxylase in three strains of R. spheroides with differentbiochemical properties. In strains Sb and Sa, which were able to grow under either light-anaerobicor dark-aerobic conditions, activities of RuDP carboxylase inthe light-grown cells were much higher than those in dark-growncells. The level of RuDP carboxylase activity in dark-growncells of the Sb strain (wild type strain) increased two to threetimes in the dark by incubating the heavy cell suspension underlow aeration, but, for a further increase in enzyme activity,a light-anaerobic condition was required. This is in contrastto the induced formation of bacteriochlorophyll which has beenshown to proceed actively in the dark as well as in the light.On the other hand, with dark-grown cells of the Sa strain, whichhad possible partial defects in the chlorophyll synthesis system,the induced synthesis of RuDP carboxylase under the light-anaerobiccondition was markedly retarded as compared to that with theSb strain. RuDP carboxylase formation was not induced in L-57(a colorless mutant) under any of these conditions. The induced formation of RuDP carboxylase, as well as of bacteriochlorophyll,under the light-anaerobic condition was considerably suppressedby hydroxyurea and mitomycin C. This suggests that the geneticcontrol systems of RuDP carboxylase synthesis may be closelyrelated with those for the formation of the photosynthetic apparatus. ¹This work was supported in part by Public Health Research GrantAM 08016 from the National Institute of Arthritis and MetabolicDiseases, U.S.A. (G. K.). ²Present address: Laboratory of Radioisotope Experiment, TohokuUniversity School of Medicine, Sendai, Japan. (Received September 6, 1968; )