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PubMed1972

Release of the -galactosidase-synthesizing system from ultraviolet catabolite repression by cyclic 3',5'-adenosine monophosphate, dark repair, photoreactivation, and cold treatment.

Recovery from the inhibitory effect of ultraviolet irradiation on the induced synthesis of beta-galactosidase was studied in Escherichia coli B/r. When irradiated cells (520 ergs/mm(2) at 254 nm) were induced and incubated in minimal medium supplemented with Casamino Acids (conditions of catabolite repression), the ability to form enzyme was greatly reduced for about 100 min and then recovery began. The inhibition observed immediately after ultraviolet irradiation was partially reversed by cyclic 3',5'-adenosine monophosphate (cyclic AMP) or by photoreactivation treatment. Inhibition was reduced if the cells were given cold treatment (5 C) before or during irradiation; the kinetics of induced enzyme formation in each case were similar to those of irradiated cells receiving cyclic AMP. These kinetics suggest that the cold treatments, like cyclic AMP, cause the release of the beta-galactosidase-synthesizing system from catabolite repression. When irradiated cells were incubated for various times before cyclic AMP or photoreactivation treatment, some reversal of the inhibition of induced enzyme formation was obtained, but by 100 min the treatments were ineffective. Because 100 min was also the time at which dark recovery of enzyme formation began, the recovery process was interpreted to be the result of completion of DNA repair, which, in turn, released the beta-galactosidase-synthesizing system from catabolite repression.

Amino Acids↗
PubMed1972

Construction and properties of Escherichia coli strains exhibiting -complementation of -galactosidase fragments in vivo.

In vivo alpha-complementation of beta-galactosidase was demonstrated in 16 Z gene terminator (nonsense) mutant strains of Escherichia coli upon introduction of the episome F'M15 which specifies production of a mutant Z gene polypeptide containing a small deletion in the N-terminal region of the enzyme monomer. Genetic and biochemical analyses of the merodiploids showed that restoration of enzyme activity was due to their terminator/F'M15 genetic constitution resulting in the production of two enzymatically inactive polypeptides which associate in vivo to reconstitute active, stable beta-galactosidase. The prematurely terminated polypeptide fragments known to be rapidly degraded in haploid cells were shown by phenotypic and biochemical studies to be stabilized (i.e., protected) in merodiploids by formation of complemented enzyme complexes with the M15 protein. Phenotypic properties of complementing diploids are described and are discussed in relation to in vitro determination of beta-galactosidase activity.

Coliphages↗
PubMed1973

Influence of polyamine limitation on the chain growth rates of beta-galactosidase and of its messenger ribonucleic acid.

The rates of elongation of beta-galactosidase and its messenger ribonucleic acid (RNA) were estimated in a polyamine-deficient mutant of Escherichia coli through an analysis of the kinetics of enzyme induction. The chain growth of beta-galactosidase was calculated from the time required after the appearance of an amino terminal fragment of 60 amino acids (auto-alpha) until completed enzyme began to accumulate. The elongation rate of beta-galactosidase messenger RNA was estimated from the time after induction at which streptolydigen-resistant, enzyme-forming capacity first appeared. Upon polyamine starvation, the rate of polypeptide elongation slowed from 17 to 10 amino acids per s and the messenger RNA elongation rate decreased from 47 to 30 nucleotides per s. These reductions in polymerization rates were proportional to the decrease in cellular growth rate produced by polyamine starvation. It was concluded that, although it is quite unlikely that polyamine levels are involved in regulation of cell growth, they may be acting as cofactors in the synthesis of RNA or protein, or both.

Drug Stability↗
PubMed1975

Two types of glucose effects on beta-galactosidase synthesis in a membrane fraction of Escherichia coli: correlation with repression observed in intact cells.

A membrane fraction obtained from an osmotic lysate of Escherichia coli spheroplasts retains capability to synthesize beta-galactosidase. The system also retains cellular regulatory functions, one of which is known as catabolite repression. Two types of repression of beta-galactosidase synthesis were observed in this membrane system: one was caused by the addition of 2-deoxyglucose or glucose at a low concentration (3 times 10- minus 4 M), and the other was caused by glucose-6-phosphate or glucose at a high concentration (3 times 10- minus 2 M). In the presence of cyclic adenosine 3',5'-monophosphate (10 mM), repression caused by the former was completely reversed, whereas repression by the latter was only partially reversed. Conditions in intact cells causing transient and permanent repression were also investigated. Upon addition of 2-deoxyglucose or glucose at a low concentration to intact cells, only transient repression of beta-galactosidase synthesis was observed. Glucose at a high concentration caused both transient and subsequent permanent repression, and intensity of permanent repression depended upon glucose concentration, whereas duration and intensity of transient repression were independent of glucose concentration. Mutants deficient in phosphoenolpyruvate-phosphotransferase system (Hpr minus and enzyme I minus) showed transient repression but failed to show permanent repression. In mutants deficient in glucose catabolism beyond glucose-6-phosphate, both transient and permanent repression were observed. Correlation between the observations in the membrane system and in intact cells is discussed. The results obtained here strongly suggest that transient repression is caused by glucose itself, and that permanent repression is caused by glucose-6-phosphate of high intracellular levels of glucose.

Cell Fractionation↗
PubMed1977

Induction and general properties of beta-galactosidase and beta-galactoside permease in Pseudomonas BAL-31.

The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside (ONPG) by BAL-31, a marine Pseudomonas that acts as a host for bacteriophage PM2, was studied with intact cells and with cell-free extracts. A transport system for ONPG in whole cells and a beta-galactosidase activity in extracts were evident for cells grown on lactose minimal medium. It was found that the addition of isopropylthio-beta-D-galactopyranoside (IPTG) to cells growing in rich medium induced an ONPG hydrolytic activity detectable in cell extracts but cryptic in whole cells. The existence of a transport system for IPTG, which remained cryptic for ONPG, became apparent from studies of the rates of induction of beta-galactosidase as a function of cell mass at different concentrations of IPTG. The main properties of beta-galactosidase and the lactose transport system of BAL-31 were studied in terms of how they were affected by pH, temperature, or by the presence of several sugars. IPTG competitively inhibits the hydrolysis of ONPG by cell extracts. In cells pregrown on lactose, IPTG slightly inhibits the transport of ONPG. Glucose, and with less efficiency lactose, also inhibits the hydrolysis of ONPG in cell extracts. The growth of cells on lactose minimal medium was inhibited by the addition of IPTG. A mechanism for this inhibition and for the inhibition of ONPG transport by IPTG is discussed.

Carbohydrate Metabolism↗
PubMed1992

Expression of the thermostable beta-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisiae and characterization of a new inducible promoter for heterologous expression.

The lacS gene from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus encodes an enzyme with beta-galactosidase activity that, like other enzymes from this organism, is exceptionally thermophilic (optimal activity above 90 degrees C), thermostable, and resistant to common protein denaturants and proteases. Expression of the gene in mesophilic hosts is needed to uncover the molecular nature of these features. We have obtained expression of beta-galactosidase in Saccharomyces cerevisiae under the control of the galactose-inducible upstream activating sequence of the yeast genes GAL1 and GAL10. The expressed enzyme is identical in molecular mass, thermostability, and thermophilicity to the native enzyme, showing that these features are intrinsic to the primary structure of the enzyme. We also present a new promoter for the expression of thermostable proteins in S. cerevisiae. This promoter contains a sequence isolated from the nematode Caenorhabditis elegans that works as a strong, heat-inducible upstream activating sequence in S. cerevisiae. Transcription of the lacS gene under the control of this sequence is rapidly and efficiently induced by heat shock. The availability of a plate assay for monitoring beta-galactosidase activity in S. cerevisiae may allow screening for mutants affecting the efficiency and activity of the enzyme.

Animals↗
PubMed1998

Cloning, expression, and catabolite repression of a gene encoding beta-galactosidase of Bacillus megaterium ATCC 14581.

A gene encoding beta-galactosidase, designated mbgA, was isolated from Bacillus megaterium ATCC 14581. Chromosomal beta-galactosidase production could be dramatically induced by lactose but not by isopropyl-beta-D-thiogalactopyranoside (IPTG) and was subject to catabolite repression by glucose. Disruption of mbgA in the B. megaterium chromosome resulted in loss of lactose-inducible beta-galactosidase production. A 27-bp inverted repeat was found to overlap the mbgA promoter sequence. Two partially overlapping catabolite-responsive elements (CREs) were identified within the inverted repeat. Base substitutions within CRE-I and/or CRE-II caused partial relief from catabolite repression. The results suggest that the 27-bp inverted repeat may serve as a target for a catabolite repressor(s).

Bacillus megaterium↗
PubMed2000

The Streptococcus pneumoniae beta-galactosidase is a surface protein.

The beta-galactosidase gene of Streptococcus pneumoniae, bgaA, encodes a putative 2,235-amino-acid protein with the two amino acid motifs characteristic of the glycosyl hydrolase family of proteins. In addition, an N-terminal signal sequence and a C-terminal LPXTG motif typical of surface-associated proteins of gram-positive bacteria are present. Trypsin treatment of cells resulted in solubilization of the enzyme, documenting that it is associated with the cell envelope. In order to obtain defined mutants suitable for lacZ reporter experiments, the bgaA gene was disrupted, resulting in a complete absence of endogenous beta-galactosidase activity. The results are consistent with beta-galactosidase being a surface protein that seems not to be involved in lactose metabolism but that may play a role during pathogenesis.

Amino Acid Sequence↗
PubMed2002

alpha-Galactosidase Aga27A, an enzymatic component of the Clostridium josui cellulosome.

The Clostridium josui aga27A gene encodes the cellulosomal alpha-galactosidase Aga27A, which comprises a catalytic domain of family 27 of glycoside hydrolases and a dockerin domain responsible for cellulosome assembly. The catalytic domain is highly homologous to those of various alpha-galactosidases of family 27 of glycoside hydrolases from eukaryotic organisms, especially plants. The recombinant Aga27A alpha-galactosidase devoid of the dockerin domain preferred highly polymeric galactomannan as a substrate to small saccharides such as melibiose and raffinose.

Amino Acid Sequence↗
PubMed1966

Transfer and incorporation of genes controlling beta-D-galactosidase synthesis from Hfr and F' donors of Escherichia coli.

Ganesan, Ann K. (Syntex Institute of Molecular Biology, Palo Alto, Calif.), and Boris Rotman. Transfer and incorporation of genes controlling beta-d-galactosidase synthesis from Hfr and F' donors of Escherichia coli. J. Bacteriol. 92:1378-1382. 1966.-Comparisons were made between Hfr(1) and F(13) donors with respect to the frequency of transfer and incorporation of genes controlling beta-d-galactosidase synthesis. The Hfr(1) donor transfers these genes as part of the chromosome, and the F(13) donor transfers them by F-duction. The criterion used for gene transfer was the acquisition by recipient cells of the ability to synthesize the enzyme, beta-d-galactosidase, measured by fluorogenic assays at the single-cell level. The criterion for incorporation was the formation of lac(+) recombinant colonies. It was found that the two types of donor showed the same frequency of gene transfer, but the probability of incorporation was 10-fold higher in F(13) matings than in Hfr(1) matings. In the former, between 46 and 97% of the merozygotes produced recombinant colonies; in the latter, 2 to 6% did so.

DNA, Bacterial↗
PubMed1967

Purification and properties of Streptococcus lactis beta-galactosidase.

beta-Galactosidase of Streptococcus lactis 7962 was partially purified, and its properties were studied. Enzyme from only this strain of numerous lactic streptococci tested was stable in cell exudates prepared by various means. Cell-free extracts of the 7962 strain were prepared by sonic treatment of washed cells previously grown in presence of lactose to fully induce enzyme synthesis. Protamine sulfate precipitation of the nucleic acids and ammonium sulfate precipitation of protein were used for partial purification of the enzyme. The resulting enzyme, when resuspended in cold (5 C) phosphate buffer, was extremely labile. However, ammonium sulfate in high concentrations (0.85 m) stabilized and stimulated beta-galactosidase activity. Sephadex G-200 gel filtration was used to achieve further purification and to monitor homogeneity of the enzyme. Separation of the beta-galactosidase in buffer at 5 C yielded an enzyme elution pattern showing two peaks of activity. However, addition of the enzyme solution in 0.85 m ammonium sulfate to the column equilibrated with the same salt concentration yielded only one peak of enzyme activity. The data suggested that the native enzyme was dissociating into active subunits which were stabilized in the presence of the ammonium sulfate.

Chromatography, Gel↗
PubMed1967

Comparative inductive responses of two beta-galactosidases of Neurospora.

The activities of the two beta-galactosidases of Neurospora increased in response to the presence of lactose or galactose. d-Xylose preferentially induced higher levels of activity in one of these enzymes. The initial period of the inductive response was examined in cultures transferred from noninducing to inducing media. Variations in the ratio of activities of these enzymes during this induction period indicate that their induction was not coordinate. The induction of the beta-galactosidases of Neurospora was less sensitive to low concentrations of inducers than was the induction of the beta-galactosidase of bacteria; the time lag of induction was greater in the eucaryotic organism, and the maximal induction of active enzyme was less than that observed in bacteria.

Enzyme Induction↗
PubMed1967

Lysis of Escherichia coli by ethylenediaminetetraacetate and phospholipases as measured by beta-galactosidase activity.

A permeaseless mutant of Escherichia coli, which produces beta-galactosidase constitutively, was treated briefly with ethylenediaminetetraacetate and then with the phospholipases of Bacillus cereus. Cell lysis occurred, as indicated by an increase in beta-galactosidase activity and a decrease in absorbancy of the cell suspension. The susceptibility of the cells to attack by ethylenediaminetetraacetate and the phospholipases was markedly affected by the age of the cells when harvested. The results suggest that permeability changes may be associated with the activity of a phospholipase that specifically degrades phosphatidyl ethanolamine. A sonic-treatment method for determining the total beta-galactosidase content of E. coli cells, which is independent of their age when harvested, is described.

Bacillus cereus↗
PubMed1967

An upper limit on beta-galactosidase transfer in bacterial conjugation.

An upper limit for beta-galactosidase transfer between mating F(+) and F(-)Escherichia coli has been determined by a new technique which relies on selective lysis of the donor strain by heat induction of a thermo-inducible strain of lambda, accompanied by chymotryptic digestion of the released beta-galactosidase. No significant transfer of beta-galactosidase during mating between F(+) and F(-) cells has been observed: 0.05 +/- 0.05% of the enzyme originally present in the male cells is found in the female cells after 1 hr of mating at 37 C.

Bacteriolysis↗
PubMed1968

Analysis of melibiose mutants deficient in alpha-galactosidase and thiomethylgalactoside permease II in Escherichia coli K-12.

Three types of mutants (mel(-)) unable to metabolize the alpha-d-galactoside, melibiose, were derived from Escherichia coli K-12. One type lacked alpha-galactosidase; another lacked a specific transport system, termed thiomethylgalactoside (TMG) permease II; and the third lacked both of these functions. The mutational sites were genetically mapped by recombination frequency with different markers and by determination of chromosomal transfer in interrupted-mating experiments. All three mel(-) mutant types mapped in a cluster near to the metA marker on the E. coli chromosome and were cotransducible. Induction studies revealed that the three alpha-d-galactosides, melibiose, melibiitol, and galactinol, induced alpha-galactosidase and TMG permease II coordinately; d-galactose also induced them but only in a galactokinaseless mutant. These data suggest that alpha-galactosidase and TMG permease II may be components of a common operon.

Conjugation, Genetic↗
PubMed1969

Effect of galactose on beta-galactosidase synthesis in Escherichia coli K-12.

In wild-type strains of Escherichia coli K-12, the rate of thiomethylgalactoside (TMG)-induced beta-galactosidase synthesis is decreased in the presence of galactose or glucose. A spontaneous mutant of a K-12 strain, 58-161, which synthesizes beta-galactosidase at a low rate was isolated. In this mutant, galactose, after a lag of about one generation time, evoked the same final differential rate of enzyme synthesis as did the gratuitous inducer TMG. However, constitutive, TMG-induced and galactose-induced synthesis in the mutant were subject to inhibition by exogenous glucose. It is concluded that repression of beta-galactosidase synthesis derived from glucose is distinct from the inhibition derived from galactose.

Enzyme Induction↗
PubMed1969

Role of lac genes in induction of beta-galactosidase synthesis by galactose.

Strain BL1003, a lacO mutant, synthesizes beta-galactosidase constitutively at a low rate. The enzyme is further inducible by d-galactose to the same differential rate as is seen in the presence of an optimal concentration of thiomethylgalactoside. lacY Mutants derived from strain BL1003 are not inducible by galactose, although they synthesize beta-galactosidase at the low constitutive rate characteristic of the parent. Galactose is a weak inducer of beta-galactosidase synthesis in wild-type Escherichia coli K-12, but it is more effective when the wild type has been preinduced with isopropyl-beta-d-thiogalactoside. Nevertheless, the rise in the differential rate of synthesis in response to galactose in a preinduced wild-type culture is much lower than in strain BL1003. Thus, two factors are involved in the induction of strain BL1003 by galactose: the mutant operator and the constitutive permease. The operator has an altered sensitivity to the i product-galactose complex. The low constitutive level of permease enabled the cells, at the high concentrations of galactose used (5 x 10(-2)m), to maintain a sufficient internal concentration for further induction.

Carbon Isotopes↗
PubMedDate not supplied

Purification and characterization of rat epididymal neutral beta-galactosidase and its changes during in vivo development.

A neutral beta-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A--agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with alpha-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral beta-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral beta-galactosidase during sexual maturity of epididymis in vivo.

Animals↗