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PubMed1991

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli.

Amino Acid Sequence↗

PubMed2002

Thermoadaptation of alpha-galactosidase AgaB1 in Thermus thermophilus.

The evolutionary potential of a thermostable alpha-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria. For this purpose, hybrid alpha-galactosidase genes of agaA and agaB from Bacillus stearothermophilus KVE39, designated agaA1 and agaB1, were cloned into an autonomously replicating Thermus vector and introduced into Thermus thermophilus OF1053GD (DeltaagaT) by transformation. This selector strain is unable to metabolize melibiose (alpha-galactoside) without recombinant alpha-galactosidases, because the native alpha-galactosidase gene, agaT, has been deleted. Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded agaA1 gene but unable when harboring a plasmid-encoded agaB1 gene. With incubation of the agaB1 plasmid-harboring strain under selective pressure at a restrictive temperature (67 degrees C) in a minimal melibiose medium, spontaneous mutants as well as N-methyl-N'-nitro-N-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated. The mutant alpha-galactosidase genes were amplified by PCR, cloned in Escherichia coli, and sequenced. A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant agaB1 genes. The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability. To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell.

Adaptation, Physiological↗

PubMed1966

Purification and characterization of the beta-galactosidase of Aeromonas formicans.

Rohlfing, S. R. (Western Reserve University, Cleveland, Ohio), and I. P. Crawford. Purification and characterization of the beta-galactosidase of Aeromonas formicans. J. Bacteriol. 91:1085-1097. 1966.-The beta-galactosidase of Aeromonas formicans was purified by diethylaminoethyl cellulose chromatography and gel filtration on Sephadex G-200. The properties of the enzyme molecule were compared with purified beta-galactosidase from Escherichia coli. The sedimentation coefficients and electrophoretic mobilities of the two enzymes were not significantly different; the electrophoretic mobility of urea-produced subunits of the two enzymes was also similar. The stabilities of the two enzymes to denaturing agents provided measurable differences; E. coli beta-galactosidase is relatively more heat-stable and more resistant to the action of urea. The amino acid compositions of the two proteins revealed significant differences in several amino acids, particularly alanine, arginine, glycine, and leucine. The comparisons cited suggest that A. formicans and E. coli are not completely unrelated, for their beta-galactosidases show considerable structural similarity.

Aeromonas↗

PubMed1966

Nature of the effector of catabolite repression of beta-galactosidase in Escherichia coli.

Loomis, William F., Jr. (Massachusetts Institute of Technology, Cambridge, Mass.), and Boris Magasanik. Nature of the effector of catabolite repression of beta-galactosidase in Escherichia coli. J. Bacteriol. 92:170-177. 1966.-Many carbon sources were found to give rise to catabolite repression of beta-galactosidase in a mutant strain of Escherichia coli lacking hexose phosphate isomerase activity. Compounds containing glucose or galactose cannot be formed from several of these carbon sources in this mutant strain, and, therefore, appear not to be required for catabolite repression of beta-galactosidase. Glucose was observed to elicit catabolite repression of beta-galactosidase in another mutant strain under conditions in which the formation of compounds of the citric acid cycle is inhibited. If catabolite repression of the lac operon is mediated by a single compound, it appears that the compound is related to the pentoses and trioses of intermediary metabolism. The repression of beta-galactosidase by galactose in galactokinase negative strains was shown to be independent of the gene, CR, which determines catabolite sensitivity of the lac operon, and to be dependent on a functional i gene.

Carbohydrates↗

PubMed1981

Transcriptional regulation of the Kluyveromyces lactis beta-galactosidase gene.

We examined the molecular basis for beta-D-galactosidase (EC 3.2.1.23) induction in the yeast Kluyveromyces lactis. The protein synthesis inhibitor anisomycin effectively blocked both protein synthesis and enzyme induction by lactose. Further, hybridization analysis with the cloned beta-galactosidase gene indicated coordinate increases in the concentration of beta-galactosidase messenger ribonucleic acid and enzyme activity. The half-life of beta-galactosidase messenger ribonucleic acid was the same (4.8 +/- 0.4 min) when measured both before and at succeeding times during enzyme induction. These results strongly support the hypothesis that expression of the yeast beta-galactosidase gene is subject to transcriptional regulation.

Anisomycin↗

PubMed1976

Evidence against the involvement of adenosine 3',5'-cyclic monophosphate in glucose inhibition of beta-galactosidase induction in Bacillus megaterium.

When Bacillus megaterium cells are grown on D-galactose as the sole carbon source, the cells actively synthesize beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23). However, D-galactose, when added to a glucose-grown culture, did not induce beta-galactosidase, apparently because of the glucose inhibition of the transport of galactose. On the other hand, when glucose was added to a galactose-grown culture, the transport of galactose continued at a reduced but significate rate, whereas further synthesis of beta-galactosidase was halted. Adenosine 3',5'-cyclic monophosphate (camp) or guanosine 3',5'-cyclic monophosphate (Cgmp) did not relieve the glucose inhibition of beta-galactosidase synthesis in the preinduced culture. A method which gave a reproducible assay of c[32P]AMP in Escherichia coli did not detect cAMP or cGMP in a B. megaterium culture undergoing beta-galactosidase induction, but revealed the extracellular accumulation of two unknown phosphorylated compounds. Cell-free extracts prepared from galactose-grown cells did not catalyze the degradation of cAMP or cGMP.

Bacillus megaterium↗

PubMed2003

Molecular genetic analyses of potential beta-galactosidase genes in Xanthomonas campestris.

Xanthomonas campestris pv. campestris, which displays no significant beta-1,4-D-galactopyranosidase activity, has three annotated beta-galactosidase genes in the sequenced genome, designated galA, galB and galC herein. GalA and GalB are similar to glycosyl hydrolase (GH) family 2 enzymes, including Escherichia coli LacZ. galA and galB cannot express detectable activity even after being cloned in-frame and driven by the vector's promoter. GalC is a GH35 enzyme homologous to the Xanthomonas axonopodis pv. manihotis Bga. The latter cleaves beta,1-3-linked galactose 1,000 times faster than beta,1-4-linked galactose and is not responsible for lactose utilization. In X. campestris pv. campestris cells, GalC is readily detectable by Western blotting, and the levels can be increased by cloning the gene under the control of the vector's promoter. Results of insertional mutation, transcriptional fusion assay and Western blotting indicated that galC, clustered with several GH genes, is cotranscribed with the upstream gene(s) and is expressed constitutively. Xc17L is a previously isolated mutant with elevated beta-galactosidase activity and a greatly improved ability to grow on lactose. Results of DNA sequencing of Xc17L galA, galB and galC, enzyme assays of galA, galB and galC mutants derived from Xc17L, and Western blotting of GalC in Xc17L indicated that the three beta-galactosidase genes do not encode the elevated beta-galactosidase activity in Xc17L. The presence of a fourth beta-galactosidase gene is proposed.

Amino Acid Motifs↗

PubMed2000

Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.

Animals↗

PubMed1992

Galactosylation at side chains of branched cyclodextrins by various beta-galactosidases.

The galactosyl transfer reaction to branched cyclodextrins (CDs) was investigated using lactose as a donor substrate and branched CDs as acceptors by various beta-galactosidases. Bacillus circulans beta-galactosidase synthesized galactosyl transfer products to branched CDs, of which the galactose residues were linked at side chains of branched CDs, not directly at CD rings. Aspergillus oryzae and Penicillium multicolor beta-galactosidases also produced derivatives galactosylated at side chains of branched CDs. The structures of main transgalactosylation products of branched CDs by these beta-galactosidases seem to be different from those by B. circulans beta-galactosidase, judging from the retention times on high performance liquid chromatography.

Bacillus↗

PubMed1997

Purification, characterization, and cDNA cloning of a novel alpha-galactosidase from Mortierella vinacea.

A novel alpha-galactosidase, designated alpha-galactosidase II, was isolated from the culture filtrate of Mortierella vinacea. The molecular size of the purified enzyme estimated by gel filtration was 60 kDa, which agreed with that, 51-62 kDa, estimated by SDS-PAGE. The enzyme was thermolabile at neutral pH, but the addition of BSA to the enzyme solution at the concentration of 0.01% increased its stability considerably. The enzyme appears to be novel because it showed a distinct substrate specificity from other microbial alpha-galactosidases on galactomanno-oligosaccharides, prepared from galactomannan, that is, the enzyme liberated not only side-chain alpha-galactosyl residue from 6(3)-mono-alpha-D-galactopyranosyl-beta-1,4-D-mannotetraose but also terminal alpha-galactosyl residue from 6(3)-mono-alpha-D-galactopyranosyl-beta-1,4-D-mannotriose. In addition, the enzyme acted on galactomannans effectively. alpha-Galactosidase II cDNA was cloned and its nucleotides sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 376 amino acid residues with a molecular mass of 41,334 Da. The derived amino acid sequence of the enzyme showed 31-49% sequence similarity with those of alpha-galactosidases from other origins.

Amino Acid Sequence↗

PubMed2004

Regioselectivity in beta-galactosidase-catalyzed transglycosylation for the enzymatic assembly of D-galactosyl-D-mannose.

The regioselectivity of beta-galactosidase derived from Bacillus circulans ATCC 31382 (beta-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for beta-1,3-galactosidase but also for beta-galactosidases of different origins. The relative hydrolysis rate of Gal beta-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of beta-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of beta-1,3-, beta-1,4- and beta-1,6-linked D-galactosyl-D-mannose was subsequently established.

Bacillus↗

PubMed2001

Humoral immune responses of black-footed penguins (Spheniscus demersus) after DNA-mediated immunization with a beta-galactosidase reporter gene.

Humoral immune responses of black-footed penguins (Spheniscus demersus) to DNA-mediated immunization with a beta-galactosidase reporter gene expression plasmid were evaluated. Six male and 6 female adult penguins received either test plasmid, pCMV-beta, containing the beta-galactosidase gene or control plasmid, pCI, lacking a gene for expression. Three birds from each group were used previously in a diluent control group and given one injection of sterile saline. All samples were screened for anti-beta-galactosidase antibodies by indirect enzyme-linked immunosorbent assay with anti-chicken immunoglobulin G as secondary antibody. Antibodies to beta-galactosidase were detected in the sera of pCMV-beta-inoculated penguins, with a peak response on day 21. Antibody titers of the test plasmid group versus both control groups on days 21, 28, and 42 differed significantly. These results demonstrate that black-footed penguins can be safely transfected with the gene encoding beta-galactosidase and will mount a humoral response against the in vivo-expressed protein. Knowledge from this initial study can be applied to the development of DNA-mediated vaccines against specific infectious diseases of penguins.

Animals↗

PubMed1996

Cloning, DNA sequence, and regulation of expression of a gene encoding beta-galactosidase from Lactococcus lactis.

The beta-galactosidase from Escherichia coli is one of the most important enzymes in molecular biology. Here we report the cloning and sequencing of a gene encoding beta-galactosidase from Lactococcus lactis and compare the predicted amino acid sequence to that from other organisms. The beta-galactosidase from L. lactis was found to be a protein of 996 residues with 68.7% similarity to the E. coli enzyme and 65.8% similarity to the enzyme from Klebsiella pneumoniae. The lactococcal beta-galactosidase has lower similarity (approx 55%) to the enzymes from other lactic acid bacteria and no significant similarity to the beta-galactosidase enzymes from Agrobacterium radiobacter, Bacillus stearothermophilus, or Clostridium thermosulfurogenes. Expression of the lacZ gene from L. lactis was found to be higher when cells were grown in medium containing lactose than when grown in glucose, and expression was higher when cells were grown at 30 degrees C than at 35 degrees C.

Amino Acid Sequence↗

PubMed1995

Dendritic morphology of cardiac related medullary neurons defined by circuit-specific infection by a recombinant pseudorabies virus expressing beta-galactosidase.

The transneuronal herpesvirus tracer, pseudorabies virus (PRV) was used to determine the dendritic architecture of cardiac-related neurons. We constructed a derivative of the Bartha strain of PRV called PRV-BaBlu, that carries the lacZ gene of E. coli. Expression of beta-galactosidase by this recombinant virus enabled us to define the dendritic morphology of motoneurons and interneurons that innervate the heart. beta-galactosidase antigen filled dendritic processes that were clearly revealed by antibodies to beta-galactosidase. In contrast, the standard enzymatic reaction for detection of beta-galactosidase activity stained the cell soma well, but was inferior for labeling dendrites. Following PRV-BaBlu cardiac injection, infected neurons were clearly defined and labeled dendrites could be traced for long distances, sometimes greater than 800 microns from the cell body. Labeled dendrites of cardiomotor neurons primarily located in the nucleus ambiguus (NA) were extensive and sometimes intertwined with dendrites from other labeled motoneurons. Dendrites of labeled neurons in the dorsal motor nucleus of the vagus (DMV) typically extended in the mediolateral direction in the transverse plane. Transynaptically labeled interneurons interposed between the cardiorespiratory region of the nucleus tractus solitarius (NTS) and the NA were primarily located in the NA region and the reticular arc, the area between the DMV and NA. These interneurons had long dendrites extending along the reticular arc in the transverse plane. The dendritic arborizations of infected cardiac-related neurons in the NTS were variable in extent. We conclude that antibody detection of beta-galactosidase expressed by PRV-BaBlu after infection of neural cardiac circuits provides a superior method to define the dendrites and dendritic fields of cardiac-related motoneurons and interneurons.

Animals↗

PubMed1993

Influence of bile on cellular integrity and beta-galactosidase activity of Lactobacillus acidophilus.

The influence of bile on beta-galactosidase activity, cellular integrity, cellular retention of beta-galactosidase, and cellular permeability of five strains of Lactobacillus acidophilus was investigated. The five strains were also compared for bile tolerance. Two strains, 223 and 4356, were significantly less resistant to bile than the others (107, NCFM, and 606). beta-Galactosidase activity of all five strains was significantly higher in the presence of .3% oxgall than in its absence. Strain 107 showed the highest increase of enzyme activity in the presence of oxgall. Cells were not lysed in the presence of .3% oxgall, and beta-galactosidase was retained inside the cell even after extended incubation (60 min) in the presence of .3% oxgall. However, material that absorbed light at 260 nm leaked from the cells in the presence of oxgall. We concluded that, in the presence of bile, the permeability of cells of L. acidophilus increased, permitting more substrate to enter the cells, thus increasing the beta-galactosidase activity of whole cells.

Bile↗

PubMed1994

Influence of bile on beta-galactosidase activity of component species of yogurt starter cultures.

The influence of bile on beta-galactosidase activity and the cellular integrity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus were tested. Two strains (OS 1 and OS 2) of L. delbrueckii ssp. bulgaricus and three strains (HC 15, 17, and 18) of S. salivarius ssp. thermophilus were studied. In the presence of .15% oxgall, beta-galactosidase activity of whole cells of both species was significantly increased. However, at a higher concentration (.3% oxgall), the beta-galactosidase activity was significantly less than in the presence of .15% oxgall. The presence of oxgall did not promote the cell lysis of any of the strains of either species. Additionally, the presence of oxgall did not cause the leakage of the enzyme from the cells. Thus, the presence of oxgall increased the cellular permeability to allow more substrate to enter the cells, thereby increasing beta-galactosidase activity, and, at higher concentrations, was inhibitory to beta-galactosidase activity of both species.

Bile↗

PubMed1976

Inhibitory activity of pyridindolol on beta-galactosidase.

The activity of pyridindolol in inhibiting beta-galactosidases obtained from various sources has been studied. Whereas acid bovine liver beta-galactosidase (optimal pH 4.0) was not affected by this compound, neutral bovine liver beta-galactosidase (pH-optimum = 7.0) was inhibited by pyridindolol in reaction mixtures of pH 4.0 approximately 5.0. There was no inhibition at pH 7.0. The type of inhibition is non-competitive by formation of a pyridindolol-enzyme complex. Since beta-galactosidases from other sources are not affected by pyridindolol, the inhibitory action of this compound seems to be rather specific for neutral bovine liver beta-galactosidase.

Animals↗

PubMed1975

Inactivation of normal beta-D-galactosidase by antibodies to defective forms of the enzyme.

A counterpart of the antibody-mediated activation of genetically defective enzymes is reported here. Antibodies elicited by certain mutant forms of beta-D-galactosidase (EC 3.2.1.23) of Escherichia coli were found to inactivate the normal form of the enzyme. (Antibodies elicited by normal beta-D-galactosidase do not affect the enzyme's catalytic activity.) We present evidence that the inactivating antibodies are directed against one or a few determinants of the enzyme. The level of inactivation caused by the antibodies was independent of temperature below 25 degrees and increased with temperature above 25 degrees. The inactivation was proportional to the concentration of antiserum until a maximum level of 50% inactivation was reached. Antibodies capable of inactivating up to 87% of the activity were obtained after the antiserum was partially absorbed in an affinity column. This antibody preparation showed a 10-fold enrichment of inactivating antibodies over other antibodies direct against the enzyme. The antibody-mediated inactivation caused a reduction in the Vmax of beta-D-galactosidase without affecting the apparent Km of the enzyme. In contrast to antibodies to normal beta-D-galactosidase, inactivating antibodies changed the response of the enzyme to cations. To explain these results, we present a model in which there is a temperature-dependent equilibrium between two active forms of beta-D-galactosidase. Inactivation results from a conformational change induced by the binding of inactivating antibodies to only one of these two forms.

Antibodies↗