Characterization and Nucleotide Sequence of the Cryptic Cel Operon of Escherichia Coli K12

Wild-type Escherichia coli are not able to utilize beta-glucoside sugars because the genes for utilization of these sugars are cryptic. Spontaneous mutations in the cel operon allow its expression and enable the organism to ferment cellobiose, arbutin and salicin. In this report we describe the structure and nucleotide sequence of the cel operon. The cel operon consists of five genes: celA, whose function is unknown; celB and celC which encode phosphoenolpyruvate-dependent phosphotransferase system enzyme IIcel and enzyme IIIcel, respectively, for the transport and phosphorylation of beta-glucoside sugars; celD, which encodes a negative regulatory protein; and celF, which encodes a phospho-beta-glucosidase that acts on phosphorylated cellobiose, arbutin and salicin. The mutationally activated cel operon is induced in the presence of its substrates, and is repressed in their absence. A comparison of proteins encoded by the cel operon with functionally equivalent proteins of the bgl operon, another cryptic E. coli gene system responsible for the catabolism of beta-glucoside sugars, revealed no significant homology between these two systems despite common functional characteristics. The celD and celF encoded repressor and phospho-beta-glucosidase proteins are homologous to the melibiose regulatory protein and to the melA encoded alpha-galactosidase of E. coli, respectively. Furthermore, the celC encoded PEP-dependent phosphotransferase system enzyme IIIcel is strikingly homologous to an enzyme IIIlac of the Gram-positive organism Staphylococcus aureus. We conclude that the genes for these two enzyme IIIs diverged much more recently than did their hosts, indicating that E. coli and S. aureus have undergone relatively recent exchange of chromosomal genes.     

Inducible system for the utilization of beta-glucosides in Escherichia coli. II. Description of mutant types and genetic analysis

Two types of mutants obtained by treating beta-gl(+) cells with nitrosoguanidine are described. One type, beta-gl(+)c, is constitutive for the biosynthesis of the aryl beta-glucoside splitting enzyme(s) and for the beta-glucoside permease; the other (beta-gl(+)sal(-)) has lost the capacity to ferment salicin, but has retained the capacity to ferment arbutin and other aryl beta-glucosides. By two successive mutational steps, beta-gl(+)sal(-)c double mutants can be obtained. Determinations of the enzymatic splitting of salicin and p-nitrophenyl beta-glucoside by beta-gl(+)sal(-) cells and extracts showed that these mutants have lost the capacity to split salicin but do split p-nitrophenyl beta-glucoside; they possess the beta-glucoside permease, and in them salicin is a gratuitous inducer for enzyme and permease biosynthesis. Studies on a beta-gl(+) strain, which splits salicin as well as p-nitrophenyl beta-glucoside, have shown that the splitting of salicin is more temperature-sensitive than that of p-nitrophenyl beta-glucoside and other beta-glucosides. Other properties of the two activities are similar. Interrupted mating experiments and cotransduction with P1kc phage showed that the genetic determinants of the beta-glucoside system map between the pyrE and ile loci. Three distinct mutational sites were found and are presumed to have the following functions: beta-glA, a structural gene for an aryl beta-glucoside splitting enzyme; beta-glB, either the structural gene for the beta-glucoside-permease or a regulatory gene; and beta-glC, a regulatory gene (or site). Escherichia coli wild-type strains are of the genotype A(+) B(-) C(+). The beta-gl(+) mutation determining the ability to ferment beta-glucosides is considered to be a permease or regulatory mutation, and the resulting genotype is A(+) B(+) C(+). The beta-gl(+)sal(-) phenotype results from a mutation in the beta-glA gene (genotype A' B(+) C(+)), and the constitutive phenotype results from a mutation in the beta-glC gene, the genotypes A(+) B(+)C(a) and A' B(+)C(a) corresponding to the phenotypes beta-gl(+)c and beta-gl(+)sal(-)c.     

Inducible System for the Utilization of β-Glucosides in Escherichia coli I. Active Transport and Utilization of β-Glucosides

Wild-type Escherichia coli strains (beta-gl(-)) do not split beta-glucosides, but inducible mutants (beta-gl(+)) can be isolated which do so. This inducible system consists of a beta-glucoside permease and an aryl beta-glucoside splitting enzyme. Both can be induced by aryl and alkyl beta-glucosides. In beta-gl(-) and noninduced beta-gl(+) cells, C(14)-labeled thioethyl beta-glucoside (TEG) is taken up by a constitutive permease, apparently identical with a glucose permease (GP). This permease has a high affinity for alpha-methyl glucoside and a low affinity for aryl beta-glucosides. No accumulation of TEG occurs in a beta-gl(-) strain lacking glucose permease (GP(-)). In induced beta-gl(+) strains, there appears a second beta-glucoside permease with low affinity for alpha-methyl glucoside and high affinity for aryl beta-glucosides. Autoradiography shows that TEG is accumulated by the beta-glucoside permease and glucose permease in two different forms (one being identical with TEG, the other probably phosphorylated TEG). In GP(+) beta-gl(+) strains with high GP activity, alkyl beta-glucosides induce the enzyme and the beta-glucoside permease after a prolonged induction lag, and they competitively inhibit the induction by aryl beta-glucosides. The induction lag and competition do not exist in GP(-) beta-gl(+) strains. It is assumed that phosphorylated alkyl and thioalkyl beta-glucosides inhibit the induction, and that this inhibition is responsible for the induction lag.     

Directed evolution of cellobiose utilization in Escherichia coli K12

The cellobiose catabolic system of Escherichia coli K12 is being used to study the role of cryptic genes in evolution of new functions. Escherichia coli does not use beta-glucoside sugars; however, mutations in several loci can activate the cryptic bgl operon and permit growth on the beta-glucoside sugars arbutin and salicin. Such Bgl+ mutants do not use cellobiose, which is the most common beta-glucoside in nature. We have isolated a Cel+ (cellobiose-utilizing) mutant from a Bgl+ mutant of E. coli K12. The Cel+ mutant grows well on cellobiose, arbutin, and salicin. Genes for utilization of these beta-glucosides are located at 37.8 min on the E. coli map. The genes of the bgl operon are not involved in cellobiose utilization. Introduction of a deletion covering bgl does not affect the ability to utilize cellobiose, arbutin, or salicin, indicating that the new Cel+ genes provide all three functions. Spontaneous cellobiose negative mutants also become arbutin and salicin negative. Analysis of beta-glucoside positive revertants of these mutants indicates that there are separate loci for utilization of each of the beta-glucoside sugars. The genes are closely linked and may be activated from a single locus. A fourth gene at an unknown location increases the growth rate on cellobiose. The cel genes constitute a second cryptic system for beta-glucoside utilization in E. coli K12.      

Involvement of Streptococcus gordonii beta-glucoside metabolism systems in adhesion, biofilm formation, and in vivo gene expression

Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides. Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci. Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.     

Biotransformation of isoeugenol and eugenol by cultured cells of Eucalyptus perriniana.

Three new biotransformation products, eugenyl beta-rutinoside, and isoeugenyl beta-gentiobioside and beta-rutinoside, together with eugenyl beta-glucoside and beta-gentiobioside, and isoeugenyl beta-glucoside, were isolated from jar fermentor culture of Eucalyptus perriniana following administration of eugenol and isoeugenol, respectively. This is the first report of rhamnosylation in a biotransformation catalysed by cultured cells of E. perriniana.     

Circular dichroism studies of the mitochondrial channel, VDAC, from Neurospora crassa.

The protein that forms the voltage-gated channel VDAC (or mitochondrial porin) has been purified from Neurospora crassa. At room temperature and pH 7, the circular dichoism (CD) spectrum of VDAC suspended in octyl beta-glucoside is similar to those of bacterial porins, consistent with a high beta-sheet content. When VDAC is reconstituted into phospholipid liposomes at pH 7, a similar CD spectrum is obtained and the liposomes are rendered permeable to sucrose. Heating VDAC in octyl beta-glucoside or in liposomes results in thermal denaturation. The CD spectrum irreversibly changes to one consistent with total loss of beta-sheet content, and VDAC-containing liposomes irreversibly lose sucrose permeability. When VDAC is suspended at room temperature in octyl beta-glucoside at pH < 5 or in sodium dodecyl sulfate at pH 7, its CD spectrum is consistent with partial loss of beta-sheet content. The sucrose permeability of VDAC-containing liposomes is decreased at low pH and restored at pH 7. Similarly, the pH-dependent changes in the CD spectrum of VDAC suspended in octyl beta-glucoside also are reversible. These results suggest that VDAC undergoes a reversible conformational change at low pH involving reduced beta-sheet content and loss of pore-forming activity.     

The Biosynthesis of Ethyl-β-glucoside in Extracts of Pea Seedlings

An enzyme, which lacks cellobiase activity, responsible for the synthesis of ethyl-beta-glucoside has been found in the extracts of pea hooks (1-centimeter length of the apical portion of epicotyl) and has been partially purified by ammonium sulfate fractionation. The enzyme can transfer the glucosyl moiety from a group of phenolic beta-glucosides to ethanol. A specific beta-glucosyl donor, isosuccinimide beta-glucoside, isolated from the extracts of pea seedlings shows the highest activity. The characteristics of the enzyme which synthesizes ethyl-beta-glucoside and the glucosyl donor, isosuccinimide beta-glucoside, have been studied. The significance of this system (enzyme and isosuccinimide beta-glucoside) has been discussed.     

A new method of synthesis of alkyl beta-glycosides using sucrose as sugar donor

Cellobiose phosphorylase from Clostridium thermocellum catalyzed the beta-anomer-selective synthesis of alkyl glucosides from cellobiose. Synthesis of alkyl beta-glucoside from inexpensive sucrose using cellobiose phosphorylase and sucrose phosphorylase from Pseudomonas saccharophilia was investigated. By combined use of these two phosphorylases, alkyl beta-glucoside was anomer-selectively synthesized from sucrose and alkyl alcohol.     

Cryptic Genes for Cellobiose Utilization in Natural Isolates of Escherichia coli

The ECOR collection of natural Escherichia coli isolates was screened to determine the proportion of strains that carried functional, cryptic and nonfunctional genes for utilization of the three beta-glucoside sugars, arbutin, salicin and cellobiose. None of the 71 natural isolates utilized any of the beta-glucosides. Each strain was subjected to selection for utilization of each of the sugars. Only five of the isolates were incapable of yielding spontaneous beta-glucoside-utilizing mutants. Forty-five strains yielded cellobiose+ mutants, 62 yielded arbutin+ mutants, and 58 strains yielded salicin+ mutants. A subset of the mutants was screen by mRNA hybridization to determine whether they were expressing either the cel or the bgl beta-glucoside utilization operons of E. coli K12. Two cellobiose+ and two arbutin+-salicin+ strains failed to express either of these known operons. It is concluded that there are at least four gene clusters specifying beta-glucoside utilization functions in E. coli populations, and that all of these are normally cryptic. It is estimated that in any random isolate the probability of any particular cluster having been irreversibly inactivated by the accumulation of random mutations is about 0.5.     

Beta-glucoside kinase (BglK) from Klebsiella pneumoniae. Purification,properties, and preparative synthesis of 6-phospho-beta-D-glucosides

ATP-dependent beta-glucoside kinase (BglK) has been purified from cellobiose-grown cells of Klebsiella pneumoniae. In solution, the enzyme (EC ) exists as a homotetramer composed of non-covalently linked subunits of M(r) approximately 33,000. Determination of the first 28 residues from the N terminus of the protein allowed the identification and cloning of bglK from genomic DNA of K. pneumoniae. The open reading frame (ORF) of bglK encodes a 297-residue polypeptide of calculated M(r) 32,697. A motif of 7 amino acids (AFD(7)IG(9)GT) near the N terminus may comprise the ATP-binding site, and residue changes D7G and G9A yielded catalytically inactive proteins. BglK was progressively inactivated (t(12) approximately 19 min) by N-ethylmaleimide, but ATP afforded considerable protection against the inhibitor. By the presence of a centrally located signature sequence, BglK can be assigned to the ROK (Repressor, ORF, Kinase) family of proteins. Preparation of (His6)BglK by nickel-nitrilotriacetic acid-agarose chromatography provided high purity enzyme in quantity sufficient for the preparative synthesis (200-500 mg) of ten 6-phospho-beta-d-glucosides, including cellobiose-6'-P, gentiobiose-6'-P, cellobiitol-6-P, salicin-6-P, and arbutin-6-P. These (and other) derivatives are substrates for phospho-beta-glucosidase(s) belonging to Families 1 and 4 of the glycosylhydrolase superfamily. The structures, physicochemical properties, and phosphorylation site(s) of the 6-phospho-beta-d-glucosides have been determined by fast atom bombardment-negative ion spectrometry, thin-layer chromatography, and (1)H and (13)C NMR spectroscopy. The recently sequenced genomes of two Listeria species, L. monocytogenes EGD-e and L. innocua CLIP 11262, contain homologous genes (lmo2764 and lin2907, respectively) that encode a 294-residue polypeptide (M(r) approximately 32,200) that exhibits approximately 58% amino acid identity with BglK. The protein encoded by the two genes exhibits beta-glucoside kinase activity and cross-reacts with polyclonal antibody to (His6)BglK from K. pneumoniae. The location of lmo2764 and lin2907 within a beta-glucoside (cellobiose):phosphotransferase system operon, may presage both enzymatic (kinase) and regulatory functions for the BglK homolog in Listeria species.     

Biotransformation of thymol, carvacrol, and eugenol by cultured cells of Eucalyptus perriniana

The biotransformations of aroma compounds of spices, such as thymol (1), carvacrol (2), and eugenol (3), were investigated using cultured plant cells of Eucalyptus perriniana. Besides a beta-glucoside product (4, 3%), a biotransformation product, i.e., 5-methyl-2-(1-methylethyl)phenyl 6-O-(beta-d-glucopyranosyl)-beta-d-glucopyranoside (5, beta-gentiobioside, 87%), was isolated from the suspension cells after the five-day incubation of 1. On administration of 2, a beta-glucoside (6, 5%) and a beta-gentiobioside, i.e., 2-methyl-5-(1-methylethyl)phenyl 6-O-(beta-d-glucopyranosyl)-beta-d-glucopyranoside (7, 56%), were produced. Furthermore, E. perriniana cells also converted 3 into the corresponding beta-glucoside (8, 7%) and beta-gentiobioside (9, 58%). The cultured cells of E. perriniana are able to convert these aroma compounds of spices into glycosides which are accumulated in the cells.     

The beta-glucosidases of porcine kidney.

Some of the properties of a partially purified particle bound and soluble beta-glucosidase (EC 3.2.1.21) from pig kidney were compared. The soluble beta-glucosidase (1) hydrolyzed 4-methylumbelliferyl-beta-D-glucoside (4-MU-beta-D-glucoside) 17 alpha-estradiol 3beta-glucoside. 17 alpha-estradiol 17beta-glucoside, and salicin, but not glucosylceramide, (2) possessed a broad pH optimum (5.5-7.0), (3) had an isoelectric point of 4.9, and (4) was inhibited by Triton X-100. Several compounds were found to be competitive inhibitors of its hydrolytic activity, gluconolactam and estrone beta-glucoside being the most effective. In contrast, a particulate beta-glucodidase purified from the same tissue (1) had an acidic pH optimum (5.0), (2) was stimulated by sodium taurocholate and 'Gaucher's factor' for the hydrolysis of both 4-MU-beta-glucosidase and glucosylceramide, and (3) was capable of catalyzing a transglucosylation reaction employing 4-MU-beta-D-glucoside or glucosylceramide as the glucosyl donor, and [14C]ceramide as acceptor.     

Phospho-β-glucosidases and β-Glucoside Permeases in Streptococcus, Bacillus, and Staphylococcus

The presence of phospho-beta-glucosidases and beta-glucoside permeases was found in strains of Streptococcus, Bacillus, and Staphylococcus. In streptococci, the phospho-beta-glucosidase activity depends on the antigenic group. The highest activity was found in strains of group D. In group D strains, phospho-beta-glucosidase activity is induced by beta-methyl glucoside and cellobiose but not by thiophenyl beta-glucoside (TPG). With the exception of four strains isolated in Japan, all strains of B. subtilis tested possess an inducible phospho-beta-glucosidase activity, beta-methyl glucoside, cellobiose, and TPG acting as inducers. S. aureus strains possess phospho-beta-glucosidase A but not phospho-beta-glucosidase B, whereas most S. albus strains show no detectable phospho-beta-glucosidase activity. The prompt fermentation of beta-methyl glucoside by S. aureus strains could serve as an additional criterion for their differentiation from S. albus. A comparative investigation of the active uptake of (14)C-TPG showed that a Streptococcus group D strain and a B. subtilis strain posses two inducible permeases with characteristics similar to the beta-glucoside permeases I and II of Enterobacteriaceae. In S. aureus, TPG is accumulated by a constitutive permease with high affinity for aromatic beta-glucosides and glucose. The active uptake of TPG by S. aureus appears to depend on the activity of the phosphoenol pyruvate-dependent phosphotransferase system.     

Study of a corn (Zea mays L.) mutant (blue fluorescent-1) which accumulates anthranilic acid and its β-glucoside

A corn (Zea mays L.) mutant, blue fluorescent-1 (bf), is described that shows ultraviolet light induced blue fluorescence in young seedling leaves if homozygous for the mutant gene, and in anthers if either homozygous or heterozygous. The blue fluorescent compounds were extracted with acetone and separated by paper chromatography. Anthranilic acid was present and the beta-glucoside was also identified by paper chromatography and beta-glucosdase and acid treatment. A third major fluorescent compound was not identified, but it was convertible to anthranilic acid by acid treatment. Anthranilate synthetase from mutant plants was 3-40 times more active and was also more resistant to feedback inhibition by tryptophan than was the enzyme from normal plants. The high activity and feedback resistance would both lead to anthranilate accumulation. Anthranilate-phosphoribosylpyrophosphate phosphoribosyltransferase (PR transferase), the enzyme which usually utilizes anthranilate in the tryptophan pathway, was inhibited by the beta-glucoside of anthranilic acid in a noncompetitive manner and showed very little activity in the mutant plant extract. This inhibition of the enzyme which utilizes anthranilate would also lead to accumulation. Apparently the oversynthesis of anthranilate leads to the formation of the beta-glucoside, which inhibits anthranilate utilization. The fluorescent compounds are absent in seed, but form on germination. The levels decrease with age after 35 days postgermination, but are still present in leaves during grain filling.     

Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification

A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).