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.     

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.     

Analysis of the Erwinia chrysanthemi arb genes, which mediate metabolism of aromatic beta-glucosides.

Erwinia chrysanthemi is one of the few members of the family Enterobacteriaceae that is capable of metabolizing most of the naturally occurring beta-glucosides. We previously isolated the clb genes, which allow the use of the disaccharide cellobiose as well as the aromatic beta-glucosides arbutin and salicin. We report here the isolation of the arb genes, which permit fermentation of the aromatic beta-glucosides only. Establishment of a functional Arb system in Escherichia coli depended on the presence of the phosphotransferase system and on the activation by the cyclic AMP-cyclic AMP receptor protein complex. Strains carrying mini-Mu-induced LacZ fusions to the arb genes were used to analyze arb genes organization and function. Three arb genes (arbG, arbF, and arbB) were identified and organized in this order. Genetic and structural evidence allowed us to assign a phospho-beta-glucosidase and a permease activity to the ArbB and ArbF proteins, respectively. Several Lac+ arb-lacZ insertions were introduced into the E. chrysanthemi chromosome. Both ArbG- and ArbF- strains were unable to ferment the aromatic beta-glucosides, whereas ArbB- strains were impaired only in salicin fermentation. None of the mutations in the arb genes affected cellobiose metabolism. The expression of the arb genes was substrate inducible and required the ArbF permease and, possibly, the ArbG protein. Collectively, our results underline the resemblance between the naturally expressed E. chrysanthemi arbGFB and the cryptic E. coli bglGFB operons, yet the arbG gene product seemed unable to activate E. coli bgl operon expression.     

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.     

Cryptic Operon for β-Glucoside Metabolism in ESCHERICHIA COLI K12: Genetic Evidence for a Regulatory Protein

Escherichia coli K12 does not metabolize beta-glucosides such as arbutin and salicin because of lack of expression of the bglBSRC operon, which contains structural genes for transport (bglC) and hydrolysis (bglB) of phospho-beta-glucosides. Mutants carrying lesions in the cis-acting regulatory site bglR metabolize beta-glucosides as a consequence of expression of this cryptic operon (Prasad and Schaefler 1974). We isolated mutations promoting beta-glucoside metabolism that were unlinked to bglR; some of these mutations were shown to be amber. All of them were mapped at 27 min on the E. coli K12 linkage map and appeared to define a single gene, for which we propose the designation bglY. Utilization of beta-glucosides in bglY mutants appeared to be a consequence of expression of the bglBSRC operon, since bglB bglR and bglB bglY double mutants had the same phenotype. All bglY mutations analyzed were recessive to the wild-type bglY+ allele. Phospho-beta-glucosidase B and beta-glucoside transport activities are inducible in bglY mutants, as they are in bglR mutants. Metabolism of beta-glucosides in both bglR and bglY mutants required cyclic AMP. We propose that bglY encodes a protein acting as a repressor of the bglBSRC operon, active in both the presence and absence of beta-glucosides, whose recognition site would be within the bglR locus.     

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.     

The beta-glucoside genes of Klebsiella aerogenes: conservation and divergence in relation to the cryptic bgl genes of Escherichia coli

The ability to metabolize aromatic beta-glucosides such as salicin and arbutin varies among members of the Enterobacteriaceae. The ability of Escherichia coli to degrade salicin and arbutin appears to be cryptic, subject to activation of the bgl genes, whereas many members of the Klebsiella genus can metabolize these sugars. We have examined the genetic basis for beta-glucoside utilization in Klebsiella aerogenes. The Klebsiella equivalents of bglG, bglB and bglR have been cloned using the genome sequence database of Klebsiella pneumoniae. Nucleotide sequencing shows that the K. aerogenes bgl genes show substantial similarities to the E. coli counterparts. The K. aerogenes bgl genes in multiple copies can also complement E. coli mutants deficient in bglG encoding the antiterminator and bglB encoding the phospho-beta-glucosidase, suggesting that they are functional homologues. The regulatory region bglR of K. aerogenes shows a high degree of similarity of the sequences involved in BglG-mediated regulation. Interestingly, the regions corresponding to the negative elements present in the E. coli regulatory region show substantial divergence in K. aerogenes. The possible evolutionary implications of the results are discussed.     

Use of pyrrolidonyl peptidase to distinguish Citrobacter from Salmonella

In the routine testing of foods for Salmonella, Citrobacter and other members of the Enterobacteriaceae often produce colonies which are almost indistinguishable from Salmonella on commonly used selective agars. Biochemical confirmation of such colonies can be expensive and time-consuming. It has been suggested that the enzyme pyrrolidonyl peptidase (PYRase) could be used as a rapid test to distinguish Citrobacter colonies (PYRase-positive) from Salmonella (PYRase-negative). Pure cultures of Salmonella, Citrobacter and other Enterobacteriaceae were tested for PYRase activity; all strains of Salmonella tested were PYRase-negative, and all Citrobacter tested were PYRase-positive. Inoculated and naturally contaminated food samples were tested for the presence of Salmonella by a standard cultural method. A PYR test was used to test Salmonella-like colonies isolated on selective agar and potentially, eliminate PYR-positive isolates from further biochemical testing. The test was able to screen out 6% of colonies selected from samples inoculated with Salmonella, and 43% of colonies selected from uninoculated samples.     

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.      

Identification of catalytic residues in the beta-glucoside permease of Escherichia coli by site-specific mutagenesis and demonstration of interdomain cross-reactivity between the beta-glucoside and glucose systems

beta-Glucoside Enzyme II (IIBgl) of the Escherichia coli phosphotransferase system transports and phosphorylates beta-glucosides, whereas the glucose Enzyme II-III pair (IIGlc-IIIGlc) transports and phosphorylates glucose as well as certain aliphatic alpha- and beta-glucosides. Comparisons of their respective amino acid sequences previously revealed that both systems are homologous and must be evolutionarily related. To gain more insight into the details of the transport mechanism, we made use of the observed homologies among phosphotransferase system permeases to design a suitable set of site-specific mutants within the gene encoding IIBgl. This set was used to study in vivo fermentation and to analyze in vitro P-enolpyruvate-dependent sugar phosphorylation as well as sugar phosphate-dependent sugar transphosphorylation. The following results were obtained. (i) IIBgl transports and phosphorylates glucose as well as aryl- and alkyl-beta-glucosides; (ii) histidyl 547 is essential for the phosphorylation of IIBgl by the histidine-containing phosphoryl carrier protein of the phosphotransferase system (HPr) (first phosphorylation site); (iii) both cysteyl 24 and histidyl 306 are essential for the transfer of the phosphoryl group to the sugar; (iv) replacement of Cys-24 by serine leads to uncoupling of sugar transport from phosphorylation; and (v) histidyl 183 is important for substrate specificity. Our studies also revealed heterologous phosphoryl transfer between the beta-glucoside and glucose permease components which probably occurs as follows: 1) HPr-P----IIBgl (His-547)----IIGlc----alkyl-alpha- or -beta-glucosides or glucose (but not aryl-beta-glucosides) and 2) HPr-P----IIIGlc----IIBgl (Cys-24 or His-306)----alkyl- or aryl-beta-glucosides or glucose (but not methyl-alpha-glucoside). In addition to the essential residues noted above, several residues in IIBgl were identified which when mutated reduced the in vitro catalytic efficiency of the enzyme more than 10-fold. Thus, aspartyl 551 and arginyl 625 appeared to function together with histidyl 547 in phosphoryl transfer involving the first phosphorylation site in the permease, whereas histidyl 183 appeared to function together with cysteyl 24 and histidyl 306 in phosphoryl transfer involving the second phosphorylation site in the permease.     

Delayed lactose fermentation by enterobacteriaceae

Goodman, R. E. (University of California, Los Angeles), and M. J. Pickett. Delayed lactose fermentation by Enterobacteriaceae. J. Bacteriol. 92:318-327. 1966.-When 171 Citrobacter freundii strains and 14 Paracolobactrum arizonae strains examined, 51 of the C. freundii strains and 13 of the P. arizonae strains were found to be delayed or negative lactose fermenters. Of the slow fermenters, 65% yielded rapidly fermenting mutants in cultures undergoing delayed fermentation. Lactose fermentation could generally be hastened by increasing lactose concentrations. Many organisms which fermented lactose slowly grew readily on a medium containing lactose as the sole carbon source. Regardless of their ability to ferment lactose, all strains of C. freundii and P. arizonae investigated could be shown to possess beta-galactosidase. Delayed fermenters failed to take up lactose from the culture medium, whereas prompt fermenters did so readily. The beta-galactosidases of 12 strains of enteric bacteria were studied in crude cell extracts with respect to specific activity, stability, and activity at varying substrate (o-nitrophenyl-beta-d-galactopyranoside) concentrations, at varying pH, and in the presence of sodium, potassium, and magnesium. The widely varying specific activities and the approximate similarity of the Michaelis constants (about 2 x 10(-4)m) suggested that the strains investigated produced differing amounts of beta-galactosidase. Moreover, qualitative differences in the enzymes provided evidence that these strains synthesized different molecular forms of beta-galactosidase. The results suggested that organisms which ferment lactose only after a prolonged delay do so because they possess multiple defects in their lactose-metabolizing machinery.     

Biochemical Genetics of the Cryptic Gene System for 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 microbial evolution. Wild-type E. coli K12 do not utilize the beta-glucoside sugars, arbutin, salicin and cellobiose. A Cel+ (cellobiose utilizing) mutant which grows on cellobiose, arbutin, and salicin was isolated previously from wild-type E. coli K12. Biochemical assays indicate that a cel structural gene (celT) specifies a single transport protein that is a beta-glucoside specific enzyme of the phosphoenolpyruvate-dependent phosphotransferase system. The transport protein phosphorylates beta-glucosides at the expense of phosphoenolpyruvate. A single phosphoglucosidase, specified by celH, hydrolyzes phosphorylated cellobiose, arbutin, and salicin. The genes of the cel system are expressed constitutively in the Cel+ mutant, whereas they are not expressed at a detectable level in the wild-type strain. The transport and hydrolase genes are simultaneously silenced or simultaneously expressed and thus constitute an operon. Cel+ strains which fail to utilize one or more beta-glucosides express the transport system at a lower level than do Cel+ strains which grow on all three beta-glucosides. Other strains inducibly express a gene which specifies transport of arbutin but not the other beta-glucosides. The arbutin transport gene, arbT, maps outside of the cel locus.     

Characterization of the Enterobacteriaceae isolated from an artisanal Italian ewe's cheese (Pecorino Abruzzese)

AIMS: To evaluate some physiological characteristics of the Enterobacteriaceae isolated from Pecorino cheese. METHODS AND RESULTS: The production of organic acids, secondary volatile compounds, biogenic amines (BA) and the lipolytic and proteolytic activities of Citrobacter braakii, Enterobacter sakazakii, Escherichia coli, Kluyvera spp., Salmonella enterica ssp. arizonae and Serratia odorifera strains were determined in skim milk after 48 h of fermentation at 30 degrees C. The proteolytic activity observed only in Ser. odorifera and Kluyvera spp. was confirmed by the peptide profiles of the pH 4.6-insoluble fraction using RP-HPLC; however, the lipase activity was evidenced in all the isolates of E. coli, Kluyvera spp. and Salm. enterica ssp. arizonae. During fermentation, all the strains utilized citric acid and produced significant quantities of putrescine followed by histamine, spermine and spermidine as well as acetic and lactic acid. Moreover, the major volatile compounds produced were ethanol, 2,3-butanedione, 3-hydroxy-2-butanone, 2-heptanone and acetone. CONCLUSIONS: The Enterobacteriaceae of dairy origin possess many metabolic activities that could affect the sensory quality of the cheese in which they grow during ripening. SIGNIFICANCE AND IMPACT OF THE STUDY: The important physiological characteristics possessed by Enterobacteriaceae confirm the complexity of the microbiota of Pecorino Abruzzese cheese, which influences the typical sensory properties of this product.     

Phenotypic variability of beta-glucoside utilization and its correlation to pathogenesis process in a few enteric bacteria

Utilization of beta-glucosides is markedly variable in the members of the family Enterobacteriaceae. The results presented here provide molecular clues for evolutionary events that resulted in the phenotypic variability seen amongst the members of these species. The genomic hybridization of selected Enterobacteriaceae members with the Escherichia coli bgl and cel genes resulted in detection of a complete homolog of the bgl and cel operons in Shigella sonnei, a member that is evolutionarily closest to E. coli. However, the Salmonella group of organisms have been shown to carry only a homolog of bglR and bglG regions and the deletions of the bglF and bglB genes. Similarly, Proteus mirabilis, Enterobacter aerogenes and a non-enteric Gram-negative bacterium Pseudomonas aeruginosa have been shown to carry a homolog of the bglR and bglG regions and deletions of the bglF and bglB genes. The homolog of the cel operon could be identified in S. sonnei and Salmonella groups of organisms. Possible implications of these observations, in connection with the phenotypic variability seen in beta-glucoside utilization amongst these members, are discussed.     

Evaluation of novel chromogenic substrates for the detection of bacterial beta-glucosidase

AIMS: To evaluate three previously unreported substrates for the detection of beta-glucosidase activity in clinically relevant bacteria and to compare their performance with a range of known substrates in an agar medium. METHODS AND RESULTS: The performance of 11 chromogenic beta-glucosidase substrates was compared using 109 Enterobacteriaceae strains, 40 enterococci and 20 strains of Listeria spp. Three previously unreported beta-glucosides were tested including derivatives of alizarin, 3',4'-dihydroxyflavone and 3-hydroxyflavone. These were compared with esculin and beta-glucoside derivatives of 3,4-cyclohexenoesculetin, 8-hydroxyquinoline and five indoxylics. All substrates yielded coloured precipitates upon hydrolysis in agar. Alizarin-beta-D-glucoside was the most sensitive substrate tested and detected beta-glucosidase activity in 72% of Enterobacteriaceae strains and all enterococci and Listeria spp. The two flavone derivatives showed poor sensitivity with Gram-negative bacteria but excellent sensitivity with enterococci and Listeria spp. CONCLUSIONS: Alizarin-beta-d-glucoside is a highly sensitive substrate for detection of bacterial beta-glucosidase and compares favourably with existing substrates. beta-glucosides of 3',4'-dihydroxyflavone and 3-hydroxyflavone are effective substrates for the detection of beta-glucosidase in enterococci and Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented allow for informed decisions to be made regarding the optimal choice of beta-glucosidase substrate for detection of pathogenic and/or indicator bacteria.     

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.     

Demonstration of cel operon expression of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa at elevated temperatures refractory to their growth.

When Escherichia coli was incubated at the growth-refractory temperatures of 48 and 54 degrees C, expression of the cel operon was demonstrated by phospho-beta-glucosidase activity. This enzyme activity was also detected at the growth-refractory temperatures in Salmonella typhimurium and Pseudomonas aeruginosa. Thermotolerant and mesothermophilic mutants of E. coli, S. typhimurium, and P. aeruginosa, able to grow with generation times of 30 to 40 min at 48 and 54 degrees C, exhibited phospho-beta-glucosidase activity at their growth temperatures of 48 and 54 degrees C. Thus, the cel operon previously described as a cryptic operon in E. coli and S. typhimurium was found to be expressed at growth-refractory temperatures of the mesophilic parent and growth-permissive temperatures (48 and 54 degrees C) of the thermotolerant and mesothermophilic mutants.