Mutations that activate the silent bgl operon of Escherichia coli confer a growth advantage in stationary phase

Wild-type strains of Escherichia coli are unable to utilize aromatic beta-glucosides such as arbutin and salicin because the major genetic system that encodes the functions for their catabolism, the bgl operon, is silent and uninducible. We show that strains that carry an activated bgl operon exhibit a growth advantage over the wild type in stationary phase in the presence of the rpoS819 allele that causes attenuated rpoS regulon expression. Our results indicate a possible evolutionary advantage in retaining the silent bgl operon by wild-type bacteria.     

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.      

Effect of Increased beta-Glucosidase Activity on Virulence of Erwinia amylovora

Plant tissues often contain beta-glucosides that can be enzymatically hydrolyzed to produce toxic aglycones. It has been suggested that the low beta-glucosidase activity found in Erwinia amylovora contributes to bacterial virulence by allowing the bacteria to infect plants that contain beta-glucosides without inducing the formation of toxic aglycones. To test this suggestion, we created strains of E. amylovora which had high beta-glucosidase activities and studied the ability of these strains to cause fire blight disease in pears (Pyrus communis). We isolated spontaneous mutants that were able to utilize beta-glucosides as the sole carbon source and showed that one class had about 10 times as much beta-glucosidase activity as the wild-type strain. In addition, we constructed several plasmids that carry the Escherichia coli bgl operon under the control of a transposon Tn5 promoter that is expressed in E. amylovora. These plasmids were introduced in E. amylovora by transformation. Pathogenesis studies in immature Bartlett pear fruits, etiolated sprouts, and young shoots showed that a 100-fold increase in beta-glucosidase activity does not interfere with normal development of fire blight disease in these model systems.     

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.     

Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes.

Wild-type Escherichia coli cells are unable to grow on beta-glucosides. Spontaneous mutants arise, however, which are able to utilize certain aromatic beta-glucosides such as salicin or arbutin as carbon sources, revealing the presence of a cryptic operon called bgl. Mutations activating the operon map within (or close to) the promoter region of the operon and are due to the transposition of an IS1 or IS5 insertion element into this region. This operon was reported to consist of three genes coding for a phospho-beta-glucosidase, a specific transport protein (enzyme IIBgl), and a positively regulating protein. We have defined the extent and location of three structural genes, bglC, bglS, and bglB, and have determined their DNA sequence. The amino acid sequences deduced from the open reading frames together with deletion and subcloning analyses suggest that the first gene, bglC, codes for the regulatory protein, the second, bglS, codes for the transport protein, and the third, bglB, for phospho-beta-glucosidase. A fourth gene may exist which codes for a product of unknown function. We discuss structural features of the DNA sequence which may bear on the regulation of the operon. Homologies to sequences preceding the gene for an excreted levansucrase of Bacillus subtilis, which are known to be involved in the regulation of this gene, and to sequences preceding the gene for an excreted beta-endoglucanase of B. subtilis, for which data pertaining to regulation are not yet available, suggest a close evolutionary relationship among the regulatory components of all three systems.     

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.     

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.     

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.     

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.     

Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations.

In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control.