Bgl Operon Research Articles

A bacterial gene involved in transcription antitermination: Regulation at a rho-independent terminator in the bgl operon of E. coli

We have investigated the mechanism of regulation of the bgl operon in Escherichia coli K-12. A regulatory region has been located downstream of the bgl promoter, revealing a 130 base leader containing a sequence from +64 to +112 characteristic of a rho-independent terminator. In vitro, over 90% of the transcripts initiated at the bgl promoter terminate within the leader, at the 3′ end of the terminator. Transcriptional fusions containing the terminator require the bglC gene in trans for expression; fusions deleted for the sequence show bglC-independent expression. A mutation resulting in partially constitutive expression of the fusion maps within the terminator. We propose that the bglC gene product mediates positive regulation of the bgl operon by functioning as an antiterminator at the rho-independent terminator located within the leader.

Positive and negative regulation of the bgl operon in Escherichia coli.

We have analyzed the functions encoded by the bgl operon in Escherichia coli K-12. Based on the ability of cloned regions of the operon to complement a series of Bgl- point mutations, we show that the three bgl structural genes, bglC, bglS, and bglB, are located downstream of the regulatory locus bglR in the order indicated. Using a bgl-lacZ transcriptional fusion, we show that bglC and bglS are involved in regulating operon expression. The presence of the bglC gene in trans is absolutely required for the expression of the fusion, which is constitutive when only the bglC gene is present. When the bglC and the bglS genes are both present in the cell, expression of the fusion requires a beta-glucoside inducer. From these observations, we conclude that (i) the bglC gene encodes a positive regulatory of bgl operon expression and (ii) the bglS gene encodes a negative regulator of operon expression, causing the requirement for a beta-glucoside inducer. These conclusions are supported by our observations that (i) a majority of bglC mutants exhibits a Bgl- phenotype, whereas rare trans-dominant mutations in bglC result in constitutive expression of the bgl operon and the fusion, and (ii) mutations in the bglS gene lead to constitutive expression of the fusion. Based on several lines of evidence presented, we propose that the bglS gene product has an additional role as a component of the beta-glucoside transport system.

Functional genes for cellobiose utilization in natural isolates of Escherichia coli.

The genes for utilization of cellobiose are normally cryptic in both laboratory strains and natural isolates of Escherichia coli. A survey of natural isolates of E. coli reveals that functional genes for cellobiose utilization, while rare, are present. The fraction of E. coli that utilized cellobiose ranged from less than 0.01% in human fecal samples to 7% in fecal samples obtained from horses. Samples obtained from sheep, cows, dogs, and pigs contained 0.1 to 0.5% cellobiose-positive E. coli. Neither the previously identified cel genes nor the bgl genes from E. coli K-12 were expressed during growth on cellobiose by any of the 14 naturally occurring Cel+ isolates that were tested. All of the naturally occurring Cel+ isolates possessed a cel operon, but all were deleted for the major portion of the bgl operon. The functional cel+ genes from these natural isolates differed from the mutationally activated cel+ genes obtained in earlier studies in that (i) the mutationally activated cel+ genes were temperature sensitive, while the functional genes were not, and (ii) transport of cellobiose was inducible in the strains carrying functional cel+ genes, while it was expressed constitutively in strains carrying mutationally activated genes.

Enhancement of bacterial gene expression by insertion elements or by mutation in a CAP-cAMP binding site

The regulatory region ( bglR) of the cryptic bgl operon was characterized by DNA sequence analysis and transcription mapping. Bgl −-specific transcription was found to occur in both the wild-type Bgl − and mutant Bgl + cells. However, the steady-state level of bgl RNA was much higher in the Bgl + mutant than in the wild-type. Activation of the bgl operon by insertion sequence-mediated bglR mutations or point mutations in bglR is therefore the result of increased transcription. The ethylmethane sulfonate-induced point mutations in bglR are alterations in a single base in the cAMP binding protein (CAP) binding site, leading to a stronger binding of the CAP-cAMP complex. The IS1 and IS5-mediated bglR mutations analyzed show that the insertion sequences can activate the bgl operon by integration 78 to 125 base-pairs upstream from the transcription initiation site. The role of the insertion sequences in activation of the bgl operon is discussed.

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.

Insertion of DNA activates the cryptic bgl operon in E. coli K12.

Spontaneous mutations which activate the cryptic bgl operon of Escherichia coli K12 are caused by insertion of DNA at a site, bglR, within the operon. Two insertion elements, IS1 and IS5, have been observed to effect this activation. Once the activating insertion has occurred the operon is inducible by βglucosides in a cyclic AMP-dependent manner.

Regulation of the beta-glucoside system in Escherchia coli K-12.

In Escherichia coli wild-type cells, a mutation at the beta-glucoside regulatory gene (bglR(+) to bglR(-)) leads to simultaneous expression of inducible phospho-beta-glucosidase B (bglB(+)) and a beta-glucoside-specific species of enzyme II (beta-glucoside transport I [bglC(+)]); an additional mutation (bglS(+) to bglS4) allows these enzymes to be formed constitutively. The bgl alleles have been mapped in the following order: pyrE, bglA, bglB, bglS, bglR, bglC, ilvD. The back mutation in the regulatory allele (bglR(-) to bglR(+)) caused the cessation of the expression of the bglB(+), bglS(+) or bglS4, bglC(+) alleles. However, a mutation in a strain with bglB(+), bglS4, bglR8, bglC(+) alleles, at the ini site that lies between the bglS4 and the bglR8 allele, allowed the expression of the bglS4 and bglB(+) alleles, but showed no affect on the expression of the bglC(+) allele. It is suggested that the ini mutation possesses a promotor-type function that in the absence of regulatory allele function (bglR8) renews the functioning of only the bglS4 and bglB(+) alleles. The complementation studies have shown that the bglB(+), bglS(+) or bglS4, bglC(+) alleles are expressed only in cis to the bglR(-) allele. In the constitutive strain (bglB(+), bglS4, bglR(-), bglC(+)), the expressed bglS4 allele formed a soluble product that acts in trans over the bglB(+) and bglC(+) alleles and that appears effective only when the bglB(+) and the bglC(+) alleles are expressed in cis to the bglR(-) allele. It thus showed that the constitutive biosynthesis of phospho-beta-glucosidase B and beta-glucoside transport I is under positive control. Since the regulatory allele bglR(-) lies between the bglS4 and the blgC(+) alleles, and acts in cis, it appears that the mutation (bglR(+) to bglR(-)) allows the initiation of transcription in one direction to express the bglS4, bglB(+) alleles and in the other to express the bglC(+) allele. The structural genes bglB and bglC lie adjacent to the regulatory genes bglR and bglS, and the structural genes are coordinately controlled by the regulatory genes. It is, therefore, proposed that the bglB, bglS, bglR, bglC genes form a bgl operon.