Cryptic Bgl Operon Research Articles

IS1 transposition is enhanced by translation errors and by bacterial growth at extreme glucose levels.

Transposition of insertion sequences (IS) is an enzyme-mediated process that only occurs in a minority of cells within a bacterial culture. Transposition is thus a rare event, but transposition frequency may vary depending on experimental conditions. For instance in a rich broth, IS elements are known to transpose during stationary phase but not during exponential growth. Using a reporter system which involves the activation of the cryptic bgl operon in Escherichia coli, we show that the frequency of IS1 transposition is a function of glucose concentration in the growth medium, it is increased by streptomycin amounts that are below minimum inhibitory concentration (sub-MIC) and is inhibited in an rpsL150 strain with high translation accuracy. Since starved cells are known to enhance ribosome frameshifting, our data suggests that growth conditions applied in this study could affect IS1 transposition by increasing translation infidelity.

Adaptive mutation in Escherichia coli.

In 1988 John Cairns, Julie Overbaugh, and Stephan Miller published a paper entitled “The origin of mutants,” in which they suggested that bacteria could choose which mutations to make (11). Shortly thereafter, Cairns and I began collaborating on a National Science Foundation-funded project to investigate the genetic basis of what was popularly called “directed mutation” (although at the time we were calling it “selection-dependent mutation,” which now seems as good a name as any). Our plan was to mutagenize an appropriate strain of Escherichia coli and identify and characterize variants defective in selection-dependent mutation. Cairns and coworkers had been investigating a strain called SM195, described in their original paper (11). However, the mechanisms of mutation in this strain had proved to be complicated, and we wanted a new experimental subject. We were setting up to investigate selection-induced activation of the cryptic bgl operon (36), when Jeffrey Miller gave us a Lac− strain, called α45, that he said had a high rate of reversion to Lac+ after plating on minimum lactose medium. The lac allele in this strain is a fusion of lacI to lacZ that eliminates the lac regulatory region as well as several residues of lacI and lacZ; transcription of the fusion is constitutive, initiating at the lacI promoter (7). α45 has a +1 frameshift mutation, lacI33, in the lacI coding region (12). Miller and coworkers have used several similar strains to study various mutational mechanisms (52, 67). As in many of the strains that originated with Jacob and Monod, proAB and lac are deleted from the chromosome and carried on an episome, F′128. This arrangement greatly facilitates genetic manipulations and turned out to be important to adaptive mutation. We mated the episome from α45 into a Δ(lac-pro) recipient that we had made rifampin resistant and named the new strain FC40 (Foster and Cairns #40).

Requirement for the molecular adapter function of StpA at the Escherichia coli bgl promoter depends upon the level of truncated H-NS protein.

Truncated derivatives of the Escherichia coli nucleoid-associated protein H-NS that lack the DNA-binding domain remain competent for silencing of the cryptic bgl operon in vivo. Previous studies have provided evidence for the involvement of either the homologous nucleoid protein StpA or the alternative sigma factor RpoS in this unusual silencing mechanism. Here, we rationalize this apparent discrepancy. We show that two hns alleles (hns-205::Tn10 and hns60), which produce virtually identical amino-terminal fragments of H-NS, have very different requirements for StpA to mediate bgl silencing. The hns60 allele produces a high level of truncated H-NS, which can overcome the absence of StpA, whereas the lower level expressed by hns-205::Tn10 requires StpA for silencing. Reversing the relative levels of the two H-NS fragments reverses their requirement for StpA to silence bgl transcription. This suggests that the amino-terminal fragment of H-NS can be targeted to DNA to mediate silencing by multiple protein-protein interactions. The high-specificity interaction with StpA can function at low levels of truncated H-NS, whereas an alternative mechanism, perhaps involving lower specificity interactions with another protein(s), is only functional when truncated H-NS is abundant. These findings have important implications for the involvement of other proteins in H-NS-dependent transcriptional repression.

A putative sigma factor from Streptomyces sp. strain A21 can activate the expression of the cryptic operon bgl in Escherichia coli K-12.

Streptomyces sp A21 is a cellulolytic strain isolated from soil which was assigned to the genus Streptomyces on the basis of distinctive morphological features. A genomic library of A21 DNA has been constructed and transformed into Escherichia coli K-12 using a high-copy-number vector. One of the recombinant plasmids activates the cryptic bgl operon when inserted into appropriate strains. The complete sequence of the 1629-bp A21 DNA fragment has been determined. The analysis revealed the presence of an ORF whose putative product shows a high degree of similarity to RNA polymerase sigma factors; we therefore designated the gene psfS (Putative sigma factor, Streptomyces). Mapping of the 5' terminus of transcript by primer extension indicated that PsfS induces transcription initiation within the bgl promoter-silencer region.

Activation of the bgl operon by adaptive mutation.

In growing Escherichia coli K12 cells, the cryptic bgl operon is activated 98% of the time by insertions of IS1 or IS5 into the control region, designated bglR. The activated bgl operon permits utilization of the beta-glucoside sugar arbutin as a sole carbon and energy source. The bgl operon is also activated by late-occurring mutations during prolonged selection on arbutin. The late-occurring mutations that occurred during prolonged carbon starvation in the presence of arbutin were "adaptive mutations" because they were specific to the presence of arbutin, and they did not occur during prolonged starvation in the absence of arbutin. The spectrum of late-arising mutations differed from that of early-arising, growth-dependent mutations in that 20% of the late-arising mutants resulted from mutations at the hns locus. This provides the first direct evidence for adaptive mutagenesis mediated by the insertion of IS elements. Because no special genetic background is required to select Bgl+ mutants, this affords the opportunity to study IS-element-mediated adaptive mutagenesis in a variety of genetic backgrounds, including the backgrounds of natural isolates of E. coli.

Histone-like proteins are required for cell growth and constraint of supercoils in DNA

The gene hns of Escherichia coli K-12, which encodes the histone-like protein H-NS, was inactivated by insertion of a DNA (gene hph) encoding hygromycin phosphotransferase. The growth rates of two mutants lacking either one or the other of the histone-like proteins, HU and IHF, were not affected by introduction of the hns mutation. However, cells depleted of HU, IHF and H-NS simultaneously, could not be constructed by P1 phage-mediated transduction. These results, together with our previous finding that cells deficient in both HU and IHF are viable at 30–37°C [Kano and Imamoto, Gene 89 (1990) 133–137] showed that E. coli cells deficient in any two of these three histone-like proteins are viable at 30–37°C, and suggested that simultaneous deficiency of all three of the proteins is lethal. There were no detectable differences in the levels of superhelicity of the reporter plasmids isolated from cells deficient in either IHF or H-NS, or from wild-type cells, but about 15% decrease in negative superhelicity was detected for the reporter plasmid isolated from cells lacking HU and lacking both HU and H-NS. However, the cryptic bgl operon, whose expression was reported to be regulated through topological changes of cellular DNA, could not be activated in cells depleted of HU or IHF. The bgl operon was expressed in cells depleted of both HU and H-NS as well as in cells depleted of H-NS. These results indicate that expression of the bgl operon is not affected by a change in superhelical density of the cellular DNA and suggest that its expression depends on some physiological processes other than topological alteration of DNA.

IS5: a mobile enhancer of transcription in Escherichia coli.

The cryptic bgl operon of Escherichia coli is activated by the spontaneous insertion of mobile DNA elements. Screening of a collection of such mutations revealed insertion of the 1195-base-pair element IS5 into various positions both upstream and downstream of the bgl promoter P0. Activation of the operon was in all cases attributable to enhancement of P0 activity. Introduction of internal deletions into IS5 almost completely abolished P0 enhancement, demonstrating that enhancement is not simply the result of mutational inactivation of some inhibitory sequences. Intact copies of IS5 in trans restored the enhancing activity of the deletion derivatives. The trans-activator is encoded by IS5 gene ins5A, an essential transposition function. Activation of gene expression by means of interaction of a defective mobile element in cis with functions encoded by a nondefective element in trans has so far been described only for a maize controlling element.

Activation of a cryptic gene by excision of a DNA fragment.

The cryptic bgl operon in Escherichia coli K-12 strain 1011A contains a 1.4-kilobase-pair fragment of foreign DNA within the bglF structural gene. The active allele found in its descendant strain, MK1, required the precise excision of that insertion for its activation. Molecular and genetic approaches have shown that strain 1011A possessed an active (bglR+) rather than a silent wild-type (bglR0) allele of the regulatory region and that this change was caused by a point mutation. Our model for the retention of cryptic genes (B. G. Hall, S. Yokoyama, and D. H. Calhoun, Mol. Biol. Evol. 1:109-124, 1983) suggested that the insertion might have been selected to silence a disadvantageous bglR+ allele. We examined the genealogy of strain MK1 and found that the insertion of foreign DNA was not selected for that reason, since it preceded the change to bglR+. This means that the change to bglR+ was also not selected, since the presence of the insertion would not allow expression of the operon. We have calculated the probability of isolating a bglR+ mutation by chance alone as less than 10(-8). We suggest that mutation rates estimated under the usual conditions of exponential growth may be irrelevant to the frequencies of these events under natural conditions.

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.