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.      

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.     

A Fourth Escherichia Coli Gene System with the Potential to Evolve β-Glucoside Utilization

Escherichia coli K12 is being used to study the potential for adaptive evolution that is present in the genome of a single organism. Wild-type E. coli K12 do not utilize any of the beta-glucoside sugars arbutin, salicin or cellobiose. It has been shown that mutations at three cryptic loci allow utilization of these sugars. Mutations in the bgl operon allow inducible growth on arbutin and salicin while cel mutations allow constitutive utilization of cellobiose as well as arbutin and salicin. Mutations in a third cryptic locus, arbT, allow the transport of arbutin. A salicin+ arbutin+ cellobiose+ mutant has been isolated from a strain which is deleted for the both the bgl and cel operons. Because the mutant utilized salicin and cellobiose as well as arbutin, it is unlikely it is the result of a mutation in arbT. A second step mutant exhibited enhanced growth on salicin and a third step mutant showed better growth on cellobiose. A fourfold level of induction in response to arbutin and a twofold level of induction in response to salicin was observed when these mutants were assayed on the artificial substrate p-nitrophenyl-beta-D-glucoside. Although growth on cellobiose minimal medium can be detected after prolonged periods of time, these strains are severely inhibited by cellobiose in liquid medium. This system has been cloned and does not hybridize to either bgl or cel specific probes. We have designated this gene system the sac locus. The sac locus is a fourth set of genes with the potential for evolving to provide beta-glucoside utilization.     

A new experimental system for study on adaptive mutations

A super-repressed mutant of purR (purR^S), which encodes a repressor protein controlling expression of purine biosynthetic genes inSalmonella typhimurium, grew very slowly on NCE medium with 10 μg/mL Ade and lactose as sole carbon source (cannot form colonies). However, a phenomenon of late-arising mutations was observed when purR^S mutants were spread on NCE+lactose plates and subjected to a prolonged non-lethal selection. The reconstruction experiments of revertants showed that the late-arising “lac⁺” mutants are not slow growing mutants. Statistical analysis indicated that the distribution of late-arising mutants is Poisson distribution, showing that reversion occurred after plating. The result of co-transductional analysis preliminarily showed that late-arising mutation occurred at selected genepurR or 16 bp PUR box,cis element of structural genepurD. The above results suggest that the phenomenon of late-arising mutation observed by our system is a result of adaptive mutations which are different from random mutations. This is the first time to extend target genes at which adaptive mutations could occur from structural genes involved in carbon metabolism and amino acid biosynthesis totrans regulatory gene coding repressor protein. Our results have provided not only a new proof for generality of adaptive mutations but also a new system for study on adaptive mutations.     

A new experimental system for study on adaptive mutations

A super-repressed mutant of purR (purRs), which encodes a repressor protein controlling expression of purine biosynthetic genes in Salmonella typhimurium, grew very slowly on NCE medium with 10 μg/mL Ade and lactose as sole carbon source (cannot form colonies). However, a phenomenon of late-arising mutations was observed when purRs mutants were spread on NCE+lactose plates and subjected to a prolonged non-lethal selection. The reconstruction experiments of revertants showed that the late-arising "lac+" mutants are not slow growing mutants. Statistical analysis indicated that the distribution of late-arising mutants is Poisson distribution, showing that reversion occurred after plating. The result of co-transductional analysis preliminarily showed that late-arising mutation occurred at selected gene purR or 16 bp PUR box, cis element of structural gene purD. The above results suggest that the phenomenon of late-arising mutation observed by our system is a result of adaptive mutations which are different     

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.     

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.     

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 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.     

Adaptive Evolution That Requires Multiple Spontaneous Mutations. I. Mutations Involving an Insertion Sequence

Escherichia coli K12 strain chi 342LD requires two mutations in the bgl (beta-glucosidase) operon, bglR0----bglR+ and excision of IS103 from within bglF, in order to utilize salicin. In growing cells the two mutations occur at rates of 4 x 10(-8) per cell division and less than 2 x 10(-12) per cell division, respectively. In 2-3-week-old colonies on MacConkey salicin plates the double mutants occur at frequencies of 10(-8) per cell, yet the rate of an unselected mutation, resistance to valine, is unaffected. The two mutations occur sequentially. Colonies that are 8-12 days old contain from 1% to about 10% IS103 excision mutants, from which the Sal+ secondary bglR0----bglR+ mutants arise. It is shown that the excision mutants are not advantageous within colonies; thus, they must result from a burst of independent excisions late in the life of the colony. Excision of IS103 occurs only on medium containing salicin, despite the fact that the excision itself confers no detectable selective advantage and serves only to create the potential for a secondary selectively advantageous mutation.     

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.     

Nucleotide sequence, function, activation, and evolution of the cryptic asc operon of Escherichia coli K12.

The cryptic asc (previous called "SAC") operon of Escherichia coli K12 has been completely sequenced. It encodes a repressor (ascG); a PTS enzyme IIasc for the transport of arbutin, salicin, and cellobiose (ascF); and a phospho-beta-glucosidase that hydrolyzes the sugars which are phosphorylated during transport (ascB). ascG and ascFB are transcribed from divergent promoters. The cryptic operon is activated by the insertion of IS186 into the ascG (repressor) gene. The ascFB genes are paralogous to the cryptic bglFB genes, and ascG is paralogous to galR. The duplications that gave rise to these paralogous genes are estimated to have occurred approximately 320 Mya, a time that predates the divergence of E. coli and Salmonella typhimurium.