Methyl galactosidase activity: an alternative evolutionary destination for the ebgA0 gene.

Previous studies (Campbell et al., 1973; Hall and Hartl, 1974; Hall and Hartl, 1975) have shown that the ebgA0 gene, whose product does not hydrolyze lactose may evolve so that its product does hydrolyze lactose; i.e., lactase activity is one evolutionary destination of the ebgA0 gene. Beginning with a strain that synthesizes ebgA0 gene product constitutively and grows extremely slowly (doubling time, 30 to 50 h) on methyl-beta-D-galactopyranoside (MG), a derivative was selected capable of growth on MG at a moderate rate (doubling time, 5.9 h). Genetic evidence is presented showing that the gene that permits growth on MG is an allele of ebgA. A comparison among strains bearing several alleles of ebgA shows that the new allele, termed ebgAmg, synthesizes a product specific for MG and thus represents a true alternative evolutionary destination for the ebgA0 gene.     

Regulation of Newly Evolved Enzymes. III Evolution of the ebg Repressor during Selection for Enhanced Lactase Activity

The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg+ strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.     

Isolation and characterization of the newly evolved ebg beta-galactosidase of Escherichia coli K-12.

The ebg beta-galactosidase of Escherichia coli K-12 strain LC110 has been purified and characterized. Strain LC110 is a Lac+ revertant of a mutant with a deletion of the lacZ beta-galactosidase gene. Its new ebg beta-galactosidase activity was shown to be due to a discrete protein, immunologically unrelated to lacZ beta-galactosidase. Its kinetics of action conformed to those of a simple conventional enzyme. With o-nitrophenyl-beta-D-galactoside as substrate, the Vmax was 11,200 nmol/min per mg of enzyme, the Km was 5 mM, and the activation energy was 12,400 cal/mol. Corresponding values for lacZ beta-galactosidase of wild-type E. coli K-12 were 350,000 nmol/min per mg of enzyme, 1.3 mM, and 8,000 cal/mol. A series of sugars has been examined as competitive inhibitors of ebg beta-galactosidase. Kinetic analyses suggest that ebg beta-galactosidase has a particularly high affinity for galactosamine and gamma-galactonolactone, binds galatose more tightly than lactose, and shows a general preference for monosaccharides rather than beta-galactosides. We conclude that the ebg beta-galactosidase may have arisen by modification of a gene involved with the metabolism of a monosaccharide, possibly a 2-amino sugar.     

Toluene transposons Tn4651 and Tn4653 are class II transposons

The toluene degradative transposon Tn4651 is included within another transposon, Tn4653, and both of these elements are members of the Tn3 family. The tnpA gene product of each element mediates formation of cointegrates as intermediate products of transposition, and the tnpS and tnpT gene products encoded by Tn4651 take part in resolution of both Tn4651- and Tn4653-mediated cointegrates. Sequence analysis demonstrated that Tn4651 and Tn4653 have 46- and 38-base-pair terminal inverted repeats, respectively, and that both elements generate 5-base-pair duplication of the target sequence upon transposition. Complementation tests of the Tn4651- and Tn4653-encoded transposition functions with those of Tn3, Tn21, and Tn1721 showed that (i) the trans-acting transposition functions encoded by Tn4651 were not interchangeable with those encoded by the four other transposons, (ii) the Tn4653 tnpA function was interchangeable with the Tn1721 function, and (iii) Tn4653 coded for a resolvase (tnpR gene product) that complemented the tnpR mutations of Tn21 and Tn1721. The Tn4653 tnpR gene was located just 5' upstream of the tnpA gene and shared extensive sequence homology with the Tn1721 tnpR gene. The res region was located adjacent to the tnpR gene, and sequence analysis indicated that failure of the Tn4653 tnpR product to resolve the Tn4653-mediated cointegrates is ascribed to an incomplete structure of the res region.     

Sequence of the ebgA gene of Escherichia coli: comparison with the lacZ gene

We have sequenced the ebgA (evolved beta-galactosidase) gene of Escherichia coli K12. The sequence shows 50% nucleotide identity with the E. coli lacZ gene, demonstrating that the two genes are related by descent from a common ancestral gene. Comparison of the two sequences suggests that the ebgA gene has recently been under selection. A significant excess of identical, rather than synonymous, codons used to encode identical amino acids at the same positions in the aligned sequences implies that some form of selection is operating directly at the DNA level. This selection is independent of, and in addition to, selection based on codon usage or on function of the gene products.      

Evolution of a regulated operon in the laboratory

The evolution of new metabolic functions is being studied in the laboratory using the EBG system of E. coli as a model system. It is demonstrated that the evolution of lactose utilization by lacZ deletion strains requires a series of structural and regulatory gene mutations. Two structural gene mutations act to increase the activity of ebg enzyme toward lactose, and to permit ebg enzyme to convert lactose into allolactose, an inducer of the lac operon. A regulatory mutation increases the sensitivity of the ebg repressor to lactose, and permits sufficient ebg enzyme activity for growth. The resulting fully evolved ebg operon regulates its own expression, and also regulates the synthesis of the lactose permease.     

The catalytic consequences of experimental evolution. Transition-state structure during catalysis by the evolved beta-galactosidases of Escherichia coli (ebg enzymes) changed by a single mutational event.

1. The first chemical step in the hydrolysis of galactosylpyridinium ions by the evolvant ebg enzyme is less sensitive to leaving-group acidity than in the case of the wild-type ebg enzyme, implying less glycone-aglycone-bond fission at the transition state. 2. The first chemical step in the hydrolysis of aryl galactosides by ebg enzyme is probably less sensitive to leaving-group acidity than in the case of ebg enzyme, possibly as a consequence of resulting in more effective proton donation to the leaving aglycone. 3. alpha-Deuterium kinetic isotope effects of 1.1(0) and beta-deuterium kinetic isotope effects of 1.0(0) were measured for the hydrolysis of galactosyl-enzyme intermediates derived from ebg and ebg enzymes: these effects are not compatible with reaction of the sugar ring through a 4C1-like conformation, or with an ionic glycosyl-enzyme intermediate. 4. The variation with pH of steady-state kinetic parameters for hydrolysis of p-nitrophenyl galactoside by ebg and ebg enzymes and of 3-methylphenyl beta-galactoside, 3,4-dinitrophenyl beta-galactoside and beta-galactosyl-3-bromopyridinium ion by ebg enzyme was measured. The steep, non-classical, fall in activity against p-nitrophenyl galactoside at low pH observed with ebg and ebg enzymes is not observed with ebg enzymes.     

DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721

DNA sequences that encode the tnpR genes and internal resolution (res) sites of transposons Tn21 and Tn501, and the res site and the start of the tnpR gene of Tn1721 have been determined. There is considerable homology between all three sequences. The homology between Tn21 and Tn501 extends further than that between Tn1721 and Tn501 (or Tn21), but in the homologous regions, Tn1721 is 93% homologous with Tn501, while Tn21 is only 72-73% homologous. The tnpR genes of Tn21 and Tn501 encode proteins of 186 amino acids which show homology with the tnpR gene product of Tn3 and with other enzymes that carry out site-specific recombination. However, in all three transposons, and in contrast to Tn3, the tnpR gene is transcribed towards tnpA gene, and the res site is upstream of both. The res site of Tn3 shows no obvious homology with the res regions of these three transposons. Just upstream of the tnpR gene and within the region that displays common homology between the three elements, there is a 50 bp deletion in Tn21, compared to the other two elements. A TnpR- derivative of Tn21 was complemented by Tn21, Tn501 and Tn1721, but not by Tn3.     

Identification and characterization of genes involved in excision of the Lactococcus lactis conjugative transposon Tn5276.

The 70-kb transposon Tn5276, originally detected in Lactococcus lactis NIZO R5 and carrying the genes for nisin production and sucrose fermentation, can be conjugally transferred to other L. lactis strains. Sequence analysis and complementation studies showed that the right end of Tn5276 contains two genes, designated xis and int, which are involved in excision. The 379-amino-acid int gene product shows high (up to 50%) similarity with various integrases, including that of the Tn916-related conjugative transposons. The xis gene product, like almost all known excisionase (Xis) proteins, is a small (68-residue), basic protein. Expression of both the Tn5276 int and xis genes is required for efficient excision of the ends of Tn5276 in Escherichia coli that appeared to be circularized in the excision process. Mutational analysis of the xis and int genes showed that excision efficiency is dependent on the integrity of the int gene but that an intact xis gene is also required for efficient excision.     

Regulation of Newly Evolved Enzymes. IV. Directed Evolution of the EBG Repressor

In Escherichia coli, the wild-type repressor of ebg (evolved beta-galactosidase) enzyme synthesis, specified by the ebgR+ gene, responds very weakly to lactulose (fructose-beta-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 X 10(-10) . EBGR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.     

Number of mutations required to evolve a new lactase function in Escherichia coli.

The frequency of mutation of the ebgAo allele to ebgA+ was compared with the frequency of mutation of strA+ to strA-. The observation that both spontaneous and ethyl methane sulfonate-induced mutations to ebgA+ occurred more frequently than mutations to strA- suggests that ebgA+ mutants arise as the result of single-point mutations.     

The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons.

The nucleotide sequence of a 2.5 kb segment of the pKM101 (R46) genome has been determined. The 1.3 kb from a BamHI site at 153 to base 1440 differs by only 2 bases from a part of the published sequence of the aadB (gentamicin resistance) gene region including the coding region for the N-terminal 70 amino acids of the predicted aadB product. The same sequence has been found 5'-to the dhfrII gene of R388 and to the aadA gene of Tn21 (R538-1). Three open reading frames are located in this region, two on the same strand as the resistance genes and one on the complementary strand. The latter predicts a polypeptide of 337 amino acids, whose N-terminal segment is 40% homologous to the predicted product of an open reading frame of 179 amino acids located next to the dhfrI gene of Tn7. The oxa2 (oxacillin resistance) gene predicts a long polypeptide commencing with (the N-terminal) 70 amino acids of the aadB product. A similar arrangement is found in the aadA gene of R538-1. The N-terminal segment of an aadA gene is located 3'- to oxa2, separated by 36 bases. Sequences surrounding the BamHI site are identical to sequences 5'- to the tnpM gene of Tn21 and homology ceases where homology between Tn21 and Tn501 commences. The possibility that this antibiotic resistance segment is a discrete mobile DNA element is discussed.     

DNA Sequence Analysis of Artificially Evolved Ebg Enzyme and Ebg Repressor Genes

The ebg system has been used as a model to study the artificial selection of new catalytic functions of enzymes and of inducer specificities of repressors. A series of mutant enzymes with altered catalytic specificities were previously characterized biochemically as were the changes in inducer specificities of mutant, but fully functional, repressors. The wild type ebg operon has been sequenced, and the sequence differences of the mutant enzymes and repressors have been determined. We now report that, contrary to our previous understanding, ebg enzyme contains 180-kD alpha-subunits and 20-kD beta-subunits, both of which are required for full activity. Mutations that dramatically affect substrate specificity and catalytic efficiency lie in two distinct regions, both well outside of the active site region. Mutations that affect inducer specificity of the ebg repressor lie within predicted sugar binding domains. Comparisons of the ebg beta-galactosidase and repressor with homologous proteins of the Escherichia coli and Klebsiella pneumoniae lac operons, and with the galactose operon repressor, suggest that the ebg and lac operons diverged prior to the divergence of E. coli from Klebsiella. One case of a triple substitution as the consequence of a single event is reported, and the implications of that observation for mechanisms of spontaneous mutagenesis are discussed.     

Transposon Tn554 encodes three products required for transposition.

Tn554 is a high-frequency, site-specific, transposable element having integrative properties resembling those of lysogenic bacteriophages. Nucleotide sequence analysis indicates that Tn554 has three transposition genes, designated tnpA, tnpB and tnpC. Mutations in each of these were complemented efficiently in trans by clones containing internal fragments of Tn554; thus the products of these genes function in trans. Elements carrying deletions of the Tn554 termini could not be complemented. The product of tnpC is not absolutely required for transposition, since deletion mutations encompassing 80% of tnpC, as well as frameshift mutations located near the amino terminus of tnpC, transposed at frequencies as high as 2% of that observed with wild-type Tn554. However, such mutations affected the orientation of insertion. With wild-type Tn554 insertion occurs in a single orientation regardless of the orientation of the donor. In tnpC mutants insertion orientation was dictated by the orientation of Tn554 in the donor molecule. A mutant lacking the carboxy-terminal 59 residues of tnpB also exhibited altered insertion orientation. Thus it appears that the tnpC gene product is required for correct orientation of the element upon insertion and that this protein may interact with the carboxy-terminal portion of the tnpB gene product.     

Participation of the uvrD gene in the precise excision of the Tn9 transposon

HfeTn5-(04,06) and IfeTn9 mutations increase efficiency of precise excision of Tn5, Tn10 and decrease that of Tn9. These mutations have been mapped in uvrD gene. In LFETn9 and UVRE502 mutants, the multicopy plasmid pEM61 carrying the cloned uvrD gene complements LFETn9- phenotype (Low Frequency Excision of Tn9). These results indicate that the uvrD gene product plays different role in excision of transposons with long and short inverted repeats. The mechanism of this effect is discussed.     

Experimental Evolution of a New Enzymatic Function. II. Evolution of Multiple Functions for EBG Enzyme in E. COLI

The evolution of ebgo enzyme of Escherichia coli, an enzyme which is unable to hydrolyze lactose, lactulose, lactobionate, or galactose-arabinoside effectively, has been directed in successive steps so that the evolved enzyme is able to hydrolyze these galactosides effectively. I show that in order for a strain of E. coli with a lacZ deletion to evolve the ability to use lactobionate as a carbon source, a series of mutations must occur in the ebg genes, and that these mutations must be selected in a particular order. The ordered series of mutations constitutes an obligatory evolutionary pathway for the acquisition of a new function for ebgo enzyme. A comparison of newly evolved strains with parental strains shows that when ebg enzyme acquires a new function, its old functions often suffer; but that in several cases old functions are either unaffected or are improved. I conclude that divergence of functions catalyzed by an enzyme need not require gene duplication.     

Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.

Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product.