Predicting evolutionary potential: in vitro evolution accurately reproduces natural evolution of the tem beta-lactamase

To evaluate the validity of our in vitro evolution method as a model for natural evolutionary processes, the TEM-1 beta-lactamase gene was evolved in vitro and was selected for increased resistance to cefotaxime, cefuroxime, ceftazadime, and aztreonam, i.e., the "extended-spectrum" phenotype. The amino acid substitutions recovered in 10 independent in vitro evolvants were compared with the amino acid substitutions in the naturally occurring extended-spectrum TEM alleles. Of the nine substitutions that have arisen multiple times in naturally occurring extended-spectrum TEM alleles, seven were recovered multiple times in vitro. We take this result as evidence that our in vitro evolution technique accurately mimics natural evolution and can therefore be used to predict the results of natural evolutionary processes. Additionally, our results predict that a phenotype not yet observed among TEM beta-lactamases in nature-resistance to cefepime-is likely to arise in nature.     

Regulatory changes in the fucose system associated with the evolution of a catabolic pathway for propanediol in Escherichia coli.

Wild-type strains of Escherichia coli are unable to use L-1,2-propanediol as a carbon and energy source. Strain 3, a mutant selected for the ability to grow on this compound at progressively more rapid rates, synthesizes constitutively a nicotinamide adenine dinucleotide-linked propanediol oxidoreductase. This enzyme is normally synthesized during anaerobic growth on L-fucose when it functions as a lactaldehyde reductase. Propanediol, the end product of this fermentation process, escapes irretrievably into the medium. The propanediol-utilizing mutant can no longer grow on fucose in either the presence or absence of molecular oxygen. In the present study nine independent lines of propanediol-positive mutants were characterized. One mutant, strain 418, attained a propanediol growth rate close to that of strain 3 without loss of the ability to grow on fucose. In all cases examined, however, prolonged selection on propanediol did result in the emergence of fucose-negative mutants. All of these mutants had enzyme patterns similar to that of strain 3; namely, fucose permease, fucose isomerase, and fuculose kinase were noninducible, whereas fuculose 1-phosphate aldolase was constitutive. In strain 418 and in the fucose-positive predecessors of the other mutants, the first four enzymes in the pathway remained inducible, as in the wild-type strain. Improvements in the growth rate on propanediol appeared to reflect principally the increased activity level of the oxidoreductase during the early stages of evolution. According to transductional analysis, the mutations affecting the ability to grow on propanediol and those that affect the expression of the first enzymes in the fucose pathway were very closely linked. The loss of the ability to grow on fucose is thought to be a mechanistic consequence incidental to the remodeling of the regulatory system in favor of the utilization of the novel carbon source.     

Experimental evolution of evolutionary potential in fluctuating environments

Variation is the raw material for evolution. Evolutionary potential is determined by the amount of genetic variation, but evolution can also alter the visibility of genetic variation to natural selection. Fluctuating environments are suggested to maintain genetic variation but they can also affect environmental variance, and thus, the visibility of genetic variation to natural selection. However, experimental studies testing these ideas are relatively scarce. In order to determine differences in evolutionary potential we quantified variance attributable to population, genotype and environment for populations of the bacterium Serratia marcescens. These populations had been experimentally evolved in constant and two fluctuating environments. We found that strains that evolved in fluctuating environments exhibited larger environmental variation suggesting that adaptation to fluctuations has decreased the visibility of genetic variation to selection.     

Characterization of a novel L-serine transport system in Escherichia coli.

A novel transport system for L-serine was found in Escherichia coli cells grown on medium containing amino acid mixture. This novel system is distinguishable from the known three transport systems for L-serine, namely, the serine-threonine system, one of the leucine-isoleucine-valine systems, and the glycine-alanine system. Uptake of L-serine via this novel system was inhibited by none of the amino acids tested, indicating that it is highly specific for L-serine. This system was induced by L-leucine, but not by L-serine. The Km for L-serine was 50 microM, and the Vmax was 23 nmol/min per mg of cell protein. Transport of L-serine via this system was strongly inhibited by KCN, an inhibitor of the respiratory chain, or by carbonyl cyanide m-chlorophenylhydrazone, an H+ conductor. Uptake of H+ was induced by L-serine influx. These results indicate that an H+-serine cotransport mechanism is operative in this novel L-serine transport system.     

Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle.

Fifteen-fold overexpression of phosphoenolpyruvate synthase (Pps) (EC 2.7.9.2) in Escherichia coli stimulated oxygen consumption in glucose minimal medium. A further increase in Pps overexpression to 30-fold stimulated glucose consumption by approximately 2-fold and resulted in an increased excretion of pyruvate and acetate. Insertion of two codons at the PvuII site in the pps gene abolished the enzymatic activity and eliminated the above-described effects. Both the active and the inactive proteins were detected at the predicted molecular weight by polyacrylamide gel electrophoresis. Therefore, the observed physiological changes were due to the activity of Pps. The higher specific rates of consumption of oxygen and glucose indicate a potential futile cycle between phosphoenolpyruvate (PEP) and pyruvate. A model for the stimulation of glucose uptake is presented; it involves an increased PEP/pyruvate ratio caused by the overexpressed Pps activity, leading to a stimulation of the PEP:sugar phosphotransferase system.     

The membrane potential in everted vesicles of Escherichia coli.

Ion-selective electrodes were used to measure the equilibration of thiocyanate across the membrane of everted (“inside-out”) vesicles of Escherichia coli W1485. Membrane potentials, vesicle interior positive, generated by the oxidation of NADH, succinate, and d-lactate, or by the hydrolysis of ATP, fell in the range of 100–150 mV depending on the carbon source for cell growth and the substrate used to energize the membranes. There was no relationship between the rate of oxidation of different substrates and the membrane potential they generated. The membrane potential generated by oxidation of NADH was relatively constant between pH 7.0 and 8.5. Somewhat lower values obtained at pH 5.5 to 6.5 were attributed to the effect of pH on substrate oxidation.     

Genetics of the glutamine transport system in Escherichia coli.

The active transport of glutamine by Escherichia coli occurs via a single osmotic shock-sensitive transport system which is known to be dependent upon a periplasmic binding protein specific for glutamine. We obtained a mutant that had elevated levels of glutamine transport and overproduced the glutamine binding protein. From this strain many point mutants and deletion-carrying strains defective in glutamine transport were isolated by a variety of techniques. The genetic locus coding for the glutamine transport system, glnP, and the regulatory mutation which causes overproduction of the transport system were both shown to map at 17.7 min on the E. coli chromosome, and it was demonstrated that the glnP locus contains the structural gene for the glutamine binding protein. Evidence was also obtained that the glutamine transport system, by an unknown mechanism, plays a direct role in the catabolism of glutamate and, hence, of glutamine and proline as well.     

Identification of potential active-site residues in the Escherichia coli leader peptidase.

Leader peptidase of Escherichia coli cleaves the leader sequence from the amino terminus of membrane and secreted proteins after these proteins insert across the membrane. Despite considerable research, the mechanism of catalysis of leader peptidase remains unknown. This peptidase cannot be classified using protease inhibitors to the serine, cysteine, aspartic acid, or metallo- classes of proteases (Zwizinski, C., Date, T., and Wickner, W. (1981) J. Biol. Chem. 256, 3593-3597). Using site-directed mutagenesis, we have attempted to place leader peptidase in one of these groups. We found that leader peptidase, lacking all of the cysteine residues, can cleave the leader peptide from procoat, the precursor to bacteriophage M13 coat protein. Replacement of each histidine residue with an alanyl residue was without effect on catalysis. Among all the serine and aspartic acid residues, serine 90 and serine 185 as well as aspartic acid 99, 153, 273, and 276 are necessary to cleave procoat in a detergent extract. However, only serine 90 and aspartic acid 153 were required for processing using a highly sensitive in vivo assay. In addition to the residues directly affecting catalysis, aspartic acid 99 plays a role in maintaining the structure of leader peptidase. Replacement of this residue with alanine results in a very unstable leader peptidase protein. This study thus defines two critical residues, serine 90 and aspartic acid 153, that may be directly involved in catalysis and provides evidence that leader peptidase belongs to a novel class of serine proteases.     

Specialized peptide transport system in Escherichia coli.

Trileucine is utilized as a source of leucine for growth of strains of Escherichia coli K-12 that are deficient in the oligopeptide transport system (Opp). Trithreonine is toxic to E. coli K-12. Opp- mutants of E. coli K-12 retain complete sensitivity to this tripeptide. Moreover, E. coli W, which is resistant to trithreonine, can utlize this tripeptide as a threonine source and this capability is fully maintained in E. coli W (Opp-). A spontaneous trithreonine-resistant mutant of E. coli K-12 (Opp-) has been isolated that has an impaired growth response to trileucine and is resistant to trithreonine. Trileucine competes with the uptake of trithreonine as measured by its ability to relieve trithreonine toxicity in E. coli K-12. It is concluded that trileucine as well as trithreonine are transported into E. coli K-12 or W by a common uptake system that is distinct from the Opp system. Trimethionine can act as a competitor of trileucine or trithreonine-supported growth and as an antagonist of trithreonine toxicity in Opp- mutants. It is concluded that trimethionine is recognized by the trileucine-trithreonine transport system. Trithreonine, trimethionine, and trileucine are also transported by the Opp system, as they all relieve triornithine toxicity towards E. coli W and compete with tetralysine utilization as lysine source for growth of a lysine auxotroph of this strain.     

Catabolite and transient repression in Escherichia coli do not require enzyme I of the phosphotransferase system.

Transient and catabolite repression with changes in intracellular concentrations of cyclic adenosine 3',5-monophosphate is produced by glycerol and by glucose-6-phosphate in a strain with a partial deletion of the structural gene for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.     

Specificity of the Escherichia coli proline transport system.

The presence of both the carbonyl portion of the carboxyl group at position 2 of the pyrrolidine ring and a secondary amine was essential for uptake of a compound by the proline permease of Escherichia coli. The permease possessed a high affinity for azetidine-2-carboxylic acid and for compounds with ring structures smaller than the pyrrolidine ring. Pipecolic acid, the higher homologue of proline, and its derivatives were not transported. Cis- and trans-3,4-methano-prolines, also six-membered ring structures, behaved anomolously in that they possessed a high affinity for the permease. The difference between the methano-prolines and other six-membered ring structures probably resides in the fact that the former exist in the "boat" configuration whereas the latter possess the "chair" configuration. In general, substituted prolines in the cis configuration displayed a higher affinity for the permease than did corresponding trans isomers, though the affinity for substituted prolines was influenced by the position, size, and polar or nonpolar nature of the substituent group. At O C many analogues with affinity for proline permease exchanged with intracellular proline, but some analogues, notably trans-3-methyl- and trans-4-methyl-L-prolines, though possessing high affinity for the permease, showed an almost complete inability to exchange with intracellular proline.     

Directed evolution of trimethoprim resistance in Escherichia coli

Directed evolution is a useful tool in the study of enzymes. It is used in this study to investigate the means by which resistance to the antibiotic trimethoprim develops in dihyrofolate reductase from Escherichia coli. Mutants with clinical levels of resistance were obtained after only three generations. After four generations of directed evolution, several mutants were characterized, along with some point mutants made to investigate amino acid changes of interest. Several mutations were found to grant resistance to trimethoprim, both by reducing the binding affinity of the enzyme for the drug, and by increasing the activity of the enzyme.