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.     

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.     

The evolution of DNA sequences in Escherichia coli

It is proposed that certain families of transposable elements originally evolved in plasmids and functioned in forming replicon fusions to aid in the horizontal transmission of non-conjugational plasmids. This hypothesis is supported by the finding that the transposable elements Tn3 and gamma delta are found almost exclusively in plasmids, and also by the distribution of the unrelated insertion sequences IS4 and IS5 among a reference collection of 67 natural isolates of Escherichia coli. Each insertion sequence was found to be present in only about one-third of the strains. Among the ten strains found to contain both insertion sequences, the number of copies of the elements was negatively correlated. With respect to IS5, approximately half of the strains containing a chromosomal copy of the insertion element also contained copies within the plasmid complement of the strain.     

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.     

A Darwinian evolutionary system. II. Experiments on protein evolution and evolutionary aspects of the genetic code

On the basis of the principles of Darwinian evolutionary systems laid out earlier, a system is constructed which simulates protein evolution. Two types of situations are studied: adaptation to highest possible alkalinity (“alkalinity model”), and adaptation to an arbitrary sequence (“sequence model”). No restrictions in adaptability were found for the (comparably special) alkalinity model, but severe restrictions were found for the sequence model. Approximately 15% of all possible evolutionary paths from one amino acid to another turned out to be impossible, in the sense that no chain of intermediate steps exists which leads to a higher fitness level, in this case an increased chemical similarity of the two amino acids.The evolutionary efficiency of the natural genetic code was also investigated by comparing it with two classes of artificially constructed codes: semi-random and random codes. It was found that the natural code possesses the highest evolutionary efficiency, given by the mean number of generations required to reach identity in 5 of 10 sites, if originally all 10 were different. Closest to the natural code in evolutionary efficiency were the random codes, next, the semi-random codes.This pattern could be explained by a theoretical measure, called the code efficiency. The most important component of the code efficiency is the percentage of impossible paths. The natural code is far superior to the other code types in this respect. However, the random codes are superior to the natural code with respect to the mean shortest path length of the possible paths, the other important component of the code efficiency.It is suggested that the natural genetic code might have arisen from a semi-random code during a process of optimizing several of its features, of which the evolutionary efficiency is a very important one; or that the natural code is the most efficient edition of a large variety of semi-random codes which originated by chance.     

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.     

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.     

Electrochemical potential releases a membrane-bound secretion intermediate of maltose-binding protein in Escherichia coli.

A secretionary intermediate of the Escherichia coli maltose-binding protein accumulated in the inner membrane when the membrane electrochemical potential was reduced and the cytosolic ATP concentration was normal. The intermediate was mature in size, but maintained a conformation similar to the cytosolic precursor form, and not the mature periplasmic protein, as measured by differences in susceptibility to proteinase K in vitro. The intermediate was located on the periplasmic side of the inner membrane. Restoration of the membrane electrochemical potential resulted in the movement of the intermediate from the inner membrane to the periplasm. In other experiments in which the ATP concentration was reduced by 96% and the electrochemical potential remained normal, no intermediate accumulated. Thus, the final step in the export of maltose-binding protein requires the electrochemical potential of the inner membrane and does not require ATP.     

RNase I*, a form of RNase I, and mRNA degradation in Escherichia coli.

A previously unreported endoRNase present in the spheroplast fraction of Escherichia coli degraded homoribopolymers and small RNA oligonucleotides but not polymer RNA. Like the periplasmic endoRNase, RNase I, the enzyme cleaved the phosphodiester bond between any nucleotides; however, RNase I degraded polymer RNA as fast as homopolymers or oligomers. Both enzymes migrated as 27-kDa polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and could not be separated by various chromatographic procedures. In rna insertion mutants, both enzymes were completely missing; the spheroplast enzyme is called RNase I*, since it must be a form of RNase I. The two forms could be distinguished by physical treatments. RNase I could be activated by Zn2+, while RNase I* was inactive in the presence of Zn2+. RNase I was inactivated very slowly at 100 degrees C over a wide pH range, while RNase I* was inactivated slowly by heat at pH 4.0 but much more rapidly as the pH was increased to 8.0. In the presence of a thiol-binding agent, the inactivation at the higher pH values was much slower. These results suggest that RNase I*, but not RNase I, has free sulfhydryl groups. RNase I* activity in the cell against a common substrate was estimated to be several times that of RNase I. All four 2',3'-phosphomonoribonucleotides were identified in the soluble pools of growing cells. Such degradative products must arise from RNase I* activity. The activity would be suited for the terminal step in mRNA degradation, the elimination of the final oligonucleotide fragments, without jeopardizing the cell RNA. An enzyme with very similar specificity was found in Saccharomyces cerevisiae, suggesting that the activity may be widespread in nature.     

Escherichia coli from animal reservoirs as a potential source of human extraintestinal pathogenic E. coli

Extraintestinal pathogenic Escherichia coli (ExPEC) are an important cause of urinary tract infections, neonatal meningitis and septicaemia in humans. Animals are recognized as a reservoir for human intestinal pathogenic E. coli, but whether animals are a source for human ExPEC is still a matter of debate. Pathologies caused by ExPEC are reported for many farm animals, especially for poultry, in which colibacillosis is responsible for huge losses within broiler chickens. Cases are also reported for companion animals. Commensal E. coli strains potentially carrying virulence factors involved in the development of human pathologies also colonize the intestinal tract of animals. This review focuses on the recent evidence of the zoonotic potential of ExPEC from animal origin and their potential direct or indirect transmission from animals to humans. As antimicrobials are commonly used for livestock production, infections due to antimicrobial-resistant ExPEC transferred from animals to humans could be even more difficult to treat. These findings, combined with the economic impact of ExPEC in the animal production industry, demonstrate the need for adapted measures to limit the prevalence of ExPEC in animal reservoirs while reducing the use of antimicrobials as much as possible.