Evolution and functional characterization of the RH50 gene from the ammonia-oxidizing bacterium Nitrosomonas europaea

The family of ammonia and ammonium channel proteins comprises the Amt proteins, which are present in all three domains of life with the notable exception of vertebrates, and the homologous Rh proteins (Rh50 and Rh30) that have been described thus far only in eukaryotes. The existence of an RH50 gene in bacteria was first revealed by the genome sequencing of the ammonia-oxidizing bacterium Nitrosomonas europaea. Here we have used a phylogenetic approach to study the evolution of the N. europaea RH50 gene, and we show that this gene, probably as a component of an integron cassette, has been transferred to the N. europaea genome by horizontal gene transfer. In addition, by functionally characterizing the Rh50_Ne protein and the corresponding knockout mutant, we determined that NeRh50 can mediate ammonium uptake. The RH50_Ne gene may thus have replaced functionally the AMT gene, which is missing in the genome of N. europaea and may be regarded as a case of nonorthologous gene displacement.     

Causes and consequences of DNA repair activity modulation during stationary phase in Escherichia coli

Escherichia coli responds to nutrient exhaustion by entering a state commonly referred to as the stationary phase. Cells entering the stationary phase redirect metabolic circuits to scavenge any available nutrients and become resistant to different stresses. However, many DNA repair pathways are downregulated in stationary-phase cells, which results in increased mutation rates. DNA repair activity generally depends on consumption of energy and often requires de novo proteins synthesis. Consequently, unless stringently regulated during stationary phase, DNA repair activities may lead to an irreversible depletion of energy sources and, therefore to cell death. Most stationary phase morphological and physiological modifications are regulated by an alternative RNA polymerase sigma factor RpoS. However, nutrient availability, and the frequency and nature of stresses, are different in distinct environmental niches, which impose conflicting choices that result in selection of the loss or of the modification of RpoS function. Consequently, DNA repair activity, which is partially controlled by RpoS, is differently modulated in different environments. This results in the variable mutation rates among different E. coli ecotypes. Hence, the polymorphism of mutation rates in natural E. coli populations can be viewed as a byproduct of the selection for improved fitness.     

Caenorhabditis elegans as a simple model to study phenotypic and genetic virulence determinants of extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause disease by invading normally sterile niches within the host body, e.g., urinary tract, blood and cerebrospinal fluid. Infections due to ExPEC strains, in particular urinary tract infections, cause considerable morbidity and significant health-care costs. The goal of our study is to evaluate whether Caenorhabditis elegans can be used as a model to study phenotypic and genetic virulence determinants of ExPEC strains. For this purpose, we used a collection of 31 E. coli strains isolated during acute extra-intestinal infections or from the feces of healthy individuals. For all strains, the phylogeny, the presence of ExPEC virulence factors, the resistance to biologically relevant stressors (bile, human serum and lysozyme), the motility, the growth rate, the virulence in C. elegans and in a murine septicaemia model has been established. The results show that there is a strong link between virulence in C. elegans and certain phenotypic and genetic virulence predictors of ExPEC strains determinable in vitro. Furthermore, there is a significant correlation between virulence of different ExPEC strains in C. elegans and in the murine model. Therefore, our results suggest that C. elegans can be used as a model to study virulence determinants of ExPEC strains.     

Antibiotic-mediated recombination: ciprofloxacin stimulates SOS-independent recombination of divergent sequences in Escherichia coli

The widespread use and abuse of antibiotics as therapeutic agents has produced a major challenge for bacteria, leading to the selection and spread of antibiotic resistant variants. However, antibiotics do not seem to be mere selectors of these variants. Here we show that the fluoroquinolone antibiotic ciprofloxacin, an inhibitor of type II DNA topoisomerases, stimulates intrachromosomal recombination of DNA sequences. The stimulation of recombination between divergent sequences occurs via either the RecBCD or RecFOR pathways and is, surprisingly, independent of SOS induction. Additionally, this stimulation also occurs in a hyperrecombinogenic mismatch repair mutS mutant. It is worth noting that ciprofloxacin also stimulates the conjugational recombination of an antibiotic resistance gene. Finally, we demonstrate that Escherichia coli is able to recover from treatments with recombination-stimulating concentrations of the antibiotic. Thus, fluoroquinolones can increase genetic variation by the stimulation of the recombinogenic capability of treated bacteria (via an SOS-independent mechanism) and consequently may favour the acquisition, evolution and spread of antibiotic resistance determinants.     

Posttranslational modification of 6-phosphofructo-1-kinase as an important feature of cancer metabolism

Background

Human cancers consume larger amounts of glucose compared to normal tissues with most being converted and excreted as lactate despite abundant oxygen availability (Warburg effect). The underlying higher rate of glycolysis is therefore at the root of tumor formation and growth. Normal control of glycolytic allosteric enzymes appears impaired in tumors; however, the phenomenon has not been fully resolved.

Methodology/Principal Findings

In the present paper, we show evidence that the native 85-kDa 6-phosphofructo-1-kinase (PFK1), a key regulatory enzyme of glycolysis that is normally under the control of feedback inhibition, undergoes posttranslational modification. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter 47-kDa fragment was formed that was insensitive to citrate and ATP inhibition. In tumorigenic cell lines, only the short fragments but not the native 85-kDa PFK1 were detected by immunoblotting. Similar fragments were detected also in a tumor tissue that developed in mice after the subcutaneous infection with tumorigenic B16-F10 cells. Based on limited proteolytic digestion of the rabbit muscle PFK-M, an active citrate inhibition-resistant shorter form was obtained, indicating that a single posttranslational modification step was possible. The exact molecular masses of the active shorter PFK1 fragments were determined by inserting the truncated genes constructed from human muscle PFK1 cDNA into a pfk null E. coli strain. Two E. coli transformants encoding for the modified PFK1s of 45,551 Da and 47,835 Da grew in glucose medium. The insertion of modified truncated human pfkM genes also stimulated glucose consumption and lactate excretion in stable transfectants of non-tumorigenic human HEK cell, suggesting the important role of shorter PFK1 fragments in enhancing glycolytic flux.

Conclusions/Significance

Posttranslational modification of PFK1 enzyme might be the pivotal factor of deregulated glycolytic flux in tumors that in combination with altered signaling mechanisms essentially supports fast proliferation of cancer cells.     

Intermediate mutation frequencies favor evolution of multidrug resistance in Escherichia coli

In studying the interplay between mutation frequencies and antibiotic resistance among Escherichia coli natural isolates, we observed that modest modifications of mutation frequency may significantly influence the evolution of antibiotic resistance. The strains having intermediate mutation frequencies have significantly more antibiotic resistances than strains having low and high mutation frequencies.     

citrate inhibition-resistant form of 6-phosphofructo-1-kinase from Aspergillus niger

Two forms of Aspergillus niger 6-phosphofructo-1-kinase (PFK1) have been described recently, the 85-kDa native enzyme and 49-kDa shorter fragment that is formed from the former by posttranslational modification. So far, kinetic characteristics have never been determined on the enzyme purified to near homogeneity. For the first time, kinetic parameters were determined for individual enzymes with respect to citrate inhibition. The native 85-kDa enzyme was found to be moderately inhibited by citrate, with the Ki value determined to be 1.5 mM, in the system with 5 mM Mg2+ ions, while increasing magnesium concentrations relieved the negative effect of citrate. An identical inhibition coefficient was determined also in the presence of ammonium ions, although ammonium acted as a strong activator of enzyme activity. On the other hand, the shorter fragment of PFK1 proved to be completely resistant to inhibition by citrate. Allosteric citrate binding sites were most probably lost after the truncation of the C-terminal part of the native protein, in which region some binding sites for inhibitor are known to be located. At near physiological conditions, characterized by low fructose-6-phosphate concentrations, a much higher efficiency of the shorter fragment was observed during an in vitro experiment. Since the enzyme became more susceptible to the positive control by specific ligands, while the negative control was lost after posttranslational modification, the shorter PFK1 fragment seems to be the enzyme most responsible for generating undisturbed metabolic flow through glycolysis in A. niger cells.     

Generation of DNA-free Escherichia coli cells by 2-aminopurine requires mismatch repair and nonmethylated DNA

Undirected mismatch repair initiated by the incorporation of the base analog 2-aminopurine kills DNA-methylation-deficient Escherichia coli dam cells by DNA double-strand breakage. Subsequently, the chromosomal DNA is totally degraded, resulting in DNA-free cells.     

Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair

Mismatch repair (MMR) is an evolutionarily conserved DNA repair system, which corrects mismatched bases arising during DNA replication. MutS recognizes and binds base pair mismatches, while the MutL protein interacts with MutS-mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination site on the DNA. The mechanism of communication between these two distal sites on the DNA is not known. We used functional fluorescent MMR proteins, MutS and MutL, in order to investigate the formation of the fluorescent MMR protein complexes on mismatches in real-time in growing Escherichia coli cells. We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci. MutL foci were, on average, 2.7 times more intense than the MutS foci co-localized on individual mismatches. A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold. This indicates that MutL accumulates from the mismatch site toward strand discrimination site along the DNA. Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.