SUMMARY Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments. Moreover, the pros and cons of different antigen display methods and the use of naïve versus immunized phage display antibody libraries is discussed, and selected examples from the field of antivenom research are highlighted. This review thus provides in-depth knowledge on the principles and use of phage display technology with a special focus on discovery of antibodies that target animal toxins.

Phenotype switching is commonly observed in nature. This prevalence has allowed the elucidation of a number of underlying molecular mechanisms. However, little is known about how phenotypic switches arise and function in their early evolutionary stages. The first opportunity to provide empirical insight was delivered by an experiment in which populations of the bacterium Pseudomonas fluorescens SBW25 evolved, de novo, the ability to switch between two colony phenotypes. Here we unravel the molecular mechanism behind colony switching, revealing how a single nucleotide change in a gene enmeshed in central metabolism (carB) generates such a striking phenotype. We show that colony switching is underpinned by ON/OFF expression of capsules consisting of a colanic acid-like polymer. We use molecular genetics, biochemical analyses, and experimental evolution to establish that capsule switching results from perturbation of the pyrimidine biosynthetic pathway. Of central importance is a bifurcation point at which uracil triphosphate is partitioned towards either nucleotide metabolism or polymer production. This bifurcation marks a cell-fate decision point whereby cells with relatively high pyrimidine levels favour nucleotide metabolism (capsule OFF), while cells with lower pyrimidine levels divert resources towards polymer biosynthesis (capsule ON). This decision point is present and functional in the wild-type strain. Finally, we present a simple mathematical model demonstrating that the molecular components of the decision point are capable of producing switching. Despite its simple mutational cause, the connection between genotype and phenotype is complex and multidimensional, offering a rare glimpse of how noise in regulatory networks can provide opportunity for evolution. Phenotype switching—the ability to switch rapidly between phenotypic states—is an evolutionary survival strategy commonly used by organisms in the face of unpredictable environmental conditions. However, little is known about how phenotype switches emerge and function in their early evolutionary stages. A previous study observed the evolutionary emergence of colony morphology switching in Pseudomonas fluorescens populations in response to fluctuating selection. Here we describe the underlying molecular basis of this colony switching, providing the first account of the mechanism behind a real-time evolved phenotype switch. We show that colony switching in this instance is underpinned at the cellular level by high frequency ON/OFF expression of colanic acid-like capsules in response to varying levels of a metabolite. Biochemical assays revealed that capsule switching results from mutations that reduce concentrations of intermediates in a central metabolic pathway—the pyrimidine biosynthetic pathway. Of key importance is the partitioning of these metabolic resources between polymer production (leading to capsulation) and cell division (leading to noncapsulation); this bifurcation marks a decision point whereby cells with low metabolite levels divert resources towards polymer production, increasing the likelihood of switching to the capsulated state. As a greater proportion of cells become capsulated, colony switching emerges. These findings show that, while colony switching evolved with relative ease, the underlying molecular mechanism is surprisingly complex.

Phage therapy has regained interest in recent years due to the alarming spread of antibiotic resistance. Whilst phage cocktails are commonly sold in pharmacies in countries such as Georgia and Russia, this is not the case in western countries due to western regulatory agencies requiring a thorough characterization of the drug. Here, DNA sequencing of constituent biological entities constitutes a first step. The pyophage (PYO) cocktail is one of the main commercial products of the Georgian Eliava Institute of Bacteriophage, Microbiology and Virology and is used to cure skin infections. Since its first production in the 1930s, the composition of the cocktail has been periodically modified to add phages effective against emerging pathogenic strains. In this paper, we compared the composition of three PYO cocktails from 1997 (PYO97), 2000 (PYO2000) and 2014 (PYO2014). Based on next generation sequencing, de novo assembly and binning of contigs into draft genomes based on tetranucleotide distance, thirty and twenty-nine phage draft genomes were predicted in PYO97 and PYO2014, respectively. Of these, thirteen and fifteen shared high similarity to known phages. Eleven draft genomes were found to be common in the two cocktails. One of these showed no similarity to publicly available phage genomes. Representatives of phages targeting E. faecalis, E. faecium, E. coli, Proteus, P. aeruginosa and S. aureus were found in both cocktails. Finally, we estimated larger overlap of the PYO2000 cocktail to PYO97 compared to PYO2014. Using next generation sequencing and metagenomics analysis, we were able to characterize and compare the content of PYO cocktails separated by 17 years in time. Even though the cocktail composition is upgraded every six months, we found it to remain relatively stable over the years.

Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis . It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.

In the genome of the gram-positive bacterium Lactococcus lactis MG1363, we have identified three genes ( clpC , clpE , and clpB ) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The clpE gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX ₂ CX ₂₂ CX ₂ C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded by clpB was strongly induced by heat shock and, while clpC mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in clpB , clpC , or clpE , we found that although the genes are part of the L. lactis heat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins, clpE mutant cells formed smaller colonies than wild-type cells and clpB and clpC mutant cells. Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in L. lactis , could be necessary for degrading proteins generated by certain types of stress.