Prokaryotic Selectivity Research Articles

Phylogenetic profiling in eukaryotes: The effect of species, orthologous group, and interactome selection on protein interaction prediction.

Phylogenetic profiling in eukaryotes is of continued interest to study and predict the functional relationships between proteins. This interest is likely driven by the increased number of available diverse genomes and computational methods to infer orthologies. The evaluation of phylogenetic profiles has mainly focussed on reference genome selection in prokaryotes. However, it has been proven to be challenging to obtain high prediction accuracies in eukaryotes. As part of our recent comparison of orthology inference methods for eukaryotic genomes, we observed a surprisingly high performance for predicting interacting orthologous groups. This high performance, in turn, prompted the question of what factors influence the success of phylogenetic profiling when applied to eukaryotic genomes. Here we analyse the effect of species, orthologous group and interactome selection on protein interaction prediction using phylogenetic profiles. We select species based on the diversity and quality of the genomes and compare this supervised selection with randomly generated genome subsets. We also analyse the effect on the performance of orthologous groups defined to be in the last eukaryotic common ancestor of eukaryotes to that of orthologous groups that are not. Finally, we consider the effects of reference interactome set filtering and reference interactome species. In agreement with other studies, we find an effect of genome selection based on quality, less of an effect based on genome diversity, but a more notable effect based on the amount of information contained within the genomes. Most importantly, we find it is not merely selecting the correct genomes that is important for high prediction performance. Other choices in meta parameters such as orthologous group selection, the reference species of the interaction set, and the quality of the interaction set have a much larger impact on the performance when predicting protein interactions using phylogenetic profiles. These findings shed light on the differences in reported performance amongst phylogenetic profiles approaches, and reveal on a more fundamental level for which types of protein interactions this method has most promise when applied to eukaryotes.

Design, Synthesis and Antibacterial Studies of Novel Cationic Amphipathic Cyclic Undecapeptides and Their Linear Counterparts against Virulent Bacterial Strains

Bacteria have acquired resistance against almost all antibiotics because of the misuse of antibacterial agents and long periods of treatment. Antimicrobial peptides (AMPs) are one of the most encouraging candidates to solve this problem, as they possess high prokaryotic selectivity, and affect the bacteria by a unique mode of action. Novel cyclic undecapeptides (QNRNFYFNRNQ and QNRNFHFNRNQ) and their linear counterparts were investigated for their antibacterial activity against virulent strains. The minimal inhibitory concentration (MIC) values showed that tyrosine and histidine AMPs have promising antibacterial activity against virulent bacteria. The MIC values against the P. aeruginosa PA14, E. coli O157:H7 CR3, S. aureus 209P, and B. subtilis ATCC 6633 bacterial strains were evaluated for the cyclic peptide containing tyrosine, and their values were 6.25, 12.5, 12.5, and 12.5 µM, respectively. Meanwhile, for the linear form, they were 9.3, 12.5, 12.5, and 12.5 µM, respectively. The cyclic-peptide–containing histidines’ MIC values were 6.25, 3.1, 6.25, and 3.1 µM, respectively. Meanwhile, for the linear form, they were 3.1, 3.1, 3.1, and 6.25 µM, respectively. The antibacterial activities of the new AMPs were compared with that of gentamicin sulfate, and showed relatively higher potencies. Time-inhibition studies demonstrated the rapid antibacterial effects of the novel AMPs, which were more likely to be concentration-dependent, rather than time-dependent. At double the MIC concentration, all of the tested peptides exhibited relatively stable antibacterial effects up to 24 h, especially the peptides containing tyrosine, which showed an improved antibacterial effect.

Design and Synthesis of a New Amphipathic Cyclic Decapeptide with Rapid, Stable, and Continuous Antibacterial Effects

Pathogens can acquire high resistance against even the most powerful antibiotics because of the long periods of treatment and high usage of antimicrobial agents. In addition, the severe side effects of commonly used antibiotics can initiate secondary diseases or may lead to death. Antimicrobial peptides (AMPs) have been reported to exhibit prokaryotic selectivity and low microbial resistance. Furthermore, AMPs show a good ability to penetrate the cell walls of microorganisms. In this study, a cyclic decapeptide and its linear counterpart were synthesized by a standard solid phase peptide synthesis method (SPPS) in a quantitative yield of the linear decapeptide (97%) and a good yield of the cyclic form (45%). Antibacterial studies were performed using Escherichia coli (a widespread Gram-negative pathogen) and Bacillus thuringiensis as a representative Gram-positive pathogen. The minimal inhibitory concentration (MIC) values were evaluated by the broth microdilution method. The cyclic peptide and its linear counterpart exhibited MIC values of 0.16 and 0.3 mg/mL, respectively, against Escherichia coli. Against Bacillus thuringiensis, the peptides had the same MIC value of 0.24 mg/mL. Time-kill studies were performed using E. coli, which indicated a fast killing effect of both peptides (≥ 99% of the bacterial cells) after 1 h of incubation using a concentration of two times the MIC value for each peptide. Moreover, bacterial cell viability studies against E. coli carried out using a high bacterial concentration showed that both peptides have a maximum killing effect of more than 80% of the tested bacterial cells.

Structure-Guided Enhancement of Selectivity of Chemical Probe Inhibitors Targeting Bacterial Seryl-tRNA Synthetase.

Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high-resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than 2 orders of magnitude compared to their human homologue, demonstrating a route to the selective chemical inhibition of these bacterial targets.

Surface Phenomena Enhancing the Antibacterial and Osteogenic Ability of Nanocrystalline Hydroxyapatite, Activated by Multiple-Ion Doping.

The present work describes a novel nanocrystalline, multidoped hydroxyapatite featuring excellent eukaryotic versus prokaryotic cell selectivity, attested by excellent osteoinductive character and evaluated with human stem cells, and anti-infective ability, tested against different pathogens. Physicochemical analysis and transmission electron microscopy (TEM)/scanning STEM observations highlighted that such enhanced biological features are related to the lower crystallinity level and increased surface charge of hydroxyapatite, both induced by multiple-ion doping. Specifically, the lattice substitution of Ca2+ with Zn2+ promotes the segregation of Ca2+ and doping Mg2+ cations to a less-ordered surface layer, thus promoting dynamic ion absorption/release acting as bioactive signals for cells and exerting an antiproliferative effect on all tested pathogens. These findings open the design of new biodevices, combining regenerative ability and effective microbial inhibition without using any antibiotic drugs. This is extremely important to circumvent bacterial resistance to antibiotics, which is today considered as one of the biggest threats to global health.

Calcium Phosphate Nanoparticles as Intrinsic Inorganic Antimicrobials: The Antibacterial Effect.

Cheap and simple to make, calcium phosphate (CP), thanks to its unusual functional pleiotropy, belongs to the new wave of abundant and naturally accessible nanomaterials applicable as a means to various technological ends. It is used in a number of industries, including the biomedical, but its intrinsic antibacterial activity in the nanoparticle form has not been sufficiently explored to date. In this study, we report on this intrinsic antibacterial effect exhibited by two distinct CP phases: an amorphous CP (ACP) and hydroxyapatite (HAp). The effect is prominent against a number of regular bacterial species, including Staphylococcus aureus, Staphylococcus epidermis, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa, but also their multidrug-resistant (MDR) analogues. Although ACP and HAp displayed similar levels of activity against Gram-negative organisms, ACP proved to be more effective against the Gram-positive ones, with respect to which HAp was mostly inert, yet this trend became reversed for the MDR strains. In addition to the intrinsic antimicrobial effect of CP nanoparticles, we have also observed a synergistic effect between the nanoparticles and certain antibiotics. Both forms of CP were engaged in a synergistic relationship with a variety of concomitantly delivered antibiotics, including ampicillin, kanamycin, oxacillin, vancomycin, minocycline, erythromycin, linezolid, and clindamycin, and enabled even antibiotics completely ineffective against particular bacterial strains to significantly suppress their growth. This relationship was complex; depending on a particular CP phase, bacterial strain and antibiotic, the antibacterial activity (i) intensified proportionally to the nanoparticle concentration, (ii) plateaued immediately after the introduction of nanoparticles in minute amounts, or (iii) exhibited concentration-dependent minima due to stress-induced biofilm formation. These findings present grounds for the further optimization of CP properties and maximization of this intriguing effect, which could in the long run make this material comparable in activity to the inorganics of choice for this application, including silver, copper, or zinc oxide, while retaining its superb safety profile and positive eukaryotic versus prokaryotic cell selectivity.

Cis-regulatory evolution in prokaryotes revealed by interspecific archaeal hybrids

The study of allele-specific expression (ASE) in interspecific hybrids has played a central role in our understanding of a wide range of phenomena, including genomic imprinting, X-chromosome inactivation, and cis-regulatory evolution. However across the hundreds of studies of hybrid ASE, all have been restricted to sexually reproducing eukaryotes, leaving a major gap in our understanding of the genomic patterns of cis-regulatory evolution in prokaryotes. Here we introduce a method to generate stable hybrids between two species of halophilic archaea, and measure genome-wide ASE in these hybrids with RNA-seq. We found that over half of all genes have significant ASE, and that genes encoding kinases show evidence of lineage-specific selection on their cis-regulation. This pattern of polygenic selection suggested species-specific adaptation to low phosphate conditions, which we confirmed with growth experiments. Altogether, our work extends the study of ASE to archaea, and suggests that cis-regulation can evolve under polygenic lineage-specific selection in prokaryotes.

Characterisation of three alpha-helical antimicrobial peptides from the venom of Scorpio maurus palmatus

Scorpion venoms provide a rich source of anti-microbial peptides. Here we characterise three from the venom of Scorpion maurus palmatus. Smp13 is biologically inactive, despite sharing homology with other antimicrobial peptides, probably because it lacks a typically charged structure. Both Smp-24 and Smp-43 have broad spectrum antimicrobial activity, disrupting bacterial membranes. In addition, there is evidence that Smp24 may inhibit DNA synthesis in Bacillus subtilis. Smp24 haemolysed red blood cells but in contrast, Smp43 was non-haemolytic. The introduction of a flexible Gly-Val-Gly hinge into the middle of Smp24 did not alter the haemolytic activity of Smp24 (as might have been predicted from earlier studies with Pandinin2 (Pin2), although C-terminal truncation of Smp-24 reduced its haemolytic activity, in agreement with earlier Pin 2 studies. Smp24 and its derivatives, as well as Smp-43, were all cytotoxic (ATP release assay) toward mammalian HepG2 liver cells. Our results highlight the beneficial effect of helical-hinge-helical conformation on promoting prokaryotic selectivity of long chain scorpion AMPs, as well as the importance of examining a wide range of mammalian cell types in cytotoxicity testing.

De Novo Design and Synthesis of Ultra-Short Peptidomimetic Antibiotics Having Dual Antimicrobial and Anti-Inflammatory Activities

BackgroundMuch attention has been focused on the design and synthesis of potent, cationic antimicrobial peptides (AMPs) that possess both antimicrobial and anti-inflammatory activities. However, their development into therapeutic agents has been limited mainly due to their large size (12 to 50 residues in length) and poor protease stability.Methodology/Principal FindingsIn an attempt to overcome the issues described above, a set of ultra-short, His-derived antimicrobial peptides (HDAMPs) has been developed for the first time. Through systematic tuning of pendant hydrophobic alkyl tails at the N(π)- and N(τ)-positions on His, and the positive charge of Arg, much higher prokaryotic selectivity was achieved, compared to human AMP LL-37. Additionally, the most potent HDAMPs showed promising dual antimicrobial and anti-inflammatory activities, as well as anti–methicillin-resistant Staphylococcus aureus (MRSA) activity and proteolytic resistance. Our results from transmission electron microscopy, membrane depolarization, confocal laser-scanning microscopy, and calcein-dye leakage experiments propose that HDAMP-1 kills microbial cells via dissipation of the membrane potential by forming pore/ion channels on bacterial cell membranes.Conclusion/SignificanceThe combination of the ultra-short size, high-prokaryotic selectivity, potent anti-MRSA activity, anti-inflammatory activity, and proteolytic resistance of the designed HDAMP-1, -3, -5, and -6 makes these molecules promising candidates for future antimicrobial therapeutics.

Prokaryotic Selectivity, Bactericidal Mechanism and LPS-neutralizing Activity of Lys-linked Dimeric Peptide of Indolicidin C-terminal Hexapeptide

Indolicidin (Ind) is a cationic antimicrobial peptide (AMP) derived from the granules of bovine neutrophils. It is a short, 13 amino acid peptide (ILPWKWPWWPWRR-NH2) that is amidated at the C-terminus and contains the highest proportion of Trp and Pro residues. Like many other AMPs, it exhibits a broad spectrum of antimicrobial activity against bacteria, fungi and protozoa. It has been shown that Ind adopts a disordered conformation in solution and a membrane-interacting structure containing coils and turns. However, Ind exhibits some toxicity to human erythrocytes and mouse T lymphocytes. In the present study, to investigate the effect of Lys-linked dimerization of Ind C-terminal hexapeptide (WWPWRRNH2) on prokaryotic selectivity, bactericidal mechanism and LPS-neutralizing activity and to develop a novel short AMP having prokaryotic selectivity and LPS-neutralizing activity, we synthesized a Lys-linked dimeric peptide (di-Ind-6) of Ind C-terminal hexapeptide (Ind-6) (Table 1). We examined the antimicrobial activity of the peptides against a representative set of bacterial strains, including three Gram-negative bacteria (Escherichia coli [KCTC 1682], Pseudomonas aeruginosa [KCTC 1637] and Salmonella typhimurium [KCTC 1926]) and three Gram-positive bacteria (Bacillus subtilis [KCTC 3068], Staphylococcus epidermidis [KCTC 1917] and Staphylococcus aureus [KCTC 1621]). The MIC values of the peptides are shown in Table 2. A Lys-linked dimeric peptide (di-Ind-6) displayed 2to 4-fold increased antimicrobial activity compared to monomeric peptide (Ind6). Furthermore, Di-Ind-6 displayed a 2-fold enhanced antimicrobial activity against Gram-negative bacteria as compare to Ind. Next, the cytotoxicity of the peptides to mammalian cells was determined by measuring their hemolytic activity toward human red blood cells (h-RBCs) Concentrationresponse curves for the hemolytic activity of the peptides are shown in Figure 1. For a quantitative measure of the hemolytic activity of the peptides, we introduced the hemolytic concentration (HC50) defined as the lowest peptide concentration that produces 50% hemolysis (Table 3). The HC50 value for Ind and di-Ind-6 was 160 μg/mL and 192 μg/mL, respectively. Di-Ind-6 was somewhat less hemolytic than Ind. In contrast, Ind-6 did not induce hemolysis even at the highest peptide concentration tested (200 μg/mL). The therapeutic potential of peptide antimicrobial drugs lies in their prokaryotic selectivity to effectively kill bacterial cells without exhibiting significant cytotoxicity toward mammalian cells such as human red blood cells. To determine prokaryotic selectivity of the peptides, we calculated their therapeutic index (TI) (Table 3). The TI is a widely employed parameter to represent the specificity of antimicrobial agents. The TI of the peptides is calculated as the ratio of the HC50 to GM (geometric mean of MICs against six selected Table 1. Amino acid sequences and calculated and observed molecular masses of the peptides

Basis of Prokaryotic Selectivity in the Antibiotic Paromomycin

Ribosomes are responsible for protein synthesis and are a major target of antibiotics. While translation is a universally conserved cellular process, the ability of drugs to target prokaryotic ribosomes depends on subtle variations from eukaryotic ribosomes. The ribosome is composed of ribosomal RNA (rRNA) and protein. The small ribosomal subunit, called 30s in prokaryotes, contains 21 proteins and one rRNA (16S) and the large subunit, called 50S, contains 31 proteins and two rRNAs (23S and 5S). Recent crystal structures reveal that the rRNAs adopt a 3D fold generating (I) decoding center for codon‐anticodon recognition, (II) a peptidyl‐transfer center (PTC) for a peptide bond formation and (III) an exit tunnel through which the nascent protein emerges. Paromomycin, used in the treatment of intestinal infections, inhibits prokaryotic ribosomes at the decoding site. Paromomycin physically restructures helix H44 of the 16S rRNA, preventing proper rotation of A1492 and A1493 during anticodon:codon recognition, decreasing tRNA selection accuracy in prokaryotic ribosomes. However, paromomycin fails to affect eukaryotes due to an A to G transition at position 1408. The Brookfield Academy SMART Team (Students Modeling A Research Topic) modeled a prokaryotic ribosome, highlighting nucleotides responsible for the prokaryotic specificity of paromomycin. (Supported by a grant from the NIH‐CTSA UL1RR031973)

Design of Short Indolicidin Analogs with Enhanced Prokaryotic Selectivity

Indolicidin (ID) is a 13-residue Trp-rich antimicrobial peptide (AMP) isolated from bovine neutrophils. In addition to having a high antimicrobial potency, it is also toxic to mammalian cells. To develop novel ID-derived AMPs with shorter lengths and enhanced prokaryotic selectivities (meaning potent antimicrobial activity against bacterial cells without toxicity against mammalian cells) over the parental ID, several ID analogs were designed and synthesized. Finally, 10-residue ID analogs (SI, SI-PA, SI-WF and SI-WL) with much higher prokaryotic selectivity than the parental ID were developed. Our results suggest that the hydrophobic and aromatic amino acids at the central position of the analog SI with the highest prokaryotic selectivity are important for potent antimicrobial activity, but two Pro residues do not affect antimicrobial activity. The order of prokaryotic selectivity for ID and its designed analogs was SI > SI-PA > SI-WF > SI-WL > ID > SI-WA. Taken together, our designed short ID analogs could be developed as therapeutic agents for treating bacterial infections.

Prokaryotic Selectivity, Anti-endotoxic Activity and Protease Stability of Diastereomeric and Enantiomeric Analogs of Human Antimicrobial Peptide LL-37

LL-37 is the only antimicrobial peptide (AMP) of the human cathelicidin family. In addition to potent antimicrobial activity, LL-37 is known to have the potential to inhibit lipolysaccharide (LPS)-induced endotoxic effects. To provide the stability to proteolytic digestion and increase prokaryotic selectivity and/or anti-endotoxic activity of two Lys/Trp-substituted 19-meric antimicrobial peptides (a4-W1 and a4-W2) designed from IG-19 (residues 13-31 of LL-37), we synthesized the diastereomeric peptides (a4-W1-D and a4-W2-D) with D-amino acid substitution at positions 3, 7, 10, 13 and 17 of a4-W1 and a4-W2, respectively and the enantiomeric peptides (a4-W1-E and a4-W2-E) composed D-amino acids. The diastereomeric peptides exhibited the best prokaryotic selectivity and effective protease stability, but no or less anti-endotoxic activity. In contrast, the enantiomeric peptides had not only prokaryotic selectivity and anti-endotoxic activity but also protease stability. Our results suggest that the hydrophobicity and -helicity of the peptide is important for anti-endotoxic activity. In particular, the enantiomeric peptides showed potent anti-endotoxic and LPS-neutralizing activities comparable to that of LL-37. Taken together, both a4-W1-E and a4-W2-E holds promise as a template for the development of peptide antibiotics for the treatment of endotoxic shock and sepsis.

Prokaryotic selectivity and LPS-neutralizing activity of short antimicrobial peptides designed from the human antimicrobial peptide LL-37

To develop novel antimicrobial peptides (AMPs) with shorter lengths, improved prokaryotic selectivity and retained lipolysaccharide (LPS)-neutralizing activity compared to human cathelicidin AMP, LL-37, a series of amino acid-substituted analogs based on IG-19 (residues 13–31 of LL-37) were synthesized. Among the IG-19 analogs, the analog a4 showed the highest prokaryotic selectivity, but much lower LPS-neutralizing activity compared to parental LL-37. The analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed LPS-neutralizing activity comparable to that of LL-37, but much lesser prokaryotic selectivity. These results indicate that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. Furthermore, to increase LPS-neutralizing activity of the analog a4 without a remarkable decrease in prokaryotic selectivity, we synthesized Trp-substituted analogs (a4-W1 and a4-W2), in which Phe5 or Phe15 of a4 is replaced by Trp. Despite their same prokaryotic selectivity, a4-W2 displayed much higher LPS-neutralizing activity compared to a4-W1. When compared with parental LL-37, a4-W2 showed retained LPS-neutralizing activity and 2.8-fold enhanced prokaryotic selectivity. These results suggest that the effective site for Trp-substitution when designing novel AMPs with higher LPS-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. Taken together, the analog a4-W2 can serve as a promising template for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection.

The Logic of Evolution: Review of the Logic of Chance by Eugene V. Koonin

The Logic of Evolution: Review of the Logic of Chance by Eugene V. Koonin

Antimicrobial decapeptide KSL-W enhances neutrophil chemotaxis and function

Mammalian cationic antimicrobial peptides have received increased attention over the last decade, due to their prokaryotic selectivity and decreased risk of microbial resistance. In addition, antimicrobial peptides display differential biological effects on mammalian immune cell function, such as migration, adhesion, and modulation of respiratory burst, which make them even more attractive as therapeutic agents. Synthetic combinatorial libraries provide a time-efficient and cost-effective source for these diverse molecules. The novel synthetic antimicrobial peptide, KSLW (KKVVFWVKFK-NH2), has been shown to display a broad spectrum of antimicrobial activity against Gram (+) and Gram (−) bacteria, fungi and viruses. In this study, we evaluated the alternative biological activity of the decapeptide on neutrophil migration and function. KSLW was demonstrated to be chemotactic for neutrophils in micromolar amounts, and neutrophil treatment with KSLW, after 1min, resulted in significant increases in F-actin polymerization. KSLW was shown to inhibit oxygen radical production in PMA- and LPS-stimulated neutrophils. Future studies, to determine if KSLW regulates neutrophil phagocytosis, adhesion, and apoptosis, or examining the effect of KSLW on other mammalian cell types, such as cell populations of healing-impaired wounds, would provide significant insight for the potential therapeutic strategies offered by antimicrobial peptides.

Translational Selection Is Ubiquitous in Prokaryotes

Codon usage bias in prokaryotic genomes is largely a consequence of background substitution patterns in DNA, but highly expressed genes may show a preference towards codons that enable more efficient and/or accurate translation. We introduce a novel approach based on supervised machine learning that detects effects of translational selection on genes, while controlling for local variation in nucleotide substitution patterns represented as sequence composition of intergenic DNA. A cornerstone of our method is a Random Forest classifier that outperformed previous distance measure-based approaches, such as the codon adaptation index, in the task of discerning the (highly expressed) ribosomal protein genes by their codon frequencies. Unlike previous reports, we show evidence that translational selection in prokaryotes is practically universal: in 460 of 461 examined microbial genomes, we find that a subset of genes shows a higher codon usage similarity to the ribosomal proteins than would be expected from the local sequence composition. These genes constitute a substantial part of the genome—between 5% and 33%, depending on genome size—while also exhibiting higher experimentally measured mRNA abundances and tending toward codons that match tRNA anticodons by canonical base pairing. Certain gene functional categories are generally enriched with, or depleted of codon-optimized genes, the trends of enrichment/depletion being conserved between Archaea and Bacteria. Prominent exceptions from these trends might indicate genes with alternative physiological roles; we speculate on specific examples related to detoxication of oxygen radicals and ammonia and to possible misannotations of asparaginyl–tRNA synthetases. Since the presence of codon optimizations on genes is a valid proxy for expression levels in fully sequenced genomes, we provide an example of an “adaptome” by highlighting gene functions with expression levels elevated specifically in thermophilic Bacteria and Archaea.

The genetic basis of laboratory adaptation in Caulobacter crescentus.

The dimorphic bacterium Caulobacter crescentus has evolved marked phenotypic changes during its 50-year history of culture in the laboratory environment, providing an excellent system for the study of natural selection and phenotypic microevolution in prokaryotes. Combining whole-genome sequencing with classical molecular genetic tools, we have comprehensively mapped a set of polymorphisms underlying multiple derived phenotypes, several of which arose independently in separate strain lineages. The genetic basis of phenotypic differences in growth rate, mucoidy, adhesion, sedimentation, phage susceptibility, and stationary-phase survival between C. crescentus strain CB15 and its derivative NA1000 is determined by coding, regulatory, and insertion/deletion polymorphisms at five chromosomal loci. This study evidences multiple genetic mechanisms of bacterial evolution as driven by selection for growth and survival in a new selective environment and identifies a common polymorphic locus, zwf, between lab-adapted C. crescentus and clinical isolates of Pseudomonas aeruginosa that have adapted to a human host during chronic infection.

Differential Selectivity of Natural and Synthetic Aminoglycosides towards the Eukaryotic and Prokaryotic Decoding A Sites

The lack of absolute prokaryotic selectivity of natural antibiotics is widespread and is a significant clinical problem. The use of this disadvantage of aminoglycoside antibiotics for the possible treatment of human genetic diseases is extremely challenging. Here, we have used a combination of biochemical and structural analysis to compare and contrast the molecular mechanisms of action and the structure-activity relationships of a new synthetic aminoglycoside, NB33, and a structurally similar natural aminoglycoside apramycin. The data presented herein demonstrate the general molecular principles that determine the decreased selectivity of apramycin for the prokaryotic decoding site, and the increased selectivity of NB33 for the eukaryotic decoding site. These results are therefore extremely beneficial for further research on both the design of new aminoglycoside-based antibiotics with diminished deleterious effects on humans, as well as the design of new aminoglycoside-based structures that selectively target the eukaryotic ribosome.

The fidelity of translation initiation: reciprocal activities of eIF1, IF3 and YciH

Eukaryotic initiation factor eIF1 and the functional C-terminal domain of prokaryotic initiation factor IF3 maintain the fidelity of initiation codon selection in eukaryotes and prokaryotes, respectively, and bind to the same regions of small ribosomal subunits, between the platform and initiator tRNA. Here we report that these nonhomologous factors can bind to the same regions of heterologous subunits and perform their functions in heterologous systems in a reciprocal manner, discriminating against the formation of initiation complexes containing codon-anticodon mismatches. We also show that like IF3, eIF1 can influence initiator tRNA selection, which occurs at the stage of ribosomal subunit joining after eIF5-induced hydrolysis of eIF2-bound GTP. The mechanisms of initiation codon and initiator tRNA selection in prokaryotes and eukaryotes are therefore unexpectedly conserved and likely involve related conformational changes induced in the small ribosomal subunit by factor binding. YciH, a prokaryotic eIF1 homologue, could perform some of IF3's functions, which justifies the possibility that YciH and eIF1 might have a common evolutionary origin as initiation factors, and that IF3 functionally replaced YciH in prokaryotes.