Methionyl-tRNA synthetase synthetic and proofreading activities are determinants of antibiotic persistence.

Bacterial antibiotic persistence is a phenomenon where bacteria are exposed to an antibiotic and the majority of the population dies while a small subset enters a low metabolic, persistent, state and are able to survive. Once the antibiotic is removed the persistent population can resuscitate and continue growing. Several different molecular mechanisms and pathways have been implicated in this phenomenon. A common mechanism that may underly bacterial antibiotic persistence is perturbations in protein synthesis. To investigate this mechanism, we characterized four distinct metG mutants for their ability to increase antibiotic persistence. Two metG mutants encode changes near the catalytic site of MetRS and the other two mutants changes near the anticodon binding domain. Mutations in metG are of particular interest because MetRS is responsible for aminoacylation both initiator tRNAMet and elongator tRNAMet indicating that these mutants could impact translation initiation and/or translation elongation. We observed that all the metG mutants increased the level of antibiotic persistence as did reduced transcription levels of wild type metG. Although, the MetRS variants did not have an impact on MetRS activity itself, they did reduce translation rates. It was also observed that the MetRS variants affected the proofreading mechanism for homocysteine and that these mutants' growth is hypersensitive to homocysteine. Taken together with previous findings, our data indicate that both reductions in cellular Met-tRNAMet synthetic capacity and reduced proofreading of homocysteine by MetRS variants are positive determinants for bacterial antibiotic persistence.

Deacylated tRNA Accumulation Is a Trigger for Bacterial Antibiotic Persistence Independent of the Stringent Response.

Bacterial antibiotic persistence occurs when bacteria are treated with an antibiotic and the majority of the population rapidly dies off, but a small subpopulation enters into a dormant, persistent state and evades death. Diverse pathways leading to nucleoside triphosphate (NTP) depletion and restricted translation have been implicated in persistence, suggesting alternative redundant routes may exist to initiate persister formation. To investigate the molecular mechanism of one such pathway, functional variants of an essential component of translation (phenylalanyl-tRNA synthetase [PheRS]) were used to study the effects of quality control on antibiotic persistence. Upon amino acid limitation, elevated PheRS quality control led to significant decreases in aminoacylated tRNAPhe accumulation and increased antibiotic persistence. This increase in antibiotic persistence was most pronounced (65-fold higher) when the relA-encoded tRNA-dependent stringent response was inactivated. The increase in persistence with elevated quality control correlated with ∼2-fold increases in the levels of the RNase MazF and the NTPase MazG and a 3-fold reduction in cellular NTP pools. These data reveal a mechanism for persister formation independent of the stringent response where reduced translation capacity, as indicated by reduced levels of aminoacylated tRNA, is accompanied by active reduction of cellular NTP pools which in turn triggers antibiotic persistence. IMPORTANCE Bacterial antibiotic persistence is a transient physiological state wherein cells become dormant and thereby evade being killed by antibiotics. Once the antibiotic is removed, bacterial persisters are able to resuscitate and repopulate. It is thought that antibiotic bacterial persisters may cause reoccurring infections in the clinical setting. The molecular triggers and pathways that cause bacteria to enter into the persister state are not fully understood. Our results suggest that accumulation of deacylated tRNA is a trigger for antibiotic persistence independent of the RelA-dependent stringent response, a pathway thought to be required for persistence in many organisms. Overall, this provides a mechanism where changes in translation quality control in response to physiological cues can directly modulate bacterial persistence.

Did Amino Acid Side Chain Reactivity Dictate the Composition and Timing of Aminoacyl-tRNA Synthetase Evolution?

The twenty amino acids in the standard genetic code were fixed prior to the last universal common ancestor (LUCA). Factors that guided this selection included establishment of pathways for their metabolic synthesis and the concomitant fixation of substrate specificities in the emerging aminoacyl-tRNA synthetases (aaRSs). In this conceptual paper, we propose that the chemical reactivity of some amino acid side chains (e.g., lysine, cysteine, homocysteine, ornithine, homoserine, and selenocysteine) delayed or prohibited the emergence of the corresponding aaRSs and helped define the amino acids in the standard genetic code. We also consider the possibility that amino acid chemistry delayed the emergence of the glutaminyl- and asparaginyl-tRNA synthetases, neither of which are ubiquitous in extant organisms. We argue that fundamental chemical principles played critical roles in fixation of some aspects of the genetic code pre- and post-LUCA.

Indirect tRNA aminoacylation during accurate translation and phenotypic mistranslation.

The fact that most bacteria do not contain a full set of aminoacyl-tRNA synthetases (aaRS) is often underappreciated. In the absence of asparaginyl-tRNA and/or glutaminyl-tRNA synthetase (AsnRS and GlnRS), Asn-tRNAAsn and/or Gln-tRNAGln are produced by an indirect tRNA aminoacylation pathway that relies on misacylation of these two tRNAs by two different misacylating aaRSs, followed by transamidation by an amidotransferase (GatCAB in bacteria). This review highlights the central importance of indirect tRNA aminoacylation to accurate protein translation, mechanistic peculiarities that appear to be unique to this system, and the newly recognized connection between indirect tRNA aminoacylation and mistranslation as a strategy used by bacteria to respond to environmental stressors like antibiotics.

Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells.

Many plasmids used for gene cloning and heterologous protein expression in Escherichia coli cells are low copy number or single copy number plasmids. The extraction of these types of plasmids from small bacterial cell cultures produces low DNA yields. In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium. TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB. By contrast, increasing ampicillin concentration to enhance plasmid retention did not improve plasmid DNA recovery. These experiments demonstrate that yields of low and single copy number plasmid DNAs from minipreps can be strongly enhanced using simple and inexpensive media.

Characterization of tunnel mutants reveals a catalytic step in ammonia delivery by an aminoacyl-tRNA amidotransferase.

The Helicobacter pylori Asp-tRNA(A) (sn) /Glu-tRNA(G) (ln) amidotransferase (GatCAB) utilizes an uncommonly hydrophilic, ~ 40 Å ammonia tunnel for ammonia/ammonium transport between isolated active sites. Hydrophilicity of this tunnel requires a distinct ammonia transport mechanism, which hypothetically occurs through a series of deprotonation and protonation steps. To explore the initiation of this relay mechanism, the highly conserved tunnel residue D185 (in the GatA subunit) was enzymatically and computationally investigated by comparing D185A, D185N, and D185E mutant enzymes to wild-type GatCAB. Our results indicate that D185 acts as an acid/base residue, participating directly in catalysis. To our knowledge, this is the first example of acid/base chemistry in a glutamine-dependent amidotransferase ammonia tunnel.

Enhancement of plasmid DNA transformation efficiencies in early stationary-phase yeast cell cultures.

Chemical-based methods have been developed for transformation of DNA into log-phase cells of the budding yeast Saccharomyces cerevisiae with high efficiency. Transformation of early stationary-phase cells, e.g. cells grown in overnight liquid cultures or as colonies on plates, is less efficient than log-phase cells but is simpler and more adaptable to high-throughput projects. In this study we have tested different approaches for transformation of early stationary-phase cell cultures and identified a method utilizing polyethylene glycol (PEG), lithium acetate and dimethyl sulphoxide (DMSO) as the most efficient. Plasmid DNA transformations using this method could be improved modestly by allowing cells to recover from the chemical treatment in rich broth before plating to selective media. Strong increases in transformation efficiencies were observed when cells were treated briefly with dithiothreitol (DTT). Tests using several different yeast strain backgrounds indicated that DTT treatment could enhance transformation efficiencies by up to 40-fold. Evaluation of multiple parameters affecting the efficiency of the method led to development of an optimized protocol achieving > 50 000 transformants/µg DNA in most backgrounds tested.