Expression of Bacillus subtilis ABCF antibiotic resistance factor VmlR is regulated by RNA polymerase pausing, transcription attenuation, translation attenuation and (p)ppGpp.

Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms.

Analysis of nucleotide pools in bacteria using HPLC-MS in HILIC mode.

Nucleotides, nucleosides and their derivatives are present in all cells at varying concentrations that change with the nutritional, and energetic status of the cell. Precise measurement of the concentrations of these molecules is instrumental for understanding their regulatory effects. Such measurement is challenging due to the inherent instability of these molecules and, despite many decades of research, the reported values differ widely.

HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp.

Here we describe an HPLC-based method to quantify bacterial housekeeping nucleotides and the signaling messengers ppGpp and pppGpp. We have replicated and tested several previously reported HPLC-based approaches and assembled a method that can process 50 samples in three days, thus making kinetically resolved experiments feasible. The method combines cell harvesting by rapid filtration, followed by acid extraction, freeze-drying with chromatographic separation.

Small Alarmone Synthetases as novel bacterial RNA-binding proteins.

The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, antibiotic tolerance and pathogenicity. We have recently shown that the Small Alarmone Synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis has an RNA-binding activity (Beljantseva et al. 2017).

Molecular mutagenesis of ppGpp: turning a RelA activator into an inhibitor.

The alarmone nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p)ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC in the sub-mM range.

Cationic bactericidal peptide 1018 does not specifically target the stringent response alarmone (p)ppGpp.

The bacterial stringent response is a key regulator of bacterial virulence, biofilm formation and antibiotic tolerance, and is a promising target for the development of new antibacterial compounds. The intracellular nucleotide (p)ppGpp acts as a messenger orchestrating the stringent response. A synthetic peptide 1018 was recently proposed to specifically disrupt biofilms by inhibiting the stringent response via direct interaction with (p)ppGpp (de la Fuente-Núñez et al.

Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors.

The stringent response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the stringent response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the stringent response hold great promise as molecular tools for disarming and pacifying bacterial pathogens.

Persisters-as elusive as ever.

Persisters-a drug-tolerant sub-population in an isogenic bacterial culture-have been featured throughout the last decade due to their important role in recurrent bacterial infections. Numerous investigations detail the mechanisms responsible for the formation of persisters and suggest exciting strategies for their eradication. In this review, we argue that the very term "persistence" is currently used to describe a large and heterogeneous set of physiological phenomena that are functions of bacterial species, strains, growth conditions, and antibiotics used in the experiments.

Composition of the outgrowth medium modulates wake-up kinetics and ampicillin sensitivity of stringent and relaxed Escherichia coli.

The transition of Escherichia coli from the exponential into the stationary phase of growth induces the stringent response, which is mediated by the rapid accumulation of the alarmone nucleotide (p)ppGpp produced by the enzyme RelA. The significance of RelA's functionality during the transition in the opposite direction, i.e.

Aim-less translation: loss of Saccharomyces cerevisiae mitochondrial translation initiation factor mIF3/Aim23 leads to unbalanced protein synthesis.

The mitochondrial genome almost exclusively encodes a handful of transmembrane constituents of the oxidative phosphorylation (OXPHOS) system. Coordinated expression of these genes ensures the correct stoichiometry of the system's components. Translation initiation in mitochondria is assisted by two general initiation factors mIF2 and mIF3, orthologues of which in bacteria are indispensible for protein synthesis and viability.