Adaptive Resistance Mutations at Suprainhibitory Concentrations Independent of SOS Mutagenesis.

Emergence of resistant bacteria during antimicrobial treatment is one of the most critical and universal health threats. It is known that several stress-induced mutagenesis and heteroresistance mechanisms can enhance microbial adaptation to antibiotics. Here, we demonstrate that the pathogen Bartonella can undergo stress-induced mutagenesis despite the fact it lacks error-prone polymerases, the rpoS gene and functional UV-induced mutagenesis. We demonstrate that Bartonella acquire de novo single mutations during rifampicin exposure at suprainhibitory concentrations at a much higher rate than expected from spontaneous fluctuations. This is while exhibiting a minimal heteroresistance capacity. The emerged resistant mutants acquired a single rpoB mutation, whereas no other mutations were found in their whole genome. Interestingly, the emergence of resistance in Bartonella occurred only during gradual exposure to the antibiotic, indicating that Bartonella sense and react to the changing environment. Using a mathematical model, we demonstrated that, to reproduce the experimental results, mutation rates should be transiently increased over 1,000-folds, and a larger population size or greater heteroresistance capacity is required. RNA expression analysis suggests that the increased mutation rate is due to downregulation of key DNA repair genes (mutS, mutY, and recA), associated with DNA breaks caused by massive prophage inductions. These results provide new evidence of the hazard of antibiotic overuse in medicine and agriculture.

Relacin, a novel antibacterial agent targeting the Stringent Response.

Finding bacterial cellular targets for developing novel antibiotics has become a major challenge in fighting resistant pathogenic bacteria. We present a novel compound, Relacin, designed to inhibit (p)ppGpp production by the ubiquitous bacterial enzyme RelA that triggers the Stringent Response. Relacin inhibits RelA in vitro and reduces (p)ppGpp production in vivo. Moreover, Relacin affects entry into stationary phase in Gram positive bacteria, leading to a dramatic reduction in cell viability. When Relacin is added to sporulating Bacillus subtilis cells, it strongly perturbs spore formation regardless of the time of addition. Spore formation is also impeded in the pathogenic bacterium Bacillus anthracis that causes the acute anthrax disease. Finally, the formation of multicellular biofilms is markedly disrupted by Relacin. Thus, we establish that Relacin, a novel ppGpp analogue, interferes with bacterial long term survival strategies, placing it as an attractive new antibacterial agent.

ppGpp analogues inhibit synthetase activity of Rel proteins from Gram-negative and Gram-positive bacteria.

A prominent feature of the stringent response is the accumulation of two unusual phosphorylated derivatives of GTP and GDP (pppGpp: 5'-triphosphate-3'-diphosphate, and ppGpp: 5'-3'-bis-diphosphate), collectively called (p)ppGpp, within a few seconds after the onset of amino-acid starvation. The synthesis of these 'alarmone' compounds is catalyzed by RelA homologues. Other features of the stringent response include inhibition of stable RNA synthesis and modulation of transcription, replication, and translation. (p)ppGpp accumulation is important for virulence induction, differentiation and antibiotic resistance. We have synthesized a group of (p)ppGpp analogues and tested them as competitive inhibitors of Rel proteins in vitro. 2'-Deoxyguanosine-3'-5'-di(methylene bisphosphonate) [compound (10)] was found as an inhibitor that reduces ppGpp formation in both Gram-negative and Gram-positive bacteria. In silico docking together with competitive inhibition analysis suggests that compound (10) inhibits activity of Rel proteins by competing with GTP/GDP for its binding site. As Rel proteins are completely absent in mammalians, this appears to be a very attractive approach for the development of novel antibacterial agents.

Escherichia coli RelA Regulation via Its C-Terminal Domain.

One of the most important stress responses in bacteria is the stringent response. The main player in this response is the signal molecule (p)ppGpp, which is synthesized by a Rel family protein. In Escherichia coli, RelA is the main synthetase of (p)ppGpp in response to amino acid starvation. Although the synthetic activity of RelA is well-understood, its regulation is not yet fully characterized. The C-terminus domain (CTD) of the E. coli RelA is responsible for the regulation of the protein and for its complete dependency on wild-type (WT) ribosome. The CTD contains three Cysteine residues, positioned in a very conserved order. Together with our previous results, we show in vitro the negative dominant effect of a part of the WT CTD (AA 564-744) named YG4 on RelA synthetic activity. This effect is abolished using mutated YG4 (YG4-638). In vitro and mass spectrometry (MS)-MS analysis of the native RelA and the mutated RelA in Cys-638 (Rel638) in the presence of the native and mutated YG4 (YG4-638) reveals that RelA forms a homodimer via its CTD by the formation of a disulfide bond between the two Cys-638 residues. This supports our previous data which showed, using a two-hybrid system, interactions between RelA proteins via the CTD. Finally, we show in vitro that excess of the native YG4 inhibited RelA synthetic activity but did not affect the amount of RelA bound to the ribosome. Our results suggest that the regulatory mechanism of RelA is by the dimerization of the protein via disulfide bonds in the CTD. Upon amino-acid starvation, the dimer changes its conformation, thus activating the stringent response in the cell.

Design, synthesis and structure-activity relationship of novel Relacin analogs as inhibitors of Rel proteins.

Rel proteins in bacteria synthesize the signal molecules (p)ppGpp that trigger the Stringent Response, responsible for bacterial survival. Inhibiting the activity of such enzymes prevents the Stringent Response, resulting in the inactivation of long-term bacterial survival strategies, leading to bacterial cell death. Herein, we describe a series of deoxyguanosine-based analogs of the Relacin molecule that inhibit in vitro the synthetic activity of Rel proteins from Gram positive and Gram negative bacteria, providing a deeper insight on the SAR for a better understanding of their potential interactions and inhibitory activity. Among the inhibitors evaluated, compound 2d was found to be more effective and potent than our previously reported Relacin.

HipA-mediated antibiotic persistence via phosphorylation of the glutamyl-tRNA-synthetase.

Bacterial persistence has been shown to be an underlying factor in the failure of antibiotic treatments. Although many pathways, among them the stringent response and toxin-antitoxin modules, have been linked to antibiotic persistence, a clear molecular mechanism for the growth arrest that characterizes persistent bacteria remained elusive. Here, we screened an expression library for putative targets of HipA, the first toxin linked to persistence, and a serine/threonine kinase. We found that the expression of GltX, the glutamyl-tRNA-synthetase, reverses the toxicity of HipA and prevents persister formation. We show that upon HipA expression, GltX undergoes phosphorylation at Ser239, its ATP-binding site. This phosphorylation leads to accumulation of uncharged tRNA(Glu) in the cell, which results in the activation of the stringent response. Our findings demonstrate a mechanism for persister formation by the hipBA toxin-antitoxin module and provide an explanation for the long-observed connection between persistence and the stringent response.

Energetics of MazG unfolding in correlation with its structural features.

MazG is a homodimeric alpha-helical protein that belongs to the superfamily of all-alpha NTP pyrophosphatases. Its function has been connected to the regulation of the toxin-antitoxin module mazEF, implicated in programmed growth arrest/cell death of Escherichia coli cells under conditions of amino acid starvation. The goal of the first detailed biophysical study of a member of the all-alpha NTP pyrophosphatase superfamily, presented here, is to improve molecular understanding of the unfolding of this type of proteins. Thermal unfolding of MazG monitored by differential scanning calorimetry, circular dichroism spectroscopy, and fluorimetry at neutral pH in the presence of a reducing agent (dithiothreitol) can be successfully described as a reversible four-state transition between a dimeric native state, two dimeric intermediate states, and a monomeric denatured state. The first intermediate state appears to have a structure similar to that of the native state while the final thermally denatured monomeric state is not fully unfolded and contains a significant fraction of residual alpha-helical structure. In the absence of dithiothreitol, disulfide cross-linking causes misfolding of MazG that appears to be responsible for the formation of multimeric aggregates. MazG is most stable at pH 7-8, while at pH <6, it exists in a molten-globule-like state. The thermodynamic parameters characterizing each step of MazG denaturation transition obtained by global fitting of the four-state model to differential scanning calorimetry, circular dichroism, and fluorimetry temperature profiles are in agreement with the observed structural characteristics of the MazG conformational states and their assumed functional role.

MazG -- a regulator of programmed cell death in Escherichia coli.

We have previously reported that mazEF, the first regulatable chromosomal 'addiction module' located on the Escherichia coli chromosome, downstream from the relA gene, plays a crucial role in the programmed cell death in bacteria under stressful conditions. It consists of a pair of genes encoding a stable toxin, MazF, and MazE, a labile antitoxin interacting with MazF to form a complex. The cellular target of MazF toxin was recently described to be cellular mRNA, which is degraded by this toxin. On the same operon, downstream to the mazEF genes, we found another open reading frame, which was called mazG. Recently, it was shown that the MazG protein has a nucleotide pyrophosphohydrolase activity. Here we show that mazG is being transcribed in the same polycistronic mRNA with mazEF. We also show that the enzymatic activity of MazG is inhibited by MazEF proteins. When the complex MazEF was added, the enzymatic activity of MazG was about 70% inhibited. We demonstrate that the enzymatic activity of MazG in vivo causes depletion of guanosine 3',5'-bispyrophosphate (ppGpp), synthesized by RelA under amino acid starvation conditions. Based on our results, we propose a model in which this third gene, which is unique for chromosomal addiction systems, has a function of limiting the deleterious activity of MazF toxin. In addition, MazG solves a frequently encountered biological problem: how to avoid the persistence of a toxic product beyond the time when its toxicity is useful to the survival of the population.

Energetics of structural transitions of the addiction antitoxin MazE: is a programmed bacterial cell death dependent on the intrinsically flexible nature of the antitoxins?

The Escherichia coli mazEF addiction module plays a crucial role in the cell death program that is triggered under various stress conditions. It codes for the toxin MazF and the antitoxin MazE, which interferes with the lethal action of the toxin. To better understand the role of various conformations of MazE in bacterial life, its order-disorder transitions were monitored by differential scanning calorimetry, spectropolarimetry, and fluorimetry. The changes in spectral and thermodynamic properties accompanying MazE dimer denaturation can be described in terms of a compensating reversible process of the partial folding of the unstructured C-terminal half (high mean net charge, low mean hydrophobicity) and monomerization coupled with the partial unfolding of the structured N-terminal half (low mean net charge, high mean hydrophobicity). At pH

Promoter protection by a transcription factor acting as a local topological homeostat.

Binding of the Escherichia coli global transcription factor FIS to the upstream activating sequence (UAS) of stable RNA promoters activates transcription on the outgrowth of cells from stationary phase. Paradoxically, while these promoters require negative supercoiling of DNA for optimal activity, FIS counteracts the increase of negative superhelical density by DNA gyrase. We demonstrate that binding of FIS at the UAS protects the rrnA P1 promoter from inactivation at suboptimal superhelical densities. This effect is correlated with FIS-dependent constraint of writhe and facilitated untwisting of promoter DNA. We infer that FIS maintains stable RNA transcription by stabilizing local writhe in the UAS. These results suggest a novel mechanism of transcriptional regulation by a transcription factor acting as a local topological homeostat.

Buffering of stable RNA promoter activity against DNA relaxation requires a far upstream sequence.

The stable RNA promoters of Escherichia coli are exquisitely sensitive to variations in the superhelical density of DNA. Previously, we have shown that binding of the DNA architectural protein FIS at the upstream activating sequences (UASs) of stable RNA promoters prevents the transcription complexes from inactivation induced by changes in the supercoiling level of DNA. Here, we identify a strong FIS binding site 89 bp upstream of the previously described cluster of FIS binding sites located between positions -64 and -150 in the rrnA P1 UAS. Binding of FIS to this 'far upstream sequence' allows the recruitment of additional FIS molecules to the region. We demonstrate that, upon DNA relaxation, the maintenance of promoter activity requires, in addition to UAS, the presence of the far upstream sequence. The far upstream sequence shows no effect in the absence of an intact cluster. This requirement for the integrity of the region encompassing the far upstream sequence and the UAS cluster is correlated with the in vitro modulation of binding of FIS to UAS and interaction of RNA polymerase with the UP element and the region around the transcriptional start point. Our results suggest that, at the rrnA P1 promoter, the entire region comprising the UAS and the far upstream sequence is involved in the assembly of the transcription initiation complex. We propose that the extensive engagement of upstream DNA in this nucleoprotein complex locally compensates for the lack of torsional strain in relaxed DNA, thus increasing the resistance of the promoter to global DNA relaxation.

Recognition of the intrinsically flexible addiction antidote MazE by a dromedary single domain antibody fragment. Structure, thermodynamics of binding, stability, and influence on interactions with DNA.

The Escherichia coli mazEF operon defines a chromosomal addiction module that programs cell death under various stress conditions. It encodes the toxic and long-lived MazF and the labile antidote MazE. The denaturation of MazE is a two-state reversible dimer-monomer transition. At lower concentrations the denatured state is significantly populated. This leads to a new aspect of the regulation of MazE concentration, which may decide about the life and death of the cell. Interactions of MazE with a dromedary antibody domain, cAbMaz1 (previously used as a crystallization aid), as well as with promoter DNA were studied using microcalorimetric and spectroscopic techniques. Unique features of cAbMaz1 enable a specific enthalpy-driven recognition of MazE and, thus, a significant stabilization of its dimeric native conformation. The MazE dimer and the MazE dimer-cAbMaz1 complex show very similar binding characteristics with promoter DNA, i.e. three binding sites with apparent affinities in micromolar range and highly exothermic binding accompanied by large negative entropy contributions. A working model for the MazE-DNA assembly is proposed on the basis of the structural and binding data. Both binding and stability studies lead to a picture of MazE solution structure that is significantly more unfolded than the structure observed in a crystal of the MazE-cAbMaz1 complex.

Crystal structure of the intrinsically flexible addiction antidote MazE.

A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to crystallize the intrinsically flexible addiction antidote MazE. Only 45% of the polypeptide chain is found ordered in the crystal. The MazE monomer consisting of two beta-hairpins connected by a short alpha-helix has no hydrophobic core on its own and represents only one half of a typical protein domain. A complete domain structure is formed by the association of two chains, creating a hydrophobic core between two four-stranded beta-sheets. This hydrophobic core consists exclusively of short aliphatic residues. The folded part of MazE contains a novel DNA binding motif. A model for DNA binding that is consistent with the available biochemical data is presented.