Biology subjects

Winther, K. S.

Publications and source records attributed to Winther, K. S..

3 recordsLinked to original sources

bioRxiv2020

The RelA hydrolase domain: a molecular switch for (p)ppGpp synthesis

Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of {gamma}-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity remain elusive. Here, we show a role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using mutagenesis screening and functional studies, we identify a loop region (residues 114-130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wildtype RelA. Our data support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

molecular biology↗

bioRxiv2020

The role of toxin:antitoxin systems and insertion sequences in the loss of virulence in Shigella sonnei

The Shigella plasmid, pINV, contains a 30 kb pathogenicity island (PAI) encoding a Type III secretion system (T3SS) which is essential for virulence. During growth in the laboratory, avirulent colonies of Shigella (which do not express a T3SS) arise spontaneously. Avirulence in Shigella flexneri mostly follows loss of the PAI, following recombination between insertion sequences (ISs) on pINV; toxin:antitoxin (TA) systems on pINV promote its retention through post-segregational killing (PSK). We show that avirulence in Shigella sonnei mainly results from plasmid loss, consistent with previous findings; IS-mediated PAI deletions can occur in S. sonnei, but through different ISs than in S. flexneri. We investigated the molecular basis for frequent loss of the S. sonnei plasmid, pINVSsonn. Introduction into pINVSsonn of CcdAB and GmvAT, toxin:antitoxin TA systems in pINV from S. flexneri but not S. sonnei, reduced plasmid loss and the emergence of avirulent bacteria. However, plasmid loss remained the leading cause of avirulence. We show that a single amino acid difference in the VapC toxin of the VapBC TA system in pINV also contributes to high frequency plasmid loss in S. sonnei compared to S. flexneri. Our findings demonstrate that the repertoire of ISs, complement of TA systems, and polymorphisms in TA systems influence plasmid dynamics and virulence loss in S. sonnei. Understanding the impact of polymorphisms should be informative about how TA systems contribute to PSK, and could be exploited for generating strains with stable plasmids.

microbiology↗

bioRxiv2019

Fatty acid starvation activates RelA by depleting lysine precursor pyruvate

Bacteria experiencing nutrient starvation induce the ubiquitous stringent response, a profound physiological change that reprograms cell physiology from fast to slow growth and stress survival. The stringent response is mediated by the secondary messengers pppGpp and ppGpp collectively referred to as (p)ppGpp or \"alarmone\". In Escherichia coli, two paralogs, RelA and SpoT, synthesize (p)ppGpp. RelA is activated by amino acid starvation whereas SpoT, which can also degrade (p)ppGpp, responds to fatty acid (FA), carbon and phosphate starvation. Here, we discover that FA starvation leads to rapid activation of RelA and reveal the underlying mechanism. We show that fatty acid starvation leads to depletion of lysine that, in turn, leads to the accumulation of uncharged tRNAlys and activation of RelA. SpoT was also activated by fatty acid starvation but to a lower level and with a delayed kinetics. Next, we discovered that pyruvate, a precursor of lysine, is depleted by FA starvation. We also propose a mechanism that explains how FA starvation leads to pyruvate depletion. Together our results indicate that many responses to nutrient depletion may ultimately result indirectly from depletion of amino acids and thereby activating RelA. Interestingly, FA starvation provoked a ~100-fold increase in relA dependent ampicillin tolerance.

microbiology↗