Biology subjects

Krueger, L.

Publications and source records attributed to Krueger, L..

3 recordsLinked to original sources

bioRxiv2020

A rendezvous of two second messengers: The c-di-AMP receptor protein DarB controls (p)ppGpp synthesis in Bacillus subtilis

Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Most likely these proteins transduce their signal by regulatory interactions with other proteins. Here, we have investigated the function of the c-di-AMP-binding protein DarB in B. subtilis, a protein consisting of two CBS (cystathionine-beta synthase) domains. We have used an unbiased search for DarB interaction partners and identified the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. Indeed, c-di-AMP inhibits the binding of DarB to Rel. The interaction results in the Rel-dependent accumulation of pppGpp. Our results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.

microbiology↗

bioRxiv2020

Resistance to serine in Bacillus subtilis: Identification of the serine transporter YbeC and of a metabolic network that links serine and threonine metabolism

The Gram-positive bacterium Bacillus subtilis uses serine not only as building block for proteins but also as an important precursor in many anabolic reactions. Moreover, a lack of serine results in the initiation of biofilm formation. However, in excess serine inhibits the growth of B. subtilis. To unravel the underlying mechanisms, we isolated suppressor mutants that can tolerate toxic serine concentrations by three targeted and non-targeted genome-wide screens. All screens as well as genetic complementation in Escherichia coli identified the so far uncharacterized permease YbeC as the major serine transporter of B. subtilis. In addition to YbeC, the threonine transporters BcaP and YbxG make minor contributions to serine uptake. A strain lacking these three transporters was able to tolerate 100 mM serine whereas the wild type strain was already inhibited by 1 mM of the amino acid. The screen for serine-resistant mutants also identified mutations that result in increased serine degradation and in increased expression of threonine biosynthetic enzymes suggesting that serine toxicity results from interference with threonine biosynthesis. Originality-Significance StatementSerine is an important precursor for many biosynthetic reactions, and lack of this amino acid can induce biofilm formation in Bacillus subtilis. However, serine is toxic for the growth of B. subtilis. To understand the reason(s) for this toxicity and to identify the so far unknown serine transporter(s) of this bacterium, we performed exhaustive mutant screens to isolate serine-resistant mutants. This screen identified YbeC, the major serine transporter of B. subtilis. Moreover, we observed an intimate link between serine and threonine metabolism that is responsible for serine toxicity by inhibiting threonine biosynthesis.

microbiology↗

bioRxiv2020

Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival

Xenotransplantation using pig organs has achieved survival times of more than 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNF were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.

microbiology↗