Biology subjects

Rothe, P.

Publications and source records attributed to Rothe, P..

4 recordsLinked to original sources

bioRxiv2022

Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes

The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen Listeria monocytogenes is intrinsically resistant towards lysozyme, mainly due to deacetylation and O-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An eslB mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less O-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of eslB phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the eslB mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-N-acetylglucosamine (UDP-GlcNAc) production or distribution.

microbiology↗

bioRxiv2022

Listeria monocytogenes gene essentiality under laboratory conditions and during macrophage infection

The Gram-positive bacterium Listeria monocytogenes occurs widespread in the environment and infects humans when ingested along with contaminated food. Such infections are particularly dangerous for risk group patients, for whom they represent a life-threatening disease. To invent novel strategies to control contamination and disease, it is important to identify those cellular processes that maintain pathogen growth in- and outside the host. We here have applied transposon insertion sequencing (Tn-Seq) to L. monocytogenes for the identification of such processes on a genome-wide scale. Our approach classified 394 open reading frames as essential for growth under standard laboratory conditions and identified 42 further genes, which become additionally essential during intracellular growth in macrophages. Most essential genes encode components of the translation machinery, act in chromosome-related processes, cell division and biosynthesis of the cellular envelope. Several cofactor biosynthesis pathways and 29 genes with unknown functions were also essential, opening novel options for the development of anti- listerial drugs. Among the genes specifically required during intracellular growth were known virulence factors, genes compensating intracellular auxotrophies and several cell division genes. Our experiments also highlight the importance of PASTA kinase signalling, glycine metabolism and chromosome segregation for efficient intracellular growth of L. monocytogenes.

microbiology↗

bioRxiv2021

Promoter repression and 3D-restructuring resolves divergent developmental gene expression in TADs

Cohesin loop extrusion facilitates precise gene expression by continuously driving promoters to sample all enhancers located within the same topologically-associated domain (TAD). However, many TADs contain multiple genes with divergent expression patterns, thereby indicating additional forces further refine how enhancer activities are utilised. Here, we unravel the mechanisms enabling a new gene, Rex1, to emerge with divergent expression within the ancient Fat1 TAD in placental mammals. We show that such divergent expression is not determined by a strict enhancer-promoter compatibility code, intra-TAD position or nuclear envelope-attachment. Instead, TAD-restructuring in embryonic stem cells (ESCs) separates Rex1 and Fat1 with distinct proximal enhancers that independently drive their expression. By contrast, in later embryonic tissues, DNA methylation renders the inactive Rex1 promoter profoundly unresponsive to Fat1 enhancers within the intact TAD. Combined, these features adapted an ancient regulatory landscape during evolution to support two entirely independent Rex1 and Fat1 expression programs. Thus, rather than operating only as rigid blocks of co-regulated genes, TAD-regulatory landscapes can orchestrate complex divergent expression patterns in evolution. HIGHLIGHTSO_LINew genes can emerge in evolution without taking on the expression pattern of their surrounding pre-existing TAD. C_LIO_LICompartmentalisation can restructure seemingly evolutionarily stable TADs to control a promoters access to enhancers. C_LIO_LILamina-associated domains neither prevent transcriptional activation nor enhancer-promoter communication. C_LIO_LIRepression rather than promoter-specificity refines when genes respond to promiscuous enhancer activities in specific tissues. C_LI

genetics↗

bioRxiv2021

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling

Gram-positive bacteria are protected by a thick mesh of peptidoglycan (PG) completely engulfing their cells. This PG network is the main component of the bacterial cell wall, it provides rigidity and acts as foundation for the attachment of other surface molecules. Biosynthesis of PG consumes a high amount of cellular resources and therefore requires careful adjustments to environmental conditions. An important switch in the control of PG biosynthesis of Listeria monocytogenes, a Gram-positive pathogen with a high infection fatality rate, is the serine/threonine protein kinase PrkA. A key substrate of this kinase is the small cytosolic protein ReoM. We have shown previously that ReoM phosphorylation regulates PG formation through control of MurA stability. MurA catalyzes the first step in PG biosynthesis and the current model suggests that phosphorylated ReoM prevents MurA degradation by the ClpCP protease. In contrast, conditions leading to ReoM dephosphorylation stimulate MurA degradation. How ReoM controls degradation of MurA and potential other substrates is not understood. Also, the individual contribution of the [~]20 other known PrkA targets to PG biosynthesis regulation is unknown. We here present murA mutants which escape proteolytic degradation. The release of MurA from ClpCP-dependent proteolysis was able to constitutively activate PG biosynthesis and further enhances the intrinsic cephalosporin resistance of L. monocytogenes. This activation required the RodA3/PBP B3 transglycosylase/transpeptidase pair as additional effectors of the PrkA signaling route. One murA escape mutation not only fully rescued an otherwise non-viable prkA mutant during growth in batch culture and inside macrophages but also overcompensated cephalosporin hypersensitivity. Our data collectively indicate that the main purpose of PrkA-mediated signaling in L. monocytogenes is control of MurA stability during extra- and intracellular growth. These findings have important implications for the understanding of PG biosynthesis regulation and {beta}-lactam resistance of L. monocytogenes and related Gram-positive bacteria. Author SummaryPeptidoglycan (PG) is the main component of the bacterial cell wall and many of the PG synthesizing enzymes are antibiotic targets. We previously have discovered a new signaling route controlling PG production in the human pathogen Listeria monocytogenes. This route also determines the intrinsic resistance of L. monocytogenes against cephalosporins, a group of {beta}-lactam antibiotics. Signaling involves PrkA, a membrane-embedded protein kinase, that is activated during cell wall stress to phosphorylate its target ReoM. Depending on its phosphorylation, ReoM activates or inactivates PG production by controlling the proteolytic stability of MurA, which catalyzes the first step in PG biosynthesis. MurA degradation depends on the ClpCP protease and we here have isolated murA mutations that escape this degradation. Using these mutants, we could show that regulation of PG biosynthesis through control of MurA stability is the primary purpose of PrkA-mediated signaling in L. monocytogenes. Further experiments identified the transglycosylase RodA and the transpeptidase PBP B3 as additional effectors of PrkA signaling. Our results suggest that both proteins act together to translate the signals received by PrkA into intensification of PG biosynthesis. These findings shed new light on the regulation of PG biosynthesis in Gram-positive bacteria with intrinsic {beta}-lactam resistance.

microbiology↗