Biology subjects

Gerdes, K.

Publications and source records attributed to Gerdes, K..

9 recordsLinked to original sources

bioRxiv2026

Phage Portal Proteins Suppress Bacterial Stringent Response to Promote Infection

Bacteria restrict viral replication not only through dedicated defense systems but also by entering global physiological states that limit cellular resources. How phages counter such host-imposed physiological barriers remains poorly understood. The stringent response (SR), mediated by the alarmones ppGpp and pppGpp, induces a growth-restrictive state that can act as a barrier to bacteriophage infection. Here we show that elevated alarmone levels delay T7-mediated host lysis, whereas (p)ppGpp-deficient cells are hypersensitive to infection, establishing alarmone signaling as a physiological constraint on phage replication. A systematic functional screen identifies the essential capsid portal protein Gp8 as a viral factor genetically linked to SR-dependent host physiology. Gp8 directly binds the alarmone synthetases RelA and SpoT, selectively inhibiting their synthetase activities in vitro and suppressing alarmone accumulation in vivo. Phages carrying interaction-defective portal mutations exhibit impaired replication, delayed lysis, and sustained (p)ppGpp elevation during infection, defects that are alleviated in SR-deficient hosts. Structural and mutational analyses reveal electrostatically mediated interfaces required for targeting RelA and SpoT. Portal proteins from diverse coliphages share similar structural features, interact with stringent-response enzymes, and display SR-linked phenotypes, indicating a broadly conserved viral strategy. Together, these findings identify phage portal proteins as a previously unrecognized class of viral counter-defense factors that target a central bacterial stress signaling pathway, revealing that essential structural virion components can moonlight as regulators of host stress physiology.

microbiology↗

bioRxiv2026

Basal ppGpp regulation by SpoT coordinates metabolic homeostasis and acid resistance

Bacteria maintain low basal levels of the alarmone ppGpp during steady-state growth, yet how this basal state is established and why it matters physiologically remain poorly understood. Here, we show that basal ppGpp represents an actively maintained regulatory state that coordinates metabolic homeostasis with stress resistance. Using a targeted perturbation of SpoT regulation in Escherichia coli, we uncover a sharp requirement for a sub-basal yet non-zero ppGpp pool to sustain growth in minimal medium and survival under extreme acid stress. Disruption of this basal state leads to misallocation of metabolic flux into arginine biosynthesis, depletion of glutamate, and collapse of the glutamate-dependent Gad acid resistance system. Through intragenic suppressor analysis, enzymatic perturbations, and protein-level feedback measurements, we further demonstrate that SpoT intrinsically tunes basal ppGpp through a distributed intramolecular regulatory network coupled to negative feedback control of SpoT abundance. This regulatory logic stabilizes ppGpp within a narrow physiological window, below the threshold of canonical stringent response activation. The requirement for SpoT-dependent basal ppGpp regulation is conserved in pathogenic Salmonella and Shigella. Together, our findings establish basal ppGpp as a distinct and actively regulated signaling regime that integrates metabolism and stress preparedness beyond acute starvation responses.

microbiology↗

bioRxiv2025

Mono and Multidomain Anti-phage Defense Toxins of the RelE/ParE Superfamily

Toxin-antitoxin (TA) modules are widely distributed across prokaryotes, often existing in large numbers despite their associated fitness costs. Most type II TA modules are bicistronic operons encoding a monodomain toxin and its cognate protein antitoxin. The RelE/ParE superfamily encompasses toxins with a conserved Barnase-EndoU-ColicinE5/D-RelE (BECR) fold, yet their cellular targets differ remarkably: RelE toxins function as ribosome-dependent RNases, while ParE toxins act as DNA gyrase inhibitors. Using a comprehensive bioinformatics approach, this study analyzed 13 BECR-fold toxin families as classified in the Pfam database. Intriguingly, the ParE family was found to include a subcluster of mRNA-cleaving toxins, challenging its conventional role as solely DNA-targeting. This study identified a novel tripartite operon encoding a PtuA-like defense ATPase, a homolog of type IV restriction endonucleases, and a RelE homolog, suggesting a coordinated role in defense mechanisms. Multidomain BECR-fold toxins, including transmembrane variants, were also discovered, extending the functional repertoire of type II TA modules to membrane-associated systems. These findings clarify the evolutionary relationships and functional diversity within the RelE/ParE superfamily, and discovers novel, putative defence systems that can now be investigated experimentally. ImportanceToxin - antitoxin modules play critical roles in prokaryotic survival and adaptation, contributing to genome stabilization and defense against phages and invading plasmids. The RelE/ParE superfamily exemplifies the structural and functional diversity of these systems, with members targeting distinct cellular processes, such as translation and DNA supercoiling. By elucidating the relationships among the 13 BECR-fold toxin families, this study enhances our understanding of microbial resistance mechanisms and reveals potential new opportunities for research into prokaryotic defense and regulation. These insights may have significant implications for medical and biotechnological applications, particularly in understanding bacterial responses to genetic invaders.

microbiology↗

bioRxiv2023

Diverse Genetic Contexts of HicA Toxin Domains Propose a Role in Anti-Phage Defense

Toxin - antitoxin (TA) modules are prevalent in prokaryotic genomes, often in substantial numbers. For instance, the Mycobacterium tuberculosis genome alone harbors close to 100 TA modules, half of which belong to a singular type. Traditionally ascribed multiple biological roles, recent insights challenge these notions and instead indicate a predominant function in phage defense. TAs are often located within Defense Islands, genomic regions that encode various defense systems. The analysis of genes within Defense Islands have unveiled a wide array of systems, including TAs that serve in anti-phage defence. Prokaryotic cells are equipped with anti-phage Viperins that, analogous to their mammalian counterparts, inhibit viral RNA transcription. Additionally, bacterial Structural Maintenance of Chromosome (SMC) proteins combat plasmid intrusion by recognizing foreign DNA signatures. This study undertakes a comprehensive bioinformatics analysis of genetic elements encoding the HicA double-stranded RNA-binding domain, complemented by protein structure modeling. The HicA toxin domains are found in at least 14 distinct contexts and thus exhibit a remarkable genetic diversity. Traditional bicistronic TA operons represent eight of these contexts, while four are characterized by monocistronic operons encoding fused HicA domains. Two contexts involve hicA adjacent to genes that encode bacterial Viperins. Notably, genes encoding RelE toxins are also adjacent to Viperin genes in some instances. This configuration hints at a synergistic enhancement of Viperin-mediated anti-phage action by HicA and RelE toxins. The discovery of a HicA domain merged with an SMC domain is compelling, prompting further investigation into its potential roles. ImportanceProkaryotic organisms harbor a multitude of Toxin - Antitoxin (TA) systems, which have long puzzled scientists as "genes in search for a function". Recent scientific advancement have shed light on a primary role of TAs as anti-phage defense mechanisms. To gain an overview of TAs it is important to analyze their genetic contexts that can give hints on function and guide future experimental inquiries. This manuscript describes a thorough bioinformatics examination of genes encoding the HicA toxin domain, revealing its presence in no fewer than 14 unique genetic arrangements. Some configurations notably align with anti-phage activities, underscoring potential roles in microbial immunity. These insights robustly reinforce the hypothesis that HicA toxins are integral components of the prokaryotic anti-phage defense repertoire. The elucidation of these genetic contexts not only advances our understanding of TAs but also contributes to a paradigm shift in how we perceive their functionality within the microbial world.

bioinformatics↗

bioRxiv2022

Structural basis for regulation of a tripartite toxin-antitoxin system by dual phosphorylation

Many bacteria encode multiple toxin-antitoxin (TA) systems targeting separate, but closely related, cellular functions. The toxin of the E. coli hipBA system, HipA, is a kinase that inhibits translation via phosphorylation of glutamyl-tRNA synthetase. Enteropathogenic E. coli (EPEC) O127:H6 encodes the hipBA-like, tripartite TA system; hipBST, in which the HipT toxin specifically targets the tryptophanyl-tRNA synthetase, TrpS. Notably, in the tripartite system, the function as antitoxin has been taken over by the third protein, HipS, but the molecular details of how activity of HipT is inhibited remain poorly understood. Here, we show that HipBST is structurally different from E. coli HipBA and that the unique HipS protein, which is homologous to the N-terminal subdomain of HipA, inhibits the kinase through insertion of a conserved Trp residue into the active site. We also show how auto-phosphorylation at two conserved sites in the kinase toxin serve different roles and affect the ability of HipS to neutralize HipT. Finally, solution structural studies show how phosphorylation affects overall TA complex flexibility.

microbiology↗

bioRxiv2021

Phylogeny Reveals Novel HipA-Homologous Kinase Families and Toxin - Antitoxin Gene Organizations

Toxin - Antitoxin modules function in the genetic stability of mobile genetic elements, bacteriophage defense, and antibiotic tolerance. A gain-of-function mutation of the Escherichia coli K-12 hipBA module can induce antibiotic tolerance in a subpopulation of bacterial cells, a phenomenon known as persistence. HipA is a Ser/Thr kinase that phosphorylates and inactivates glutamyl tRNA synthetase, inhibiting cellular translation and inducing the stringent response. Additional characterized HipA homologues include HipT from pathogenic E. coli O127 and YjjJ of E. coli K-12, which are encoded by tri-cistronic hipBST and monocistronic operons, respectively. The apparent diversity of HipA homologues in bacterial genomes inspired us to investigate overall phylogeny. Here we present a comprehensive phylogenetic analysis of the Hip kinases in bacteria and archaea that expands on this diversity by revealing seven novel kinase families. Kinases of one family, encoded by monocistronic operons, consist of an N-terminal core kinase domain, a HipS-like domain and a HIRAN (HIP116 Rad5p N-terminal) domain. HIRAN domains bind single or double-stranded DNA ends. Moreover, five types of bicistronic kinase operons encode putative antitoxins with HipS-HIRAN, HipS, {gamma}{delta}-resolvase or Stl repressor-like domains. Finally, our analysis indicates that reversion of hipBA gene-order happened independently several times during evolution. ImportanceBacterial multidrug tolerance and persistence are problems of increasing scientific and medical significance. The first gene discovered to confer persistence was hipA, encoding the kinase toxin of the hipBA toxin-antitoxin (TA) module of E. coli. HipA-homologous kinases phosphorylate and thereby inactivate specific tRNA synthetases, thus inhibiting protein translation and cell proliferation. Here, we present a comprehensive phylogenetic analysis of bacterial Hip kinases and discover seven new families with novel operon structures and domains. Overall, Hip kinases are encoded by TA modules with at least 10 different genetic organizations, seven of which have not been described before. These results open up exciting avenues for the experimental analysis of the superfamily of Hip kinases.

microbiology↗

bioRxiv2020

Proteome dynamics in persisting and resuscitating bacterial cells

During antibiotic persistence, bacterial cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of antibiotic persistence is hindered by low count of persisting cells and the need for their isolation. Here we used sustained addition of stable isotope-labeled lysine to selectively label the proteome during hipA-induced persistence and hipB-induced resuscitation of E. coli cells in minimal medium after antibiotic treatment. Time-resolved, 24-hour measurement of label incorporation allowed detection of over 500 newly synthesized proteins in viable cells, demonstrating low but widespread protein synthesis during persistance. Many essential proteins were newly synthesized and several ribosome-associated proteins, such as RaiA and Sra showed high synthesis levels, pointing to their roles in maintenance of persistence. At the onset of resuscitation, cells synthesized the ribosome-splitting GTPase HflX and various ABC transporters, restored translation machinery and resumed metabolism by inducing glycolysis and biosynthesis of amino acids.

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↗

bioRxiv2017

Nasty Prophages and the Dynamics of Antibiotic-Tolerant Persister Cells

Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and the activation of toxin-antitoxin (TA) modules. This model found support in the field, but also generated controversy as part of recent heated debates on the validity of significant parts of the literature. In this study, we therefore used our previous work as a model to critically examine common experimental procedures in order to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variation of culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage {varphi}80, a notorious laboratory contaminant, has apparently caused several phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon, but do not anymore support a role of TA modules in E. coli persister formation. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.\n\nImportanceThe recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies have identified multiple genetic pathways involved in persister formation, but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely different results based on ill-controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis of future studies on the formation and eradication of antibiotic-tolerant persisters.

microbiology↗