Biology subjects

Tamman, H.

Publications and source records attributed to Tamman, H..

5 recordsLinked to original sources

bioRxiv2025

Nanobody-mediated modulation of long RSH enzymes Rel and RelA catalysis by restriction of their conformational landscape

Long RSH enzymes, Rel and RelA, are the master regulators of (p)ppGpp alarmone levels in bacteria. Their catalytic activity is governed by transitions between a compact, hydrolysis-competent (HDON) state and an elongated, synthesis-competent (SYNTHON) state. The equilibrium between these states is modulated by factors such as "starved" ribosomes and regulatory proteins DarB, EIIANTR, ACP and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by trapping Rel/RelA enzymes in distinct conformational states. Nanobodies that lock the TGS domain of RelA and prevent its activation by deacylated tRNA on starved ribosomes, strongly inhibit (p)ppGpp synthesis and suppress the virulence of E. coli in an animal model. Nb898 stabilizes Rel in the open SYNTHON state, enhancing synthesis activity while suppressing hydrolysis. Conversely, Nb585 traps Rel in a HDON conformation, strongly inhibiting alarmone synthesis while promoting (p)ppGpp hydrolysis. Structural and biochemical analyses reveal that nanobodies, like natural allosteric regulators, act by restricting the RSH enzymes conformational landscape. These findings establish nanobodies as powerful tools for dissecting RSH function and provide potential leads for developing protein-based RSH modulators.

microbiology↗

bioRxiv2024

Isolation and characterization of a phage collection against Pseudomonas putida

The environmental bacterium Pseudomonas putida has a wide range of metabolic pathways and thus entails great promise both as cell factories in biotechnological production and for bioremediation approaches to degrade various aromatic pollutants. To thrive in the environment, P. putida has to be able to endure the constant threat posed by bacteriophages. Surprisingly, up until now, only a few phages have been isolated for the common laboratory strain P. putida KT2440, and no phage defence mechanisms have been characterized yet. Here, we introduce a novel Collection of Environmental Pseudomonas putida Phages from Estonia (CEPEST), consisting of 67 dsDNA phages that belong to 22 phage species and nine phage genera. We show that most of the phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require intact LPS for P. putida infection. Additionally, we demonstrate that cryptic prophages in the P. putida chromosome strongly protect against the infection of many phages, whereas the chromosomal toxin-antitoxin systems play no role in phage defence of P. putida.

microbiology↗

bioRxiv2022

Direct activation of an innate immune system in bacteria by a viral capsid protein

Bacteria have evolved sophisticated and diverse immunity mechanisms to protect themselves against a nearly constant onslaught of bacteriophages1-3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns (PAMPs)4, many bacterial immune systems that respond to bacteriophage infection require a phage-specific trigger to be activated. However, the identities of such triggers and the mechanistic basis of sensing remain almost completely unknown. Here, we discover and investigate the anti-phage function of a fused toxin-antitoxin (TA) system called CapRelSJ46 that protects E. coli against diverse phages. Through genetic, biochemical, and structural analysis, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both an antitoxin element and a phage-infection sensor. Following infection by certain phages, the newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to then pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for TA-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a Red Queen conflict5, revealing a new front in the intense coevolutionary battle being waged by phage and bacteria. With capsid proteins of some eukaryotic viruses known to stimulate innate immune signaling in mammalian hosts6-10, our results now reveal an ancient, deeply conserved facet of immunity.

microbiology↗

bioRxiv2020

Pseudomonas putida chromosomal toxin-antitoxin systems carry neither clear fitness benefits nor big costs

Chromosomal toxin-antitoxin (TA) systems are widespread genetic elements among bacteria, yet, despite extensive studies in the last decade, their biological importance remains ambivalent. The ability of TA-encoded toxins to affect stress tolerance supports the hypothesis of TA systems being associated with stress adaptation. However, the deletion of TA genes has usually no fitness consequences, supporting the selfish elements hypothesis. Here, we aimed to evaluate the cost and benefits of chromosomal TA systems to Pseudomonas putida. We show that multiple TA systems do not confer fitness benefits to this bacterium as deletion of 13 TA loci does not influence stress tolerance, persistence and biofilm formation. Our results instead show that TA loci are costly and decrease the competitive fitness of P. putida. Still, the cost of multiple TA systems is low and detectable in certain conditions only. Construction of antitoxin deletion strains showed that only five TA systems code for toxic proteins, while other TA loci have evolved towards reduced toxicity and encode non-toxic or moderately potent proteins. Analysis of P. putida TA systems homologs among fully sequenced Pseudomonads suggests that the TA loci have been subjected to purifying selection and that TA systems spread among bacteria by horizontal gene transfer.

microbiology↗

bioRxiv2019

Nucleotide-mediated allosteric regulation of bifunctional Rel enzymes

Bifunctional Rel stringent factors, the most broadly distributed class of RSHs, are ribosome-associated enzymes that transfer a pyrophosphate group from ATP onto the 3' of GTP or GDP to synthesize (p)ppGpp and also catalyse the 3' pyrophosphate hydrolysis of the alarmone to degrade it. The precise regulation of these enzymes seems to be a complex allosteric mechanism, and despite decades of research, it is unclear how the two opposing activities of Rel are controlled at the molecular level. Here we show that a stretch/recoil guanosine-switch mechanism controls the catalytic cycle of T. thermophilus Rel (RelTf). The binding of GDP/ATP stretches apart the NTD catalytic domains of RelTf (RelTtNTD) activating the synthetase domain and allosterically blocking the hydrolase active site. Conversely, binding of ppGpp unlocks the hydrolase domain and triggers recoil of both NTDs, which partially buries the synthetase active site and precludes the binding of synthesis precursors. This allosteric mechanism acts as an activity switch preventing futile cycles of alarmone synthesis and degradation.

molecular biology↗