Biology subjects

Johansson, M. J. O.

Publications and source records attributed to Johansson, M. J. O..

9 recordsLinked to original sources

bioRxiv2026

Characterization of six environmental coli-phages isolated in Astana, Kazakhstan, during the School of Molecular and Theoretical Biology

Bacteriophage (phage) collections are essential resources for studying virus-host interactions in bacterial species. Here, we report six Escherichia coli-infecting phages that expand the Lund Collection of Bacteriophages. These phages were isolated in 2025 within the framework of the School of Molecular and Theoretical Biology for high-school students, from samples collected in Lake Taldykol, Astana, Kazakhstan, using E. coli strains MG1655{Delta}RM and EV36 as hosts. The isolated phages comprise Taldykol (LuPh6), a member of the genus Kagunavirus; Aidakhar (LuPh7) of the genus Phapecoctavirus; Samruk (LuPh8) of the genus Tequintavirus; the T-odd-like phage Baiterek (LuPh9) of the genus Vequintavirus; and two T-even-like phages Tulpar (LuPh10) and Shurale (LuPh11) that belong to the Tequatrovirus genus. This expanded phage collection enhances the toolkit for investigating phage-host interactions and their molecular mechanisms and highlights the use of phage isolation as a component of high school research education. ImportancePhage collections are a key resource for studying phage biology, phage-bacteria interactions and bacterial immune systems. Here, we extend the Lund Phage Collection through the isolation and characterisation of six E. coli-infecting phages, including three novel species (LuPh6, LuPh8 and LuPh11) as well as a member of the genus Phapecoctavirus that not represented in widely used collections such as BASEL (LuPh7). This study expands the resources available for probing phage-host interactions and demonstrates an example of integrating phage research into education of high school students.

microbiology↗

bioRxiv2026

Structures and enzymatic mechanisms of DRT7/UG10 antiphage reverse transcriptases

The ongoing evolutionary arms race between bacteria and bacteriophages has driven the emergence of numerous anti-phage defense systems. Several of these defenses contain domains related to reverse transcriptases (RTs), DNA polymerases that utilize RNA as a template. Certain members of the Abi/UG RT family employ a unique enzymatic mechanism combining protein-primed DNA synthesis with template-independent polymerization. A subgroup, UG10 - also known as Defense-related RT 7 (DRT7) - is characterized by an enzymatic core comprising an RT-like domain and a primase domain, accompanied by a poorly characterized accessory element. This element is either C-terminally fused to the core or encoded by a separate open reading frame. Here, we present cryo-EM structures of protein-DNA conjugates from two UG10 enzymes, revealing a compact yet flexible domain architecture. We demonstrate that the poly(dT) product synthesized by the RT-like domain serves as a template for the primase domain to generate a complementary poly(A) strand. Combining biochemical experiments with structural modeling, we propose the conformational changes necessary for protein priming and coordination between the RT and primase domains. Finally, we show that DRT7 exhibits broad-spectrum anti-phage activity and demonstrate that the {lambda} phage DNA mimic protein Gam can trigger this defense system.

molecular biology↗

bioRxiv2025

Characterization of five environmental phages infecting Escherichia coli K-12 isolated during a phage biology training course

Phage collections are essential tools for discovering and dissecting bacterial anti-phage defense systems. Here, we report the isolation and characterization of five environmental Escherichia coli-infecting phages, obtained during the 2023 Fundamentals of Basic and Applied Phage Biology course at Lund University. The phages were isolated using a motile E. coli K-12 BW25113 strain, whose motility is conferred by an IS5 insertion upstream of the flhDC operon, the master regulator of flagellar synthesis. The isolated Escherichia phages include Lubas (LuPh1) and Lucat (LuPh2) of the genus Tequatrovirus; Lupin (LuPh3) and Lucris (LuPh4) of the genus Tequintavirus; and Kompetensportalen (LuPh5) of the genus Chivirus. Transmission electron microscopy confirmed myovirus and siphovirus morphologies consistent with these genera. As expected for phages in the flagellotropic Chivirus genus, LuPh5 failed to infect a poorly motile BW25113 strain lacking the IS5 element upstream of flhDC. By testing a panel of eight previously described anti-phage defense systems, we found that LuPh1 and LuPh2 are inhibited by the toxin-antitoxin-chaperone CmdTAC system; LuPh5 is inhibited by both the restriction-modification system EcoRI and the abortive infection reverse transcriptase AbiK; and all five phages are sensitive to the hybrid artificial CmdTA-HigC system. Collectively, our findings expand the toolkit for probing phage-host interactions and underscore the pedagogical value of incorporating phage isolation into practical training for emerging researchers.

microbiology↗

bioRxiv2024

Toxic Small Alarmone Synthetase FaRel2 inhibits translation by pyrophosphorylating tRNAGly and tRNAThr

Translation-targeting toxic Small Alarmone Synthetases (toxSAS) are effectors of bacterial Toxin-Antitoxin systems that pyrophosphorylate the 3-CCA end of tRNA to prevent aminoacylation. toxSAS are implicated in antiphage immunity: phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNAGly and tRNAThr. The 1st, 2nd, 4th and 5th base pairs the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRelSJ46 differ in tRNA specificity from FaRel2, and rationalise this through structural modelling: while the universal 3-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defences, we hypothesise that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

microbiology↗

bioRxiv2024

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys> isoacceptors in the P site

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

molecular biology↗

bioRxiv2024

Mechanism of phage sensing and abortion by toxin-antitoxin-chaperone systems

Toxin-antitoxins (TAs) are prokaryotic two-gene systems comprised of a toxin neutralised by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilises the antitoxin by recognising its chaperone addiction (ChAD) element. TACs have been shown to mediate antiphage defence, but the mechanisms of viral sensing and restriction are unexplored. We identify and characterise two Escherichia coli antiphage TAC systems containing HigBA and CmdTA TA units, HigBAC and CmdTAC. The HigBAC is triggered through recognition of the gpV major tail protein of phage {lambda}. Both the ChAD and gpV are recognised by the HigC chaperone through analogous aromatic molecular patterns, explaining the mechanism of activation. We show that the CmdT ADP-ribosyltransferase toxin modifies mRNA to shut down protein synthesis. We establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with the HigC chaperone phage sensor. HighlightsE. coli HigBAC and CmdTAC are translation-targeting phage immunity TAC systems HigC chaperone recognises phage {lambda} major tail protein to trigger HigBAC toxicity CmdT ADP-ribosyltransferase toxin abrogates translation through modification of mRNA HigC combined with CmdTA yields hybrid broad-spectrum antiphage defence system

microbiology↗

bioRxiv2023

The structural basis of hyperpromiscuity in a core combinatorial network of Type II toxin-antitoxin and related phage defence systems

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralising antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins) we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralisation by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralising mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralisation, while in multidomain antitoxins the neutralisation is mediated by other domains, such as PAD1, Phd-C and ZFD. We hypothesise that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 new NetFlax TA systems. We used functional domain annotations and with metabolic labelling assays to predict their potential mechanisms of toxicity (such as disruption of membrane integrity, inhibition of cell division and abrogation of protein synthesis) as well as biological functions (such as antiphage defence). The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/. Significance statementToxin-antitoxin systems are enigmatic components of microbial genomes, with their biological functions being a conundrum of debate for decades. Increasingly, TAs are being found to have a role in defence against bacteriophages. By mapping and experimentally validating a core combinatorial network of TA systems and high-throughput prediction of structural interfaces, we uncover the evolutionary scale of TA partner swapping and discover new toxic effectors. We validate the predicted toxin:antitoxin complex interfaces of four TA systems, uncovering the evolutionary malleable mechanism of toxin neutralisation by Panacea-containing PanA antitoxins. We find TAs are evolutionarily related to several other phage defence systems, cementing their role as important molecular components of the arsenal of microbial warfare.

microbiology↗

bioRxiv2019

A role for the Saccharomyces cerevisiae ABCF protein New1 during translation termination

Translation on the ribosome is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related ABCF ATPase, New1, is encoded by a non-essential gene with a cold sensitivity and ribosome assembly defect knock-out phenotype. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide ribosome assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed a combination of yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination.

biochemistry↗

bioRxiv2018

Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae

In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5 part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.

biochemistry↗