Biology subjects

Schmid, N. E.

Publications and source records attributed to Schmid, N. E..

4 recordsLinked to original sources

bioRxiv2024

Discovery of a pentose as a cytosine nucleobase modification in Shewanella phage Thanatos-1 genomic DNA mediating enhanced resistance towards host restriction systems

Co-evolution of bacterial defense systems and phage counter defense mechanisms has resulted in an intricate biological interplay between bacteriophages and their prey. To evade nuclease-based mechanisms targeting the DNA, various bacteriophages modify their nucleobases, which impedes or even inhibits recognition by endonucleases. We found that Shewanella phage Thanatos-1 DNA is insensitive to multiple restriction enzymes and, partially, also to Cas I-Fv and Cas9 cleavage. Furthermore, the phage genome shows strongly impaired basecalling with nanopore sequencing. We characterised the phage adenine methyltransferase TH1_126 in methylase-free E. coli ER3413 and derived and confirmed its recognition motif 5-ATC-3. Moreover, the data pointed to an additional, much more substantial nucleobase modification. Using LC-MS, we identified a deoxypentose of unknown configuration attached to cytosine as a yet undiscovered phage DNA modification, which is present in Thanatos-1 genomic DNA, likely mediates the observed resistance to restriction endonucleases, as well as a strong reduction in Cas nuclease activity. To elucidate the underlying enzyme functions, we determined structural homologs of Thanatos-1 proteins among known glycosyltransferase folds and experimentally proved a UDP-xylose pyrophosphorylase function of phage protein TH1_063 by in vitro enzyme assays.

molecular biology↗

bioRxiv2024

Protein aggregation is a consequence of the dormancy-inducing membrane toxin TisB in Escherichia coli

Bacterial dormancy is a valuable strategy to survive stressful conditions. Toxins from chromosomal toxin-antitoxin systems have the potential to halt cell growth, induce dormancy and eventually promote a stress-tolerant persister state. Due to their potential toxicity when overexpressed, sophisticated expression systems are needed when studying toxin genes. Here, we present an optimized plasmid expression system for toxin genes based on an artificial 5 untranslated region. We applied the system to induce expression of the toxin gene tisB from the chromosomal type I toxin- antitoxin system tisB/istR-1 in Escherichia coli. TisB is a small hydrophobic protein that targets the inner membrane, resulting in depolarization and ATP depletion. We analyzed TisB-producing cells by RNA- sequencing and revealed several genes with a role in recovery from TisB-induced dormancy, including the chaperone genes ibpB, spy and cpxP. The importance of chaperone genes suggested that TisB- producing cells are prone to protein aggregation, which was validated by an in vivo fluorescent reporter system. We moved on to show that TisB is an essential factor for protein aggregation upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin in E. coli wild-type cells. The occurrence of protein aggregates correlates with an extended dormancy duration, which underscores their importance for the life cycle of TisB-dependent persister cells. ImportanceProtein aggregates occur in all living cells due to misfolding of proteins. In bacteria, protein aggregation is associated with cellular inactivity, which is related to dormancy and tolerance to stressful conditions, including the exposure to antibiotics. In Escherichia coli, the membrane toxin TisB is an important factor for dormancy and antibiotic tolerance upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin. Here, we show that TisB provokes protein aggregation, which in turn promotes a deeper state of cellular dormancy. Our study suggests that protein aggregation is a consequence of membrane toxins with the potential to affect the duration of dormancy and the outcome of antibiotic therapy.

microbiology↗

bioRxiv2024

Identifying the components of the Shewanella phage LambdaSo lysis system

Phage-induced lysis of Gram-negative bacterial hosts usually requires a set of phage lysis proteins, a holin, an endopeptidase and a spanin system, to disrupt each of the three cell envelope layers. Genome annotations and previous studies identified a gene region in the Shewanella oneidensis prophage LambdaSo, which comprises potential holin- and endolysin-encoding genes but lacks an obvious spanin system. By a combination of candidate approaches, mutant screening, characterization and microscopy we found that LambdaSo uses a pinholin/signal-anchor-release (SAR) endolysin system to induce proton-leakage and degradation of the cell wall. Between the corresponding genes we found that two extensively nested open reading frames encode a two-component spanin module Rz/Rz1. Unexpectedly, we identified another factor strictly required for LambdaSo-induced cell lysis, the phage protein Lcc6. Lcc6 is a transmembrane protein of 65 amino acid residues with hitherto unknown function, which acts at the level of holin in the cytoplasmic membrane to allow endolysin release. Thus, LambdaSo-mediated cell lysis requires at least four protein factors (pinholin, SAR-endolysin, spanin, Lcc6). The findings further extend the known repertoire of phage proteins involved in host lysis and phage egress. SignificanceFor the release of the assembled virions, phages have to breach the cell envelope. For Gram-negatives, this requires the disruption of three layers, the outer and inner membrane and the cell wall. In most cases, the lysis systems of phages infecting Gram-negatives comprises holins to disrupt or depolarize the membrane, thereby releasing or activating endolysins, which then degrade the cell wall. This, in turn, allows the spanins to become active and fuse outer and inner membrane, completing cell envelope disruption and allowing phage egress. Here we show that the presence of these three components may not be sufficient to allow cell lysis, implicating that also in known phages further factors may be required.

microbiology↗

bioRxiv2023

A rolling circle-replicating plasmid as an Inovirus phage satellite

Bacterial viruses (phages) are potent agents of lateral gene transfer and thus are important drivers of evolution. A group of mobile genetic elements (MGEs), referred to as phage satellites, exploit phages to disseminate their own genetic material. Here we isolated a novel member of the genus Inovirus, Shewanella phage Dolos, along with an autonomous rolling circle-replicating plasmid, pDolos. Dolos causes a chronic infection in its host Shewanella oneidensis by phage production with only minor effects on the host cell proliferation. When present, plasmid pDolos hijacks Dolos functions to be predominantly packaged into phage virions and released into the environment. pDolos can disseminate further genetic material encoding, e.g., resistances, fluorophores, and metabolically active proteins, to host cells sensitive to Dolos infection. Given the rather simple requirements of a plasmid for takeover of an inovirus, the wide distribution of phages of this group and the broad spectrum of rolling circle-replicating plasmids, we speculate that similar phage-satellite systems are common among bacteria.

microbiology↗