Biology subjects

Severinov, K.

Publications and source records attributed to Severinov, K..

30 records · Page 2Linked to original sources

bioRxiv2022

Cell-free mutant analysis combined with structure prediction of a lasso peptide biosynthetic protein B2

Biochemical and structural analyses of purified proteins are essential for the understanding of their properties. However, many proteins are unstable and difficult to purify, hindering their characterization. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The B2 proteins are poorly soluble and no experimentally-solved structures are available. Here, we performed a rabid semi-comprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida fusca (TfuB2) using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2. Analysis of 34 TfuB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, TfuB1. Our results suggest that the combination of rapid cell-free mutant analyses with precise structure predictions can greatly accelerate structure-function research of proteins for which no structures are available.

biochemistry↗

bioRxiv2022

Cells with Stochastically Increased Methyltransferase to Restriction Endonuclease Ratio Provide an Entry for Bacteriophage into Protected Cell Population

The action of type II restriction-modification (RM) systems depends on restriction endonuclease (REase), which cleaves foreign DNA at specific sites, and methyltransferase (MTase), which protects host genome from restriction by methylating the same sites. We show that protection from phage infection increases as the copy number of plasmids carrying the Esp1396l RM system is increased. However, since increased plasmid copy number leads to both increased absolute intracellular REase and MTase levels and decreased MTase to REase ratio, it is impossible to determine which factor determines resistance/susceptibility to infection. By controlled expression of Esp1396I MTase or REase genes in cells carrying the Esp1396I system, we show that a shift in the MTase to REase ratio caused by overproduction of MTase or REase leads, respectively, to decreased or increased protection from infection. Consistently, due to stochastic variation of MTase and REase amount in individual cells, bacterial cells that are productively infected by bacteriophage have significantly higher MTase to REase ratios than cells that ward off the infection. Our results suggest that cells with transiently increased MTase to REase ratio at the time of infection serve as entry points for unmodified phage DNA into protected bacterial populations.

microbiology↗

bioRxiv2022

Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages

RNA sequencing has become the method of choice to study the transcriptional landscape of phage-infected bacteria. However, short-read RNA sequencing approaches generally fail to capture the primary 5 and 3 boundaries of transcripts, confounding the discovery of key transcription initiation and termination events as well as operon architectures. Yet, the elucidation of these elements is crucial for the understanding of the strategy of transcription regulation during the infection process, which is currently lacking beyond a handful of model phages. To this end, we developed ONT-cappable-seq, a specialized long-read RNA sequencing technique that allows end-to-end sequencing of primary prokaryotic transcripts using the Nanopore sequencing platform. We applied ONT-cappable-seq to study transcription of Pseudomonas aeruginosa phage LUZ7, obtaining a comprehensive genome-wide map of viral transcription start sites, terminators, and complex operon structures that fine-regulate gene expression. Our work provides new insights in the RNA biology of a non-model phage, unveiling distinct promoter architectures, putative small non-coding viral RNAs, and the prominent regulatory role of terminators during infection. The robust workflow presented here offers a framework to obtain a global, yet fine-grained view of phage transcription and paves the way for standardized, in depth transcription studies for microbial viruses or bacteria in general.

genomics↗

bioRxiv2021

tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA1,2. In addition to the CRISPR RNA (crRNA)-guided, sequence-specific binding and cleavage of target RNAs, such as phage transcripts, the type VI effector, Cas13, causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from phage spread3,4. We show here that the principal form of collateral RNA degradation elicited by Cas13a protein from Leptotrichia shahii upon target RNA recognition is the cleavage of anticodons of multiple tRNA species, primarily those with anticodons containing uridines. This tRNA cleavage is necessary and sufficient for bacterial dormancy induction by Cas13a. In addition, Cas13a activates the RNases of bacterial toxin-antitoxin modules, thus indirectly causing mRNA and rRNA cleavage, which could provide a back-up defense mechanism. The identified mode of action of Cas13a resembles that of bacterial anticodon nucleases involved in antiphage defense5, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module6,7 encompassing an anticodon nuclease.

microbiology↗

bioRxiv2021

Host-associated phages disperse across the extraterrestrial analogue Antarctica

Extreme Antarctic conditions provide one of the closest analogues of extraterrestrial environments. Since air and snow samples, especially from polar regions, yield DNA amounts in the lower picogram range, binning of prokaryotic genomes is challenging and renders studying the dispersal of biological entities across these environments difficult. Here, we hypothesized that dispersal of host-associated bacteriophages (adsorbed, replicating or prophages) across the Antarctic continent can be tracked via their genetic signatures aiding our understanding of virus and host dispersal across long distances. Phage genome fragments (PGFs) reconstructed from surface snow metagenomes of three Antarctic stations were assigned to four host genomes, mainly Betaproteobacteria including Ralstonia spp. We reconstructed the complete genome of a temperate phage with near-complete alignment to a prophage in the reference genome of Ralstonia pickettii 12D. PGFs from different stations were related to each other at the genus level and matched similar hosts. Metagenomic read mapping and nucleotide polymorphism analysis revealed a wide dispersal of highly identical PGFs, 13 of which were detected in seawater from the Western Antarctic Peninsula in distance of 5538 km to the snow sampling stations. Our results suggest that host-associated phages, especially of Ralstonia sp., disperse over long distances despite harsh conditions of the Antarctic continent. Given that 14 phages associated with two R. pickettii draft genomes isolated from space equipment were identified, we conclude that Ralstonia phages are ideal mobile genetic elements to track dispersal and contamination in ecosystems relevant for Astrobiology. ImportanceHost-associated phages of the bacterium Ralstonia identified in snow samples can be used to track microbial dispersal over thousands of kilometers across the Antarctic continent, which functions as an extraterrestrial analogue because of its harsh environmental conditions. Due to presence of these bacteria carrying genome-integrated prophages on space-related equipment, and the here demonstrated potential for dispersal of host-associated phages, our work has implications for Planetary Protection, a discipline in Astrobiology interested in preventing contamination of celestial bodies with alien biomolecules or forms of life.

microbiology↗

bioRxiv2021

Interaction Between Transcribing RNA Polymerase and Topoisomerase I Prevents R-loop Formation in E. coli

Bacterial topoisomerase I (TopoI) removes excessive negative supercoiling and is thought to relax DNA molecules during transcription, replication and other processes. Using ChIP-Seq, we show that TopoI of Escherichia coli (EcTopoI) is co-localized, genome-wide, with RNA polymerase (RNAP) in transcription units. Treatment with transcription elongation inhibitor rifampicin leads to EcTopoI relocation to promoter regions, where RNAP also accumulates. When a 14 kDa RNAP-binding EcTopoI C-terminal domain (CTD) is overexpressed, co-localization of EcTopoI and RNAP along the transcription units is reduced. Pull-down experiments directly show that the two enzymes interact in vivo. Using ChIP-Seq and Topo-Seq, we demonstrate that EcTopoI is enriched and in and upstream (within up to 12-15 Kbs) of highly-active transcription units, indicating that EcTopoI relaxes negative supercoiling generated by transcription. Uncoupling of the RNAP-EcTopoI interaction by either overexpression of EcTopoI CTD or deletion of EcTopoI domains involved in the interaction is toxic for cells and leads to excessive negative plasmid supercoiling. Moreover, the CTD overexpression leads to R-loops accumulation genome-wide, indicating that the RNAP-EcTopoI interaction is required for prevention of R-loops formation. Article HighlightsO_LITopoI colocalizes genome-wide and interacts with RNAP in E. coli C_LIO_LIDisruption of the interaction between TopoI and RNAP decreases cells viability, leads to hypernegative DNA supercoiling, and R-loops accumulation C_LIO_LITopoI and DNA gyrase are enriched, respectively, upstream and downstream of transcription units in accordance with twin-domain model of Liu and Wang C_LIO_LITopoI recognizes its cleavage sites through a specific motif and by sensing negative supercoiling C_LI

molecular biology↗

bioRxiv2021

Bacteriostatic antibiotics promote the evolution of CRISPR-Cas immunity

Phage therapy can be used in combination with antibiotics to combat infections with bacterial pathogens1-3. However, bacteria can rapidly evolve phage resistance via receptor mutation, or using their CRISPR-Cas adaptive immune systems4, which insert short phage-derived sequences into CRISPR loci in the bacterial genome5 to guide sequence-specific cleavage of cognate sequences6. Unlike CRISPR-Cas immunity, mutation of the phage receptor leads to attenuated virulence when the opportunistic pathogen Pseudomonas aeruginosa is infected with its phage DMS3vir7, which underscores the need to predict how phage resistance evolves under clinically relevant conditions. Here, using eight antibiotics with various modes of action, we show that bacteriostatic antibiotics (which inhibit cell growth without killing) specifically promote evolution of CRISPR-Cas immunity in P. aeruginosa by slowing down phage development and providing more time for cells to acquire phage-derived sequences and mount an immune response. Our data show that some antimicrobial treatments can contribute to the evolution of phage-resistant pathogens with high virulence.

microbiology↗

bioRxiv2021

Long-Term Persistence of Plasmids Targeted by CRISPR Interference in Bacterial Populations

CRISPR-Cas systems provide prokaryotes with an RNA-guided defense against foreign mobile genetic elements (MGEs) such as plasmids and viruses. A common mechanism by which MGEs avoid interference by CRISPR consists of acquisition of escape mutations in regions targeted by CRISPR. Here, using microbiological, live microscopy, and microfluidics analyses we demonstrated that plasmids can persist in Escherichia coli cells at conditions of continuous targeting by the type I-E CRISPR-Cas system without acquiring any genetic alterations. We used mathematical modeling to show how plasmid persistence in a subpopulation of cells mounting CRISPR interference is achieved due to the stochastic nature of CRISPR interference and plasmid replication events. We hypothesize that the observed complex dynamics provides bacterial populations with long-term benefits due to the presence of mobile genetic elements in some cells, leading to diversification of phenotypes in the entire community and allowing rapid changes in the population structure to meet the demands of a changing environment.

microbiology↗

bioRxiv2020

Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) directing target DNA cleavage by type V-A Cas12a nucleases consist of repeat-derived 5-scaffold moiety and 3-spacer moiety. We demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by Cas12a ortholog from Acidaminococcus sp (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer part only, while crRNAs split into two individual moieties (scaffold and spacer RNAs) catalyzed highly specific and efficient cleavage of target DNA. Our data also indicate that AsCas12a combined with split crRNA forms a stable complex with the target. These observations were also confirmed in lysates of human cells expressing AsCas12a. The ability of the AsCas12a nuclease to be programmed with split crRNAs opens new lines of inquiry into the mechanisms of target recognition and cleavage and will further facilitate genome editing techniques based on Cas12a nucleases.

molecular biology↗

bioRxiv2019

Phazolicin - a Novel Thiazole/Oxazole-Modified Peptide Inhibiting the Bacterial Ribosome in a Species-Specific Way.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding and largely untapped class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display an outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium sp. Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants belonging to the Rhizobiales. PHZ inhibits prokaryotic translation through the obstruction of the passage of the nascent peptide through the ribosome exit channel. The cryo-EM structure of the Escherichia coli ribosome with bound PHZ revealed that the drug interacts with the 23S rRNA and ribosomal proteins uL4 and uL22 and obstructs the exit tunnel in a way that is distinct from other compounds blocking the exit channel. We show that the sequence of uL4 ribosomal protein loop involved in PHZ binding determines the species-specificity of antibiotic interaction with its target. PHZ and its predicted homologs from other bacterial species expand the known diversity of LAPs and may be used in the future as biocontrol agents for the needs of agriculture.

molecular biology↗

bioRxiv2019

Detection of Spacer Precursors Formed In Vivo During Primed CRISPR Adaptation

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA1. Immunity involves \"adaptation,\" the integration of ~30-bp DNA fragments into the CRISPR array as \"spacer\" sequences, and \"interference,\" the targeted degradation of DNA containing a \"protospacer\" sequence mediated by a complex containing a spacer-derived CRISPR RNA (crRNA)1-4. Specificity for targeting interference to protospacers, but not spacers, occurs through recognition of a 3-bp protospacer adjacent motif (PAM)5 by the crRNA-containing complex6. Interference-driven DNA degradation of protospacer-containing DNA can be coupled with \"primed adaptation,\" ill which spacers are acquired from DNA surrounding the targeted protospacer in a bidirectional, orientation-dependent manner2,3,7. Here we construct a robust in vivo model for primed adaptation consisting of an Escherichia coli type I-E CRISPR-Cas \"self-targeting\" locus encoding a crRNA that targets a chromosomal protospacer. We develop a strand-specific, high-throughput-sequencing method for analysis of DNA fragments, \"FragSeq,\" and use this method to detect short fragments derived from DNA surrounding the targeted protospacer. The detected fragments have sequences matching spacers acquired during primed adaptation, contain ~3- to 4-nt overhangs derived from excision of genomic DNA within a PAM, are generated in a bidirectional, orientation-dependent manner relative to the targeted protospacer, require the functional integrity of machinery for interference and adaptation to accumulate, and function as spacer precursors when exogenously introduced into cells by transformation. DNA fragments with a similar structure accumulate in cells undergoing primed adaptation in a type I-F CRISPR-Cas self-targeting system. We propose the DNA fragments detected in this work are products of universal steps of spacer precursor processing in type I CRISPR-Cas systems.

microbiology↗

bioRxiv2017

Optimal Number Of Spacers In CRISPR Arrays

We estimate the number of spacers in a CRISPR array of a bacterium which maximizes its protection against a viral attack. The optimality follows from a competition between two trends: too few distinct spacers make the bacteria vulnerable to an attack by a virus with mutated corresponding protospacers, while an excessive variety of spacers dilutes the number of the CRISPR complexes armed with the most recent and thus most effective spacers. We first evaluate the optimal number of spacers in a simple scenario of an infection by a single viral species and later consider a more general case of multiple viral species. We find that depending on such parameters as the concentration of CRISPR-CAS interference complexes and its preference to arm with more recently acquired spacers, the rate of viral mutation, and the number of viral species, the predicted optimal array length lies within a range quite reasonable from the viewpoint of recent experiments.\n\nAuthor summaryCRISPR-Cas system is an adaptive immunity defense in bacteria and archaea against viruses. It works by accumulating in bacterial genome an array of spacers, or fragments of virus DNA from previous attacks. By matching spacers to corresponding parts of virus DNA called protospacers, CRISPR-Cas system identifies and destroys intruder DNA. Here we theoretically estimate the number of spacers that maximizes bacterial survival. This optimum emerges from a competition between two trends: More spacers allow a bacterium to hedge against mutations in viral protospacers. However, keeping too many spacers makes the older ones inefficient because of accumulation of mutations in corresponding protospacers in viruses. Thus, fewer CRISPR-Cas molecular machines are left armed with more efficient young spacers. We have shown that a higher efficiency of CRISPR-Cas system allows a bacterium to utilize more spacers, increasing the optimal array length. On contrary, a higher viral mutation rate makes older spacers useless and favors shorter arrays. A higher diversity in viral species reduces the efficiency of CRISPR-Cas but does not necessary lead to longer arrays. We think that our study provides a new viewpoint at a huge variety in the observed array lengths and adds relevance to evolutionary models of bacterial-phage coexistence.

systems biology↗