Transcription is the central step, and a major regulatory checkpoint of gene expression. Defective transcription regulation is the common cause of aberrant growth and development and may result in malignant transformation. Transcription is carried out by DNA-dependent RNA polymerases–large, multisubunit molecular machines. Understanding RNA polymerase (RNAP) structure and function is a key to understanding gene expression in molecular detail. The long-term objective of our research is to uncover the molecular basis of transcription mechanism and regulation through structure-functional analysis of bacterial RNAP and associated proteins. In addition, we use bacteriophage development as a model system to study temporal regulation of gene expression and to uncover novel mechanisms of transcription regulation. We also study microcins, small ribosomally-synthesized inhibitors of bacterial growth.
The following research projects were actively pursued during the last year:
Studies of bacteriophage development
Genomic sequences of several novel bacteriophages have been determined and host RNAP binding transcription factors encoded by these phages have been identified. The molecular mechanisms of these factors function, and the structural aspects of their interaction with RNAP have been studied.
Analysis of phage-host interaction has taken a new direction in 2008, concentrating on a novel mechanism of bacterial resistance to phages -- through the action of CRISPR system. CRISPR is a novel system that may be similar to siRNA system in eukaryotes. Our bioinformatic analysis of CRISPR system in plant pathogen X. oryzae have revealed a previously unrecognised degree of diversity of CRISPR machinery in bacteria and showed that CRISPR systems likely target the genomes of infecting phages, not their RNA. Ongoing work on this intriguing system is aimed at elucidation of details of CRISPR genes transcription and of processing of the CRISPR transcript into short RNAs that are required for phage resistance to be manifested.
Structure-functional analysis of RNAP
We continued our studies of various aspects of bacterial RNAP structure and function. The most significant findings included studies of the mechanism of transcription inhibition by antibiotic streptolydigin, elucidation of the mechanism of RNAP stalling when it transcribes through DNA triplexes, and mapping RNAP interaction sites with several transcription factors encoded by novel bacteriophages.
Structure-activity analysis of Microcin C, an inhibitor of translation
Microcin C, a peptide-nucleotide antibiotic, is a potent inhibitor of growth of some Gram-negative bacteria. Microcin C is a Trojan-horse inhibitor: upon entry into sensitive cells microcin C is processed with the release of processed McC -- a modified non-hydrolizable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase. During the last year, improved methods of total synthesis of microcin C analogues have been developed (a collaboration with Dr. Arthur Van Aerschot, Leuven, Belgium) and a series of chemical derivatives of microcin C that target all cellular tRNA synthetases, as well as compounds with varying length of the peptide part of microcin C have been prepared and evaluated. New studies of microcin C resistance uncovered a novel mechanism that involves acetylation of processed microcin C by cellular acetylases of the Rim family.
Studies of bacterial diversity in Antarctica
We are interested in planetary-scale mechanisms responsible for global spread of at least some microbes and their viruses. The ice-covered expanse of Antarctica can be considered as a giant trap for microbes deposited from air due to Aeolian effects. We took part in the 54th and 55-th Russian Antarctic Expeditions and collected large samples of snow from several sites in Antarctica. The samples were melted, concentrated, and are currently being studied by metagenomic and microbiological tool to identify microbes that accumulated on the Antarctic snow surface during the 2009-10 nd 2010-11th seasons.
Complete list of publications: [Pubmed]
Natural diversity of CRISPR spacers of Thermus: evidence of local spacer acquisition and global spacer exchange
Lopatina A, Medvedeva S, Artamonova D, Kolesnik M, Sitnik V, Ispolatov Y, Severinov K. Natural diversity of CRISPR spacers of Thermus: evidence of local spacer acquisition and global spacer exchange Philosophical Transactions of the Royal Society B: Biological Sciences, 2019; 374 (1772): 20180092
We investigated the diversity of CRISPR spacers of Thermus communities from two locations in Italy, two in Chile and one location in Russia. Among the five sampling sites, a total of more than 7200 unique spacers belonging to different CRISPR-Cas systems types and subtypes were identified. Most of these spacers are not found in CRISPR arrays of sequenced Thermus strains. Comparison of spacer sets revealed that samples within the same area (separated by few to hundreds of metres) have similar spacer sets, which appear to be largely stable at least over the course of several years. While at further distances (hundreds of kilometres and more) the similarity of spacer sets is decreased, there are still multiple common spacers in Thermus communities from different continents. The common spacers can be reconstructed in identical or similar CRISPR arrays, excluding their independent appearance and suggesting an extensive migration of thermophilic bacteria over long distances. Several new Thermus phages were isolated in the sampling sites. Mapping of spacers to bacteriophage sequences revealed examples of local acquisition of spacers from some phages and distinct patterns of targeting of phage genomes by different CRISPR-Cas systems.
Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation.
Musharova O, Sitnik V, Vlot M, Savitskaya E, Datsenko KA, Krivoy A, Fedorov I, Semenova E, Brouns SJJ, Severinov K. Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation. Mol Microbiol. 2019 Mar 15.
CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» - acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference, and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids.
Bayfield OW, Klimuk E, Winkler DC, Hesketh EL, Chechik M, Cheng N, Dykeman EC, Minakhin L, Ranson NA, Severinov K, Steven AC, Antson AA. Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids. Proc Natl Acad Sci U S A. 2019 Feb 26;116(9):3556-3561.
Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.
Architecture of Microcin B17 Synthetase: An Octameric Protein Complex Converting a Ribosomally Synthesized Peptide into a DNA Gyrase Poison.
Ghilarov D, Stevenson CEM, Travin DY, Piskunova J, Serebryakova M, Maxwell A, Lawson DM, Severinov K. Architecture of Microcin B17 Synthetase: An Octameric Protein Complex Converting a Ribosomally Synthesized Peptide into a DNA Gyrase Poison. Mol Cell. 2019 Feb 21;73(4):749-762.e5. Epub 2019 Jan 17.
The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.
Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome.
Sutormin D, Rubanova N, Logacheva M, Ghilarov D, Severinov K. Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome. Nucleic Acids Res. 2019 Feb 20.
Xenogeneic Regulation of the Bacterial Transcription Machinery.
Tabib-Salazar A, Mulvenna N, Severinov K, Matthews SJ, Wigneshweraraj S. Xenogeneic Regulation of the Bacterial Transcription Machinery. J Mol Biol. 2019 Feb 15. pii: S0022-2836(19)30085-3.
The parasitic life cycle of viruses involves the obligatory subversion of the host's macromolecular processes for efficient viral progeny production. Viruses that infect bacteria, bacteriophages (phages), are no exception and have evolved sophisticated ways to control essential biosynthetic machineries of their bacterial prey to benefit phage development. The xenogeneic regulation of bacterial cell function is a poorly understood area of bacteriology. The activity of the bacterial transcription machinery, the RNA polymerase (RNAP), is often regulated by a variety of mechanisms involving small phage-encoded proteins. In this review, we provide a brief overview of known phage proteins that interact with the bacterial RNAP and compare how two prototypical phages of Escherichia coli, T4 and T7, use small proteins to "puppeteer" the bacterial RNAP to ensure a successful infection.
BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site.
Gordeeva J, Morozova N, Sierro N, Isaev A, Sinkunas T, Tsvetkova K, Matlashov M, Truncaite L, Morgan RD, Ivanov NV, Siksnys V, Zeng L, Severinov K. BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site. Nucleic Acids Res. 2019 Jan 10;47(1):253-265.
Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage λ infection, induction of λ prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.
Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference.
You L, Ma J, Wang J, Artamonova D, Wang M, Liu L, Xiang H, Severinov K, Zhang X, Wang Y. Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference. Cell. 2019 Jan 10;176(1-2):239-253.e16.
Effects of Population Dynamics on Establishment of a Restriction-Modification System in a Bacterial Host.
Graovac S, Rodic A, Djordjevic M, Severinov K, Djordjevic M. Effects of Population Dynamics on Establishment of a Restriction-Modification System in a Bacterial Host. Molecules. 2019 Jan 7;24(1). pii: E198.
Biosynthesis of the RiPP trojan horse nucleotide antibiotic microcin C is directed by the N-formyl of the peptide precursor.
Dong SH, Kulikovsky A, Zukher I, Estrada P, Dubiley S, Severinov K, Nair SK. Biosynthesis of the RiPP trojan horse nucleotide antibiotic microcin C is directed by the N-formyl of the peptide precursor. Chem Sci. 2018 Dec 26;10(8):2391-2395.
Microcin C7 (McC) is a peptide antibiotic modified by a linkage of the terminal isoAsn amide to AMP via a phosphoramidate bond. Post-translational modification on this ribosomally produced heptapeptide precursor is carried out by MccB, which consumes two equivalents of ATP to generate the N-P linkage. We demonstrate that MccB only efficiently processes the precursor heptapeptide that retains the N-formylated initiator Met (fMet). Binding studies and kinetic measurements evidence the role of the N-formyl moiety. Structural data show that the N-formyl peptide binding results in an ordering of residues in the MccB "crossover loop", which dictates specificity in homologous ubiquitin activating enzymes. The N-formyl peptide exhibits substrate inhibition, and cannot be displaced from MccB by the desformyl counterpart. Such substrate inhibition may be a strategy to avert unwanted McC buildup and avert toxicity in the cytoplasm of producing organisms.
Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation.
Musharova O, Vyhovskyi D, Medvedeva S, Guzina J, Zhitnyuk Y, Djordjevic M, Severinov K, Savitskaya E. Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation. MBio. 2018 Dec 4;9(6). pii: e02169-18.
CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA. Here, we used a combination of bioinformatics and experimental approaches to understand factors affecting the efficiency of acquisition of spacers by the Escherichia coli type I-E CRISPR-Cas system, for which two modes of CRISPR adaptation have been described: naive and primed. We found that during primed adaptation, efficiency of spacer acquisition is strongly negatively affected by the presence of an AAG trinucleotide-a consensus PAM-within the sequence being selected. No such trend is observed during naive adaptation. The results are consistent with a unidirectional spacer selection process during primed adaptation and provide a specific signature for identification of spacers acquired through primed adaptation in natural populations.IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. Different fragments are acquired into CRISPR arrays with widely different efficiencies, but the factors responsible are not known. We analyzed the frequency of spacers acquired during primed adaptation in an E. coli CRISPR array and found that AAG motif was depleted from highly acquired spacers. AAG is also a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery. These results are important because they provide new information on the mechanism of primed spacer acquisition. They add to other previous evidence in the field that pointed out to a "directionality" in the capture of new spacers. Our data strongly suggest that the recognition of an AAG PAM by the interference machinery components prior to spacer capture occludes downstream AAG sequences, thus preventing their recognition by the adaptation machinery.
CRISPR-Cas molecular beacons as tool for studies of assembly of CRISPR-Cas effector complexes and their interactions with DNA.
Mekler V, Kuznedelov K, Minakhin L, Murugan K, Sashital DG, Severinov K. CRISPR-Cas molecular beacons as tool for studies of assembly of CRISPR-Cas effector complexes and their interactions with DNA. Methods Enzymol. 2019;616:337-363. Epub 2018 Dec 1.
CRISPR-Cas systems protect prokaryotic cells from invading phages and plasmids by recognizing and cleaving foreign nucleic acid sequences specified by CRISPR RNA spacer sequences. Several CRISPR-Cas systems have been widely used as tool for genetic engineering. In DNA-targeting CRISPR-Cas nucleoprotein effector complexes, the CRISPR RNA forms a hybrid with the complementary strand of foreign DNA, displacing the noncomplementary strand to form an R-loop. The DNA interrogation and R-loop formation involve several distinct steps the molecular details of which are not fully understood. This chapter describes a recently developed fluorometric Cas beacon assay that may be used for measuring of specific affinity of various CRISPR-Cas complexes for unlabeled target DNA and model DNA probes. The Cas beacon approach also can provide a sensitive method for monitoring the kinetics of assembly of CRISPR-Cas complexes.
Controller protein of restriction-modification system Kpn2I affects transcription of its gene by acting as a transcription elongation roadblock.
Klimuk E, Bogdanova E, Nagornykh M, Rodic A, Djordjevic M, Medvedeva S, Pavlova O, Severinov K. Controller protein of restriction-modification system Kpn2I affects transcription of its gene by acting as a transcription elongation roadblock. Nucleic Acids Res. 2018 Nov 16;46(20):10810-10826.
C-proteins control restriction-modification (R-M) systems' genes transcription to ensure sufficient levels of restriction endonuclease to allow protection from foreign DNA while avoiding its modification by excess methyltransferase. Here, we characterize transcription regulation in C-protein dependent R-M system Kpn2I. The Kpn2I restriction endonuclease gene is transcribed from a constitutive, weak promoter, which, atypically, is C-protein independent. Kpn2I C-protein (C.Kpn2I) binds upstream of the strong methyltransferase gene promoter and inhibits it, likely by preventing the interaction of the RNA polymerase sigma subunit with the -35 consensus element. Diminished transcription from the methyltransferase promoter increases transcription from overlapping divergent C-protein gene promoters. All known C-proteins affect transcription initiation from R-M genes promoters. Uniquely, the C.Kpn2I binding site is located within the coding region of its gene. C.Kpn2I acts as a roadblock stalling elongating RNA polymerase and decreasing production of full-length C.Kpn2I mRNA. Mathematical modeling shows that this unusual mode of regulation leads to the same dynamics of accumulation of R-M gene transcripts as observed in systems where C-proteins act at transcription initiation stage only. Bioinformatics analyses suggest that transcription regulation through binding of C.Kpn2I-like proteins within the coding regions of their genes may be widespread.
Ultrahigh-throughput functional profiling of microbiota communities.
Terekhov SS, Smirnov IV, Malakhova MV, Samoilov AE, Manolov AI, Nazarov AS, Danilov DV, Dubiley SA, Osterman IA, Rubtsova MP, Kostryukova ES, Ziganshin RH, Kornienko MA, Vanyushkina AA, Bukato ON, Ilina EN, Vlasov VV, Severinov KV, Gabibov AG, Altman S. Ultrahigh-throughput functional profiling of microbiota communities. Proc Natl Acad Sci U S A. 2018 Sep 18;115(38):9551-9556.
Microbiome spectra serve as critical clues to elucidate the evolutionary biology pathways, potential pathologies, and even behavioral patterns of the host organisms. Furthermore, exotic sources of microbiota represent an unexplored niche to discover microbial secondary metabolites. However, establishing the bacterial functionality is complicated by an intricate web of interactions inside the microbiome. Here we apply an ultrahigh-throughput (uHT) microfluidic droplet platform for activity profiling of the entire oral microbial community of the Siberian bear to isolate Bacillus strains demonstrating antimicrobial activity against Staphylococcus aureus Genome mining allowed us to identify antibiotic amicoumacin A (Ami) as responsible for inhibiting the growth of S. aureus Proteomics and metabolomics revealed a unique mechanism of Bacillus self-resistance to Ami, based on a subtle equilibrium of its deactivation and activation by kinase AmiN and phosphatase AmiO, respectively. We developed uHT quantitative single-cell analysis to estimate antibiotic efficacy toward different microbiomes and used it to determine the activity spectra of Ami toward human and Siberian bear microbiota. Thus, uHT microfluidic droplet platform activity profiling is a powerful tool for discovering antibiotics and quantifying external influences on a microbiome.
Defining the seed sequence of the Cas12b CRISPR-Cas effector complex.
Jain I, Minakhin L, Mekler V, Sitnik V, Rubanova N, Severinov K, Semenova E. Defining the seed sequence of the Cas12b CRISPR-Cas effector complex. RNA Biol. 2018 Aug 17:1-10.
Target binding by CRISPR-Cas ribonucleoprotein effectors is initiated by the recognition of double-stranded PAM motifs by the Cas protein moiety followed by destabilization, localized melting, and interrogation of the target by the guide part of CRISPR RNA moiety. The latter process depends on seed sequences, parts of the target that must be strictly complementary to CRISPR RNA guide. Mismatches between the target and CRISPR RNA guide outside the seed have minor effects on target binding, thus contributing to off-target activity of CRISPR-Cas effectors. Here, we define the seed sequence of the Type V Cas12b effector from Bacillus thermoamylovorans. While the Cas12b seed is just five bases long, in contrast to all other effectors characterized to date, the nucleotide base at the site of target cleavage makes a very strong contribution to target binding. The generality of this additional requirement was confirmed during analysis of target recognition by Cas12b effector from Alicyclobacillus acidoterrestris. Thus, while the short seed may contribute to Cas12b promiscuity, the additional specificity determinant at the site of cleavage may have a compensatory effect making Cas12b suitable for specialized genome editing applications.
New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System.
Maikova A, Severinov K, Soutourina O. New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System. Front Microbiol. 2018 Jul 31;9:1740.
Over the last decades the enteric bacterium Clostridium difficile (novel name Clostridioides difficile) - has emerged as an important human nosocomial pathogen. It is a leading cause of hospital-acquired diarrhea and represents a major challenge for healthcare providers. Many aspects of C. difficile pathogenesis and its evolution remain poorly understood. Efficient defense systems against phages and other genetic elements could have contributed to the success of this enteropathogen in the phage-rich gut communities. Recent studies demonstrated the presence of an active CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) subtype I-B system in C. difficile. In this mini-review, we will discuss the recent advances in characterization of original features of the C. difficile CRISPR-Cas system in laboratory and clinical strains, as well as interesting perspectives for our understanding of this defense system function and regulation in this important enteropathogen. This knowledge will pave the way for the development of promising biotechnological and therapeutic tools in the future. Possible applications for the C. difficile strain monitoring and genotyping, as well as for CRISPR-based genome editing and antimicrobials are also discussed.
Konstantin Severinov, Ph.D., D.Sc., is a Principal Investigator at the Waksman Institute and a Professor of Molecular Biology and Biochemistry at Rutgers University. In Russia, he is the Director of the Center of Life Sciences at the Skolkovo Institute of Science and Technology in Skolkovo, and also heads laboratories at the Russian Academy of Sciences Institutes of Molecular Genetics and Gene Biology in Moscow. He is an American Academy of Microbiology Fellow, a Senior Fulbright Scholar, member of the Science and Technology Council of the Russian Nanotechnology corporation (RUSNANO) and a consultant for 7 biotech companies and several Russian government agencies; Scientific interests include the mechanisms of gene expression in bacteria and development of new antibiotics. Dr. Severinov has published over 200 papers in top scientific journals and holds 6 international patents.
Konstantin works on genetic, biochemical, and structural analyses of transcription mechanism and regulation in bacteria. He also studies the regulation of macromolecular synthesis during the process of bacteriophage infection and various molecular interactions between bacteriophages and their host bacteria. Another main direction of his research involves analysis of antibacterial peptides called microcins: their structures, mechanisms of function, evolution, and the use as new antibiotics.