Ekaterina Bogdanova

Transcription, processing and function of CRISPR cassettes in Escherichia coli

CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laboratory strains of Escherichia coli contain a functional CRISPR/Cas system (as judged by appearance of phage resistance at conditions of artificial co‐overexpression of Cas genes and a CRISPR cassette engineered to target a λ‐phage), no natural phage resistance due to CRISPR system function was observed in this best‐studied organism and no E. coli CRISPR spacer matches sequences of well‐studied E. coli phages. To better understand the apparently ‘silent’E. coli CRISPR/Cas system, we systematically characterized processed transcripts from CRISPR cassettes. Using an engineered strain with genomically located spacer matching phage λ we show that endogenous levels of CRISPR cassette and cas genes expression allow only weak protection against infection with the phage. However, derepression of the CRISPR/Cas system by disruption of the hns gene leads to high level of protection.

Transcription regulation of restriction-modification system Esp1396I

The convergently transcribed restriction (R) and methylase (M) genes of the Restriction–Modification system Esp1396I are tightly regulated by a controller (C) protein that forms part of the CR operon. We have mapped the transcriptional start sites from each promoter and examined the regulatory role of C.Esp1396I in vivo and in vitro. C-protein binding at the CR and M promoters was analyzed by DNA footprinting and a range of biophysical techniques. The distal and proximal C-protein binding sites at the CR promoter are responsible for activation and repression, respectively. In contrast, a C-protein dimer binds to a single site at the M-promoter to repress the gene, with an affinity much greater than for the CR promoter. Thus, during establishment of the system in a naïve host, the activity of the M promoter is turned off early, preventing excessive synthesis of methylase. Mutational analysis of promoter binding sites reveals that the tetranucleotide inverted repeats long believed to be important for C-protein binding to DNA are less significant than previously thought. Instead, symmetry-related elements outside of these repeats appear to be critical for the interaction and are discussed in terms of the recent crystal structure of C.Esp139I bound to the CR promoter.

Transcription regulation of the EcoRV restriction–modification system

When a plasmid containing restriction–modification (R–M) genes enters a naïve host, unmodified host DNA can be destroyed by restriction endonuclease. Therefore, expression of R–M genes must be regulated to ensure that enough methyltransferase is produced and that host DNA is methylated before the endonuclease synthesis begins. In several R–M systems, specialized Control (C) proteins coordinate expression of the R and the M genes. C proteins bind to DNA sequences called C-boxes and activate expression of their cognate R genes and inhibit the M gene expression, however the mechanisms remain undefined. Here, we studied the regulation of gene expression in the C protein-dependent EcoRV system. We map the divergent EcoRV M and R gene promoters and we define the site of C protein-binding that is sufficient for activation of the EcoRV R transcription.

Normalization of genomic DNA using duplex-specific nuclease.

An application of duplex-specific nuclease (DSN) normalization technology to whole-genome shotgun sequencing of genomes with a large proportion of repetitive DNA is described. The method uses a thermostable DSN from the Kamchatka crab that specifically hydrolyzes dsDNA. In model experiments on human genomic DNA, we demonstrated that DSN normalization of double-stranded DNA formed during C0t analysis is effective against abundant repetitive sequences with high sequence identity, while retaining highly divergent repeats and coding regions at base-line levels. Thus, DSN normalization applied to C0t analysis can be used to eliminate evolutionarily young repetitive elements from genomic DNA before sequencing, and should prove invaluable in studies of large eukaryotic genomes, such as those of higher plants.

Regulation of gene expression in restriction-modification system Eco29kI

The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR–eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.

Regulation of gene expression in a type II restriction-modification system

Type II restriction-modification systems are comprised of a restriction endonuclease and methyltransferase. The enzymes are coded by individual genes and recognize the same DNA sequence. Endonuclease makes a double-stranded break in the recognition site, and methyltransferase covalently modifies DNA bases within the recognition site, thereby preventing cleavage by the endonuclease. The concerted action of these enzymes plays the role of a primitive immune system and protects the bacterial host cell from invasion by foreign (for example, viral) DNA. However, uncontrolled expression of restriction-modification system genes can result in the death of a bacterial host cell because of endonuclease cleavage of the host DNA. In the present review, data on the regulation of expression of the type II restriction-modification enzymes genes are discussed.

Controller protein of restriction–modification system Kpn2I affects transcription of its gene by acting as a transcription elongation roadblock

Abstract 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.