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1.  CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli 
Nucleic Acids Research  2014;43(1):530-543.
Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5′ handle (8 nt) and a 3′ handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5′ handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.
PMCID: PMC4288178  PMID: 25488810
2.  The C-Terminal Part of Microcin B Is Crucial for DNA Gyrase Inhibition and Antibiotic Uptake by Sensitive Cells 
Journal of Bacteriology  2014;196(9):1759-1767.
Microcin B (McB) is a ribosomally synthesized antibacterial peptide. It contains up to nine oxazole and thiazole heterocycles that are introduced posttranslationally and are required for activity. McB inhibits the DNA gyrase, a validated drug target. Previous structure-activity analyses indicated that two fused heterocycles located in the central part of McB are important for antibacterial action and gyrase inhibition. Here, we used site-specific mutagenesis of the McB precursor gene to assess the functional significance of the C-terminal part of McB that is located past the second fused heterocycle and contains two single heterocycles as well as an unmodified four-amino-acid C-terminal tail. We found that removal of unmodified C-terminal amino acids of McB, while having no effect on fused heterocycles, has a very strong negative effect on activity in vivo and in vitro. In fact, even nonconservative point substitutions in the last McB amino acid have a very strong effect by simultaneously decreasing uptake and ability to inhibit the gyrase. The results highlight the importance of unmodified McB amino acids for function and open the way for creation of recombinant McB derivatives with an altered or expanded spectrum of antibacterial action.
PMCID: PMC3993328  PMID: 24563033
3.  Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C 
Nucleic Acids Research  2014;42(19):11891-11902.
Microcin C (McC) is a peptide–nucleotide antibiotic produced by Escherichia coli cells harboring a plasmid-borne operon mccABCDE. The heptapeptide MccA is converted into McC by adenylation catalyzed by the MccB enzyme. Since MccA is a substrate for MccB, a mechanism that regulates the MccA/MccB ratio likely exists. Here, we show that transcription from a promoter located upstream of mccA directs the synthesis of two transcripts: a short highly abundant transcript containing the mccA ORF and a longer minor transcript containing mccA and downstream ORFs. The short transcript is generated when RNA polymerase terminates transcription at an intrinsic terminator located in the intergenic region between the mccA and mccB genes. The function of this terminator is strongly attenuated by upstream mcc sequences. Attenuation is relieved and transcription termination is induced when ribosome binds to the mccA ORF. Ribosome binding also makes the mccA RNA exceptionally stable. Together, these two effects—ribosome-induced transcription termination and stabilization of the message—account for very high abundance of the mccA transcript that is essential for McC production. The general scheme appears to be evolutionary conserved as ribosome-induced transcription termination also occurs in a homologous operon from Helicobacter pylori.
PMCID: PMC4231749  PMID: 25274735
4.  Molecular basis of RNA polymerase promoter specificity switch revealed through studies of Thermus bacteriophage transcription regulator 
Bacteriophage  2014;4:e29399.
Transcription initiation is the central point of gene expression regulation. Understanding of molecular mechanism of transcription regulation requires, ultimately, the structural understanding of consequences of transcription factors binding to DNA-dependent RNA polymerase (RNAP), the enzyme of transcription. We recently determined a structure of a complex between transcription factor gp39 encoded by a Thermus bacteriophage and Thermus RNAP holoenzyme. In this addendum to the original publication, we highlight structural insights that explain the ability of gp39 to act as an RNAP specificity switch which inhibits transcription initiation from a major class of bacterial promoters, while allowing transcription from a minor promoter class to continue.
PMCID: PMC4124052  PMID: 25105059
bacterial RNA polymerase; bacteriophage; inhibitor; sigma factor; transcription regulation
5.  Enzymatic Synthesis of Bioinformatically Predicted Microcin C-Like Compounds Encoded by Diverse Bacteria 
mBio  2014;5(3):e01059-14.
The Trojan horse Escherichia coli antibiotic microcin C (McC) consists of a heptapeptide attached to adenosine through a phosphoramidate linkage. McC is synthesized by the MccB enzyme, which terminally adenylates the ribosomally synthesized heptapeptide precursor MccA. The peptide part is responsible for McC uptake; it is degraded inside the cell to release a toxic nonhydrolyzable aspartyl-adenylate. Bionformatic analysis reveals that diverse bacterial genomes encoding mccB homologues also contain adjacent short open reading frames that may encode MccA-like adenylation substrates. Using chemically synthesized predicted peptide substrates and recombinant cognate MccB protein homologs, adenylated products were obtained in vitro for predicted MccA peptide-MccB enzyme pairs from Helicobacter pylori, Streptococcus thermophilus, Lactococcus johnsonii, Bartonella washoensis, Yersinia pseudotuberculosis, and Synechococcus sp. Some adenylated products were shown to inhibit the growth of E. coli by targeting aspartyl-tRNA synthetase, the target of McC.
Our results prove that McC-like adenylated peptides are widespread and are encoded by both Gram-negative and Gram-positive bacteria and by cyanobacteria, opening ways for analyses of physiological functions of these compounds and for creation of microcin C-like antibiotics targeting various bacteria.
PMCID: PMC4010828  PMID: 24803518
6.  High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli 
RNA Biology  2013;10(5):716-725.
In Escherichia coli, the acquisition of new CRISPR spacers is strongly stimulated by a priming interaction between a spacer in CRISPR RNA and a protospacer in foreign DNA. Priming also leads to a pronounced bias in DNA strand from which new spacers are selected. Here, ca. 200,000 spacers acquired during E. coli type I-E CRISPR/Cas-driven plasmid elimination were analyzed. Analysis of positions of plasmid protospacers from which newly acquired spacers have been derived is inconsistent with spacer acquisition machinery sliding along the target DNA as the primary mechanism responsible for strand bias during primed spacer acquisition. Most protospacers that served as donors of newly acquired spacers during primed spacer acquisition had an AAG protospacer adjacent motif, PAM. Yet, the introduction of multiple AAG sequences in the target DNA had no effect on the choice of protospacers used for adaptation, which again is inconsistent with the sliding mechanism. Despite a strong preference for an AAG PAM during CRISPR adaptation, the AAG (and CTT) triplets do not appear to be avoided in known E. coli phages. Likewise, PAM sequences are not avoided in Streptococcus thermophilus phages, indicating that CRISPR/Cas systems may not have been a strong factor in shaping host-virus interactions.
PMCID: PMC3737330  PMID: 23619643
CRISPR adaptation; CRISPR/Cas systems; Escherichia coli; bacteriophage; high-throughput sequencing
7.  Pervasive generation of oppositely oriented spacers during CRISPR adaptation 
Nucleic Acids Research  2014;42(9):5907-5916.
During the process of prokaryotic CRISPR adaptation, a copy of a segment of foreign deoxyribonucleic acid referred to as protospacer is added to the CRISPR cassette and becomes a spacer. When a protospacer contains a neighboring target interference motif, the specific small CRISPR ribonucleic acid (crRNA) transcribed from expanded CRISPR cassette can protect a prokaryotic cell from virus infection or plasmid transformation and conjugation. We show that in Escherichia coli, a vast majority of plasmid protospacers generate spacers integrated in CRISPR cassette in two opposing orientations, leading to frequent appearance of complementary spacer pairs in a population of cells that underwent CRISPR adaptation. When a protospacer contains a spacer acquisition motif AAG, spacer orientation that generates functional protective crRNA is strongly preferred. All other protospacers give rise to spacers oriented in both ways at comparable frequencies. This phenomenon increases the repertoire of available spacers and should make it more likely that a protective crRNA is formed as a result of CRISPR adaptation.
PMCID: PMC4027179  PMID: 24728991
8.  A non-bacterial transcription factor inhibits bacterial transcription by a multipronged mechanism 
RNA Biology  2013;10(4):495-501.
The process of transcription initiation is the major target for regulation of gene expression in bacteria and is performed by a multi-subunit RNA polymerase enzyme (RNAp). A complex network of regulatory elements controls the activity of the RNAp to fine-tune transcriptional output. Thus, RNAp is a nexus for controlling bacterial gene expression at the transcription level. Many bacteriophages, viruses that infect bacteria, encode transcription factors that specifically target and modulate the activity of the host RNAp and, thereby, facilitate the acquisition of the host bacteria by the phage. Here, we describe the modus operandi of a T7 bacteriophage-encoded small protein called Gp2 and define Gp2 as a non-bacterial regulator of bacterial transcription.
PMCID: PMC3710356  PMID: 23558648
Gp2; RNA polymerase; T7 inhibition; bacterial transcription regulation; bacteriophage; σ factor
9.  Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature 
Natural product reports  2013;30(1):108-160.
This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
PMCID: PMC3954855  PMID: 23165928
10.  The sabotage of the bacterial transcription machinery by a small bacteriophage protein 
Bacteriophage  2014;4:e28520.
Many bacteriophages produce small proteins that specifically interfere with the bacterial host transcription machinery and thus contribute to the acquisition of the bacterial cell by the bacteriophage. We recently described how a small protein, called P7, produced by the Xp10 bacteriophage inhibits bacterial transcription initiation by causing the dissociation of the promoter specificity sigma factor subunit from the host RNA polymerase holoenzyme. In this addendum to the original publication, we present the highlights of that research.
PMCID: PMC3962504  PMID: 24701369
bacterial RNA polymerase; bacteriophage; inhibitor; sigma factor; transcription
11.  Structure of Microcin B-Like Compounds Produced by Pseudomonas syringae and Species Specificity of Their Antibacterial Action 
Journal of Bacteriology  2013;195(18):4129-4137.
Escherichia coli microcin B (Ec-McB) is a posttranslationally modified antibacterial peptide containing multiple oxazole and thiazole heterocycles and targeting the DNA gyrase. We have found operons homologous to the Ec-McB biosynthesis-immunity operon mcb in recently sequenced genomes of several pathovars of the plant pathogen Pseudomonas syringae, and we produced two variants of P. syringae microcin B (Ps-McB) in E. coli by heterologous expression. Like Ec-McB, both versions of Ps-McB target the DNA gyrase, but unlike Ec-McB, they are active against various species of the Pseudomonas genus, including human pathogen P. aeruginosa. Through analysis of Ec-McB/Ps-McB chimeras, we demonstrate that three centrally located unmodified amino acids of Ps-McB are sufficient to determine activity against Pseudomonas, likely by allowing specific recognition by a transport system that remains to be identified. The results open the way for construction of McB-based antibacterial molecules with extended spectra of biological activity.
PMCID: PMC3754757  PMID: 23852863
12.  Antibiotic Streptolydigin Requires Noncatalytic Mg2+ for Binding to RNA Polymerase 
Multisubunit RNA polymerase, an enzyme that accomplishes transcription in all living organisms, is a potent target for antibiotics. The antibiotic streptolydigin inhibits RNA polymerase by sequestering the active center in a catalytically inactive conformation. Here, we show that binding of streptolydigin to RNA polymerase strictly depends on a noncatalytic magnesium ion which is likely chelated by the aspartate of the bridge helix of the active center. Substitutions of this aspartate may explain different sensitivities of bacterial RNA polymerases to streptolydigin. These results provide the first evidence for the role of noncatalytic magnesium ions in the functioning of RNA polymerase and suggest new routes for the modification of existing and the design of new inhibitors of transcription.
PMCID: PMC3957892  PMID: 24342645
13.  A bacteriophage transcription regulator inhibits bacterial transcription initiation by σ-factor displacement 
Nucleic Acids Research  2014;42(7):4294-4305.
Bacteriophages (phages) appropriate essential processes of bacterial hosts to benefit their own development. The multisubunit bacterial RNA polymerase (RNAp) enzyme, which catalyses DNA transcription, is targeted by phage-encoded transcription regulators that selectively modulate its activity. Here, we describe the structural and mechanistic basis for the inhibition of bacterial RNAp by the transcription regulator P7 encoded by Xanthomonas oryzae phage Xp10. We reveal that P7 uses a two-step mechanism to simultaneously interact with the catalytic β and β’ subunits of the bacterial RNAp and inhibits transcription initiation by inducing the displacement of the σ70-factor on initial engagement of RNAp with promoter DNA. The new mode of interaction with and inhibition mechanism of bacterial RNAp by P7 underscore the remarkable variety of mechanisms evolved by phages to interfere with host transcription.
PMCID: PMC3985653  PMID: 24482445
14.  Analysis of the mechanism of nucleosome survival during transcription 
Nucleic Acids Research  2013;42(3):1619-1627.
Maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. Our previous data identified a key intermediate (a small intranucleosomal DNA loop, Ø-loop) that is likely required for nucleosome survival during transcription by RNA polymerase II (Pol II) through chromatin, and suggested that strong nucleosomal pausing guarantees efficient nucleosome survival. To evaluate these predictions, we analysed transcription through a nucleosome by different, structurally related RNA polymerases and mutant yeast Pol II having different histone-interacting surfaces that presumably stabilize the Ø-loop. The height of the nucleosomal barrier to transcription and efficiency of nucleosome survival correlate with the net negative charges of the histone-interacting surfaces. Molecular modeling and analysis of Pol II-nucleosome intermediates by DNase I footprinting suggest that efficient Ø-loop formation and nucleosome survival are mediated by electrostatic interactions between the largest subunit of Pol II and core histones.
PMCID: PMC3919589  PMID: 24234452
15.  N-Alkylated Aminoacyl sulfamoyladenosines as Potential Inhibitors of Aminoacylation Reactions and Microcin C Analogues Containing D-Amino Acids 
PLoS ONE  2013;8(11):e79234.
Microcin C analogues were recently envisaged as important compounds for the development of novel antibiotics. Two issues that may pose problems to these potential antibiotics are possible acquisition of resistance through acetylation and in vivo instability of the peptide chain. N-methylated aminoacyl sulfamoyladenosines were synthesized to investigate their potential as aminoacyl tRNA synthetase inhibitors and to establish whether these N-alkylated analogues would escape the natural inactivation mechanism via acetylation of the alpha amine. It was shown however, that these compounds are not able to effectively inhibit their respective aminoacyl tRNA synthetase. In addition, we showed that (D)-aspartyl-sulfamoyladenosine (i.e. with a (D)-configuration for the aspartyl moiety), is a potent inhibitor of aspartyl tRNA synthetase. However, we also showed that the inhibitory effect of (D)- aspartyl-sulfamoyladenosine is relatively short-lasting. Microcin C analogues with (D)-amino acids throughout from positions two to six proved inactive. They were shown to be resistant against metabolism by the different peptidases and therefore not able to release the active moiety. This observation could not be reversed by incorporation of (L)-amino acids at position six, showing that none of the available peptidases exhibit endopeptidase activity.
PMCID: PMC3817062  PMID: 24223911
16.  12-Fold symmetry of the putative portal protein from the Thermus thermophilus bacteriophage G20C determined by X-ray analysis 
Crystal data on a putative portal protein from the thermostable bacteriophage G20C indicate that it forms a 12-subunit assembly.
In tailed bacteriophages and several animal viruses, the portal protein forms the gateway through which viral DNA is translocated into the head structure during viral particle assembly. In the mature virion the portal protein exists as a dodecamer, while recombinant portal proteins from several phages, including SPP1 and CNPH82, have been shown to form 13-subunit assemblies. A putative portal protein from the thermostable bacteriophage G20C has been cloned, overexpressed and purified. Crystals of the protein diffracted to 2.1 Å resolution and belonged to space group P4212, with unit-cell parameters a = b = 155.3, c = 115.4 Å. The unit-cell content and self-rotation function calculations indicate that the protein forms a circular 12-subunit assembly.
PMCID: PMC3818042  PMID: 24192358
putative portal protein; Thermus thermophilus; bacteriophage G20C
17.  Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition 
PLoS Genetics  2013;9(9):e1003742.
Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.
Author Summary
CRISPR loci and their associated genes form a diverse set of adaptive immune systems that are widespread among prokaryotes. In these systems, the CRISPR-associated genes (cas) encode for proteins that capture fragments of invading DNA and integrate these sequences between repeat sequences of the host's CRISPR locus. This information is used upon re-infection to degrade invader genomes. Storing invader sequences in host genomes necessitates a mechanism to differentiate between invader sequences on invader genomes and invader sequences on the host genome. CRISPR-Cas of Staphylococcus epidermidis (Type III-A system) is inhibited when invader sequences are flanked by repeat sequences, and this prevents targeting of the CRISPR locus on the host genome. Here we demonstrate that Escherichia coli CRISPR-Cas (Type I-E system) is not inhibited by repeat sequences. Instead, this system is specifically activated by the presence of bona fide Protospacer Adjacent Motifs (PAMs) in the target. PAMs are conserved sequences adjoining invader sequences on the invader genome, and these sequences are never adjacent to invader sequences within host CRISPR loci. PAM recognition is not affected by base pairing potential of the target with the crRNA. As such, the Type I-E system lacks the ability to specifically recognize self DNA.
PMCID: PMC3764190  PMID: 24039596
18.  The putative small terminase from the thermophilic dsDNA bacteriophage G20C is a nine-subunit oligomer 
The putative small terminase protein from the thermostable bacteriophage G20C has been produced, purified and crystallized.
The assembly of double-stranded DNA bacteriophages is dependent on a small terminase protein that normally plays two important roles. Firstly, the small terminase protein specifically recognizes viral DNA and recruits the large terminase protein, which makes the initial cut in the dsDNA. Secondly, once the complex of the small terminase, the large terminase and the DNA has docked to the portal protein, and DNA translocation into a preformed empty procapsid has begun, the small terminase modulates the ATPase activity of the large terminase. Here, the putative small terminase protein from the thermostable bacteriophage G20C, which infects the Gram-negative eubacterium Thermus thermophilus, has been produced, purified and crystallized. Size-exclusion chromatography–multi-angle laser light scattering data indicate that the protein forms oligomers containing nine subunits. Crystals diffracting to 2.8 Å resolution have been obtained. These belonged to space group P212121, with unit-cell parameters a = 94.31, b = 125.6, c = 162.8 Å. The self-rotation function and Matthews coefficient calculations are consistent with the presence of a nine-subunit oligomer in the asymmetric unit.
PMCID: PMC3729163  PMID: 23908032
putative small terminase; Thermus thermophilus; bacteriophage G20C
19.  Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis 
Journal of molecular biology  2012;420(0):366-383.
Microcin C (McC) is heptapeptide-adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine-peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its AMP-complex reveal specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance.
PMCID: PMC3690760  PMID: 22516613
MccF; serine peptidase; nucleophilic elbow; catalytic triad (Ser-His-Glu); substrate binding loop
20.  Cooperativity and interaction energy threshold effects in recognition of the −10 promoter element by bacterial RNA polymerase 
Nucleic Acids Research  2013;41(15):7276-7285.
RNA polymerase (RNAP) melts promoter DNA to form transcription-competent open promoter complex (RPo). Interaction of the RNAP σ subunit with non-template strand bases of a conserved −10 element (consensus sequence T−12A−11T−10A−9A−8T−7) is an important source of energy-driving localized promoter melting. Here, we used an RNAP molecular beacon assay to investigate interdependencies of RNAP interactions with −10 element nucleotides. The results reveal a strong cooperation between RNAP interactions with individual −10 element non-template strand nucleotides and indicate that recognition of the −10 element bases occurs only when free energy of the overall RNAP −10 element binding reaches a certain threshold level. The threshold-like mode of the −10 element recognition may be related to the energetic cost of attaining a conformation of the −10 element that is recognizable by RNAP. The RNAP interaction with T/A−12 base pair was found to be strongly stimulated by RNAP interactions with other −10 element bases and with promoter spacer between the −10 and −35 promoter elements. The data also indicate that unmelted −10 promoter element can impair RNAP interactions with promoter DNA upstream of the −11 position. We suggest that cooperativity and threshold effects are important factors guiding the dynamics and selectivity of RPo formation.
PMCID: PMC3753650  PMID: 23771146
21.  CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3 
Molecular Cell  2012;46(5):595-605.
The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR-interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a Protospacer Adjacent Motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R-loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg2+-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization.
PMCID: PMC3372689  PMID: 22521689
22.  Genome-Wide Identification of Regulatory RNAs in the Human Pathogen Clostridium difficile 
PLoS Genetics  2013;9(5):e1003493.
Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA–seq and differential 5′-end RNA–seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA–based regulation of gene expression in this emergent enteropathogen.
Author Summary
The emergent human pathogen Clostridium difficile is a major cause of nosocomial diarrhea associated with antibiotic therapy. During the last few years, severe forms of C. difficile infections became more frequent due to the emergence of hypervirulent isolates. Despite intensive studies, many questions regarding the mechanisms controlling C. difficile virulence remain unanswered. We hypothesized that C. difficile, a member of an ancient group of bacteria, might widely use ancestral RNA–based mechanisms to control its gene expression for better adaptation to host conditions. Indeed, using next-generation sequencing technology, we identified a great number and a large diversity of potential RNA regulators in this pathogen. We obtained experimental evidence for regulatory roles of a particular class of regulatory RNAs responding to c-di-GMP, a universal bacterial signaling molecule regulating motility, biofilm formation, and virulence. We also detected abundant small RNA products of recently discovered adaptive prokaryotic immunity CRISPR-Cas systems that might be important for C. difficile survival in gut communities. Our findings suggest that small RNA molecules may play a major role in regulatory processes during C. difficile infection cycle and as such are promising targets of new therapeutic strategies.
PMCID: PMC3649979  PMID: 23675309
23.  Temporal Regulation of Gene Expression of the Escherichia coli Bacteriophage phiEco32 
Journal of Molecular Biology  2012;416(3):389-399.
Escherichia coli phage phiEco32 encodes two proteins that bind to host RNA polymerase — gp79, a novel protein, and gp36, a distant homolog of σ70 family proteins. Here, we investigated the temporal pattern of phiEco32 and host gene expression during the infection. Host transcription shut-off and three distinct bacteriophage temporal gene classes – early, middle, and late – were revealed. A combination of bioinformatic and biochemical approaches allowed identification of phage promoters recognized by host RNA polymerase holoenzyme containing the σ70 factor. These promoters are located upstream of early phage genes. A combination of macroarray data, primer extension, and in vitro transcription analyses allowed identification of six promoters recognized by RNA polymerase holoenzyme containing gp36. These promoters are characterized by a single consensus element tAATGTAtA and are located upstream of the middle and late phage genes. Curiously, gp79, an inhibitor of host and early phage transcription by σ70-holoenzyme, activated transcription by the gp36-holoenzyme in vitro.
PMCID: PMC3275717  PMID: 22261232
bacteriophage; genome; RNA polymerase; sigma factor; transcription regulation
24.  Overexpression of Escherichia coli udk mimics the absence of T7 Gp2 function and thereby abrogates successful infection by T7 phage 
Microbiology  2013;159(Pt 2):269-274.
Successful infection of Escherichia coli by bacteriophage T7 relies upon the transcription of the T7 genome by two different RNA polymerases (RNAps). The bacterial RNAp transcribes early T7 promoters, whereas middle and late T7 genes are transcribed by the T7 RNAp. Gp2, a T7-encoded transcription factor, is a 7 kDa product of an essential middle T7 gene 2, and is a potent inhibitor of the host RNAp. The essential biological role of Gp2 is to inhibit transcription of early T7 genes that fail to terminate efficiently in order to facilitate the coordinated usage of the T7 genome by both host and phage RNAps. Overexpression of the E. coli udk gene, which encodes a uridine/cytidine kinase, interferes with T7 infection. We demonstrate that overexpression of udk antagonizes Gp2 function in E. coli in the absence of T7 infection and thus independently of T7-encoded factors. It seems that overexpression of udk reduces Gp2 stability and functionality during T7 infection, which consequently results in inadequate inhibition of host RNAp and in the accumulation of early T7 transcripts. In other words, overexpression of udk mimics the absence of Gp2 during T7 infection. Our study suggests that the transcriptional regulation of the T7 genome is surprisingly complex and might potentially be affected at many levels by phage- and host-encoded factors.
PMCID: PMC3709561  PMID: 23242801
25.  Genomic Analysis of Pseudomonas putida Phage tf with Localized Single-Strand DNA Interruptions 
PLoS ONE  2012;7(12):e51163.
The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure – a blunt right end and a 4-nucleotide 3′-protruding left end – was observed. Secondly, 14 single-chain interruptions (nicks) were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5′-TACT/RTGMC-3′. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages.
PMCID: PMC3517423  PMID: 23236447

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