[Show abstract][Hide abstract] ABSTRACT: CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR–Cas systems two
modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved
Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers
incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching
target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers
capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR–Cas system. We observe both naïve
and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation
shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent
with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition
and illustrate surprising mechanistic diversity of related CRISPR–Cas systems.
Preview · Article · Nov 2015 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits
aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound.
MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the
N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable
aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered
a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular
processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable
aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides,
either exogenous or those generated inside the cell during normal metabolism.
Full-text · Article · Jul 2014 · Journal of Bacteriology
[Show abstract][Hide abstract] ABSTRACT: 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.
IMPORTANCE 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.
[Show abstract][Hide abstract] ABSTRACT: 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 an
No preview · Article · Apr 2014 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Apr 2014 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Genetic interaction networks are especially useful for functional assignment of genes and gaining new insights into the systems-level organization of the cell. While studying interactions of nonessential genes can be relatively straight-forward via use of deletion mutants, different approaches must be used to reveal interactions of essential genes due to their indispensability. One method shown to be useful for revealing interactions of essential genes requires tagging the query protein. However, this approach can be complicated by mutational effects of potential hypomorphic alleles. Here, we describe a pilot study for a new scheme of systematically studying the interactions of essential genes. Our method uses a low-copy, F-based, complementing plasmid, pFE604T, from which the essential gene is conditionally expressed. The essential gene is expressed at lower levels, producing a moderate growth defect in a query host. Secondary mutations are introduced into the query host by conjugation and the resultant exconjugants are scored for growth by imaging them over time. We report results from studying five essential query genes: dnaN, ftsW, trmD, yrfF and yjgP, showing (on average) interactions with nearly 80 nonessential genes. This system should prove useful for genome-wide analyses of other essential genes in E. coli K-12.
Preview · Article · Aug 2013 · Genes & Genetic Systems
[Show abstract][Hide abstract] ABSTRACT: CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) is a small RNA-based adaptive prokaryotic immunity system that functions by acquisition of short fragments of DNA (mainly from foreign invaders such as viruses and plasmids) and subsequent destruction of DNA with sequences matching acquired fragments. Some mutations in foreign DNA that affect the match prevent CRISPR/Cas defensive function. Here we show that matching sequences that are no longer able to elicit defense, still guide the CRISPR/Cas acquisition machinery to foreign DNA, thus making the spacer acquisition process adaptive and leading to restoration of CRISPR/Cas-mediated protection. We present evidence suggesting that after initial recognition of partially matching foreign DNA, the CRISPR/Cas acquisition machinery moves along the DNA molecule, occasionally selecting fragments to be incorporated into the CRISPR locus. Our results explain how adaptive CRISPR/Cas immunity becomes specifically directed towards foreign DNA, allowing bacteria to efficiently counter individual viral mutants that avoid CRISPR/Cas defense.
No preview · Article · Jul 2012 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Amadoriases are discovered in distant groups of organisms ranging from bacteria to humans. These enzymes catalyze the degradation of Amadori products which are formed in the early stage a non-enzymatic reaction between reducing sugars and primary amines called the Maillard reaction or glycation. The physiological role of amadoriases is debatable and perhaps not constrained to one cellular process only. Among other functions amadoriases are proposed to ensure enzymatic defense against the deleterious consequences of protein glycation. A decade ago an amadoriase enzyme (the FrlB amadoriase) has been discovered in Escherichia coli catalyzing the removal of Amadori products form the.-amino group of free lysine. The E. coli FrlB enzyme has been suggested to catabolize glycated lysine released in the human intestine upon digestion of food proteins. In the present study we demonstrate that the E. coli FrlB amadoriase is capable of catalyzing in the reverse reaction the formation of Amadori products on a number of free amino acids and on polypeptides as well. Based on these results we suggest that in the forward reaction the FrlB enzyme might serve to deglycate E. coli proteins thus helping bacterial cells to withstand the protein glycation burden.
No preview · Article · Oct 2011 · Biotechnology & Biotechnological Equipment
[Show abstract][Hide abstract] ABSTRACT: Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.
Full-text · Article · Jun 2011 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Microcin C (McC), a natural antibacterial compound consisting of a heptapeptide attached to a modified adenosine, is actively
taken up by the YejABEF transporter, after which it is processed by cellular aminopeptidases, releasing the nonhydrolyzable
aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC analogues with variable length of the peptide moiety were
synthesized and evaluated in order to characterize the substrate preferences of the YejABEF transporter. It was shown that
a minimal peptide chain length of 6 amino acids and the presence of an N-terminal formyl-methionyl-arginyl sequence are required
Full-text · Article · May 2011 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Several plasmids that contain genetically marked variants of the TnBP transposon isolated from Bordetella pertussis have been constructed based on a thermosensitive plasmid. The frequencies of the integration of these plasmids into an E. coli K12 chromosome at a nonpermissive temperature (42°C) have been studied. A decline in the frequency of RSBP-induced and HPr-
or enzyme I-deficient cointegrate formation between the plasmid and the K12 bacterial chromosome were detected. The introduction
of the bvgAS operon of B. pertussis at the trans-position into the mutant E. coli K12 bacteria restores the frequency of the formation and resolution of RSBP-induced cointegrates.
No preview · Article · Mar 2010 · Molecular Genetics Microbiology and Virology