Moran G. Goren

Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called ‘adaptation’, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.

CRISPR adaptation biases explain preference for acquisition of foreign DNA.

CRISPR–Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA (‘spacers’) are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

Transfer of carbapenem-resistant plasmid from Klebsiella pneumoniae ST258 to Escherichia coli in patient.

Klebsiella pneumoniae carbapenemase (KPC) 3–producing Escherichia coli was isolated from a carrier of KPC-3–producing K. pneumoniae. The KPC-3 plasmid was identical in isolates of both species. The patients gut flora contained a carbapenem-susceptible E. coli strain isogenic with the KPC-3–producing isolate, which suggests horizontal interspecies plasmid transfer.

Molecular epidemiology, sequence types, and plasmid analyses of KPC-producing Klebsiella pneumoniae strains in Israel.

ABSTRACT Sporadic isolates of carbapenem-resistant KPC-2-producing Klebsiella pneumoniae were isolated in Tel Aviv Medical Center during 2005 and 2006, parallel to the emergence of the KPC-3-producing K. pneumoniae sequence type 258 (ST 258). We aimed to study the molecular epidemiology of these isolates and to characterize their blaKPC-carrying plasmids and their origin. Ten isolates (8 KPC-2 and 2 KPC-3 producing) were studied. All isolates were extremely drug resistant. They possessed the blaKPC gene and varied in their additional beta-lactamase contents. The KPC-2-producing strains belonged to three different sequence types: ST 340 (n = 2), ST 277 (n = 2), and a novel sequence type, ST 376 (n = 4). Among KPC-3-producing strains, a single isolate (ST 327) different from ST 258 was identified, but both strains carried the same plasmid (pKpQIL). The KPC-2-encoding plasmids varied in size (45 to 95 kb) and differed among each of the STs. Two of the Klebsiella blaKPC-2-carrying plasmids were identical to plasmids from Escherichia coli, suggesting a common origin of these plasmids. These data indicate that KPC evolution in K. pneumoniae is related to rare events of interspecies spread of blaKPC-2-carrying plasmids from E. coli followed by limited clonal spread, whereas KPC-3 carriage in this species is related almost strictly to clonal expansion of ST 258 carrying pKpQIL.

High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system.

Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.

DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins constitute a recently identified prokaryotic defense system against invading nucleic acids. DNA segments, termed protospacers, are integrated into the CRISPR array in a process called adaptation. Here, we establish a PCR-based assay that enables evaluating the adaptation efficiency of specific spacers into the type I-E Escherichia coli CRISPR array. Using this assay, we provide direct evidence that the protospacer adjacent motif along with the first base of the protospacer (5′-AAG) partially affect the efficiency of spacer acquisition. Remarkably, we identified a unique dinucleotide, 5′-AA, positioned at the 3′ end of the spacer, that enhances efficiency of the spacers acquisition. Insertion of this dinucleotide increased acquisition efficiency of two different spacers. DNA sequencing of newly adapted CRISPR arrays revealed that the position of the newly identified motif with respect to the 5′-AAG is important for affecting acquisition efficiency. Analysis of approximately 1 million spacers showed that this motif is overrepresented in frequently acquired spacers compared with those acquired rarely. Our results represent an example of a short nonprotospacer adjacent motif sequence that affects acquisition efficiency and suggest that other as yet unknown motifs affect acquisition efficiency in other CRISPR systems as well.

Plasmid-encoded OXA-48 carbapenemase in Escherichia coli from Israel

Sir, Carbapenem resistance among Enterobacteriaceae in Israel emerged in 2004 and was observed mainly in Klebsiella pneumoniae, but also in Enterobacter species and Escherichia coli. Since its emergence, carbapenem resistance in these species, both in clinical and colonizing isolates, has been rendered by the production of plasmid-mediated K. pneumoniae carbapenemase (KPC). In late 2007, a woman in her early thirties previously diagnosed with acute lymphoblastic leukaemia, was admitted to the haematology ward in Tel Aviv Sourasky Medical Center. The patient had arrived in Israel from Jordan in order to undergo chemotherapy and, later, bone marrow transplantation. During the first month after transplantation the patient was intermittently febrile and was treated with various antimicrobials, including piperacillin/tazobactam, amikacin, ciprofloxacin, vancomycin, imipenem and voriconazole. Two months after admission, while being treated with imipenem and voriconazole, the patient suffered from fever, dyspnoea and renal failure, and was transferred to the intensive care unit. During that period a carbapenem-resistant E. coli strain (E. coli 1736) was isolated from a Hickman catheter, leading to removal of the catheter and further treatment with ceftazidime and colistin. Treatment with these antibacterial agents cleared the OXA-48-producing E. coli, yet unfortunately the patient died 3 months later from a systemic Pseudomonas infection. E. coli 1736 was multidrug resistant, showing resistance to penicillins, piperacillin/tazobactam, aminoglycosides, quinolones and carbapenems, but susceptibility to all cephalosporins, aztreonam, tigecycline and colistin (Table 1). Analytical isoelectric focusing (IEF) performed on crude enzyme preparations revealed the presence of two b-lactamases with pIs of 5.4 and 7.2 (data not shown). PCR screening for the presence of b-lactamases in E. coli 1736 indicated the presence of blaTEM-1 and blaOXA-48 genes corresponding to the pIs of the b-lactamases visualized by IEF. Plasmid analysis of E. coli 1736 revealed four plasmids, three of around ≤50 kb in size and a larger plasmid of around 100 kb. Plasmid DNA was purified and transformed into E. coli DH10B. Transformant colonies that were screened positive for blaOXA-48 by PCR harboured the 50 kb plasmid. Southern analysis of plasmid DNA derived from these transformants using a blaOXA-48-labelled probe demonstrated the presence of blaOXA-48 on the acquired 50 kb plasmid. Acquisition of this plasmid increased the MICs of imipenem, meropenem and ertapenem without conferring full resistance (Table 1). PCR mapping of the genetic environment surrounding blaOXA-48 was performed in collaboration with the laboratory of Professor P. Nordmann (Hospital de Bicetre, Paris, France). blaOXA-48 was found to be located inside Tn1999.2, similar to the structure described for other enteric strains, such as the E. coli strain from Turkey and the K. pneumoniae strain from Lebanon. E. coli 1736 was not resistant to all b-lactam antibiotics as reported for other OXA-48-producing strains, yet it presented a high level of resistance to the commonly used carbapenems (MICs ≥16 mg/L), higher than usually seen in KPC-producing E. coli strains isolated in our hospital. These high carbapenem MICs suggested the presence of additional resistance mechanisms together with OXA-48 carbapenemase. Outer membrane protein (OMP) produced by E. coli 1736 was determined by PCR and sequencing of ompA, ompC and ompF genes. Further OMP analysis was performed by protein extraction and separation on Tris–Tricine gels using SDS-PAGE followed by mass spectrometry (performed in the Biological Mass Spectrometry Facility at the Weizmann Institute of Science). Both methods indicated the absence of at least one major porin, OmpC. Until the isolation of E. coli 1736, carbapenem resistance in E. coli in our country was exclusively attributed to the Ambler class A carbapenemase KPC. This is the first identified Enterobacteriaceae isolate in our country possessing a carbapenem-hydrolysing oxacillinase. To seek the possible origin of this strain we performed multilocus sequence typing (MLST; http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi. html), which genotyped the strain as sequence type (ST) 2, an E. coli ST that has never been recorded previously in our Research letters

Carbapenem-Resistant KPC-2-Producing Escherichia coli in a Tel Aviv Medical Center, 2005 to 2008

ABSTRACT All of the carbapenem-resistant Escherichia coli (CREC) isolates identified in our hospital from 2005 to 2008 (n = 10) were studied. CREC isolates were multidrug resistant, all carried blaKPC-2, and six of them were also extended-spectrum beta-lactamase producers. Pulsed-field gel electrophoresis indicated six genetic clones; within the same clone, similar transferable blaKPC-2-containing plasmids were found whereas plasmids differed between clones. Tn4401 elements were identified in all of these plasmids.

The Bacterial CRISPR/Cas System as Analog of the Mammalian Adaptive Immune System

Bacteria, like mammals, have to constantly defend themselves from viral attack. Like mammals, they use both innate and adaptive defense mechanisms. In this point of view we highlight the commonalities between defense systems of bacteria and mammals. Our focus is on the recently discovered bacterial adaptive immune system, the clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas). We suggest that fundamental aspects of CRISPR/Cas immunity may be viewed in light of the vast accumulated knowledge on the mammalian immune system, and propose that further insights will be revealed by thorough comparison between the systems.

Extending the Host Range of Bacteriophage Particles for DNA Transduction

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.