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1.  CRISPR adaptation biases explain preference for acquisition of foreign DNA 
Nature  2015;520(7548):505-510.
In the process of CRISPR adaptation, short pieces of DNA (“spacers”) are acquired from foreign elements and integrated into the CRISPR array. It so far 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 dsDNA break repair complex. Our results suggest that in E. coli, acquisition of new spacers depends on RecBCD-mediated processing of dsDNA 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 from both high copy plasmids and phages.
PMCID: PMC4561520  PMID: 25874675
2.  Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli 
Nucleic Acids Research  2012;40(12):5569-5576.
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.
PMCID: PMC3384332  PMID: 22402487
3.  Molecular Epidemiology, Sequence Types, and Plasmid Analyses of KPC-Producing Klebsiella pneumoniae Strains in Israel▿  
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.
PMCID: PMC2897314  PMID: 20350950
4.  Carbapenem-Resistant KPC-2-Producing Escherichia coli in a Tel Aviv Medical Center, 2005 to 2008▿  
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.
PMCID: PMC2876404  PMID: 20231393
5.  Transfer of Carbapenem-Resistant Plasmid from Klebsiella pneumoniae ST258 to Escherichia coli in Patient 
Emerging Infectious Diseases  2010;16(6):1014-1017.
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 patient's gut flora contained a carbapenem-susceptible E. coli strain isogenic with the KPC-3–producing isolate, which suggests horizontal interspecies plasmid transfer.
PMCID: PMC3086234  PMID: 20507761
Horizontal gene transfer; carbapenem-resistant plasmid; KPC; bacteria; plasmids; Klebsiella pneumoniae; Escherichia coli; antibiotic resistance; Israel; dispatch

Results 1-5 (5)