We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36-to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCRgenerated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.bacterial genomics ͉ FLP recombinase ͉ FRT sites ͉ Red recombinase T he availability of complete bacterial genome sequences has provided a wealth of information on the molecular structure and organization of a myriad of genes and ORFs whose functions are poorly understood. A systematic mutational analysis of genes in their normal location can provide significant insight into their function. Although a number of general allele replacement methods (1-7) can be used to inactivate bacterial chromosomal genes, these all require creating the gene disruption on a suitable plasmid before recombining it onto the chromosome. In contrast, genes can be directly disrupted in Saccharomyces cerevisiae by transformation with PCR fragments encoding a selectable marker and having only 35 nt of flanking DNA homologous to the chromosome (8). This PCR-mediated gene replacement method has greatly facilitated the generation of specific mutants in the functional analysis of the yeast genome; it relies on the high efficiency of mitotic recombination in yeast (9). Directed disruption of chromosomal genes can also be done in Candida albicans by using similar PCR fragments with 50-to 60-nt homology extensions (10).In contrast to yeast and a few naturally competent bacteria, most bacteria are not readily transformable with linear DNA. One reason Escherichia coli is not so transformable is because of the presence of intracellular exonucleases that degrade linear DNA (11). However, recombination-proficient mutants lacking exonuclease V of the RecBCD recombination complex are transformable with linear DNA (12). Recombination can occur in recB or recC mutants carrying a suppressor (sbcA or sbcB) mutation that activates an alternative recombination pathway; sbcA activates ...

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

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.bacteriophage | RNA interference | small RNA C RISPR (clustered regularly interspaced short palindromic repeats) cassettes are present in virtually every archaeon and in approximately 40% of bacteria (1-3). A CRISPR cassette consists of almost identical direct repeats that are regularly interspersed with spacers (4). In any given cassette, the length of spacers is similar, whereas their sequences vary. CRISPR cassettes are often flanked by a diverse set of CRISPR-associated (cas) genes (2,5,6).CRISPR/Cas (CRISPR-associated sequences) functions as an adaptive immunity system by excluding viruses and other mobile genetic elements that contain sequences matching CRISPR cassette spacers (6-9). Bacterial and archaeal CRISPR/Cas systems generally target DNA (10-13), although one archaeal system has been demonstrated in vitro to interfere at the level of RNA (14). Transcription of a CRISPR cassette, followed by processing with the help of dedicated endoribonucleases, creates small CRISPR RNAs (crRNAs) that guide the Cas machinery to the target, eventually resulting in target cleavage (11,(15)(16)(17)(18)(19)(20).Although a match between a single CRISPR spacer and a foreign DNA sequence called the protospacer can provide immunity to the entry of that DNA into the host, it is not sufficient. Mutations in the conserved protospacer-adjacent motif (PAM, ref. 21) abolish CRISPR-mediated immunity even in the presence of a perfect spacer-protospacer match. Likewise, some point mutations in protospacer that introduce single mismatches with the spacer abolish CRISPR/Cas function even when the PAM is intact (22). Thus, a PAM and a match between a spacer and protospacer are both required for CRISPR/Cas function.Recently, how...

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SummaryCRISPR/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 l-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 l 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.