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Transcription, processing and function of CRISPR cassettes in Escherichia coli.

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 ?-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 ? 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.

SUBMITTER: Pougach K

PROVIDER: S-EPMC2939963 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Transcription, processing and function of CRISPR cassettes in Escherichia coli.

Pougach Ksenia K Semenova Ekaterina E Bogdanova Ekaterina E Datsenko Kirill A KA Djordjevic Marko M Wanner Barry L BL Severinov Konstantin K

Molecular microbiology 20100901 6

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 ...[more]

PMID: 20624226

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Project description:UnlabelledClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (cas) genes constitute the CRISPR-Cas systems found in the Bacteria and Archaea domains. At least in some strains they provide an efficient barrier against transmissible genetic elements such as plasmids and viruses. Two CRISPR-Cas systems have been identified in Escherichia coli, pertaining to subtypes I-E (cas-E genes) and I-F (cas-F genes), respectively. In order to unveil the evolutionary dynamics of such systems, we analyzed the sequence variations in the CRISPR-Cas loci of a collection of 131 E. coli strains. Our results show that the strain grouping inferred from these CRISPR data slightly differs from the phylogeny of the species, suggesting the occurrence of recombinational events between CRISPR arrays. Moreover, we determined that the primary cas-E genes of E. coli were altogether replaced with a substantially different variant in a minor group of strains that include K-12. Insertion elements play an important role in this variability. This result underlines the interchange capacity of CRISPR-Cas constituents and hints that at least some functional aspects documented for the K-12 system may not apply to the vast majority of E. coli strains.ImportanceEscherichia coli is a model microorganism for the study of diverse aspects such as microbial evolution and is a component of the human gut flora that may have a direct impact in everyday life. This work was undertaken with the purpose of elucidating the evolutionary pathways that have led to the present situation of its significantly different CRISPR-Cas subtypes (I-E and I-F) in several strains of E. coli. In doing so, this information offers a novel and wider understanding of the variety and relevance of these regions within the species. Therefore, this knowledge may provide clues helping researchers better understand these systems for typing purposes and make predictions of their behavior in strains that, depending on their particular genetic dotation, would result in different levels of immunity to foreign genetic elements.

Project description:BackgroundThe trisaccharide 2'-fucosyllactose (2'-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2'-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2'-FL can be produced enzymatically in vitro and in vivo. The most promising approach for a large-scale formation of 2'-FL is the whole cell biosynthesis in Escherichia coli by intracellular synthesis of GDP-L-fucose and subsequent fucosylation of lactose with an appropriate α1,2-fucosyltransferase. Even though whole cell approaches have been demonstrated for the synthesis of 2'-FL, further improvements of the engineered E. coli host are required to increase product yields. Furthermore, an antibiotic-free method of whole cell synthesis of 2'-FL is desirable to simplify product purification and to avoid traces of antibiotics in a product with nutritional purpose.ResultsHere we report the construction of the first selection marker-free E. coli strain that produces 2'-FL from lactose and glycerol. To construct this strain, recombinant genes of the de novo synthesis pathway for GDP-L-fucose as well as the gene for the H. pylori fucosyltransferase futC were integrated into the chromosome of E. coli JM109 by using the λ-Red recombineering technique. Strains carrying additional copies of the futC gene and/or the gene fkp (from Bacteroides fragilis) for an additional salvage pathway for GDP-L-fucose production were used and shown to further improve production of 2'-FL in shake flask experiments. An increase of the intracellular GDP-L-fucose concentration by expression of fkp gene as well as an additional copy of the futC gene lead to an enhanced formation of 2'-FL. Using an improved production strain, feasibility of large scale 2'-FL production was demonstrated in an antibiotic-free fed-batch fermentation (13 l) with a final 2'-FL concentration of 20.28 ± 0.83 g l(-1) and a space-time-yield of 0.57 g l(-1) h(-1).ConclusionsBy chromosomal integration of recombinant genes, altering the copy number of these genes and analysis of 2'-FL and intracellular GDP-L-fucose levels, we were able to construct and improve the first selection marker-free E. coli strain which is capable to produce 2'-FL without the use of expression plasmids. Analysis of intracellular GDP-L-fucose levels identified the de novo synthesis pathway of GDP-L-fucose as one bottleneck in 2'-FL production. In antibiotic-free fed-batch fermentation with an improved strain, scale-up of 2'-FL could be demonstrated.

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Transcription, processing and function of CRISPR cassettes in Escherichia coli.

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Transcription, processing and function of CRISPR cassettes in Escherichia coli.

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