Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

Nucleotide sequence of the second psbG gene in Synechocystis 6803. Possible implications for psbG function as a NAD(P)H dehydrogenase subunit gene

Nucleotide sequencing of the second Synechocystis 6803 psbG gene, psbG2 shows the predicted polypeptide to be 219 amino acids long. It is less similar to chloroplast psbG genes than is the Synechocystis psbG1 copy. Alignment with seven other psbG protein sequences, including that from the Paramecium mitochondrial genome reveals a central highly conserved region common to each. This is discussed as evidence supporting the proposal that the psbG polypeptide is a NAD(P)H dehydrogenase (complex I) subunit in cyanobacteria, chloroplasts and mitochondria.     

Isolation and characterization of a glycoprotein from the stonefly, Pteronarcys californica, which binds cadmium

The present study reports the isolation and characterization of a cadmium-containing glycoprotein from the water-soluble fraction of an aquatic insect. The isolated glycoprotein contained 0·67% cadmium, 62·1% carbohydrate, and 37·2% protein. The glycoprotein appears to be involved in the detoxification of cadmium, because species insensitive to cadmium contain five times the amount of the glycoprotein as do species sensitive to cadmium.