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Abstract: An improved method for the electrotransformation of Lactobacillus plantarum CECT 220 (ATCC 8014) with plasmid DNA isolated from Escherichia coli is described. The two main modifications with respect to existing methods are: (i) isolation of plasmid DNA from E. coli JM110 grown in minimal medium and (ii) in vitro modification of the DNA by cell-free extracts of the host L. plantarum. Optimal electrotransformation was obtained with exponentially growing cells of L. plantarum concentrated to 6×109 cfuml−1, with electric pulses of 13 kVcm−1 in cuvettes with 1 mm inter-electrode distance. We consider that this method constitutes a useful tool for routine manipulation of L. plantarum, and can probably be extended to other lactic acid bacteria. [Copyright &y& Elsevier]

In addition to core Cas proteins, CRISPR-Cas loci often encode ancillary proteins that modulate the activity of the respective effectors in interference. Subtype VI-B1 CRISPR-Cas systems encode the Csx27 protein that down-regulates the activity of Cas13b when the type VI-B locus is expressed in Escherichia coli. We show that Csx27 belongs to an expansive family of proteins that contain four predicted transmembrane helices and are typically encoded in predicted operons with components of the bacterial natural transformation machinery, multidomain proteins that consist of components of the ubiquitin signaling system and proteins containing the ligand-binding WYL domain and a helix-turn-helix domain. The Csx27 family proteins are predicted to form membrane channels for ssDNA that might comprise the core of a putative novel, Ub-regulated system for DNA uptake and, possibly, degradation. In addition to these associations, a distinct subfamily of the Csx27 family appears to be a part of a novel, membrane-associated system for DNA modification. In Bacteroidetes, subtype VI-B1 systems might degrade nascent transcripts of foreign DNA in conjunction with its uptake by the bacterial cell. These predictions suggest several experimental directions for the study of type VI CRISPR-Cas systems and distinct mechanisms of foreign DNA uptake and degradation in bacteria. [ABSTRACT FROM AUTHOR]