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Long inverted repeats around the chromosome replication terminus in the model strain Bacillus thuringiensis serovar israelensis BGSC 4Q7
Bacillus thuringiensis serovar israelensis is the most widely used natural biopesticide against mosquito larvae worldwide. Its lineage has been actively studied and a plasmid-free strain B . thuringiensis serovar israelensis BGSC 4Q7 (4Q7) has been produced. Previous sequencing of the genome of this strain has revealed the persistent presence of a 235 kb extrachromosomal element pBtic235 which has been shown to be an inducible prophage although three putative chromosomal prophages have been lost. Moreover a 492 kb region potentially including the standard replication terminus has also been deleted in the 4Q7 strain indicating an absence of essential genes in this area. We reanalysed the genome coverage distribution of reads for the previously sequenced variant strain and sequenced two independently maintained samples of the 4Q7 strain. A 553 kb area close to the 492 kb deletion was found to be duplicated. This duplication presumably restored the equal sizes of the replichores and a balanced functioning of replication termination. An analysis of genome assembly graphs revealed a transient association of the host chromosome with the pBtic235 element. This association may play a functional role in the replication of the bacterial chromosome and the termination of this process in particular. The genome-restructuring events detected may modify the genetic status of cytotoxic or haemolytic toxins potentially influencing strain virulence. Twelve of the single-nucleotide variants identified in 4Q7 were probably due to the procedure used for strain construction or were present in the precursor of this strain. No sequence variants were found in pBtic235 but the distribution of the corresponding 4Q7 reads indicates a significant difference from counterparts in natural B. thuringiensis serovar israelensis strains suggesting a duplication or over-replication in 4Q7. Thus the 4Q7 strain is not a pure plasmid-less offshoot but a highly genetically modified derivative of its natural ancestor. In addition to potentially influencing virulence genome-restructuring events can modify the replication termination machinery. These findings have potential implications for the conclusions of virulence studies on 4Q7 as a model but they also raise interesting fundamental questions about the functioning of the Bacillus genome.
Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin
Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs) composed of 25–50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species named here as cas1B cas5 and cas6 and also revealed a certain number of spacers that have homology with extant genes most frequently derived from phages but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements and hypothesize that they provide the cell immunity against phage infection and more generally foreign DNA expression by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.