PMC:3514855 / 19679-24171 JSONTXT

{"project":"2_test","denotations":[{"id":"23069868-8320214-25970120","span":{"begin":1652,"end":1654},"obj":"8320214"},{"id":"23069868-21294681-25970121","span":{"begin":2038,"end":2040},"obj":"21294681"},{"id":"23069868-20528693-25970121","span":{"begin":2038,"end":2040},"obj":"20528693"},{"id":"23069868-20056882-25970121","span":{"begin":2038,"end":2040},"obj":"20056882"},{"id":"23069868-20129051-25970121","span":{"begin":2038,"end":2040},"obj":"20129051"},{"id":"23069868-20072129-25970121","span":{"begin":2038,"end":2040},"obj":"20072129"},{"id":"23069868-21531607-25970121","span":{"begin":2038,"end":2040},"obj":"21531607"},{"id":"23069868-22337052-25970121","span":{"begin":2038,"end":2040},"obj":"22337052"},{"id":"23069868-19239620-25970122","span":{"begin":3041,"end":3043},"obj":"19239620"},{"id":"23069868-19239620-25970123","span":{"begin":3800,"end":3802},"obj":"19239620"}],"text":"3.4. Larger indels and plasmids\nIn addition to this relatively large number of SNPs, only seven larger deletions were found on the chromosome and two plasmids. Compared with the reference, a deletion of 0.6 kb exists in the gene slr1753 (#4 in Table 1), which encodes, according to our data, a giant protein comprising 1549 amino acids that probably is transported to the cell surface. However, we found this deletion in our verification also in ‘GT-Kazusa’ and ‘GT-V’. Moreover, the deleted/inserted region consists of long series of DNA repeats (Fig. 1), an evidence for a possible assembly or annotation error in the original sequence analysis.\nFigure 1. Alignment of the possible indel region in gene slr1753. The sequence obtained in the verification experiment is aligned with that of the ‘GT-Kazusa’ reference. Two types of DNA repeats are indicated by the filled and non-filled lozenges.\nGiven the very scarce available information concerning biological functions of the plasmids in Synechocystis sp. PCC 6803, it was interesting that all seven plasmids were detected during our analysis. Two, pCC5.2 and pCB2.4, were initially not found. However, as they were amplified easily by PCR, we re-inspected the unmapped sequencing reads, but still could not detect a single read matching these plasmids. This observation may relate to a lower copy number of these compared with the other plasmids, but this was not tested in the current study. Analysing the plasmid sequences, we observed a remarkable genetic stability. In addition to a single-base substitution in the plasmid pCA2.4 that might rather constitute an error in the reference sequence37 (see above) and a missing mobile element on the plasmid pSYSM, two mutations were observed, both in the plasmid pSYSA.\nTwo major mutations affect the clustered regularly interspaced short palindrome repeats-CRISPR-associated proteins (CRISPR-Cas) system, located on the plasmid pSYSA. CRISPR-Cas systems provide in many archaea and bacteria an adaptive immunity against invading DNA.38–44 The plasmid pSYSA encodes the three independent systems CRISPR1, CRISPR2 and CRISPR3. A 2399-bp deletion encompassing the spacer-repeat regions 15–47 of CRISPR1 was detected in ‘PCC-M’ (#43), which also eliminated the relatively short genes ssr7018, ssl7019, ssl7020 and ssl7021, annotated within the spacer-repeat array of CRISPR1. However, the theoretical protein sequences of these gene products show no conservation at all and might not constitute real genes. Nevertheless, the deletion of spacer-repeat regions 15–47 of CRISPR1 is severe, since compared with the reference, it has eliminated two-thirds, 33 of its 49 spacer-repeat units. The sequence analysis suggests that the recombination events leading to the deletion of spacer-repeat regions 15–47 must have occurred within the direct repeats. Thus, this recombination is in agreement with previous observations that the downstream ends of the repeat clusters are conserved such that deletions and recombination events occur internally.45\nA very different type of deletion was noticed for the CRISPR2 system located on the same plasmid. In this case, 159 bp were deleted (event #44 in Table 1). These 159 deleted bases correspond to positions 71 499–71 657 in the reference. The deletion encompasses two repeats including the spacer 41 in between. It is very surprising that the recombination did not occur within the repeat sections but in the adjacent spacers 40 and 42, thus generating a new ‘hybrid’ spacer 40 at positions 69 082–69 111 in the pSYSA plasmid of ‘PCC-M’ (Fig. 2). As a result, spacers 40, 41 and 42 of the original sequence are missing and became replaced by this hybrid sequence. The vast majority of described deletions in the CRISPR system occur between the direct repeats.45 Non-homologous recombination between two different spacers is rare, the deletion observed here in CRISPR2 of the plasmid pSYSA is generating additional sequence diversity in the CRISPR system. Due to the two deletions in the plasmid pSYSA, we determined its total length as 100 749 bp, compared with 103 307 bp for the reference.\nFigure 2. Non-homologous recombination in the plasmid pSYSA affecting spacers 40, 41 and 42 of CRISPR2. As a result of the 159-bp deletion in ‘PCC-M’ compared with ‘GT-Kazusa’, a novel hybrid spacer 40 was generated. The direct repeats are presented as squares and the nucleotide positions in the ‘GT-Kazusa’ are given according to the GenBank file NC_005230."}