PMC:3514855 / 7983-25065 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3514855","sourcedb":"PMC","sourceid":"3514855","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3514855","text":"3. Results\n\n3.1. Overview\nSequencing of the Synechocystis sp. PCC 6803 ‘Moscow’ substrain ‘PCC-M’ by Illumina (Solexa) yielded an average 1100-fold coverage of the chromosome and five of the seven plasmids. The existence of the two remaining plasmids was verified individually by PCR. Following assembly of sequences, mapping to the reference strain sequences and annotation, the obtained genome and plasmid sequences were deposited in the GenBank database with the accession numbers CP003265–CP003272.\nAltogether, we found 45 differences (36 SNPs and 9 indels \u003e1 bp) between the investigated substrain ‘PCC-M’ and the published sequences of the ‘GT-Kazusa’ chromosome2 and plasmids20 used here as references (Table 1). From these differences, 41 are located in the chromosome and four in the plasmids pSYSA, pSYSM and pCA2.4. For verification, about one-third of these differences were randomly chosen and confirmed independently by PCR and Sanger sequencing of the respective regions in substrain ‘PCC-M’, but no misidentified mutations were found. These DNA regions were, in addition, amplified and compared with the sequences from substrains ‘GT-Kazusa’ and ‘GT-V’ for control and comparison, respectively. The GT ‘GT-V’ was chosen for comparison as is widely used for the dissection and analysis of photosynthetic mutants. Fully segregated PSI, PSII and Chl biosynthesis mutants were successfully generated in this genetic background21,22 and some of these mutants could not be obtained in other substrains.23\nTable 1. Location and effects of SNPs and indels found in ‘PCC-M’ compared with the nucleotide sequence of ‘GT-Kazusa’ in the database\nThe events are numbered (column #), the type of mutation (M) is indicated as S, substitution, D, deletion or I, insertion, together with the respective start and end positions in the ‘GT-Kazusa’ reference sequence. For each event the respective nucleotide change is indicated on the forward strand, together with the resulting codon modification (Ref. → Mut) and amino acid change, if any. Highlighted in italics are four instances of missing ISY203 copies and in bold all SNPs affecting intergenic spacer regions (IGR). aIndicate errors in the database.\nThe number of differences between ‘PCC-M’ and ‘GT-Kazusa’ are almost twice as many as reported by Tajima et al.10 for the GT (GT-S) ‘Kazusa’ strain, where a total of 22 differences from the published sequence were found.10 All but 3 of those 22 differences were also detected in the ‘PCC-M’ strain studied here. The three unique differences in the ‘GT-S’ and 26 differences between ‘PCC-M’ and ‘GT-Kazusa’ underline the existence of lineage splitting in the Synechocystis substrains. Moreover, we found seven SNPs (#5, 13, 15, 16, 27, 32 and 33 in Tables 1 and 2) and one larger indel (#6 in Tables 1 and 2) specifically shared between the ‘PCC-M’ and the ‘PCC-N and PCC-P’ substrains, indicating that ‘PCC-M’ belongs to the ‘PCC’ group of motile substrains.9 ‘PCC-M and PCC-P’ are strains that both exhibit the native positive phototaxis, whereas ‘PCC-N’ strain shows negative phototaxis.24\nTable 2. Comparison of SNPs and indels found in the chromosome of ‘PCC-M’ with sequences from other substrains\nAll events are numbered (column #) as in Table 1. The presence of the respective ‘PCC-M’ mutation in the different substrains is indicated by the check marks. aThe deletion of 0.6 kb in the gene slr1753 compared with the reference was also verified here in ‘GT-Kazusa’.\n\n3.2. SNPs in protein-coding genes\nOf the total of 36 SNPs in ‘PCC-M’ compared with ‘GT-Kazusa’, all except 1 are located in the chromosome. The single base substitution that was found on the plasmid pCA2.4 within the repA gene (#42 in Table 1) seems to be no mutation but an error in the published sequence of ‘GT-Kazusa’, since in our PCR-control experiments, the sequence was identical in the three strains ‘GT-Kazusa’, ‘PCC-M’ and ‘GT-V’. Of the 35 chromosomal SNPs compared with ‘GT-Kazusa’, 5 are silent base substitutions, 14 substitutions lead to amino acid substitutions, in 6 cases a single basepair is deleted and in 2 cases (#23 and #28) one basepair was inserted within an ORF, causing a frameshift mutation. Furthermore, five substitutions, two single basepair insertions and one single basepair deletion were observed in intergenic regions (IGR) of ‘PCC-M’ compared with the reference (Table 1).\nSeven SNPs are specifically shared between the ‘PCC-M’, ‘PCC-N and PCC-P’ substrains. These are in slr1865 (#13), encoding a hypothetical protein, in sll1951 (#15), encoding a haemolysin-like protein, in slr1983 (#16), encoding a two-component hybrid sensor and regulator protein, in slr0222 (#27), encoding the histidine kinase Hik25, a silent mutation in slr0302 (#32), encoding a PAS/PAC and GAF sensors-containing diguanylate cyclase, one missing basepair, leaving the spkA gene intact (#5) and, finally, in ssr1176 (#33), encoding a transposase (Tables 1 and 2).\nThe gene for a cell surface-localized haemolysin-like protein, HlyA (sll1951), reported to function as a barrier against the adsorption of toxic compounds,25,26 is lacking one nucleotide in ‘PCC-M’ compared with the reference (difference #15). In the ‘GT-Kazusa’, ‘GT-V’ as well as the ‘GT-I’ and ‘GT-S’ strains,9 the presence of the additional A leads to the fusion of two ORFs that are separate in ‘PCC-M’, ‘PCC-N’ and ‘PCC-P’ substrains.9 As a result, Sll1951 is 1741 amino acids in the former and only 1437 residues in the latter.\nIn our data, some other previously published mutations8,10 are confirmed. For instance, spkA (sll1574; #5), a regulator of cellular motility via phosphorylation of membrane proteins,11,27 is disrupted by a 1-bp insertion in the non-motile ‘GT-Kazusa’ and ‘GT-V’ strains, whereas it is intact in the motile ‘PCC-M’ strain (Table 1). Similarly, the pilC gene (slr0162/3) required for pili assembly has been reported to carry a frameshift mutation in the ‘GT-Kazusa’ and ‘GT-S’ sequences.8,10,28 We found an intact pilC gene in ‘PCC-M’ (#20), as well as in the ‘GT-V’ substrain.\nAnother SNP (G–A) exists in psaA (slr1834; #9), encoding the photosynthetic P700 apoprotein subunit Ia; however, in accordance with Tajima et al.10, we believe this is an annotation error in the database as we found an A in the respective position in all three strains dealt with in this work (Table 1). Similarly, ycf22 (sll0751; #26) is here suggested to be fused to the downstream reading frame sll0752. Indeed, in blastp comparisons, both proteins together match against a single, widely distributed, larger protein of 452 amino acids. This protein possesses a Ttg2C domain (COG1463), which is found in an ABC-type transport system involved in resistance to organic solvents. The acronym ycf stands for hypothetical chloroplast reading frames, meaning proteins conserved in chloroplasts and also cyanobacteria. The 1-bp shorter version, which is splitted into sll0751/sll0752, is a database error in the case of ‘GT-Kazusa’ as well.\n\n3.2.1. SNPs unique to ‘PCC-M’\nSix of the 10 SNPs unique to ‘PCC-M’ are located within coding regions and cause amino acid substitutions or alter the length of the respective reading frame.\nA single basepair transversion in the gene sigF (slr1564; #39 in Table 1) is leading to a M231K substitution within the −35 element DNA-binding region29 of a group 3 sigma factor required for phototactic movement30 and salt-stress response.31 This SNP cannot lead to impaired motility as ‘PCC-M’ is motile but it might influence the DNA–protein interaction of SigF because positively charged residues such as lysine located in this part of the σ4.2 region can directly interact with DNA.29\nAnother transversion, in argB (slr1898; #8 in Table 1), leads to an S2N amino acid substitution in N-acetylglutamate kinase, the enzyme performing the first committed step of Arg biosynthesis. Transitions in sll1359 and slr1609 (#11 and #3 in Table 1) result in an N–K substitution at a very conserved position within a predicted cytochrome and an L608S (L548S) substitution in the long-chain acyl-CoA-synthetase Slr1609 that has been found crucial for fatty acid activation and the biosynthesis of alkanes.32 Interestingly, an unrelated SNP exists at position 488 923 within the slr1609 coding sequence in a strain ‘YF’, leading to a G546L (G486L) substitution.17 It should be noted that the slr1609 reading frame has been annotated 60 codons shorter (636 instead of 696 amino acids) during recent re-sequencing analyses,9,10 compared with the original annotation of ‘GT-Kazusa’ (numbers in brackets). The shorter Slr1609 protein of 636 amino acids is also consistent with the mapped start site of transcription at position 487 352,33 located 115 nt upstream of the revised start codon.\nA transition in slr0753 (#41 in Table 1) leads to a P113L substitution in a putative chloride efflux transport protein involved in maintaining the chloride ion concentration homoeostasis as required for a functional photosystem II.34\nA single basepair deletion in sll1496 (#38 in Table 1), encoding mannose-1-phosphate guanyltransferase, causes a frameshift and premature stop of the gene in ‘PCC-M’. The resulting protein is with 515 instead of 643 amino acids severely truncated and may be rendered function-less.\n\n3.3. Point mutations in IGRs\nCompared with the reference, eight SNPs are located in IGRs, three of these (#7, 24 and 36) are ‘PCC-M’ specific. One of these (#36 in Table 1) SNPs is predicted to affect one of the recently reported cis-antisense RNAs.33 The additional A between positions 3194022 and 3194023 is located in the IGR between genes slr0533 and slr0534, encoding histidine kinase 10 (Hik10) and the soluble lytic transglycosylase Slt. On the reverse strand, the additional T falls within the predicted −10 element of the slr0534_as3 promoter. Instead of the high-scoring CATAAT,33 the motif is changed to ATTAAT. Hence, a modulation of slr0534_as3 expression compared with the reference is possible. In contrast to its designation, this cis-antisense RNA overlaps the 3′ end of genes slr0533 and hik10 (due to an error in the annotation used as the reference). In microarray analyses, slr0534_as3 of strain ‘PCC-M’ was found to be moderately to highly expressed under four tested conditions. Compared with the accumulation of the hik10 mRNA, it appeared even stronger.33 A function for Hik10 has been found in the perception of salt stress or transduction of the signal.35 The slr0534_as3 transcript may play a silencing role with regard to hik10 under non-inducing conditions. Mutation of its promoter element may hence cause a physiological effect in the salt stress response.\nTwo other SNPs (at positions 831 647 and 2 400 722; #7 and #24 in Table 1) could have an impact on the promoter strength or the regulation of the genes infA and glcP. For glcP, the initiation site of transcription was mapped to position 2 400 66633 and for infA to position 831 635 (unpublished). Thus, these two SNPs are located 12 and 56 nt upstream of the respective initiation site of transcription. In the case of the infA promoter, the transition replaces a nucleotide within the putative −10 element, changing it from TGTGAT to TATGAT, a much more typical motif for a −10 element in Synechocystis.33 The mutation 56 nt upstream of the initiation site of transcription of glcP might be functionally relevant as well. The gene product, a glucose transporter, is directly relevant for the physiological ability to use glucose; its gene expression is affected by mutation of the gene for the AbrB-type transcription factor Sll0822.36 The region at position −56 might well be part of the recognized sequence.\n\n3.4. 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.\n\n3.5. Mobile elements\nAs can be seen in Tables 1 and 2 (differences #12, 17, 40 and 45), the ‘PCC-M’ substrain lacks four insertion elements of the ISY203 type present in ‘GT-Kazusa’.7 These elements are ISY203b, e and g on the chromosome and ISY203j on the plasmid pSYSM. These four indels have the exact same size of 1183 bp, only one is 1185 bp.\nIn the ‘GT-S’ substrain re-sequenced by Tajima et al.10 one of these four elements, ISY203e, is already present, placing this strain (in accordance with Ikeuchi and Tabata)8 before ‘GT-Kazusa’ in the strain history. The absence of ISY203b, e and g in ‘PCC-M’ is further shared with the strains ‘GT-I’, ‘PCC-N’ and ‘PCC-P’,9 whereas no statement is possible with regard to the possible presence of ISY203j on the plasmid pSYSM in the latter.\nWith respect to the described mobile elements, ‘PCC-M’ appears as one of the least-derived substrains.","divisions":[{"label":"title","span":{"begin":4,"end":11}},{"label":"sec","span":{"begin":13,"end":3479}},{"label":"title","span":{"begin":19,"end":27}},{"label":"p","span":{"begin":28,"end":504}},{"label":"p","span":{"begin":505,"end":2207}},{"label":"table caption","span":{"begin":1517,"end":2207}},{"label":"p","span":{"begin":1526,"end":1651}},{"label":"p","span":{"begin":1653,"end":2173}},{"label":"p","span":{"begin":2174,"end":2207}},{"label":"p","span":{"begin":2207,"end":3480}},{"label":"table 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