Microevolution in cyanobacteria: re-sequencing a motile substrain of Synechocystis sp. PCC 6803.
ABSTRACT: Synechocystis sp. PCC 6803 is a widely used model cyanobacterium for studying photosynthesis, phototaxis, the production of biofuels and many other aspects. Here we present a re-sequencing study of the genome and seven plasmids of one of the most widely used Synechocystis sp. PCC 6803 substrains, the glucose tolerant and motile Moscow or 'PCC-M' strain, revealing considerable evidence for recent microevolution. Seven single nucleotide polymorphisms (SNPs) specifically shared between 'PCC-M' and the 'PCC-N and PCC-P' substrains indicate that 'PCC-M' belongs to the 'PCC' group of motile strains. The identified indels and SNPs in 'PCC-M' are likely to affect glucose tolerance, motility, phage resistance, certain stress responses as well as functions in the primary metabolism, potentially relevant for the synthesis of alkanes. Three SNPs in intergenic regions could affect the promoter activities of two protein-coding genes and one cis-antisense RNA. Two deletions in 'PCC-M' affect parts of clustered regularly interspaced short palindrome repeats-associated spacer-repeat regions on plasmid pSYSA, in one case by an unusual recombination between spacer sequences.
Project description:The cyanobacterium, Synechocystis sp. PCC 6803, was the first photosynthetic organism whose genome sequence was determined in 1996 (Kazusa strain). It thus plays an important role in basic research on the mechanism, evolution, and molecular genetics of the photosynthetic machinery. There are many substrains or laboratory strains derived from the original Berkeley strain including glucose-tolerant (GT) strains. To establish reliable genomic sequence data of this cyanobacterium, we performed resequencing of the genomes of three substrains (GT-I, PCC-P, and PCC-N) and compared the data obtained with those of the original Kazusa strain stored in the public database. We found that each substrain has sequence differences some of which are likely to reflect specific mutations that may contribute to its altered phenotype. Our resequence data of the PCC substrains along with the proposed corrections/refinements of the sequence data for the Kazusa strain and its derivatives are expected to contribute to investigations of the evolutionary events in the photosynthetic and related systems that have occurred in Synechocystis as well as in other cyanobacteria.
Project description:Synechocystis sp. PCC 6803 is a widely used model cyanobacterium, whose substrains can vary on both genotype and phenotype levels. Previously described phenotypic variations include ability of mixotrophic growth, ability of movement on agar plates and variations in pigments composition or cell size. In this study, we report for the first time significant variation among Synechocystis substrains in complex cellular traits such as growth rate, photosynthesis efficiency, cellular dry weight and cellular composition (including protein or carbohydrates content). We also confirmed previously reported differences in cell size. Synechocystis cultures were cultivated in controlled environment of flat panel photobioreactors under red, blue and white light of intensities up to 790 ?mol(photons) m-2 s-1, temperatures 23°C-60°C, input CO2 concentrations ranging from 400 to 15 000 ppm and in BG11 cultivation medium with and without addition of NaCl. Three Synechocystis substrains were used for the comparative experiments: GT-L, GT-B (Brno, CZ) and PCC-B (Brno, CZ). Growth rates of Synechocystis GT-B were inhibited under high intensities of red light (585-670 nm), and growth rates of both substrains GT-B and PCC-B were inhibited under photons of wavelengths 485-585 nm and 670-700 nm. Synechocystis GT-B was more sensitive to low temperatures than the other two tested substrains, and Synechocystis GT-L was sensitive to the presence of NaCl in the cultivation media. The results suggest that stress sensitivity of commonly used Synechocystis substrains can strongly vary, similarly as glucose tolerance or motility as reported previously. Our study further supports the previous statement that emphasizes importance of proper Synechocystis substrains selection and awareness of phenotypical differences among Synechocystis substrains which is crucial for comparative and reproducible research. This is highly relevant for studies related to stress physiology and development of sustainable biotechnological applications.
Project description:Cyanobacterium Synechocystis PCC 6803 represents a favored model organism for photosynthetic studies. Its easy transformability allowed construction of a vast number of Synechocystis mutants including many photosynthetically incompetent ones. However, it became clear that there is already a spectrum of Synechocystis "wild-type" substrains with apparently different phenotypes. Here, we analyzed organization of photosynthetic membrane complexes in a standard motile Pasteur collection strain termed PCC and two non-motile glucose-tolerant substrains (named here GT-P and GT-W) previously used as genetic backgrounds for construction of many photosynthetic site directed mutants. Although, both the GT-P and GT-W strains were derived from the same strain constructed and described by Williams in 1988, only GT-P was similar in pigmentation and in the compositions of Photosystem II (PSII) and Photosystem I (PSI) complexes to PCC. In contrast, GT-W contained much more carotenoids but significantly less chlorophyll (Chl), which was reflected by lower level of dimeric PSII and especially trimeric PSI. We found that GT-W was deficient in Chl biosynthesis and contained unusually high level of unassembled D1-D2 reaction center, CP47 and especially CP43. Another specific feature of GT-W was a several fold increase in the level of the Ycf39-Hlip complex previously postulated to participate in the recycling of Chl molecules. Genome re-sequencing revealed that the phenotype of GT-W is related to the tandem duplication of a large region of the chromosome that contains 100 genes including ones encoding D1, Psb28, and other PSII-related proteins as well as Mg-protoporphyrin methylester cyclase (Cycl). Interestingly, the duplication was completely eliminated after keeping GT-W cells on agar plates under photoautotrophic conditions for several months. The GT-W strain without a duplication showed no obvious defects in PSII assembly and resembled the GT-P substrain. Although, we do not exactly know how the duplication affected the GT-W phenotype, we hypothesize that changed stoichiometry of protein components of PSII and Chl biosynthetic machinery encoded by the duplicated region impaired proper assembly and functioning of these multi-subunit complexes. The study also emphasizes the crucial importance of a proper control strain for evaluating Synechocystis mutants.
Project description:Bacterial toxin-antitoxin (TA) systems are genetic elements, which are encoded by plasmid as well as chromosomal loci and mediate plasmid and genomic island maintenance through post-segregational killing mechanisms. TA systems exist in surprisingly high numbers in all prokaryotes, but cyanobacterial TA systems have been only very poorly experimentally characterized so far. Cyanobacteria are the only prokaryotes that perform oxygenic photosynthesis. As such, cyanobacteria are of high ecological importance and are considered promising for the production of biofuels. Here, we present the molecular characterization of the sll7003/ssl7004 TA system encoded on plasmid pSYSA of the model cyanobacterium Synechocystis sp. PCC 6803 as involving a Mg(2+)-dependent RNA endonuclease activity targeting single-stranded RNA regions and demonstrate the functionality of four more TA systems encoded on this 100,749-bp plasmid. Furthermore, one additional type I, one additional type II, and three freestanding TA system components are predicted on pSYSA, all of which appear active judged by their expression. By harboring at least seven simultaneously active TA systems, pSYSA appears as the plasmid most strongly selected for among all plasmids studied in this respect thus far. These results point to a high biological relevance of pSYSA, whose coding capacity is 75% devoted to three distinct clustered regularly interspaced short palindromic repeats (CRISPR) systems mediating antiviral defense.
Project description:Cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 show similar changes in the metabolic response to changed CO2 conditions but exhibit significant differences at the transcriptomic level. This study employs a systems biology approach to investigate the difference in metabolic regulation of Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803. Presented multi-level kinetic model for Synechocystis sp. PCC 6803 is a new approach integrating and analysing metabolomic, transcriptomic and fluxomics data obtained under high and ambient CO2 levels. Modelling analysis revealed that higher number of different isozymes in Synechocystis 6803 improves homeostatic stability of several metabolites, especially 3PGA by 275%, against changes in gene expression, compared to Synechococcus sp. PCC 7942. Furthermore, both cyanobacteria have the same amount of phosphoglycerate mutases but Synechocystis 6803 exhibits only ~20% differences in their mRNA levels after shifts from high to ambient CO2 level, in comparison to ~500% differences in the case of Synechococcus sp. PCC 7942. These and other data imply that the biochemical control dominates over transcriptional regulation in Synechocystis 6803 to acclimate central carbon metabolism in the environment of variable inorganic carbon availability without extra cost carried by large changes in the proteome.
Project description:Cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism in basic research and biofuel biotechnology application. Here, we report the genomic sequence of chromosome and seven plasmids of a glucose-tolerant, non-motile strain originated from ATCC 27184, GT-G, in use at Guangzhou. Through high-throughput genome re-sequencing and verification by Sanger sequencing, eight novel variants were identified in its chromosome and plasmids. The eight novel variants, especially the five non-silent mutations might have interesting effects on the phenotype of GT-G strains, for example the truncated Sll1895 and Slr0322 protein. These resequencing data provide background information for further research and application based on the GT-G strain and also provide evidence to study the evolution and divergence of Synechocystis 6803 globally.
Project description:Background:Cyanobacteria, oxygenic photoautotrophic prokaryotes, can be engineered to produce various valuable chemicals from solar energy and CO2 in direct processes. The concept of photosynthetic production of isobutanol, a promising chemical and drop-in biofuel, has so far been demonstrated for Synechocystis PCC 6803 and Synechococcus elongatus PCC 7942. In Synechocystis PCC 6803, a heterologous expression of ?-ketoisovalerate decarboxylase (Kivd) from Lactococcus lactis resulted in an isobutanol and 3-methyl-1-butanol producing strain. Kivd was identified as a bottleneck in the metabolic pathway and its activity was further improved by reducing the size of its substrate-binding pocket with a single replacement of serine-286 to threonine (KivdS286T). However, isobutanol production still remained low. Results:In the present study, we report on how cultivation conditions significantly affect the isobutanol production in Synechocystis PCC 6803. A HCl-titrated culture grown under medium light (50 ?mol photons m-2 s-1) showed the highest isobutanol production with an in-flask titer of 194 mg l-1 after 10 days and 435 mg l-1 at day 40. This corresponds to a cumulative isobutanol production of 911 mg l-1, with a maximal production rate of 43.6 mg l-1 day-1 observed between days 4 and 6. Additional metabolic bottlenecks in the isobutanol biosynthesis pathway were further addressed. The expression level of KivdS286T was significantly affected when co-expressed with another gene downstream in a single operon and in a convergent oriented operon. Moreover, the expression of the ADH encoded by codon-optimized slr1192 and co-expression of IlvC and IlvD were identified as potential approaches to further enhance isobutanol production in Synechocystis PCC 6803. Conclusion:The present study demonstrates the importance of a suitable cultivation condition to enhance isobutanol production in Synechocystis PCC 6803. Chemostat should be used to further increase both the total titer as well as the rate of production. Furthermore, identified bottleneck, Kivd, should be expressed at the highest level to further enhance isobutanol production.
Project description:The effect of phycobilisome antenna-truncation in the cyanobacterium Synechocystis sp. PCC 6803 on biomass production and glycogen accumulation have not yet been fully clarified. To investigate these effects here, the apcE gene, which encodes the anchor protein linking the phycobilisome to the thylakoid membrane, was deleted in a glucose tolerant strain of Synechocystis sp. PCC 6803. Biomass production of the apcE-deleted strain under photoautotrophic and atmospheric air conditions was 1.6 times higher than that of strain PCC 6803 (1.32?±?0.01 versus 0.84?±?0.07 g cell-dry weight L(-1), respectively) after 15 days of cultivation. In addition, the glycogen content of the apcE-deleted strain (24.2?±?0.7%) was also higher than that of strain PCC 6803 (11.1?±?0.3%). Together, these results demonstrate that antenna truncation by deleting the apcE gene was effective for increasing biomass production and glycogen accumulation under photoautotrophic and atmospheric air conditions in Synechocystis sp. PCC 6803.
Project description:NADP+-isocitrate dehydrogenase (NADP+-IDH) activity and protein levels in crude extracts from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 and the filamentous, dinitrogen-fixing Anabaena sp. strain PCC 7120 were determined under different nitrogen conditions. The highest NADP+-IDH activity and protein accumulation were found under dinitrogen-fixing conditions for the Anabaena strain and under nitrogen starvation for Synechocystis sp. PCC 6803. The icd gene that encodes the NADP+-IDH from Synechocystis sp. strain PCC 6803 was cloned by heterologous hybridization with the previously isolated icd gene from Anabaena sp. strain PCC 7120. The two cyanobacterial icd genes show 81% sequence identity and share a typical 44-amino-acid region different from all the other icd genes sequenced so far. The icd gene seems to be essential for Synechocystis growth since attempts to generate a completely segregated icd mutant were unsuccessful. Transcripts of 2.0 and 1.6 kb were detected by Northern (RNA) blot analysis, for the Anabaena and Synecho-cystis icd genes, respectively. Maximal icd mRNA accumulation was reached after 5 It of nitrogen starvation in Synechocystis cells and under dinitrogen-fixing conditions in Anabaena cells. Primer extension analysis showed that the structure of the Synechocystis icd gene promoter resembles those of the NtcA-regulated promoters. In addition, mobility shift assays demonstrated that purified Synechocystis NtcA protein binds to the promoter of the icd gene. All these data suggest that the expression of the icd gene from Synechocystis sp. strain PCC 6803 may be subjected to nitrogen control mediated by the positively acting regulatory protein NtcA.
Project description:Many organisms harbor circadian clocks with periods close to 24 h. These cellular clocks allow organisms to anticipate the environmental cycles of day and night by synchronizing circadian rhythms with the rising and setting of the sun. These rhythms originate from the oscillator components of circadian clocks and control global gene expression and various cellular processes. The oscillator of photosynthetic cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, linked to a complex regulatory network. Synechocystis sp. strain PCC 6803 possesses the standard cyanobacterial kaiABC gene cluster plus multiple kaiB and kaiC gene copies and antisense RNAs for almost every kai transcript. However, there is no clear evidence of circadian rhythms in Synechocystis sp. PCC 6803 under various experimental conditions. It is also still unknown if and to what extent the multiple kai gene copies and kai antisense RNAs affect circadian timing. Moreover, a large number of small noncoding RNAs whose accumulation dynamics over time have not yet been monitored are known for Synechocystis sp. PCC 6803. Here we performed a 48-h time series transcriptome analysis of Synechocystis sp. PCC 6803, taking into account periodic light-dark phases, continuous light, and continuous darkness. We found that expression of functionally related genes occurred in different phases of day and night. Moreover, we found day-peaking and night-peaking transcripts among the small RNAs; in particular, the amounts of kai antisense RNAs correlated or anticorrelated with those of their respective kai target mRNAs, pointing toward the regulatory relevance of these antisense RNAs. Surprisingly, we observed that the amounts of 16S and 23S rRNAs in this cyanobacterium fluctuated in light-dark periods, showing maximum accumulation in the dark phase. Importantly, the amounts of all transcripts, including small noncoding RNAs, did not show any rhythm under continuous light or darkness, indicating the absence of circadian rhythms in Synechocystis.