Upregulation of Plasmid Genes during Stationary Phase in Synechocystis sp. Strain PCC 6803, a Cyanobacterium

Abstract

We analyzed DNA microarrays to identify highly expressed genes during stationary-phase growth of Synechocystis sp. PCC 6803. Many identified genes are on endogenous plasmids, with copy numbers between 0.4 and 7 per chromosome. The promoters of such genes will be useful for synthetic biology applications with this phototrophic host. Bacterial cultures enter stationary phase when either nutrient limitation or a buildup of growth by-products ceases cell division. However, this does not necessarily imply that cells become metabolically inactive. Artificial "leaves" have been constructed from Rhodopseudomonas palustris cells embedded in latex that can produce H 2 photoheterotrophically for over 5 months without cell growth (5). Pfic in Escherichia coli was recently used to produce a high titer of a bacteriotoxin at stationary phase without any inducer and during exponential phase without detectable growthlimiting toxin during exponential phase (1).Cultures and microarray analysis. We used DNA microarray analysis to identify genes and promoters active during stationary phase in Synechocystis sp. PCC 6803. This naturally competent cyanobacterium is a widely used host organism for autotrophic synthetic biology (3,4,13,18). Synthetic biology is an emerging field in which genes, promoters, and other units of genetic code either taken from across the diversity of life or created entirely from scratch are mixed and matched in a host organism (chassis) to improve existing cellular functions or create entirely new ones. Such studies demand the use of a wide variety of promoters that are active under different conditions in the chassis organism.We grew replicate cultures of Synechocystis PCC 6803 in BG11 medium bubbled with air plus 5% CO 2 (autotrophic) or with air plus 5 mM glucose (mixotrophic). The temperature was maintained at 30°C, and light intensity was 100 E m Ϫ2 s Ϫ1 from cool white fluorescent lamps. Cell growth was monitored by measurement of the optical density at 730 nm on a BioTek Quant plate reader (BioTek, VT). Cultures were sampled for microarrays in exponential phase and twice during stationary phase ( Fig. 1) and analyzed as described previously (20). Briefly, 2 replicate microarrays were analyzed for each of 2 replicate cultures for each nutritional condition. Data were LOWESS normalized by using the MATLAB bioinformatics toolbox. Normalized probe intensities were grouped by genes and t tested to determine significant up-or downregulation (P Ͻ 0.05). SPPS.To quantify the activity of potential promoters at stationary phase, we calculated a stationary-phase promoter score (SPPS) for each open reading frame (ORF), based on the following equation: SPPS ϭ log 2 (fold change) ϩ log 2 (normalized expression).The changes were averaged across nutritional conditions. Nor-

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

“…The NSP1 mutant expressed 8 to 14 times more EYFP than NSC1 and NSC2, and that ratio increased from the exponential to the stationary phase. This agrees with our earlier observations of plasmid copy number (14) and shows that expression of heterologous genes from pCC5.2 can result in a highly favorable expression profile for the use of Synechocystis PCC 6803 as a microbial cell factory that first produces cellular biomass and later produces products of interest in stationary phase. As also shown in Fig.…”

“…Moreover, the small endogenous plasmids (pCA2.4, pCB2.4, pCC5.2) of Synechocystis PCC 6803 have been shown to increase their copy numbers at stationary phase to ca. 3 to 8 copies per chromosome (14). This could make these genomic loci attractive as insertion sites for the expression of heterologous genes during stationary phase.…”

“…Since there are a limited number of self-replicating exogenous plasmids for the expression of heterologous genes in Synechocystis PCC 6803 (12) and other cyanobacteria, these endogenous plasmids may prove to be attractive parts for synthetic biology (13). Additionally, these plasmids increase their copy numbers during the transition from the exponential-growth phase to the stationary phase (14). Thus, we have hypothesized that the expression of heterologous genes from sites in these plasmids could be autoinduced by such a growth transition.…”

A Novel Mechanism, Linked to Cell Density, Largely Controls Cell Division in Synechocystis

“…Considering that the metabolism does not control the rate of cell division, we tested whether already known factors were responsible, such as (1) nutrient starvation (Lazazzera, 2000;Berla and Pakrasi, 2012), (2) quorum sensing (Sharif et al, 2008), and (3) reduction of light quality and intensity available to the cells due to high cell density (self-shading; Raven and Kübler, 2002;Ort and Melis, 2011;Lea-Smith et al, 2014).…”

Synthetic biology of cyanobacteria: unique challenges and opportunities

“…Synechocystis sp. PCC 6803 also has plasmids whose copy numbers span a similar range [from ~0.4–8 per chromosome (Berla and Pakrasi, 2012)]. The origins of replication from these plasmids constitute a source of genetic parts that could be used to generate cyanobacterial expression plasmids having a range of copy numbers, and which could potentially be modified to create higher or lower-copy plasmids that are compatible with existing plasmids in various cyanobacterial systems.…”