Engineering Synechococcus elongatus PCC 7942 for Continuous Growth under Diurnal Conditions

Synechococcus elongatus strain PCC 7942 strictly depends upon the generation of photosynthetically derived energy for growth and is incapable of biomass increase in the absence of light energy. Obligate phototrophs'' core metabolism is very similar to that of heterotrophic counterparts exhibiting diverse trophic behavior. Most characterized cyanobacterial species are obligate photoautotrophs under examined conditions. Here we determine that sugar transporter systems are the necessary genetic factors in order for a model cyanobacterium, Synechococcus elongatus PCC 7942, to grow continuously under diurnal (light/dark) conditions using saccharides such as glucose, xylose, and sucrose. While the universal causes of obligate photoautotrophy may be diverse, installing sugar transporters provides new insight into the mode of obligate photoautotrophy for cyanobacteria. Moreover, cyanobacterial chemical production has gained increased attention. However, this obligate phototroph is incapable of product formation in the absence of light. Thus, converting an obligate photoautotroph to a heterotroph is desirable for more efficient, economical, and controllable production systems.     

High-Throughput Functional Analysis of the Synechococcus elongatus PCC 7942 Genome

Synechococcus elongatus PCC 7942 was the first cyanobacterialstrain to be reliably transformed by exogenously added DNA andhas become the model organism for cyanobacterial circadian rhythms.With a small genome (2.7 Mb) and well-developed genetic tools,PCC 7942 provides an exceptional opportunity to elucidate thecircadian mechanism through genetics. We describe a projectto create mutations in every locus of the genome, both to assayeach locus for its potential contribution to the circadian clockand to archive data for the cyanobacterial community. Cosmidclones that carry inserts of PCC 7942 DNA are saturated withtransposon insertions in vitro to provide sequencing templatesand substrates for mutagenesis of the PCC 7942 genome via homologousrecombination. We have mutagenized 53% of the chromosome from50 chromosome-bearing cosmids and identified the positions ofinsertions in 31 of those cosmids and the 46 kb plasmid, pANL.PCC 7942 mutants defective for 490 different genes have beenscreened for circadian phenotypes. Mutagenesis of three apparentlyessential loci, including clpPIIclpX, resulted in circadianphenotypes. We developed an effective antisense suppressionmethod to further the analysis of essential genes. When completed,the set of comprehensive mutations will provide the communitywith a unique resource whose impact will extend beyond circadianresearch.     

Phase determination of circadian gene expression in Synechococcus elongatus PCC 7942

The authors analyzed the upstream regulatory region of purF, a gene that is expressed in a minority phase that peaks at dawn (class 2 circadian phasing) in Synechococcus elongatus, to determine whether specific cis elements are responsible for this characteristic expression pattern. Fusions of various promoter-bearing fragments to luciferase reporter genes showed that normal class 2 phasing of purF expression was correlated with promoter strength. No specific cis element that is separable from the promoter was responsible for determining phase. Very weak promoter activity of unstable phasing was mapped to a 50-bp segment. Inclusion of sequences that flank this minimal promoter either upstream or downstream increased the promoter strength and stabilized the phase in class 2, but neither segment was individually necessary. Because the data suggested a role for the overall promoter context rather than a specific "phase element," the authors proposed that DNA topology is important in the phase determination of circadian gene expression in S. elongatus. To test this hypothesis, they fused the well-characterized DNA topology-dependent Escherichia coli fis promoter to luciferase and showed that it acts as a class 2 promoter in S. elongatus.     

HtpG,the prokaryotic homologue of Hsp90, stabilizes a phycobilisome protein in the cyanobacterium Synechococcus elongatus PCC 7942

HtpG, a homologue of HSP90, is essential for thermotolerance in cyanobacteria. It is not known how it plays this important role. We obtained evidence that HtpG interacts with linker polypeptides of phycobilisome in the cyanobacterium Synechococcus elongatus PCC 7942. In an htpG mutant, the 30 kDa rod linker polypeptide was reduced. In vitro studies with purified HtpG and phycobilisome showed that HtpG interacts with the linker polypeptide as well as other linker polypeptides to suppress their thermal aggregation with a stoichiometry of one linker polypeptide/HtpG dimer. We constructed various domain‐truncated derivatives of HtpG to identify putative chaperone sites at which HtpG binds linker polypeptides. The middle domain and the N‐terminal domain, although less efficiently, prevented the aggregation of denatured polypeptides, while the C‐terminal domain did not. Truncation of the C‐terminal domain that is involved in the dimerization of HtpG led to decrease in the anti‐aggregation activity, while fusion of the N‐terminal domain to the middle domain lowered the activity. In vitro studies with HtpG and the isolated 30 kDa rod linker polypeptide provided basically similar results to those with HtpG and phycobilisome. ADP inhibited the anti‐aggregation activity, indicating that a compact ADP conformational state provides weaker aggregation protection compared with the others.     

Enhancement of acetyl-CoA flux for photosynthetic chemical production by pyruvate dehydrogenase complex overexpression in Synechococcus elongatus PCC 7942

Genetic manipulation in cyanobacteria enables the direct production of valuable chemicals from carbon dioxide. However, there are still very few reports of the production of highly effective photosynthetic chemicals. Several synthetic metabolic pathways (e.g., isopropanol, acetone, isoprene, and fatty acids) have been constructed by branching from acetyl-CoA and malonyl-CoA, which are key intermediates for photosynthetic chemical production downstream of pyruvate decarboxylation. Recent reports of the absolute determination of cellular metabolites in Synechococcus elongatus PCC 7942 have shown that its acetyl-CoA levels corresponded to about one hundredth of the pyruvate levels. In short, one of the reasons for lower photosynthetic chemical production from acetyl-CoA and malonyl-CoA was the smaller flux to acetyl-CoA. Pyruvate decarboxylation is a primary pathway for acetyl-CoA synthesis from pyruvate and is mainly catalyzed by the pyruvate dehydrogenase complex (PDHc). In this study, we tried to enhance the flux toward acetyl-CoA from pyruvate by overexpressing PDH genes and, thus, catalyzing the conversion of pyruvate to acetyl-CoA via NADH generation. The overexpression of PDH genes cloned from S. elongatus PCC 7942 significantly increased PDHc enzymatic activity and intracellular acetyl-CoA levels in the crude cell extract. Although growth defects were observed in overexpressing strains of PDH genes, the combinational overexpression of PDH genes with the synthetic metabolic pathway for acetate or isopropanol resulted in about 7-fold to 9-fold improvement in its production titer, respectively (9.9 mM, 594.5 mg/L acetate, 4.9 mM, 294.5 mg/L isopropanol). PDH genes overexpression would, therefore, be useful not only for the production of these model chemicals, but also for the production of other chemicals that require acetyl-CoA as a key precursor.     

Characterization of dnaJ multigene family in the cyanobacterium Synechococcus elongatus PCC 7942

The genome of the cyanobacterium Synechococcus elongatus PCC 7942 contains four dnaJ homologs, which are classified into three types based on domain structure. Among these, dnaJ1, dnaJ2, and dnaJ3 are essential for normal growth, and hence we analyzed them with a view to characterizing their specificity. Expression analysis indicated that dnaJ2, which encodes type II DnaJ protein, exhibited typical responses to heat and high-light stresses. Their localization and ability to prevent aggregation of luciferase were also diverse, suggesting a possible functional differentiation of these proteins. Since the expression of dnaJ1, which belongs to conserved type I DnaJ, down-regulated under heat stress, the unique structure of DnaJ2 may be involved in stress responses of S. elongatus. Based on phylogenetic analysis, the diverse dnaJ family was assumed to have evolved its own specific functions in each cyanobacterial species.     

Synechococcus elongatus PCC 7942 is more tolerant to chromate as compared to Synechocystis sp. PCC 6803

Two unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 showed contrasting responses to chromate stress with EC₅₀ of 12 ± 2 and 150 ± 15 μM potassium dichromate respectively. There was no depletion of chromate in growth medium in both the cases. Using labeled chromate, very low accumulation (<1 nmol/10⁸ cells) was observed in Synechocystis after incubation for 24 h in light. No accumulation of chromate could be observed in Synechococcus under these conditions. Chromate oxyanion is known to enter the cells using sulfate uptake channels. Therefore, inhibition of sulfate uptake caused by chromate was monitored using ³⁵S labeled sulfate. IC₅₀ values of chromate for ³⁵sulfate uptake were higher in Synechococcus as compared to Synechocystis. The results suggested that the sulfate transporters in Synechococcus have lower affinity to chromate than those from Synechocystis possibly due to differences in affinity of sulfate receptors for chromate. Bioinformatic analyses revealed presence of sulfate and chromate transporters with considerable similarity; however, minor differences in these may play a role in their differential response to chromate. In both cases the IC₅₀ values decreased when sulfate concentration was reduced in the medium indicating competitive inhibition of sulfate uptake by chromate. Interestingly, Synechococcus showed stimulation of growth at concentrations of chromate less than 100 μM, which affected its cell size without disturbing the ultrastructure and thylakoid organization. In Synechocystis, growth with 12 μM potassium dichromate damaged the ultrastructure and thylakoid organization with slight elongation of the cells. The results suggested that Synechococcus possesses efficient strategies to prevent entry and to remove chromate from the cell as compared to Synechocystis. This is the first time a differential response of Synechococcus 7942 and Synechocystis 6803 to chromate is reported. The contrasting characteristics observed in the two cyanobacteria will be useful in understanding the basis of resistance or susceptibility to chromate.     

sll1961 is a novel regulator of phycobilisome degradation during nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803

The sll1961 gene was reported to encode a regulatory factor of photosystem stoichiometry in the cyanobacterium Synechocystis sp. PCC 6803. We here show that the sll1961 gene is also essential for the phycobilisome degradation during nitrogen starvation. The defect in phycobilisome degradation was observed in the sll1961 mutant despite the increased expression of nblA, a gene involved in phycobilisome degradation during nitrogen starvation. Photosystem stoichiometry is not affected by nitrogen starvation in the sll1961 mutant nor in the wild-type. The results indicate the presence of a novel pathway for phycobilisome degradation control independent of nblA expression.     

Expression analysis of multiple dnaK genes in the cyanobacterium Synechococcus elongatus PCC 7942

We analyzed the stress responses of three dnaK homologues (dnaK1, dnaK2, and dnaK3) in the cyanobacterium Synechococcus elongatus PCC 7942. A reporter assay showed that under stress conditions the expression of only the dnaK2 gene was induced, suggesting a functional assignment of these homologues. RNA blot hybridization indicated a typical stress response of dnaK2 to heat and high-light stress. Primer extension mapping showed that dnaK2 was transcribed from similar sites under various stress conditions. Although no known sequence motif was detected in the upstream region, a 20-bp sequence element was highly conserved in dnaK2; it was essential not only for the stress induction but also for the basal expression of dnaK2. The ubiquitous upstream localization of this element in each heat shock gene suggests its important role in the cyanobacterial stress response.     

Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942

Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b₆f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.     

Observation of polyphosphate bodies and DNA during the cell division cycle of Synechococcus elongatus PCC 7942

Although most cyanobacterial cells contain prominent polyphosphate bodies in the central cytoplasmic area enclosed by the peripheral thylakoid membranes, their roles are not fully understood. Storing phosphate for nucleotide production might be one of their important roles in survival of the cells. As a step towards identifying a possible contribution of the polyphosphate bodies to DNA synthesis, the relationship between polyphosphate bodies and DNA throughout cell division cycle of Synechococcus elongatus PCC 7942 cells cultured under light/dark cycles was investigated with light and electron microscopy. During the dark period, the average size of polyphosphate bodies increased gradually without significant change in their number and distribution. However, during the light period, the number of polyphosphate bodies increased, while the size of each polyphosphate body decreased and cells elongated until the end of the light period, when most cells divided. The ratio of the content of polyphosphate bodies to cell length increased gradually during the dark period and decreased during the light period. Hoechst 33342‐stained DNA appeared diffuse during the dark period, but in the light period it became condensed and eventually formed a wavy, rope‐like structure prior to cell division. Close association between fibres containing DNA and polyphosphate bodies was demonstrated by TEM using DNA‐specific staining and BrdU labelling. These regular coordinated changes of polyphosphate bodies and DNA shape during the cell division cycle, together with their intimate interaction, imply a role of polyphosphate bodies in supplying material for DNA.     

The endogenous redox rhythm is controlled by a central circadian oscillator in cyanobacterium Synechococcus elongatus PCC7942

Photosynthesis Research - The intracellular redox and the circadian clock in photosynthetic organisms are two major regulators globally affecting various biological functions. Both of the global...