Qingfang He

The High Light-inducible Polypeptides inSynechocystis PCC6803 EXPRESSION AND FUNCTION IN HIGH LIGHT

There are five Synechocystis PCC6803 genes encoding polypeptides with similarity to the Lhc polypeptides of plants. Four of the polypeptides, designated HliA–D (Dolganov, N. A. M., Bhaya, D., and Grossman, A. R. (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 636–640) (corresponding to ScpC, ScpD, ScpB, and ScpE in Funk, C., and Vermaas, W. (1999) Biochemistry 38, 9397–9404) contain a single transmembrane domain. The fifth polypeptide (HemH) represents a fusion between a ferrochelatase and an Hli-like polypeptide. By using an epitope tag to identify specifically the different Hli polypeptides, the accumulation of each (excluding HemH) was examined under various environmental conditions. The levels of all of the Hli polypeptides were elevated in high light and during nitrogen limitation, whereas HliA, HliB, and HliC also accumulated to high levels following exposure to sulfur deprivation and low temperature. The temporal pattern of accumulation was significantly different among the different Hli polypeptides. HliC rapidly accumulated in high light, and its level remained high for at least 24 h. HliA and HliB also accumulated rapidly, but their levels began to decline 9–12 h following the imposition of high light. HliD was transiently expressed in high light and was not detected 24 h after the initiation of high light exposure. These results demonstrate that there is specificity to the accumulation of the Hli polypeptides under a diverse range of environmental conditions. Furthermore, mutants for the individual and combinations of the hli genes were evaluated for their fitness to grow in high light. Although all of the mutants grew as fast as wild-type cells in low light, strains inactivated forhliA or hliC/hliD were unable to compete with wild-type cells during co-cultivation in high light. A mutant lacking all four hli genes gradually lost its photosynthesis capacity and died in high light. Hence, the Hli polypeptides are critical for survival when Synechocystis PCC6803 is absorbing excess excitation energy and may allow the cells to cope more effectively with the production of reactive oxygen species.

Tracking the light environment by cyanobacteria and the dynamic nature of light harvesting.

In nature photosynthetic organisms cope with fluctuating light conditions. Light intensity and quality vary dramatically during the day or from one habitat to another. Photosynthetic organisms sense intensities and wavelengths of light both directly and indirectly. Because light fuels photosynthetic electron transport and CO2 fixation, it is the primary determinant of levels of NADP/ NADPH, ATP, and carbon metabolites, all of which can serve to modulate cellular processes. Light is also absorbed by photoreceptors that link light cues to cellular metabolism. However, light represents a single environmental cue, and other signals interact with light through a web of regulatory circuits that result in dynamic acclimatory responses. This review focuses on two specific aspects of light-influenced processes in Cyanobacteria; both concern changes in light harvesting structure and biosynthesis. The first part of this review discusses effects of changing wavelengths of light on the biosynthesis of the phycobilisomes (PBS), dominant light harvesting complexes of Cyanobacteria. The other discusses how Cyanobacteria tune light harvesting and photosynthetic function to both light intensity and nutrient availability and how the two responses are integrated.

Elimination of high-light-inducible polypeptides related to eukaryotic chlorophyll a/b-binding proteins results in aberrant photoacclimation in Synechocystis PCC6803.

The hli genes, present in cyanobacteria, algae and vascular plants, encode small proteins [high-light-inducible polypeptides (HLIPs)] with a single membrane-spanning alpha-helix related to the first and third helices of eukaryotic chlorophyll a/b-binding proteins. The HLIPs are present in low amounts in low light and they accumulate transiently at high light intensities. We are investigating the function of those polypeptides in a Synechocystis PCC6803 mutant lacking four of the five hli genes. Growth of the quadruple hli mutant was adversely affected by high light intensities. The most striking effect of the quadruple hli mutation was an alteration of cell pigmentation. Pigment changes associated with cell acclimation to increasing light intensity [i.e. decrease in light-harvesting pigments, accumulation of the carotenoid myxoxanthophyll and decrease in photosystem I (PSI)-associated chlorophylls] were strongly exacerbated in the quadruple hli mutant, resulting in yellowish cultures that bleached in high light and died as light intensities exceeded (>500 micromol photon m(-2) s(-1)). However, these pigment changes were not associated with an inhibition of photosynthesis, as probed by in vivo chlorophyll fluorescence, photoacoustic and O(2)-evolution measurements. On the contrary, the HLIP deficiency was accompanied by a stimulation of the photochemical activity, especially in high-light-grown cells. Western blot analyses revealed that the PSI reaction center level (PsaA/B) was noticeably reduced in the quadruple hli mutant relative to the wild type, whereas the abundance of the PSII reaction center protein D1 was comparatively little affected. The hli mutations did not enhance photoinhibition and photooxidation when cells were exposed over a short term to a very high light intensity. Together, the results of this study indicate that HLIPs are critical in the adaptation of the cyanobacterium to variations in light intensity. The data are consistent with the idea that HLIPs are involved, through a direct or indirect means, in nonphotochemical dissipation of absorbed light energy.

Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways

We have deleted a gene for a sensor histidine kinase, dspA (or hik33), in the cyanobacterium Synechocystis sp. strain PCC6803. In low and moderate light, the mutant grew slowly under photoautotrophic conditions, with a doubling time of approximately 40 h, and had severely reduced photosynthetic oxygen evolution. When the mutant was maintained in low or moderate light in the presence of glucose, its growth rate was only somewhat lower than that of wild-type cells. However, the mutant was light sensitive and rapidly died in high light. Furthermore, levels of many transcripts encoding genes associated with photosynthesis were altered in the mutant relative to wild-type Synechocystis sp. strain PCC6803 both in low light and following exposure to high light. There was constitutive expression of several high-light-inducible genes, including hli, psbAIII, and gpx2; there was little increased accumulation of sodB mRNA in high light; and the cells failed to accumulate cpcBA and psaAB mRNAs in low light in the presence of glucose, although a normal decline in the levels of these mRNAs was observed during exposure to high light. These results suggest that DspA is involved in controlling sets of photosynthetic and high-light-responsive genes, either directly or indirectly. These and other results, some of which are presented in a companion paper (C.-J. Tu, J. Shrager, R. Burnap, B. L. Postier, and A. R. Grossman, J. Bacteriol. 186:3889-3902, 2004), suggest that DspA acts as a global regulator that helps coordinate cellular metabolism with growth limitations imposed by environmental conditions.

Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p-coumaric acid

Significance The photosynthetic cyanobacteria are promising candidates for the sustainable production of a plethora of plant secondary metabolites, which are difficult to produce and purify in other systems. Many secondary metabolites are beneficial to human health. For instance, the phenylpropanoids, which are derived from p-coumaric acid, have anticancer, antiviral, and anti-inflammatory properties. Here, we constructed a strain of cyanobacterium Synechocystis 6803 that heterologously expressed a foreign gene encoding a tyrosine ammonia lyase, which converts tyrosine into p-coumaric acid and lacked a native laccase that degrades p-coumaric acid. The strain secreted ∼82.6 mg/L p-coumaric acid, which was readily extracted and purified from the culture medium. We thus show that cyanobacteria may indeed be used to sustainably produce plant secondary metabolites. p-Coumaric acid is the precursor of phenylpropanoids, which are plant secondary metabolites that are beneficial to human health. Tyrosine ammonia lyase catalyzes the production of p-coumaric acid from tyrosine. Because of their photosynthetic ability and biosynthetic versatility, cyanobacteria are promising candidates for the production of certain plant metabolites, including phenylpropanoids. Here, we produced p-coumaric acid in a strain of transgenic cyanobacterium Synechocystis sp. Pasteur Culture Collection 6803 (hereafter Synechocystis 6803). Whereas a strain of Synechocystis 6803 genetically engineered to express sam8, a tyrosine ammonia lyase gene from the actinomycete Saccharothrix espanaensis, accumulated little or no p-coumaric acid, a strain that both expressed sam8 and lacked slr1573, a native hypothetical gene shown here to encode a laccase that oxidizes polyphenols, produced ∼82.6 mg/L p-coumaric acid, which was readily purified from the growth medium.

Suppression of the Lethality of High Light to a Quadruple HLI Mutant by the Inactivation of the Regulatory Protein PfsR in Synechocystis PCC 6803

A regulatory gene, designated pfsR (photosynthesis, Fe homeostasis and stress-response regulator), was discovered by a genetic screen in Synechocystis PCC 6803. Deletion of the gene from a high light-sensitive strain lacking four hli genes (4Xhli) restored viability to the parental strain under high light conditions. The quintuple mutant pfsR-/4Xhli retained photosystem-II and oxygen evolution capacity at levels similar to the wild-type levels under high light conditions. The transcripts of the two bfr genes (encoding bacterioferritin) were found to be constitutively up-regulated, whereas the transcripts of ho1 gene (encoding a heme oxygenase) were greatly down-regulated in high light upon deletion of pfsR. Under intermediate high intensity light, the pfsR deletion strains accumulated carotenoids and chlorophyll a to a significantly higher level than their corresponding parental strains. An exacerbated, transient increase in oxygen evolution during the early hours of high light acclimation and a somewhat increased steady-state level of photosystem-II-mediated oxygen evolution observed in the 4Xhli strain were brought back to the wild-type levels upon deletion of pfsR from the strain. The pfsR deletion mutants were found to be less sensitive to iron limitation under low light conditions and to suffer less lipid peroxidation following exposure to high light. Therefore, inactivation of PfsR resulted in tighter control of iron availability, which in turn reduced oxidative stress during photosynthesis in high light. These studies have revealed a critical role of PfsR in regulation of iron homeostasis and stress response.

IsiA Is Required for the Formation of Photosystem I Supercomplexes and for Efficient State Transition in Synechocystis PCC 6803

Iron deficiency and other stress conditions strongly impact photosynthetic apparatus in photosynthetic organisms. Two novel chlorophyll (Chl)-containing supercomplexes (F4 and F5) in addition to the photosystem (PS) I trimers (F3) were observed by sucrose gradient ultracentrifugation in Synechocystis PCC 6803 under extensive iron starvation. 77K fluorescence and Western blot analyses of these supercomplexes revealed that they all contained IsiA. The F4 was identified as an IsiA-PSI-PSII supercomplex, while the F5 was assigned as an IsiA-PSI supercomplex. Deletion of isiA resulted in diminishing the PSI trimers (including the PSI trimers in iron-replete cells) and the two novel PSI supercomplexes (F4 and F5), and a significant reduction in the saturated whole-chain electron transport rate. However, the maximum PSII activities remained at levels similar to those of the wild type under various light conditions. The isiA- mutant was defective in state transition and sensitive to high light. The sensitivity of the isiA- mutant to high light was correlated with a higher level of membrane peroxidation. These results demonstrated that IsiA is required for the formation of PSI trimers and other higher complexes, and that IsiA is critical for efficient state transition.

RpaA Regulates the Accumulation of Monomeric Photosystem I and PsbA under High Light Conditions in Synechocystis sp. PCC 6803

The response regulator RpaA was examined by targeted mutagenesis under high light conditions in Synechocystis sp. PCC 6803. A significant reduction in chlorophyll fluorescence from photosystem I at 77 K was observed in the RpaA mutant cells under high light conditions. Interestingly, the chlorophyll fluorescence emission from the photosystem I trimers at 77 K are similar to that of the wild type, while the chlorophyll fluorescence from the photosystem I monomers was at a much lower level in the mutant than in the wild type under high light conditions. The RpaA inactivation resulted in a dramatic reduction in the monomeric photosystem I and the D1 protein but not the CP47 content. However, there is no significant difference in the transcript levels of psaA or psbA or other genes examined, most of which are involved in photosynthesis, pigment biosynthesis, or stress responses. Under high light conditions, the growth of the mutant was affected, and both the chlorophyll content and the whole-chain oxygen evolution capability of the mutant were found to be significantly lower than those of the wild type, respectively. We propose that RpaA regulates the accumulation of the monomeric photosystem I and the D1 protein under high light conditions. This is the first report demonstrating that inactivation of a stress response regulator has specifically reduced the monomeric photosystem I. It suggests that PS I monomers and PS I trimers can be regulated independently for acclimation of cells to high light stress.

Cyanobacteria as Cell Factories to Produce Plant Secondary Metabolites

Cyanobacteria represent a promising platform for the production of plant secondary metabolites. Their capacity to express plant P450 proteins, which have essential functions in the biosynthesis of many plant secondary metabolites, makes cyanobacteria ideal for this purpose, and their photosynthetic capability allows cyanobacteria to grow with simple nutrient inputs. This review summarizes the advantages of using cyanobacteria to transgenically produce plant secondary metabolites. Some techniques to improve heterologous gene expression in cyanobacteria are discussed.

PfsR Is a Key Regulator of Iron Homeostasis in Synechocystis PCC 6803

Iron is an essential cofactor in numerous cellular processes. The iron deficiency in the oceans affects the primary productivity of phytoplankton including cyanobacteria. In this study, we examined the function of PfsR, a TetR family transcriptional regulator, in iron homeostasis of the cyanobacterium Synechocystis PCC 6803. Compared with the wild type, the pfsR deletion mutant displayed stronger tolerance to iron limitation and accumulated significantly more chlorophyll a, carotenoid, and phycocyanin under iron-limiting conditions. The mutant also maintained more photosystem I and photosystem II complexes than the wild type after iron deprivation. In addition, the activities of photosystem I and photosystem II were much higher in pfsR deletion mutant than in wild-type cells under iron-limiting conditions. The transcripts of pfsR were enhanced by iron limitation and inactivation of the gene affected pronouncedly expression of fut genes (encoding a ferric iron transporter), feoB (encoding a ferrous iron transporter), bfr genes (encoding bacterioferritins), ho genes (encoding heme oxygenases), isiA (encoding a chlorophyll-binding protein), and furA (encoding a ferric uptake regulator). The iron quota in pfsR deletion mutant cells was higher than in wild-type cells both before and after exposure to iron limitation. Electrophoretic mobility shift assays showed that PfsR bound to its own promoter and thereby auto-regulated its own expression. These data suggest that PfsR is a critical regulator of iron homeostasis.