Untersuchungen zur Funktion multipler DnaJ-Proteine in dem Cyanobakterium Synechocystis sp. PCC 6803

Sowohl in Synechocystis sp. PCC 6803 als auch in anderen Cyanobakterien konnten multiple DnaJ-Proteine nachgewiesen werden, deren Funktion jedoch noch weitestgehend unverstanden ist. Im Rahmen dieser Arbeit wurden die Funktionen der multiplen DnaJ-Proteine von Synechocystis sp. charakterisiert. Das DnaJ-Protein, Sll0897 gehört aufgrund seiner Domänenstruktur zu den Typ I-Proteinen, Slr0093 und Sll1933 zu den Typ II-Proteinen und Sll0909, Sll1011, Sll1384 und Sll1666 zu den Typ III DnaJ-Proteinen. Durch Komplementationsstudien des E. coli ΔdnaJ-Stammes OD259 konnte eine Komplementation des Wachstumsdefekts bei höheren Temperaturen durch die Proteine Slr0093 und Sll0897 gezeigt werden. In Synechocystis war eine komplette Disruption von sll1933 nicht möglich, weshalb das Protein Sll1933 unter normalen Wachstumsbedingungen essentiell ist. Doppelte Insertionmutationen waren lediglich bei der Kombination der Gene sll0909 und sll1384 möglich. Untersuchungen des Wachstumsverhaltens der dnaJ-Disruptions-stämme unter Hitze- und Kältestressbedingungen zeigten, dass das Protein Sll0897 eine wichtige Funktion bei der Stressantwort in Synechocystis besitzt und unter Hitzestressbedingungen essentiell ist. Eine vollständige Deletion des Gens sll0897 war Synechocystis sp. bereits unter normalen Wachstumsbedingungen nicht möglich. Bei den für ein Wachstum mindestens notwendigen Domänen des Sll0897 handelt es sich um die charakteristische J-Domäne und die Glycin-Phenylalanin-reiche Domäne. Unter Hitzestressbedingungen ist das Volllängen-Protein Sll0897 für ein Wachstum essentiell. rnNeben den in vivo Wachstumsexperimenten wurde eine Methode zur heterologen Expression der sieben DnaJ-Proteine in E. coli und einer nativen Reinigung von Slr0093, Sll0897, Sll0909 und Sll1666 etabliert. Untersuchungen zur Thermostabilität der gereinigten Proteine zeigten für das Slr0093 und Sll1666 einen reversiblen Prozess, wodurch sie auch nach dem Hitzestress noch als Faltungshelfer fungieren können. Bei den Proteinen Sll0897 und Sll0909 ist der Prozess jedoch nicht reversibel, so dass sie nach Hitzestresseinwirkung neu synthetisiert oder durch Chaperoneinwirkung korrekt gefaltet werden müssen. Die Affinitäts-„Pull-Down“ Analysen lieferten keine klaren Hinweise auf die DnaK-Interaktionspartner der Proteine Slr0093, Sll0897, Sll0909 und Sll1666, weshalb weitere Untersuchungen notwendig sind. Mit Hilfe der Gelfiltrationsanalysen konnten die errechneten molaren Massen der Proteine Slr0093 und Sll1666 bestätigt und beide Proteine in einer monomeren Form nachgewiesen werden. Die DnaJ-Proteine Sll0897 und Sll0909 konnten in zwei oligomeren Zuständen detektiert werden. Analysen der ATPase-Aktivität des DnaK2-Proteins alleine und des DnaK2-Proteins zusammen mit den DnaJ-Proteinen Slr0093, Sll0897, Sll0909 und Sll1666 zeigten eine Steigerung der ATP-Hydrolyserate bei der Interaktion von DnaK und DnaJ, wobei Sll0897 die größte Steigerung der ATPase-Aktivität des DnaK2 induzierte.

Hsp70-Chaperone und ihre Rolle bei der Thylakoidmembranbiogenese im Cyanobakterium Synechocystis sp. PCC 6803

Im Genom des Cyanobakteriums Synechocystis sp. PCC6803 sind vier homologe Hsp70-Proteine kodiert. Im Rahmen dieser Arbeit konnten neue Erkenntnisse über die möglichen Funktionen der einzelnen Mitglieder der Hsp70-Proteinfamilie in dem Modellorganismus gewonnen bzw. bekannte Aufgabenbereiche erweitert werden. Wie für E. coli schon gezeigt, konnte auch für Synechocystis sp. nachgewiesen werden, dass eine Deletion des ribosomassoziierten Chaperons Trigger Factor ohne Beeinträchtigung der Zellviabilität möglich ist. Darüber hinaus war auch eine Doppeldeletion mit dnaK1 durchführbar. Als Auswirkung der Deletion ließ sich in den jeweiligen Deletionsstämmen eine veränderte Expression der homologen Hsp70-Proteine und Trigger Factor nachweisen. Mit Hilfe der Synechocystis sp.-Mutationsstämme ∆dnaK1, ∆dnaK2, ∆dnaK3, ∆tig und ∆dnaK1∆tig wurden Auswirkungen der Deletion bzw. Depletion umfassend dargestellt und daraus hervorgehende putative Funktionen eingehend diskutiert. Die Reduzierung der zellulären DnaK3-Konzentration um etwa 70 % führte im Depletionsstamm ΔdnaK3 zu weitreichenden physiologischen Änderungen hinsichtlich photosynthetischer Prozesse. Zusammen mit einer lichtabhängigen Expression, konnte DnaK3 als essentieller Faktor für die funktionelle Aufrechterhaltung der Thylakoidmembran identifiziert werden. Durch die Analyse des Proteoms und Lipidoms dunkeladaptierter Synechocystis sp.-Zellen konnte im Vergleich zu älteren Studien eine erheblich größere Anzahl von Proteinen detektiert und quantifiziert werden, womit neue Erkenntnisse über die physiologischen Veränderungen unter heterotrophem Wachstum sowie der Thylakoidmembranbiogenese gewonnen werden konnten.

A Novel Gene, pmgA, Specifically Regulates Photosystem Stoichiometry in the Cyanobacterium Synechocystis Species PCC 6803 in Response to High Light1

Previously, we identified a novel gene, pmgA, as an essential factor to support photomixotrophic growth of Synechocystis species PCC 6803 and reported that a strain in which pmgA was deleted grew better than the wild type under photoautotrophic conditions. To gain insight into the role of pmgA, we investigated the mutant phenotype of pmgA in detail. When low-light-grown (20 μE m−2 s−1) cells were transferred to high light (HL [200μE m−2 s−1]), pmgA mutants failed to respond in the manner typically associated with Synechocystis. Specifically, mutants lost their ability to suppress accumulation of chlorophyll and photosystem I and, consequently, could not modulate photosystem stoichiometry. These phenotypes seem to result in enhanced rates of photosynthesis and growth during short-term exposure to HL. Moreover, mixed-culture experiments clearly demonstrated that loss of pmgA function was selected against during longer-term exposure to HL, suggesting that pmgA is involved in acquisition of resistance to HL stress. Finally, early induction of pmgA expression detected by reverse transcriptase-PCR upon the shift to HL led us to conclude that pmgA is the first gene identified, to our knowledge, as a specific regulatory factor for HL acclimation.

Stepwise Photoinhibition of Photosystem II1 : Studies with Synechocystis Species PCC 6803 Mutants with a Modified D-E Loop of the Reaction Center Polypeptide D1

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the QB-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of QA reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified QB pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.

Light-induced expression of fatty acid desaturase genes

In cyanobacterial cells, fatty acid desaturation is one of the crucial steps in the acclimation processes to low-temperature conditions. The expression of all the four acyl lipid desaturase genes of Synechocystis PCC 6803 was studied as a function of temperature and separately as a function of light. We used cells grown at 25°C in light-activated heterotrophic growth conditions. In these cells, the production of α-linolenic acid and 18:4 fatty acids was negligible and the synthesis of γ-linolenic acid was remarkably suppressed compared with those of the cells grown photoautotrophically. The cells grown in the light in the presence of glucose showed no difference in fatty acid composition compared with cells grown photoautotrophically. The level of desC mRNA for Δ9 desaturase was not affected by either the temperature or the light. It was constitutively expressed at 25°C with and without illumination. The level of desB transcripts was negligible in the dark-grown cells and was enhanced about 10-fold by exposure of the cells to light. The maximum level of expression occurred within 15 min. The level of desA and desD mRNAs was higher in dark-grown cells than that of desB mRNA for ω3 desaturase. However, the induction of both desA and desD mRNAs for Δ12 and Δ6 desaturases, respectively, was enhanced by light about 10-fold. Rifampicin, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea completely blocked the induction of the expression of desA, desB, and desD. Consequently, we suggest the regulatory role of light via photosynthetic processes in the induction of the expression of acyl lipid desaturases.

硫代硫酸钠及葡萄糖对蓝细菌Synechocystis sP.PCC6803脂及光合器组成的影响

本文通过对蓝细菌Synechocystis sp. PCC 6803在添加葡萄糖、Na2S203的BG-11培养基中的生长特性、脂类及脂肪酸组成、细胞低温荧光、色素组成进行分析测定，总结出如下规律： 当蓝细菌Synechocystis sp. PCC 6803在添加有葡萄糖的BG-11培养基中培养时细胞出现了一种新的糖脂（记为糖脂-x），在添加果糖、麦芽糖、乳糖等其它碳源的培养基中生长的细胞中也检测到糖脂-x糖脂-x的出现经推测是与活性氧相作用的产物，当在含糖的培养基中加入活性氧猝灭剂Na2S203时能有效地抑制糖脂-x的出现。糖脂一x的出现伴随着其它脂、尤其是双半乳糖甘油二酯(DGDG)的含量下降，这可能与细胞营养代谢类型的转变相适应。糖脂-x的出现使细胞适应异养生长条件，这时藻胆体(PBS)，光系统II(PSII)，光系统I(PSD降解，叶绿素消失。 糖脂-x经1H-NMR波谱术检测证实为甘油糖脂，经气质联谱分析其脂肪酸组成中含大量的枝链脂肪酸，12-甲基十四碳酸、12-甲基十五碳酸、12-甲基十六碳酸以及两种稀有的含氮脂肪酸。这些脂肪酸在添加高浓度葡萄糖的培养基中生长的．Synechocystis sp. PCC 6803中的单半乳糖甘油二酯(MGDG)也能检测到。ESI-MS以及P-SI-MS测定结果表明糖脂．x含一分子的脂酰基侧链以及两分子的己糖，半乳糖与葡萄糖。 对．Synechocystis sp. PCC 6803生长在不同浓度的葡萄糖与Na2S203培养基中脂类组成与脂肪酸组成进行比较，发现Na2S203能有效地增加膜脂中硫代异鼠李糖二酰基甘油(SQDG)和磷脂酰甘油(PG)的百分含量，培养基中同时添加葡萄糖时能抵消Na2S203的这一效应。此外，Na2S203能显著增加单半乳糖甘油二酯(MGDG)、双半乳糖甘油二酯(DGDG)中十六碳酸(C16:0)的百分含量，这一效应也能为葡萄糖恢复。Na2S203不能显著地改变SQDG中C16:0的百分含量，加入葡萄糖时能降低C16:0的百分含量。这些结果说明Na2S203可能充当一种还原剂使膜脂处于一种低的不饱和状态，同时加入葡萄糖时能降低Na2S203的还原力。此外，Na2S203还可作为SQDG合成中的硫供体。 用HPLC测定．Synechocystis sp. PCC 6803在添加不同浓度的Na2S203，葡萄糖的BG-11培养基中生长时的叶绿素与类胡萝卜素浓度，结果表明葡萄糖表现出对叶绿素与类胡萝卜素水平的抑制效应，Na2S203在低浓度时表现出对叶绿素与类胡萝卜素水平的促进效应，但在高浓度时表现出抑制效应。因此适当浓度的Na2S203的加入有利于维持蓝细菌在培养基中添加葡萄糖的生长条件下的低水平自由基，能使葡萄糖表现出促进细胞生长的特性。 通过测定Synechocystis sp. PCC 6803生长曲线中葡萄糖、Na2S203的浓度效应，结果表明葡萄糖在低浓度（例如5 mmoI.L-l）时表现出促进细胞的生长，在相对高的浓度表现出抑制细胞生长的效应。在培养基中同时加入Na2S203时可恢复葡萄糖对细胞的生长的促进效应。单独加入Na2S203表现出对细胞生长的抑制效应。这说明葡萄糖、Na2S203对细胞的生长存在着正的协同效应。

Transcriptional and Posttranscriptional Control of mRNA from lrtA, a Light-Repressed Transcript in Synechococcus sp. PCC 70021

Transcription regulation and transcript stability of a light-repressed transcript, lrtA, from the cyanobacterium Synechococcus sp. PCC 7002 were studied using ribonuclease protection assays. The transcript for lrtA was not detected in continuously illuminated cells, yet transcript levels increased when cells were placed in the dark. A lag of 20 to 30 min was seen in the accumulation of this transcript after the cells were placed in the dark. Transcript synthesis continued in the dark for 3 h and the transcript levels remained elevated for at least 7 h. The addition of 10 μm rifampicin to illuminated cells before dark adaptation inhibited the transcription of lrtA in the dark. Upon the addition of rifampicin to 3-h dark-adapted cells, lrtA transcript levels remained constant for 30 min and persisted for 3 h. A 3-h half-life was estimated in the dark, whereas a 4-min half-life was observed in the light. Extensive secondary structure was predicted for this transcript within the 5′ untranslated region, which is also present in the 5′ untranslated region of lrtA from a different cyanobacterium, Synechocystis sp. PCC 6803. Evidence suggests that lrtA transcript stability is not the result of differences in ribonuclease activity from dark to light. Small amounts of lrtA transcript were detected in illuminated cells upon the addition of 25 μg mL−1 chloramphenicol. The addition of chloramphenicol to dark-adapted cells before illumination allowed detection of the lrtA transcript for longer times in the light relative to controls without chloramphenicol. These results suggest that lrtA mRNA processing in the light is different from that in the dark and that protein synthesis is required for light repression of the lrtA transcript.

固氮蓝藻与非固氮蓝藻谷氨酰胺酶的基因克隆、表达及其特性分析

蓝藻是唯一可以进行有氧光合作用的原核生物，是水生食物链主要的初级生产者。氮素是蓝藻细胞必需的大量营养元素之一，揭示蓝藻如何应对环境中氮素的变化、维持自身碳氮平衡的分子机理，对深刻理解蓝藻与环境的相互作用、有效促进或控制蓝藻的生长与繁殖，有重要的理论和实践意义。已有的研究发现，蓝藻细胞的碳氮平衡主要是通过调控氨同化途径中的关键酶类实现。但先前的研究主要集中在固氮蓝藻谷氨酰胺合成酶（GS）-谷氨酸合成酶(GOGAT)循环的特性分析方面，而对催化谷氨酰胺水解生成谷氨酸和氨的主要酶之一谷氨酰胺酶的报道极少，其分子特性及生理学意义尚不明了。因此，本论文以模式固氮蓝藻鱼腥藻7120 和非固氮蓝藻集胞藻6803 为材料，采用分子生物学和生物化学方法，对蓝藻谷氨酰胺酶进行体外研究，并对其生物学功能进行了初步探讨，获得了如下主要结果：1）对体外重组蛋白的酶活性检测发现，两类蓝藻基因组编码的假定性谷氨酰胺酶，均具有谷氨酰胺酶催化活性，表明基因组注释是准确的；2）固氮蓝藻重组酶（All2934、All4774）与非固氮蓝藻重组酶（Slr2079）酶学特征差异显著，具有不同的最适pH、温度及底物亲和力；3）固氮蓝藻重组酶All2934 催化活性受磷酸盐的激活，而非固氮蓝藻重组酶Slr2079 在高Na+浓度下活性更高；4）RT-PCR 分析结果表明，在正常培养条件下，两类蓝藻的谷氨酰胺酶基因在细胞内均有表达；5）在缺氮培养条件下，固氮蓝藻谷氨酰胺酶基因all2934 的表达水平发生明显变化，而all4774 保持相对稳定，表明前者可能在这类细胞应对氮饥饿过程中起重要作用；6）在正常培养条件下，非固氮蓝藻谷氨酰胺酶基因的缺失突变体(Δslr2079)与野生型表型相似，但在盐胁迫条件下，突变体生长速率及光合放氧活性均高于野生型，表明该基因可能在提高非固氮蓝藻细胞高盐耐受力方面起负调控作用。上述重要发现，不仅初步揭示了光合自氧生物谷氨酰胺酶体外重组酶的分子特征，也为进一步研究谷氨酰胺酶在蓝藻细胞内的专一性功能奠定了重要基础。

Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC alpha and beta subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

Physiologische Veränderungen bei der Thylakoidmembranbiogenese und die Funktionsweise des Nukleotidaustauschfaktors GrpE in Cyanobakterien

Bei Wachstum im Dunkeln zeigten sich rudimentäre Thylakoidstrukturen, wobei nach dem Transfer ins Licht ein vollständiges Thylakoidmembransystem erneut ausgebildet wurde. Parallel stieg, der Chlorophyllgehalt pro Zelle und das Verhältnis von Phycobilisomen zu Chlorophyll verschob sich erneut auf die Seite des Chlorophylls. Das bei Wachstum im Dunkeln als Monomer vorliegende PS II, war nicht funktional. Nach dem Transfer ins Licht, war nach etwa acht bis zwölf Stunden ein aktives PS II zu detektieren. Das PS I lag nach der Inkubation im Dunkeln, in geringerer Konzentration aber aktiv als Trimer in den Zellen vor.rnZwei Typ I Signalpeptidasen aus Synechocystis sp. PCC 6803 zeigten Unterschiede im Bezug auf ihre intrazelluläre Lokalisation. Für die Untersuchungen der Lokalisation konnte ein neues System der Fluoreszenzmikroskopie entwickelt und erfolgreich eingesetzt werden. Das LepB1 zeigte einen (auto-) proteolytischen Abbau. Für Untersuchungen zur katalytischen Aktivität wurden Vorläuferproteine als Substrate für LepB2 identifiziert.rnDie Funktionsweise der GrpE-Proteine aus verwandten Cyanobakterien zeigt Unterschiede. Bei beiden GrpE-Proteinen erfolgt der reversible Übergang von einem Dimer hin zu einem Monomer innerhalb eines physiologisch relevanten Temperaturbereichs in einem Schritt. Bei dem Protein aus Synechocystis sp. ist der N-Terminus und bei dem Protein aus dem thermophilen Bakterium Thermosynechococcus der C-Terminus für die Dimerisierung essentiell. rn

The heme oxygenase gene (pbsA) in the red alga Rhodella violacea is discontinuous and transcriptionally activated during iron limitation

Heme oxygenase (HO) catalyzes the opening of the heme ring with the release of iron in both plants and animals. In cyanobacteria, red algae, and cryptophyceae, HO is a key enzyme in the synthesis of the chromophoric part of the photosynthetic antennae. In an attempt to study the regulation of this key metabolic step, we cloned and sequenced the pbsA gene encoding this enzyme from the red alga Rhodella violacea. The gene is located on the chloroplast genome, split into three distant exons, and is presumably expressed by a trans-splicing mechanism. The deduced polypeptide sequence is homologous to other reported HOs from organisms containing phycobilisomes (Porphyra purpurea and Synechocystis sp. strain PCC 6803) and, to a lesser extent, to vertebrate enzymes. The expression is transcriptionally activated under iron deprivation, a stress condition frequently encountered by algae, suggesting a second role for HO as an iron-mobilizing agent in photosynthetic organisms.

Structural model of cytochrome b559 in photosystem II based on a mutant with genetically fused subunits

Photosystem II is a reaction center protein complex located in photosynthetic membranes of plants, algae, and cyanobacteria. Using light energy, photosystem II catalyzes the oxidation of water and the reduction of plastoquinone, resulting in the release of molecular oxygen. A key component of photosystem II is cytochrome b559, a membrane-embedded heme protein with an unknown function. The cytochrome is unusual in that a heme links two separate polypeptide subunits, α and β, either as a heterodimer (αβ) or as two homodimers (α2 and β2). To determine the structural organization of cytochrome b559 in the membrane, we used site-directed mutagenesis to fuse the coding regions of the two respective genes in the cyanobacterium Synechocystis sp. PCC 6803. In this construction, the C terminus of the α subunit (9 kDa) is attached to the N terminus of the β subunit (5 kDa) to form a 14-kDa αβ fusion protein that is predicted to have two membrane-spanning α-helices with antiparallel orientations. Cells containing the αβ fusion protein grow photoautotrophically and assemble functional photosystem II complexes. Optical spectroscopy shows that the αβ fusion protein binds heme and is incorporated into photosystem II. These data support a structural model of cytochrome b559 in which one heme is coordinated to an α2 homodimer and a second heme is coordinated to a β2 homodimer. In this model, each photosystem II complex contains two cytochrome b559 hemes, with the α2 heme located near the stromal side of the membrane and the β2 heme located near the lumenal side.

Immunocytochemical localization of acyl-lipid desaturases in cyanobacterial cells: evidence that both thylakoid membranes and cytoplasmic membranes are sites of lipid desaturation.

There are four acyl-lipid desaturases in the cyanobacterium Synechocystis sp. PCC 6803. Each of these desaturases introduces a double bond at a specific position, such as the Delta6, Delta9, Delta12, or omicron3 position, in C18 fatty acids. The localization of the desaturases in cyanobacterial cells was examined immunocytochemically with antibodies raised against synthetic oligopeptides that corresponded to the carboxyl-terminal regions of the desaturases. All four desaturases appeared to be located in the regions of both the cytoplasmic and the thylakoid membranes. These findings suggest that fatty acid desaturation of membrane lipids takes place in the thylakoid membranes as well as in the cytoplasmic membranes.