Bifunctional enzyme FBPase/SBPase is essential for photoautotrophic growth in cyanobacterium Synechocystis sp PCC 6803

From a random insertion mutant library of Synechocystis sp. PCC 6803, a mutant defective in photoautotrophic growth was obtained. The interrupted gene was identified to be slr2094 (rbpl), which encodes the fructose-1,6-biphosphatase (FBPase)/sedoheptulose-1,7-biphosphatase (SBPase) bifunctional enzyme (F-I). Two other independently constructed slr2094 mutants showed an identical phenotype. The FBPase activity was found to be virtually lacking in an slr2094 mutant, which was sensitive to light under mixotrophic growth conditions. These results indicate that slr2094 is the only active FBPase-encoding gene in this cyanobacterium. Inactivation of photosystem II by interrupting psbB in slr2094 mutant alleviated the sensitiveness to light. This report provides the direct genetic evidence for the essential role of F-I in the photosynthesis of Synechocystis sp. PCC 6803. (c) 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Identification of a gene, ccr-1 (sll1242), required for chill-light tolerance and growth at 15 degrees C in Synechocystis sp PCC 6803

Synechocystis sp. PCC 6803 exposed to chill (5 degrees C)-light (100 mu mol photons m(-2) s(-1)) stress loses its ability to reinitiate growth. From a random insertion mutant library of Synechocystis sp. PCC 6803, a sll1242 mutant showing increased sensitivity to chill plus light was isolated. Mutant reconstruction and complementation with the wild-type gene confirmed the role of sll1242 in maintaining chill-light tolerance. At 15 degrees C, the autotrophic and mixotrophic growth of the mutant were both inhibited, paralleled by decreased photosynthetic activity. The expression of sll1242 was upregulated in Synechocystis sp. PCC 6803 after transfer from 30 to 15 degrees C at a photosynthetic photon flux density of 30 mu mol photons m(-2) S-1. sll1242, named ccr (cyanobacterial cold resistance gene)- 1, may be required for cold acclimation of cyanobacteria in light.

Phycobilisome-Deficient Strains of Synechocystis sp. PCC 6803 Have Reduced Size and Require Carbon-Limiting Conditions to Exhibit Enhanced Productivity.

Reducing excessive light harvesting in photosynthetic organisms may increase biomass yields by limiting photoinhibition and increasing light penetration in dense cultures. The cyanobacterium Synechocystis sp. PCC 6803 harvests light via the phycobilisome, which consists of an allophycocyanin core and six radiating rods, each with three phycocyanin (PC) discs. Via targeted gene disruption and alterations to the promoter region, three mutants with two (pcpcT→C) and one (ΔCpcC1C2:pcpcT→C) PC discs per rod or lacking PC (olive) were generated. Photoinhibition and chlorophyll levels decreased upon phycobilisome reduction, although greater penetration of white light was observed only in the PC-deficient mutant. In all strains cultured at high cell densities, most light was absorbed by the first 2 cm of the culture. Photosynthesis and respiration rates were also reduced in the ΔCpcC1C2:pcpcT→C and olive mutants. Cell size was smaller in the pcpcT→C and olive strains. Growth and biomass accumulation were similar between the wild-type and pcpcT→C under a variety of conditions. Growth and biomass accumulation of the olive mutant were poorer in carbon-saturated cultures but improved in carbon-limited cultures at higher light intensities, as they did in the ΔCpcC1C2:pcpcT→C mutant. This study shows that one PC disc per rod is sufficient for maximal light harvesting and biomass accumulation, except under conditions of high light and carbon limitation, and two or more are sufficient for maximal oxygen evolution. To our knowledge, this study is the first to measure light penetration in bulk cultures of cyanobacteria and offers important insights into photobioreactor design.

The functional divergence of two glgP homologues in Synechocystis sp PCC 6803

Glycogen phosphorylase (GlgP, EC 2.4.1.1) catalyzes the cleavage of glycogen into glucose-1-phosphate (Glc-1-P), the first step in glycogen catabolism. Two glgP homologues are found in the genome of Synechocystis sp. PCC 6803, a unicellular cyanobacterium: sll1356 and slr1367. We report on the different functions of these glgP homologues. sll1356, rather than slr1367, is essential for growth at high temperatures. On the other hand, when CO2-fixation and the supply of glucose are both limited, slr1367 is the key factor in glycogen metabolism. In cells growing autotrophically, sll1356 plays a more important role in glycogen digestion than slr1367. This functional divergence is also supported by a phylogenetic analysis of glgP homologues in cyanobacteria.

A TPR-family membrane protein gene is required for light-activated heterotrophic growth of the cyanobacterium Synechocystis sp PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC6803 can grow heterotrophically in complete darkness, given that a brief period of illumination is supplemented every day (light-activated heterotrophic growth, LAHG), or under very weak ( < 0.5 mumol m(-2) s(-1)) but continuous light. By random insertion of the genome with an antibiotic resistance cassette, mutants defective in LAHG were generated. In two identical mutants, sll0886, a tetratricopeptide repeat (TPR)-family membrane protein gene, was disrupted. Targeted insertion of sll0886 and three downstream genes showed that the phenotype was not due to a polar effect. The sll0886 mutant shows normal photoheterotrophic growth when the light intensity is at 2.5 mumol m(-2) s(-1) or above, but no growth at 0.5 mumol m(-2) s(-1). Homologs to sll0886 are also present in cyanobacteria that are not known of LAHG. sll0886 and homologs may be involved in controlling different physiological processes that respond to light of low fluence. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Three-piece-ligation PCR and application in disruption of chlorophyll synthesis genes in Synechocystis sp PCC 6803

Linear DNA, consisting of a drug-resistance marker and long flanking sequences, was synthesized by one-step polymerase chain reaction after a three-piece ligating reaction. Chlorophyll synthesis genes, chlH and chIL in Synechocystis sp. PCC 6803, were replaced by a kanamycin-resistance marker through double recombinations with flanking homology regions. Under LAHG conditions, the chIL but not chlH mutant stopped chlorophyll synthesis, while both synthesized chlorophyll in the light.

Construction and analysis of a high-CO2-requiring mutant of the cyanobacterium Anabaena sp strain PCC7120

A mutant of Anabaena sp. strain PCC7120 requiring high CO2 was generated using Tn5 mutagenesis. This is the first data for a filamentous cyanobacterium. The mutant was capable of growing at 5% CO2, but incapable of growing at air levels of CO2. Southern hybridization analysis indicated that the Anabaena genome was inserted by the transposon at one site. The apparent photosynthetic affinity of the mutant to external dissolved inorganic carbon (DIC) was about 300 times lower that of the wild type (WT), and the medium alkalization rate as well as the carboxysomal carbonic anhydrase activity of the mutant was also lower than those of the WT. When the mutant was transferred from the culture medium bubbled with 5% CO2 to higher DIC (8.4% CO2) or 1% CO2, it showed similar responses to the WT. However, aberrant carboxysomes were found in the mutant cells through ultrastructural analysis, indicating it was most probably the wrong organization of the carboxysomes that eventually led to the inefficient operation of carboxysomal carbonic anhydrase and the subsequent defectiveness of the mutant in utilizing DIC.

Functional characterization of sll0659 from Synechocystis sp PCC 6803

Synchocystis sp. PCC 6803 lacks a gene for the any known types of lycopene cyclase. Recently, we reported that sll0659 (unknown for its function) from Synechocystis sp. PCC6803 shows similarity in sequence to a lycopene cyclase gene-CruA from Chlorobium tepidum. To test, whether Sll0659 encoded protein serves as lycopene cyclase, in this study, we investigated the carotenoids of the wild types ans mutants, In the sll0659 deleted mutant, there is no blockage at the lycopene cyclization step. Our results demonstrate that sll0659 does not affect lycopene cyclization. However, the ultrastructure of mutants suggests the involvement or necessity of sll0659 in the cell division.

Synechococcus sp. PCC 7002 CpcB lyase null mutations alter phycocyanin chromophore function and, consequently, affect the redistribution of excitation energy via the light state transition /

Phycobilisomes are the major light harvesting complexes for cyanobacteria and phycocyanin is the primary phycobiliprotein of the phycobilisome rod. The phycocyanobilin lyases responsible for chromophorylating the phycocyanin p subunit (CpcB) have been recently identified in the cyanobacterium Synechococcus sp. PCC 7002. Surprisingly, mutants missing the CpcB lyases were nevertheless capable of producing pigmented phycocyanin. 10K absorbance measurements revealed that the energy states of the p phycocyanin chromophores were only subtly shifted; however, 77K steady state fluorescence emission spectroscopy showed excitation energy transfer involving the targeted chromophores to be highly disrupted. Such evidence suggests that phycobilin orientation within the binding domain is specifically modified. We hypothesized that alternate, less specific lyases are able to act on the p binding sites. A phycocyanin linker-polypeptide deficient mutant was similarly characterized. The light state transition, a short term adaptation of the photosynthetic light harvesting apparatus resulting in the redistribution of excitation energy among the photo systems, was shown to be dominated by the reallocation of phycocyanin-absorbed excitation energy. Treatment with a high M phosphate buffer effectively prevented the redistribution of both chlorophyll a- and phycobilisome- absorbed excitation energy, suggesting that the two effects are not strictly independent. The mutant strains required a larger redistribution of excitation energy between light states, perhaps to compensate for their loss in phycobilisome antenna function.

Characterization of the phosphorylation of thylakoid membrane proteins from cyanobacterium synechococcus sp. PCC 6301 in light state 1 and light state 2

The distribution of excitation energy between the two photosystems (PSII and PSI) of photosynthesis is regulated by the light state transition. Three models have been proposed for the mechanism of the state transition in phycobilisome (PBS) containing organisms, two involving protein phosphorylation. A procedure for the rapid isolation of thylakoid membranes and PBS fractions from the cyanobacterium Synechococcus m. PCC 6301 in light state 1 and light state 2 was developed. The phosphorylation of thylakoid and soluble proteins rapidly isolated from intact cells in state 1 and state 2 was investigated. 77 K fluorescence emission spectra revealed that rapidly isolated thylakoid membranes retained the excitation energy distribution characteristic of intact cells in state 1 and state 2. Phosphoproteins were identified by gel electrophoresis of both thylakoid membrane and phycobilisome fractions isolated from cells labelled with 32p orthophosphate. The results showed very close phosphoprotein patterns for either thylakoid membrane or PBS fractions in state 1 and state 2. These results do not support proposed models for the state transition which required phosphorylation of PBS or thylakoid membrane proteins.

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.

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.

Abundancia y distribución de cianobacterias (Microcystis sp, Anabaena sp, Oscillatoria sp) en el Lago de Ilopango, El Salvador

Las cianobacterias, organismos procariotas fotosintéticos, pueden formar parte del fitoplancton de lagos y embalses y que en algunas ocasiones, si las condiciones de temperatura, pH y disponibilidad de luz son favorables y abundan los nutrientes, principalmente el fósforo y el nitrógeno, pueden dar lugar a proliferaciones (“blooms”). Los efectos negativos que se producen como consecuencia del crecimiento masivo de estas poblaciones, entre estas la producción de potentes toxinas por parte de algunas especies y teniendo en cuenta la extraordinaria importancia ecológica del lago de Ilopango, la realización de estudios que permitan conocer la abundancia, distribución y variación de cianobacterias potencialmente tóxicas (Anabaena sp, Oscillatoria sp y Microcystis sp), así como su relación con diversos parámetros físico-químicos como temperatura, pH, disponibilidad de luz, fósforo y nitrógeno, no sólo estaría plenamente justificada, sino que se revela como un instrumento totalmente imprescindible, debido a las diversas actividades que se desarrollan o están próximas a desarrollarse en el lago, entre estas la potabilización del agua. Con este fin se realizaron muestreos mensuales de cianobacterias (Anabaena sp, Microcystis sp y Oscillatoria sp), nitrógeno, fósforo, pH superficial, temperatura superficial del agua y penetración de la luz en los meses de noviembre de 2012 a febrero de 2013 a diferentes profundidades de la columna de agua en 7 puntos de muestreo previamente establecidos. Para determinar las diferencias significativas entre los puntos de muestreo y los meses muestreados se utilizó una Anova, asimismo se utilizó una correlación de Pearson para determinar la relación existente entre la abundancia de las cianobacterias y los parámetros muestreados. Los datos encontrados sugieren que la proliferación de cianobacterias en el lago de Ilopango es un fenómeno que no se presenta en época seca, probablemente por las bajas concentraciones de nitrógeno durante los meses muestreados, claramente relacionado con, la escasa precipitación que evita la llegada de nutrientes al lago por escorrentía; mientras que la influencia de los demás parámetros físico-químicos por sí solos, no constituyen un factor determinante en la proliferación de estos microorganismos.

Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions

Heterocyst differentiation in the filamentous cyanobacterium Anabaena PCC 7120 requires a functional hetR gene. Increased expression of the hetR gene is seen in developing and mature heterocysts in response to fixed nitrogen limitation. We mapped four likely transcriptional start sites for hetR and identified a specific transcript that is positively autoregulated. By using the copper-responsive petE promoter from Anabaena PCC 7120 to drive hetR expression, we show that ectopic expression of hetR increases heterocyst frequency and induces heterocyst differentiation under fully repressing conditions. Coexpression of a reporter gene shows that expression from the petE promoter is smoothly induced depending on the amount of copper supplied. In the heterocyst pattern mutant PatA, where terminally positioned heterocysts are formed almost exclusively, expression of the petE∷hetR fusion does not result in the formation of intercalary heterocysts. These results suggest that although the intracellular concentration of HetR has to be elevated for the differentiation decision, PatA plays a role as well. This role may be in the form of posttranslational modification of HetR, because PatA is a member of the response regulator family of proteins.