Differential requirement of two homologous proteins encoded by sll1214 and sll1874 for the reaction of Mg protoporphyrin monomethylester oxidative cyclase under aerobic and micro-oxic growth conditions

AbstractThe two open reading frames in the Synechocystis sp. PCC 6803 genome, sll1214 and sll1874, here designated cycI and cycII, respectively, encode similar proteins, which are involved in the Mg protoporphyrin monomethylester (MgProtoME) cyclase reaction. The impairment of tetrapyrrole biosynthesis was examined by separate inactivation of both cyclase encoding genes followed by analysis of chlorophyll contents, MgProtoME levels and several enzyme activities of tetrapyrrole biosynthesis. We additionally addressed the question, whether the two isoforms can complement cyclase deficiency under normal aerobic and micro-oxic growth conditions in light. A cycII knock-out mutant grew without any adverse symptoms at normal air conditions, but showed MgProtoME accumulation at growth under low oxygen conditions. A complete deletion of cycI failed in spite of mixotrophic growth and low light at both ambient and low oxygen, but resulted in accumulation of 150 and 28 times more MgProtoME, respectively, and circa 60% of the wild-type chlorophyll content. The CycI deficiency induced a feedback-controlled limitation of the metabolic flow in the tetrapyrrole biosynthetic pathway by reduced ALA synthesis and Fe chelatase activity. Ectopic expression of the CycI protein restored the wild-type phenotype in cycI− mutant cells under ambient air as well as micro-oxic growth conditions. Overexpressed CycII protein could not compensate for cycI− mutation under micro-oxic and aerobic growth conditions, but complemented the cycII knock-out mutant as indicated by wild-type MgProtoME and chlorophyll levels. Our findings indicate the essential contribution of CycI to the cyclase reaction at ambient and low oxygen conditions, while low oxygen conditions additionally require CycII for the cyclase activity

Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803

BACKGROUND: The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. RESULTS: The transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d(-1) and 0.0303% (v/v) ethanol d(-1) were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. CONCLUSIONS: Changes in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected

Small RNAs Establish Delays and Temporal Thresholds in Gene Expression

Non-coding RNAs are crucial regulators of gene expression in prokaryotes and eukaryotes, but it remains poorly understood how they affect the dynamics of transcriptional networks. We analyzed the temporal characteristics of the cyanobacterial iron stress response by mathematical modeling and quantitative experimental analyses, and focused on the role of a recently discovered small non-coding RNA, IsrR. We found that IsrR is responsible for a pronounced delay in the accumulation of isiA mRNA encoding the late-phase stress protein, IsiA, and that it ensures a rapid decline in isiA levels once external stress triggers are removed. These kinetic properties allow the system to selectively respond to sustained (as opposed to transient) stimuli, and thus establish a temporal threshold, which prevents energetically costly IsiA accumulation under short-term stress conditions. Biological information is frequently encoded in the quantitative aspects of intracellular signals (e.g., amplitude and duration). Our simulations reveal that competitive inhibition and regulated degradation allow intracellular regulatory networks to efficiently discriminate between transient and sustained inputs

Untersuchungen zu Funktion und Struktur des Regulatorproteins Hfq in Synechocystis sp. PCC 6803

Das phylogenetisch weit verbreitete RNA-bindende Protein Hfq ist an einer Vielzahl von Prozessen innerhalb des bakteriellen RNA-Metabolismus, insbesondere im Rahmen der post-transkriptionellen Genregulation durch kleine RNAs (sRNAs) beteiligt. Hfq-Proteine zählen zu der Familie der Sm- und Lsm-Proteine und zeichnen sich strukturell durch die funktionelle Ausbildung ringförmiger Homohexamere aus. Cyanobakterielle Orthologe zeigen gegenüber den gut untersuchten Hfq-Proteinen aus E. coli und anderen Proteobakterien eine schwache Sequenzkonservierung und bieten auch daher einen interessanten Ansatzpunkt für die Untersuchung riboregulatorischer Prozesse in diesen Organismen. In der vorliegenden Arbeit werden einleitende Untersuchungen zu Funktion und Struktur des orthologen Hfq-Proteins aus dem einzelligen Modell-Cyanobakterium Synechocystis sp. PCC 6803 vorgestellt. Die Inaktivierung des hfq-Gens (ssr3341) führte in diesem Organismus zum Verlust der phototaktischen Motilität. Mithilfe elektronenmikroskopischer Analysen konnte dieser Phänotyp auf das Fehlen von Typ IV Pili zurückgeführt werden. Microarray-Analysen wiesen in der deltahfq-Mutante für 31 Gene eine veränderte, in den meisten Fällen reduzierte Transkriptakkumulation nach. Am stärksten betroffenen waren Gene bzw. Operone, welche dem Regulon des cAMP-Rezeptorproteins Sycrp1 zugeordnet werden und zum Teil nachweislich an der Motilität von Synechocystis-Zellen beteiligt sind. Weitere vergleichende Expressionsanalysen identifizierten mithilfe eines speziellen Tiling-arrays ferner zwei „intergenisch“ kodierte potenzielle sRNAs, Hpr1 und Hpr3, deren Transkriptmengen signifikant von der hfq-Inaktivierung beeinflusst werden. Kristallstrukturdaten deuten zusammen mit den Ergebnissen aus in vitro-Bindungsstudien und genetischen Komplementierungsexperimenten - trotz starker Konservierung zentraler struktureller Charakteristika - neuartige biochemische und funktionelle Eigenschaften des Hfq-Proteins aus Synechocystis sp. PCC 6803 an. Funktionelle Implikationen werden im strukturellen und phylogenetischen Kontext diskutiert.The phylogenetically conserved RNA binding protein Hfq is a key player in bacterial RNA metabolism, particularly with regard to sRNA-mediated post-transcriptional gene regulation. Hfq proteins belong to the well-conserved family of Sm- and Lsm proteins and are characterized by the formation of homo-hexameric ring-shaped structures. In comparison with well-studied Hfq proteins from E.coli and other proteobacteria the cyanobacterial orthologues show rather poor sequence conservation. Therefore, they provide a quite interesting background for analyzing riboregulatory processes in these organisms. In this work, the orthologous Hfq protein from the unicellular model cyanobacterium Synechocystis sp. PCC 6803 has been initially characterized on the functional and structural level. Insertional inactivation of the hfq gene (ssr3341) led to a non-phototactic phenotype that was due to the loss of type IV pili on the cell surface, as demonstrated by electron microscopy. Microarray analyses revealed a set of 31 genes with altered transcript levels in the knock-out mutant. Among the most strongly affected genes, there were members of two operons that had previously been shown to be involved in motility, controlled by the cAMP receptor protein Sycrp1. Further comparative transcriptional analyses using custom tiling arrays revealed two putative sRNAs (Hpr1 and Hpr3) from intergenic regions, whose transcript levels appeared to be significantly affected by hfq-inactivation. Structural analyses, genetic complementation as well as RNA-binding studies in vitro indicate that the Hfq orthologue from Synechocystis sp. PCC 6803 exhibits novel biochemical and functional properties, though retaining general structural features of its proteobacterial counterparts. Functional implications are discussed with regard to structural und phylogenetic considerations

Towards controlled biosynthesis of plant triterpenes in cyanobacteria

A presentation I gave during my visit at the Terauchi lab at Ritsumeikan University, Japan, in June 2017

Synthetic RNA-based devices

Presentation I gave at the ESF conference 'Biology of Plastids - Towards a Blueprint for Synthetic Organelles' in 2014 in Pultusk, Poland. It was right after the kick-off of my participation in the EU-funded Ribonets project at the Institute for Synthetic Microbiology (@synmibi) in Düsseldorf. <div><br></div