Transcriptional Analysis of the Unicellular, Diazotrophic Cyanobacterium Cyanothece ATCC 51142 Grown Under Short Day/Night Cycles.

Cyanothece sp. strain ATCC 51142 is a unicellular, diazotrophic cyanobacterium that demonstrates extensive metabolic periodicities of photosynthesis, respiration, and nitrogen fixation when grown under N2-fixing conditions. We have performed a global transcription analysis of this organism using 6 h light:dark (L:D) cycles in order to determine the response of the cell to these conditions and to differentiate between diurnal and circadian-regulated genes. In addition, we used a context-likelihood of relatedness (CLR) analysis with these data and those from 2 d L:D and L:D plus continuous light experiments to better differentiate between diurnal and circadian-regulated genes. Cyanothece sp. acclimated in several ways to growth under short L:D conditions. Nitrogen was fixed in every second dark period and only once in each 24 h period. Nitrogen fixation was strongly correlated to the energy status of the cells and glycogen breakdown, and high respiration rates were necessary to provide appropriate energy and anoxic conditions for this process. We conclude that glycogen breakdown is a key regulatory step within these complex processes. Our results demonstrated that the main metabolic genes involved in photosynthesis, respiration, nitrogen fixation, and central carbohydrate metabolism have strong (or total) circadian-regulated components. The short L:D cycles enable us to identify transcriptional differences among the family of psbA genes, as well as the differing patterns of the hup genes, which follow the same pattern as nitrogenase genes, relative to the hoxgenes, which displayed a diurnal, dark-dependent gene expression

Generation of synthetic shuttle vectors enabling modular genetic engineering of cyanobacteria

Cyanobacteria have raised great interest in biotechnology due to their potential for a sustainable, photosynthesis-driven production of fuels and value-added chemicals. This has led to a concomitant development of molecular tools to engineer the metabolism of those organisms. In this regard, however, even cyanobacterial model strains lag behind compared to their heterotrophic counterparts. For instance, replicative shuttle vectors that allow gene transfer independent of recombination into host DNA are still scarce. Here, we introduce the pSOMA shuttle vector series comprising 10 synthetic plasmids for comprehensive genetic engineering of Synechocystis sp. PCC 6803. The series is based on the small endogenous plasmids pCA2.4 and pCB2.4, each combined with a replicon from Escherichia coli, different selection markers as well as features facilitating molecular cloning and the insulated introduction of gene expression cassettes. We made use of genes encoding green fluorescent protein (GFP) and a Baeyer–Villiger monooxygenase (BVMO) to demonstrate functional gene expression from the pSOMA plasmids in vivo. Moreover, we demonstrate the expression of distinct heterologous genes from individual plasmids maintained in the same strain and thereby confirmed compatibility between the two pSOMA subseries as well as with derivatives of the broad-host-range plasmid RSF1010. We also show that gene transfer into the filamentous model strain Anabaena sp. PCC 7120 is generally possible, which is encouraging to further explore the range of cyanobacterial host species that could be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector series displays an attractive alternative to existing plasmid series and thus meets the current demand for the introduction of complex genetic setups and to perform extensive metabolic engineering of cyanobacteria

Construction and evaluation of a whole genome microarray of Chlamydomonas reinhardtii

Toepel J, Albaum S, Arvidsson S, et al. Construction and evaluation of a whole genome microarray of Chlamydomonas reinhardtii. BMC Genomics. 2011;12(1): 579.ABSTRACT: BACKGROUND: Chlamydomonas reinhardtii is widely accepted as a model organism regarding photosynthesis, circadian rhythm, cell mobility, phototaxis, and biotechnology. The complete annotation of the genome allows transcriptomic studies, however a new microarray platform was needed. Based on the completed annotation of Chlamydomonas reinhardtii a new microarray on an Agilent platform was designed using an extended JGI 3.1 genome data set which included 15000 transcript models. RESULTS: In total 44000 probes were determined (3 independent probes per transcript model) covering 93% of the transcriptome. Alignment studies with the recently published AUGUSTUS 10.2 annotation confirmed 11000 transcript models resulting in a very good coverage of 70% of the transcriptome (17000). Following the estimation of 10000 predicted genes in Chlamydomonas reinhardtii our new microarray, nevertheless, covers the expected genome by 90-95%. CONCLUSIONS: To demonstrate the capabilities of the new microarray, we analyzed transcript levels for cultures grown under nitrogen as well as sulfate limitation, and compared the results with recently published microarray and RNA-seq data. We could thereby confirm previous results derived from data on nutrient-starvation induced gene expression of a group of genes related to protein transport and adaptation of the metabolism as well as genes related to efficient light harvesting, light energy distribution and photosynthetic electron transport

Differential Transcriptional Analysis of the Cyanobacterium Cyanothece sp. Strain ATCC 51142 during Light-Dark and Continuous-Light Growth▿ †

We analyzed the metabolic rhythms and differential gene expression in the unicellular, diazotrophic cyanobacterium Cyanothece sp. strain ATCC 51142 under N2-fixing conditions after a shift from normal 12-h light-12-h dark cycles to continuous light. We found that the mRNA levels of ∼10% of the genes in the genome demonstrated circadian behavior during growth in free-running (continuous light) conditions. The genes for N2 fixation displayed a strong circadian behavior, whereas photosynthesis and respiration genes were not as tightly regulated. One of our main objectives was to determine the strategies used by these cells to perform N2 fixation under normal day-night conditions, as well as under the greater stress caused by continuous light. We determined that N2 fixation cycled in continuous light but with a lower N2 fixation activity. Glycogen degradation, respiration, and photosynthesis were also lower; nonetheless, O2 evolution was about 50% of the normal peak. We also demonstrated that nifH (encoding the nitrogenase Fe protein), nifB, and nifX were strongly induced in continuous light; this is consistent with the role of these proteins during the assembly of the enzyme complex and suggested that the decreased N2 fixation activity was due to protein-level regulation or inhibition. Many soluble electron carriers (e.g., ferredoxins), as well as redox carriers (e.g., thioredoxin and glutathione), were strongly induced during N2 fixation in continuous light. We suggest that these carriers are required to enhance cyclic electron transport and phosphorylation for energy production and to maintain appropriate redox levels in the presence of elevated O2, respectively

A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus <i>Hormoconis resinae</i>

<p>A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus <i>Hormoconis resinae</i>, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify <i>H. resinae</i> in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of <i>H. resinae</i> in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms.</p