Cyanobacteria are a group of photosynthetic prokaryotes that have a diverse morphology, minimal nutritional requirements and metabolic plasticity that has made them attractive organisms to use in biotechnological applications. The use of these organisms as cell factories requires the knowledge of their physiology and metabolism at a systems level. For the quantification of gene transcripts real-time quantitative polymerase chain reaction (RT-qPCR) is the standard technique. However, to obtain reliable RT-qPCR results the use and validation of reference genes is mandatory. Towards this goal we have selected and analyzed twelve candidate reference genes from three morphologically distinct cyanobacteria grown under routinely used laboratory conditions. The six genes exhibiting less variation in each organism were evaluated in terms of their expression stability using geNorm, NormFinder and BestKeeper. In addition, the minimum number of reference genes required for normalization was determined. Based on the three algorithms, we provide a list of genes for cyanobacterial RT-qPCR data normalization. To our knowledge, this is the first work on the validation of reference genes for cyanobacteria constituting a valuable starting point for future works.

The taxonomic positions and phylogenetic relationships of two new methylotrophic isolates from Lake Washington (USA) sediment, FAM5^Tand 500, and the previously described methylotrophic strain EHg5 isolated from contaminated soil in Estarreja (Portugal) were investigated. All three strains were facultative methylotrophs capable of growth on a variety of C₁and multicarbon compounds. Optimal growth occurred at pH 7.5–8 and 30–37 °C. The major fatty acids were C_16 : 1ω7cand C_16 : 0. The major quinone was ubiquinone Q8. Neither methanol dehydrogenase nor methanol oxidase activities were detectable in cells grown on methanol, suggesting an alternative, as-yet unknown, mechanism for methanol oxidation. The isolates assimilated C₁units at the level of formaldehyde, via the serine cycle. The DNA G+C content of the strains ranged between 64 and 65 mol%. 16S rRNA gene sequence similarity between the three new isolates was 99.85–100 %, but was below 94 % with other members of theBetaproteobacteria, indicating that the isolates represent a novel taxon. Based on physiological, phenotypic and genomic characteristics of the three isolates, a new genus,Methyloversatilisgen. nov., is proposed within the familyRhodocyclaceae. The type strain ofMethyloversatilis universalisgen. nov., sp. nov. is FAM5^T(=CCUG 52030^T=JCM 13912^T).

The use of microorganisms as cell factories frequently requires extensive molecular manipulation. Therefore, the identification of genomic neutral sites for the stable integration of ectopic DNA is required to ensure a successful outcome. Here we describe the genome mapping and validation of five neutral sites in the chromosome of Synechocystis sp. PCC 6803, foreseeing the use of this cyanobacterium as a photoautotrophic chassis. To evaluate the neutrality of these loci, insertion/deletion mutants were produced, and to assess their functionality, a synthetic green fluorescent reporter module was introduced. The constructed integrative vectors include a BioBrick-compatible multiple cloning site insulated by transcription terminators, constituting robust cloning interfaces for synthetic biology approaches. Moreover, Synechocystis mutants (chassis) ready to receive purpose-built synthetic modules/circuits are also available. This work presents a systematic approach to map and validate chromosomal neutral sites in cyanobacteria, and that can be extended to other organisms.

Cyanobacteria are promising “low-cost” cell factories since they have minimal nutritional requirements, high metabolic plasticity, and can use sunlight and CO₂ as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the “green” E. coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the SEVA repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference P_rnpB, three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene (encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol) and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the glucosylglycerol in a ggpS deficient mutant validating the functionality of the tools/device developed in this study.