The ‘Standard European Vector Architecture’ database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

SignificanceCyanobacteria were responsible for the origin of oxygenic photosynthesis, and have since come to colonize almost every environment on Earth. Here we show that their ecological range is not limited by the presence of sunlight, but also extends down to the deep terrestrial biosphere. We report the presence of microbial communities dominated by cyanobacteria in the continental subsurface using microscopy, metagenomics, and antibody microarrays. These cyanobacteria were related to surface rock-dwelling lineages known for their high tolerance to environmental and nutritional stress. We discuss how these adaptations allow cyanobacteria to thrive in the dark underground, a lifestyle that might trace back to their nonphotosynthetic ancestors.

The genome of the soil bacterium Pseudomonas putida KT2440 encodes singular orthologues of genes crp (encoding the catabolite repression protein, Crp) and cyaA (adenylate cyclase) of Escherichia coli. The levels of cAMP formed by P. putida cells were below detection with a Dictyostelium biosensor in vivo. The cyaA(P. putida) gene was transcribed in vivo but failed to complement the lack of maltose consumption of a cyaA mutant of E. coli, thereby indicating that cyaA(P. putida) was poorly translated or rendered non-functional in the heterologous host. Yet, generation of cAMP by CyaA(P. putida) could be verified by expressing the cyaA(P. putida) gene in a hypersensitive E. coli strain. On the other hand, the crp(P. putida) gene restored the metabolic capacities of an equivalent crp mutant of E. coli, but not in a double crp/cyaA strain, suggesting that the ability to regulate such functions required cAMP. In order to clarify the breadth of the Crp/cAMP system in P. putida, crp and cyaA mutants were generated and passed through a battery of phenotypic tests for recognition of gross metabolic properties and stress-endurance abilities. These assays revealed that the loss of each gene led in most (but not all) cases to the same phenotypic behaviour, indicating a concerted functionality. Unexpectedly, none of the mutations affected the panel of carbon compounds that can be used by P. putida as growth substrates, the mutants being impaired only in the use of various dipeptides as N sources. Furthermore, the lack of crp or cyaA had little influence on the gross growth fingerprinting of the cells. The poor physiological profile of the Crp-cAMP system of P. putida when compared with E. coli exposes a case of regulatory exaptation, i.e. the process through which a property evolved for a particular function is co-opted for a new use.

In a given habitat, bacterial cells often experience recurrent exposures to the same environmental stimulus. The ability to memorize the past event and to adjust current behaviors can lead to efficient adaptation to the recurring stimulus. Here we demonstrate that the versatile bacterium Pseudomonas aeruginosa adopts a virulence phenotype after serial passage in the invertebrate model host Galleria mellonella. The virulence phenotype was not linked to the acquisition of genetic variations and was sustained for several generations, despite cultivation of the ex vivo virulence-adapted P. aeruginosa cells under rich medium conditions in vitro. Transcriptional reprogramming seemed to be induced by a host-specific food source, as reprogramming was also observed upon cultivation of P. aeruginosa in rich medium supplemented with polyunsaturated long-chain fatty acids. The establishment of induced memory responses adds a time dimension and seems to fill the gap between long-term evolutionary genotypic adaptation and short-term induced individual responses. Efforts to unravel the fundamental mechanisms that underlie the carry-over effect to induce such memory responses will continue to be of importance as hysteretic behavior can serve survival of bacterial populations in changing and challenging habitats.

Here we report the chemical and microbial characterization of the surface water of a CO2-rich hydrothermal vent known in Costa Rica as Borbollones, located at Tenorio Volcano National Park. The Borbollones showed a temperature surrounding 60 ºC, a pH of 2.4 and the gas released has a composition of ~97% CO2, ~0.07% H2S, ~2.3% N2 and ~0.12% CH4. Other chemical species such as sulfate and iron were found at high levels with respect to typical fresh water bodies. Analysis by 16S rRNA gene metabarcoding revealed that in Borbollones predominates an archaeon from the order Thermoplasmatales and one bacterium from the genus Sulfurimonas. Other sulfur-(genera Thiomonas, Acidithiobacillus, Sulfuriferula and Sulfuricurvum) and ironoxidizing bacteria (genera Sideroxydans, Gallionella, Ferrovum) were identified. Our results show that CO2-influenced surface water of Borbollones contain microorganisms that are usually found in acid rock drainage environments or sulfur-rich hydrothermal vents. To our knowledge, this is the first microbiological characterization of a CO2-dominated hydrothermal spring from Central America and expands our understanding of those extreme ecosystems. _____________________________________________________________________________ Compliance with ethical standards Conflict of interest. The authors declare that there are no conflicts of interest. Ethical approval. This study does not describe any experimental work related to human.

Summary The ability of Pseudomonas species to thrive in all major natural environments (i.e. terrestrial, freshwater and marine) is based on its exceptional capability to adapt to physicochemical changes. Thus, environmental bacteria have to tightly control the maintenance of numerous physiological traits across different conditions. The intracellular pH (pHi) homoeostasis is a particularly important feature, since the pHi influences a large portion of the biochemical processes in the cell. Despite its importance, relatively few reliable, easy‐to‐implement tools have been designed for quantifying in vivo pHi changes in Gram‐negative bacteria with minimal manipulations. Here we describe a convenient, non‐invasive protocol for the quantification of the pHi in bacteria, which is based on the ratiometric fluorescent indicator protein PHP (pH indicator for Pseudomonas). The DNA sequence encoding PHP was thoroughly adapted to guarantee optimal transcription and translation of the indicator in Pseudomonas species. Our PHP‐based quantification method demonstrated that pHi is tightly regulated over a narrow range of pH values not only in Pseudomonas, but also in other Gram‐negative bacterial species such as Escherichia coli. The maintenance of the cytoplasmic pH homoeostasis in vivo could also be observed upon internal (e.g. redirection of glucose consumption pathways in P. putida) and external (e.g. antibiotic exposure in P. aeruginosa) perturbations, and the PHP indicator was also used to follow dynamic changes in the pHi upon external pH shifts. In summary, our work describes a reliable method for measuring pHi in Pseudomonas, allowing for the detailed investigation of bacterial pHi homoeostasis and its regulation.

Although the genome of Pseudomonas putida KT2440 encodes an orthologue of the crp gene of Escherichia coli (encoding the cAMP receptor protein), the regulatory scope of this factor seems to be predominantly co-opted in this bacterium for controlling non-metabolic functions. In order to investigate the reasons for such a functional divergence in otherwise nearly identical proteins, the Crp regulator of P. putida (Crp(P. putida)) was purified to apparent homogeneity and subject to a battery of in vitro assays aimed at determining its principal physicochemical properties. Analytical ultracentrifugation indicated effector-free Crp(P. putida) to be a dimer in solution that undergoes a significant change in its hydrodynamic shape in the presence of cAMP. Such a conformational transition was confirmed by limited proteolysis of the protein in the absence or presence of the inducer. Thermodynamic parameters calculated by isothermal titration calorimetry revealed a tight cAMP-Crp(P. putida) association with an apparent K(D) of 22.5 ± 2.8 nM, i.e. much greater affinity than that reported for the E. coli's counterpart. The regulator also bound cGMP, but with a K(D) = 2.6 ± 0.3 µM. An in vitro transcription system was then set up with purified P. putida's RNA polymerase for examining the preservation of the correct protein-protein architecture that makes Crp to activate target promoters. These results, along with cognate gel retardation assays indicated that all basic features of the reference Crp(E. coli) protein are kept in the P. putida's counterpart, albeit operating under a different set of parameters, the extraordinarily high affinity for cAMP being the most noticeable.

Whether the extreme conditions of acidity and heavy metal pollution of streams and rivers originating in pyritic formations are caused primarily by mining activities or by natural activities of metal-oxidizing microbes living within the geological formations is a subject of considerable controversy. Most microbiological studies of such waters have so far focused on acid mine drainage sites, which are heavily human-impacted environments, so it has been problematic to eliminate the human factor in the question of the origin of the key metal compounds. We have studied the physico-chemistry and microbiology of the Río Sucio in the Braulio Carrillo National Park of Costa Rica, 22 km from its volcanic rock origin. Neither the remote origin, nor the length of the river to the sampling site, have experienced human activity and are thus pristine. The river water had a characteristic brownish-yellow color due to high iron-dominated minerals, was slightly acidic, and rich in chemolithoautotrophic iron- and sulfur-oxidizing bacteria, dominated by Gallionella spp. Río Sucio is thus a natural acid-rock drainage system whose metal-containing components are derived primarily from microbial activities.