Choosing a healthy and sustainable diet: A three-level approach for understanding the drivers of the Italians' dietary regime over time.

Dietary patterns play key roles in health promotion and in preserving the environment. A growing number of studies show the importance of individual factors on food consumption choices, such as socio-economic status, lifestyle variables and contextual and social factors that characterize the geographical area in which individuals reside. The Mediterranean Diet is a sustainable diet that respects the environment, thus reducing per capita emissions from food production in respect to less sustainable diet. The aim of this paper is to determine the Italians' prevailing food patterns using a composite indicator and to identify which factors determine a higher adherence to the Mediterranean Diet in Italy. By using 15 waves of the ISTAT "Aspect of Daily Life" survey, we constructed an original data set and referred to the multilevel approach which enabled us to distinguish between temporal and cross-sectional effects thus providing valuable insights to policy makers and stakeholders in order to promote the Mediterranean Diet and reap environmental and public health. The results show that education plays an important role in determining food consumption behavior while the tendency to practice sports on a regular basis and to have breakfast and lunch at home positively influence people's adherence to this diet.

Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes.

Bacterial biofilms outperform planktonic counterparts in whole-cell biocatalysis. The transition between planktonic and biofilm lifestyles of the platform strain Pseudomonas putida KT2440 is ruled by a regulatory network controlling the levels of the trigger signal cyclic di-GMP (c-di-GMP). This circumstance was exploited for designing a genetic device that over-runs the synthesis or degradation of c-di-GMP--thus making P. putida to form biofilms at user's will. For this purpose, the transcription of either yedQ (diguanylate cyclase) or yhjH (c-di-GMP phoshodiesterase) from Escherichia coli was artificially placed under the tight control of a cyclohexanone-responsive expression system. The resulting strain was subsequently endowed with a synthetic operon and tested for 1-chlorobutane biodegradation. Upon addition of cyclohexanone to the culture medium, the thereby designed P. putida cells formed biofilms displaying high dehalogenase activity. These results show that the morphologies and physical forms of whole-cell biocatalysts can be genetically programmed while purposely designing their biochemical activity.

The private life of environmental bacteria: pollutant biodegradation at the single cell level.

Bacteria display considerable cell-to-cell heterogeneity in a number of genetic and physiological traits. Stochastic differences in regulatory patterns (e.g. at the transcriptional level) propagate into the metabolic and physiological status of otherwise isogenic cells, which ultimately results in appearance of sub-populations within the community. As new technologies emerge and because novel single cell strategies are constantly being refined, our knowledge on microbial individuality is in burgeoning and constant expansion. These approaches encompass not only molecular biology tools (e.g. fluorescent-protein based reporters) but also a suite of sophisticated, non-invasive technologies to gain insight into the metabolic state of individual cells. Defining the role of individual heterogeneities is thus instrumental for the population-level understanding of macroscopic processes in both environmental and industrial set-ups. The present article reviews the state-of-the-art methodologies for the investigation of single bacteria at both the genetic and metabolic level, and places the application of currently available tools in the context of microbial ecology and environmental microbiology. As a case example, we examine the stochastic and multi-stable behaviour of the TOL-encoded pathway of Pseudomonas putida mt-2 for the biodegradation of aromatic compounds. Bet-hedging strategies and division of labour are considered as factors pushing forward the evolution of environmental microorganisms.

Standardization of regulatory nodes for engineering heterologous gene expression: a feasibility study.

The potential of LacI/Ptrc , XylS/Pm , AlkS/PalkB , CprK/PDB3 and ChnR/PchnB regulatory nodes, recruited from both Gram-negative and Gram-positive bacteria, as the source of parts for formatting expression cargoes following the Standard European Vector Architecture (SEVA) has been examined. The five expression devices, which cover most known regulatory configurations in bacteria were assembled within exactly the same plasmid backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance was then analysed in an Escherichia coli strain of reference through the readout of a fluorescence reporter gene that contained strictly identical translation signal elements. This approach allowed us to describe and compare the cognate expression systems with quantitative detail. The constructs under scrutiny diverged considerably in their capacity, expression noise, inducibility and ON/OFF ratios. Inspection of such a variance exposed the different constraints that rule the optimal arrangement of functional DNA segments in each case. The data highlighted also the ease of standardizing inducer-responsive devices subject to transcriptional activation as compared to counterparts based on repressors. The study resulted in a defined collection of formatted expression cargoes lacking any cross talk while offering a panoply of choices to potential users and help interoperability of the specific constructs.

Deconvolution of Gene Expression Noise into Spatial Dynamics of Transcription Factor-Promoter Interplay.

Gene expression noise is not only the mere consequence of stochasticity, but also a signal that reflects the upstream physical dynamics of the cognate molecular machinery. Soil bacteria facing recalcitrant pollutants exploit noise of catabolic promoters to deploy beneficial phenotypes such as metabolic bet-hedging and/or division of biochemical labor. Although the role of upstream promoter-regulator interplay in the origin of this noise is little understood, its specifications are probably ciphered in flow cytometry data patterns. We studied Pm promoter activity of the environmental bacterium Pseudomonas putida and its cognate regulator XylS by following expression of Pm-gfp fusions in single cells. Using mathematical modeling and computational simulations, we determined the kinetic properties of the system and used them as a baseline code to interpret promoter activity in terms of upstream regulator dynamics. Transcriptional noise was predicted to depend on the intracellular physical distance between regulator source (where XylS is produced) and the target promoter. Experiments with engineered bacteria in which this distance is minimized or enlarged confirmed the predicted effects of source/target proximity on noise patterns. This approach allowed deconvolution of cytometry data into mechanistic information on gene expression flow. It also provided a basis for selecting programmable noise levels in synthetic regulatory circuits.

Data on the standardization of a cyclohexanone-responsive expression system for Gram-negative bacteria.

Engineering of robust microbial cell factories requires the use of dedicated genetic tools somewhat different from those traditionally used for laboratory-adapted microorganisms. We have edited and formatted the ChnR/P chnB regulatory node of Acinetobacter johnsonii to ease the targeted engineering of ectopic gene expression in Gram-negative bacteria. The proposed compositional standard was thoroughly verified with a monomeric and superfolder green fluorescent protein (msf•GFP) in Escherichia coli. The expression data presented reflect a tightly controlled transcription initiation signal in response to cyclohexanone. Data in this article are related to the research paper "Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes" [1].

Transcription factor levels enable metabolic diversification of single cells of environmental bacteria.

Transcriptional noise is a necessary consequence of the molecular events that drive gene expression in prokaryotes. However, some environmental microorganisms that inhabit polluted sites, for example, the m-xylene degrading soil bacterium Pseudomonas putida mt-2 seem to have co-opted evolutionarily such a noise for deploying a metabolic diversification strategy that allows a cautious exploration of new chemical landscapes. We have examined this phenomenon under the light of deterministic and stochastic models for activation of the main promoter of the master m-xylene responsive promoter of the system (Pu) by its cognate transcriptional factor (XylR). These analyses consider the role of co-factors for Pu activation and determinants of xylR mRNA translation. The model traces the onset and eventual disappearance of the bimodal distribution of Pu activity along time to the growth-phase dependent abundance of XylR itself, that is, very low in exponentially growing cells and high in stationary. This tenet was validated by examining the behaviour of a Pu-GFP fusion in a P. putida strain in which xylR expression was engineered under the control of an IPTG-inducible system. This work shows how a relatively simple regulatory scenario (for example, growth-phase dependent expression of a limiting transcription factor) originates a regime of phenotypic diversity likely to be advantageous in competitive environmental settings.

Mining Environmental Plasmids for Synthetic Biology Parts and Devices.

The scientific and technical ambition of contemporary synthetic biology is the engineering of biological objects with a degree of predictability comparable to those made through electric and industrial manufacturing. To this end, biological parts with given specifications are sequence-edited, standardized, and combined into devices, which are assembled into complete systems. This goal, however, faces the customary context dependency of biological ingredients and their amenability to mutation. Biological orthogonality (i.e., the ability to run a function in a fashion minimally influenced by the host) is thus a desirable trait in any deeply engineered construct. Promiscuous conjugative plasmids found in environmental bacteria have evolved precisely to autonomously deploy their encoded activities in a variety of hosts, and thus they become excellent sources of basic building blocks for genetic and metabolic circuits. In this article we review a number of such reusable functions that originated in environmental plasmids and keep their properties and functional parameters in a variety of hosts. The properties encoded in the corresponding sequences include inter alia origins of replication, DNA transfer machineries, toxin-antitoxin systems, antibiotic selection markers, site-specific recombinases, effector-dependent transcriptional regulators (with their cognate promoters), and metabolic genes and operons. Several of these sequences have been standardized as BioBricks and/or as components of the SEVA (Standard European Vector Architecture) collection. Such formatting facilitates their physical composability, which is aimed at designing and deploying complex genetic constructs with new-to-nature properties.

Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida.

The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable genetic constructs. In this work, we have established a standardized heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida's chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments and flow cytometry with single-copy promoter-GFP constructs revealed a robust, constitutive behavior of these promoters, whose strengths and properties could be faithfully compared. This standardized expression device significantly extends the repertoire of tools available for reliable metabolic engineering and other genetic enhancements of P. putida.

Promoter fusions with optical outputs in individual cells and in populations.

Reporter genes are widely used to quantify promoter activity, which controls production of mRNA through the interplay with RNA polymerases and transcription factors. Some of such reporters have either diffuse (lux) or focused (GFP) optical outputs that allow description of transcriptional activity in populations and in single cells. This chapter discusses the use of a dual reporter system GFP-luxCDABE that is placed in broad-host-range plasmids having origins of replication from RK2 and pBBR1. The value of this system is shown in Pseudomonas putida by characterizing the activity of the Pb promoter, which drives an operon for benzoate biodegradation in this bacterium. To this end we compare in the same cells bioluminescence as the output signal of the whole population and single cell-bound fluorescence caused by GFP expression and revealed by flow cytometry assays.

Quantitative, non-disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system.

A dual promoter probe system based on a tandem bi-cistronic GFP-luxCDABE reporter cassette is described and implemented. This system is assembled in two synthetic, modular, broad-host range plasmids based on pBBR1 and RK2 origins of replication, allowing its utilization in an extensive number of gram-negative bacteria. We analyze the performance of this dual cassette in two hosts, Escherichia coli and Pseudomonas putida, by examining the induction properties of the lacI(q)-Ptrc expression system in the first host and the Pb promoter of the benzoate degradation pathway in the second host. By quantifying the bioluminescence signal produced through the expression of the lux genes, we explore the dynamic range of induction for the two systems (Ptrc-based and Pb-based) in response to the two inducers. In addition, by quantifying the fluorescence signals produced by GFP expression, we were able to monitor the single-cell expression profile and to explore stochasticity of the same two promoters by flow cytometry. The results provided here demonstrate the power of the dual GFP-luxCDABE cassette as a new, single-step tool to assess promoter properties at both the population and single-cell levels in gram-negative bacteria.