The dark recovery rate in the photocycle of the bacterial photoreceptor YtvA is affected by the cellular environment and by hydration

We report thermal recovery kinetics of the lit state into the parental dark state, measured for the blue light-sensing photoreceptor YtvA inside overexpressing E. coli and B. subtilis bacterial cells, performed for the wild type and several mutated proteins. Recovery was followed as a recovery of the fluorescence, as this property is only found for the parental but not for the photochemically generated lit state. When cells were deposited onto a microscope glass plate, the observed thermal recovery rate in the photocycle was found ca. ten times faster in comparison to purified YtvA in solution. When the E. coli or B. subtilis colonies were soaked in an isotonic buffer, the dark relaxation became again much slower and was very similar to that observed for YtvA in solution. The observed effects show that rate constants can be tuned by the cellular environment through factors such as hydration. Copyright

Differential single nucleotide polymorphism-based analysis of an outbreak caused by Salmonella enterica serovar Manhattan reveals epidemiological details missed by standard pulsed-field gel electrophoresis

We retrospectively analyzed a rare Salmonella enterica serovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients (n = 15) and food, feed, animal, and environmental sources (n = 24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB-BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system

In vitro digestion and stability assessment of b-lactoglobulin/riboflavin nanostructures

B-Lactoglobulin (b-Lg) is the major protein fraction of bovine whey serum and a primary gelling agent. b-Lg has a high nutritional value, is stable at low pH being highly resistant to proteolytic degradation in the stomach, besides, it has the ability of acting as an encapsulating agent. This study aims at assessing the ability of b-Lg nanostructures to associate a nutraceutical - i.e. riboflavin - and release it in a controlled manner throughout an in vitro gastrointestinal (GI) system. For this reason b-Lg nanostructures loaded with riboflavin were critically characterized in terms of their morphology (i.e. size, polydispersity, -potential and shape) by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and efficiency to associate to riboflavin through spectrofluorimetry. Furthermore, these nanocomplexes were evaluated in an in vitro GI model, simulating the physiological conditions. Stable b-Lg nanostructures were obtained at pH 6, of spherical shape, characterized by particle size of 172±1 nm, low polydispersity (i.e. PDI of 0.06±0.02), -potential of -32±3 mV and association efficiency (AE) of 26±1 %. b-Lg nanostructures showed to be stable upon their passage throughout stomach (i.e. particle size, PDI and potential of 248±10 nm, 0.18±0.03 and 18±3 mV, respectively). Concerning their passage throughout the intestine, such nanostructures were mostly degraded in the duodenum. Regarding riboflavin, a release of about 11 % was observed after their passage through stomach, while 35 %, 38 % and 5 % were the released percentages of the total riboflavin associated observed after passage through duodenum, jejunum and ileum, respectively. Hence,b-Lg nanostructures showed to be suitable carriers for riboflavin until the intestine, where their degradation occurs. b-Lg also showed to be structurally stable, under food simulant conditions (yoghurt simulant, composed of 3 % acetic acid), over 14 days, with a protective effect upon riboflavin activity, releasing it in a 7 day period.Oscar L. Ramos, Ricardo N. Pereira and Ana C. Pinheiro gratefully acknowledge their Post-Doctoral grants (SFRH/BPD/80766/2011, SFRH/BPD/81887/2011 and SFRH/BPD/101181/2014, respectively) and Ana I. Bourbon gratefully acknowledge her Doctoral grant (SFRH/BD/73178/2010) to Fundacao para a Ciencia e a Tecnologia (FCT). The authors would like to acknowledge to Francisco Figueiredo from the Institute for Molecular and Cell Biology (IBMC), University of Porto, for assistance in taking the TEM pictures. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and the project "BioInd e Biotechnology and Bioengineering for improved Industrial and Agro-Food processes", REF. NORTE-07-0124-FEDER-000028, co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte), QREN, FEDER

Distance-tree analysis, distribution and co-presence of bilin- and flavin-binding prokaryotic photoreceptors for visible light

In recent years it has become increasingly evident that prokaryotic organisms can sense and react to light stimuli via a variety of photosensory receptors and signal transduction pathways. There are two main superfamilies of non-membrane-bound photoreceptors: the bilin-binding phytochrome-related proteins based on GAF (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and transcription activator FhlA) domains (bilin-GAF proteins), and the flavin-binding proteins (FL-Blues), photoperceptive thanks to their LOV (Light, Oxygen and Voltage) and BLUF (Blue Light sensing Using Flavins) domains. In this manuscript we present a comprehensive scenario of the existence of bilin-GAF, LOV and BLUF proteins in the prokaryotic world and inspect possible phylogenetic pathways, also defining novel criteria for identifying gene (and protein) sequences based on experimentally assessed photochemical events. As a whole we have inspected almost 2000 proteins recovered in 985 bacteria and 16 archaea. For LOV and BLUF proteins, ten and, respectively, twelve superconserved amino acids have been identified, which were used as criterion for selection. A similarly strict parameter cannot be applied to the more variegate family of bilin-GAF domains. The co-presence of bilin-GAF and FL-Blues occurs in 22% of the analyzed bacteria, with emphasis on the bilin-GAF/LOV co-presence in cyanobacteria and of bilin-GAF/BLUF in the Bacteroidetes/Chlorobi group. For construction of phylogeny/distance-trees we used the neighboringmethod to obtain a branching pattern, limited to photosensing domains. We observed that in many cases organisms belonging to the same phylum are neighbors, but clustering mostly occurs according to the type of functional domain associated with the photosensing modules