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Scope: Plant phenolics, known to exert beneficial effects on human health, were supplemented to cultures of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) to assess their effect on its adhesive capacity and the abundancy of individual proteins., Methods and Results: The presence of resveratrol and ferulic acid during bacterial growth stimulated adhesion of NCFM to mucin and human intestinal HT-29 cells, while tannic acid improved adhesion only to HT-29 cells and caffeic acid had very modest effect overall. Some dosage dependence was found for the four phenolics supplemented at 100, 250, and 500 μg mL -1 to the cultures. Notably, 500 μg mL -1 ferulic acid only stimulated adhesion to mucin. Analyses of differential whole-cell as well as surface proteomes revealed relative abundancy changes for a total of 27 and 22 NCFM proteins, respectively. These changes include enzymes acting in metabolic pathways, such as glycolysis, nucleotide metabolism, and stress response, as well as known moonlighting or surface-associated proteins., Conclusion: The five plant phenolics found in various foods stimulate the adhesive capacity of NCFM in diverse ways and elicit relative abundancy changes of specific proteins, providing molecular level insight into the mechanism of the putative beneficial effects of the polyphenols., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The definition of safety and efficacy of food-employed bacteria as well as probiotic strains is a continuous, often unattended, challenge. Proteomic techniques such as 2DE, DIGE and LC/LC-MS/MS are suitable and powerful tools to reveal new aspects (positive and negative) of "known" and "unknown" strains that can be employed in food making and as nutraceutical supplements for human health. Unfortunately, these techniques are not used as extensively as it should be wise. The present report describes the most significant results obtained by our research group in 10years of study on subproteomes in bacteria, chiefly lactic acid bacteria. Production of desired and undesired metabolites, differences between strains belonging to same species but isolated from different ecological niches, the effect of cryoprotectants on survival to lyophilization as well as the adhesive capability of strains, were elucidated by analysis of cytosolic, membrane-enriched, surface and extracellular proteomes. The present review opens a window on a yet largely underexplored field and highlights the huge potential of subproteome investigations for more rational choice of microbial strains as food starters, probiotics and for production of nutraceuticals. These analyses will hopefully contribute to manufacturing safer and healthier food and food supplements in the near future. This article is part of a Special Issue entitled: HUPO 2014., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In the present study, the high isoelectric point sub-proteome of Acinetobacter radioresistens S13 grown on aromatic compounds (benzoate or phenol) was analyzed and compared to the protein pattern, in the same pI range, of acetate-grown bacteria (control condition). Analyses concerned both soluble and membrane enriched proteomes and led to the identification of 25 proteins that were differentially expressed among the growth conditions considered: most of them were up-regulated in cells grown on aromatic compounds. Up to 17 identified proteins can be, more or less directly, related to the so called "envelope stress responses": these signal transduction pathways are activated when bacterial cells are exposed to stressing environments (e.g., heat, pH stress, organic solvents, osmotic stress) causing accumulation of misfolded/unfolded cell wall proteins into the periplasmic space. For, at least, five of these proteins (a DegP-like serine protease, a peptidyl-prolyl cis-trans isomerase, a phosphatidylserine decarboxylase, a pseudouridine synthase, and a TolB-like protein) a direct induction via either the σ(E) or the Cpx alternative signalling systems mediating envelope stress responses was previously demonstrated in Gram-negative bacteria. The proteins identified in this study include periplasmic proteases, chaperones, enzymes catalyzing peptydoglycan biogenesis, proteins involved in outer membrane integrity, cell surface properties and cellular redox homeostasis. The present study brings additional information to previous works on the acidic proteome of A. radioresistens S13, thus complementing and refining the metabolic picture of this bacterial strain during growth on aromatic compounds.

Intracellular calcium signals activated by growth factors in endothelial cells during angiogenesis regulate cytosolic and nuclear events involved in survival, proliferation and motility. Among the intracellular messengers released upon proangiogenic stimulation, arachidonic acid (AA) and its metabolites play a key role, and their effects are strictly related to calcium homeostasis. In human breast tumor-derived endothelial cells (B-TECs) AA stimulates proliferation and tubulogenesis in a calcium-dependent way. Here, to characterize the proteins whose expression is regulated by AA-induced calcium entry, we used a proteomic approach (two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, 2-DE and MALDI-MS) and we compared the proteomes of B-TECs stimulated with AA in presence or in absence of calcium entry (with addition to the culture medium of the calcium chelator EGTA, which completely prevents calcium fluxes throughout the plasma membrane). We found that six proteins increased their levels of expression, all higher when AA-induced calcium entry was abolished. These proteins have been identified by mass spectrometry and database search, and their potential roles in AA-stimulated pathway and in angiogenesis are discussed.

Study of the bacterial membrane proteome, though in its early stages, is a field of growing interest in the search for information about nutrient transport and processing. We tested different strategies and chemical compounds to extract proteins from the membranes (inner and outer) of Acinetobacter radioresistens S13, a Gram-negative bacterium selected for its ability to degrade aromatics. A. radioresistens S13 was monitored under different growth substrate conditions, using acetate, benzoate or phenol as sole carbon source. Two-dimensional gel electrophoresis map analysis of membrane extracts from benzoate- and phenol-grown cells reveals differences versus controls (acetate-grown cultures). Primarily, a different pattern of spots was observed and, in particular, some proteins were only expressed in the presence of aromatic substrate. Among these, we detected a Na(+)/H(+) antiporter, whose function is likely to be regulation of intracellular pH, and an ABC type sugar transport system, probably involved in capsular polysaccharide translocation. We also identified other proteins, detectable in acetate-grown but over-expressed in aromatic-grown cells. These include: (1) an outer membrane protein ascribable to an OmpA-like protein, recently described in the literature as "alasan", a bioemulsifying agent involved in solubilizing and enhancing bioavailability of hydrocarbons; (2) a trimeric porin of the PhoE family also belonging to the outer membrane and involved in facilitating the transport of anions (especially phosphate); and (3) two glycosyl transferases probably involved in capsules and/or lipopolysaccharide biosynthesis. Study of the bacterial membrane proteome helps to elucidate the role of the membrane as modulable site enabling communication between internal and external environments.

An Acinetobacter radioresistens strain able to grow on phenol or benzoate as sole carbon and energy source through the beta-ketoadipate pathway was isolated in our laboratories. In previous research, we found a different expression of catechol-1,2-dioxygenase isoenzymes (C-1,2-O) depending on the growth substrate (phenol or benzoate). In the present study, we used proteome techniques to extend our investigation to other enzymes involved in the aromatic degradation pathway. Since the first nontoxic metabolite in this route is cis,cis-muconic acid, we focused our attention on the enzymes leading to this compound, chiefly phenol hydroxylase (PH), benzoate dioxygenase (BD), cis-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate dehydrogenase (D) and C-1,2-O. In particular, the A. radioresistens proteome was monitored under different growth substrate conditions, using acetate, benzoate, or phenol as sole carbon source. We compared the protein maps by software image analysis and detected marked differences, suggesting the inducibility of most enzymes. This research also sought to evaluate the conditions allowing the best expression of enzymes to be used in immobilized systems suitable for bioremediation. The experimental data indicate that benzoate is the best carbon source to gain the highest amount of C-1,2-O and D, while phenol is the best growth substrate to obtain PH.