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A novel method was developed for virus-like particle (VLP) extraction and characterization from biological soil crust (BSC) after microbial community reactivation. The method consisted of a single cell analysis by flow cytometry to monitor viable cells in BSC reactivated under controlled hydration, temperature, and light/dark exposure. Then, VLPs were extracted from reactivated BSCs, followed by viral DNA extraction and shotgun metagenomic analysis. The hydrated BSC under light/dark conditions showed the highest number of viable cells, and this condition was optimal for VLPs isolation. Taxonomic composition showed that families of the order of Caudovirales (Podoviridae, Myoviridae and Syphoviridiae) were the most abundant double strand DNA phages while Microviridiae were the most abundant single strand DNA phages. The isolated VLPs also carried sequences of relevant bacterial inhabiting soil. The functional categories of “phages, prophages, transposable elements, plasmids” and “clustering-base subsystem” were abundant (38 and 12%, respectively). All these data suggest viral predation as a key factor in shaping and maintaining bacterial diversity in the BSCs.

The microbiota has been shown to promote intestinal tumourigenesis, but a possible anti-tumourigenic effect has also been postulated. Here, we demonstrate that changes in the microbiota and mucus composition are concomitant with tumourigen- esis. We identified two anti-tumourigenic strains of the microbiota--Faecalibaculum rodentium and its human homologue, Holdemanella biformis--that are strongly under-represented during tumourigenesis. Reconstitution of ApcMin/+ or azoxymeth- ane- and dextran sulfate sodium-treated mice with an isolate of F. rodentium (F. PB1) or its metabolic products reduced tumour growth. Both F. PB1 and H. biformis produced short-chain fatty acids that contributed to control protein acetylation and tumour cell proliferation by inhibiting calcineurin and NFATc3 activation in mouse and human settings. We have thus identified endog- enous anti-tumourigenic bacterial strains with strong diagnostic, therapeutic and translational potential.

Yeasts are responsible for spoilage of fruit juices and beverages because of their low pH and high sugar content. Their control is generally guaranteed by thermal treatments that however can affect organoleptic and nutritional properties. A strategy to reduce thermal damage is the combination of mild treatments with natural antimicrobials. This work studied the effect of citral on thermal inactivation of a Saccharomyces cerevisiae strain isolated from spoiled beverages, also in relation to medium pH, through plate counting and flow cytometry (FCM), to assess cell viability and membrane integrity. The results of treatments at 60 °C for 30 min showed that sublethal concentrations of citral increased death kinetics, especially at lower pH. Conversely, FCM analysis performed on cells treated at 60 °C for 10 min evidenced higher mortality at pH 6.0, while at pH 4.0 the population was mainly injured. This different behavior was likely due to higher membrane permeability observed at pH 6.0. No recovery of injured cells to a viable status was observed after 3 h of incubation. Since the fate of these cells in foods is a great concern, these results are helpful to increase knowledge about the physiological response of S. cerevisiae and to optimize strategies for food stability.

Exopolysaccharides production by 3 ropy strains of Lactobacillus delbrueckii subsp. bulgaricus of dairy origin was evaluated in synthetic medium by combining different approaches: impedometric measurements, fluorescent microscopy and flow cytometry analyses. The evaluation of ΔE by impedometric measurement (E%max-E%40h) allowed the detection of EPS production in synthetic medium, but the differences in EPS production kinetic was highlighted by flow cytometry analysis and fluorescent microcopy. This approach enabled us to unravel the diversity in EPS synthesis and release into the laboratory medium during the growth of the strains. Our results showed that the maximum EPS production occurred after 8 h of incubation, when cells were in late exponential growth phase. Furthermore, flow cytometry analysis revealed that only part of the cell population could be identified as EPS producer or as EPS-bounded cell. Therefore, the combined approach used, allowed us to define at the same time the kinetics of EPS production and release by three strains belonging to the same species and, highlight that the production of EPS depends also on the number of EPS-producing cells within the same population. This approach could be useful for the selection of strains to be used as starter cultures in dairy products where EPS production is considered an important feature.

The combined effect of thymol, carvacrol and mild heat treatments against Listeria monocytogenes Scott A was assessed in buffered system by plate counting and flow cytometry (FCM). The susceptibility of cells increased when heat treatment was combined with terpenes. The data modelling with Bigelow and Weibull equations showed that the latter was more appropriate to describe the inactivation kinetics. After treatments, cells were no longer able to form colonies on plates; nevertheless, FCM indicated that most of the cells were damaged rather than dead. Treated cells were not able to recover the damage after 6 h. This opens the question if they could recover in a food matrix. FCM can be a helpful technique to better comprehend the physiological state of microorganisms. In the perspective of industrial applications, studies based on predictive microbiology, as well as a deeper comprehension of the action mechanisms of antimicrobials, play a key role for process optimization.

Plant polyphenolic compounds are considered a promising source for new antibacterial agents. In this study, we evaluated the antimicrobial activity of a collection of resveratrol-derived monomers and dimers screened as single molecules against a panel of nine foodborne pathogens. The results demonstrated that two monomers (i.e., pterostilbene 2 and (E)-3-hydroxy-4′,5-dimethoxystilbene 9) and three dimers (i.e., δ-viniferin 10, viniferifuran 14 and dehydro-δ-viniferin 15) were endowed with significant antibacterial activity against gram-positive bacteria. The exposure of gram-positive foodborne pathogens to 100 µg/mL of 2, 9 and 15 induced severe cell membrane damage, resulting in the disruption of the phospholipid bilayer. The most promising dimeric compound, dehydro-δ-viniferin 15, was tested against Listeria monocytogenes, resulting in a loss of cultivability, viability and cell membrane potential. TEM analysis revealed grave morphological modifications on the cell membrane and leakage of intracellular content, confirming that the cell membrane was the principal biological target of the tested derivative.

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-β), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1β were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production.IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

Probiotics are live microorganisms, and viability after transit through the gastrointestinal tract (GIT) is considered an inherent property of the health benefits of probiotics. The aim of the present study was to quantify the viable and total loads of Lactobacillus paracasei DG cells after passage through the GIT following the consumption of the probiotic product Enterolactis (L. casei DG®; L. paracasei CNCM I-1572; L. paracasei DG) from drinkable vials by healthy adults. We developed a novel method for discriminating and enumerating culturable L. paracasei DG cells based on the unique sticky, filamentous phenotype of this strain on MRS agar containing vancomycin and kanamycin. The identity of DG was also confirmed with strain-specific primers by colony PCR. This method was used for a recovery study of the DG strain to quantify viable cells in the fecal samples of 20 volunteers during a 1-week probiotic consumption period and a 1-week follow-up. We isolated L. paracasei DG from at least one fecal sample from all the volunteers. The highest concentration of viable DG cells [ranging from 3.6 to 6.7 log10 colony-forming unit (CFU) per gram of feces] in the feces was observed between 4 and 8 days from the beginning of Enterolactis intake and for up to 5 days after cessation of intake. As expected, the total DG count determined by real-time quantitative PCR (qPCR) was mostly higher than the viable DG cells recovered. Viable count experiments, carried out by combining ad hoc culture-based discriminative conditions and strain-specific molecular biological protocols, unambiguously demonstrated that L. paracasei DG can survive gastrointestinal transit in healthy adults when ingested as Enterolactis in drinkable vials containing no less than one billion CFU at the end of shelf life.

A sustainable and scalable process for the production of a new mixture of fructooligosaccharides (FOS) was developed using a continuous-flow approach based on an immobilized whole cells-packed bed reactor. The technological transfer from a classical batch system to an innovative flow environment allowed a significant improvement of the productivity. Moreover, the stability of this production system was ascertained by up to 7 days of continuous working. These results suggest the suitability of the proposed method for a large-scale production of the desired FOS mixture, in view of a foreseeable use as a novel prebiotic preparation.

Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system.

Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1 -like gene, named tgaA . Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.

Streptococcus thermophilus strains are used in fermented dairy products for their capacity to metabolize lactose into lactic acid. The rate of lactic acid production in milk is of major economic importance, as rapid acidification prevents growth of undesirable microorganisms. It is also of paramount significance for aroma, texture and flavor of the end product. Besides achieving customer satisfaction, improvement of production rate and operational costs incite industrials into selecting fast acidifying strains. Another important trait of S. thermophilus influencing acidification is the urease, which catabolizes urea into ammonia and has a detrimental effect on acidification. Unfortunately, most of the S. thermophilus strains possess the urease, and the urease-negative ones are necessary for industrial applications. Urease activity is a widely distributed activity in S. thermophilus species, and urease-negative strains are rare. The later are however interesting from an industrial point of view, as they may give faster acidification in dairy applications, because lactic acid is not buffered by urea-derived ammonia. Nowadays, the efforts to improve the characteristics of strains for industrial applications are based on natural strategies such as random mutagenesis. This implies the need of a screening method that is efficient in terms of time and success. In this context, the aim of this study was the development of a new medium that allows selection of urease-defective mutants based on S. thermophilus colony morphology. Discrimination capacity of the new medium was verified using previously characterized urease-negative recombinant strains. The new milk-based medium, applied to industrial S. thermophilus strains subjected to UV mutagenesis, allowed the selection of 3 mutants, partially or completely defective in urease activity. Genetic characterization of urease-defective mutants highlighted the presence of nonsense or missense mutations in the ureA, ureC and ureG genes, thus supporting their phenotype. Evaluation of milk acidification revealed increased performance for one out of three urease-defective mutants compared to wild-type strains.

The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism.

Biocides are antimicrobial compounds that are widely used in the food industry and medical environments. In this study, we determined the Minimum Inhibitory Concentrations (MICs) by the microdilution method of four different biocides for a large collection of LAB of different origins. The tested isolates belong to 11 species of the Lactobacillus genus and to Streptococcus thermophilus, Streptococcus salivarius and Lactococcus garvieae. The results obtained in this study indicate that low susceptibilities to benzalkonium chloride (BC), triclosan (Tr), chlorhexidine (Ch), and sodium hypochlorite (SH) are not frequent among LAB. Moreover, no systematic co-tolerance between two or more tested biocides was found; that is, strains displaying high MIC values and thus low sensitivity to one of the biocides did not show higher MIC values for the other biocides.

We present a simple and rapid method for screening nisin producers that directly identifies nisinogenic bacteria by induction of bioluminescence within the Lactococcus lactis NZ9800lux biosensor strain (Immonen and Karp, 2007, Biosensors and Bioelectronics 22, 1982–7). An overlay of putative nisinogenic colonies with the biosensor strain gives identification results within 1 h. Functionality and specificity of the method were verified by screening nisin producers among 144 raw milk colonies and a panel of 91 lactococcal strains. Studies performed on strains and colonies that did not induce bioluminescence but inhibited growth of the biosensor demonstrated that only nisinogenic bacteria can cause induction. Bacteria known to produce bacteriocins other than nisin failed to induce bioluminescence, further verifying the specificity of the assay. We discovered a non-inducing but inhibitory lactococcal strain harboring a modified nisin Z gene, and demonstrated that the source of the inhibitory action is not a non-inducing variant of nisin, but a bacteriocin of lower molecular weight. The concentration of nisin producers in a raw milk sample was 1.3 × 10 2 CFU/ml. We identified from raw milk a total of seven nisin Z producing L. lactis subsp. lactis colonies, which were shown by genetic fingerprinting to belong to three different groups. Among the panel of 91 lactococci, four strains were nisin A producers, and one strain harbored the modified nisin Z gene. The method presented here is robust, cost-effective and simple to perform, and avoids the pitfalls of traditional screening methods by directly specifying the identity of the inhibitory substance.

Streptococcus thermophilus is a major component of dairy starter cultures used for the manufacture of yoghurt and cheese. In this study, the CO2 metabolism of S. thermophilus DSM 20617T, grown in either a N2 atmosphere or an enriched CO2 atmosphere, was analysed using both genetic and proteomic approaches. Growth experiments performed in a chemically defined medium revealed that CO2 depletion resulted in bacterial arginine, aspartate and uracil auxotrophy. Moreover, CO2 depletion governed a significant change in cell morphology, and a high reduction in biomass production. A comparative proteomic analysis revealed that cells of S. thermophilus showed a different degree of energy status depending on the CO2 availability. In agreement with proteomic data, cells grown under N2 showed a significantly higher milk acidification rate compared with those grown in an enriched CO2 atmosphere. Experiments carried out on S. thermophilus wild-type and its derivative mutant, which was inactivated in the phosphoenolpyruvate carboxylase and carbamoyl-phosphate synthase activities responsible for fixing CO2 to organic molecules, suggested that the anaplerotic reactions governed by these enzymes have a central role in bacterial metabolism. Our results reveal the capnophilic nature of this micro-organism, underlining the essential role of CO2 in S. thermophilus physiology, and suggesting potential applications in dairy fermentation processes.

The taxonomic identification of the biological material contained in the hallucinogenic mushrooms culture media, was carried out using a DNA-based approach, thus highlighting the usefulness of this approach in the forensic identification of illegal samples also when they are present as basidiospores mixed in culture media and spore-bearing fruiting body are not present. This approach is very useful as it allows the unequivocal identification of potentially illicit material before the cultivation and it enables to stop the material to the Customs and to destroy it due to its dangerousness without cultivating the "grow-kits" and without instructing a criminal case. In fact, even if psilocin and psilocybin and the whole mushrooms are illegal in many countries, there is no specific indication in the law about the so called "grow-kits", containing the spores. To confirm the data obtained by the taxonomic identification, a simple, reliable, efficient LC-UV method, using tryptamine as internal standard, suitable for the forensic quali-quantitative determination of psilocin and psilocybin in hallucinogenic mushroom was optimized, validated and applied to the mushrooms grown after the cultivation of the grow-kits seized by the judicial authority, with the authorization of the Ministry of Health. A cation exchange column was used in a gradient elution mode (Phase A: 50mMK2HPO4; 100mM NaCl pH=3 Phase B: methanol). The developed method was linear over the calibration range with a R(2)>0.9992 for both the analytes. The detection and quantification limits were respectively 0.01 and 0.1μg/mL for psilocybin and 0.05μg/mL and 0.1μg/mL for psilocin and the intra- and inter-day precision was satisfactory (coefficients of variation

The taxonomic identification of mushrooms suspected to contain hallucinogenic active principles was carried out using a DNA-based approach, thus highlighting the usefulness of this approach in the forensic identification of illegal samples also when they are difficult to identify because the morphologic identification is prevented, due to the bad conservation of the vegetable material. To confirm the presence of the illegal active principles, the optimization of a LC/MS-MS method for the qualitativequantitative analysis of psilocin and psilocybin in mushroom samples seized by the judicial authority is described. For the quantitative determination it was necessary to identify and synthesize a proper internal standard (IS, i.e., 5-hydroxy-N,N-diethyltryptamine), endowed with chromatographic features suitable for the analysis of the active principles. LC/MS-MS analysis evidenced that the amount of psilocybin ranged from 0.5 to 1.4% while that of psilocin from 1.3 to 2.5% (w/w), confirming literature data. The concentration of psilocin was higher in the cap and in the distal part of the stem (near to the soil) than in the part of the stem proximal to the cap. On the other hand the concentration of psilocybin was higher in the cap and in the proximal part, being lower in the distal part of the stem.

Staphylococcus aureus strains harboring QacA, QacB, QacC, QacG transporters and norA promoter up-regulating mutations were characterized by phenotype microarray (PM), standard methods for susceptibility testing, and ethidium bromide efflux assays, in order to increase knowledge on phenotypes associated to efflux pumps and their substrates. PM data and standard susceptibility testing lead to the identification of new potential efflux targets, such as guanidine hydrochloride or 8-hydroxyquinoline for QacA and QacC pumps, respectively. The identification of compounds to which the presence of efflux pumps induced increased susceptibility opens new perspectives for potential adjunct anti-resistance treatment (i.e. strains bearing QacB transporters showed increased susceptibility to thioridazine, amitriptyline and orphenadrine). Although the tested isolates were characterized by high degree of heterogeneity, a hallmark of clinical isolates, direct ethidium bromide efflux assays were effective in highlighting differences in efflux efficiency among strains. These data add to characterization of substrate specificity in the different classes of staphylococcal multidrug efflux systems conferring specific substrate profiles and efflux features to each of them.

In order to preserve environmental quality, alternative strategies to chemical-intensive agriculture are strongly needed. In this study, we characterized in vitro the potential plant growth promoting (PGP) properties of a gamma-proteobacterium, named MIMR1, originally isolated from apple shoots in micropropagation. The analysis of the 16S rRNA gene sequence allowed the taxonomic identification of MIMR1 as Luteibacter rhizovicinus. The PGP properties of MIMR1 were compared to Pseudomonas chlororaphis subsp. aurantiaca DSM 19603(T), which was selected as a reference PGP bacterium. By means of in vitro experiments, we showed that L. rhizovicinus MIMR1 and P. chlororaphis DSM 19603(T) have the ability to produce molecules able to chelate ferric ions and solubilize monocalcium phosphate. On the contrary, both strains were apparently unable to solubilize tricalcium phosphate. Furthermore, the ability to produce 3-indol acetic acid by MIMR1 was approximately three times higher than that of DSM 19603(T). By using fluorescent recombinants of strains MIMR1 and DSM 19603(T), we also demonstrated that both bacteria are able to abundantly proliferate and colonize the barley rhizosphere, preferentially localizing on root tips and in the rhizoplane. Finally, we observed a negative effect of DSM 19603(T) on barley seed germination and plant growth, whereas MIMR1, compared to the control, determined a significant increase of the weight of aerial part (+22 %), and the weight and length of roots (+53 and +32 %, respectively). The results obtained in this work make L. rhizovicinus MIMR1 a good candidate for possible use in the formulation of bio-fertilizers.

Disinfectants have been used in a variety of environmental applications, in products for personal care and in the food industry. The food industry has increased the use of biocides and chemical-based disinfectants to control microbial ecology at production sites in an effort to improve hygiene measures and food safety. However, the susceptibility profile of micro-organisms to disinfectants has been largely neglected. This study therefore aimed to provide this type of information by focusing on the four most commonly used biocides in the food industry, determining their minimum inhibitory concentrations (MICs) and analysing the distribution of MICs across a variety of micro-organisms. In total, 99 different strains of Bifidobacterium spp. were studied. Results showed a unimodal distribution of MICs for chlorhexidine, triclosan (Irgasan) and sodium hypochlorite with no apparent species-specific correlation. Conversely, part of the tested bifidobacteria population (20%) showed reduced susceptibility to benzalkonium chloride compared with the susceptibility exhibited by the majority of the tested bacterial community. The highest MICs were distributed among almost all of the considered Bifidobacterium spp. In generally, the sensitivity of the studied strains to the four tested biocides appeared to be a genus-related trait.

Microorganisms display a considerable versatility, with mechanisms that govern cell bioenergetics and a large number of redox active molecules being used as electron donors or acceptors. We will not review the basis of microbial bioenergetics here, but instead focus attention on the metabolic systems that microorganisms have evolved to optimise the efficiency of cell catabolism and cell energy homeostasis. The mechanisms that act in the regulation of cell bioenergetics belong to the complexity of biological systems in which large networks of metabolic pathways interact to govern the life and responsiveness of cells towards environmental fluctuations. During growth, all microorganisms determine considerable changes in the environmental concentration of nutrients, organic acids and other molecules generated by cell catabolism. As a consequence, microorganisms are constantly faced with different environmental stimuli and stresses. The natural habitats of some microorganisms may fluctuate erratically, whereas others which are more predictable offer the opportunity to prepare in advance for the next environmental change. In this context, microorganisms may have evolved the bioenergetic machinery to anticipate environmental fluctuations by adapting to their temporal order of appearance. Food matrixes represent an example of 'predictable' fluctuating environments, generated by anthropic activities and able to drive the speciation of several microorganisms. The nutrient’s richness, and specifically the abundance of monoand disaccharides that characterise several food matrixes (such as milk and grape juice), have allowed the speciation of lactic acid bacteria (LAB) and yeasts with a high fermentation capacity. The bakers' yeast Saccharomyces cerevisiae degrades sugars to two-carbon components – in particular, ethanol – even in the presence of excess oxygen, thus using a fermentation metabolism instead of the energetically favourable respiration metabolism (2 mol versus about 32 mol of ATP per mol of glucose respectively). S. cerevisiae alcoholic fermentation has been exploited for several millennia throughout the world in a variety of food processes of crucial importance for humans, such as the making of beer, wine and bread. Moreover, LAB species have partially lost the genetic information need in order to carry out a respiratory metabolism on behalf of a homofermentative pathway in which lactic acid is the primary product, or a heterofermentative pathway in which lactic acid, CO2, acetic acid and/or ethanol are produced (Kandler, 1983). The seemingly simplistic metabolism of LAB has been exploited throughout history for the preservation of foods and beverages in nearly all societies, and dates back to the origins of agriculture. The domestication of LAB strains passed down through various

expression, gene regulation An understanding of the integrated relationships among the principal cellular functions that govern the bioenergetic reactions of an organism is necessary to determine how cells remain viable and optimise their fitness in the environment. Urease is a complex enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. While the induction of urease activity by several microorganisms has been predominantly considered a stress-response that is initiated to generate a nitrogen source in response to a low environmental pH, here we demonstrate a new role of urease in the optimisation of cellular bioenergetics. We show that urea hydrolysis increases the catabolic efficiency of Streptococcus thermophilus, a lactic acid bacterium that is widely used in the industrial manufacture of dairy products. By modulating the intracellular pH and thereby increasing the activity of β-galactosidase, glycolytic enzymes and lactate dehydrogenase, urease increases the overall change in enthalpy generated by the bioenergetic reactions. A cooperative altruistic behaviour of urease-positive microorganisms on the urease-negative microorganisms within the same environment was also observed. The physiological role of a single enzymatic activity demonstrates a novel and unexpected view of the non-transcriptional regulatory mechanisms that govern the bioenergetics of a bacterial cell, highlighting a new role for cytosol-alkalizing biochemical pathways in acidogenic microorganisms.

The aim of this study was to investigate the adhesive phenotype of the human intestinal isolate Bifidobacterium bifidum MIMBb75 to human colon carcinoma cell lines. We have previously shown that the adhesion of this strain to Caco-2 cells is mediated by an abundant surface lipoprotein named BopA. In this study, we found that this strain adheres to Caco-2 and HT-29 cells, and that its adhesion strongly depends on the environmental conditions, including the presence of sugars and bile salts and the pH. Considerably more adhesion to a Caco-2 monolayer occurred in the presence of fucose and mannose and less when MIMBb75 grew in Oxgall bile salts compared to standard environmental conditions. In particular, growth in Oxgall bile salts reduced the adhesion ability of MIMBb75 and modified the SDS-PAGE profile of the cell wall associated proteins of the strain. The pH markedly affected both adhesion to Caco-2 and bacterial autoaggregation. Finally, experiments with sodium metaperiodate suggested that not only proteinaceous determinants are involved in the adhesion process of B. bifidum. In conclusion, it seems that the colonization strategy of this bacterium can be influenced by factors varying along the gastrointestinal tract, such as the presence of specific sugars and bile salts and the pH, possibly limiting the adhesion of B. bifidum to only restricted distal sites of the gut.

Aim: The aim of the study was to study the role of carbon dioxide metabolism in Streptococcus thermophilus through investigation of the phenotype of a carbamoylphosphate synthetase-negative mutant. Methods and results: The effect of carbon dioxide on the nutritional requirements of Strep. thermophilus DSM20617T and its derivative, carbamoylphosphate synthetase-negative mutant A17(ΔcarB), was investigated by cultivating the strain in a chemically defined medium under diverse gas compositions and in milk. The results obtained revealed that CO2 depletion or carB gene inactivation determined the auxotrophy of Strep. thermophilus for l-arginine and uracil. In addition, the parent strain grew faster than the mutant, even when milk was supplemented with uracil or arginine. Conclusions: Milk growth experiments underlined that carbamoylphosphate synthetase activity was essential for the optimal growth of Strep. thermophilus in milk. Significance and impact of the study: The study of the carbon dioxide metabolism in Strep. thermophilus revealed new insights with regard to the metabolism of this species, which could be useful for the optimization of dairy fermentation processes.

We investigated the carbon dioxide metabolism of Streptococcus thermophilus , evaluating the phenotype of a phosphoenolpyruvate carboxylase-negative mutant obtained by replacement of a functional ppc gene with a deleted and inactive version, Δppc . The growth of the mutant was compared to that of the parent strain in a chemically defined medium and in milk, supplemented or not with l -aspartic acid, the final product of the metabolic pathway governed by phosphoenolpyruvate carboxylase. It was concluded that aspartate present in milk is not sufficient for the growth of S. thermophilus . As a consequence, phosphoenolpyruvate carboxylase activity was considered fundamental for the biosynthesis of l -aspartic acid in S. thermophilus metabolism. This enzymatic activity is therefore essential for growth of S. thermophilus in milk even if S. thermophilus was cultured in association with proteinase-positive Lactobacillus delbrueckii subsp. bulgaricus . It was furthermore observed that the supplementation of milk with aspartate significantly affected the level of urease activity. Further experiments, carried out with a p ureI - gusA recombinant strain, revealed that expression of the urease operon was sensitive to the aspartate concentration in milk and to the cell availability of glutamate, glutamine, and ammonium ions.

Since the discovery of Helicobacter pylori, the urease activity ofthis bacterial pathogen has been identified as the key factor ininfection and acid acclimation in the human stomach. Ureolyticactivity plays a key role in the pathogenesis of several bacteria, andurease has also been described as an emerging pathogenic factorduring fungal infection. However, urease produced by the oralbacteria community has been shown to counteract caries, andcaries-free subjects have high levels of urease activity in plaquesamples. Some lactic acid bacteria with documented probioticbehavior are urease-positive. Likewise, other lactic acid bacterialspecies that are widely used in yogurt production and otherfermented dairy products use urease activity to counteract acidstress and to feed several biosynthetic pathways with carbondioxide and ammonia derived from urea hydrolysis. Urease is alsodiffused in several species belonging to the human gut microbiota,and it is estimated that this complex microbial community is ableto hydrolyze 15%–30% of the urea synthesized in normal subjects.In this context, urease was proposed to serve as a microbialbiomarker to distinguish microbiomes based on age and geogra-phy, thus highlighting the crucial involvement of this enzymaticactivity in nitrogen recycling when dietary nitrogen is limiting. Inlight of these considerations, the designation of urease as amicrobial virulence factor would be misleading, and the proposeduse of urease as a therapeutic target to counteract microbialinfections should be carefully evaluated.