Streptococcus thermophilus is a Gram-positive bacterium belonging to the group of lactic acid bacteria, among which several genera play an essential role in manufacture of food products. Recently, a genomic consortium sequenced and annotated its entire genome, which has been demonstrated to contain 1900 coding sequences. In this study, we have revealed the expression products of almost 200 different genes using a proteomic strategy combining 2-DE plus MALDI-TOF PMF and differential 1-DE plus muLC-ESI-IT-MS/MS. Thus, a number of cellular pathways related to important physiological processes were described at the proteomic level. Almost 50 genes were related to multiple electrophoretic species, whose heterogeneity was mainly due to variability in pI values. A 2-DE reference map obtained for lactose-grown cells was compared with those obtained after heat, cold, acid, oxidative and starvation stresses. Protein up/down-regulation measurements demonstrated that adaptation to different environmental challenges may involve the contribution of unique as well as combined physiological mechanisms. Common regulatory sites in the promoter region of genes whose expression was induced after stress were identified. These results provide a better comprehension of biochemical processes related to stress resistance in S. thermophilus, allowing defining the molecular bases of adaptative responses or markers for the identification of strains with potential industrial applications.

Among the group of lactic acid bacteria, Streptococcus thermophilus has found a wide application in industrial processes used for the manufacture of dairy products. Taking advantage of different proteome extraction and subfractionation protocols, bacterial cytosolic and membrane proteins were isolated and resolved by independent gel-free and gel-based separation procedures. Whole cytosolic fraction and its acid, basic and low molecular mass protein components were separated by different resolutive 2-DE and tricine 1-DE gels and identified by MALDI-TOF PMF and/or microLC-ESI-IT-MS/MS. Membrane proteins were resolved by 2-DE and SDS-PAGE gels and similarly identified by PMF and TMS analysis. In parallel, whole extract was trypsinized and resulting peptides were identified by shotgun 2-D LC-ESI-IT-MS/MS analysis. Using this combined approach, expression products corresponding to 458 different genes were identified, which cover almost a third of the predicted vegetative proteome. Relative protein concentration and hydrophobicity affected protein detection. Broad recognition was obtained for enzymes involved in carbohydrate, fatty acid, amino acid and nucleotide metabolism, replication, transcription, translation, cell wall synthesis, as well as for proteins affecting bacterial functions important for industrial applications, i.e. milk sugar import and exopolysaccharide biosynthesis. By providing detailed reference electrophoretic/chromatographic maps to be used in future comparative proteomic investigations on bacteria grown under various experimental conditions or on different bacterial strains, our results will favour dedicated studies on S. thermophilus metabolism and its regulation or on detection of biomarkers for selection of optimal strains for industrial applications.

A pUC19-derived plasmid was constructed that coded for a hybrid cellulase with the Thermomonosporafusca E2 cellulose-binding domain at its C terminus joined to the PrevoteUla ruminicola 40.5-kDa carboxymethyl cellulase (CMCase). The hybrid enzyme was purified and characterized enzymatically. It bound tightly to cellulose, and its specific activities on carboxymethyl cellulose, amorphous cellulose, and ball-milled cellulose were 1.5, 10, and 8 times that of the 40.5-kDa CMCase, respectively. Furthermore, the modified enzyme gave synergism with an exocellulase in the degradation of filter paper, while the 40.5-kDa CMCase did not. Cellulose is the most abundant polymer in nature, but mammals do not produce enzymes which can degrade this material. Ruminant animals have developed the capacity for cellulose digestion through a symbiotic relationship with ruminal microorganisms. The animal provides a habitat for the microbial growth, and the microbes convert low-quality feeds into organic acids that the animal can utilize (11). The rumen microflora is very complex, but only a few species of ruminal bacteria are cellulolytic (2, 9). A variety of noncellulolytic ruminal bacteria can utilize cellodextrins (20), and some of these bacteria produce endoglucanases which can degrade carboxymethyl cellulose (CMC). Prevotella ruminicola (Bacteroides ruminicola) secretes two immunologically cross-reacting carboxymethyl cellulases (CMCases) into the culture supernatant (16). These enzymes also degrade xylan, but they have very low activity on native cellulose. The lack of cellulose digestion may be related to the inability of these enzymes to bind tightly to cellulose.

Tomato plants were subjected to three fertilisation treatments (M: mineral fertiliser; DMPP: mineral fertiliser + 3,4-dimethylpyrazole phosphate; OM: NKP + organic animal manure) in combination with two water regimes (100% and 50% evapotranspiration). Plant biomass, fruit production, nitrogen use efficiency (NUE) and N uptake, maximal PSII photochemical efficiency, Fv/Fm and cumulative soil N2O emission were determined. Well-watered OM plants showed higher values of biomass, fruit production, NUE and N uptake than M and DMPP plants; cumulative N2O fluxes were lower in DMPP plots than in M and OM plots. The reduced water supply determined a drop in crop biomass, fruit production, NUE and N uptake, and cumulative N2O fluxes in M and OM treatments that were higher in OM plots, whereas it determined a significant rise in cumulative N2O fluxes in DMPP plots that was lower in absolute term compared to M and OM plots recorded under well-water irrigation. It can be concluded that DMPP added-fertiliser has a good performance in semiarid environment resulting a better nitrogen source compared to conventional and organo-mineral fertilisers under reduced water supply, able to preserve crop yield and to determine soil N2O emissions (as expressed in CO2 eq) not dangerous for global environment.

Growing cultures of Fibrobacter succinogenes S85 digested cellulose at a rapid rate, but nongrowing cells and cell extracts did not have detectable crystalline cellulase activity. Cells that had been growing exponentially on cellobiose initiated cellulose digestion and succinate production immediately, and cellulose-dependent succinate production could be used as an index of enzyme activity against crystalline cellulose. Cells incubated with cellulose never produced detectable cellobiose, and cells that were preincubated for a short time with thiocellobiose lost their ability to digest cellulose (competitive inhibition [K i ] of only 0.2 mg/ml or 0.56 mM). Based on these results, the crystalline cellulases of F. succinogenes were very sensitive to feedback inhibition. Different cellulose sources bound different amounts of Congo red, and the binding capacity was HCl-regenerated cellulose > ball-milled cellulose > Sigmacel > Avicel > filter paper. Congo red binding capacity was highly correlated with the maximum rates of metabolism of cellulose digestion and inversely related to K m. Congo red (250 g/ml) did not inhibit the growth of F. succinogenes S85 on cellobiose, but this concentration of Congo red inhibited the rate of ball-milled cellulose digestion. A Lineweaver-Burk plot of ball-milled cellulose digestion rate versus the amount of cellulose indicated that Congo red was a competitive inhibitor of cellulose digestion (K i was 250 g/ml).

Multipurpose production of hemp has led to renewed interest for this crop cultivation, especially for human nutrition. To date, no information about the influence of nitrogen source on hemp seed quality is available. Hemp is also used for food and beverages due to its nutritional characteristics. This further use of hemp has led to an increase in hemp-grown areas. Therefore, it is important to get more information on the role of nitrogen on the quality production as well as to evaluate the environmental impact of the cultivation technique. In this work, we evaluate the influence of nitrogen source (i.e., NH4NO3 and urea) on the seed fatty acid composition of an edible hemp as well as on the environment in terms of soil N2O emission. Nitrogen source modified seed quality very little. Even if characterized by a lower acidic profile, seed from plants grown under urea and NH4NO3 had a ω-3/ω-6 ratio (0.3) within the optimal range from the nutritional standpoint, being considered as the optimal proportion for human metabolism and health. Urea fertilization reduced soil N2O emission. Our findings suggest that nitrogen source seems not to influence seed quality and that urea fertilizer might be more climate-friendly than NH4NO3 in terms of greenhouse gas emissions, in an extensive cultivation of hemp for industrial use.

The effect of the nitrification inhibitor 3,4-dimethylphyrazole phosphate (DMPP) on N-fertilized crop growth and soil N₂O emissions were studied at two experimental sites in Southern Italy, characterised by a Mediterranean climate and different soil texture. The experiments were a randomized block design of two treatments: crop fertilized with NH₄NO₃ (considered the control treatment) or amended with DMPP plus NH₄NO₃ (considered the DMPP treatment). ANOVA was performed to assess differences between treatments and fertilization periods whereas simple and multiple linear regressions were performed in order to assess the effect of the soil-related in-dependent variables on soil gases emissions. Growth of potato plants fertilized with DMPP-added nitrogen was enhanced compared to control plants, whereas no benefit on maize plants grown during summer was observed. N₂O emissions measured from soil to potato after the first fertilization with DMPP-added nitrogen was reduced during winter, but was higher than control after the second fertilizer application in spring, leading to comparable N₂O emission factors (EF1) between treatments. In maize N₂O emissions and EF1 were lower for DMPP compared to control treatment. The effectiveness of reduction in soil N₂O emission was influenced by soil temperature and water-filled pore space (WFPS) in both experimental sites. However, the overall effect of WFPS was contrasting as N₂O emissions were decreased in potato and enhanced in maize.

A novel 5.3-kb deletion of the alpha-globin gene cluster was observed in a family from Naples, Southern Italy. It removes the 5′ end of the alpha 2-globin gene, causing an alpha (+)-thalassemia defect. Because of the presence of the residual 3′ end of the alpha 2-globin gene, we indicated this new haplotype with the symbol (alpha)alpha 5.3. The 5′ breakpoint, the first to be reported in the intergene region of the psi alpha 2- and psi alpha 1-globin genes, is located 822 bp upstream of the cap site of the psi alpha 1-gene and about 150 bp upstream of a 300- nt Alu family member. The 3′ breakpoint is located in the IVS-1 nt 58 of the alpha 2-globin gene. The 5.3-kb deleted fragment shows particular characteristics: it contains four Alu sequences having long regions 80% complementary and the 5′-GGCC-3′ short repeat at both ends. The sequences spanning across the breakpoints on the same strand and containing this repeat on their 3′ and 5′ ends, respectively, are 17 of 25 base complementary. These particular features led us to assume the formation of a multistem-loop due to the intrastrand interaction between the complementary regions as intermediate to the deletion. The unusual localization of the 5′ breakpoint suggests that even the intergene region of the psi alpha 2- and psi alpha 1-globin genes may function as a deletion target.