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The aim of the present study was to determine the effect of including green forage in the diet of Italian Mediterranean dairy buffaloes on the ruminal microbiota, CAZymes profile, functional biomolecules and total antioxidant activity in bulk milk. Sixteen buffaloes were randomly assigned according to lactation number and daily milk production to two homogeneous groups, and for 60 days received each: Group 1, a standard total mixed ration (TMR) or group 2, TMR + ryegrass green forage (30% of diet). The diets of the two groups were iso-nitrogenous and iso-energetic and differed only in the proportion of green forage. Buffaloes that received TMR + green feed had a higher (p <.01) representation of bacteria belonging to the orders Veillonellales, Selenomonadales and Bradymonadales compared with buffaloes that received TMR. The former buffaloes also had a greater (p <.01) abundance of CAZymes of the GT class (GHT4, GT14, GT20, GT26, GT39) and AA class (AA1, AA3, AA6). The milk of buffaloes that received TMR + green feed had a higher (p <.01) antioxidant capacity and greater (p <.01) amounts of the functional biomolecules l-carnitine, propionyl-l-carnitine, acetyl-l-carnitine and δ-valerobetaine. The findings have provided evidence for metabolic and biosynthetic pathways that link green forage with rumen bacteria, CAZymes and the synthesis of amino acids and functional biomolecule in buffaloes. Highlights: Green feed diet in dairy buffaloes favours ruminal microbiota that produces CAZymes. CAZymes support the synthesis of amino acids and functional biomolecules in milk. Functional milk from animals fed natural diets will be more appreciated by consumers. [ABSTRACT FROM AUTHOR]

GerR is a sporulation-specific transcriptional factor of Bacillus subtilis that has been identified as a negative regulator of genes transcribed by [[sigma].sup.E]-containing RNA polymerase and as a positive effector of the expression of three late sporulation genes. Here we confirmed that gerR transcription is dependent on [[sigma].sup.E]-containing RNA polymerase but also observed that it requires the transcriptional regulator SpoIIID. The study of the role of GerR in regulating the expression of several late sporulation genes allowed us to observe that its effect is strongly positive on spoVIF, cotC, and cotG, weakly positive on cotB, and negative on cotU. The results of chromatin immunoprecipitation (CHIP) experiments indicated that GerR binds to the promoter regions of some, but not all, of the GerR-controlled genes, leading us to propose that GerR controls late sporulation genes in two ways: (i) directly, by acting on the transcription of cotB, cotU and spoVIF; and (ii) indirectly, through the activation of SpoVIF, which stabilizes the transcriptional activator GerE and consequently induces the expression of the GerE-dependent genes cotC and cotG. doi: 10.1128/JB.00329-10

Bacillus subtilis is a Gram-positive spore-bearing bacterium long used as a probiotic product and more recently regarded as an attractive vehicle for delivering heterologous antigens to be used for mucosal vaccination. This report describes the in vitro interaction between human macrophages and B. subtilis spores displaying the tetanus toxin fragment C or the B subunit of the heat-labile toxin of Escherichia coli on their surface in comparison to spores of the parental strain. Recombinant and parental B. subtilis spores were similarly internalized by human macrophages, at a frequency lower than 2.5%. Inside macrophages, nearly all spores germinated and were killed within 6 h. Using germination-defective spores and inhibiting spore germination inside macrophages, evidence was produced that only germinated spores were killed by human macrophages and that intracellular spore germination was mediated by an alanine-dependent pathway. The germinated spores were killed by macrophages before any round of cell duplication, as estimated by fluorescence microscopy analysis of macrophages infected with spores carrying the gfp gene fused to abrB, a B. subtilis gene shown here to be expressed at the transition between outgrowth and vegetative growth. Monitoring of macrophage infection never revealed cytotoxic effects being exerted by B. subtilis spores. These in vitro data support the hypothesis that B. subtilis spores may potentially be used as a suitable and safe vehicle for administering heterologous antigens to humans.

We report evidence that CotC and CotU, two previously identified components of the Bacillus subtilis spore coat, are produced concurrently in the mother cell chamber of the sporulating cell under the control of [[sigma].sup.l] and GerE and immediately assembled around the forming spore. In the coat, the two proteins interact to forma coat component of 23 kDa. The CotU-CotC interaction was not detected in two heterologous hosts, suggesting that it occurs only in B. subtilis. Monomeric forms of both CotU and CotC failed to be assembled at the surface of the developing spore and accumulated in the mother cell compartment of cells mutant for cotE. In contrast, neither CotU nor CotC accumulated in the mother cell compartment of cells mutant for cotH. These results suggest that CotH is required to protect both CotU and CotC in the mother cell compartment of the sporangium and that CotE is needed to allow their assembly and subsequent interaction at the spore surface.

The assembly of a multiprotein coat around the Bacillus subtilis spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains [epsilon]-([gamma]-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a spore-associated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (cotE), that are missing the inner coat and with a greatly altered outer coat (gerE), or that are lacking discernible inner and outer coat structures (cotE gerE double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a tgl insertional mutation, the tglC116A allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under [[sigma].sup.K] control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of tgl causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat.