Potatoes are cultivated in southwest Greenland without the use of pesticides and with limited crop rotation. Despite the fact that plant-pathogenic fungi are present, no severe-disease outbreaks have yet been observed. In this report, we document that a potato soil at Inneruulalik in southern Greenland is suppressive against Rhizoctonia solani Ag3 and uncover the suppressive antifungal mechanism of a highly potent biocontrol bacterium, Pseudomonas fluorescens In5, isolated from the suppressive potato soil. A combination of molecular genetics, genomics, and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) imaging mass spectrometry (IMS) revealed an antifungal genomic island in P. fluorescens In5 encoding two nonribosomal peptides, nunamycin and nunapeptin, which are key components for the biocontrol activity by strain In5 in vitro and in soil microcosm experiments. Furthermore, complex microbial behaviors were highlighted. Whereas nunamycin was demonstrated to inhibit the mycelial growth of R. solani Ag3, but not that of Pythium aphanidermatum, nunapeptin instead inhibited P. aphanidermatum but not R. solani Ag3. Moreover, the synthesis of nunamycin by P. fluorescens In5 was inhibited in the presence of P. aphanidermatum. Further characterization of the two peptides revealed nunamycin to be a monochlorinated 9-amino-acid cyclic lipopeptide with similarity to members of the syringomycin group, whereas nunapeptin was a 22-amino-acid cyclic lipopeptide with similarity to corpeptin and syringopeptin.
Replication of plasmid RI is controlled by the products of two genes, copA and copB, that act as inhibitors of replication. Here it is shown that one small RNA synthesized from the copA gene acts as replication inhibitor. This RNA molecule was identified from analyses of RNAs synthesized in Escherichia coli minicells carrying RI miniplasmids or chimeric plasmids containing the copA gene. In minicells, this RNA was found to be unstable with a half-life of less than a few minutes. Two mutant hybrid plasmids lacking the inhibitor function did not express the RNA normally made from plasmids carrying the wild-type copA allele.Nucleotide sequence analysis of one of the copA mutants showed that a base substitution had occurred within the promoter sequence in front of the copA gene. DNA sequence analysis of the other mutant showed that a putative transcription-termination sequence was affected. The DNA sequence analysis also showed that the RNA molecule synthesized from the copA gene is untranslatable but has the potential for a high degree of secondary structure.The molecular mechanisms involved in the replication control of DNA have not yet been elucidated despite extensive 'efforts. Most progress has been-obtained by using bacterial plasmids and bacteriophages as model systems, as these molecules can often be dispensed with by the host cell. So far, this has led to fine-structure mapping ofseveral replication regions ofbacterial chromosomes, plasmids, and bacteriophages (1-7), but the actual identification of the gene products involved in the regulation ofDNA replication has been performed in only afew cases (8-11) and almost nothing is yet known about the interactions among the components that are required to ensure stable and precise regulation of DNA replication. We have chosen the transferable IncFII resistance plasmid Rldrd-19 as a model system for studies of control of DNA replication. This plasmid has been shown to contain the genetic information for the control ofits own replication and for IncFII incompatibility (12). These
Three -galactosidase genes from Bifidobacterium bifidum DSM20215 and one -galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum -galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published -galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis -galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 -galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal -galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum -galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four -galactosidases were highly specific for hydrolysis of -D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.Since they were first discovered by Tissier (33), the bifidobacteria have been investigated extensively by several scientists (e.g., references 23 and 27). In recent years, bifidobacteria have attracted particular attention due to their promising health-promoting properties, for example, reduction of harmful bacteria and toxic compounds in the intestine, prevention of dental caries, reduction of total cholesterol and lipid in serum, and relief of constipation (2,5,10,17,36,41). Therefore, live probiotic bifidobacteria, which may improve the microbial balance of the human gastrointestinal tract, have been used to supplement dairy products for many years. Another approach to increase the number of beneficial bacteria in the human intestine is to selectively stimulate their growth by supplementing food with ingredients which can only be metabolized by such bacteria. Certain oligosaccharides, the so-called prebiotics, have been shown to exert this growth-stimulating effect on probiotic bacteria, including bifidobacteria.So far, most of the probiotic bacteria and the prebiotic oligosaccharides have been used in combination with dairy products, and since these products often contain large amounts of lactose, much attention has been focused on the enzyme -galactosidase (EC 3.2.1.23), which is involved in the bacterial metabolism of lactose. In addition to normal hydrolysis of the -D-galactoside linkage in lactose, some -D-galactosidase enzymes may catalyze the formation of galactooligosaccharides through transfer of one or more D-galactosyl units onto the D-galactose moiety of lactose. This transgal...
Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66T is a marine bacterium capable of hydrolyzing polysaccharides found in the cell wall of red macroalgae. In this study, we applied an approach combining genomic mining with functional analysis to uncover the potential of this bacterium to produce enzymes for the hydrolysis of complex marine polysaccharides. A special feature of P. hydrolytica S66T is the presence of a large genomic region harboring an array of carbohydrate-active enzymes (CAZymes) notably agarases and carrageenases. Based on a first functional characterization combined with a comparative sequence analysis, we confirmed the enzymatic activities of several enzymes required for red algal polysaccharide degradation by the bacterium. In particular, we report for the first time, the discovery of novel enzyme activities targeting furcellaran, a hybrid carrageenan containing both β-carrageenan and κ/β-carrageenan motifs. Some of these enzymes represent a new subfamily within the CAZy classification. From the combined analyses, we propose models for the complete degradation of agar and κ/β-type carrageenan by P. hydrolytica S66T. The novel enzymes described here may find value in new bio-based industries and advance our understanding of the mechanisms responsible for recycling of red algal polysaccharides in marine ecosystems.
We found that propionic acid secreted from propionibacteria induces expression of the NKG2D ligands MICA/B on activated T lymphocytes and different cancer cells, without affecting MICA/B expression on resting peripheral blood cells. Growth supernatant from propionibacteria or propionate alone could directly stimulate functional MICA/B surface expression and MICA promoter activity by a mechanism dependent on intracellular calcium. Deletion and point mutations further demonstrated that a GC-box motif around −110 from the MICA transcription start site is essential for propionate-mediated MICA promoter activity. Other short-chain fatty acids such as lactate, acetate, and butyrate could also induce MICA/B expression. We observed a striking difference in the molecular signaling pathways that regulate MICA/B. A functional glycolytic pathway was essential for MICA/B expression after exposure to propionate and CMV. In contrast, compounds with histone deacetylase-inhibitory activity such as butyrate and FR901228 stimulated MICA/B expression through a pathway that was not affected by inhibition of glycolysis, clearly suggesting that MICA/B is regulated through different molecular mechanisms. We propose that propionate, produced either by bacteria or during cellular metabolism, has significant immunoregulatory function and may be cancer prophylactic.
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