Replication of plasmid R1 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 R1 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.

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. IMPORTANCE Crop rotation and systematic pest management are used to only a limited extent in Greenlandic potato farming. Nonetheless, although plant-pathogenic fungi are present in the soil, the farmers do not experience major plant disease outbreaks. Here, we show that a Greenlandic potato soil is suppressive against Rhizoctonia solani , and we unravel the key biocontrol components for Pseudomonas fluorescens In5, one of the potent biocontrol bacteria isolated from this Greenlandic suppressive soil. Using a combination of molecular genetics, genomics, and microbial imaging mass spectrometry, we show that two cyclic lipopeptides, nunamycin and nunapeptin, are important for the biocontrol activity of P. fluorescens In5 both in vitro and in microcosm assays. Furthermore, we demonstrate that the synthesis of nunamycin is repressed by the oomycete Pythium aphanidermatum . Overall, our report provides important insight into interkingdom interference between bacteria and fungi/oomycetes.

The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns.

Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66^T 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 S66^T 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 S66^T. 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.

Three β-galactosidase genes from Bifidobacterium bifidum DSM20215 and one β-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum β-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published β-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis β-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 β-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 β-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum β-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 β-galactosidases were highly specific for hydrolysis of β- d -galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.