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.
Algal cell wall polysaccharides constitute a large fraction in the biomass of marine primary producers and are thus important in nutrient transfer between trophic levels in the marine ecosystem. In order for this transfer to take place, polysaccharides must be degraded into smaller mono-and disaccharide units, which are subsequently metabolized, and key components in this degradation are bacterial enzymes. The marine bacterium Colwellia echini A3 T is a potent enzyme producer since it completely hydrolyzes agar and -carrageenan. Here, we report that the genome of C. echini A3 T harbors two large gene clusters for the degradation of carrageenan and agar, respectively. Phylogenetical and functional studies combined with transcriptomics and in silico structural modeling revealed that the carrageenolytic cluster encodes furcellaranases, a new class of glycoside hydrolase family 16 (GH16) enzymes that are key enzymes for hydrolysis of furcellaran, a hybrid carrageenan containing both and -carrageenan motifs. We show that furcellaranases degrade furcellaran into neocarratetraose-43-O-monosulfate [DA-(␣1,3)-G4S-(1,4)-DA-(␣1,3)-G], and we propose a molecular model of furcellaranases and compare the active site architectures of furcellaranases, -carrageenases, -agarases, and -porphyranases. Furthermore, C. echini A3 T was shown to encode -carrageenases, -carrageenases, and members of a new class of enzymes, active only on hybrid /-carrageenan tetrasaccharides. On the basis of our genomic, transcriptomic, and functional analyses of the carrageenolytic enzyme repertoire, we propose a new model for how C. echini A3 T degrades complex sulfated marine polysaccharides such as furcellaran, -carrageenan, and -carrageenan. IMPORTANCE Here, we report that a recently described bacterium, Colwellia echini, harbors a large number of enzymes enabling the bacterium to grow on -carrageenan and agar. The genes are organized in two clusters that encode enzymes for the total degradation of -carrageenan and agar, respectively. As the first, we report on the structure/ function relationship of a new class of enzymes that hydrolyze furcellaran, a partially sulfated /-carrageenan. Using an in silico model, we hypothesize a molecular structure of furcellaranases and compare structural features and active site architectures of furcellaranases with those of other GH16 polysaccharide hydrolases, such as -carrageenases, -agarases, and -porphyranases. Furthermore, we describe a new class of enzymes distantly related to GH42 and GH160 -galactosidases and show that this new class of enzymes is active only on hybrid /-carrageenan oligosaccharides. Finally, we propose a new model for how the carrageenolytic enzyme repertoire enables C. echini to metabolize /-, -, and -carrageenan.
Summary Plant breeding for belowground traits that have a positive impact on the rhizosphere microbiome is a promising strategy to sustainably improve crop yields. Root architecture and morphology are understudied plant breeding targets despite their potential to significantly shape microbial community structure and function in the rhizosphere. In this review, we explore the relationship between various root architectural and morphological traits and rhizosphere interactions, focusing on the potential of root diameter to impact the rhizosphere microbiome structure and function while discussing the potential biological and ecological mechanisms underpinning this process. In addition, we propose three future research avenues to drive this research area in an effort to unravel the effect of belowground traits on rhizosphere microbiology. This knowledge will pave the way for new plant breeding strategies that can be exploited for sustainable and high‐yielding crop cultivars.
Potato juice, a by-product of starch processing, is a potential high-value food ingredient due to its high protein content. However, conversion from feed to human protein requires the removal of the toxic antinutritional glycoalkaloids (GAs) α-chaconine and α-solanine. Detoxification by enzymatic removal could potentially provide an effective and environmentally friendly process for potato-derived food protein production. While degradation of GAs by microorganisms has been documented, there exists limited knowledge on the enzymes involved and in particular how bacteria degrade and metabolize GAs. Here we describe a series of methods for the isolation, screening, and selection of GA-degrading bacteria. Bacterial cultures from soils surrounding greened potatoes, including the potato peels, were established and select bacterial isolates were studied. Screening of bacterial crude extracts for the ability to hydrolyze GAs was performed using a combination of thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and liquid chromatography mass spectrometry (LC-MS). Analysis of the 16S rRNA sequences revealed that bacteria within the genus Arthrobacter were among the most efficient GA-degrading strains.
Nunamycin and nunapeptin are two antimicrobial cyclic lipopeptides (CLPs) produced by Pseudomonas fluorescens In5 and synthesized by nonribosomal synthetases (NRPS) located on two gene clusters designated the nun–nup regulon. Organization of the regulon is similar to clusters found in other CLP‐producing pseudomonads except for the border regions where putative LuxR‐type regulators are located. This study focuses on understanding the regulatory role of the LuxR‐type‐encoding gene nunF in CLP production of P. fluorescens In5. Functional analysis of nunF coupled with liquid chromatography–high‐resolution mass spectrometry (LC‐HRMS) showed that CLP biosynthesis is regulated by nunF. Quantitative real‐time PCR analysis indicated that transcription of the NRPS genes catalyzing CLP production is strongly reduced when nunF is mutated indicating that nunF is part of the nun–nup regulon. Swarming and biofilm formation was reduced in a nunF knockout mutant suggesting that these CLPs may also play a role in these phenomena as observed in other pseudomonads. Fusion of the nunF promoter region to mCherry showed that nunF is strongly upregulated in response to carbon sources indicating the presence of a fungus suggesting that environmental elicitors may also influence nunF expression which upon activation regulates nunamycin and nunapeptin production required for the growth inhibition of phytopathogens.
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