Alpha-glucans from bacterial necromass indicate an intra-population loop within the marine carbon cycle.

Phytoplankton blooms provoke bacterioplankton blooms, from which bacterial biomass (necromass) is released via increased zooplankton grazing and viral lysis. While bacterial consumption of algal biomass during blooms is well-studied, little is known about the concurrent recycling of these substantial amounts of bacterial necromass. We demonstrate that bacterial biomass, such as bacterial alpha-glucan storage polysaccharides, generated from the consumption of algal organic matter, is reused and thus itself a major bacterial carbon source in vitro and during a diatom-dominated bloom. We highlight conserved enzymes and binding proteins of dominant bloom-responder clades that are presumably involved in the recycling of bacterial alpha-glucan by members of the bacterial community. We furthermore demonstrate that the corresponding protein machineries can be specifically induced by extracted alpha-glucan-rich bacterial polysaccharide extracts. This recycling of bacterial necromass likely constitutes a large-scale intra-population energy conservation mechanism that keeps substantial amounts of carbon in a dedicated part of the microbial loop.

Marine Bacteroidetes enzymatically digest xylans from terrestrial plants.

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved ß-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.

Metabolic engineering enables Bacillus licheniformis to grow on the marine polysaccharide ulvan.

BACKGROUND: Marine algae are responsible for half of the global primary production, converting carbon dioxide into organic compounds like carbohydrates. Particularly in eutrophic waters, they can grow into massive algal blooms. This polysaccharide rich biomass represents a cheap and abundant renewable carbon source. In nature, the diverse group of polysaccharides is decomposed by highly specialized microbial catabolic systems. We elucidated the complete degradation pathway of the green algae-specific polysaccharide ulvan in previous studies using a toolbox of enzymes discovered in the marine flavobacterium Formosa agariphila and recombinantly expressed in Escherichia coli. RESULTS: In this study we show that ulvan from algal biomass can be used as feedstock for a biotechnological production strain using recombinantly expressed carbohydrate-active enzymes. We demonstrate that Bacillus licheniformis is able to grow on ulvan-derived xylose-containing oligosaccharides. Comparative growth experiments with different ulvan hydrolysates and physiological proteogenomic analyses indicated that analogues of the F. agariphila ulvan lyase and an unsaturated ß-glucuronylhydrolase are missing in B. licheniformis. We reveal that the heterologous expression of these two marine enzymes in B. licheniformis enables an efficient conversion of the algal polysaccharide ulvan as carbon and energy source. CONCLUSION: Our data demonstrate the physiological capability of the industrially relevant bacterium B. licheniformis to grow on ulvan. We present a metabolic engineering strategy to enable ulvan-based biorefinery processes using this bacterial cell factory. With this study, we provide a stepping stone for the development of future bioprocesses with Bacillus using the abundant marine renewable carbon source ulvan.

Channeling C1 Metabolism toward S-Adenosylmethionine-Dependent Conversion of Estrogens to Androgens in Estrogen-Degrading Bacteria.

Bacterial degradation of endocrine disrupting and carcinogenic estrogens is essential for their elimination from the environment. Recent studies of the denitrifying, estrogen-degrading Denitratisoma strain DHT3 revealed the conversion of estrogens to androgens by a putative cobalamin-dependent methyltransferase encoded by the emtABCD genes. The methyl donor and its continuous regeneration to initiate estradiol catabolism have remained unknown. Here, large-scale cultivation of the denitrifying bacterium Denitratisoma oestradiolicum with estrogen provided the biomass required for quantitative biochemical analyses. Soluble fractions of extracts from estradiol-grown cells catalyzed the S-adenosyl-l-methionine (SAM)- and Ti(III)-citrate-dependent conversion of 17ß-estradiol/estrone to the respective androgens at 0.15 nmol min-1 mg-1 Kinetic studies of 17ß-estradiol methylation and reverse 1-dehydrotestosterone demethylation reactions indicated that the exergonic methyl transfer from SAM to the putative cobalamin drives the endergonic methyl transfer from the methylcobalamin intermediate to the phenolic ring A. Based on a high-quality circular genome from D. oestradiolicum, proteogenomic analyses identified a 17ß-estradiol-induced gene cluster comprising emtABCD genes together with genes involved in SAM regeneration via l-serine and l-methionine. Consistent with this finding, l-methionine/ATP or l-serine/ATP/tetrahydrofolate/l-homocysteine substituted for SAM as methyl donors, further confirmed by the incorporation of the 13C-methyl-group from 13C-l-methonine into methyl(III)cobalamine and the estrone methylation product androsta-1,4-diene-3-one. This work demonstrates that during bacterial estrogen catabolism, the C1 pool is channeled toward the initiating methyl transfer to ring A. The effective cellular SAM regeneration system may serve as a model for whole-cell SAM-dependent methylation reactions of biotechnological interest.IMPORTANCE Estrogens comprise a group of related hormones occurring in predominantly female vertebrates, with endocrine disrupting and carcinogenic potential. Microbial biodegradation of estrogens is essential for their elimination from surface waters and wastewater. Aerobic bacteria employ oxygenases for the initial cleavage of the aromatic ring A. In contrast, anaerobic degradation of estrogens is initiated by methyl transfer-dependent conversion into androgens involving a putative cobalamin-dependent methyltransferase system. The methyl donor for this unprecedented reaction and its stoichiometric regeneration have remained unknown. With the biomass obtained from large-scale fermentation of an estrogen-degrading denitrifying bacterium, we identified S-adenosyl-methionine (SAM) as the methyl donor for the cobalamin-mediated methyl transfer to estrogens. To continuously supply C1 units to initiate estrogen degradation, genes for SAM regeneration from estradiol-derived catabolites are highly upregulated. Data presented here shed light into biochemical processes involved in the globally important microbial degradation of estrogens.