Unveiling the role of novel carbohydrate-binding modules in laminarin interaction of multimodular proteins from marine Bacteroidota during phytoplankton blooms.

Laminarin, a ß(1,3)-glucan, serves as a storage polysaccharide in marine microalgae such as diatoms. Its abundance, water solubility and simple structure make it an appealing substrate for marine bacteria. Consequently, many marine bacteria have evolved strategies to scavenge and decompose laminarin, employing carbohydrate-binding modules (CBMs) as crucial components. In this study, we characterized two previously unassigned domains as laminarin-binding CBMs in multimodular proteins from the marine bacterium Christiangramia forsetii KT0803T, thereby introducing the new laminarin-binding CBM families CBM102 and CBM103. We identified four CBM102s in a surface glycan-binding protein (SGBP) and a single CBM103 linked to a glycoside hydrolase module from family 16 (GH16_3). Our analysis revealed that both modular proteins have an elongated shape, with GH16_3 exhibiting greater flexibility than SGBP. This flexibility may aid in the recognition and/or degradation of laminarin, while the constraints in SGBP could facilitate the docking of laminarin onto the bacterial surface. Exploration of bacterial metagenome-assembled genomes (MAGs) from phytoplankton blooms in the North Sea showed that both laminarin-binding CBM families are widespread among marine Bacteroidota. The high protein abundance of CBM102- and CBM103-containing proteins during phytoplankton blooms further emphasizes their significance in marine laminarin utilization.

Investigation of TGF-α-overexpressing mouse hepatocytes (TAMH) cultured as spheroids for use in hepatotoxicity screening of drug candidates.

The immortalized mouse liver cell line TAMH has been described as a valuable tool for studying hepatotoxic mechanisms, but until now, it has only been reported to grow as a monolayer in culture. However, culturing hepatocytes as three-dimensional (3D) spheroids has been shown to result in improved liver-specific functions (e.g., metabolic capacity) by better mimicking the in vivo environment. This approach may lead to more reliable detection of drug-induced liver injury (DILI) in the early phase of drug discovery, preventing post-marketing drug withdrawals. Here, we investigated the cultivation of TAMH as 3D spheroids, characterizing them with optical and transmission electron microscopy as well as analyzing their gene expression at mRNA level (especially drug-metabolizing enzymes) compared to TAMH monolayer. In addition, comparisons were made with spheroids grown from the human hepatoblastoma cell line HepG2, another current spheroid model. The results indicate that TAMH spheroids express hepatic structures and show elevated levels of some of the key phase I and II drug-metabolizing enzymes, in contrast to TAMH monolayer. The in vitro hepatotoxic potencies of the drugs acetaminophen and flupirtine maleate were found to be very similar between TAMH spheroidal and the monolayer cultures. Both the advantages and disadvantages of TAMH spheroids as an in vitro hepatotoxicity model compared to monolayer model are discussed.

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.

Unique alcohol dehydrogenases involved in algal sugar utilization by marine bacteria.

Marine algae produce complex polysaccharides, which can be degraded by marine heterotrophic bacteria utilizing carbohydrate-active enzymes. The red algal polysaccharide porphyran contains the methoxy sugar 6-O-methyl-D-galactose (G6Me). In the degradation of porphyran, oxidative demethylation of this monosaccharide towards D-galactose and formaldehyde occurs, which is catalyzed by a cytochrome P450 monooxygenase and its redox partners. In direct proximity to the genes encoding for the key enzymes of this oxidative demethylation, genes encoding for zinc-dependent alcohol dehydrogenases (ADHs) were identified, which seem to be conserved in porphyran utilizing marine Flavobacteriia. Considering the fact that dehydrogenases could play an auxiliary role in carbohydrate degradation, we aimed to elucidate the physiological role of these marine ADHs. Although our results reveal that the ADHs are not involved in formaldehyde detoxification, a knockout of the ADH gene causes a dramatic growth defect of Zobellia galactanivorans with G6Me as a substrate. This indicates that the ADH is required for G6Me utilization. Complete biochemical characterizations of the ADHs from Formosa agariphila KMM 3901T (FoADH) and Z. galactanivorans DsijT (ZoADH) were performed, and the substrate screening revealed that these enzymes preferentially convert aromatic aldehydes. Additionally, we elucidated the crystal structures of FoADH and ZoADH in complex with NAD+ and showed that the strict substrate specificity of these new auxiliary enzymes is based on a narrow active site. KEY POINTS: • Knockout of the ADH-encoding gene revealed its role in 6-O-methyl-D-galactose utilization, suggesting a new auxiliary activity in marine carbohydrate degradation. • Complete enzyme characterization indicated no function in a subsequent reaction of the oxidative demethylation, such as formaldehyde detoxification. • These marine ADHs preferentially convert aromatic compounds, and their strict substrate specificity is based on a narrow active site.

A new carbohydrate-active oligosaccharide dehydratase is involved in the degradation of ulvan.

Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.

Connecting Algal Polysaccharide Degradation to Formaldehyde Detoxification.

Formaldehyde is a toxic metabolite that is formed in large quantities during bacterial utilization of the methoxy sugar 6-O-methyl-d-galactose, an abundant monosaccharide in the red algal polysaccharide porphyran. Marine bacteria capable of metabolizing porphyran must therefore possess suitable detoxification systems for formaldehyde. We demonstrate here that detoxification of formaldehyde in the marine Flavobacterium Zobellia galactanivorans proceeds via the ribulose monophosphate pathway. Simultaneously, we show that the genes encoding the key enzymes of this pathway are important for maintaining high formaldehyde resistance. Additionally, these genes are upregulated in the presence of porphyran, allowing us to connect porphyran degradation to the detoxification of formed formaldehyde.

Methanosaeta and "Candidatus Velamenicoccus archaeovorus".

The phylum "Candidatus Omnitrophica" (candidate division OP3) is ubiquitous in anaerobic habitats but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiotic cells. In this study, we enriched OP3 cells by double density gradient centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population by applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in the culture community, contained empty cells, cells devoid of rRNA or of both rRNA and DNA, and dead cells according to transmission electron microscopy (TEM), thin-section TEM, scanning electron microscopy (SEM), catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), and LIVE/DEAD imaging. OP3 LiM cells were ultramicrobacteria (200 to 300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals of OP3 LiM cells attached to Bacteria and to Archaea indicated many rRNA molecules and an active metabolism, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules and an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an Rnf complex (ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin-section TEM showed distinctive structures of predation. Our study demonstrated a predatory metabolism for OP3 LiM cells, and therefore, we propose the name "Candidatus Velamenicoccus archaeovorus" gen. nov., sp. nov., for OP3 LiM. IMPORTANCE Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria. We detected sick and dead cells of the filamentous archaeon Methanosaeta in slowly growing methanogenic cultures. OP3 LiM lives as a sugar fermenter, likely on polysaccharides from outer membranes, and has the genomic potential to live as a syntroph. The predatory lifestyle of OP3 LiM was supported by its genome, the first closed genome for the phylum "Candidatus Omnitrophica," and by images of cell-to-cell contact with prey cells. We propose naming OP3 LiM "Candidatus Velamenicoccus archaeovorus." Its metabolic versatility explains the ubiquitous presence of "Candidatus Omnitrophica" 3 in anoxic habitats and gives ultramicrobacterial epibionts an important role in the recycling and remineralization of microbial biomass. The removal of polysaccharides from outer membranes by ultramicrobacteria may also influence biological interactions between pro- and eukaryotes.

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.

A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis.

Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1ß, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. KEY POINTS: • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.

Reaching out in anticipation: bacterial membrane extensions represent a permanent investment in polysaccharide sensing and utilization.

Outer membrane extensions are common in many marine bacteria. However, the function of these surface enlargements or extracellular compartments is poorly understood. Using a combined approach of microscopy and subproteome analyses, we therefore examined Pseudoalteromonas distincta ANT/505, an Antarctic polysaccharide degrading gamma-proteobacterium. P. distincta produced outer membrane vesicles (MV) and vesicle chains (VC) on polysaccharide and non-polysaccharide carbon sources during the exponential and stationary growth phase. Surface structures of carbohydrate-grown cells were equipped with increased levels of highly substrate-specific proteins. At the same time, proteins encoded in all other polysaccharide degradation-related genomic regions were also detected in MV and VC samples under all growth conditions, indicating a basal expression. In addition, two alkaline phosphatases were highly abundant under non-limiting phosphate conditions. Surface structures may thus allow rapid sensing and fast responses in nutritionally deprived environments. It may also facilitate efficient carbohydrate processing and reduce loss of substrates and enzymes by diffusion as important adaptions to the aquatic ecosystem.

Genomic and proteomic profiles of biofilms on microplastics are decoupled from artificial surface properties.

Microplastics in marine ecosystems are colonized by diverse prokaryotic and eukaryotic communities. How these communities and their functional profiles are shaped by the artificial surfaces remains broadly unknown. In order to close this knowledge gap, we set up an in situ experiment with pellets of the polyolefin polymer polyethylene (PE), the aromatic hydrocarbon polymer polystyrene (PS), and wooden beads along a coastal to estuarine gradient in the Baltic Sea, Germany. We used an integrated metagenomics/metaproteomics approach to evaluate the genomic potential as well as protein expression levels of aquatic plastic biofilms. Our results suggest that material properties had a minor influence on the plastic-associated assemblages, as genomic and proteomic profiles of communities associated with the structurally different polymers PE and PS were highly similar, hence polymer-unspecific. Instead, it seemed that these communities were shaped by biogeographic factors. Wood, on the other hand, induced the formation of substrate-specific biofilms and served as nutrient source itself. Our study indicates that, while PE and PS microplastics may be relevant in the photic zone as opportunistic colonization grounds for phototrophic microorganisms, they appear not to be subject to biodegradation or serve as vectors for pathogenic microorganisms in marine habitats.

A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan.

Marine seaweeds increasingly grow into extensive algal blooms, which are detrimental to coastal ecosystems, tourism and aquaculture. However, algal biomass is also emerging as a sustainable raw material for the bioeconomy. The potential exploitation of algae is hindered by our limited knowledge of the microbial pathways-and hence the distinct biochemical functions of the enzymes involved-that convert algal polysaccharides into oligo- and monosaccharides. Understanding these processes would be essential, however, for applications such as the fermentation of algal biomass into bioethanol or other value-added compounds. Here, we describe the metabolic pathway that enables the marine flavobacterium Formosa agariphila to degrade ulvan, the main cell wall polysaccharide of bloom-forming Ulva species. The pathway involves 12 biochemically characterized carbohydrate-active enzymes, including two polysaccharide lyases, three sulfatases and seven glycoside hydrolases that sequentially break down ulvan into fermentable monosaccharides. This way, the enzymes turn a previously unexploited renewable into a valuable and ecologically sustainable bioresource.

Publisher Correction: Oxidative demethylation of algal carbohydrates by cytochrome P450 monooxygenases.

In the version of this article originally published, the line of conditions shown for NADH in Figure 2b was shifted out of place. The error has been corrected in the HTML and PDF versions of the article.

Oxidative demethylation of algal carbohydrates by cytochrome P450 monooxygenases.

Sugar O-methylation shields algal polysaccharides against microbial hydrolytic enzymes. Here, we describe cytochrome P450 monooxygenases from marine bacteria that, together with appropriate redox-partner proteins, catalyze the oxidative demethylation of 6-O-methyl-D-galactose, which is an abundant monosaccharide of the algal polysaccharides agarose and porphyran. This previously unknown biological function extends the group of carbohydrate-active enzymes to include the class of cytochrome P450 monooxygenases.

Biotransformation of bisphenol A analogues by the biphenyl-degrading bacterium Cupriavidusbasilensis - a structure-biotransformation relationship.

Comparative analyses determined the relationship between the structure of bisphenol A (BPA) as well as of seven bisphenol analogues (bisphenol B (BPB), bisphenol C (BPC), bisphenol E (BPE), bisphenol F (BPF), bisphenol Z (BPZ), bisphenol AP (BPAP), bisphenol PH (BPPH)) and their biotransformability by the biphenyl-degrading bacterium Cupriavidus basilensis SBUG 290. All bisphenols were substrates for bacterial transformation with conversion rates ranging from 6 to 98% within 216 h and 36 different metabolites were characterized. Transformation by biphenyl-grown cells comprised four different pathways: (a) formation of ortho-hydroxylated bisphenols, hydroxylating either one or both phenols of the compounds; (b) ring fission; (c) transamination followed by acetylation or dimerization; and (d) oxidation of ring substituents, such as methyl groups and aromatic ring systems, present on the 3-position. However, the microbial attack of bisphenols by C. basilensis was limited to the phenol rings and its substituents, while substituents on the carbon bridge connecting the rings were not oxidized. All bisphenol analogues with modifications at the carbon bridge could be oxidized up to ring cleavage, while substituents at the 3-position of the phenol ring other than hydroxyl groups did not allow this reaction. Replacing one methyl group at the carbon bridge of BPA by a hydrophobic aromatic or alicyclic ring system inhibited both dimerization and transamination followed by acetylation. While most of the bisphenol analogues exhibited estrogenic activity, four biotransformation products tested were not estrogenically active.

Transcriptomic and proteomic insight into the mechanism of cyclooctasulfur- versus thiosulfate-oxidation by the chemolithoautotroph Sulfurimonas denitrificans.

Chemoautotrophic bacteria belonging to the genus Sulfurimonas (class Campylobacteria) were previously identified as key players in the turnover of zero-valence sulfur, a central intermediate in the marine sulfur cycle. S. denitrificans was further shown to be able to oxidize cyclooctasulfur (S8 ). However, at present the mechanism of activation and metabolism of cyclooctasulfur is not known. Here, we assessed the transcriptome and proteome of S. denitrificans grown with either thiosulfate or S8 as the electron donor. While the overall expression profiles under the two growth conditions were rather similar, distinct differences were observed that could be attributed to the utilization of S8 . This included a higher abundance of expressed genes related to surface attachment in the presence of S8 , and the differential regulation of the sulfur-oxidation multienzyme complex (SOX), which in S. denitrificans is encoded in two gene clusters: soxABXY 1 Z 1 and soxCDY 2 Z 2 . While the proteins of both clusters were present with thiosulfate, only proteins of the soxCDY 2 Z 2 were detected at significant levels with S8 . Based on these findings a model for the oxidation of S8 is proposed. Our results have implications for interpreting metatranscriptomic and -proteomic data and for the observed high level of diversification of soxY 2 Z 2 among sulfur-oxidizing Campylobacteria.

Microbial metal-sulfide oxidation in inactive hydrothermal vent chimneys suggested by metagenomic and metaproteomic analyses.

Metal-sulfides are wide-spread in marine benthic habitats. At deep-sea hydrothermal vents, they occur as massive sulfide chimneys formed by mineral precipitation upon mixing of reduced vent fluids with cold oxygenated sea water. Although microorganisms inhabiting actively venting chimneys and utilizing compounds supplied by the venting fluids are well studied, only little is known about microorganisms inhabiting inactive chimneys. In this study, we combined 16S rRNA gene-based community profiling of sulfide chimneys from the Manus Basin (SW Pacific) with radiometric dating, metagenome (n = 4) and metaproteome (n = 1) analyses. Our results shed light on potential lifestyles of yet poorly characterized bacterial clades colonizing inactive chimneys. These include sulfate-reducing Nitrospirae and sulfide-oxidizing Gammaproteobacteria dominating most of the inactive chimney communities. Our phylogenetic analysis attributed the gammaproteobacterial clades to the recently described Woeseiaceae family and the SSr-clade found in marine sediments around the world. Metaproteomic data identified these Gammaproteobacteria as autotrophic sulfide-oxidizers potentially facilitating metal-sulfide dissolution via extracellular electron transfer. Considering the wide distribution of these gammaproteobacterial clades in marine environments such as hydrothermal vents and sediments, microbially accelerated neutrophilic mineral oxidation might be a globally relevant process in benthic element cycling and a considerable energy source for carbon fixation in marine benthic habitats.

Characterization of a thaumarchaeal symbiont that drives incomplete nitrification in the tropical sponge Ianthella basta.

Marine sponges represent one of the few eukaryotic groups that frequently harbour symbiotic members of the Thaumarchaeota, which are important chemoautotrophic ammonia-oxidizers in many environments. However, in most studies, direct demonstration of ammonia-oxidation by these archaea within sponges is lacking, and little is known about sponge-specific adaptations of ammonia-oxidizing archaea (AOA). Here, we characterized the thaumarchaeal symbiont of the marine sponge Ianthella basta using metaproteogenomics, fluorescence in situ hybridization, qPCR and isotope-based functional assays. 'Candidatus Nitrosospongia ianthellae' is only distantly related to cultured AOA. It is an abundant symbiont that is solely responsible for nitrite formation from ammonia in I. basta that surprisingly does not harbour nitrite-oxidizing microbes. Furthermore, this AOA is equipped with an expanded set of extracellular subtilisin-like proteases, a metalloprotease unique among archaea, as well as a putative branched-chain amino acid ABC transporter. This repertoire is strongly indicative of a mixotrophic lifestyle and is (with slight variations) also found in other sponge-associated, but not in free-living AOA. We predict that this feature as well as an expanded and unique set of secreted serpins (protease inhibitors), a unique array of eukaryotic-like proteins, and a DNA-phosporothioation system, represent important adaptations of AOA to life within these ancient filter-feeding animals.

An auto-inducible phosphate-controlled expression system of Bacillus licheniformis.

BACKGROUND: A promoter that drives high-level, long-term expression of the target gene under substrate limited growth conditions in the absence of an artificial inducer would facilitate the efficient production of heterologous proteins at low cost. A novel phosphate-regulated expression system was constructed using the promoter of the phytase encoding gene phyL from Bacillus licheniformis for the overexpression of proteins in this industrially relevant host. RESULTS: It is shown that the phyL promoter enables a strong overexpression of the heterologous genes amyE and xynA in B. licheniformis when cells were subjected to phosphate limitation. Whether B. licheniformis can use phytate as an alternative phosphate source and how this substrate influences the PphyL controlled gene expression under growth conditions with limited inorganic phosphate concentrations were also investigated. It is shown that B. licheniformis cells are able to use sodium phytate as alternative phosphate source. The addition of small amounts of sodium phytate (≤ 5 mM) to the growth medium resulted in a strong induction and overexpression of both model genes in B. licheniformis cells under phosphate limited growth conditions. CONCLUSIONS: The PphyL controlled expression of the investigated heterologous genes in B. licheniformis is strongly auto-induced under phosphate limited conditions. The proposed PphyL expression system enables an overexpression of target genes in B. licheniformis under growth conditions, which can be easily performed in a fed-batch fermentation process.

Biopearling of Interconnected Outer Membrane Vesicle Chains by a Marine Flavobacterium.

Large surface-to-volume ratios provide optimal nutrient uptake conditions for small microorganisms in oligotrophic habitats. The surface area can be increased with appendages. Here, we describe chains of interconnecting vesicles protruding from cells of strain Hel3_A1_48, affiliating with Formosa spp. within the Flavobacteriia and originating from coastal free-living bacterioplankton. The chains were up to 10 µm long and had vesicles emanating from the outer membrane with a single membrane and a size of 80 to 100 nm by 50 to 80 nm. Cells extruded membrane tubes in the exponential phase, whereas vesicle chains dominated on cells in the stationary growth phase. This formation is known as pearling, a physical morphogenic process in which membrane tubes protrude from liposomes and transform into chains of interconnected vesicles. Proteomes of whole-cell membranes and of detached vesicles were dominated by outer membrane proteins, including the type IX secretion system and surface-attached peptidases, glycoside hydrolases, and endonucleases. Fluorescein-labeled laminarin stained the cells and the vesicle chains. Thus, the appendages provide binding domains and degradative enzymes on their surfaces and probably storage volume in the vesicle lumen. Both may contribute to the high abundance of these Formosa-affiliated bacteria during laminarin utilization shortly after spring algal blooms.IMPORTANCE Microorganisms produce membrane vesicles. One synthesis pathway seems to be pearling that describes the physical formation of vesicle chains from phospholipid vesicles via extended tubes. Bacteria with vesicle chains had been observed as well as bacteria with tubes, but pearling was so far not observed. Here, we report the observation of, initially, tubes and then vesicle chains during the growth of a flavobacterium, suggesting biopearling of vesicle chains. The flavobacterium is abundant during spring bacterioplankton blooms developing after algal blooms and has a special set of enzymes for laminarin, the major storage polysaccharide of microalgae. We demonstrated with fluorescently labeled laminarin that the vesicle chains bind laminarin or contain laminarin-derived compounds. Proteomic analyses revealed surface-attached degradative enzymes on the outer membrane vesicles. We conclude that the large surface area and the lumen of vesicle chains may contribute to the ecological success of this marine bacterium.