A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan

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Charakterisierung der bakteriellen Transformation von Bisphenolen als östrogenen Umweltschadstoffen durch Bacillus amyloliquefaciens und Cupriavidus basilensis

Kunststoffe durchdringen nahezu jeden Bereich unseres alltäglichen Lebens. Zur Herstellung bestimmter Plastikmaterialien (Epoxidharze und Polycarbonate) werden Bisphenole als Grundbaustein benötigt, deren Grundstruktur sich aus zwei Phenolringen zusammensetzt, die über eine substituierte Kohlstoffbrücke miteinander verbunden sind. Die einzelnen Bisphenole unterscheiden sich jeweils durch verschiedene Substituenten an der Kohlenstoffbrücke oder an den aromatischen Ringsystemen. Dabei gehen die großmaßstäbliche Produktion der Bisphenole sowie unser permanenter Kontakt mit bisphenolhaltigen Materialien mit einer verstärkten Nachweisbarkeit dieser Chemikalien in Umweltproben bzw. in menschlichen Geweben und Körperflüssigkeiten einher. Gleichzeitig stehen Bisphenole im Verdacht, aufgrund ihrer Toxizität und hormonähnlichen Wirkung eine Vielzahl von Erkrankungen zu begünstigen. Vor allem die Interaktion mit dem Östrogenrezeptor alpha ist gut verstanden. Daher wurde in dieser Arbeit die bakterielle Transformation von acht verschiedenen Bisphenolen (Bisphenol A, AP, B, C, E, F, PH und Z) durch die Umweltisolate Cupriavidus basilensis SBUG 290 und Bacillus amyloliquefaciens SBUG 1837 untersucht und näher charakterisiert. Beide Bakterienstämme waren in der Lage, alle eingesetzten Bisphenole zu transformieren. Dabei war die Transformation auf die Phenolringe oder daran befindliche Substituenten beschränkt. Ein mikrobieller Angriff an der ringverbindenden Kohlenstoffbrücke wurde nicht nachgewiesen. Während B. amyloliquefaciens die Schadstoffe ungeachtet ihrer Struktur jeweils phosphorylierte, transformierte C. basilensis die Bisphenole in Abhängigkeit von ihrer Struktur zu hydroxylierten Derivaten, Ringspaltungsprodukten, Produkten mit Acetamidstruktur oder zu Dimeren. Neben der Strukturaufklärung der mikrobiell gebildeten Produkte wurden die einzelnen Transformationswege für beide Bakterienstämme näher charakterisiert. Hierfür wurden u.a. die Produkte als Transformationssubstrate eingesetzt, der Einfluss der Kultivierung und Inkubationsmedien auf die Biotransformation untersucht sowie Proteomanalysen durchgeführt. Die Bildung von hydrophileren Transformationsprodukten durch die Bakterienstämme führte zur Detoxifizierung und Reduktion der östrogenen Aktivität der hydrophoben Bisphenole.Plastics pervade almost every sphere of our everyday life. For the production of certain plastic materials (epoxy resins and polycarbonate plastics) bisphenols are required as a basic component which consist of two phenol rings connected by a substituted carbon bridge. Individual bisphenols differ on varying substituents at the aromatic ring systems or at the carbon bridge. Large-scale bisphenol production as well as our permanent contact with bisphenol-containing materials entails an increased detectability of these chemicals in environmental samples or in human tissues and body fluids. Due to their toxicity and hormone-like effects, bisphenols are suspected to promote a variety of diseases. Their interaction with the estrogen receptor alpha is well studied. Therefore, in this work the bacterial transformation of eight different bisphenols (bisphenol A, AP, B, C, E, F, PH and Z) by the environmental isolates Cupriavidus basilensis SBUG 290 and Bacillus amyloliquefaciens SBUG 1837 was investigated and further characterized. Transformation was limited to the phenol rings or substituents attached to them. Microbial attack of the ring-connecting carbon bridge was not detected. Both bacterial strains were able to transform each applied bisphenol. While B. amyloliquefaciens phosphorylated the pollutants irrespective of their structure, C. basilensis transformed the bisphenols depending on their structure to hydroxylated derivatives, ring fission products, products with acetamide structure or dimers. In addition to the identification of the structure of the products formed, the individual transformation pathways were further characterized for both strains. Methods applied for this purpose included the use of products as transformation substrates, investigations on the influence of cultivation and incubation media on biotransformation as well as proteome analyses. Formation of these more hydrophilic products by the bacterial strains led to a detoxification and reduction of estrogenic activity of the hydrophobic bisphenols

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

Summary 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

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

Summary 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

Marine bacteroidetes use a conserved enzymatic cascade to digest diatom β-mannan

The polysaccharide β-mannan, which is common in terrestrial plants but unknown in microalgae, was recently detected during diatom blooms. We identified a β-mannan polysaccharide utilization locus (PUL) in the genome of the marine flavobacterium Muricauda sp. MAR_2010_75. Proteomics showed β-mannan induced translation of 22 proteins encoded within the PUL. Biochemical and structural analyses deduced the enzymatic cascade for β-mannan utilization. A conserved GH26 β-mannanase with endo-activity depolymerized the β-mannan. Consistent with the biochemistry, X-ray crystallography showed the typical TIM-barrel fold of related enzymes found in terrestrial β-mannan degraders. Structural and biochemical analyses of a second GH26 allowed the prediction of an exo-activity on shorter manno-gluco oligosaccharides. Further analysis demonstrated exo-α-1,6-galactosidase- and endo-β-1,4-glucanase activity of the PUL-encoded GH27 and GH5_26, respectively, indicating the target substrate is a galactoglucomannan. Epitope deletion assays with mannanases as analytic tools indicate the presence of β-mannan in the diatoms Coscinodiscus wailesii and Chaetoceros affinis. Mannanases from the PUL were active on diatom β-mannan and polysaccharide extracts sampled during a microalgal bloom at the North Sea. Together these results demonstrate that marine microorganisms use a conserved enzymatic cascade to degrade β-mannans of marine and terrestrial origin and that this metabolic pathway plays a role in marine carbon cycling

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 β-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