Selective purification of catecholate, hydroxamate and α-hydroxycarboxylate siderophores with titanium dioxide affinity chromatography

Siderophores, high affinity iron chelators, play a key role in the uptake of iron by microorganisms and regulate many biological functions. Siderophores are categorized by their chelating group, e.g., catecholates, hydroxamates, α-hydroxycarboxylates. Natural concentrations of siderophores are often either too low or sample matrices are too complex for direct analysis by, e.g., liquid chromatography – mass spectrometry. Therefore, both concentration and purification are prerequisite for reliable analyses. However, a chromatographic technique that is selective for all siderophore classes and affords high levels of purification is lacking. We developed a titanium dioxide affinity chromatography (TDAC) solid-phase extraction (SPE) that affords the selective purification of these siderophore classes from complex sample matrices with recoveries up to 82%. The one-step purification removed most non-ligand sample ‘contaminants’, therefore, affording the straightforward identification of siderophore peaks in base peak chromatograms. As a proof of concept, the bioinformatic processing, dereplication of known features and selection of significant features in the TDAC eluates afforded a fast identification of six novel siderophores (woodybactines) from bacterial supernatants. We propose TDAC SPE as a fast and cost-effective methodology to screen for known or discover novel siderophores in natural samples in combination with untargeted bioinformatic processing by, e.g., XCMS. The method is scalable and yielded large amounts of highly purified siderophores from bacterial culture supernatants, providing an effective quantitative sample clean-up for, e.g., NMR structure elucidation

Polysaccharide utilization mechanisms under permanent low-temperature conditions in the Southern Ocean

Bakterien sind die diversesten Organismen auf der Welt. Obwohl eine Vielzahl an Bakterienarten bekannt ist, so sind doch die meisten Arten bisher unerforscht. Dies liegt unter anderem daran, dass die Mehrheit der Bakterien nicht kultivierbar ist. Gerade bei marinen Vertretern gilt die Kultivierung als besonders herausfordernd. Viele dieser Bakterien ernähren sich hauptsächlich von organischen Kohlenstoffverbindungen. Dabei stellen Algenbiomassen die größte Nährstoffquelle dieser heterotrophen Bakterien dar. Algen bestehen zu 70% aus Polysacchariden, die von den Bakterien verwertet werden können. Dazu benötigen die Bakterien eine entsprechende Enzymausstattung die meist zusammenhängend in Polysaccharide Utilization Loci (PUL) genomisch codiert ist. In dieser Arbeit wurde das im Antarktischen Ozean isolierte Modelbakterium Pseudoalteromonas distincta ANT/505, auf die Ausbildung von Membranstrukturen hinsichtlich des Polysaccharidabbaus untersucht. Viele marine Bakterien produzieren Membranausstülpungen wie Membranvesikel (MV) oder Anhänge (VC). Die Funktion dieser zellulären Strukturen ist weitestgehend unbekannt. In dieser Studie wurden P. distincta Zellen auf Alginat, Pektin und Pepton kultiviert und subzelluläre Proteomanalysen durchgeführt. Über mikroskopische Verfahren konnte gezeigt werden, dass die Bildung der MV und VC von der äußeren Membran ausgeht und unabhängig von der Nährstoffquelle und Wachstumsphase ist. Proteine, die für den Transport und den initialen Abbau von Polysacchariden verantwortlich sind, konnten in den MV und VC verstärkt detektiert werden. Zusätzlich wurden unter allen Bedingungen zwei alkalische Phosphatasen in den MV und VC abundant nachgewiesen. Die Ergebnisse zeigen eine kontinuierliche Bildung dieser Enzyme, welche in einer Phosphat-limitierenden Umgebung von Vorteil sein kann. Die verstärkte Abundanz von CAZymes, Transportern und alkalischen Phosphatasen deutet darauf hin, dass es in der Zelle eine Sortierung dieser in die MV und VC gibt. Die Membranausstülpungen führen zu einer Oberflächenvergrößerung der Bakterien, um in einer nährstofflimitierten Umgebung besser mit den Nährstoffen in Kontakt zu kommen. Des Weiteren kann durch die hier abundant vorkommenden Polysaccharid-verwertenden Enzyme schneller auf Änderungen im Nährstoffangebot reagiert werden. Somit dienen die Membranausstülpungen als „Fänger“ für Nährstoffe, was in einer diffusiven aquatischen Umwelt von Vorteil ist. Der Antarktische Ozean ist einer der am wenigsten untersuchten Meeresbereiche der Welt, da unter anderem die klimatischen Bedingungen große Herausforderungen darstellen. Der Antarktische Ozean spielt jedoch eine zentrale Rolle in der Aufnahme an atmosphärischen CO2 und im globalen Kohlenstoffkreislauf. Diese Vorgänge der biologischen Pumpe der Ozeane werden maßgeblich von marinen Bakterien bestimmt. Im Zuge des Klimawandels verändert sich auch die Antarktis durch ansteigende Temperaturen. Dies bedingt, dass sich auch die bakteriellen Gemeinschaften und deren Stoffwechselwege anpassen. Um besondere Stoffwechselleistungen heterotropher mariner Bakterien im Südlichen Ozean zu bestimmen, wurden an zwei verschiedenen Standorten Wasserproben sequenziell gefiltert und deren Mikrobiome detailliert untersucht. Dazu wurden Metagenom- und Metaproteomanalysen durchgeführt. Die Zusammensetzung der bakteriellen Gemeinschaft wurde über 16S-rDNA Sequenzierung bestimmt. Die Bakterien wurden in partikel-assoziiert und frei-lebend eingeteilt. Die generelle partikel-assoziierte Gemeinschaft zeigte keine grundlegenden Unterschiede, am häufigsten waren Bacteroidetes und Gammaproteobakterien zu finden. Innerhalb der Gammaproteobakterien waren die Alteromonadaceae und Colwelliaceae am Standort 2 (S2) abundanter als an Standort 1 (S1). Die frei-lebende bakterielle Gemeinschaft wurde an S1 von den Alphaproteobakterien, genauer der SAR11 Clade I, dominiert. Mit Hilfe des Metagenoms wurden die PULs an beiden Standorden identifiziert. Die PUL Zusammensetzung und die Substratspezifität war an beiden Standorten annähernd gleich, jedoch gab es leichte Unterschiede in der taxonomischen Zuordnung der PULs. Bei den im Metaproteom identifizierten PUL-assoziierten Proteinen handelte es sich hauptsächlich um TBDRs. Die taxonomische Zuordnung der Proteine zeigte erhebliche Unterschiede an beiden Standorten. An S2 waren die Gattungen Colwellia und Arcobacter deutlich erhöht im Vergleich zu S1, wo Candidatus Pelagibacter ubique, Planktomarina und Polaribacter am häufigsten zu finden waren. Die generelle funktionelle Charakterisierung der Proteine war an beiden Standorten gleich. Die proteomischen Abweichungen in den bakteriellen Gemeinschaften an beiden Standorten können nicht durch spezifische Stoffwechselwege erklärt werden, da für die abundanten Gruppen keine speziellen metabolischen Eigenschaften nachgewiesen werden konnten. Daher können beobachtete differentielle Genexpressionsmuster nur auf unterschiedliche Umweltparameter zurückzuführen sein. Zusätzlich zu den Wasserproben wurden auch Kultivierungsexperimente von gefiltertem Oberflächenwasser mit Pektin durchgeführt. Durch diesen Ansatz konnte ein PUL für die Verwertung von Rhamnogalakturonan, ein Baustein von Pektin, an Standort 1 nachgewiesen werden. Des Weiteren wurden neue Bakterien, die Pektin verwerten können, isoliert. Bei fünf von diesen Stämmen wurde ein shotgun-Genom erstellt. Dort konnten die Gene für den Pektinabbau identifiziert werden. Daten dieser Studie zeigen, dass die Stoffwechselphysiologie und Interaktion bakterieller Gemeinschaften weiterhin nur unvollständig verstanden sind.Marine bacteria represent the most diverse organisms in the marine environment. The majority of these microbes is unknown and unculturable. Algae represent the main nutrient source for bacteria. Macro- and microalgae can consist to 70% of polysaccharides. The metabolic degradation of marine polysaccharides is underexplored and thus these mechanisms have to be investigated. These mechanisms are of high importance to generate defined oligosaccharides for the medical and pharmaceutical applications. The specific structure of marine poly- and oligosaccharides show antiviral activities, e.g. carrageenans from red algae are used for the inhibition of human papillomavirus. Another alginate derived marine polysaccharide show inhibition of the replication of the human immunodeficiency virus (HIV). The degradation mechanisms of marine CAZymes and the structure of marine polysaccharides should be further investigated for their high potential of antiviral activities and the creation of new marine drugs. Many marine bacteria produce membrane extension like membrane vesicles or appendages but the function of these is poorly understood. In order to investigate their function, especially concerning polysaccharide utilization, proteomic analyses of subcellular compartments were performed. Microscopy analyses revealed that, beside MV, P. distincta forms different appendages, vesicle chains (VC) and thin filaments which were dedicated to extracellular polymeric substance. The formation of MV and VC was independent of growth phase or carbon source. The proteomic data showed that transporters end enzymes for the initial degradation of pectin and alginate were highly abundant in these membrane extensions and that there could be a kind of sorting for proteins in the membrane extensions. Additionally, two PUL encoded alkaline phosphatases and other phosphate acquiring enzymes were abundant in the MV and VC fractions. This indicates, that P. distincta constitutively produces enzymes for phosphate uptake, which would be necessary in the phosphate-limiting environment of the Southern Ocean. On the one hand marine bacteria produce membrane extensions in order to create a larger surface in the nutrient limiting marine environment for an increased chance to get in contact to nutrients and on the other hand the results indicate an accumulation of enzymes responsible for uptake and degradation of carbohydrates and phosphates in the MV and VC. Therefore, the membrane extensions act as nutrient traps and this might be beneficial for the bacteria in the diffuse aquatic environment. The microbial community structure and the metabolism of bacteria in the Southern Ocean are very poorly investigated. The SO is a harsh environment for all organism but nevertheless, the SO is of high importance for the climate in the world due to the high carbon dioxide uptake. In this study water samples from two different sampling sites (S1 and S2) in the SO were investigated. With a metagenomic and metaproteomic approach the key players and the metabolic activity were analyzed. Additionally, the surface water was inoculated with pectin and incubated for several days in order to analyze polysaccharide utilization loci for pectin degradation and to isolate new pectin degraders. 16S-rDNA analyses revealed the bacterial community from the genomic data. Bacteria were separated in particle-associated and free-living bacteria. The overall particle associated bacterial community at both sampling sites was comparable, with Bacteroidetes and Gammaproteobacteria as the abundant phylum. Within the Gammaproteobacteria the Alteromonadaceae and Colwelliaceae were more abundant at S2 than at S1. The free-living bacteria at S1 were dominated by the Alphaproteobacteria, especially the SAR11 clade I. Metagenomic analyses showed that both sampling sites had comparable PUL composition, but taxonomical classification of PULs was differently. The metaproteome data revealed that PUL encoded enzymes were not highly abundant. Only few CAZymes were found, mostly TonB-dependent transporters belonged to the detected PUL proteins. Taxonomical classification of proteins showed differences between the sampling sites. At S2 the genus Colwellia and Arcobacter were highly increased compared to S1. At this location Candidatus Pelagibacter, Planktomarina and Polaribacter were the abundant taxa. The functional classification at both sampling sites was comparable. The only difference was the high abundance of Epsilonproteobacteria at S2 referable to the Arcobacter species. Nevertheless, the notably taxonomical differences could not be explained by the proteomic data and the functional classification, because no specific metabolic function could be highly addressed to these bacteria. These results assumed that different abundance of the key players could be explained by different environmental conditions. The pectin enriched cultured at both sampling sites were investigated for the functional potential of pectin degrading enzymes. No metaproteomic approach could be performed due to less sampling material. Only one PUL for the degradation of rhamnogalacturonan, a component of pectin, was found at S1. In contrast, bacteria grown on pectin could be isolated from these samples. Genome sequencing of five isolates showed that functional potential of pectin degradation is available. Due to the limitations of sequence alignments, it was not possible to detect a PUL responsible for pectin utilization in the metagenomic data. The results show that the polysaccharide degradation mechanism in the Southern Ocean has to be more investigated to get knowledge about the bacterial activity in the ocean’s surface and the carbon turnover in this underexplored environment

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

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