Cultivation of Flavobacteria and other in situ abundant bacteria from the North Sea

The isolation and cultivation of heterotrophic marine bacteria opens possibilities to study their physiology and genomes with respect to their function in the marine environment. In the pelagic marine realm bacteria remineralize more than half of the photosynthetically produced biomass, and thus play an important role in the biogeochemical cycling of elements. Flavobacteria are abundant of up to 30% in the North Sea. In previous studies marine Gammaproteobacteria, Alphaproteobacteria and Actinobacteria were predominantly cultivated, but cultures of Flavobacteria were infrequently obtained. This thesis addresses the isolation of phylogenetically diverse marine Flavobacteria using three new approaches. First, samples were retrieved from various pelagic and benthic habitats of the North Sea. Second, a new marine artificial seawater HaHa medium was developed to facilitate the growth of Flavobacteria. This medium was supplemented with saccharides and proteins as carbon sources at a concentration of 2 g/L. Third, a specific 16S rRNA gene PCR assay was applied to identify Flavobacteria-Cytophagia among the colonies. The molecular screen was preferred over the identification by cell and colony morphology, since the latter has predominantly resulted in the isolation of strains of the genera Arenibacter, Cellulophaga and Maribacter. The 375 Flavobacteriaceae strains isolated on agar plates comprised (i) seven presumably novel genera, (ii) 42 presumably novel species in 22 validly described Flavobacteriaceae genera and (iii) many isolates that were so far not distinguishable from 37 type strains in 16 genera. Thus, in contrast to previous studies, we could show that phylogenetically diverse Flavobacteria from the North Sea can be cultivated on solid medium. The isolation of representative strains of the genera Formosa, Polaribacter, and Reinekea from the North Sea was attempted. In previous studies these bacterial populations were proposed to be of importance during coastal diatom-dominated phytoplankton blooms, based on their high abundance of 15% to 25% of the bacterioplankton and their potential capability to decompose algae derived polysaccharides. A new medium was devised which had the same composition as the marine HaHa medium, but with environmental-like micromolar carbon, nitrogen, and phosphate concentrations. Aerobic dilution cultivation in the HaHa medium led to a high culturability of 35% of the bacterioplankton in spring 2010 and 27% of the bacterioplankton in summer 2010. 23 strains of Flavobacteria, Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were obtained directly by dilution cultivation of single cell inocula. One strain that belonged to the genus Reinekea was isolated by generating co-cultivatures of randomly mixed bacterial populations which potentially had a positive effect on the growth of Reinekea. Strains that affiliated with Polaribacter , Formosa, Gillisia (Flavobacteria), the Roseobacter clade associated (RCA) lineage (Alphaproteobacteria), Reinekea, and the OM182 clade (Gammaproteobacteria) had 16S rRNA gene sequence identities of >99.9% with 16S rRNA clones of the bacterioplankton from the North Sea in spring 2009. In addition, draft genomes of Formosa, Polaribacter , and Reinekea strains were used to recruit reads of metagenomes of the bacterioplankton in spring 2009. Thereby, reads of >95% nucleotide identity covered the draft genomes of the Formosa clade B strain to 94%, of Reinekea sp. to 90% and of Polaribacter sp. to 50%. Based on these results we argue that the novel species of Formosa, Polaribacter , and Reinekea are representatives of ecologically relevant clades catalyzing the remineralization of coastal diatom-dominated phytoplankton biomass. The physiological characteristics of the strains were investigated focusing on the growth on different mono- and polysaccharides, to provide further evidence that Formosa, Polaribacter and Reinekea species could prevail in different ecological niches during algae decay. Interestingly, Polaribacter strains grew heterotrophically on all tested sulfated (e.g. agar, carrageenan) and non-sulfated polysaccharides (e.g. cellulose, laminarin), whereas Formosa strains grew only on non-sulfated polysaccharides. In contrast, Reinekea sp. did not grow on polysaccharides but on all tested mono-, di-, and trisaccharides including N-acetylneuraminic acid. Finally, I proposed for these novel species the names Formosa flavarachnoidea , Formosa forsetii , Polaribacter forsetii , Polaribacter frigidimaris , Polaribacter adhaesivus , and Reinekea forsetii

Mycofactocin Is Associated with Ethanol Metabolism in Mycobacteria

Tuberculosis is caused by Mycobacterium tuberculosis, and the increasing emergence of multidrug-resistant strains renders current treatment options ineffective. Although new antimycobacterial drugs are urgently required, their successful development often relies on complete understanding of the metabolic pathways—e.g., cholesterol assimilation—that are critical for persistence and for pathogenesis of M. tuberculosis. In this regard, mycofactocin (MFT) function appears to be important because its biosynthesis genes are predicted to be essential for M. tuberculosisin vitro growth in cholesterol. In determining the metabolic basis of this genetic requirement, our results unexpectedly revealed the essential function of MFT in ethanol metabolism. The metabolic dysfunction thereof was found to affect the mycobacterial growth in cholesterol which is solubilized by ethanol. This knowledge is fundamental in recognizing the bona fide function of MFT, which likely resembles the pyrroloquinoline quinone-dependent ethanol oxidation in acetic acid bacteria exploited for industrial production of vinegar.Mycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in many Actinobacteria. Mycobacterium tuberculosis assimilates cholesterol during chronic infection, and its in vitro growth in the presence of cholesterol requires most of the MFT biosynthesis genes (mftA, mftB, mftC, mftD, mftE, and mftF), although the reasons for this requirement remain unclear. To identify the function of MFT, we characterized MFT biosynthesis mutants constructed in Mycobacterium smegmatis, M. marinum, and M. tuberculosis. We found that the growth deficit of mft deletion mutants in medium containing cholesterol—a phenotypic basis for gene essentiality prediction—depends on ethanol, a solvent used to solubilize cholesterol. Furthermore, functionality of MFT was strictly required for growth of free-living mycobacteria in ethanol and other primary alcohols. Among other genes encoding predicted MFT-associated dehydrogenases, MSMEG_6242 was indispensable for M. smegmatis ethanol assimilation, suggesting that it is a candidate catalytic interactor with MFT. Despite being a poor growth substrate, ethanol treatment resulted in a reductive cellular state with NADH accumulation in M. tuberculosis. During ethanol treatment, mftC mutant expressed the transcriptional signatures that are characteristic of respirational dysfunction and a redox-imbalanced cellular state. Counterintuitively, there were no differences in cellular bioenergetics and redox parameters in mftC mutant cells treated with ethanol. Therefore, further understanding of the function of MFT in ethanol metabolism is required to identify the cause of growth retardation of MFT mutants in cholesterol. Nevertheless, our results establish the physiological role of MFT and also provide new insights into the specific functions of MFT homologs in other actinobacterial systems

Proteiniphilum saccharofermentans str. M3/6T isolated from a laboratory biogas reactor is versatile in polysaccharide and oligopeptide utilization as deduced from genome-based metabolic reconstructions

Proteiniphilum saccharofermentans str. M3/6T is a recently described species within the family Porphyromonadaceae (phylum Bacteroidetes), which was isolated from a mesophilic laboratory-scale biogas reactor. The genome of the strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding biomass degradation and fermentation pathways. The P. saccharofermentans str. M3/6T genome consists of a 4,414,963 bp chromosome featuring an average GC-content of 43.63%. Genome analyses revealed that the strain possesses 3396 protein-coding sequences. Among them are 158 genes assigned to the carbohydrate-active-enzyme families as defined by the CAZy database, including 116 genes encoding glycosyl hydrolases (GHs) involved in pectin, arabinogalactan, hemicellulose (arabinan, xylan, mannan, β-glucans), starch, fructan and chitin degradation. The strain also features several transporter genes, some of which are located in polysaccharide utilization loci (PUL). PUL gene products are involved in glycan binding, transport and utilization at the cell surface. In the genome of strain M3/6T, 64 PUL are present and most of them in association with genes encoding carbohydrate-active enzymes. Accordingly, the strain was predicted to metabolize several sugars yielding carbon dioxide, hydrogen, acetate, formate, propionate and isovalerate as end-products of the fermentation process. Moreover, P. saccharofermentans str. M3/6T encodes extracellular and intracellular proteases and transporters predicted to be involved in protein and oligopeptide degradation. Comparative analyses between P. saccharofermentans str. M3/6T and its closest described relative P. acetatigenes str. DSM 18083T indicate that both strains share a similar metabolism regarding decomposition of complex carbohydrates and fermentation of sugars. © 2018 The Author

Complete Genome Sequence of a New Firmicutes Species Isolated from Anaerobic Biomass Hydrolysis

A new Firmicutes isolate, strain HV4-6-A5C, was obtained from the hydrolysis stage of a mesophilic and anaerobic two-stage lab-scale leach-bed system for biomethanation of fresh grass. It is assumed that the bacterial isolate contributes to plant biomass degradation. Here, we report a draft annotated genome sequence of this organism. © 2017 Abendroth et al

Förderung von Vögeln der Agrarlandschaft durch die Neuanlage von Brut- und Nahrungshabitaten

Die zunehmende Verschlechterung von Brut- und Nahrungshabitaten für Vögel der Agrarlandschaft zählt heute zu den wichtigsten Ursachen für deren steten Rückgang. Mit der Bildung großflächiger Bewirtschaftungseinheiten mit einseitigen Fruchtfolgen verschlechtern sich die Nahrungsbedingungen für die Vögel auf dem Feld. Nach der politischen Wende bestand schon Anfang der 1990er Jahre Bedarf an der Neustrukturierung ausgeräumter Agrarflächen, besonders in Ostdeutschland. 1993 wurde auf einer Ackerfläche am südlichen Stadtrand von Berlin die sogenannte “Brandenburger Schichtholzhecke” als Modellprojekt angelegt, um einen ökologischen und ökonomischen Weg zur Neuanlage von Hecken und Feldrainen aufzuzeigen. Diese naturnahen Kleinstrukturen bieten in ihrer Kombination sowohl Brut- als auch ganzjährig Nahrungshabitate und zeigen insbesondere unter den heutigen Bedingungen Möglichkeiten auf, um die Lebensbedingungen der Agrarvögel zu verbessern. Dabei ist das Konzept der „Benjeshecke“ modifiziert worden, indem zwischen zwei parallel zueinander verlaufenden Gestrüppwällen aus Totholz heimische Bäume und Sträucher einreihig gepflanzt wurden. Im Frühjahr 1994 ist ein mindestens 5 m breiter Wildkräuterstreifen zwischen buhnenförmigen Querwällen aus Totholz entlang der 575 m langen Hecke etabliert worden und beendete damit die Gestaltungsphase. Regelmäßige Vogelbestandserfassungen von 1995 bis 1998 und im Jahr 2004 zeigten die kontinuierliche Nutzung der Hecke als Lebensraum durch Agrarvögel. Schon 1994 konnten die ersten Neuntöter an den Totholzwällen beobachtet werden. 1995 diente der Saum als Brutrevier für Goldammer, Neuntöter, Steinschmätzer und Schafstelze. 1998 waren insbesondere die Gehölzstrukturen schon so weit entwickelt, dass die Dorngrasmücke erstmalig in der Hecke nistete. Im Jahr 2004, zehn Jahre nach Anlage der Hecke, brüteten 7 Vogelarten mit 13 Brutpaaren im Saum (Goldammer, Neuntöter, Steinschmätzer, Schafstelze, Dorngrasmücke, Stieglitz, Rotkehlchen). Die Goldammer mit fünf Brutpaaren war der häufigste Brutvogel. Insgesamt erreichte die Zahl der Brutreviere einen Wert von 2,3 je 100 lfd. Meter Hecke. Untersuchungen zum Auftreten von Schwebfliegen im Saum und dem angrenzenden Feld haben gezeigt, dass deren Aktivität auf den Krautstreifen 1995 fünf mal und 2004 sieben mal höher war als im 5 m Bereich der angrenzenden Ackerkultur. Da Schwebfliegen den Hauptbestandteil der Nestlingsnahrung z. B. für die Goldammer darstellen, belegen diese Zahlen die Bedeutung des Saumes als Nahrungshabitat.Stichwörter: Vögel, Hecken, Feldraine, NahrungPromotion of farmland birds by recreation of nesting and feeding habitatsAbstractThe increasing degradation of nesting and feeding habitats for farmland birds, is one of the main causes of their steady decline. With the increasing formation of large-scale fields with uniform crops, the food conditions deteriorate for the birds on the field. After the political turn in the beginning of the 90ies, there was also a need for the restructuring of cleared agricultural landscape in East Germany. In 1993, the so-called ‚Brandenburg stackedwood hedge‘ was created on a fi eld south to Berlin as a model to show an ecological and economic way for the replanting of hedges and fi eld margins. These small structures providing in their combination of both breeding and foraging habitat throughout the year. Under the current condition they are showing a way to improve the living conditions for birds on farmland. The ‚Brandenburg stacked-wood hedge‘ consists of two brush barriers of dead wood arranged in parallel. Between them, there is one row of native-species trees and shrubs planted. The 575 m long stacked hedge is adjoined by a five-metre-wide herbaceous strip established by sowing a suitable mixture of seed in 1994. Regular recording of birds from 1995 to 1998 and in 2004 shows the continuous use of the hedge as a habitat for birds. Already in 1994, the first red-backed shrikes were observed in the dead wood. In 1995, the dead wood provided a breeding ground for yellow-hammer, red-backed shrike, northern wheatear und yellow wagtail. In 1998, the shrubs had grown to a suitable size to shelter for the fi rst time white throat. In 2004, ten years after establishment of the hedgerow, 13 bird pairs of 7 species nested in the hedgerow - yellowhammer, red-backed shrike, northern wheatear, yellow wagtail, whitethroat, eurasian goldfi nch, european robin. The yellow-hammer occurred most frequently with fi ve pairs. Density of breeding birds totalled 2.3 pairs per 100 m hedgerow. Investigations on the occurrence of hoverflies in the edge biotop and the adjacent field have shown, that there activity on the wild herb strips were five times higher in 1995 and seven times higher in 2004 than in the 5 m region of the adjacent arable crop. Since hoverflies representing the main part of the nestling food (yellowhammer), these figures demonstrate the importance of the edge biotop as foraging habitat.Keywords: birds, hedges, field margins, food sourc

Complete genome sequence of a new clostridium sp. isolated from anaerobic digestion and biomethanation

Here, we present the genome sequence and annotation of the bacterial strain HV4-5-A1G, a potentially new Clostridium species. Based on its genomic data, this strain may act as a keystone microorganism in the hydrolysis of complex polymers, as well as in the different acidogenesis and acetogenesis steps during anaerobic digestion. © 2020 Hahnke et al

Draft Genome Sequence of a New Oscillospiraceae Bacterium Isolated from Anaerobic Digestion of Biomass

Here, we present the genome sequence and annotation of the novel bacterial strain HV4-5-C5C, which may represent a new genus within the family Oscillospiraceae (order Eubacteriales). This strain is a potential keystone species in the hydrolysis of complex polymers during anaerobic digestion of biomass. © 2020 Pascual et al

Complete Genome Sequence of a New Ruminococcaceae Bacterium Isolated from Anaerobic Biomass Hydrolysis

A new Ruminococcaceae bacterium, strain HV4-5-B5C, participating in the anaerobic digestion of grass, was isolated from a mesophilic two-stage laboratoryscale leach bed biogas system. The draft annotated genome sequence presented in this study and 16S rRNA gene sequence analysis indicated the affiliation of HV4-5- B5C with the family Ruminococcaceae outside recently described genera. © 2018 Hahnke et al

Complete genome sequence of a new Bacteroidaceae bacterium isolated from anaerobic biomass digestion

Here, we present the genome sequence and annotation of HV4-6-C5C, a bacterial strain isolated from a mesophilic two-stage laboratory-scale leach bed biogas reactor system. Strain HV4-6-C5C may represent a new genus of the family Bacteroidaceae and may have a key role in acidogenesis and acetogenesis steps during anaerobic biomass digestion. © 2019 Hahnke et al

The novel oligopeptide utilizing species Anaeropeptidivorans aminofermentans M3/9T, its role in anaerobic digestion and occurrence as deduced from large-scale fragment recruitment analyses

Research on biogas-producing microbial communities aims at elucidation of correlations and dependencies between the anaerobic digestion (AD) process and the corresponding microbiome composition in order to optimize the performance of the process and the biogas output. Previously, Lachnospiraceae species were frequently detected in mesophilic to moderately thermophilic biogas reactors. To analyze adaptive genome features of a representative Lachnospiraceae strain, Anaeropeptidivorans aminofermentans M3/9T was isolated from a mesophilic laboratory-scale biogas plant and its genome was sequenced and analyzed in detail. Strain M3/9T possesses a number of genes encoding enzymes for degradation of proteins, oligo- and dipeptides. Moreover, genes encoding enzymes participating in fermentation of amino acids released from peptide hydrolysis were also identified. Based on further findings obtained from metabolic pathway reconstruction, M3/9T was predicted to participate in acidogenesis within the AD process. To understand the genomic diversity between the biogas isolate M3/9T and closely related Anaerotignum type strains, genome sequence comparisons were performed. M3/9T harbors 1,693 strain-specific genes among others encoding different peptidases, a phosphotransferase system (PTS) for sugar uptake, but also proteins involved in extracellular solute binding and import, sporulation and flagellar biosynthesis. In order to determine the occurrence of M3/9T in other environments, large-scale fragment recruitments with the M3/9T genome as a template and publicly available metagenomes representing different environments was performed. The strain was detected in the intestine of mammals, being most abundant in goat feces, occasionally used as a substrate for biogas production.Peer Reviewe