The ecophysiology of nitrite-oxidizing bacteria in the genus Nitrospira

Ein Kernprozess des biogeochemischen Stickstoffzyklus in natürlichen Lebensräumen und technischen Anlagen (z. B. Kläranlagen) ist die Nitrifikation. Dieser aerobe Prozess untergliedert sich in zwei Stufen, der oxidativen Umwandlung von Ammoniak zu Nitrit bzw. von Nitrit zu Nitrat. Zwei verschiedene funktionelle Gruppen chemolithotropher Mikroorganismen sind dabei katalytisch an der Stoffumsetzung beteiligt: die Ammoniak oxidierenden Bakterien (AOB) bzw. Archaeen (AOA) und die Nitrit oxidierenden Bakterien (NOB). Ein Hauptaugenmerk in dieser Doktorarbeit wurde auf NOB der Gattung Nitrospira gelegt. Diese Gruppe von Bakterien ist maßgeblich an der Stickstoffeleminierung in Kläranlagen beteiligt und ist darüber hinaus weit verbreitet in den unterschiedlichsten aquatischen und terrestrischen Lebensräumen. Bisher ist wenig bekannt über die Ökophysiologie dieser meist unkultivierbaren NOB. In dieser Arbeit konnte zum ersten Mal auf Metagenomsequenzen dieser Bakteriengruppe zurückgegriffen werden und zusammen mit dem Einsatz verschiedener molekularbiologischer Techniken erschlossen sich bis dato unbekannte ökophysiologische Charakteristika dieser NOB. Der gekoppelte Einsatz von Fluoreszenz in situ Hybridisierung (FISH) und quantitativer Bildanalyse ergab Hinweise für eine Nischendifferenzierung zweier Nitrospira Populationen in einem Biofilm aufgrund ihrer Präferenz für verschiedene Nitritkonzentrationen. Weitere physiologische Adaptationen dieser NOB ergaben sich aus Metagenomanalysen. Nitrospira besitzt das detoxifizierende Enzym Chloritdismutase. Es ist gut möglich, dass Nitrospira den Abbau von Chlor-Komponenten mit der Nitrifizierung an kontaminierten Standorten koppelt. Die Genomdaten lieferten darüber hinaus erste Einblicke in das bisher unbekannte Nitrit-oxidierende System dieser NOB. Diese Doktorarbeit erweitert unser bisheriges Verständnis über die Ökophysiologie Nitrit-oxidierender Nitrospiren. Der gekoppelte Einsatz neuer molekularer Methoden mit ersten Metagenomdaten ermöglichte eine detaillierte Beschreibung dieser langsam wachsenden, meist unkultivierbaren NOB.Nitrification is a key process of the biogeochemical nitrogen-cycle in natural and engineered habitats. The two steps of aerobic nitrification, the oxidation of ammonia to nitrite and subsequently from nitrite to nitrate, are catalysed by two functional groups of chemolithotrophic prokaryotes: the ammonia-oxidizing bacteria (AOB) and archaea (AOA), and the nitrite-oxidizing bacteria (NOB). This thesis focused on the genus Nitrospira, one of the important but yet less intensively studied groups of NOB. Nitrospira-like bacteria are the dominant NOB in wastewater treatment plants and the most diverse known NOB in nature, being widespread in various aquatic and terrestrial habitats. However, ecophysiological and genomic data of these mostly uncultured NOB are either scarce or lacking. Molecular and, for the first time metagenomic data retrieved in the course of this thesis brought forward novel ecophysiological traits of these NOB. Fluorescence in situ hybridization and quantitative image analysis provided indication of a different spatial arrangement of members of two Nitrospira sub-populations in a biofilm suggesting a niche differentiation of the coexisting NOB with respect to their preferred concentrations of nitrite. Metagenome analyses revealed further ecophysiological adaptations of these NOB. Nitrospira harbours a gene encoding the detoxifying enzyme chlorite dismutase. Therefore, Nitrospira might link the bioremediation of chloroxo compounds with nitrogen turnover at contaminated sites. Moreover, the metagenomic data uncovered a unique nitrite-oxidizing system in this NOB. This thesis extends our knowledge of the ecophysiology of nitrite-oxidizing Nitrospira-like bacteria. Combining novel molecular methods with metagenomic data allowed in-depth characterization of this slow-growing, mostly uncultured NOB

Monitoring of total contents of copper in organically and conventionally managed soils. Part 1: Study plan and preliminary sampling of copper and other anthropogenic induced heavy metal contents in vineyard soils

The project aims to develop a survey designed for the representative assessment of copper contents in vineyard soils. The method development for the sampling of adequate monitoring areas is based on a preliminary survey of load distribution as well as on exposure assessment on selected areas of some typical vinery sites in Rheinland-Pfalz, differently managed and with different use history. Additionally to the sampling of selected areas, which reflects the spectrum of the load situation in viticulture, chemical quantitative analysis of soil samples collected from areas of known mode of cultivation and history followed by biological standard tests will be conducted. Obtained findings will serve for further method development, for preselection of adequate monitoring areas and for first indications of hazard potential. The project is a prerequisite for the performance of the long-term study “Development of current data of effects of copper contents in organic soils in viticulture, pomiculture and hopculture on soil life in terms of the strategypaper concerning further copper application in agriculture, especially in organic farming.

probeBase—an online resource for rRNA-targeted oligonucleotide probes: new features 2007

probeBase is a curated database of annotated rRNA-targeted oligonucleotide probes and supporting information. Rapid access to probe, microarray and reference data is achieved by powerful search tools and via different lists that are based on selected categories such as functional or taxonomic properties of the target organism(s) or the hybridization format (fluorescence in situ hybridization or microarray) in which the probes were applied. Additional information on probe coverage and specificity is available through direct submissions of probe sequences from probeBase to RDP-II and Greengenes, two major rRNA sequence databases. A freely editable user comments field for each probe entry allows any user to add, modify or remove information or to report errors in real-time. probeBase entries increased from 700 to more than 1200 during the past three years. Several options for submission of single probes or entire probe sets, even prior to publication of newly developed probes, should further contribute to keeping probeBase an up-to-date and useful resource. probeBase is freely accessible at . Email correspondence can be addressed to [email protected]

Tuberkulózis nyomai késő neolitikum - kora rézkori magyarországi emberi maradványokban (Alsónyék-Bátaszék, Dél-Magyarország)

Alsónyék-Bátaszék in Southern Hungary is one of the largest late Neolithic settlements and cemeteries excavated in Central Europe. In total, 2,359 burials from the Late Neolithic – Early Copper Age Lengyel culture were found between 2006 and 2009. Anthropological investigations previously carried out on individuals from this site revealed an interesting paleopathological case of tuberculosis in the form of Pott’s disease dated to the early 5th millennium BC. In this study, selected specimens from this osteoarcheological series were subjected to paleomicrobiological analysis to establish the presence of MTBC bacteria. As all individuals showing clear osteological signs of TB infection belonged to a single grave group, 38 individuals from this grave group were analysed. The sample included the case of Pott’s disease as well as individuals both with and without osseous TB manifestations. The presence of TB DNA in the individual with Pott’s disease was established, confirming the occurrence of TB in Neolithic populations of Europe. Moreover, our molecular analysis indicated that several other individuals of the same grave group were also infected with TB, opening the possibility for further analyses of this unique Neolithic skeletal series

Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge

Previously uncultured nitrite-oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira-like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira-like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 µg ml−1. The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA-targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira-like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as ‘Candidatus Nitrospira defluvii’

Metagenomic analysis of ancient dental calculus reveals unexplored diversity of oral archaeal Methanobrevibacter

Background Dental calculus (mineralised dental plaque) preserves many types of microfossils and biomolecules, including microbial and host DNA, and ancient calculus are thus an important source of information regarding our ancestral human oral microbiome. In this study, we taxonomically characterised the dental calculus microbiome from 20 ancient human skeletal remains originating from Trentino-South Tyrol, Italy, dating from the Neolithic (6000–3500 BCE) to the Early Middle Ages (400–1000 CE). Results We found a high abundance of the archaeal genus Methanobrevibacter in the calculus. However, only a fraction of the sequences showed high similarity to Methanobrevibacter oralis, the only described Methanobrevibacter species in the human oral microbiome so far. To further investigate the diversity of this genus, we used de novo metagenome assembly to reconstruct 11 Methanobrevibacter genomes from the ancient calculus samples. Besides the presence of M. oralis in one of the samples, our phylogenetic analysis revealed two hitherto uncharacterised and unnamed oral Methanobrevibacter species that are prevalent in ancient calculus samples sampled from a broad range of geographical locations and time periods. Conclusions We have shown the potential of using de novo metagenomic assembly on ancient samples to explore microbial diversity and evolution. Our study suggests that there has been a possible shift in the human oral microbiome member Methanobrevibacter over the last millennia

Genetic Predisposition of Atherosclerotic Cardiovascular Disease in Ancient Human Remains

Background: Several computed tomographic studies have shown the presence of atherosclerosis in ancient human remains. However, while it is important to understand the development of atherosclerotic cardiovascular disease (ASCVD), genetic data concerning the prevalence of the disease-associated single nucleotide polymorphisms (SNPs) in our ancestors are scarce. Objective: For a better understanding of the role of genetics in the evolution of ASCVD, we applied an enrichment capture sequencing approach to mummified human remains from different geographic regions and time periods. Methods: Twenty-two mummified individuals were analyzed for their genetic predisposition of ASCVD. Next-generation sequencing methods were applied to ancient DNA (aDNA) samples, including a novel enrichment approach specifically designed to capture SNPs associated with ASCVD in genome-wide association studies of modern humans. Findings: Five out of 22 ancient individuals passed all filter steps for calculating a weighted polygenic risk score (PRS) based on 87 SNPs in 56 genes. PRSs were correlated to scores obtained from contemporary people from around the world and cover their complete range. The genetic results of the ancient individuals reflect their phenotypic results, given that the only two mummies showing calcified atherosclerotic arterial plaques on computed tomography scans are the ones exhibiting the highest calculated PRSs. Conclusions: These data show that alleles associated with ASCVD have been widespread for at least 5,000 years. Despite some limitations due to the nature of aDNA, our approach has the potential to lead to a better understanding of the interaction between environmental and genetic influences on the development of ASCVD

Sensitive lipid biomarker detection for tuberculosis in late Neanderthal skeletons from Subalyuk Cave, Hungary

Skeletal remains of two Neanderthal individuals, a 25-35 year-old woman and a 3-4 year-old child, were discovered in a Subalyuk Cave in North-Eastern Hungary. Radiocarbon dating of the female and child remains revealed an age of 39,732-39,076 and 36,117-35,387 cal BP, respectively. Paleopathological studies of these Neanderthal remains revealed probable evidence of skeletal mycobacterial infection, including in the sacrum of the adult specimen and the endocranial surface of the child's skull. Application of PCR amplification to the juvenile cranium and a vertebra gave a positive result (IS6110) for tuberculosis, backed up by spoligotyping. Lipid biomarker analyses of the same two specimens revealed definitive signals for C32 mycoserosates, a very characteristic component of the Mycobacterium tuberculosis complex (MTBC). A vertebra from the adult provided weak evidence for mycocerosate biomarkers. The correlation of probable skeletal lesions with characteristic amplified DNA fragments and a proven lipid biomarker points to the presence of tuberculosis in these Neanderthals. In particular, the closely similar biomarker profiles, for two distinct juvenile cranial and vertebral bones, strengthen this diagnosis. </p