Biology subjects

Schmitz, R. A.

Publications and source records attributed to Schmitz, R. A..

28 records · Page 2Linked to original sources

bioRxiv2022

Methanotrophs are vigorous H2S oxidizers using a sulfide:quinone oxidoreductase and a ba3-type terminal oxidase

Hydrogen sulfide (H2S) is produced in a wide range of anoxic environments where sulfate (SO42-) reduction is coupled to decomposition of organic matter. In the same environments, methane (CH4) is the end product of an anaerobic food chain and both H2S and CH4 diffuse upwards into oxic zones where aerobic microorganisms can utilize these gases. Methane-oxidizing bacteria are known to oxidize a major part of the produced CH4 in these ecosystems, mitigating the emissions of this potent greenhouse gas to the atmosphere. However, how methanotrophy is affected by toxic H2S is largely unexplored. Here, we show that a single microorganism can oxidize CH4 and H2S simultaneously. By oxidizing H2S, the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV can alleviate the inhibitory effects on CH4 oxidation. In response to H2S, strain SolV upregulated a type III sulfide:quinone oxidoreductase (SQR) and a sulfide-insensitive ba3-type terminal oxidase to dissipate the reducing equivalents derived from H2S oxidation. Through extensive chemostat cultivation of M. fumariolicum SolV we demonstrate that it converts high loads of H2S to elemental sulfur (S0). Moreover, we show chemolithoautotrophy by tracing 13CO2 fixation into new biomass by using H2S as sole energy source. Molecular surveys revealed several putative SQR sequences in a range of proteobacterial methanotrophs from various environments, suggesting that H2S detoxification is much more widespread in methanotrophs than previously assumed, enabling them to connect carbon and sulfur cycles in new ways.

microbiology↗

bioRxiv2022

An alternative tetrahydrofolate pathway for formaldehyde oxidation in verrucomicrobial methanotrophs: Primer design for folD and ftfL and transformation of E. coli

Methylotrophs make a living by using one-carbon compounds as energy and carbon source. Methanol (CH3OH) is utilized by various methylotrophs and is oxidized by a methanol dehydrogenase. The calcium-dependent methanol dehydrogenase MxaFI converts methanol to formaldehyde (CH2O). In addition to MxaFI, the lanthanide-dependent methanol dehydrogenase XoxF is found in a wide range of bacteria. XoxF isolated from the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV possesses an unusually high affinity for both methanol and formaldehyde and converts methanol to formate (HCOOH) in vitro. However, genomic analyses and biochemical studies on related XoxF methanol dehydrogenases have questioned whether these enzymes are dedicated to the conversion of formaldehyde to formate in vivo. Instead, the genes encoding the bifunctional enzyme FolD and the enzyme FtfL, which we detected in all verrucomicrobial methanotrophs, were proposed to form a formaldehyde oxidation pathway utilizing tetrahydrofolate as C1-carrier. folD and ftfL are expressed in M. fumariolicum SolV and most closely related to homologues found in the phyla Verrucomicrobia and Proteobacteria, respectively. Here, we designed primers targeting Mf-folD and Mf-ftfL and amplified these genes through PCR. The amplified genes were ligated into pET30a(+) vectors which were subsequently used for the successful transformation of E. coli XL1-Blue cells. The vectors can be used for heterologous expression and subsequent His-tag purification to biochemically investigate whether FolD and FtfL could form an alternative tetrahydrofolate pathway for formaldehyde oxidation in verrucomicrobial methanotrophs.

microbiology↗

bioRxiv2022

Bacteroides muris sp. nov. isolated from the cecum of wild-derived house mice

Two bacterial strains, KH365_2T and KH569_7, were isolated from the cecum contents of wild-derived house mice. The strains were characterized as Gram-negative, rod-shaped, strictly anaerobic, and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains were most closely related to Bacteroides uniformis ATCC 8492T. Whole genome sequences of KH365_2T and KH569_7 strains have a DNA G+C content of 46.02% and 46.03% mol, respectively. Most morphological and biochemical characteristics did not differ between the newly isolated strains and classified Bacteroides strains. However, the average nucleotide identity (ANI) and dDNA-DNA hybridization (dDDH) values clearly distinguished the two strains from described members of the genus Bacteroides. Here, we present the phylogeny, morphology, and physiology of a novel species of the genus Bacteroides and propose the name Bacteroides muris sp. nov., with KH365_2T (DSM XXX = CCUG XXX) as type strain.

microbiology↗

bioRxiv2021

Label-free Raman microspectroscopy for identifying virocells

Raman microspectroscopy has been thoroughly used to assess growth dynamics and heterogeneity of prokaryotic cells. Yet, little is known about how the chemistry of individual cells changes during infection with lytic viruses, resulting in so-called virocells. Here, we investigate biochemical changes of bacterial and archaeal cells of three different species in laboratory cultures before and after addition of their respective viruses using single-cell Raman microspectroscopy. By applying multivariate statistics, we identified significant differences in the spectra of single cells and cells after addition of lytic phage (phi6) for Pseudomonas syringae. A general ratio of wavenumbers that contributed the greatest differences in the recorded spectra was defined as an indicator for virocells. Based on reference spectra, this difference is likely attributable to an increase in nucleic acid vs. protein ratio of virocells. This method proved also successful for identification of Bacillus subtilis cells infected with phi29 displaying a decrease in respective ratio but failed for archaeal virocells (Methanosarcina mazei with Methanosarcina Spherical Virus) due to autofluorescence. Multivariate and univariate analyses suggest that Raman spectral data of infected cells can also be used to explore the complex biology behind viral infections of bacteria. Using this method, we confirmed the previously described two-stage infection of P. syringaes phi6 and that infection of B. subtilis by phi29 results in a stress response within single cells. We conclude that Raman microspectroscopy is a promising tool for chemical identification of Gram-positive and Gram-negative virocells undergoing infection with lytic DNA or RNA viruses. ImportanceViruses are highly diverse biological entities shaping many ecosystems across Earth. Yet, understanding the infection of individual microbial cells and the related biochemical changes remains limited. Using Raman microspectroscopy in conjunction with univariate and unsupervised machine learning approaches, we established a marker for identification of infected Gram-positive and Gram-negative bacteria. This non-destructive, label-free analytical method at single-cell resolution paves the way for future studies geared towards analyzing complex biological changes of virus-infected cells in pure culture and natural ecosystems.

microbiology↗

bioRxiv2020

Microbial regulation of hexokinase 2 links mitochondrial metabolism and cell death in colitis

Hexokinases (HK) catalyze the first step of glycolysis and thereby limit its pace. HK2 is highly expressed in the gut epithelium, plays a role in immune responses and is upregulated in inflammation and ulcerative colitis 1-3. Here, we examined the microbial regulation of HK2 and its impact on intestinal inflammation by generating mice lacking HK2 specifically in intestinal epithelial cells (Hk2{Delta}IEC). Hk2{Delta}IEC mice were less susceptible to acute intestinal inflammation upon challenge with dextran sodium sulfate (DSS). Analyzing the epithelial transcriptome from Hk2{Delta}IEC mice during acute colitis revealed downregulation of cell death signaling and mitochondrial dysfunction dependent on loss of HK2. Using intestinal organoids derived from Hk2{Delta}IEC mice and Caco-2 cells lacking HK2, we identified peptidyl-prolyl cis-trans isomerase (PPIF) as a key target of HK2-mediated regulation of mitochondrial permeability and repression of cell-death during intestinal inflammation. The microbiota strongly regulated HK2 expression and activity. The microbially-derived short-chain fatty acid (SCFA) butyrate repressed HK2 expression and oral supplementation protected wildtype but not Hk2{Delta}IEC mice from DSS colitis. Our findings define a novel mechanism how butyrate may act as a protective factor for intestinal barrier homeostasis and suggest targeted HK2 inhibition as a promising therapeutic avenue in intestinal inflammation.

immunology↗

bioRxiv2020

A comprehensive analysis of the global human gut archaeome from a thousand genome catalogue

The human gut microbiome plays an important role in health and disease, but the archaeal diversity therein remains largely unexplored. Here we report the pioneering analysis of 1,167 non-redundant archaeal genomes recovered from human gastrointestinal tract microbiomes across countries and populations. We identified three novel genera and 15 novel species including 52 previously unknown archaeal strains. Based on distinct genomic features, we warrant the split of the Methanobrevibacter smithii clade into two separate species, with one represented by the novel Candidatus M. intestini. Patterns derived from 1.8 million proteins and 28,851 protein clusters coded in these genomes showed substantial correlation with socio-demographic characteristics such as age and lifestyle. We infer that archaea are actively replicating in the human gastrointestinal tract and are characterized by specific genomic and functional adaptations to the host. We further demonstrate that the human gut archaeome carries a complex virome, with some viral species showing unexpected host flexibility. Our work furthers our current understanding of the human archaeome, and provides a large genome catalogue for future analyses to decipher its role and impact on human physiology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/392621v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@3e0060org.highwire.dtl.DTLVardef@8adb45org.highwire.dtl.DTLVardef@41c087org.highwire.dtl.DTLVardef@c6fdf9_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG HIGHLIGHTSO_LIThe human gut archaeome analysis reveals a previously unseen active diversity C_LIO_LIThe most abundant methanogen, Methanobrevibacter smithii, splits into two species C_LIO_LIArchaeal protein catalogue can predict geography, demographics and health aspects C_LIO_LIHost-associated and environmental archaea show distinct genomic & functional traits C_LI

bioinformatics↗

bioRxiv2020

Small protein 26 interacts and enhances glutamine synthetase activity in Methanosarcina mazei

Small ORFs (sORF) encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding and non-coding predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved within numerous Methanosarcina strains on the amino acid as well as on nucleotide level strongly arguing for a cellular function of the small protein. spRNA26 is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. His-tagged sP26 was heterologously expressed and purified by fractionated ammonium sulfate precipitation, affinity chromatography and size exclusion centrifugation. Using independent biochemical approaches (pull-down by affinity chromatography followed by MS analysis, revers pull-down, microscale thermophoresis and size exclusion chromatography) we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1) with high affinity (app. KD = 45 +/- 14 {micro}M). Upon interaction with sP26, GlnA1 activity was significantly stimulated independently and in addition to the known activation by the metabolite 2-oxoglutarate. Besides strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (KD= 1.4 {micro}M +/- 0.9 {micro}M). On the basis of these findings, we hypothesize that in addition to 2-oxoglutarate, sP26 activates GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1.

biochemistry↗

bioRxiv2020

Histone variants in archaea and the evolution of combinatorial chromatin complexity

Nucleosomes in eukaryotes act as platforms for the dynamic integration of epigenetic information. Post-translational modifications are reversibly added or removed and core histones exchanged for paralogous variants, in concert with changing demands on transcription and genome accessibility. Histones are also common in archaea. Their role in genome regulation, however, and the capacity of individual paralogs to assemble into histone-DNA complexes with distinct properties remain poorly understood. Here, we combine structural modelling with phylogenetic analysis to shed light on archaeal histone paralogs, their evolutionary history and capacity to generate complex combinatorial chromatin states through hetero-oligomeric assembly. Focusing on the human commensal Methanosphaera stadtmanae as a model archaeal system, we show that the heteromeric complexes that can be assembled from its seven histone paralogs vary substantially in DNA binding affinity and tetramer stability, occupying a large but densely populated chromatin state space. Using molecular dynamics simulations, we go on to identify unique paralogs in M. stadtmanae and Methanobrevibacter smithii that are characterized by unstable dimer:dimer interfaces. We propose that these paralogs act as capstones that prevent stable tetramer formation and extension into longer oligomers characteristic of model archaeal histones. Importantly, we provide evidence from phylogeny and genome architecture that these capstones, as well as other paralogs in the Methanobacteriales, have been maintained for hundreds of millions of years following ancient duplication events. Taken together, our findings indicate that at least some archaeal histone paralogs have evolved to play distinct and conserved functional roles, reminiscent of eukaryotic histone variants. We conclude that combinatorially complex histone-based chromatin is not restricted to eukaryotes and likely predates their emergence.

evolutionary biology↗

bioRxiv2019

Comparative analysis of amplicon and metagenomic sequencing methods reveals key features in the evolution of animal metaorganisms

BackgroundThe interplay between hosts and their associated microbiome is now recognized as a fundamental basis of the ecology, evolution and development of both players. These interdependencies inspired a new view of multicellular organisms as \"metaorganisms\". The goal of the Collaborative Research Center \"Origin and Function of Metaorganisms\" is to understand why and how microbial communities form long-term associations with hosts from diverse taxonomic groups, ranging from sponges to humans in addition to plants.\n\nMethodsIn order to optimize the choice of analysis procedures, which may differ according to the host organism and question at hand, we systematically compared the two main technical approaches for profiling microbial communities, 16S rRNA gene amplicon- and metagenomic shotgun sequencing across our panel of ten host taxa. This includes two commonly used 16S rRNA gene regions and two amplification procedures, thus totaling five different microbial profiles per host sample.\n\nConclusionWhile 16S rRNA gene-based analyses are subject to much skepticism, we demonstrate that many aspects of bacterial community characterization are consistent across methods and that metagenomic shotgun results are largely dependent on the employed pipeline. The resulting insight facilitates the selection of appropriate methods across a wide range of host taxa. Finally, by contrasting taxonomic and functional profiles and performing phylogenetic analysis, we provide important and novel insight into broad evolutionary patterns among metaorganisms, whereby the transition of animals from an aquatic to a terrestrial habitat marks a major event in the evolution of host-associated microbial composition.

microbiology↗

bioRxiv2018

The Neutral Metaorganism

Almost all animals and plants are inhabited by diverse communities of microorganisms, the microbiota. The hosts often pose highly selective environments, which only a subset of the environmentally available microbes are able to colonize. From the hosts perspective it seems useful to shape the community composition of these allowed microbes to promote a beneficial host-microbe symbiosis. In contrast to this, neutral models assume that the structure of the microbiota is entirely shaped by random population dynamics and dispersal. We here show that microbiota community structure from a wide range of host organisms, in particular including previously understudied invertebrates, is in almost all cases consistent with neutral expectations. Moreover, we demonstrate that apparent discrepancies with the neutral model can be due to transient community states rather than host selection. In conclusion, even though hosts are often assumed to control microbiota composition to ensure beneficial interactions, our broad-scale analysis highlights that following colonization, it could rather be neutral processes that determine microbial community structure.

ecology↗