Biology subjects

van Loosdrecht, M.

Publications and source records attributed to van Loosdrecht, M..

3 recordsLinked to original sources

bioRxiv2020

A general approach to explore prokaryotic protein glycosylation reveals the unique surface layer modulation of an anammox bacterium.

The enormous chemical diversity and strain variability of prokaryotic protein glycosylation makes a large-scale exploration exceptionally challenging. Therefore, despite the universal relevance of protein glycosylation across all domains of life, the understanding of their biological significance and the evolutionary forces shaping oligosaccharide structures remains highly limited. Here, we report on a newly established mass binning glycoproteomics approach that establishes the chemical identity of the carbohydrate components and performs untargeted exploration of prokaryotic oligosaccharides from large-scale proteomics data directly. We demonstrate our approach by exploring an enrichment culture of the globally relevant anaerobic ammonium-oxidizing bacterium Ca. Kuenenia stuttgartiensis. By doing so we resolved a remarkable array of oligosaccharides, produced by two entirely unrelated glycosylation machineries targeting the same surface-layer protein (SLP) simultaneously. More intriguingly, the investigated strain also accomplished modulation of highly specialized sugars, supposedly in response to its energy metabolism--the anaerobic oxidation of ammonium --which depends on the acquisition of substrates of opposite charge. Ultimately, we provide a systematic approach for the compositional exploration of prokaryotic protein glycosylation, and reveal for the first time a remarkable balance between maximising cellular protection through a complex array of oligosaccharides and adhering to the requirements of the metabolic lifestyle.

systems biology↗

bioRxiv2020

Effects of light/dark diel cycles on the photoorganoheterotrophic metabolism of Rhodopseudomonas palustris for differential electron allocation to PHAs and H2

Light/dark cycles can impact the electron distribution in Rhodopseudomonas palustris, a hyperversatile photoorganoheterotrophic purple non-sulfur bacterium (PNSB). Dynamic conditions during diel cycles are important for the physiology of PNSB, but the coupling between illumination patterns and redox balancing has not been extensively studied. For survival and growth, Rhodopseudomonas has developed different mechanisms to allocate electrons under dynamic growth conditions. Products such as hydrogen and poly-{beta}-hydroxyalkanoates (PHAs) can form alternative electron sinks. A continuous culture, fed with a balanced nutrients medium, was exposed to three different conditions: 24 h continuous infrared illumination, 16h light/8h dark, and 8h light/16h dark. Light and dark phase durations in a cycle determined the energy availability level (light) and the attainment of a stationary state. Under long dark phases, the acetate substrate accumulated to levels that could not be depleted by growth in the light. Under short dark phases, acetate was rapidly consumed in the light with most of the phototrophic growth occurring under acetate-limiting conditions. Under diel cycles, substrate uptake and growth were unbalanced and Rhodopseudomonas shunted the excess of carbon and electron flow first toward PHAs production. Only secondarily, when PHA storage got saturated, the electron excess was redirected toward H2. A numerical model described well the dynamics of biomass and nutrients during the different light/dark cycle regimes. The model simulations allowed determination of stoichiometric and kinetic parameters for conversion by Rhodopseudomonas. Understanding the inherent process dynamics of diel light cycles in purple sulfur bacteria cultures would enable optimization procedures for targeted bioproduct formation. ImportancePurple non-sulfur bacteria (PNSB) are important anoxygenic phototrophic microorganisms that take part in numerous environmental processes, based on their metabolic versatility. Rhodopseudomonas palustris is a model photosynthetic bacterium of the PNSB guild. Light cycles influence deeply its physiology. Poly-{beta}-hydroxyalkanoates (PHAs) and biohydrogen are two of the most studied metabolic products of Rhodopseudomonas, because of their biotechnology potential besides involvement in carbon and electron allocations in its metabolism. Their production mechanisms have often been described as competitive, but the rationale behind the production of one or the other compound has not been elucidated. Here, we found that under light / dark cycles an excess of organic substrate was first directed toward PHAs production, and only when this pathway was saturated H2 was produced. Understanding the dynamics of carbon and electron allocation under intermittent light cycles enhances our knowledge on PNSB metabolisms and paves ways to manage the formation of targeted bioproducts.

microbiology↗

bioRxiv2020

A novel method to isolate free-floating extracellular DNA from wastewater for quantitation and metagenomic profiling of mobile genetic elements and antibiotic resistance genes

Antibiotic resistant genes (ARGs) and mobile genetic elements (MGEs) can be found in the free-floating extracellular DNA (eDNA) fraction of microbial systems. These xenogenic components can generate bacterial cells resistant to one or more antibiotics by natural transformation. Because of low concentration in wastewater, the obtaining of a high quality and a high yield of eDNA extract is challenging. We developed a method using chromatography to isolate eDNA without causing cell lysis (often unchecked) from complex wastewater matrices. The chromatographic step involved a diethylaminoethyl-cellulose-monolithic column to capture the eDNA found in cell-free filtered wastewater samples (e.g. influent wastewater, activated sludge and treated effluent wastewaster). Free-floating eDNA yields from 1 L of influent, activated sludge and treated effluent water reached 12.5 {+/-} 1.9 g, 12.3 {+/-} 1 g and 5.6 {+/-} 2.9 g of raw eDNA and 9.0 {+/-} 0.7 g, 5.6 {+/-} 0.46 g and 2.6 {+/-} 1.3 g of purified eDNA, respectively. In order to check the suitability of free-floating eDNA extracts for molecular analysis, qPCR and metagenomics were performed. eDNA extracts from treated effluent water were analyzed by qPCR to quantify a selected panel of ARGs and MGEs. Microbiome, resistome, and mobilome profiles from activated sludge free-floating eDNA were measured by metagenomic sequencing. Between iDNA and eDNA fractions, qPCR showed differences of 0.94, 1.11, 1.92 and 1.32 log10 gene copies mL-1 for sulfonamides resistant genes (sul1 and sul2), {beta}-lactamase resistance gene blaCTXM, and the class 1 integron-integrase (intI1) MGE, respectively. These differences highlighted the crucial need for an isolation method to discern both iDNA and eDNA to understand ARGs persistence and quantity in complex cultures. The eDNA yields obtained from 1 L of activated sludge (3.6 g of total suspended solids L-1) samples were substantially higher than the amount of DNA template needed for high-throughput sequencing (>1 g) in service facilities. Subsystems classification showed that the eDNA metagenome was mainly composed by MGEs (65.1%). The 35.9% rest related to traditional functional genetic signatures. It was the first time the resistome from the eDNA fraction was analyzed showing lower number of primary aligned reads when compared to the iDNA and a predominance of aminoglycosides and {beta}-lactamams. Metagenome results showed that eDNA can not be discarded as a pool of ARGs and MGEs for horizontal gene transfer. This novel isolation method was powerful to elucidate the molecular compositions of free-floating eDNA fractions in complex environmental samples such as wastewater environments at different microbial densities. Data obtained using this extraction method will foster xenogenic and microbial risk assessments across urban and natural water systems. This will support water authorities in the delineation of measures to adopt at wastewater treatment plants to remove them and safeguard environmental and public health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/072397v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1905305org.highwire.dtl.DTLVardef@3a774borg.highwire.dtl.DTLVardef@6e1262org.highwire.dtl.DTLVardef@790c42_HPS_FORMAT_FIGEXP M_FIG Picture created with BioRender C_FIG

molecular biology↗