Biology subjects

Arntzen, M. O.

Publications and source records attributed to Arntzen, M. O..

9 recordsLinked to original sources

bioRxiv2024

Self-immunity towards a novel competence-induced streptococcal murein hydrolase is mediated by a Fem-transferase-like protein

Murein hydrolases (or peptidoglycan hydrolases) play diverse roles in bacteria, from controlled remodeling of the bacterial cell wall to lytic agents. In streptococci, a subset of these hydrolases is associated with competence-induced fratricide, a process where bacteria kill closely related cells to release DNA that can be taken up during natural transformation. Here, we characterize ScrM, a competence-induced murein hydrolase from Streptococcus dysgalactiae comprising a CHAP domain, an SH3b domain and an uncharacterized C-terminal domain (CCD). ScrM displayed lytic activity against pyogenic and salivarius group streptococci. Microscopy analysis of fluorescent fusions revealed that ScrM specifically localizes to the division zone of sensitive cells, with binding and localization mediated primarily by CCD. Upon competence induction, cells became immune to ScrM due to expression of ScrI, a Fem-transferase-like protein. We show by LC-MS/MS that ScrI incorporates Thr in place of Ala into the interpeptide bridges of peptidoglycan, which in turn prevents ScrM binding to the division zone, thereby protecting the cells from self-lysis during competence. ScrM and ScrI are conserved among pyogenic streptococcal pathogens and represent new players in the cell wall biogenesis of these bacteria that may form a platform for development of novel antimicrobial strategies.

microbiology↗

bioRxiv2024

Microbial consortia driving lignocellulose transformation in agricultural woodchip bioreactors

Freshwater ecosystems can be largely affected by neighboring agriculture fields where potential fertilizer nitrate run-off may leach into surrounding water bodies. To counteract this eutrophic driver, farmers in certain areas are utilizing denitrifying woodchip bioreactors (WBRs) in which a consortium of microorganisms convert the nitrate into nitrogen-gases in anoxia, fueled by the degradation of lignocellulose. Polysaccharide-degrading strategies have been well-described for various aerobic and anaerobic systems, including the use of carbohydrate-active enzymes, utilization of lytic polysaccharide monooxygenases (LPMOs) and other redox enzymes, as well as the use of cellulosomes and polysaccharide utilization loci (PULs). However, for denitrifying microorganisms, the lignocellulose-degrading strategies remain largely unknown. Here, we have applied a combination of enrichment techniques, gas measurements, multi-omics approaches, and amplicon sequencing of fungal ITS and procaryotic 16S rRNA genes to identify microbial drivers for lignocellulose transformation in woodchip bioreactors, and their active enzymes. Our findings highlight a microbial community enriched for lignocellulose-degrading denitrifiers with key players from Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota). A wide substrate specificity is observed among the many expressed carbohydrate active enzymes (CAZymes) including PULs from Bacteroidetes. This suggests a broad degradation of lignocellulose subfractions, even including enzymes with auxiliary activities whose functionality is still puzzling under strict anaerobic conditions. ImportanceFreshwater ecosystems face significant threats from agricultural runoff, which can lead to eutrophication and subsequent degradation of water quality. One solution to mitigate this issue is using denitrifying woodchip bioreactors (WBRs), where microorganisms convert nitrate into nitrogen gases utilizing lignocellulose as a carbon source. Despite the well-documented polysaccharide-degrading strategies in various systems, the mechanisms employed by denitrifying microorganisms in WBRs remain largely unexplored. This study fills a critical knowledge gap by revealing the degrading strategies of denitrifying microbial communities in WBRs. By integrating state-of-the-art techniques, we have identified key microbial drivers including Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota) playing significant roles in lignocellulose transformation and showcases a broad substrate specificity and complex metabolic capability. Our findings advance the understanding of microbial ecology in WBRs and by revealing the enzymatic activities, this research may inform efforts to improving water quality, protecting aquatic ecosystems, and reducing greenhouse gas emissions from WBRs.

microbiology↗

bioRxiv2022

Denitrifying bradyrhizobia retain strong N2O reduction during periods of starvation

Rhizobia living as microsymbionts inside nodules have stable access to carbon substrates, but also have to survive as free-living bacteria in soil where they are starved for carbon and energy most of the time. Many rhizobia can denitrify, thus switch to anaerobic respiration under low O2 tension using N-oxides as electron acceptors. The cellular machinery regulating this transition is relatively well-known from studies under optimal laboratory conditions, while little is known about this regulation in starved organisms. It is, for example, not known if the strong preference for N2O-over NO3 --reduction in bradyrhizobia is retained under carbon limitation. Here we show that starved cultures of a Bradyrhizobium strain with respiration rates 1-18% of well-fed cultures, reduced all available N2O before touching provided NO3-. Proteomics showed similar abundance of Nap (periplasmic NO3 - reductase) and NosZ (N2O reductase), suggesting that competition between electron pathways to Nap and NosZ favoured N2O reduction also in starved cells, similar to well-fed cultures. This contrasts the general notion that NosZ activity diminishes under carbon limitation. The results suggest that bradyrhizobia carrying NosZ can act as strong sinks for N2O under natural conditions and that this criterion should be considered in the development of biofertilizers. ImportanceLegume cropped farmlands account for substantial N2O emissions globally. Legumes are commonly inoculated with N2-fixing bacteria, rhizobia, to improve crop yields. Rhizobia belonging to Bradyrhizobium, the micro-symbionts of several economically important legumes, are generally capable of denitrification but many lack genes encoding N2O reductase and will be N2O sources. Bradyrhizobia with complete denitrification will instead act as sinks since N2O-reduction efficiently competes for electrons over nitrate reduction in these organisms. This phenomenon has only been demonstrated under optimal conditions and it is not known how carbon substrate limitation, which is the common situation in most soils, affects the denitrification phenotype. Here we demonstrate that bradyrhizobia retain their strong preference for N2O under carbon starvation. The findings add basic knowledge about mechanisms controlling denitrification and support the potential for developing novel methods for greenhouse gas mitigation based on legume inoculants with the dual capacity to optimize N2-fixation and minimize N2O emission.

microbiology↗

bioRxiv2021

The fish pathogen Aliivibrio salmonicida LFI1238 can degrade and metabolize chitin despite major gene loss in the chitinolytic pathway

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that Al. salmonicida grew efficiently on N-acetylglucosamine (GlcNAc) and chitobiose ((GlcNAc)2), the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate compared to the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both - and {beta}-chitin to (GlcNAc)2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity compared to other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on {beta}-chitin, whereas the LPMO gene-deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of Al. salmonicida LFI1238 growth on {beta}-chitin showed expression of all three chitinolytic enzymes, and intriguingly also three of the disrupted chitinases. In conclusion, our results show that Al. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability. IMPORTANCEThe ability to utilize chitin as a source of nutrients is important for the survival and spread of marine microbial pathogens in the environment. One such pathogen is Aliivibrio (Vibrio) salmonicida, the causative agent of cold water vibriosis. Due to extensive gene decay, many key enzymes in the chitinolytic pathway have been disrupted, putatively rendering this bacterium incapable of chitin degradation and utilization. In the present study we demonstrate that Al. salmonicida can degrade and metabolize chitin, the most abundant biopolymer in the ocean. Our findings shed new light on the environmental adaption of this fish pathogen.

biochemistry↗

bioRxiv2021

Critical Assessment of Metaproteome Investigation (CAMPI): a Multi-Lab Comparison of Established Workflows

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carried out the first community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluated the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observed that variability at the peptide level was predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappeared at the protein group level. While differences were observed for predicted community composition, similar functional profiles were obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

microbiology↗

bioRxiv2020

Competition for electrons favors N2O reduction in denitrifying Bradyrhizobium isolates

Bradyrhizobia are common members of soil microbiomes and known as N2-fixing symbionts of economically important legumes. Many are also denitrifiers, which can act as sinks or sources for N2O. Inoculation with compatible rhizobia is often needed for optimal N2-fixation, but the choice of inoculant may also have consequences for N2O emission. Here, we analyzed the phylogeny and denitrification capacity of Bradyrhizobium strains, most of them isolated from peanut-nodules. All were dinitrifiers, but only ~1/3 could reduce N2O while most others were net N2O producers. The N2O-reducing isolates showed strong preference for N2O- over NO3--reduction. Such preference was also observed in a study of other bradyrhizobia and tentatively ascribed to competition between the electron pathways to Nap (periplasmic NO3- reductase) and Nos (N2O reductase). Another possible explanation is lower abundance of Nap than Nos. Here, proteomics revealed that Nap was instead more abundant than Nos, supporting the hypothesis that the electron pathway to Nos outcompetes that to Nap. In contrast, Paracoccus denitrificans, which has membrane-bond NO3- reductase (Nar), reduced N2O and NO3- simultaneously. We propose that the control at the metabolic level, favoring N2O reduction over NO3- reduction, applies also to other denitrifiers carrying Nos and Nap but lacking Nar. Originality-Significance StatementThis study extends the current knowledge on denitrification in bradyrhizobia, which mostly originates from studies of one model strain, by investigating the denitrification phenotypes of a diverse collection of Bradyrhizobium isolates. Only 1/3 of them could reduce N2O while the others were net sources for this potent greenhouse gas. All N2O-reducers showed strong preference for N2O over NO3-. We revealed by proteomics that this was not explained by differences in the abundances of Nap (periplasmic nitrate reductase) and Nos (N2O reductase), which strengthens our hypothesis (Mania et al., 2020) of a metabolic control mechanism by which Nos competes efficiently with Nap for electrons, making these organisms strong sinks for N2O. The findings highlight the potential importance of these organisms as N2O sinks in natural and agricultural ecosystems and pinpoint the need to take N2O reduction into account, along with N2-fixation effectiveness, when searching for strains suitable for production of inoculants.

microbiology↗

bioRxiv2020

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 hours in nylon bags within the rumen of cannulated dairy cows. Across a gene catalogue covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of polysaccharides such as hemicellulose, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8 and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggests that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

microbiology↗

bioRxiv2019

Integration of absolute multi-omics reveals translational and metabolic interplay in mixed-kingdom microbiomes.

Microbiology is founded on well-known model organisms. For example, the majority of our fundamental knowledge regarding the quantitative levels of DNA, RNA, and protein backdates to keystone pure culture-based studies. Nowadays, meta-omic approaches allow us to directly access the molecules that constitute microbes and microbial communities, however due to a lack of absolute measurements, many original culture-derived "microbiology statutes" have not been updated or adapted to more complex microbiome settings. Within a cellulose-degrading and methanogenic consortium, we temporally measured genome-centric absolute RNA and protein levels per gene, and obtained a protein-to-RNA ratio of 102-104 for bacterial populations, whereas Archaeal RNA/protein dynamics (103-105: Methanothermobacter thermoautotrophicus) were more comparable to Eukaryotic representatives humans and yeast. The linearity between transcriptome and proteome had a population-specific change over time, highlighting a minimal subset of four functional carriers (cellulose degrader, fermenter, syntrophic acetate-oxidizer and methanogen) that coordinated their respective metabolisms, cumulating in the overarching community phenotype of converting polysaccharides to methane. Our findings show that upgrading multi-omic toolkits with traditional absolute measurements unlocks the scaling of core biological questions to dynamic and complex microbiomes, creating a deeper insight into inter-organismal relationships that drive the greater community function.

microbiology↗

bioRxiv2019

Proteome analysis of xylose metabolism in Rhodotorula toruloides during lipidproduction

BackgroundRhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids.\n\nResultsR. toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose.\n\nConclusionsThe results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.

microbiology↗