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Basidiomycete fungi are the main decomposers of dead wood with an impact on the global carbon cycle. Their degradative mechanisms have been well-studied under aerobic conditions. Here, we study their activity in oxygen-depleted environments. We use metaproteomics in a field study to identify active wood-decomposing fungi and their enzymes at different depths from the wood surface, including in oxygen-depleted conditions. In vitro, we observe that the brown-rot fungus Fomitopsis pinicola can grow on wood in complete anoxia. Using 13 C solid-state NMR, we demonstrate the degradation of plant cell-wall polysaccharides and fungal growth in the absence of oxygen. Proteomic analyses reveal that F. pinicola switches from a Fenton chemistry-based process under aerobic conditions to the secretion of plant cell wall-active enzymes in anoxia. Our finding that wood decay fungi can thrive in complete anoxia provides a deeper understanding of lignocellulose degradation mechanisms in nature and raises opportunities for the development of bio-inspired anaerobic processes. The degradation of dead wood by basidiomycete fungi relies on Fenton chemistry under aerobic conditions. Here, Röllig et al. show that these fungi can also thrive and degrade wood in anoxia, switching from a Fenton chemistry-based process to the secretion of plant cell wall-active enzymes.

Abstract Pseudomonas species have evolved dynamic and intricate regulatory networks to fine-tune gene expression, with complex regulation occurring at every stage in the processing of genetic information. This approach enables Pseudomonas to generate precise individual responses to the environment in order to improve their fitness and resource economy. The weak correlations we observe between RNA and protein abundance highlight the significant regulatory contribution of a series of intersecting post-transcriptional pathways, influencing mRNA stability, translational activity and ribosome function, to Pseudomonas environmental responses. This review examines our current understanding of three major post-transcriptional regulatory systems in Pseudomonas spp.; Gac/Rsm, Hfq and RimK, and presents an overview of new research frontiers, emerging genome-wide methodologies, and their potential for the study of global regulatory responses in Pseudomonas. Pseudomonas species have evolved dynamic and intricate regulatory networks to quickly fine-tune gene expression in response to environmental stimuli.

Background Staphylococcus aureus is the predominant pathogen isolated in diabetic foot infections. Recently, the skin commensal bacterium, Helcococcus kunzii, was found to modulate the virulence of this pathogen in an in vivo model. This study aims to elucidate the molecular mechanisms underlying the interaction between these two bacterial species, using a proteomic approach. Results Our results reveal that H. kunzii can coexist and proliferate alongside S. aureus in a Chronic Wound Media (CWM), thereby mimicking an in vitro chronic wound environment. We noted that the secreted proteome of H. kunzii induced a transcriptional effect on S. aureus virulence, resulting in a decrease in the expression level of agrA, a gene involved in quorum sensing. The observed effect could be ascribed to specific proteins secreted by H. kunzii including polysaccharide deacetylase, peptidoglycan DD-metalloendopeptidase, glyceraldehyde-3-phosphate dehydrogenase, trypsin-like peptidase, and an extracellular solute-binding protein. These proteins potentially interact with the agr system, influencing S. aureus virulence. Additionally, the virulence of S. aureus was notably affected by modifications in iron-related pathways and components of cell wall architecture in the presence of H. kunzii. Furthermore, the overall metabolism of S. aureus was reduced when cocultured with H. kunzii. Conclusion Future research will focus on elucidating the role of these excreted factors in modulating virulence.

Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions as a rapid-response mechanism that tunes global gene translation in response to environmental signals.

Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome.

The colonization and persistence of Pseudomonas aeruginosa in chronically diseased lungs are driven by various virulence factors. However, pulmonary infections in cystic fibrosis (CF) patients are predominantly polymicrobial. While Achromobacter xylosoxidans is an opportunistic pathogen in these patients, its impact on P. aeruginosa virulence during co-infection remains largely unknown. This study investigated P. aeruginosa interaction with two clonally related A. xylosoxidans strains, Ax 198 and Ax 200, co-isolated from CF sputum. We found that the interaction was strain-dependent, with Ax 200 significantly reducing P. aeruginosa virulence in a zebrafish model, providing the first in vivo evidence of this interaction. Proteomic analysis revealed that P. aeruginosa proteome was differently impacted by the two A. xylosoxidans strains, with Ax 200 altering proteins involved in biofilm formation, swimming motility, iron acquisition, and secretion systems. These findings were validated by phenotypic assays, confirming that A. xylosoxidans affected major P. aeruginosa virulence phenotypes, including biofilm formation, swimming motility, and siderophore production. Genetic analysis confirmed that distinct regulatory mechanisms, including iron cycle pathways, may account for the strain-dependent effects. These findings reveal a novel multi-target competitive mechanism through which A. xylosoxidans significantly disrupts P. aeruginosa virulence.

One Health seeks to integrate and balance the health of humans, animals, and environmental systems, which are intricately linked through microbiomes. These microbial communities exchange microbes and genes, influencing not only human and animal health but also key environmental, agricultural, and biotechnological processes. Preventing the emergence of pathogens as well as monitoring and controlling the composition of microbiomes through microbial effectors including virulence factors, toxins, antibiotics, non-ribosomal peptides, and viruses holds transformative potential. However, the mechanisms by which these microbial effectors shape microbiomes and their broader functional consequences for host and ecosystem health remain poorly understood. Metaproteomics offers a novel methodological framework as it provides insights into microbial dynamics by quantifying microbial biomass composition, metabolic functions, and detecting effectors like viruses, antimicrobial resistance proteins, and non-ribosomal peptides. Here, we highlight the potential of metaproteomics in elucidating microbial effectors and their impact on microbiomes and discuss their potential for modulating microbiomes to foster desired functions. Graphical Abstract Word Cloud showing the abundance of keywords in combination with the “Microbiome” in PubMed NCBI. As abundance values, the rounded logarithm with the base of 2 hits were used and submitted to https://wordart.com/create . For microbiome, the number without any combination was used for calculation. The word cloud displays different aspects of microbiome research: (i.) sources of microbiomes (green), (ii.) interactions (purple), (iii.) involved taxa (red), (iv.) applied experimental approaches (blue), and (vi.) societal effects and recent or future applications (gray). FN-L_zSKWXUyi3FV_c3WKh Video Abstract

Effective regulation of primary carbon metabolism is critically important for bacteria to successfully adapt to different environments. We have identified an uncharacterised transcriptional regulator; RccR, that controls this process in response to carbon source availability. Disruption of rccR in the plant-associated microbe Pseudomonas fluorescens inhibits growth in defined media, and compromises its ability to colonise the wheat rhizosphere. Structurally, RccR is almost identical to the Entner-Doudoroff (ED) pathway regulator HexR, and both proteins are controlled by the same ED-intermediate; 2-keto-3-deoxy-6-phosphogluconate (KDPG). Despite these similarities, HexR and RccR control entirely different aspects of primary metabolism, with RccR regulating pyruvate metabolism (aceEF), the glyoxylate shunt (aceA, glcB, pntAA) and gluconeogenesis (pckA, gap). RccR displays complex and unusual regulatory behaviour; switching repression between the pyruvate metabolism and glyoxylate shunt/gluconeogenesis loci depending on the available carbon source. This regulatory complexity is enabled by two distinct pseudo-palindromic binding sites, differing only in the length of their linker regions, with KDPG binding increasing affinity for the 28 bp aceA binding site but decreasing affinity for the 15 bp aceE site. Thus, RccR is able to simultaneously suppress and activate gene expression in response to carbon source availability. Together, the RccR and HexR regulators enable the rapid coordination of multiple aspects of primary carbon metabolism, in response to levels of a single key intermediate.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and is continuing to spread rapidly around the globe. No effective vaccine is currently available to prevent COVID-19, and intense efforts are being invested worldwide into vaccine development. In this context, all technology platforms must overcome several challenges resulting from the use of an incompletely characterized new virus. These include finding the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles. Here, we describe a shotgun tandem mass spectrometry workflow, the data produced can be used to guide optimization of the conditions for viral amplification. In parallel, we analysed the changes occurring in the host cell proteome following SARS-CoV-2 infection to glean information on the biological processes modulated by the virus that could be further explored as potential drug targets to deal with the pandemic.

Metaproteomics enables functional insight into microbial communities by identifying and quantifying proteins in complex samples. Yet, heterogeneous analytical workflows and the lack of standardization across experimental and bioinformatics stages hinder reproducibility and comparability, limiting integration with other omics data. We here present a community-developed reporting checklist tailored to the specific needs of metaproteomics. We also outline current efforts to enable structured and interoperable metadata capture, drawing on standards from proteomics and microbiome research wherever possible. By promoting transparent reporting and advancing metadata practices, our recommendations aim to align metaproteomics more closely with FAIR principles and support reproducible and interoperable research practices. AKqcSWMLpN1xDKxGK6XHvB Video Abstract