Porphyromonas gingivalis is a keystone periodontal pathogen implicated in a variety of diseases ranging from periodontitis to neurodegenerative conditions. A hallmark of P. gingivalis is the constitutive production of a peptidyl-arginine deiminase (PPAD), which converts positively charged arginine residues into charge-neutral citrulline residues. The PPAD enzyme is known to be localized to the bacterial outer membrane and outer membrane vesicles (OMVs). Our present study was aimed at dissecting the roles of PPAD in the bacterial evasion of human macrophages and passage of cellular barriers in vitro. Here, we show that PPAD is required for activity of the arginine-specific proteases RgpA and RgpB, which are critical virulence factors of P. gingivalis. Furthermore, we show that the absence of PPAD leads to an increased OMV charge. Both changes impact the immune evasion strategy of P. gingivalis, as the PPAD-deficient bacteria display increased biofilm formation while their ability to evade and kill human macrophages is strongly reduced. In fact, PPAD-deficient bacteria and OMVs are more effectively internalized by macrophages and they elicit altered inflammatory macrophage responses, as demonstrated by reduced secretion of pro-inflammatory cytokines and major changes in the macrophage proteome. Intriguingly, we show that P. gingivalis OMVs have a high propensity for in vitro blood-brain barrier passage through transcytosis, which is significantly increased by PPAD-deficiency. This implies that OMV citrullination is a potentially critical determinant for trafficking of P. gingivalis-derived vesicles within the human host. Altogether, our present in vitro findings explain how PPAD shapes the immunomodulatory and invasive properties of P. gingivalis and its OMVs.

Fructans are ubiquitous in terrestrial ecosystems, however, these glycans are underexplored in the marine environment. We have discovered that the Antarctic gammaproteobacterium Pseudoalteromonas distincta is highly adapted to the degradation of fructose-containing substrates. This is enabled by proteins encoded in several genomic regions, including a fructan polysaccharide utilization locus (PUL). In addition to a glycoside hydrolase from family 32 (GH32), the fructan PUL encodes two proteins that have been described as specific for the phylum Bacteroidota and were previously unknown for the class Gammaproteobacteria (phylum Pseudomonadota): a glycan-binding SusD-like protein and a SusC-like TonB-dependent transporter (TBDT), which work as a complex in glycan import in Bacteroidota. Proteome, biochemical, sequence, and structural analyses indicate that the SusD-like protein and SusC-like TBDT of P. distincta mediate the uptake of inulin-type fructans, followed by degradation by a periplasmic exo-active GH32. In contrast, P. distincta likely degrades levan-type fructans via an extracellular endo-acting GH32 that is not encoded in the fructan PUL. Comparative genomics identified further SusD-like proteins and SusC-like TBDTs in Gammaproteobacteria, most of which are co-encoded with GH32s, indicative of fructan PULs, and are frequently associated with the marine habitat. Our study thus suggests that SusC/D-like complexes are not exclusive to the phylum Bacteroidota. It further shows that fructans contribute to the marine glycan pool and are targeted by specialized marine communities.

Microbial coexistence in complex communities requires mechanisms that minimize competition and optimize resource use. However, the mechanisms by which these ecological strategies are executed remain poorly understood. Here we show that bacteria modulate protein abundance in response to specific community members, reducing functional redundancy and promoting metabolic complementarity. Using synthetic gut-derived consortia exposed to distinct carbon sources, we systematically profiled proteomic responses of individual species across isolate, pairwise and 4-member communities. We found that biotic interactions, rather than abiotic conditions, were the dominant drivers of proteomic variation. These interactions led to reproducible, partner-specific expression shifts that significantly reduced functional overlap and were frequently associated with increased community productivity. Together, these findings highlight gene expression as a means by which microbes implement ecological strategies in community contexts. Through this regulatory plasticity, microbes dynamically reshape their realized niche through protein abundance modulation, enabling them to partition metabolic space and stabilize community structure.

Clostridioides difficile infection (CDI), primarily mediated by toxin B (TcdB), is a leading cause of healthcare-associated and community-acquired diarrhea worldwide. Although this genus exhibits substantial genomic diversity, functional studies have focused mainly on a limited set of isolates, leaving other genomospecies understudied in this regard. Here, we characterized five isolates derived from five distinct patients with suspected CDI cases that tested negative for the tcdC PaLoc marker. Integrated genomic, proteomic, and phenotypic analyses confirm that these strains constitute three novel toxin-producing species and their distinct carbon utilization profiles, exoproteomes, and spore protein repertoires suggest niche specialization within the human gut ecosystem. These species harbour monotoxin PaLoc architectures encoding TcdB7 or TcdB11, located on either chromosomal or extrachromosomal elements, which frequently co-occur with binary toxin loci. Functional assays confirmed secretion of active TcdB, cytotoxicity in mammalian cells, and moderate enterotoxicity in a murine ileal loop model, although virulence was attenuated relative to C. difficile R20291 from Clade 2. Collectively, these findings expand the taxonomy of toxigenic Clostridioides, highlight the dynamic evolution of TcdB-mediated pathogenicity, and emphasize the importance of refining diagnostic workflows and surveillance strategies to address emerging diarrheal diseases. The three novel species were designated as Clostridioides cryptodifficilis sp. nov., Clostridioides divergens sp. nov., and Clostridioides subdifficilis sp. nov.

SliP4 is a small, 37 amino acids protein that is strongly induced when the cyanobacterium Synechocystis sp. PCC 6803 is exposed to high-light (HL) conditions. Deletion mutants manifest a light-sensitive phenotype due to impaired cyclic electron flow and state transitions. In this study, we aimed to investigate the consequences of SliP4 deficiency on the process of high-light acclimation on systems level. Transcriptomic data revealed that the deletion mutant ΔsliP4 exhibited a wild-type-like gene regulatory response 30 minutes after the light intensity was increased from 50 to 250 μmol photons m-2 s-1, a process that is controlled by the RpaB-PsrR1 system. Proteome analysis showed consistent expression changes of many HL-regulated proteins. Metabolome analysis provided hints for a changed N and C metabolism in mutant cells compared to wild type. In addition, the mutant increased the production of extracellular polysaccharides causing the mutant cells to aggregate after the shift to HL. This effect corresponds to the upregulated expression of xssA-E and xssN-P genes for the production of the sulfated exopolysaccharide synechan. We interpret these observations as a response that counteracts the potential light stress effects caused by the impaired capacity for cyclic electron flow and state transitions in the ΔsliP4 mutant. Our results demonstrate that the unicellular cyanobacterium Synechocystis compensates for the loss of SliP4 and its crucial role by activating a genetic program for a population-level response that helps the cells to cope with HL conditions.

Infections caused by carbapenem-resistant Acinetobacter baumannii (A. baumannii; CRAb) are associated with high patient morbidity and mortality. The serious threat for human health imposed by CRAb was recently underscored by identification of close-to-untouchable carbapenem- and tetracycline-resistant isolates. Since outer membrane vesicles (OMVs) of Gram-negative bacteria may contribute to antimicrobial resistance, our present study was aimed at investigating OMVs produced by the first two carbapenem- and tetracycline-resistant A. baumannii isolates in Europe. These isolates, denoted CRAb1 and CRAb2, contain large, nearly identical plasmids that specify multiple resistances. Both isolates produce OMVs that were analyzed by differential light scattering, transmission electron microscopy and proteomics. By comparison with OMVs from the plasmid-free non-carbapenem-resistant A. baumannii isolate Ab1, which is an isogenic ancestor of the CRAb1 isolate, we show that plasmid carriage by the CRAb1 and CRAb2 isolates leads to an increased OMV size that is accompanied by increased diversity of the OMV proteome. Our analyses show that OMVs from CRAb1 and CRAb2 are major reservoirs of proteins involved in antimicrobial resistance, including the plasmid-encoded carbapenemases New Delhi metallo-β-lactamase-1 (NDM-1), and carbapenem-hydrolyzing oxacillinase OXA-97 (OXA-97). Here we report that these OMV-borne carbapenemases hydrolyze imipenem and protect otherwise carbapenem-sensitive A. baumannii and Escherichia coli (E. coli) isolates against this antibiotic. In conclusion, our findings demonstrate that OMVs from highly drug-resistant CRAb confer protection against last-resort antibiotics to non-resistant bacterial pathogens.

Marine sediments harbor extremely diverse microbial communities that contribute to global biodiversity and play an essential role in the functioning of ecosystems. However, the metaproteome of marine sediments is still poorly understood. The extraction of proteins from environmental samples is still a challenge, especially from marine sediments, due to the complexity of the matrix. Therefore, methods for protein extraction from marine sediments need to be improved. To develop an effective workflow for protein extraction for clayey sediments, we compared, combined and enhanced different protein extraction methods. The workflow presented here includes blocking of protein binding sites on sediment particles with high concentrations of amino acids, effective cell lysis by ultrasonic capture, electro-elution, and simultaneous fractionation of proteins. To test the protocol's efficacy, we added Escherichia coli cells to sediment samples before protein extraction. By using our refined workflow, we were able to identify a comparable number of E. coli proteins from the supplemented sediment to those from pure E. coli cultures. This new protocol will enable future studies to identify active players in clay-rich marine sediments and accurately determine functional biodiversity based on their respective protein complements.

Bacillus subtilis is a Gram-positive bacterium widely used in biotechnology due to its efficient secretion systems. Among these, the twin-arginine (Tat) pathway facilitates the export of fully folded cofactor-containing proteins across the cytoplasmic membrane. The TatAyCy translocase, which is expressed constitutively, is key to this process. Previous studies showed that this translocase not only consists of the TatAy and TatCy subunits, but that it also recruits the LiaH protein upon overexpression. Presumably, the recruitment of LiaH represents an intrinsic protective mechanism against the potentially detrimental effects of facilitating the membrane passage of large, fully folded proteins. However, to date the full spectrum of physiological consequences of protein translocation via TatAyCy has remained elusive. In this study, we employed a 14N/15N metabolic labeling approach combined with subcellular fractionation to quantitatively analyze proteomic changes in the cytoplasm, membrane, and extracellular milieu upon TatAyCy overexpression. Our findings show that high-level TatAyCy expression leads to a prolonged vegetative state and disrupts key cellular processes, including genetic competence, motility, chemotaxis, and biofilm formation. Notably, arginine metabolism emerges as a central factor in the cellular adaptation to TatAyCy-induced stress.

Bacterial small proteins impact diverse physiological processes, however, technical challenges posed by small size hampered their systematic identification and biochemical characterization. In our quest to uncover small proteins relevant for Salmonella pathogenicity, we previously identified YjiS, a 54 amino acid protein, which is strongly induced during this pathogen's intracellular infection stage. Here, we set out to further characterize the role of YjiS. Cell culture infection assays with Salmonella mutants lacking or overexpressing YjiS suggested this small protein to delay bacterial escape from macrophages. Mutant scanning of the protein's conserved, arginine-rich DUF1127 domain excluded a major effect of single amino acid substitutions on the infection phenotype. A comparative dual RNA-seq assay uncovered the molecular footprint of YjiS in the macrophage response to infection, with host effects related to oxidative stress and the cell cortex. Bacterial cell fractionation experiments demonstrated YjiS to associate with the inner membrane and proteins interacting with YjiS in pull-down experiments were enriched for inner membrane processes. Among the YjiS interactors was the two-component system SsrA/B, the master transcriptional activator of intracellular virulence genes and a suppressor of flagellar genes. Indeed, in the absence of YjiS, we observed elevated expression of motility genes and an increased number of flagella per bacterium. Together, our study points to a role for Salmonella YjiS as a membrane-associated timer of pathogen dissemination.

The hyphal nature of filamentous streptomycetes poses unique challenges to their multicellular lifestyle, since it requires organizing cellular functions at the scale of hundreds of micrometers length. Streptomycetes exhibit a strong and patchy autofluorescence of so far unknown origin in their hyphae that, as we demonstrate, is a natural property of filamentous actinobacteria. The foci are dynamic and evenly distributed throughout the hyphae, including the hyphal tips, where they are cell membrane-associated. Here, we resolved the high spatiotemporal dynamics of these foci during spore germination and vegetative growth in Streptomyces venezuelae. We isolated a fluorescent protein band and identified the responsible protein as dihydrolipoyl dehydrogenase LpdA. An lpdA deletion mutant lacked these fluorescent foci and showed minor deficiencies in growth and development. Heterologous LpdA production in E. coli and characterization of the enzyme verified that a flavin cofactor is responsible for the green autofluorescence. LpdA is highly conserved in actinomycetes as a part of multienzyme complexes involved in central metabolism. Such delocalized metabolic centers provide a potential solution to mycelial multicellular lifestyle, where diffusion becomes a major challenge. ### Competing Interest Statement The authors have declared no competing interest.

The accurate construction of computational models in systems biology heavily relies on the availability of quantitative proteomics data, specifically, absolute protein abundances. However, the complex nature of proteomics data analysis necessitates specialised expertise, making the integration of this data into models challenging. Therefore, the development of software tools that ease the analysis of proteomics data and bridge between disciplines is crucial for advancing the field of systems biology. We developed an open access Python-based software tool available either as downloadable library or as web-based graphical user interface (GUI). The pipeline simplifies the extraction and calculation of protein abundances from unprocessed proteomics data, accommodating a range of experimental approaches based on label-free quantification. Our tool was conceived as a versatile and robust pipeline designed to ease and simplify data analysis, thereby improving reproducibility between researchers and institutions. Moreover, the robust modular structure of Alpaca allows its integration with other software tools.

Phytoplankton blooms create a substrate-rich environment that supports the growth of bacterial planktonic heterotrophs. Previously, we studied the dynamics of such bacterioplankton at a long-term ecological research site near the coast of Helgoland Island (North Sea) once a day. Here, we present a novel dataset (available under the PRIDE-ID: PXD055396) indicating significant differences at the protein level in a semi-diurnal analysis. Using metaproteomics, we studied changes in the free-living (0.2-3 µm) bacterial community that occurred between early (7 am) and late (9 pm) sampling over 3 days. The results highlight the sensitivity, robustness, and reproducibility of mass spectrometry-based metaproteomic analyses to assess changes in the activities of the bacterioplankton communities. Taxonomic analyses revealed significant changes in the abundance of 65 bacterial genera. Particularly, proteins from the flavobacterial genera Candidatus Prosiliicoccus and Aurantivirga were significantly more abundant in the late samples. This comprehensive dataset highlights semi-diurnal changes in bacterial community composition and metabolic activity during a phytoplankton bloom that would have remained undetected with a once-per-day sampling approach.

Degradation of complex dietary fiber by gut microbes is essential for colonic fermentation, short-chain fatty acid production, and microbiome function. Ruminococcus bromii is the primary resistant starch (RS) degrader in humans, which relies on the amylosome, a specialized cell-bound enzymatic complex. To unravel its architecture, function, and the interplay among its components, we applied a holistic multilayered approach: Cryo-electron tomography reveals that the amylosome comprises a constitutive extracellular layer extending toward the RS substrate. Proteomics demonstrates remodeling of its contents across different growth conditions, with Amy4 and Amy16 comprising 60% of the amylosome in response to RS. Structural and biochemical analyses reveal complementarity and synergistic RS degradation by these enzymes. We demonstrate that amylosome composition and RS degradation are regulated at two levels: structural constraints and expression-driven shifts in enzyme proportions enforce enzyme proximity, which allows R. bromii to fine-tune its adaptation to dietary fiber and shape colonic metabolism.

The computational analysis of large proteomics datasets from gradient profiling or spatially resolved proteomics is often as crucial as experimental design. We present RAPDOR, a tool for intuitive analyzing and visualizing such datasets, based on the Jensen-Shannon distance and analysis of similarities between replicates, applied to the identification of RNA-binding proteins (RBPs) and spatial proteomics. First, we examine the in-gradient distribution profiles of protein complexes with or without RNase treatment (GradR) to identify RBPs in the cyanobacterium Synechocystis 6803. RBPs play pivotal regulatory and structural roles. Although numerous RBPs are well characterized, the complete set of RBPs remains unknown for any species. RAPDOR identifies 165 potential RBPs, including ribosomal proteins, RNA-modifying enzymes, and proteins not previously associated with RNA binding. High-ranking putative RBPs, such as ribosome hibernation factor LrtA/RaiA, phosphoglucomutase Sll0726, antitoxin Ssl2245, and preQ(1) synthase QueF predicted by RAPDOR but not the TriPepSVM algorithm, are experimentally validated, indicating the existence of uncharacterized RBP domains. These data are available online, providing a resource for RNase-sensitive protein complexes in cyanobacteria. We then show by reanalyzing existing datasets that RAPDOR effectively examines the intracellular redistribution of proteins upon growth factor stimulation. RAPDOR is a generic, non-parametric tool for analyzing highly complex datasets.

The yeast Komagataella phaffii (syn. Pichia pastoris) is a highly effective and well-established host for the production of recombinant proteins. The redox balance of its secretory pathway, which is multi-organelle dependent, is of high importance for producing secretory proteins. Redox imbalance and oxidative stress can significantly influence protein folding and secretion. Glutathione serves as the main redox buffer of the cell and cellular redox conditions can be assessed through the status of the glutathione redox couple (GSH-GSSG). Previous research often focused on the redox potential of the endoplasmic reticulum (ER), where oxidative protein folding and disulphide bond formation occur. In this study, in vivo measurements of the glutathione redox potential were extended to different subcellular compartments by targeting genetically encoded redox sensitive fluorescent proteins (roGFPs) to the cytosol, ER, mitochondria and peroxisomes. Using these biosensors, the impact of oxygen availability on the redox potentials of the different organelles was investigated in non-producing and producing K. phaffii strains in glucose-limited chemostat cultures. It was found that the transition from normoxic to hypoxic conditions affected the redox potential of all investigated organelles, while the exposure to hyperoxic conditions did not impact them. Also, as reported previously, hypoxic conditions led to increased recombinant protein secretion. Finally, transcriptome and proteome analyses provided novel insights into the short-term response of the cells from normoxic to hypoxic conditions.

As one of the most-consumed drugs worldwide, ibuprofen (IBU) reaches the environment in considerable amounts as environmental pollutant, necessitating studies of its biotransformation as potential removal mechanism. Here, we screened bacteria with known capabilities to degrade aromatic environmental pollutants, belonging to the genera Bacillus, Priestia (formerly also Bacillus), Paenibacillus, Mycobacterium, and Cupriavidus, for their ability to transform ibuprofen. We identified seven transformation products, namely 2-hydroxyibuprofen, carboxyibuprofen, ibuprofen pyranoside, 2-hydroxyibuprofen pyranoside, 4-carboxy-α-methylbenzene-acetic acid, 1-[4-(2-hydroxy-2-methylpropyl)phenyl]ethanone, and 2-hydroxyibuprofenmethyl ester. Based on our screening results, we focused on ibuprofen biotransformation by Priestia megaterium SBUG 518, to identify structures of transformation products, and to shed light on the drug’s impact on bacterial physiology. Biotransformation reactions by P. megaterium SBUG 518 involved (A) the hydroxylation of the isobutyl side chain at two positions, and (B) conjugate formation via esterification with a sugar molecule of the carboxylic group of ibuprofen and an ibuprofen hydroxylation product. Glycosylation seems to be a detoxification process, since the ibuprofen conjugate (ibuprofen pyranoside) was considerably less toxic than the parent compound to P. megaterium SBUG 518. Based on proteome profile changes and inhibition assays, cytochrome P450 systems appear to be central for ibuprofen transformation in P. megaterium SBUG 518. The toxic effect of ibuprofen appears to be caused by interference of the drug with different physiological pathways, especially sporulation.

Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative oral pathogen associated with periodontitis and systemic diseases. Seven serotypes of Aa are known, with serotypes a, b and c being most prevalent worldwide. Interestingly, serotype a, b and c isolates present differences in virulence. This focuses interest on their secreted virulence factors. Gram-negative bacteria evolved a specific protein secretion mechanism, based on the release of outer membrane vesicles (OMVs) with a protein cargo. The present study was therefore aimed at investigating whether differences in the protein cargo of OMVs could be associated with the differential virulence of Aa serotypes a, b or c. Accordingly, the different OMV proteomes were defined by mass spectrometry and infection assays were performed with human neutrophils that represent the main innate defense against oral pathogens like Aa. Subsequently, we correlated the OMV proteome data with the observed OMV-neutrophil interactions. A total of 276 OMV-associated proteins was identified, including 53 known virulence factors. Interestingly, OMVs from Aa isolates with different serotypes displayed similar protein cargo, but the relative quantities differed. OMVs of serotype a isolates were exceptional in carrying CRISPR proteins with a potential role in virulence. Intriguingly, Aa OMVs mostly coated the neutrophil surface, triggering formation of neutrophil extracellular traps (NETs). Conversely, the NETs captured Aa OMVs. Since the observed OMV-neutrophil interplay will occur at a distance from the OMV-producing bacteria, we postulate that it allows the bacteria to evade capture and elimination by neutrophils.

One of the most common post-translational modifications occurring in bacteria is proteolytic cleavage. Particularly, in secretory proteins this cleavage is observed for specific protein segments, named pro-peptides, which are frequently located between the signal peptide and the mature protein. Pro-peptides are known to serve two main functions, namely a chaperone function in protein folding and an enzyme-inhibiting function. To date, the contribution of pro-peptides to protein secretion is not fully understood. Therefore, the present study was aimed at assessing possible roles of the N-terminal pro-peptides from the S8 class subtilisin-like serine proteases Bpr, Vpr and WprA of Bacillus subtilis in protein secretion. To this end, the signal peptides of these proteases were fused to the alkaline phosphatase PhoA of Escherichia coli with or without the cognate pro-peptides. Subsequently, secretion of the PhoA protein and its activity were assessed in the B. subtilis type strain 168 or the genome-reduced strain IIG-Bs27-39 and correlated to the respective pro-peptide structure as predicted by AlphaFold. Altogether, the obtained results suggest that the contributions of the investigated pro-peptides to protein secretion in B. subtilis are related to a combination of their functions in protein folding, their own folding state, and the applied strain.

The successful sustainable cultivation of the well-known medicinal plant sundew on rewetted peatlands not only leads to the preservation of natural populations, but also provides a basis for the sustainable pharmaceutical use of the plant. The bioactive compounds of sundew, flavonoids and naphthoquinones, show biofilm-inhibiting properties against multidrug-resistant, ESBL-producing E. coli strains and open up new therapeutic possibilities. This study investigates the molecular mechanisms of these compounds in biofilm inhibition through proteomic analyses. Specific fractions of flavonoids and naphthoquinones, as well as individual substances like 7-methyljuglone and 2″-O-galloylhyperoside, are analyzed. Results show that naphthoquinones appear to act via central regulatory proteins such as OmpR and alter the stress response while flavonoids likely affect biofilm formation by creating an iron-poor environment through iron complexation and additionally influence polyamine balance, reducing intracellular spermidine levels. Further investigations including assays for iron complexation and analysis of polyamines confirmed the proteomic data. Safety evaluations through cytotoxicity tests in 3D cell cultures and the Galleria mellonella in vivo model confirm the safety of the extracts used. These findings highlight sundew as a promising candidate for new phytopharmaceuticals.

Carbohydrate esterases modify polysaccharides by removing different ester moieties thereby affecting their physicochemical properties and their accessibility by glycoside hydrolases. We determined the full-length structures of two members (Fl8CE20_II and PpCE20_II) from the carbohydrate esterase family 20 (CE20) by X-ray crystallography that feature an ancillary domain, inserted into the catalytic SGNH-hydrolase domain. Detailed structural analysis identifies a so far undescribed catalytic triad architecture which lacks the typical aspartate for polarization of the histidine but instead reveals a precisely coordinated water molecule mediating contact between the His and Asp. This coordinated water in the Ser-His-(H 2 O-Asp/Asn) motif, as further confirmed by mutational studies and by determination of kinetic constants, is crucial for catalytic activity. We therefore term this active site architecture a water-mediated catalytic triad.