Acquisition of Phage Sensitivity by Bacteria through Exchange of Phage Receptors.

Bacteriophages (phages) typically exhibit a narrow host range, yet they tremendously impact horizontal gene transfer (HGT). Here, we investigate phage dynamics in communities harboring phage-resistant (R) and sensitive (S) bacteria, a common scenario in nature. Using Bacillus subtilis and its lytic phage SPP1, we demonstrate that R cells, lacking SPP1 receptor, can be lysed by SPP1 when co-cultured with S cells. This unanticipated lysis was triggered in part by phage lytic enzymes released from nearby infected cells. Strikingly, we discovered that occasionally phages can invade R cells, a phenomenon we termed acquisition of sensitivity (ASEN). We found that ASEN is mediated by R cells transiently gaining phage attachment molecules from neighboring S cells and provide evidence that this molecular exchange is driven by membrane vesicles. Exchange of phage attachment molecules could even occur in an interspecies fashion, enabling phage adsorption to non-host species, providing an unexplored route for HGT. VIDEO ABSTRACT.

Antiviral activity of bacterial TIR domains via immune signalling molecules.

The Toll/interleukin-1 receptor (TIR) domain is a canonical component of animal and plant immune systems1,2. In plants, intracellular pathogen sensing by immune receptors triggers their TIR domains to generate a molecule that is a variant of cyclic ADP-ribose3,4. This molecule is hypothesized to mediate plant cell death through a pathway that has yet to be resolved5. TIR domains have also been shown to be involved in a bacterial anti-phage defence system called Thoeris6, but the mechanism of Thoeris defence remained unknown. Here we show that phage infection triggers Thoeris TIR-domain proteins to produce an isomer of cyclic ADP-ribose. This molecular signal activates a second protein, ThsA, which then depletes the cell of the essential molecule nicotinamide adenine dinucleotide (NAD) and leads to abortive infection and cell death. We also show that, similar to eukaryotic innate immune systems, bacterial TIR-domain proteins determine the immunological specificity to the invading pathogen. Our results describe an antiviral signalling pathway in bacteria, and suggest that the generation of intracellular signalling molecules is an ancient immunological function of TIR domains that is conserved in both plant and bacterial immunity.

Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status.

Potential Mycobacterium tuberculosis (Mtb) transmission during different pulmonary tuberculosis (TB) disease states is poorly understood. We quantified viable aerosolized Mtb from TB clinic attendees following diagnosis and through six months' follow-up thereafter. Presumptive TB patients (n=102) were classified by laboratory, radiological, and clinical features into Group A: Sputum-Xpert Ultra-positive TB (n=52), Group B: Sputum-Xpert Ultra-negative TB (n=20), or Group C: TB undiagnosed (n=30). All groups were assessed for Mtb bioaerosol release at baseline, and subsequently at 2 wk, 2 mo, and 6 mo. Groups A and B were notified to the national TB program and received standard anti-TB chemotherapy; Mtb was isolated from 92% and 90% at presentation, 87% and 74% at 2 wk, 54% and 44% at 2 mo and 32% and 20% at 6 mo, respectively. Surprisingly, similar numbers were detected in Group C not initiating TB treatment: 93%, 70%, 48% and 22% at the same timepoints. A temporal association was observed between Mtb bioaerosol release and TB symptoms in all three groups. Persistence of Mtb bioaerosol positivity was observed in ~30% of participants irrespective of TB chemotherapy. Captured Mtb bacilli were predominantly acid-fast stain-negative and poorly culturable; however, three bioaerosol samples yielded sufficient biomass following culture for whole-genome sequencing, revealing two different Mtb lineages. Detection of viable aerosolized Mtb in clinic attendees, independent of TB diagnosis, suggests that unidentified Mtb transmitters might contribute a significant attributable proportion of community exposure. Additional longitudinal studies with sputum culture-positive and -negative control participants are required to investigate this possibility.

Alternating access of a bacterial homolog of neurotransmitter: sodium symporters determined from AlphaFold2 ensembles and DEER spectroscopy.

Neurotransmitter:sodium symporters (NSSs) play critical roles in neural signaling by regulating neurotransmitter uptake into cells powered by sodium electrochemical gradients. Bacterial NSSs orthologs, including MhsT from Bacillus halodurans, have emerged as model systems to understand the structural motifs of alternating access in NSSs and the extent of conservation of these motifs across the family. Here, we apply a computational/experimental methodology to illuminate the conformational landscape of MhsT alternating access. Capitalizing on our recently developed method, Sampling Protein Ensembles and Conformational Heterogeneity with AlphaFold2 (SPEACH_AF), we derived clusters of MhsT models spanning the transition from inward-facing to outward-facing conformations. Systematic application of double electron-electron resonance (DEER) spectroscopy revealed ligand-dependent movements of multiple structural motifs that underpin MhsT's conformational cycle. Remarkably, comparative DEER analysis in detergent micelles and lipid nanodiscs highlights the profound effect of the environment on the energetics of conformational changes. Through experimentally derived selection of collective variables, we present a model of ion and substrate-powered transport by MhsT consistent with the conformational cycle derived from DEER. Our findings not only advance the understanding of MhsT's function but also uncover motifs of conformational dynamics conserved within the broader context of the NSS family and within the LeuT-fold class of transporters. Importantly, our methodological blueprint introduces an approach that can be applied across a diverse spectrum of transporters to describe their conformational landscapes.

The quorum-sensing peptidic inhibitor rescues host immune system eradication: A novel infectivity mechanism.

Subverting the host immune system is a major task for any given pathogen to assure its survival and proliferation. For the opportunistic human pathogen Bacillus cereus (Bc), immune evasion enables the establishment of potent infections. In various species of the Bc group, the pleiotropic regulator PlcR and its cognate cell-cell signaling peptide PapR7 regulate virulence gene expression in response to fluctuations in population density, i.e., a quorum-sensing (QS) system. However, how QS exerts its effects during infections and whether PlcR confers the immune evading ability remain unclear. Herein, we report how interception of the QS communication in Bc obliterates the ability to affect the host immune system. Here, we designed a peptide-based QS inhibitor that suppresses PlcR-dependent virulence factor expression and attenuates Bc infectivity in mouse models. We demonstrate that the QS peptidic inhibitor blocks host immune system-mediated eradication by reducing the expression of PlcR-regulated major toxins similarly to the profile that was observed for isogenic strains. Our findings provide evidence that Bc infectivity is regulated by QS circuit-mediated destruction of host immunity, thus reveal a interesting strategy to limit Bc virulence and enhance host defense. This peptidic quorum-quenching agent constitutes a readily accessible chemical tool for studying how other pathogen QS systems modulate host immunity and forms a basis for development of anti-infective therapeutics.

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX.

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.

Bacillus B2 promotes root growth and enhances phosphorus absorption in apple rootstocks by affecting MhMYB15.

Due to the chelation of phosphorus in the soil, it becomes unavailable for plant growth and development. The mechanisms by which phosphorus-solubilizing bacteria activate immobilized phosphorus to promote the growth and development of woody plants, as well as the intrinsic molecular mechanisms, are not clear. Through the analysis of microbial communities in the rhizosphere 16S V3-V4 and a homologous gene encoding microbial alkaline phosphomonoesterase (phoD) in phosphate-efficient (PE) and phosphate-inefficient apple rootstocks, it was found that PE significantly enriched beneficial rhizobacteria. The best phosphorus-solubilizing bacteria, Bacillus sp. strain 7DB1 (B2), was isolated, purified, and identified from the rhizosphere soil of PE rootstocks. Incubating with Bacillus B2 into the rhizosphere of apple rootstocks significantly increased the soluble phosphorus and flavonoid content in the rhizosphere soil. Simultaneously, this process stimulates the root development of the rootstocks and enhances plant phosphorus uptake. After root transcriptome sequencing, candidate transcription factor MhMYB15, responsive to Bacillus B2, was identified through heatmap and co-expression network analysis. Yeast one-hybrid, electrophoretic mobility shift assay, and LUC assay confirmed that MhMYB15 can directly bind to the promoter regions of downstream functional genes, including chalcone synthase MhCHS2 and phosphate transporter MhPHT1;15. Transgenic experiments with MhMYB15 revealed that RNAi-MhMYB15 silenced lines failed to induce an increase in flavonoid content and phosphorus levels in the roots under the treatment of Bacillus B2, and plant growth was slower than the control. In conclusion, MhMYB15 actively responds to Bacillus B2, regulating the accumulation of flavonoids and the uptake of phosphorus, thereby influencing plant growth and development.

A non-helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition.

MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+ -dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.

Genetic and Structural Analyses of RRNPP Intercellular Peptide Signaling of Gram-Positive Bacteria.

Bacteria use diffusible chemical messengers, termed pheromones, to coordinate gene expression and behavior among cells in a community by a process known as quorum sensing. Pheromones of many gram-positive bacteria, such as Bacillus and Streptococcus, are small, linear peptides secreted from cells and subsequently detected by sensory receptors such as those belonging to the large family of RRNPP proteins. These proteins are cytoplasmic pheromone receptors sharing a structurally similar pheromone-binding domain that functions allosterically to regulate receptor activity. X-ray crystal structures of prototypical RRNPP members have provided atomic-level insights into their mechanism and regulation by pheromones. This review provides an overview of RRNPP prototype signaling; describes the structure-function of this protein family, which is spread widely among gram-positive bacteria; and suggests approaches to target RRNPP systems in order to manipulate beneficial and harmful bacterial behaviors.

Evaluation of probiotic properties of a Brevibacillus laterosporus strain.

Brevibacillus laterosporus is a strain of probiotic bacteria that has been widely used in pest control, cash crop, and other production areas. However, few studies have been conducted on its use as a feed additive in animals. Therefore, the probiotic potential of B. laterosporus PBC01 was evaluated by characterizing hydrophobicity, auto-aggregation activity, bile salt and simulated gastrointestinal fluid tolerance, bienzymatic, and antibacterial activity. Antibiotic susceptibility, hemolysis assays, and supplemental feeding of mice were also performed to evaluate safety features. Our results showed that B. laterosporus PBC01 had moderate hydrophobicity, high auto-agglutination ability. Meanwhile, B. laterosporus PBC01 had good tolerance to bile salt and simulated gastrointestinal fluid. It had the ability to secrete protease, cellulase, and to inhibit various pathogens. In addition, B. laterosporus PBC01 was sensitive to many antibiotics, and did not produce hemolysin. In the safety assessment of mice, it did not cause any deaths, nor did it affect the cell components of blood, antioxidant capacity, and reproductive health. The study indicated the great probiotic characteristics and safety of B. laterosporus PBC01. This may provide a theoretical basis for the clinical application and development of probiotic-based feed additives.

High Bacillary Burden and the ESX-1 Type VII Secretion System Promote MHC Class I Presentation by Mycobacterium tuberculosis-Infected Macrophages to CD8 T Cells.

We used a mouse model to study how Mycobacterium tuberculosis subverts host defenses to persist in macrophages despite immune pressure. CD4 T cells can recognize macrophages infected with a single bacillus in vitro. Under identical conditions, CD8 T cells inefficiently recognize infected macrophages and fail to restrict M. tuberculosis growth, although they can inhibit M. tuberculosis growth during high-burden intracellular infection. We show that high intracellular M. tuberculosis numbers cause macrophage death, leading other macrophages to scavenge cellular debris and cross-present the TB10.4 Ag to CD8 T cells. Presentation by infected macrophages requires M. tuberculosis to have a functional ESX-1 type VII secretion system. These data indicate that phagosomal membrane damage and cell death promote MHC class I presentation of the immunodominant Ag TB10.4 by macrophages. Although this mode of Ag presentation stimulates cytokine production that we presume would be host beneficial, killing of uninfected cells could worsen immunopathology. We suggest that shifting the focus of CD8 T cell recognition to uninfected macrophages would limit the interaction of CD8 T cells with infected macrophages and impair CD8 T cell-mediated resolution of tuberculosis.

A novel Bacillus aerolatus CX253 attenuates inflammation induced by Streptococcus pneumoniae in childhood and pregnant rats by regulating gut microbiome.

Streptococcus pneumoniae (Spn) is the predominant pathogen responsible for community-acquired pneumonia (CAP) in children under five years old, and it can induce over 17% of pregnant women. However, no more effective measures exist to prevent infection induced by Spn in these two special populations. The beneficial microbes can antagonize Spn and provide new targets for preventing pneumococcal infections. This study used 16S rRNA gene sequencing and targeted metabolomics to evaluate the role of the Bacillus aerolatus CX253 (CX253) in alleviating Spn infection. Additionally, the colonization of CX253 was observed in nose, trachea, and lung by using confocal laser scanning microscopy and fluorescent labeling techniques. Compared with the model group, the expression level of interleukin-1ß was dropped 1.81-fold and 2.22-fold, and interleukin-6 was decreased 2.39-fold and 1.84-fold. The express of tumor necrosis factor-α was down 2.30-fold and 3.84-fold in prevention group of childhood and pregnant rats, respectively. The 16S rRNA sequencing results showed that CX253 administration alone significantly increased the abundance of Lactobacillus, Limosilactobacillus, and Prevotella in the gut of childhood and pregnant rats. Furthermore, the CX253 increased propionate in the gut of childhood rats and increased propionate and butyrate in the gut of pregnant rats to inhibit pulmonary inflammation. In summary, CX253 attenuated Spn-induced inflammation by regulating the gut microbiota and SCFAs. The research provides valuable information for the prevention of pneumonia.

Genome-wide transformation reveals extensive exchange across closely related Bacillus species.

Bacterial transformation is an important mode of horizontal gene transfer that helps spread genetic material across species boundaries. Yet, the factors that pose barriers to genome-wide cross-species gene transfer are poorly characterized. Here, we develop a replacement accumulation assay to study the effects of genomic distance on transfer dynamics. Using Bacillus subtilis as recipient and various species of the genus Bacillus as donors, we find that the rate of orthologous replacement decreases exponentially with the divergence of their core genomes. We reveal that at least 96% of the B. subtilis core genes are accessible to replacement by alleles from Bacillus spizizenii. For the more distantly related Bacillus atrophaeus, gene replacement events cluster at genomic locations with high sequence identity and preferentially replace ribosomal genes. Orthologous replacement also creates mosaic patterns between donor and recipient genomes, rearranges the genome architecture, and governs gain and loss of accessory genes. We conclude that cross-species gene transfer is dominated by orthologous replacement of core genes which occurs nearly unrestricted between closely related species. At a lower rate, the exchange of accessory genes gives rise to more complex genome dynamics.

Signal binding at both modules of its dCache domain enables the McpA chemoreceptor of Bacillus velezensis to sense different ligands.

Bacteria have evolved multiple signal transduction systems that permit an adaptation to changing environmental conditions. Chemoreceptor-based signaling cascades are very abundant in bacteria and are among the most complex signaling systems. Currently, our knowledge on the molecular features that determine signal recognition at chemoreceptors is limited. Chemoreceptor McpA of Bacillus velezensis SQR9 has been shown to mediate chemotaxis to a broad range of different ligands. Here we show that its ligand binding domain binds directly 13 chemoattractants. We provide support that organic acids and amino acids bind to the membrane-distal and membrane-proximal module of the dCache domain, respectively, whereas binding of sugars/sugar alcohols occurred at both modules. Structural biology studies combined with site-directed mutagenesis experiments have permitted to identify 10 amino acid residues that play key roles in the recognition of multiple ligands. Residues in membrane-distal and membrane-proximal regions were central for sensing organic acids and amimo acids, respectively, whereas all residues participated in sugars/sugar alcohol sensing. Most characterized chemoreceptors possess a narrow and well-defined ligand spectrum. We propose here a sensing mechanism involving both dCache modules that allows the integration of very diverse signals by a single chemoreceptor.

Insights into the biocontrol and plant growth promotion functions of Bacillus altitudinis strain KRS010 against Verticillium dahliae.

BACKGROUND: Verticillium wilt, caused by the fungus Verticillium dahliae, is a soil-borne vascular fungal disease, which has caused great losses to cotton yield and quality worldwide. The strain KRS010 was isolated from the seed of Verticillium wilt-resistant Gossypium hirsutum cultivar "Zhongzhimian No. 2." RESULTS: The strain KRS010 has a broad-spectrum antifungal activity to various pathogenic fungi as Verticillium dahliae, Botrytis cinerea, Fusarium spp., Colletotrichum spp., and Magnaporthe oryzae, of which the inhibition rate of V. dahliae mycelial growth was 73.97% and 84.39% respectively through confrontation test and volatile organic compounds (VOCs) treatments. The strain was identified as Bacillus altitudinis by phylogenetic analysis based on complete genome sequences, and the strain physio-biochemical characteristics were detected, including growth-promoting ability and active enzymes. Moreover, the control efficiency of KRS010 against Verticillium wilt of cotton was 93.59%. After treatment with KRS010 culture, the biomass of V. dahliae was reduced. The biomass of V. dahliae in the control group (Vd991 alone) was 30.76-folds higher than that in the treatment group (KRS010+Vd991). From a molecular biological aspect, KRS010 could trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Its extracellular metabolites and VOCs inhibited the melanin biosynthesis of V. dahliae. In addition, KRS010 had been characterized as the ability to promote plant growth. CONCLUSIONS: This study indicated that B. altitudinis KRS010 is a beneficial microbe with a potential for controlling Verticillium wilt of cotton, as well as promoting plant growth.

Structural and Functional Characterization of a Novel Class A Flavin Monooxygenase from Bacillus niacini.

A gene cluster responsible for the degradation of nicotinic acid (NA) in Bacillus niacini has recently been identified, and the structures and functions of the resulting enzymes are currently being evaluated to establish pathway intermediates. One of the genes within this cluster encodes a flavin monooxygenase (BnFMO) that is hypothesized to catalyze a hydroxylation reaction. Kinetic analyses of the recombinantly purified BnFMO suggest that this enzyme catalyzes the hydroxylation of 2,6-dihydroxynicotinic acid (2,6-DHNA) or 2,6-dihydroxypyridine (2,6-DHP), which is formed spontaneously by the decarboxylation of 2,6-DHNA. To understand the details of this hydroxylation reaction, we determined the structure of BnFMO using a multimodel approach combining protein X-ray crystallography and cryo-electron microscopy (cryo-EM). A liganded BnFMO cryo-EM structure was obtained in the presence of 2,6-DHP, allowing us to make predictions about potential catalytic residues. The structural data demonstrate that BnFMO is trimeric, which is unusual for Class A flavin monooxygenases. In both the electron density and coulomb potential maps, a region at the trimeric interface was observed that was consistent with and modeled as lipid molecules. High-resolution mass spectral analysis suggests that there is a mixture of phosphatidylethanolamine and phosphatidylglycerol lipids present. Together, these data provide insights into the molecular details of the central hydroxylation reaction unique to the aerobic degradation of NA in Bacillus niacini.

Maize Root Exudates Promote Bacillus sp. Za Detoxification of Diphenyl Ether Herbicides by Enhancing Colonization and Biofilm Formation.

Diphenyl ether herbicides are extensively utilized in agricultural systems, but their residues threaten the health of sensitive rotation crops. Functional microbial strains can degrade diphenyl ether herbicides in the rhizosphere of crops, facilitating the restoration of a healthy agricultural environment. However, the interplay between microorganisms and plants in diphenyl ether herbicides degradation remains unclear. Thus, the herbicide-degrading strain Bacillus sp. Za and the sensitive crop, maize, were employed to uncover the interaction mechanism. The degradation of diphenyl ether herbicides by strain Bacillus sp. Za was promoted by root exudates. The strain induced root exudate re-secretion in diphenyl ether herbicide-polluted maize. We further showed that root exudates enhanced the rhizosphere colonization and the biofilm biomass of strain Za, augmenting its capacity to degrade diphenyl ether herbicide. Root exudates regulated gene fliZ, which is pivotal in biofilm formation. Wild-type strain Za significantly reduced herbicide toxicity to maize compared to the ZaΔfliZ mutant. Moreover, root exudates promoted strain Za growth and chemotaxis, which was related to biofilm formation. This mutualistic relationship between the microorganisms and the plants demonstrates the significance of plant-microbe interactions in shaping diphenyl ether herbicide degradation in rhizosphere soils. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

In silico biotechnological potential of Bacillus sp. strain MHSD_37 bacterial endophyte.

BACKGROUND: Endophytic bacteria possess a range of unique characteristics that enable them to successfully interact with their host and survive in adverse environments. This study employed in silico analysis to identify genes, from Bacillus sp. strain MHSD_37, with potential biotechnological applications. RESULTS: The strain presented several endophytic lifestyle genes which encode for motility, quorum sensing, stress response, desiccation tolerance and root colonisation. The presence of plant growth promoting genes such as those involved in nitrogen fixation, nitrate assimilation, siderophores synthesis, seed germination and promotion of root nodule symbionts, was detected. Strain MHSD_37 also possessed genes involved in insect virulence and evasion of defence system. The genome analysis also identified the presence of genes involved in heavy metal tolerance, xenobiotic resistance, and the synthesis of siderophores involved in heavy metal tolerance. Furthermore, LC-MS analysis of the excretome identified secondary metabolites with biological activities such as anti-cancer, antimicrobial and applications as surfactants. CONCLUSIONS: Strain MHSD_37 thereby demonstrated potential biotechnological application in bioremediation, biofertilisation and biocontrol. Moreover, the strain presented genes encoding products with potential novel application in bio-nanotechnology and pharmaceuticals.

Probiotic Bacillus spp. enhance TLR3-mediated TNF signalling in macrophages.

Probiotics have been reported to have immunomodulatory properties in the context of infectious disease and inflammation, although the underlying mechanisms are not fully understood. Here, we aimed to determine how different probiotic bacterial strains modulated macrophage function during TLR3 stimulation mimicking viral infection. We screened 14 different strains for their ability to modulate TNF-α, IL-6 IL-10, IFN-α, IFN-ß and IFN-γ secretion in RAW 264.7 macrophages with or without poly(I:C) stimulation. Seven strains were selected for further analysis using primary porcine alveolar macrophages. In-depth transcriptomic analysis on alveolar macrophages was conducted for two strains. Most strains induced a synergistic effect when co-incubated with poly(I:C) resulting in increased levels of IL-6 and TNF-α secretion from RAW 264.7 cells. This synergistic effect was found to be TLR2 independent. Only strains of Bacillus spp. could induce this effect in alveolar macrophages. Transcriptomic analysis indicated that the increased TNF-α secretion in alveolar macrophages after co-incubation with poly(I:C) correlated with significant upregulation of TNF and IL23A-related pathways. Collectively, our data show that probiotic bacteria possess strain-dependent immunomodulatory properties that may be harnessed to enhance innate immune responses to pathogens.

Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization.

The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The ß-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE3F3 cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.