In wastewater treatment plants (WWTPs), complex microbial communities process diverse chemical compounds from sewage. Secreted proteins are critical because many are the first to interact with or degrade external (macro)molecules. To better understand microbial functions in WWTPs, we predicted secreted proteomes of WWTP microbiota from more than 1,000 high-quality metagenome-assembled genomes (MAGs) from 23 Danish WWTPs with biological nutrient removal. Focus was placed on examining secreted catabolic exoenzymes that target major classes of macromolecules. We demonstrate that Bacteroidota has a high potential to digest complex polysaccharides, but also proteins and nucleic acids. Poorly understood activated sludge members of Acidobacteriota and Gemmatimonadota also have high capacities for extracellular polysaccharide digestion. Secreted nucleases are encoded by 61% of MAGs indicating an importance for extracellular DNA and/or RNA digestion in WWTPs. Secreted lipases were the least common macromolecule-targeting enzymes predicted, encoded mainly by Gammaproteobacteria and Myxococcota. In contrast, diverse taxa encode extracellular peptidases, indicating that proteins are widely used nutrients. Diverse secreted multi-heme cytochromes suggest capabilities for extracellular electron transfer by various taxa, including some Bacteroidota that encode undescribed cytochromes with >100 heme-binding motifs. Myxococcota have exceptionally large secreted protein complements, probably related to predatory lifestyles and/or complex cell cycles. Many Gammaproteobacteria MAGs (mostly former Betaproteobacteria) encode few or no secreted hydrolases, but many periplasmic substrate-binding proteins and ABC- and TRAP-transporters, suggesting they are mostly sustained by small molecules. Together, this study provides a comprehensive overview of how WWTPs microorganisms interact with the environment, providing new insights into their functioning and niche partitioning.IMPORTANCEWastewater treatment plants (WWTPs) are critical biotechnological systems that clean wastewater, allowing the water to reenter the environment and limit eutrophication and pollution. They are also increasingly important for the recovery of resources. They function primarily by the activity of microorganisms, which act as a \"living sponge,\" taking up and transforming nutrients, organic material, and pollutants. Despite much research, many microorganisms in WWTPs are uncultivated and poorly characterized, limiting our understanding of their functioning. Here, we analyzed a large collection of high-quality metagenome-assembled genomes from WWTPs for encoded secreted enzymes and proteins, with special emphasis on those used to degrade organic material. This analysis showed highly distinct secreted proteome profiles among different major phylogenetic groups of microorganisms, thereby providing new insights into how different groups function and co-exist in activated sludge. This knowledge will contribute to a better understanding of how to efficiently manage and exploit WWTP microbiomes.

Pesticides have increased crop yield but severely impacted ecosystems and non-target organisms. Flubendiamide, a new generation pesticide, targets insect larvae but also affects non-target organisms. This study examines the effects of lowest observed effect concentration of technical grade flubendiamide (0.5 µg/µL) flubendiamide on chick liver, focusing on cytochrome P450 (CYP) enzyme expression, oxidative stress, and liver damage. Chick embryos treated with flubendiamide showed significant alterations in CYP mRNA and protein levels, indicating increased toxicant accumulation. Elevated CYP3A4, CYP1A1, CYP1A2, and CYP2C19 levels were noted, suggesting enhanced biotransformation and detoxification processes. However, increased oxidative byproducts led to oxidative stress, as evidenced by decreased glutathione (GSH) levels and elevated superoxide dismutase (SOD) and catalase activities. DCFDA staining confirmed increased hydrogen peroxide (H2O2) levels, indicating heightened reactive oxygen species (ROS). Liver function tests revealed significant increases in serum ALP, ALT, and AST levels, indicating acute liver damage. Histopathological analysis showed structural liver damage, including expanded sinusoidal spaces, impaired portal veins, and compromised hepatocyte architecture. These findings underscore flubendiamide\'s potential hepatotoxicity in non-target organisms, emphasizing the need for cautious pesticide use to minimize environmental impacts.

Cannabidiol (CBD) has gained widespread popularity; however, its pharmacological and toxicological profiles in the context of human genetic diversity remain largely unexplored. Here, we investigated the variability in metabolism and toxicity of CBD-rich cannabis extract (CRCE) in genetically diverse mouse models: C57BL/6J, B6C3F1/J, and NZO/HlLtJ strains. Mice received a single dose of CRCE containing 57.9% CBD at dosages of 0, 246, 738, and 2460 mg/kg of CBD. At 24 h after treatment, no appreciable histomorphological changes were detected in the liver. Plasma bilirubin levels increased markedly in all strains at the highest CBD dose. Mice in all treatment groups displayed significant but distinct increases in ALT and AST levels. While B6C3F1/J and NZO/HlLtJ mice had negligible plasma CBD levels at 738 mg/kg, C57BL/6J mice exhibited levels exceeding 7000 ng/mL. At 2460 mg/kg, high CBD concentrations were found in B6C3F1/J and C57BL/6J mice, but markedly lower levels were seen in NZO/HlLtJ mice. Gene expression profiling showed significant increases in Cyp2b10 across all strains but varying responses in Cyp1a1 expression, indicating strain-specific CYP dysregulation. Genetically diverse mice exhibited differential pharmacological and toxicological responses to CRCE, suggesting a high potential for inter-individual variability in the pharmacology and toxicology of CBD in humans.

To investigate their roles in extracellular electron transfer (EET), the porin-cytochrome (pcc) gene clusters Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 of the Gram-negative bacterium Geobacter metallireducens were deleted. Failure to delete all pcc gene clusters at the same time suggested their essential roles in extracellular reduction of Fe(III)-citrate by G. metallireducens. Deletion of Gmet0825-0828 had no impact on bacterial reduction of Fe(III)-citrate but diminished bacterial reduction of ferrihydrite and abolished anode reduction and direct interspecies electron transfer (DIET) to Methanosarcina barkeri and Geobacter sulfurreducens. Although it had no impact on the bacterial reduction of Fe(III)-citrate, deletion of Gmet0908-0910 delayed ferrihydrite reduction, abolished anode reduction, and diminished DIET. Deletion of Gmet0911-0913 had little impact on DIET but diminished bacterial reductions of Fe(III)-citrate, ferrihydrite, and anodes. Most importantly, deletions of both Gmet0825-0828 and Gmet0908-0910 restored bacterial reduction of ferrihydrite and anodes and DIET. Enhanced expression of Gmet0911-0913 in this double mutant when grown in coculture with G. sulfurreducens ΔhybLΔfdnG suggested that this cluster might compensate for impaired EET functions of deleting Gmet0825-0828 and Gmet0908-0910. Thus, these pcc gene clusters played essential, distinct, overlapping, and compensatory roles in EET of G. metallireducens that are difficult to characterize as deletion of some clusters affected expression of others. The robustness of these pcc gene clusters enabled G. metallireducens to mediate EET to different acceptors for anaerobic growth even when two of its three pcc gene clusters were inactivated by mutation. The results from this investigation provide new insights into the roles of pcc gene clusters in bacterial EET.
OBJECTIVE: The Gram-negative bacterium Geobacter metallireducens is of environmental and biotechnological significance. Crucial to the unique physiology of G. metallireducens is its extracellular electron transfer (EET) capability. This investigation sheds new light on the robust roles of the three porin-cytochrome (pcc) gene clusters, which are directly involved in EET across the bacterial outer membrane, in the EET of G. metallireducens. In addition to their essential roles, these gene clusters also play distinct, overlapping, and compensatory roles in the EET of G. metallireducens. The distinct roles of the pcc gene clusters enable G. metallireducens to mediate EET to a diverse group of electron acceptors for anaerobic respirations. The overlapping and compensatory roles of the pcc gene clusters enable G. metallireducens to maintain and restore its EET capability for anaerobic growth when one or two of its three pcc gene clusters are deleted from the genome.

Heme consists of a tetrapyrrole ring ligating an iron ion and has important roles in biological systems. While well-known as the oxygen-binding molecule within hemoglobin of mammals, heme is also cofactor for several enzymes and a major iron source for bacteria within the host. The enterococci are a diverse group of Gram-positive bacteria that exist primarily within the gastrointestinal tract of animals. However, some species within this genus can transform into formidable opportunistic pathogens, largely owing to their extraordinary adaptability to hostile environments. Although enterococci cannot synthesize heme nor depend on heme to grow, several species within the genus encode proteins that utilize heme as a cofactor, which appears to increase their fitness and ability to thrive in challenging environments. This includes more efficient energy generation via aerobic respiration and protection from reactive oxygen species. Here, we review the significance of heme to enterococci, primarily the major human pathogen Enterococcus faecalis, use bioinformatics to assess the prevalence of hemoproteins throughout the genus, and highlight recent studies that underscore the central role of the heme-E. faecalis relationship in host-pathogen dynamics and interspecies bacterial interactions.

BACKGROUND: Cytochrome bd complexes are respiratory oxidases found exclusively in prokaryotes that are important during infection for numerous bacterial pathogens.
METHODS: In silico docking was employed to screen approved drugs for their ability to bind to the quinol site of Escherichia coli cytochrome bd-I. Respiratory inhibition was assessed with oxygen electrodes using membranes isolated from E. coli and methicillin-resistant Staphylococcus aureus strains expressing single respiratory oxidases (ie, cytochromes bd, bo\', or aa3). Growth/viability assays were used to measure bacteriostatic and bactericidal effects.
RESULTS: The steroid drugs ethinylestradiol and quinestrol inhibited E. coli bd-I activity with median inhibitory concentration (IC50) values of 47 ± 28.9 µg/mL (158 ± 97.2 µM) and 0.2 ± 0.04 µg/mL (0.5 ± 0.1 µM), respectively. Quinestrol inhibited growth of an E. coli \"bd-I only\" strain with an IC50 of 0.06 ± 0.02 µg/mL (0.2 ± 0.07 µM). Growth of an S. aureus \"bd only\" strain was inhibited by quinestrol with an IC50 of 2.2 ± 0.43 µg/mL (6.0 ± 1.2 µM). Quinestrol exhibited potent bactericidal effects against S. aureus but not E. coli.
CONCLUSIONS: Quinestrol inhibits cytochrome bd in E. coli and S. aureus membranes and inhibits the growth of both species, yet is only bactericidal toward S. aureus.

Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b558, heme b595 and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O2 reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer. Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b558 is pH dependent, with a maximum rate constant (~104 s-1) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pKa ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58B➔Asn and Asp105B➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105B➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105B in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pKa proton donor, and the conservation pattern of the Asp-105B in the cyt. bd superfamily.

Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to delineate the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.

Cytochrome bd-I from Escherichia coli belongs to the superfamily of prokaryotic bd-type oxygen reductases. It contains three hemes, b558, b595 and d, and couples oxidation of quinol by dioxygen with the generation of a proton-motive force. The enzyme exhibits resistance to various stressors and is considered as a target protein for next-generation antimicrobials. By using electronic absorption and MCD spectroscopy, this work shows that cyanide binds to heme d2+ in the isolated fully reduced cytochrome bd-I. Cyanide-induced difference absorption spectra display changes near the heme d2+ α-band, a minimum at 633 nm and a maximum around 600 nm, and a W-shaped response in the Soret region. Apparent dissociation constant (Kd) of the cyanide complex of heme d2+ is ∼0.052 M. Kinetics of cyanide binding is monophasic, indicating the presence of a single ligand binding site in the enzyme. Consistently, MCD data show that cyanide binds to heme d2+ but not to b5582+ or b5952+. This agrees with the published structural data that the enzyme\'s active site is not a di-heme site. The observed rate of binding (kobs) increases as the concentration of cyanide is increased, giving a second-order rate constant (kon) of ∼0.1 M-1 s-1.

Multiheme cytochromes located in different compartments are crucial for extracellular electron transfer in the bacterium Geobacter sulfurreducens to drive important environmental processes and biotechnological applications. Recent studies have unveiled that for particular sets of electron terminal acceptors, discrete respiratory pathways selectively recruit specific cytochromes from both the inner and outer membranes. However, such specificity was not observed for the abundant periplasmic cytochromes, namely the triheme cytochrome family PpcA-E. In this work, the distinctive NMR spectroscopic signatures of these proteins in different redox states were explored to monitor pairwise interactions and electron transfer reactions between each pair of cytochromes. The results showed that the five proteins interact transiently and can exchange electrons between each other revealing intra-promiscuity within the members of this family. This discovery is discussed in the light of the establishment of an effective electron transfer network by this pool of cytochromes. This network is advantageous to the bacteria as it enables the maintenance of the functional working potential redox range within the cells.