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ABSTRACTExtracellular electron transfer (EET) is a metabolic process that frequently uses quinones to couple intracellular redox reactions with extracellular electron acceptors. The physiological relevance of this metabolism for microorganisms capable of EET but unable to synthesize their own quinones remains to be determined. To address this question, we investigated quinone utilization by Lactiplantibacillus plantarum, a microorganism required for food fermentations, that performs EET and is also a quinone auxotroph. L. plantarum selectively used 1,4-dihydroxy-2-naphthoic acid (DHNA) and more hydrophilic naphthoquinones for EET reduction of insoluble iron (ferrihydrite). However, quinones used for EET also inhibited L. plantarum growth in non-aerated conditions. Transcriptomic analysis showed that DHNA-induced oxidative stress in L. plantarum, but this was alleviated when the electron acceptor, ferric ammonium citrate (FeAC), was included in the growth medium. Although DHNA and FeAC induced L. plantarum EET, this metabolism was still dependent on direct access to environmental electron shuttles. To determine whether quinone-producing food fermentation bacteria could be sources of those electron shuttles, L. plantarum EET was measured after incubation with Lactococcus lactis and Leuconostoc mesenteroides. Quinone-producing L. lactis, but not a quinone-deficient L. lactis ΔmenC mutant, increased L. plantarum ferrihydrite reduction and medium acidification through an EET-dependent mechanism. L. plantarum EET was also stimulated by L. mesenteroides, resulting in greater environmental acidification and transient increases in L. plantarum cell numbers. Our findings show that L. plantarum overcomes the toxic effects of exogenous quinones to use those compounds for EET-conferred, ecological advantages during the early stages of food fermentations.IMPORTANCEWhile quinones are essential for respiratory microorganisms, their importance for microbes that rely on fermentation metabolism is not understood. This gap in knowledge hinders our understanding of anaerobic microbial habitats, such in mammalian digestive tracts and fermented foods. We show that Lactiplantibacillus plantarum, a model fermentative lactic acid bacteria species abundant in human, animal, and insect microbiomes and fermented foods, uses multiple exogenous, environmental quinones as electron shuttles for a hybrid metabolism involving EET. Interestingly, quinones both stimulate this metabolism as well as cause oxidative stress when extracellular electron acceptors are absent. We also found that quinone-producing, lactic acid bacteria species commonly enriched together with L. plantarum in food fermentations accelerate L. plantarum growth and medium acidification through a mainly quinone- and EET-dependent mechanism. Thus, our work provides evidence of quinone cross-feeding as a key ecological feature of anaerobic microbial habitats.

Nitrification is a core process in the global nitrogen (N) cycle mediated by ammonia-oxidizing microorganisms, including ammonia-oxidizing archaea (AOA) as a key player. Although much is known about AOA abundance and diversity across environments, the genetic drivers of the ecophysiological adaptations of the AOA are often less clearly defined. This is especially true for AOA within the genus Nitrosocosmicus , which have several unique physiological traits (e.g., high substrate tolerance, low substrate affinity, and large cell size). To better understand what separates the physiology of Nitrosocosmicus AOA, we performed comparative genomics with genomes from 39 cultured AOA, including five Nitrosocosmicus AOA. The absence of a canonical high-affinity type ammonium transporter and typical S-layer structural genes was found to be conserved across all Nitrosocosmicus AOA. In agreement, cryo-electron tomography confirmed the absence of a visible outermost S-layer structure, which has been observed in other AOA. In contrast to other AOA, the cryo-electron tomography highlighted the possibility that Nitrosocosmicus AOA may possess a glycoprotein or glycolipid-based glycocalyx cell covering outer layer. Together, the genomic, physiological, and metabolic properties revealed in this study provide insight into niche adaptation mechanisms and the overall ecophysiology of members of the Nitrosocosmicus clade in various terrestrial ecosystems., Importance: Nitrification is a vital process within the global biogeochemical nitrogen cycle but plays a significant role in the eutrophication of aquatic ecosystems and the production of the greenhouse gas nitrous oxide (N 2 O) from industrial agriculture ecosystems. While various types of ammonia-oxidizing microorganisms play a critical role in the N cycle, ammonia-oxidizing archaea (AOA) are often the most abundant nitrifiers in natural environments. Members of the genus Nitrosocosmicus are one of the prevalent AOA groups detected in undisturbed terrestrial ecosystems and have previously been reported to possess a range of physiological characteristics that set their physiology apart from other AOA species. This study provides significant progress in understanding these unique physiological traits and their genetic drivers. Our results highlight how physiological studies based on comparative genomics-driven hypotheses can contribute to understanding the unique niche of Nitrosocosmicus AOA., Competing Interests: The authors declare no conflict of interest.

Benzene is a widespread groundwater contaminant that persists under anoxic conditions. The aim of this study was to more accurately investigate anaerobic microbial degradation pathways to predict benzene fate and transport. Preliminary genomic analysis of Geotalea daltonii strain FRC-32, isolated from contaminated groundwater, revealed the presence of putative aromatic-degrading genes. G. daltonii was subsequently shown to conserve energy for growth on benzene as the sole electron donor and fumarate or nitrate as the electron acceptor. The hbs gene, encoding for 3-hydroxybenzylsuccinate synthase (Hbs), a homolog of the radical-forming, toluene-activating benzylsuccinate synthase (Bss), was upregulated during benzene oxidation in G. daltonii , while the bss gene was upregulated during toluene oxidation. Addition of benzene to the G. daltonii whole-cell lysate resulted in toluene formation, indicating that methylation of benzene was occurring. Complementation of σ 54 - (deficient) E. coli transformed with the bss operon restored its ability to grow in the presence of toluene, revealing bss to be regulated by σ 54 . Binding sites for σ 70 and the transition state regulator AbrB were identified in the promoter region of the σ 54 -encoding gene rpoN, and binding was confirmed. Induced expression of abrB during benzene and toluene degradation caused G. daltonii cultures to transition to the death phase. Our results suggested that G. daltonii can anaerobically oxidize benzene by methylation, which is regulated by σ 54 and AbrB. Our findings further indicated that the benzene, toluene, and benzoate degradation pathways converge into a single metabolic pathway, representing a uniquely efficient approach to anaerobic aromatic degradation in G. daltonii ., Importance: The contamination of anaerobic subsurface environments including groundwater with toxic aromatic hydrocarbons, specifically benzene, toluene, ethylbenzene, and xylene, has become a global issue. Subsurface groundwater is largely anoxic, and further study is needed to understand the natural attenuation of these compounds. This study elucidated a metabolic pathway utilized by the bacterium Geotalea daltonii capable of anaerobically degrading the recalcitrant molecule benzene using a unique activation mechanism involving methylation. The identification of aromatic-degrading genes and AbrB as a regulator of the anaerobic benzene and toluene degradation pathways provides insights into the mechanisms employed by G. daltonii to modulate metabolic pathways as necessary to thrive in anoxic contaminated groundwater. Our findings contribute to the understanding of novel anaerobic benzene degradation pathways that could potentially be harnessed to develop improved strategies for bioremediation of groundwater contaminants., Competing Interests: The authors declare no conflict of interest.

Tupanviruses, members of the family Mimiviridae , infect phagocytic cells. Particle uncoating begins inside the phagosome, with capsid opening via the stargate. The mechanism through which this opening takes place is unknown. Once phagocytized, metal ion flux control and ROS are induced to inactivate foreign particles, including viruses. Here, we studied the effect of iron ions, copper ions, and H 2 O 2 on Tupanvirus particles. Such treatments induced stargate opening in vitro , as observed by different microscopy techniques. Metal-treated viruses were found to be non-infectious, leading to the hypothesis that stargate opening likely resulted in the release of the viral seed, which is required for infection initiation. To the best of our knowledge, this is the first description of a giant virus capsid morphological change induced by transition metals and H 2 O 2 , which may be important to describe new virulence factors and capsid uncoating mechanisms., Competing Interests: The authors declare no conflict of interest.

Clostridioides difficile , the major cause of antibiotic-associated diarrhea, is a strict anaerobic, sporulating Firmicutes. However, during its infectious cycle, this anaerobe is exposed to low oxygen (O 2 ) tensions, with a longitudinal decreasing gradient along the gastrointestinal tract and a second lateral gradient with higher O 2 tensions in the vicinity of the cells. A plethora of enzymes involved in oxidative stress detoxication has been identified in C. difficile , including four O 2 -reducing enzymes: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). Here, we investigated the role of the four O 2 -reducing enzymes in the tolerance to increasing physiological O 2 tensions and air. The four enzymes have different, yet overlapping, spectra of activity. revRbr2 is specific to low O 2 tensions (<0.4%), FdpA to low and intermediate O 2 tensions (0.4%-1%), revRbr1 has a wider spectrum of activity (0.1%-4%), and finally FdpF is more specific to tensions > 4% and air. These different O 2 ranges of action partly arise from differences in regulation of expression of the genes encoding those enzymes. Indeed, we showed that revrbr2 is under the dual control of σ A and σ B . We also identified a regulator of the Spx family that plays a role in the induction of fdp and revrbr genes upon O 2 exposure. Finally, fdpF is regulated by Rex, a regulator sensing the NADH/NAD + ratio. Our results demonstrate that the multiplicity of O 2 -reducing enzymes of C. difficile is associated with different roles depending on the environmental conditions, stemming from a complex multi-leveled network of regulation., Importance: The gastrointestinal tract is a hypoxic environment, with the existence of two gradients of O 2 along the gut, one longitudinal anteroposterior decreasing gradient and one proximodistal increasing from the lumen to the epithelial cells. O 2 is a major source of stress for an obligate anaerobe such as the enteropathogen C. difficile . This bacterium possesses a plethora of enzymes capable of scavenging O 2 and reducing it to H 2 O. In this work, we identified the role of the four O 2 -reducing enzymes in the tolerance to the physiological O 2 tensions faced by C. difficile during its infectious cycle. These four enzymes have different spectra of action and protect the vegetative cells over a large range of O 2 tensions. These differences are associated with a distinct regulation of each gene encoding those enzymes. The complex network of regulation is crucial for C. difficile to adapt to the various O 2 tensions encountered during infection., Competing Interests: The authors declare no conflict of interest.

Manganese-oxidizing bacteria (MnOB) produce Mn oxide minerals that can be used by humans for bioremediation, but the purpose for the bacterium is less clear. This study describes the isolation and characterization of cold-tolerant MnOB strains isolated from a compost pile in Morris, Minnesota, USA: Pseudomonas sp. MS-1 and DSV-1. The strains were preliminarily identified as members of species Pseudomonas psychrophila by 16S rRNA analysis and a multi-locus phylogenetic study using a database of 88 genomes from the Pseudomonas genus. However, the average nucleotide identity between these strains and the P. psychrophila sp. CF149 type strain was less than 93%. Thus, the two strains are members of a novel species that diverged from P. psychrophila . DSV-1 and MS-1 are cold tolerant; both grow at 4°C but faster at 24°C. Unlike the mesophilic MnOB P. putida GB-1, both strains are capable of robustly oxidizing Mn at low temperatures. Both DSV-1 and MS-1 genomes contain homologs of several Mn oxidation genes found in P. putida GB-1 ( mnxG , mcoA , mnxS1 , mnxS2, and mnxR ). Random mutagenesis by transposon insertion was successfully performed in both strains and identified genes involved in Mn oxidation that were similar to those found in P. putida GB-1. Our results show that MnOB can be isolated from compost, supporting a role for Mn oxidation in plant waste degradation. The novel isolates Pseudomonas spp. DSV-1 and MS-1 both can oxidize Mn at low temperature and likely employ similar mechanisms and regulation as P. putida GB-1.IMPORTANCEBiogenic Mn oxides have high sorptive capacity and are strong oxidants. These two characteristics make these oxides and the microbes that make them attractive tools for the bioremediation of wastewater and contaminated environments. Identifying MnOB that can be used for bioremediation is an active area of research. As cold-tolerant MnOB, Pseudomonas sp. DSV-1 and MS-1 have the potential to expand the environmental conditions in which biogenic Mn oxide bioremediation can be performed. The similarity of these organisms to the well-characterized MnOB P. putida GB-1 and the ability to manipulate their genomes raise the possibility of modifying them to improve their bioremediation ability., Competing Interests: The authors declare no conflict of interest.

This study explores the genetic landscape of nitrous oxide (N 2 O) reduction in wastewater treatment plants (WWTPs) by profiling 1,083 high-quality metagenome-assembled genomes (HQ MAGs) from 23 Danish full-scale WWTPs. The focus is on the distribution and diversity of nitrous oxide reductase ( nosZ ) genes and their association with other nitrogen metabolism pathways. A custom pipeline for clade-specific nosZ gene identification with higher sensitivity revealed 503 nosZ sequences in 489 of these HQ MAGs, outperforming existing Kyoto Encyclopedia of Genes and Genomes (KEGG) module-based methods. Notably, 48.7% of the total 1,083 HQ MAGs harbored nosZ genes, with clade II being predominant, accounting for 93.7% of these genes. Taxonomic profiling highlighted the prevalence of nosZ -containing taxa within Bacteroidota and Pseudomonadota. Chloroflexota exhibited unexpected affiliations with both the sec and tat secretory pathways, and all were found to contain the accessory nosB gene, underscoring the importance of investigating the secretory pathway. The majority of non-denitrifying N 2 O reducers were found within Bacteroidota and Chloroflexota . Additionally, HQ MAGs with genes for dissimilatory nitrate reduction to ammonium and assimilatory nitrate reduction frequently co-occurred with the nosZ gene. Traditional primers targeting nosZ often focus on short-length amplicons. Therefore, we introduced custom-designed primer sets targeting near-full-length nosZ sequences. These new primers demonstrate efficacy in capturing diverse and well-characterized sequences, providing a valuable tool with higher resolution for future research. In conclusion, this comprehensive analysis enhances our understanding of N 2 O-reducing organisms in WWTPs, highlighting their potential as N 2 O sinks with the potential for optimizing wastewater treatment processes and mitigating greenhouse gas emissions., Importance: This study provides critical insights into the genetic diversity of nitrous oxide reductase (nosZ) genes and the microorganisms harboring them in wastewater treatment plants (WWTPs) by exploring 1,083 high-quality metagenome-assembled genomes (MAGs) from 23 Danish full-scale WWTPs. Despite the pivotal role of nosZ-containing organisms, their diversity remains largely unexplored in WWTPs. Our custom pipeline for detecting nosZ provides near-full-length genes with detailed information on secretory pathways and accessory nos genes. Using these genes as templates, we developed taxonomically diverse clade-specific primers that generate nosZ amplicons for phylogenetic annotation and gene-to-MAG linkage. This approach improves detection and expands the discovery of novel sequences, highlighting the prevalence of non-denitrifying N 2 O reducers and their potential as N 2 O sinks. These findings have the potential to optimize nitrogen removal processes and mitigate greenhouse gas emissions from WWTPs by fully harnessing the capabilities of the microbial communities., Competing Interests: The authors declare no conflict of interest.

The acetogen Acetobacterium woodii couples caffeate reduction with ferredoxin reduction and NADH oxidation via electron bifurcation, providing additional reduced ferredoxin for energy conservation and cell synthesis. Caffeate is first activated by an acyl-CoA synthetase (CarB), which ligates CoA to caffeate at the expense of ATP. After caffeoyl-CoA is reduced to hydrocaffeoyl-CoA, the CoA moiety in hydrocaffeoyl-CoA could be recycled for caffeoyl-CoA synthesis by an ATP-independent CoA transferase (CarA) to save energy. However, given that CarA and CarB are co-expressed, it was not well understood how ATP could be saved when both two competitive pathways of caffeate activation are present. Here, we reported a dual feedback inhibition of the CarB-mediated caffeate activation by the intermediate hydrocaffeoyl-CoA and the end-product hydrocaffeate. As the product of CarA, hydrocaffeate inhibited CarB-mediated caffeate activation by serving as another substrate of CarB with hydrocaffeoyl-CoA produced. It effectively competed with caffeate even at a concentration much lower than caffeate. Hydrocaffeoyl-CoA formed in this process can also inhibit CarB-mediated caffeate activation. Thus, the dual feedback inhibition of CarB, together with the faster kinetics of CarA, makes the ATP-independent CarA-mediated CoA loop the major route for caffeoyl-CoA synthesis, further saving ATP in the caffeate-dependent electron-bifurcating pathway. A genetic architecture similar to carABC has been found in other anaerobic bacteria, suggesting that the feedback inhibition of acyl-CoA ligases could be a widely employed strategy for ATP conservation in those pathways requiring substrate activation by CoA., Importance: This study reports a dual feedback inhibition of caffeoyl-CoA synthetase by two downstream products, hydrocaffeate and hydrocaffeoyl-CoA. It elucidates how such dual feedback inhibition suppresses ATP-dependent caffeoyl-CoA synthesis, hence making the ATP-independent route the main pathway of caffeate activation. This newly discovered mechanism contributes to our current understanding of ATP conservation during the caffeate-dependent electron-bifurcating pathway in the ecologically important acetogen Acetobacterium woodii . Bioinformatic mining of microbial genomes revealed contiguous genes homologous to carABC within the genomes of other anaerobes from various environments, suggesting this mechanism may be widely used in other CoA-dependent electron-bifurcating pathways., Competing Interests: The authors declare no conflict of interest.

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli . This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H ( Ec FDH-H). Using an Ec FDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active Ec FDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using Ec FDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 Ec FDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.IMPORTANCEOxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO 2 and N 2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications., Competing Interests: The authors declare no conflict of interest.

Leptothrix ochracea creates distinctive iron-mineralized mats that carpet streams and wetlands. Easily recognized by its iron-mineralized sheaths, L. ochracea was one of the first microorganisms described in the 1800s. Yet it has never been isolated and does not have a complete genome sequence available, so key questions about its physiology remain unresolved. It is debated whether iron oxidation can be used for energy or growth and if L. ochracea is an autotroph, heterotroph, or mixotroph. To address these issues, we sampled L. ochracea -rich mats from three of its typical environments (a stream, wetlands, and a drainage channel) and reconstructed nine high-quality genomes of L. ochracea from metagenomes. These genomes contain iron oxidase genes cyc2 and mtoA, showing that L. ochracea has the potential to conserve energy from iron oxidation. Sox genes confer potential to oxidize sulfur for energy. There are genes for both carbon fixation (RuBisCO) and utilization of sugars and organic acids (acetate, lactate, and formate). In silico stoichiometric metabolic models further demonstrated the potential for growth using sugars and organic acids. Metatranscriptomes showed a high expression of genes for iron oxidation; aerobic respiration; and utilization of lactate, acetate, and sugars, as well as RuBisCO, supporting mixotrophic growth in the environment. In summary, our results suggest that L. ochracea has substantial metabolic flexibility. It is adapted to iron-rich, organic carbon-containing wetland niches, where it can thrive as a mixotrophic iron oxidizer by utilizing both iron oxidation and organics for energy generation and both inorganic and organic carbon for cell and sheath production., Importance: Winogradsky's observations of L. ochracea led him to propose autotrophic iron oxidation as a new microbial metabolism, following his work on autotrophic sulfur-oxidizers. While much culture-based research has ensued, isolation proved elusive, so most work on L. ochracea has been based in the environment and in microcosms. Meanwhile, the autotrophic Gallionella became the model for freshwater microbial iron oxidation, while heterotrophic and mixotrophic iron oxidation is not well-studied. Ecological studies have shown that Leptothrix overtakes Gallionella when dissolved organic carbon content increases, demonstrating distinct niches. This study presents the first near-complete genomes of L. ochracea , which share some features with autotrophic iron oxidizers, while also incorporating heterotrophic metabolisms. These genome, metabolic modeling, and transcriptome results give us a detailed metabolic picture of how the organism may combine lithoautotrophy with organoheterotrophy to promote Fe oxidation and C cycling and drive many biogeochemical processes resulting from microbial growth and iron oxyhydroxide formation in wetlands., Competing Interests: The authors declare no conflict of interest.

Enhancing the availability of representative isolates from hydrothermal vents (HTVs) is imperative for comprehending the microbial processes that propel the vent ecosystem. In recent years, Campylobacteria have emerged as the predominant and ubiquitous taxon across both shallow and deep-sea vent systems. Nevertheless, only a few isolates have been cultured, primarily originating from deep-sea HTVs. Presently, no cultivable isolates of Campylobacteria are accessible in shallow water vent systems (<200 m), which exhibit markedly distinct environmental conditions from their deep-sea counterparts. In this study, we enriched a novel isolate (genus Sulfurospirillum , Campylobacteria) from shallow-water HTVs of Kueishan Island. Genomic and physiological analysis revealed that this novel Campylobacteria species grows on a variety of substrate and carbon/energy sources. The pan-genome and phenotypic comparisons with 12 previously isolated Sulfurospirillum species from different environments supported the identification of functional features in Sulfurospirillum genomes crucial for adaptation to vent environments, such as sulfur oxidation, carbon fixation, biofilm formation, and benzoate/toluene degradation, as well as diverse genes related with signal transportation. To conclude, the metabolic characteristics of this novel Campylobacteria augment our understanding of Campylobacteria spanning from deep-sea to shallow-water vent systems.IMPORTANCECampylobacteria emerge as the dominant and ubiquitous taxa within vent systems, playing important roles in the vent ecosystems. However, isolated representatives of Campylobacteria have been mainly from the deep-sea hydrothermal fields, leaving a significant knowledge gap regarding the functions, activities, and adaptation strategies of the vent microorganisms in shallow-water hydrothermal vents (HTVs). This study bridges this gap by providing insights into the phenomics and genomic diversity of genus Sulfurospirillum (order Campylobacterales, class Campylobacteria) based on data derived from a novel isolate obtained from shallow-water HTVs. Our mesophilic isolate of Sulfurospirillum not only augments the genus diversity of Campylobacteria pure cultures derived from vent systems but also serves as the inaugural reference isolate for Campylobacteria in shallow-water environments., Competing Interests: The authors declare no conflict of interest.

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 Δ hybL Δ fdnG 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., Importance: 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., Competing Interests: The authors declare no conflict of interest.

The Soudan Underground Mine State Park, found in the Vermilion Iron Range in northern Minnesota, provides access to a ~ 2.7 billion-year-old banded iron formation. Exploratory boreholes drilled between 1958 and 1962 on the 27th level (713 m underground) of the mine intersect calcium and iron-rich brines that have recently been subject to metagenomic analysis and microbial enrichments. Using concentrated brine samples pumped from a borehole depth of up to 55 m, a novel Gram-positive bacterium was enriched under anaerobic, acetate-oxidizing, and Fe(III) citrate-reducing conditions. The isolated bacterium, designated strain MK1, is non-motile, rod-shaped, spore-forming, anaerobic, and mesophilic, with a growth range between 24°C and 30°C. The complete circular MK1 genome was found to be 3,720,236 bp and encodes 25 putative multiheme cytochromes, including homologs to inner membrane cytochromes in the Gram-negative bacterium Geobacter sulfurreducens and cytoplasmic membrane and periplasmic cytochromes in the Gram-positive bacterium Thermincola potens . However, MK1 does not encode homologs of the peptidoglycan (CwcA) and cell surface-associated (OcwA) multiheme cytochromes proposed to be required by T. potens to perform extracellular electron transfer. The 16S rRNA gene sequence of MK1 indicates that its closest related isolate is Desulfitibacter alkalitolerans strain sk.kt5 (91% sequence identity), which places MK1 in a novel genus within the Desulfitibacteraceae family and Moorellales order. Within the Moorellales order, only Calderihabitans maritimus strain KKC1 has been reported to reduce Fe(III), and only D. alkalitolerans can also grow in temperatures below 40°C. Thus, MK1 represents a novel species within a novel genus, for which we propose the name " Metallumcola ferriviriculae" strain MK1, and provides a unique opportunity to study a cytochrome-rich, mesophilic, Gram-positive, spore-forming Fe(III)-reducing bacterium.IMPORTANCEThe Soudan Underground Mine State Park gives access to understudied regions of the deep terrestrial subsurface that potentially predate the Great Oxidation Event. Studying organisms that have been relatively unperturbed by surface conditions for as long as 2.7 billion years may give us a window into ancient life before oxygen dominated the planet. Additionally, studying microbes from anoxic and iron-rich environments can help us better understand the requirements of life in analogous environments, such as on Mars. The isolation and characterization of " Metallumcola ferriviriculae" strain MK1 give us insights into a novel genus and species that is distinct both from its closest related isolates and from iron reducers characterized to date. " M. ferriviriculae" strain MK1 may also act as a model organism to study how the processes of sporulation and germination are affected by insoluble extracellular acceptors, as well as the impact of spores in the deep terrestrial biosphere., Competing Interests: The authors declare no conflict of interest.

Cable bacteria are filamentous bacteria that couple the oxidation of sulfide in sediments to the reduction of oxygen via long-distance electron transport over centimeter distances through periplasmic wires. However, the capability of cable bacteria to perform extracellular electron transfer to acceptors, such as electrodes, has remained elusive. In this study, we demonstrate that living cable bacteria actively move toward electrodes in different bioelectrochemical systems. Carbon felt and carbon fiber electrodes poised at +200 mV attracted live cable bacteria from the sediment. When the applied potential was switched off, cable bacteria retracted from the electrode. qPCR and scanning electron microscopy corroborated this finding and revealed cable bacteria in higher abundance present on the electrode surface compared with unpoised controls. These experiments raise new possibilities to study metabolism of cable bacteria and cultivate them in bioelectrochemical devices for bioelectronic applications, such as biosensing and bioremediation., Importance: Extracellular electron transfer is a metabolic function associated with electroactive bacteria wherein electrons are exchanged with external electron acceptors or donors. This feature has enabled the development of several applications, such as biosensing, carbon capture, and energy recovery. Cable bacteria are a unique class of long, filamentous microbes that perform long-distance electron transport in freshwater and marine sediments. In this study, we demonstrate the attraction of cable bacteria toward carbon electrodes and demonstrate their potential electroactivity. This finding enables electronic control and monitoring of the metabolism of cable bacteria and may, in turn, aid in the development of bioelectronic applications., Competing Interests: The authors declare no conflict of interest.

Bacteria perform diverse redox chemistries in the periplasm, cell wall, and extracellular space. Electron transfer for these extracytosolic activities is frequently mediated by proteins with covalently bound flavins, which are attached through post-translational flavinylation by the enzyme ApbE. Despite the significance of protein flavinylation to bacterial physiology, the basis and function of this modification remain unresolved. Here we apply genomic context analyses, computational structural biology, and biochemical studies to address the role of ApbE flavinylation throughout bacterial life. We identify ApbE flavinylation sites within structurally diverse protein domains and show that multi-flavinylated proteins, which may mediate longer distance electron transfer via multiple flavinylation sites, exhibit substantial structural heterogeneity. We identify two novel classes of flavinylation substrates that are related to characterized proteins with non-covalently bound flavins, providing evidence that protein flavinylation can evolve from a non-covalent flavoprotein precursor. We further find a group of structurally related flavinylation-associated cytochromes, including those with the domain of unknown function DUF4405, that presumably mediate electron transfer in the cytoplasmic membrane. DUF4405 homologs are widespread in bacteria and related to ferrosome iron storage organelle proteins that may facilitate iron redox cycling within ferrosomes. These studies reveal a complex basis for flavinylated electron transfer and highlight the discovery power of coupling comparative genomic analyses with high-quality structural models., Importance: This study explores the mechanisms bacteria use to transfer electrons outside the cytosol, a fundamental process involved in energy metabolism and environmental interactions. Central to this process is a phenomenon known as flavinylation, where a flavin molecule-a compound related to vitamin B2-is covalently attached to proteins, to enable electron transfer. We employed advanced genomic analysis and computational modeling to explore how this modification occurs across different bacterial species. Our findings uncover new types of proteins that undergo this modification and highlight the diversity and complexity of bacterial electron transfer mechanisms. This research broadens our understanding of bacterial physiology and informs potential biotechnological applications that rely on microbial electron transfer, including bioenergy production and bioremediation., Competing Interests: The authors declare no conflict of interest.

Certain members of the family Sulfolobaceae represent the only archaea known to oxidize elemental sulfur, and their evolutionary history provides a framework to understand the development of chemolithotrophic growth by sulfur oxidation. Here, we evaluate the sulfur oxidation phenotype of Sulfolobaceae species and leverage comparative genomic and transcriptomic analysis to identify the key genes linked to sulfur oxidation. Metabolic engineering of the obligate heterotroph Sulfolobus acidocaldarius revealed that the known cytoplasmic components of sulfur oxidation alone are not sufficient to drive prolific sulfur oxidation. Imaging analysis showed that Sulfolobaceae species maintain proximity to the sulfur surface but do not necessarily contact the substrate directly. This indicates that a soluble form of sulfur must be transported to initiate cytoplasmic sulfur oxidation. Conservation patterns and transcriptomic response implicate an extracellular tetrathionate hydrolase and putative thiosulfate transporter in a newly proposed mechanism of sulfur acquisition in the Sulfolobaceae.IMPORTANCESulfur is one of the most abundant elements on earth (2.9% by mass), so it makes sense that the earliest biology found a way to use sulfur to create and sustain life. However, beyond evolutionary significance, sulfur and the molecules it comprises have important technological significance, not only in chemicals such as sulfuric acid and in pyritic ores containing critical metals but also as a waste product from oil and gas production. The thermoacidophilic Sulfolobaceae are unique among the archaea as sulfur oxidizers. The trajectory for how sulfur biooxidation arose and evolved can be traced using experimental and bioinformatic analyses of the available genomic data set. Such analysis can also inform the process by which extracellular sulfur is acquired and transported by thermoacidophilic archaea, a phenomenon that is critical to these microorganisms but has yet to be elucidated., Competing Interests: The authors declare no conflict of interest.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite that infects warm-blooded vertebrates across the world. In humans, seropositivity rates of T. gondii range from 10% to 90% across communities. Despite its prevalence, few studies address how T. gondii infection changes the metabolism of host cells. In this study, we investigate how T. gondii manipulates the host cell metabolic environment by monitoring the metabolic response over time using noninvasive autofluorescence lifetime imaging of single cells, metabolite analysis, extracellular flux analysis, and reactive oxygen species (ROS) production. Autofluorescence lifetime imaging indicates that infected host cells become more oxidized and have an increased proportion of bound NAD(P)H compared to uninfected controls. Over time, infected cells also show decreases in levels of intracellular glucose and lactate, increases in oxygen consumption, and variability in ROS production. We further examined changes associated with the pre-invasion "kiss and spit" process using autofluorescence lifetime imaging, which also showed a more oxidized host cell with an increased proportion of bound NAD(P)H over 48 hours compared to uninfected controls, suggesting that metabolic changes in host cells are induced by T. gondii kiss and spit even without invasion.IMPORTANCEThis study sheds light on previously unexplored changes in host cell metabolism induced by T. gondii infection using noninvasive, label-free autofluorescence imaging. In this study, we use optical metabolic imaging (OMI) to measure the optical redox ratio (ORR) in conjunction with fluorescence lifetime imaging microscopy (FLIM) to noninvasively monitor single host cell response to T. gondii infection over 48 hours. Collectively, our results affirm the value of using autofluorescence lifetime imaging to noninvasively monitor metabolic changes in host cells over the time course of a microbial infection. Understanding this metabolic relationship between the host cell and the parasite could uncover new treatment and prevention options for T. gondii infections worldwide., Competing Interests: The authors declare no conflict of interest.

Climate change is rapidly transforming Arctic landscapes where increasing soil temperatures speed up permafrost thaw. This exposes large carbon stocks to microbial decomposition, possibly worsening climate change by releasing more greenhouse gases. Understanding how microbes break down soil carbon, especially under the anaerobic conditions of thawing permafrost, is important to determine future changes. Here, we studied the microbial community dynamics and soil carbon decomposition potential in permafrost and active layer soils under anaerobic laboratory conditions that simulated an Arctic summer thaw. The microbial and viral compositions in the samples were analyzed based on metagenomes, metagenome-assembled genomes, and metagenomic viral contigs (mVCs). Following the thawing of permafrost, there was a notable shift in microbial community structure, with fermentative Firmicutes and Bacteroidota taking over from Actinobacteria and Proteobacteria over the 60-day incubation period. The increase in iron and sulfate-reducing microbes had a significant role in limiting methane production from thawed permafrost, underscoring the competition within microbial communities. We explored the growth strategies of microbial communities and found that slow growth was the major strategy in both the active layer and permafrost. Our findings challenge the assumption that fast-growing microbes mainly respond to environmental changes like permafrost thaw. Instead, they indicate a common strategy of slow growth among microbial communities, likely due to the thermodynamic constraints of soil substrates and electron acceptors, and the need for microbes to adjust to post-thaw conditions. The mVCs harbored a wide range of auxiliary metabolic genes that may support cell protection from ice formation in virus-infected cells., Importance: As the Arctic warms, thawing permafrost unlocks carbon, potentially accelerating climate change by releasing greenhouse gases. Our research delves into the underlying biogeochemical processes likely mediated by the soil microbial community in response to the wet and anaerobic conditions, akin to an Arctic summer thaw. We observed a significant shift in the microbial community post-thaw, with fermentative bacteria like Firmicutes and Bacteroidota taking over and switching to different fermentation pathways. The dominance of iron and sulfate-reducing bacteria likely constrained methane production in the thawing permafrost. Slow-growing microbes outweighed fast-growing ones, even after thaw, upending the expectation that rapid microbial responses to dominate after permafrost thaws. This research highlights the nuanced and complex interactions within Arctic soil microbial communities and underscores the challenges in predicting microbial response to environmental change., Competing Interests: The authors declare no conflict of interest.

Nitrification by aquarium biofilters transforms ammonia waste (NH 3 /NH 4 + ) to less toxic nitrate (NO 3 - ) via nitrite (NO 2 - ). Prior to the discovery of complete ammonia-oxidizing ("comammox" or CMX) Nitrospira , previous research revealed that ammonia-oxidizing archaea (AOA) dominated over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Here, we profiled aquarium biofilter microbial communities and quantified the abundance of all three known ammonia oxidizers using 16S rRNA gene sequencing and quantitative PCR (qPCR), respectively. Biofilter and water samples were each collected from representative residential and commercial freshwater and saltwater aquaria. Distinct biofilter microbial communities were associated with freshwater and saltwater biofilters. Comammox Nitrospira amoA genes were detected in all 38 freshwater biofilter samples (average CMX amoA genes: 2.2 × 10 3 ± 1.5 × 10 3 copies/ng) and dominant in 30, whereas AOA were present in 35 freshwater biofilter samples (average AOA amoA genes: 1.1 × 10 3 ± 2.7 × 10 3 copies/ng) and only dominant in 7 of them. The AOB were at relatively low abundance within biofilters (average of 3.2 × 10 1 ± 1.1 × 10 2 copies of AOB amoA genes/ng of DNA), except for the aquarium with the highest ammonia concentration. For saltwater biofilters, AOA or AOB were differentially abundant, with no comammox Nitrospira detected. Additional sequencing of Nitrospira amoA genes revealed differential distributions, suggesting niche adaptation based on water chemistry (e.g., ammonia, carbonate hardness, and alkalinity). Network analysis of freshwater microbial communities demonstrated positive correlations between nitrifiers and heterotrophs, suggesting metabolic and ecological interactions within biofilters. These results demonstrate that comammox Nitrospira plays a previously overlooked, but important role in home aquarium biofilter nitrification., Importance: Nitrification is a crucial process that converts toxic ammonia waste into less harmful nitrate that occurs in aquarium biofilters. Prior research found that ammonia-oxidizing archaea (AOA) were dominant over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Our study profiled microbial communities of aquarium biofilters and quantified the abundance of all currently known groups of aerobic ammonia oxidizers. The findings reveal that complete ammonia-oxidizing (comammox) Nitrospira were present in all freshwater aquarium biofilter samples in high abundance, challenging our previous understanding of aquarium nitrification. We also highlight niche adaptation of ammonia oxidizers based on salinity. The network analysis of freshwater biofilter microbial communities revealed significant positive correlations among nitrifiers and other community members, suggesting intricate interactions within biofilter communities. Overall, this study expands our understanding of nitrification in aquarium biofilters, emphasizes the role of comammox Nitrospira , and highlights the value of aquaria as microcosms for studying nitrifier ecology., Competing Interests: Subsequent to when this research was conducted, J.D.N. received a research grant from the Natural Sciences and Engineering Research Council of Canada (NSERC), with industry partner Hagen Industries, to explore the possibility of using AOA and comammox Nitrospira to improve existing aquarium biological supplements.

Life depends on a conserved set of chemical energy currencies that are relics of early biochemistry. One of these is ATP, a molecule that, when paired with a divalent metal ion such as Mg 2+ , can be hydrolyzed to support numerous cellular and molecular processes. Despite its centrality to extant biochemistry, it is unclear whether ATP supported the function of ancient enzymes. We investigate the evolutionary necessity of ATP by experimentally reconstructing an ancestral variant of the N 2 -reducing enzyme nitrogenase. The Proterozoic ancestor is predicted to be ~540-2,300 million years old, post-dating the Great Oxidation Event. Growth rates under nitrogen-fixing conditions are ~80% of those of wild type in Azotobacter vinelandii . In the extant enzyme, the hydrolysis of two MgATP is coupled to electron transfer to support substrate reduction. The ancestor has a strict requirement for ATP with no other nucleotide triphosphate analogs (GTP, ITP, and UTP) supporting activity. Alternative divalent metal ions (Fe 2+ , Co 2+ , and Mn 2+ ) support activity with ATP but with diminished activities compared to Mg 2+ , similar to the extant enzyme. Additionally, it is shown that the ancestor has an identical efficiency in ATP hydrolyzed per electron transferred to the extant of two. Our results provide direct laboratory evidence of ATP usage by an ancient enzyme.IMPORTANCELife depends on energy-carrying molecules to power many sustaining processes. There is evidence that these molecules may predate the rise of life on Earth, but how and when these dependencies formed is unknown. The resurrection of ancient enzymes provides a unique tool to probe the enzyme's function and usage of energy-carrying molecules, shedding light on their biochemical origins. Through experimental reconstruction, this research investigates the ancestral dependence of a nitrogen-fixing enzyme on the energy carrier ATP, a requirement for function in the modern enzyme. We show that the resurrected ancestor does not have generalist nucleotide specificity. Rather, the ancestor has a strict requirement for ATP, like the modern enzyme, with similar function and efficiency. The findings elucidate the early-evolved necessity of energy-yielding molecules, delineating their role in ancient biochemical processes. Ultimately, these insights contribute to unraveling the intricate tapestry of evolutionary biology and the origins of life-sustaining dependencies., Competing Interests: The authors declare no conflict of interest.

Botrytis cinerea is a typical necrotrophic plant pathogenic fungus which can deliberately acidify host tissues and trigger oxidative bursts therein to facilitate its virulence. The white collar complex (WCC), consisting of BcWCL1 and BcWCL2, is recognized as the primary light receptor in B. cinerea . Nevertheless, the specific mechanisms through which the WCC components, particularly BcWCL2 as a GATA transcription factor, control virulence are not yet fully understood. This study demonstrates that deletion of BcWCL2 results in the loss of light-sensitive phenotypic characteristics. Additionally, the Δ bcwcl2 strain exhibits reduced secretion of citrate, delayed infection cushion development, weaker hyphal penetration, and decreased virulence. The application of exogenous citric acid was found to restore infection cushion formation, hyphal penetration, and virulence of the Δ bcwcl2 strain. Transcriptome analysis at 48 h post-inoculation revealed that two citrate synthases, putative citrate transporters, hydrolytic enzymes, and reactive oxygen species scavenging-related genes were down-regulated in Δ bcwcl2 , whereas exogenous citric acid application restored the expression of the above genes involved in the early infection process of Δ bcwcl2 . Moreover, the expression of Bcvel1 , a known regulator of citrate secretion, tissue acidification, and secondary metabolism, was down-regulated in Δ bcwcl2 but not in Δ bcwcl1 . ChIP-qPCR and electrophoretic mobility shift assays revealed that BcWCL2 can bind to the promoter sequences of Bcvel1 . Overexpressing Bcvel1 in Δ bcwcl2 was found to rescue the mutant defects. Collectively, our findings indicate that BcWCL2 regulates the expression of the global regulator Bcvel1 to influence citrate secretion, tissue acidification, redox homeostasis, and virulence of B. cinerea .IMPORTANCEThis study illustrated the significance of the fungal blue light receptor component BcWCL2 protein in regulating citrate secretion in Botrytis cinerea . Unlike BcWCL1, BcWCL2 may contribute to redox homeostasis maintenance during infection cushion formation, ultimately proving to be essential for full virulence. It is also demonstrated that BcWCL2 can regulate the expression of Bcvel1 to influence host tissue acidification, citrate secretion, infection cushion development, and virulence. While the role of organic acids secreted by plant pathogenic fungi in fungus-host interactions has been recognized, this paper revealed the importance, regulatory mechanisms, and key transcription factors that control organic acid secretion. These understanding of the pathogenetic mechanism of plant pathogens can provide valuable insights for developing effective prevention and treatment strategies against fungal diseases., Competing Interests: The authors declare no conflict of interest.

Sulfur-oxidizing bacteria (SOB) have developed distinct ecological strategies to obtain reduced sulfur compounds for growth. These range from specialists that can only use a limited range of reduced sulfur compounds to generalists that can use many different forms as electron donors. Forming intimate symbioses with animal hosts is another highly successful ecological strategy for SOB, as animals, through their behavior and physiology, can enable access to sulfur compounds. Symbioses have evolved multiple times in a range of animal hosts and from several lineages of SOB. They have successfully colonized a wide range of habitats, from seagrass beds to hydrothermal vents, with varying availability of symbiont energy sources. Our extensive analyses of sulfur transformation pathways in 234 genomes of symbiotic and free-living SOB revealed widespread conservation in metabolic pathways for sulfur oxidation in symbionts from different host species and environments, raising the question of how they have adapted to such a wide range of distinct habitats. We discovered a gene family expansion of soxY in these genomes, with up to five distinct copies per genome. Symbionts harboring only the "canonical" soxY were typically ecological "specialists" that are associated with specific host subfamilies or environments (e.g., hydrothermal vents, mangroves). Conversely, symbionts with multiple divergent soxY genes formed versatile associations across diverse hosts in various marine environments. We hypothesize that expansion and diversification of the soxY gene family could be one genomic mechanism supporting the metabolic flexibility of symbiotic SOB enabling them and their hosts to thrive in a range of different and dynamic environments.IMPORTANCESulfur metabolism is thought to be one of the most ancient mechanisms for energy generation in microorganisms. A diverse range of microorganisms today rely on sulfur oxidation for their metabolism. They can be free-living, or they can live in symbiosis with animal hosts, where they power entire ecosystems in the absence of light, such as in the deep sea. In the millions of years since they evolved, sulfur-oxidizing bacteria have adopted several highly successful strategies; some are ecological "specialists," and some are "generalists," but which genetic features underpin these ecological strategies are not well understood. We discovered a gene family that has become expanded in those species that also seem to be "generalists," revealing that duplication, repurposing, and reshuffling existing genes can be a powerful mechanism driving ecological lifestyle shifts., Competing Interests: The authors declare no conflict of interest.

Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers' deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus's central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS's function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion.IMPORTANCEBrown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of "omics" platforms has recently uncovered the key "oxidative-hydrolytic" mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes' catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system's use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts., Competing Interests: The authors declare no conflict of interest.

Extracellular cytochrome filaments are proposed to serve as conduits for long-range extracellular electron transfer. The primary functional physiological evidence has been the reported inhibition of Geobacter sulfurreducens Fe(III) oxide reduction when the gene for the filament-forming cytochrome OmcS is deleted. Here we report that the OmcS-deficient strain from that original report reduces Fe(III) oxide as well as the wild-type, as does a triple mutant in which the genes for the other known filament-forming cytochromes were also deleted. The triple cytochrome mutant displayed filaments with the same 3 nm diameter morphology and conductance as those produced by Escherichia coli heterologously expressing the G. sulfurreducens PilA pilin gene. Fe(III) oxide reduction was inhibited when the pilin gene in cytochrome-deficient mutants was modified to yield poorly conductive 3 nm diameter filaments. The results are consistent with the concept that 3 nm diameter electrically conductive pili (e-pili) are required for G. sulfurreducens long-range extracellular electron transfer. In contrast, rigorous physiological functional evidence is lacking for cytochrome filaments serving as conduits for long-range electron transport., Importance: Unraveling microbial extracellular electron transfer mechanisms has profound implications for environmental processes and advancing biological applications. This study on Geobacter sulfurreducens challenges prevailing beliefs on cytochrome filaments as crucial components thought to facilitate long-range electron transport. The discovery of an OmcS-deficient strain's unexpected effectiveness in Fe(III) oxide reduction prompted a reevaluation of the key conduits for extracellular electron transfer. By exploring the impact of genetic modifications on G. sulfurreducens ' performance, this research sheds light on the importance of 3-nm diameter electrically conductive pili in Fe(III) oxide reduction. Reassessing these mechanisms is essential for uncovering the true drivers of extracellular electron transfer in microbial systems, offering insights that could revolutionize applications across diverse fields., Competing Interests: The authors declare no conflict of interest.

Mycobacterium tuberculosis ( Mtb ), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum ( Mm ) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3 , a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD + , NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria., Importance: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para -hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH . Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH 's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis., Competing Interests: The authors declare no conflict of interest.

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In Escherichia coli , disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins, while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR, which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/VKOR, represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using E. coli cells expressing a periplasmic β-Galactosidase sensor (β-Gal dbs ), which is only active when disulfide bond formation is inhibited. Here, we report the construction of plasmids that allows fine-tuning of the expression of the β-Gal dbs sensor and can be mobilized into other gram-negative organisms. As an example, when expressed in Pseudomonas aeruginosa UCBPP-PA14, which harbors two DsbB homologs, β-Gal dbs behaves similarly as in E. coli , and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these β-Gal dbs reporter plasmids provide a basis to identify novel inhibitors of DsbA and DsbB/VKOR in multidrug-resistant gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria., Importance: Disulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria because inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens., Competing Interests: The authors declare no conflict of interest.

Marine oxygen-deficient zones (ODZs) are portions of the ocean where intense nitrogen loss occurs primarily via denitrification and anammox. Despite many decades of study, the identity of the microbes that catalyze nitrogen loss in ODZs is still being elucidated. Intriguingly, high transcription of genes in the same family as the nitric oxide dismutase ( nod ) gene from Methylomirabilota has been reported in the anoxic core of ODZs. Here, we show that the most abundantly transcribed nod genes in the Eastern Tropical North Pacific ODZ belong to a new order (UBA11136) of Alphaproteobacteria , rather than Methylomirabilota as previously assumed. Gammaproteobacteria and Planctomycetia also transcribe nod , but at lower relative abundance than UBA11136 in the upper ODZ. The nod -transcribing Alphaproteobacteria likely use formaldehyde and formate as a source of electrons for aerobic respiration, with additional electrons possibly from sulfide oxidation. They also transcribe multiheme cytochrome (here named ptd ) genes for a putative porin-cytochrome protein complex of unknown function, potentially involved in extracellular electron transfer. Molecular oxygen for aerobic respiration may originate from nitric oxide dismutation via cryptic oxygen cycling. Our results implicate Alphaproteobacteria order UBA11136 as a significant player in marine nitrogen loss and highlight their potential in one-carbon, nitrogen, and sulfur metabolism in ODZs.IMPORTANCEIn marine oxygen-deficient zones (ODZs), microbes transform bioavailable nitrogen to gaseous nitrogen, with nitric oxide as a key intermediate. The Eastern Tropical North Pacific contains the world's largest ODZ, but the identity of the microbes transforming nitric oxide remains unknown. Here, we show that highly transcribed nitric oxide dismutase ( nod ) genes belong to Alphaproteobacteria of the novel order UBA11136, which lacks cultivated isolates. These Alphaproteobacteria show evidence for aerobic respiration, using oxygen potentially sourced from nitric oxide dismutase, and possess a novel porin-cytochrome protein complex with unknown function. Gammaproteobacteria and Planctomycetia transcribe nod at lower levels. Our results pinpoint the microbes mediating a key step in marine nitrogen loss and reveal an unexpected predicted metabolism for marine Alphaproteobacteria., Competing Interests: The authors declare no conflict of interest.

Avilamycins, which possess potent inhibitory activity against Gram-positive bacteria, are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes . Among these structurally related oligosaccharide antibiotics, avilamycin A serves as the main bioactive component in veterinary drugs and animal feed additives, which differs from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. However, the mechanisms underlying assembly and modification of the oligosaccharide chain to diversify individual avilamycins remain poorly understood. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C . Remarkably, the ratio of these two components produced by AviZ1 depends on the utilization of specific redox cofactors, namely NADH/NAD + or NADPH/NADP + . These findings are inspired by gene disruption and complementation experiments and are further supported by in vitro enzymatic activity assays, kinetic analyses, and cofactor affinity studies on AviZ1-catalyzed redox reactions. Additionally, the results from sequence analysis, structure prediction, and site-directed mutagenesis of AviZ1 validate it as an NADH/NAD + -favored aldo-keto reductase that primarily oxidizes avilamycin C to form avilamycin A by utilizing abundant NAD + in vivo . Building upon the biological function and catalytic activity of AviZ1, overexpressing AviZ1 in S. viridochromogenes is thus effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study represents, to our knowledge, the first characterization of biochemical reactions involved in avilamycin biosynthesis and contributes to the construction of high-performance strains with industrial value.IMPORTANCEAvilamycins are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes , which can be used as veterinary drugs and animal feed additives. Avilamycin A is the most bioactive component, differing from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. Currently, the biosynthetic pathway of avilamycins is not clear. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C . More importantly, AviZ1 exhibits a unique NADH/NAD + preference, allowing it to efficiently catalyze the oxidation of avilamycin C to form avilamycin A using abundant NAD + in cells. Thus, overexpressing AviZ1 in S. viridochromogenes is effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study serves as an enzymological guide for rational strain design, and the resulting high-performance strains have significant industrial value., Competing Interests: The authors declare no conflict of interest.

Chloroflexota bacteria are abundant and globally distributed in various deep-sea ecosystems. It has been reported based on metagenomics data that two deep-sea Chloroflexota lineages (the SAR202 group and Dehalococcoidia class) have the potential to drive sulfur cycling. However, the absence of cultured Chloroflexota representatives is a significant bottleneck toward understanding their contribution to the deep-sea sulfur cycling. In this study, we find that Phototrophicus methaneseepsis ZRK33 isolated from deep-sea sediment has a heterotrophic lifestyle and can assimilate sulfate and thiosulfate. Using combined physiological, genomic, proteomic, and in situ transcriptomic methods, we find that strain ZRK33 can perform assimilatory sulfate reduction in both laboratory and deep-sea conditions. Metabolism of sulfate or thiosulfate by strain ZRK33 significantly promotes the transport and degradation of various macromolecules and thereby stimulates the energy production. In addition, metagenomic results show that genes associated with assimilatory and dissimilatory sulfate reduction are ubiquitously distributed in the metagenome-assembled genomes of Chloroflexota members derived from deep-sea sediments. Metatranscriptomic results also show that the expression levels of related genes are upregulated, strongly suggesting that Chloroflexota bacteria may play undocumented roles in deep-sea sulfur cycling., Importance: The cycling of sulfur is one of Earth's major biogeochemical processes and is closely related to the energy metabolism of microorganisms living in the deep-sea cold seep and hydrothermal vents. To date, some of the members of Chloroflexota are proposed to play a previously unrecognized role in sulfur cycling. However, the sulfur metabolic characteristics of deep-sea Chloroflexota bacteria have never been reported, and remain to be verified in cultured deep-sea representatives. Here, we show that the deep-sea Chloroflexota bacterium ZRK33 can perform sulfate assimilation in both laboratory and deep-sea conditions, which expands our knowledge of the sulfur metabolic potential of deep-sea Chloroflexota bacteria. We also show that the genes associated with assimilatory and dissimilatory sulfate reduction ubiquitously distribute in the deep-sea Chloroflexota members, providing hints to the roles of Chloroflexota bacteria in deep-sea sulfur biogeochemical cycling., Competing Interests: The authors declare no conflict of interest.

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H 2 O 2 ), to degrade lignocellulose in wood. H 2 O 2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO 2 ) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H 2 18 O 2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H 2 O 2 ) on secreted proteins remains unclear. This study aims at evaluating the effect of H 2 O 2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H 2 O 2 . The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown., Competing Interests: The authors declare no conflict of interest.

Geobacter sp. strain SVR uses antimonate [Sb(V)] as a terminal electron acceptor for anaerobic respiration. Here, we visualized a possible key enzyme, periplasmic Sb(V) reductase (Anr), via active staining and non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that a novel dimethyl sulfoxide (DMSO) reductase family protein, WP&#95;173201954.1, is involved in Anr. This protein was closely related with AnrA, a protein suggested to be the catalytic subunit of a respiratory Sb(V) reductase in Desulfuribacillus stibiiarsenatis . The anr genes of strain SVR ( anrXSRBAD ) formed an operon-like structure, and their transcription was upregulated under Sb(V)-respiring conditions. The expression of anrA gene was induced by more than 1 µM of antimonite [Sb(III)]; however, arsenite [As(III)] did not induce the expression of anrA gene. Tandem mass tag-based proteomic analysis revealed that, in addition to Anr proteins, proteins in the following categories were upregulated under Sb(V)-respiring conditions: (i) Sb(III) efflux systems such as Ant and Ars; (ii) antioxidizing proteins such as ferritin, rubredoxin, and thioredoxin; (iii) protein quality control systems such as HspA, HslO, and DnaK; and (iv) DNA repair proteins such as UspA and UvrB. These results suggest that strain SVR copes with antimony stress by modulating pleiotropic processes to resist and actively metabolize antimony. To the best of our knowledge, this is the first report to demonstrate the involvement of AnrA in Sb(V) respiration at the protein level. Furthermore, this is the first example to show high expression of the Ant system proteins in the Sb(V)-respiring bacterium.IMPORTANCEAntimony (Sb) exists mainly as antimonite [Sb(III)] or antimonate [Sb(V)] in the environment, and Sb(III) is more toxic than Sb(V). Recently, microbial involvement in Sb redox reactions has received attention. Although more than 90 Sb(III)-oxidizing bacteria have been reported, information on Sb(V)-reducing bacteria is limited. Especially, the enzyme involved in dissimilatory Sb(V) reduction, or Sb(V) respiration, is unclear, despite this pathway being very important for the circulation of Sb in nature. In this study, we demonstrated that the Sb(V) reductase (Anr) of an Sb(V)-respiring bacterium ( Geobacter sp. SVR) is a novel member of the dimethyl sulfoxide (DMSO) reductase family. In addition, we found that strain SVR copes with Sb stress by modulating pleiotropic processes, including the Ant and Ars systems, and upregulating the antioxidant and quality control protein levels. Considering the abundance and diversity of putative anr genes in the environment, Anr may play a significant role in global Sb cycling in both marine and terrestrial environments., Competing Interests: The authors declare no conflict of interest.

Nitrogen availability limits the net primary productivity in alpine meadows on the Qinghai-Tibetan Plateau, which is regulated by ammonia-oxidizing microorganisms. However, little is known about the elevational patterns of soil ammonia oxidizers in alpine meadows. Here, we investigated the potential nitrification rate (PNR), abundance, and community diversity of soil ammonia-oxidizing microorganisms along the altitudinal gradient between 3,200 and 4,200 m in Qinghai-Tibetan alpine meadows. We found that both PNR and amoA gene abundance declined from 3,400 to 4,200 m but lowered at 3,200 m, possibly due to intense substrate competition and biological nitrification inhibition from grasses. The primary contributors to soil nitrification were ammonia-oxidizing archaea (AOA), and their proportionate share of soil nitrification increased with altitude in comparison to ammonia-oxidizing bacteria (AOB). The alpha diversity of AOA increased by higher temperature and plant richness at low elevations, while decreased by higher moisture and low legume biomass at middle elevations. In contrast, the alpha diversity of AOB increased along elevation. The elevational patterns of AOA and AOB communities were primarily driven by temperature, soil moisture, and vegetation. These findings suggest that elevation-induced climate changes, such as shifts in temperature and water conditions, could potentially alter the soil nitrification process in alpine meadows through changes in vegetation and soil properties, which provide new insights into how soil ammonia oxidizers respond to climate change in alpine meadows.IMPORTANCEThe importance of this study is revealing that elevational patterns and nitrification contributions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) communities were primarily driven by temperature, soil moisture, and vegetation. Compared to AOB, the relative contribution of AOA to soil nitrification increased at higher elevations. The research highlights the potential impact of elevation-induced climate change on nitrification processes in alpine meadows, mediated by alterations in vegetation and soil properties. By providing new insights into how ammonia oxidizers respond to climate change, this study contributes valuable knowledge to the field of microbial ecology and helps predict ecological responses to environmental changes in alpine meadows., Competing Interests: The authors declare no conflict of interest.

Aerobic ammonia oxidizers (AOs) are prokaryotic microorganisms that contribute to the global nitrogen cycle by performing the first step of nitrification, the oxidation of ammonium to nitrite and nitrate. While aerobic AOs are found ubiquitously, their distribution is controlled by key environmental conditions such as substrate (ammonium) availability. Ammonia-oxidizing archaea (AOA) and complete ammonia oxidizers (comammox) are generally found in oligotrophic environments with low ammonium availability. However, whether AOA and comammox share these habitats or outcompete each other is not well understood. We assessed the competition for ammonium between an AOA and comammox enriched from the freshwater Lake Burr Oak. The AOA enrichment culture (AOA-BO1) contained Nitrosarchaeum sp. BO1 as the ammonia oxidizer and Nitrospira sp. BO1 as the nitrite oxidizer. The comammox enrichment BO4 (cmx-BO4) contained the comammox strain Nitrospira sp. BO4. The competition experiments were performed either in continuous cultivation with ammonium as a growth-limiting substrate or in batch cultivation with initial ammonium concentrations of 50 and 500 µM. Regardless of the ammonium concentration, Nitrospira sp. BO4 outcompeted Nitrosarchaeum sp. BO1 under all tested conditions. The dominance of Nitrospira sp. BO4 could be explained by the ability of comammox to generate more energy through the complete oxidation of ammonia to nitrate and their more efficient carbon fixation pathway-the reductive tricarboxylic acid cycle. Our results are supported by the higher abundance of comammox compared to AOA in the sediment of Lake Burr Oak., Importance: Nitrification is a key process in the global nitrogen cycle. Aerobic ammonia oxidizers play a central role in the nitrogen cycle by performing the first step of nitrification. Ammonia-oxidizing archaea (AOA) and complete ammonia oxidizers (comammox) are the dominant nitrifiers in environments with low ammonium availability. While AOA have been studied for almost 20 years, comammox were only discovered 8 years ago. Until now, there has been a gap in our understanding of whether AOA and comammox can co-exist or if one strain would be dominant under ammonium-limiting conditions. Here, we present the first study characterizing the competition between freshwater AOA and comammox under varying substrate concentrations. Our results will help in elucidating the niches of two key nitrifiers in freshwater lakes., Competing Interests: The authors declare no conflict of interest.

Acetate is a major intermediate in the anaerobic digestion of organic waste to produce CH 4 . In methanogenic systems, acetate degradation is carried out by either acetoclastic methanogenesis or syntrophic degradation by acetate oxidizers and hydrogenotrophic methanogens. Due to challenges in the isolation of syntrophic acetate-oxidizing bacteria (SAOB), the diversity and metabolism of SAOB and the mechanisms of their interactions with methanogenic partners are not fully characterized. In this study, the in situ activity and metabolic characteristics of potential SAOB and their interactions with methanogens were elucidated through metagenomics and metatranscriptomics. In addition to the reported SAOB classified in the genera Tepidanaerobacter , Desulfotomaculum , and Thermodesulfovibrio , we identified a number of potential SAOB that are affiliated with Clostridia , Thermoanaerobacteraceae, Anaerolineae, and Gemmatimonadetes. The potential SAOB possessing the glycine-mediated acetate oxidation pathway dominates SAOB communities. Moreover, formate appeared to be the main product of the acetate degradation by the most active potential SAOB. We identified the methanogen partner of these potential SAOB in the acetate-fed chemostat as Methanosarcina thermophila . The dominated potential SAOB in each chemostat had similar metabolic characteristics, even though they were in different fatty-acid-fed chemostats. These novel syntrophic lineages are prevalent and may play critical roles in thermophilic methanogenic reactors. This study expands our understanding of the phylogenetic diversity and in situ biological functions of uncultured syntrophic acetate degraders and presents novel insights into how they interact with methanogens.IMPORTANCECombining reactor operation with omics provides insights into novel uncultured syntrophic acetate degraders and how they perform in thermophilic anaerobic digesters. This improves our understanding of syntrophic acetate degradation and contributes to the background knowledge necessary to better control and optimize anaerobic digestion processes., Competing Interests: The authors declare no conflict of interest.

The rapid degradation of short-chain fatty acids (SCFAs) is an essential issue of anaerobic digestion (AD), in which SCFA oxidizers could generally metabolize in syntrophy with methanogens. The dynamic responses of active metagenome-assembled genomes to low concentrations of propionate and acetate were analyzed to identify specific syntrophic SCFA oxidizers and their metabolic characteristics in continuous-flow AD systems treating waste activated sludge with and without hydrochar. In this study, hydrochar increased methane production by 19%, possibly due to hydrochar enhancing acidification and methanogenesis processes. A putative syntrophic propionate oxidizer and two acetate oxidizers contributed substantially to the syntrophic degradation of SCFAs, and hydrochar positively regulated their functional gene expressions. A significant relationship was established between the replication rate of SCFA oxidizers and their stimulation-related transcriptional activity. Acetate was degraded in the hydrochar group, which might be mainly through the syntrophic acetate oxidizer from the genus Desulfallas and methanogens from the genus Methanosarcina .IMPORTANCEShort-chain fatty acid (SCFA) degradation is an important process in the methanogenic ecosystem. However, current knowledge of this microbial mechanism is mainly based on studies on a few model organisms incubated as mono- or co-cultures or in enrichments, which cannot provide appropriate evidence in complex environments. Here, this study revealed the microbial mechanism of a hydrochar-mediated anaerobic digestion (AD) system promoting SCFA degradation at the species level and identified key SCFA oxidizing bacteria. Our analysis provided new insights into the SCFA oxidizers involved in the AD of waste activated sludge facilitated by hydrochar., Competing Interests: The authors declare no conflict of interest.

A genome scale metabolic model of the bacterium Paracoccus denitrificans has been constructed. The model containing 972 metabolic genes, 1,371 reactions, and 1,388 unique metabolites has been reconstructed. The model was used to carry out quantitative predictions of biomass yields on 10 different carbon sources under aerobic conditions. Yields on C1 compounds suggest that formate is oxidized by a formate dehydrogenase O, which uses ubiquinone as redox co-factor. The model also predicted the threshold methanol/mannitol uptake ratio, above which ribulose biphosphate carboxylase has to be expressed in order to optimize biomass yields. Biomass yields on acetate, formate, and succinate, when NO 3 - is used as electron acceptor, were also predicted correctly. The model reconstruction revealed the capability of P. denitrificans to grow on several non-conventional substrates such as adipic acid, 1,4-butanediol, 1,3-butanediol, and ethylene glycol. The capacity to grow on these substrates was tested experimentally, and the experimental biomass yields on these substrates were accurately predicted by the model.IMPORTANCE Paracoccus denitrificans has been broadly used as a model denitrifying organism. It grows on a large portfolio of carbon sources, under aerobic and anoxic conditions. These characteristics, together with its amenability to genetic manipulations, make P. denitrificans a promising cell factory for industrial biotechnology. This paper presents and validates the first functional genome-scale metabolic model for P. denitrificans , which is a key tool to enable P. denitrificans as a platform for metabolic engineering and industrial biotechnology. Optimization of the biomass yield led to accurate predictions in a broad scope of substrates., Competing Interests: The authors declare no conflict of interest.

Many Acinetobacter species can grow on n -alkanes of varying lengths (≤C40). AlmA, a unique flavoprotein in these Acinetobacter strains, is the only enzyme proven to be required for the degradation of long-chain (LC) n -alkanes, including C32 and C36 alkanes. Although it is commonly presumed to be a terminal hydroxylase, its role in n -alkane degradation remains elusive. In this study, we conducted physiological, biochemical, and bioinformatics analyses of AlmA to determine its role in n -alkane degradation by Acinetobacter baylyi ADP1. Consistent with previous reports, gene deletion analysis showed that almA was vital for the degradation of LC n -alkanes (C26-C36). Additionally, enzymatic analysis revealed that AlmA catalyzed the conversion of aliphatic 2-ketones (C10-C16) to their corresponding esters, but it did not conduct n -alkane hydroxylation under the same conditions, thus suggesting that AlmA in strain ADP1 possesses Baeyer-Villiger monooxygenase (BVMO) activity. These results were further confirmed by bioinformatics analysis, which revealed that AlmA was closer to functionally identified BVMOs than to hydroxylases. Altogether, the results of our study suggest that LC n -alkane degradation by strain ADP1 possibly follows a novel subterminal oxidation pathway that is distinct from the terminal oxidation pathway followed for short-chain n -alkane degradation. Furthermore, our findings suggest that AlmA catalyzes the third reaction in the LC n -alkane degradation pathway.IMPORTANCEMany microbial studies on n -alkane degradation are focused on the genes involved in short-chain n -alkane (≤C16) degradation; however, reports on the genes involved in long-chain (LC) n -alkane (>C20) degradation are limited. Thus far, only AlmA has been reported to be involved in LC n -alkane degradation by Acinetobacter spp.; however, its role in the n -alkane degradation pathway remains elusive. In this study, we conducted a detailed characterization of AlmA in A. baylyi ADP1 and found that AlmA exhibits Baeyer-Villiger monooxygenase activity, thus indicating the presence of a novel LC n -alkane biodegradation mechanism in strain ADP1., Competing Interests: The authors declare no conflict of interest.

Biodesulfurization (BD) systems that treat sour gas employ mixtures of haloalkaliphilic sulfur-oxidizing bacteria to convert sulfide to elemental sulfur. In the past years, these systems have seen major technical innovations that have led to changes in microbial community composition. Different studies have identified and discussed the microbial communities in both traditional and improved systems. However, these studies do not identify metabolically active community members and merely focus on members' presence/absence. Therefore, their results cannot confirm the activity and role of certain bacteria in the BD system. To investigate the active community members, we determined the microbial communities of six different runs of a pilot-scale BD system. 16S rRNA gene-based amplicon sequencing was performed using both DNA and RNA. A comparison of the DNA- and RNA-based sequencing results identified the active microbes in the BD system. Statistical analyses indicated that not all the existing microbes were actively involved in the system and that microbial communities continuously evolved during the operation. At the end of the run, strains affiliated with Alkalilimnicola ehrlichii and Thioalkalivibrio sulfidiphilus were confirmed as the most active key bacteria in the BD system. This study determined that microbial communities were shaped predominantly by the combination of hydraulic retention time (HRT) and sulfide concentration in the anoxic reactor and, to a lesser extent, by other operational parameters.IMPORTANCEHaloalkaliphilic sulfur-oxidizing bacteria are integral to biodesulfurization (BD) systems and are responsible for converting sulfide to sulfur. To understand the cause of conversions occurring in the BD systems, knowing which bacteria are present and active in the systems is essential. So far, only a few studies have investigated the BD system's microbial composition, but none have identified the active microbial community. Here, we reveal the metabolically active community, their succession, and their influence on product formation., Competing Interests: The authors declare no conflict of interest.

Importance: In waterlogged soils, iron plaque forms a reactive barrier between the root and soil, collecting phosphate and metals such as arsenic and cadmium. It is well established that iron-reducing bacteria solubilize iron, releasing these associated elements. In contrast, microbial roles in plaque formation have not been clear. Here, we show that there is a substantial population of iron oxidizers in plaque, and furthermore, that these organisms ( Sideroxydans and Gallionella ) are distinguished by genes for plant colonization and nutrient fixation. Our results suggest that iron-oxidizing and iron-reducing bacteria form and remodel iron plaque, making it a dynamic system that represents both a temporary sink for elements (P, As, Cd, C, etc.) as well as a source. In contrast to abiotic iron oxidation, microbial iron oxidation results in coupled Fe-C-N cycling, as well as microbe-microbe and microbe-plant ecological interactions that need to be considered in soil biogeochemistry, ecosystem dynamics, and crop management., Competing Interests: The authors declare no conflict of interest.

Importance: Rhodospirillum rubrum vast metabolic versatility places it as a remarkable model bacterium and an excellent biotechnological chassis. The key component of photosynthesis (PS) studied in this work (HP1) stands out among the other members of PpaA/AerR anti-repressor family since it lacks the motif they all share: the cobalamin B-12 binding motif. Despite being reduced and poorly conserved, HP1 stills controls PS as the other members of the family, allowing a fast response to changes in the redox state of the cell. This work also shows that HP1 absence affects genes from relevant biological processes other than PS, including nitrogen fixation and stress response. From a biotechnological perspective, HP1 could be manipulated in approaches where PS is not necessary, such as hydrogen or polyhydroxyalkanoates production, to save energy., Competing Interests: The authors declare no conflict of interest.

Importance: Knowledge on microbial iron oxidation is important for understanding the cycling of iron, carbon, nitrogen, nutrients, and metals. The current study yields important insights into the niche sharing, diversification, and Fe(III) oxyhydroxide morphology of Ghiorsea , an iron- and hydrogen-oxidizing Zetaproteobacteria representative belonging to Zetaproteobacteria operational taxonomic unit 9. The study proposes that Ghiorsea exhibits a more extensive morphology of Fe(III) oxyhydroxide than previously observed. Overall, the results increase our knowledge on potential drivers of Zetaproteobacteria diversity in iron microbial mats and can eventually be used to develop strategies for the cultivation of sheath-forming Zetaproteobacteria., Competing Interests: The authors declare no conflict of interest.

Importance: Neutrophilic iron-oxidizing bacteria (FeOB) produce copious iron (oxyhydr)oxides that can profoundly influence biogeochemical cycles, notably the fate of carbon and many metals. To fully understand environmental microbial iron oxidation, we need a thorough accounting of iron oxidation mechanisms. In this study, we show the Gallionellaceae FeOB genomes encode both characterized iron oxidases as well as uncharacterized multiheme cytochromes (MHCs). MHCs are predicted to transfer electrons from extracellular substrates and likely confer metabolic capabilities that help Gallionellaceae occupy a range of different iron- and mineral-rich niches. Gallionellaceae appear to specialize in iron oxidation, so it would be advantageous for them to have multiple mechanisms to oxidize various forms of iron, given the many iron minerals on Earth, as well as the physiological and kinetic challenges faced by FeOB. The multiple iron/mineral oxidation mechanisms may help drive the widespread ecological success of Gallionellaceae., Competing Interests: The authors declare no conflict of interest.

Importance: Nitrification, the microbial conversion of ammonia to nitrate via nitrite, plays a pivotal role in the global nitrogen cycle. However, the excessive use of ammonium-based fertilizers in agriculture has disrupted this cycle, leading to groundwater pollution and greenhouse gas emissions. In this study, we have demonstrated the inhibitory effects of plant-derived juglone and related 1,4-naphthoquinones on the nitrification process in Nitrosomonas europaea . Notably, the inhibition mechanism is elucidated in which 1,4-naphthoquinones interact with hydroxylamine oxidoreductase, disrupting the electron transfer to cytochrome c 554 , a physiological electron acceptor. These findings support the notion that phytochemicals can impede nitrification by interfering with the essential electron transfer process in ammonia oxidation. The findings presented in this article offer valuable insights for the development of strategies aimed at the management of nitrification, reduction of fertilizer utilization, and mitigation of greenhouse gas emissions., Competing Interests: NARO holds a patent (WO2017126542A1) for the method of measuring oxidoreductase activity which inventors are T.Y and Y.N.

Microorganisms are essential drivers of earth's geochemical cycles. However, the significance of elemental redox cycling mediated by microorganisms is often underestimated beyond the most well-studied nutrient cycles. Phosphite, (per)chlorate, and iodate are each considered esoteric substrates metabolized by microorganisms. However, recent investigations have indicated that these metabolisms are widespread and ubiquitous, affirming a need to continue studying the underlying microbiology to understand their biogeochemical effects and their interface with each other and our biosphere. This review focuses on combining canonical techniques of culturing microorganisms with modern omic approaches to further our understanding of obscure metabolic pathways and elucidate their importance in global biogeochemical cycles. Using these approaches, marker genes of interest have already been identified for phosphite, (per)chlorate, and iodate using traditional microbial physiology and genetics. Subsequently, their presence was queried to reveal the distribution of metabolic pathways in the environment using publicly available databases. In conjunction with each other, computational and experimental techniques provide a more comprehensive understanding of the location of these microorganisms, their underlying biochemistry and genetics, and how they tie into our planet's geochemical cycles., Competing Interests: The authors declare no conflict of interest.

Importance: While quinones are essential for respiratory microorganisms, their importance for microbes that rely on fermentation metabolism is not understood. This gap in knowledge hinders our understanding of anaerobic microbial habitats, such in mammalian digestive tracts and fermented foods. We show that Lactiplantibacillus plantarum, a model fermentative lactic acid bacteria species abundant in human, animal, and insect microbiomes and fermented foods, uses multiple exogenous, environmental quinones as electron shuttles for a hybrid metabolism involving EET. Interestingly, quinones both stimulate this metabolism as well as cause oxidative stress when extracellular electron acceptors are absent. We also found that quinone-producing, lactic acid bacteria species commonly enriched together with L. plantarum in food fermentations accelerate L. plantarum growth and medium acidification through a mainly quinone- and EET-dependent mechanism. Thus, our work provides evidence of quinone cross-feeding as a key ecological feature of anaerobic microbial habitats., Competing Interests: The authors declare no conflict of interest.

Importance: In 2022, it was estimated that 10.6 million people fell ill, and 1.6 million people died from tuberculosis (TB). Available treatment is lengthy and requires a multi-drug regimen, which calls for new strategies to cure Mycobacterium tuberculosis ( Mtb ) infections more efficiently. We have previously shown that simultaneous inactivation of type 1 (Ndh-1) and type 2 (Ndh-2) NADH dehydrogenases kills Mtb . NADH dehydrogenases play two main physiological roles: NADH oxidation and electron entry into the respiratory chain. Here, we show that this bactericidal effect is a consequence of impaired NADH oxidation. Importantly, we demonstrate that Ndh-1/Ndh-2 synthetic lethality can be achieved through simultaneous chemical inhibition, which could be exploited by TB drug development programs., Competing Interests: The authors declare no conflict of interest.

Importance: Synthetic nitrification inhibitors are routinely used with nitrogen fertilizers to reduce nitrogen losses from agroecosystems, despite having drawbacks like poor efficiency, cost, and entry into the food chain. Plant-derived BNIs constitute a more environmentally conducive alternative. Knowledge on the activity of BNIs to soil nitrifiers is largely based on bioassays with a single Nitrosomonas europaea strain which does not constitute a dominant member of the community of ammonia-oxidizing microorganisms (AOM) in soil. We determined the activity of several plant-derived molecules reported as having activity, including the recently discovered maize-isolated BNI, zeanone, and its natural analog, 2-methoxy-1,4-naphthoquinone, on a range of ecologically relevant AOM and one nitrite-oxidizing bacterial culture, expanding our knowledge on the intrinsic inhibition potential of BNIs toward AOM and highlighting the necessity for a deeper understanding of the effect of BNIs on the overall soil microbiome integrity before their further use in agricultural settings., Competing Interests: The authors declare no conflict of interest.

Comamonas testosteroni TA441 is capable of aerobically degrading steroids through the aromatization and cleavage of the A- and B-rings, followed by D- and C-ring cleavage via β-oxidation. While most of the degradation steps have been previously characterized, a few intermediate compounds remained unidentified. In this study, we proposed that the cleavage of the D-ring at C13-17 required the ScdY hydratase, followed by C-ring cleavage via the ScdL1L2 transferase. The anticipated reaction was expected to yield 6-methyl-3,7-dioxo-decane-1,10-dioic acid-coenzyme A (CoA) ester. To confirm this hypothesis, we constructed a plasmid enabling the induction of targeted genes in TA441 mutant strains. Induction experiments of ScdL1L2 revealed that the major product was 3-hydroxy-6-methyl-7-oxo-decane-1,10-dioic acid-CoA ester. Similarly, induction experiments of ScdY demonstrated that the substrate of ScdY was a geminal diol, 17-dihydroxy-9-oxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid-CoA ester. These findings suggest that ScdY catalyzes the addition of a water molecule at C14 of 17-dihydroxy-9-oxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid-CoA ester, leading to D-ring cleavage at C13-17. Subsequently, the C9 ketone of the D-ring cleavage product is converted to a hydroxyl group, followed by C-ring cleavage, resulting in the production of 3-hydroxy-6-methyl-7-oxo-decane-1,10-dioic acid-CoA ester.IMPORTANCEStudies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain substrates for steroid drugs. In recent years, the role of steroid-degrading bacteria in relation to human health has gained significant attention, as emerging evidence suggests that the intestinal microflora plays a crucial role in human health. Furthermore, cholic acid, a major component of bile acid secreted in the intestines, is closely associated with the gut microbiota. While Comamonas testosteroni TA441 is recognized as the leading bacterial model for aerobic steroid degradation, the involvement of aerobic steroid degradation in the intestinal microflora remains largely unexplored. Nonetheless, the presence of C. testosteroni in the cecum suggests the potential influence of aerobic steroid degradation on gut microbiota. To establish essential information about the role of these bacteria, here, we identified the missing compounds and propose more details of C-, and D-ring cleavage, which have remained unclear until now., Competing Interests: The authors declare no conflict of interest.