Genetic manipulation of Patescibacteria provides mechanistic insights into microbial dark matter and the epibiotic lifestyle.

Patescibacteria, also known as the candidate phyla radiation (CPR), are a diverse group of bacteria that constitute a disproportionately large fraction of microbial dark matter. Its few cultivated members, belonging mostly to Saccharibacteria, grow as epibionts on host Actinobacteria. Due to a lack of suitable tools, the genetic basis of this lifestyle and other unique features of Patescibacteira remain unexplored. Here, we show that Saccharibacteria exhibit natural competence, and we exploit this property for their genetic manipulation. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth, and a transposon-insertion sequencing (Tn-seq) genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii, as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.

The Science of Antibiotic Discovery.

We are experiencing an antimicrobial resistance (AMR) crisis, brought on by the drying up of the antibiotic discovery pipeline and the resulting unchecked spread of resistant pathogens. Traditional methods of screening environmental isolates or compound libraries have not produced a new drug in over 30 years. Antibiotic discovery is uniquely difficult due to a highly restrictive penetration barrier and other mechanisms that allow bacteria to survive in the presence of toxic compounds. In this Perspective, we analyze the challenges facing discovery and discuss an emerging new platform for antibiotic discovery. The penetration barrier makes screening conventional synthetic compound libraries largely impractical, and actinomycetes, the main source of natural product compounds, have been overmined. The emerging platform is based on understanding the rules that guide the permeation of molecules into bacteria and on advances in microbiology, which enable us to identify and access attractive groups of secondary metabolite producers. Establishing this platform will enable reliable production of lead compounds to combat AMR.

Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.

Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.

Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion.

Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increase in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.

Host remodeling of the gut microbiome and metabolic changes during pregnancy.

Many of the immune and metabolic changes occurring during normal pregnancy also describe metabolic syndrome. Gut microbiota can cause symptoms of metabolic syndrome in nonpregnant hosts. Here, to explore their role in pregnancy, we characterized fecal bacteria of 91 pregnant women of varying prepregnancy BMIs and gestational diabetes status and their infants. Similarities between infant-mother microbiotas increased with children's age, and the infant microbiota was unaffected by mother's health status. Gut microbiota changed dramatically from first (T1) to third (T3) trimesters, with vast expansion of diversity between mothers, an overall increase in Proteobacteria and Actinobacteria, and reduced richness. T3 stool showed strongest signs of inflammation and energy loss; however, microbiome gene repertoires were constant between trimesters. When transferred to germ-free mice, T3 microbiota induced greater adiposity and insulin insensitivity compared to T1. Our findings indicate that host-microbial interactions that impact host metabolism can occur and may be beneficial in pregnancy.

Systems biology elucidates the distinctive metabolic niche filled by the human gut microbe Eggerthella lenta.

Human gut bacteria perform diverse metabolic functions with consequences for host health. The prevalent and disease-linked Actinobacterium Eggerthella lenta performs several unusual chemical transformations, but it does not metabolize sugars and its core growth strategy remains unclear. To obtain a comprehensive view of the metabolic network of E. lenta, we generated several complementary resources: defined culture media, metabolomics profiles of strain isolates, and a curated genome-scale metabolic reconstruction. Stable isotope-resolved metabolomics revealed that E. lenta uses acetate as a key carbon source while catabolizing arginine to generate ATP, traits which could be recapitulated in silico by our updated metabolic model. We compared these in vitro findings with metabolite shifts observed in E. lenta-colonized gnotobiotic mice, identifying shared signatures across environments and highlighting catabolism of the host signaling metabolite agmatine as an alternative energy pathway. Together, our results elucidate a distinctive metabolic niche filled by E. lenta in the gut ecosystem. Our culture media formulations, atlas of metabolomics data, and genome-scale metabolic reconstructions form a freely available collection of resources to support further study of the biology of this prevalent gut bacterium.

An engineered PET depolymerase to break down and recycle plastic bottles.

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.

Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling.

Addressing the ongoing antibiotic crisis requires the discovery of compounds with novel mechanisms of action that are capable of treating drug-resistant infections1. Many antibiotics are sourced from specialized metabolites produced by bacteria, particularly those of the Actinomycetes family2. Although actinomycete extracts have traditionally been screened using activity-based platforms, this approach has become unfavourable owing to the frequent rediscovery of known compounds. Genome sequencing of actinomycetes reveals an untapped reservoir of biosynthetic gene clusters, but prioritization is required to predict which gene clusters may yield promising new chemical matter2. Here we make use of the phylogeny of biosynthetic genes along with the lack of known resistance determinants to predict divergent members of the glycopeptide family of antibiotics that are likely to possess new biological activities. Using these predictions, we uncovered two members of a new functional class of glycopeptide antibiotics-the known glycopeptide antibiotic complestatin and a newly discovered compound we call corbomycin-that have a novel mode of action. We show that by binding to peptidoglycan, complestatin and corbomycin block the action of autolysins-essential peptidoglycan hydrolases that are required for remodelling of the cell wall during growth. Corbomycin and complestatin have low levels of resistance development and are effective in reducing bacterial burden in a mouse model of skin MRSA infection.

A treasure trove of 1034 actinomycete genomes.

Filamentous Actinobacteria, recently renamed Actinomycetia, are the most prolific source of microbial bioactive natural products. Studies on biosynthetic gene clusters benefit from or require chromosome-level assemblies. Here, we provide DNA sequences from >1000 isolates: 881 complete genomes and 153 near-complete genomes, representing 28 genera and 389 species, including 244 likely novel species. All genomes are from filamentous isolates of the class Actinomycetia from the NBC culture collection. The largest genus is Streptomyces with 886 genomes including 742 complete assemblies. We use this data to show that analysis of complete genomes can bring biological understanding not previously derived from more fragmented sequences or less systematic datasets. We document the central and structured location of core genes and distal location of specialized metabolite biosynthetic gene clusters and duplicate core genes on the linear Streptomyces chromosome, and analyze the content and length of the terminal inverted repeats which are characteristic for Streptomyces. We then analyze the diversity of trans-AT polyketide synthase biosynthetic gene clusters, which encodes the machinery of a biotechnologically highly interesting compound class. These insights have both ecological and biotechnological implications in understanding the importance of high quality genomic resources and the complex role synteny plays in Actinomycetia biology.

The nuclease-associated short prokaryotic Argonaute system nonspecifically degrades DNA upon activation by target recognition.

Prokaryotic Argonautes (pAgos) play a vital role in host defense by utilizing short nucleic acid guides to recognize and target complementary nucleic acids. Despite being the majority of pAgos, short pAgos have only recently received attention. Short pAgos are often associated with proteins containing an APAZ domain and a nuclease domain including DUF4365, SMEK, or HNH domain. In contrast to long pAgos that specifically cleave the target DNA, our study demonstrates that the short pAgo from Thermocrispum municipal, along with its associated DUF4365-APAZ protein, forms a heterodimeric complex. Upon RNA-guided target DNA recognition, this complex is activated to nonspecifically cleave DNA. Additionally, we found that the TmuRE-Ago complex shows a preference for 5'-OH guide RNA, specifically requires a uridine nucleotide at the 5' end of the guide RNA, and is sensitive to single-nucleotide mismatches between the guide RNA and target DNA. Based on its catalytic properties, our study has established a novel nucleic acid detection method and demonstrated its feasibility. This study not only expands our understanding of the defense mechanism employed by short pAgo systems but also suggests their potential applications in nucleic acid detection.

Structural insights into the transcription activation mechanism of the global regulator GlnR from actinobacteria.

In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate metabolism in actinobacteria. Although many researchers have attempted to elucidate the mechanisms of GlnR-dependent transcription activation, progress is impeded by lacking of an overall structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a co-crystal structure of the C-terminal DNA-binding domain of GlnR (GlnR_DBD) in complex with its regulatory cis-element DNA and a cryo-EM structure of GlnR-TAC which comprises Mycobacterium tuberculosis RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures illustrate how four GlnR protomers coordinate to engage promoter DNA in a head-to-tail manner, with four N-terminal receiver domains of GlnR (GlnR-RECs) bridging GlnR_DBDs and the RNAP core enzyme. Structural analysis also unravels that GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved ß flap, σAR4, αCTD, and αNTD domains of RNAP, which are further confirmed by our biochemical assays. Taken together, these results reveal a global transcription activation mechanism for the master regulator GlnR and other OmpR/PhoB subfamily proteins and present a unique mode of bacterial transcription regulation.

Species invasions shift microbial phenology in a two-decade freshwater time series.

Invasive species impart abrupt changes on ecosystems, but their impacts on microbial communities are often overlooked. We paired a 20 y freshwater microbial community time series with zooplankton and phytoplankton counts, rich environmental data, and a 6 y cyanotoxin time series. We observed strong microbial phenological patterns that were disrupted by the invasions of spiny water flea (Bythotrephes cederströmii) and zebra mussels (Dreissena polymorpha). First, we detected shifts in Cyanobacteria phenology. After the spiny water flea invasion, Cyanobacteria dominance crept earlier into clearwater; and after the zebra mussel invasion, Cyanobacteria abundance crept even earlier into the diatom-dominated spring. During summer, the spiny water flea invasion sparked a cascade of shifting diversity where zooplankton diversity decreased and Cyanobacteria diversity increased. Second, we detected shifts in cyanotoxin phenology. After the zebra mussel invasion, microcystin increased in early summer and the duration of toxin production increased by over a month. Third, we observed shifts in heterotrophic bacteria phenology. The Bacteroidota phylum and members of the acI Nanopelagicales lineage were differentially more abundant. The proportion of the bacterial community that changed differed by season; spring and clearwater communities changed most following the spiny water flea invasion that lessened clearwater intensity, while summer communities changed least following the zebra mussel invasion despite the shifts in Cyanobacteria diversity and toxicity. A modeling framework identified the invasions as primary drivers of the observed phenological changes. These long-term invasion-mediated shifts in microbial phenology demonstrate the interconnectedness of microbes with the broader food web and their susceptibility to long-term environmental change.

Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycete-specific transcription factor.

Mycobacterium tuberculosis (Mtb) has evolved diverse cellular processes in response to the multiple stresses it encounters within the infected host. We explored available TnSeq datasets to identify transcription factors (TFs) that are essential for Mtb survival inside the host. The analysis identified a single TF, Rv1332 (AosR), conserved across actinomycetes with a so-far uncharacterized function. AosR mitigates phagocyte-derived oxidative and nitrosative stress, thus promoting mycobacterial growth in the murine lungs and spleen. Oxidative stress induces formation of a single intrasubunit disulphide bond in AosR, which in turn facilitates AosR interaction with an extracytoplasmic-function sigma factor, SigH. This leads to the specific upregulation of the CysM-dependent non-canonical cysteine biosynthesis pathway through an auxiliary intragenic stress-responsive promoter, an axis critical in detoxifying host-derived oxidative and nitrosative radicals. Failure to upregulate AosR-dependent cysteine biosynthesis during the redox stress causes differential expression of 6% of Mtb genes. Our study shows that the AosR-SigH pathway is critical for detoxifying host-derived oxidative and nitrosative radicals to enhance Mtb survival in the hostile intracellular environment.

Architecture of the mycobacterial type VII secretion system.

Host infection by pathogenic mycobacteria, such as Mycobacterium tuberculosis, is facilitated by virulence factors that are secreted by type VII secretion systems1. A molecular understanding of the type VII secretion mechanism has been hampered owing to a lack of three-dimensional structures of the fully assembled secretion apparatus. Here we report the cryo-electron microscopy structure of a membrane-embedded core complex of the ESX-3/type VII secretion system from Mycobacterium smegmatis. The core of the ESX-3 secretion machine consists of four protein components-EccB3, EccC3, EccD3 and EccE3, in a 1:1:2:1 stoichiometry-which form two identical protomers. The EccC3 coupling protein comprises a flexible array of four ATPase domains, which are linked to the membrane through a stalk domain. The domain of unknown function (DUF) adjacent to the stalk is identified as an ATPase domain that is essential for secretion. EccB3 is predominantly periplasmatic, but a small segment crosses the membrane and contacts the stalk domain. This suggests that conformational changes in the stalk domain-triggered by substrate binding at the distal end of EccC3 and subsequent ATP hydrolysis in the DUF-could be coupled to substrate secretion to the periplasm. Our results reveal that the architecture of type VII secretion systems differs markedly from that of other known secretion machines2, and provide a structural understanding of these systems that will be useful for the design of antimicrobial strategies that target bacterial virulence.

Multikingdom interactions govern the microbiome in subterranean cultural heritage sites.

SignificanceThe conservation of historical relics against microbial biodeterioration is critical to preserving cultural heritages. One major challenge is our limited understanding of microorganisms' dispersal, colonization, and persistence on relics after excavation and opening to external environments. Here, we investigate the ecological and physiological profiles of the microbiome within and outside the Dahuting Han Dynasty Tomb with a 1,800-y history. Actinobacteria dominate the microbiome in this tomb. Via interkingdom signaling mutualism, springtails carry Actinobacteria as one possible source into the tomb from surrounding environments. Subsequently, Actinobacteria produce cellulases combined with antimicrobial substances, which helps them to colonize and thrive in the tomb via intrakingdom competition. Our findings unravel the ecology of the microbiomes colonizing historical relics and provide help for conservation practices.

Discovery of unusual dimeric piperazyl cyclopeptides encoded by a Lentzea flaviverrucosa DSM 44664 biosynthetic supercluster.

Rare actinomycetes represent an underexploited source of new bioactive compounds. Here, we report the use of a targeted metabologenomic approach to identify piperazyl compounds in the rare actinomycete Lentzea flaviverrucosa DSM 44664. These efforts to identify molecules that incorporate piperazate building blocks resulted in the discovery and structural elucidation of two dimeric biaryl-cyclohexapeptides, petrichorins A and B. Petrichorin B is a symmetric homodimer similar to the known compound chloptosin, but petrichorin A is unique among known piperazyl cyclopeptides because it is an asymmetric heterodimer. Due to the structural complexity of petrichorin A, solving its structure required a combination of several standard chemical methods plus in silico modeling, strain mutagenesis, and solving the structure of its biosynthetic intermediate petrichorin C for confident assignment. Furthermore, we found that the piperazyl cyclopeptides comprising each half of the petrichorin A heterodimer are made via two distinct nonribosomal peptide synthetase (NRPS) assembly lines, and the responsible NRPS enzymes are encoded within a contiguous biosynthetic supercluster on the L. flaviverrucosa chromosome. Requiring promiscuous cytochrome p450 crosslinking events for asymmetric and symmetric biaryl production, petrichorins A and B exhibited potent in vitro activity against A2780 human ovarian cancer, HT1080 fibrosarcoma, PC3 human prostate cancer, and Jurkat human T lymphocyte cell lines with IC50 values at low nM levels. Cyclic piperazyl peptides and their crosslinked derivatives are interesting drug leads, and our findings highlight the potential for heterodimeric bicyclic peptides such as petrichorin A for inclusion in future pharmaceutical design and discovery programs.

A conserved signal-peptidase antagonist modulates membrane homeostasis of actinobacterial sortase critical for surface morphogenesis.

Most Actinobacteria encode a small transmembrane protein, whose gene lies immediately downstream of the housekeeping sortase coding for a transpeptidase that anchors many extracellular proteins to the Gram-positive bacterial cell wall. Here, we uncover the hitherto unknown function of this class of conserved proteins, which we name SafA, as a topological modulator of sortase in the oral Actinobacterium Actinomyces oris. Genetic deletion of safA induces cleavage and excretion of the otherwise predominantly membrane-bound SrtA in wild-type cells. Strikingly, the safA mutant, although viable, exhibits severe abnormalities in cell morphology, pilus assembly, surface protein localization, and polymicrobial interactions-the phenotypes that are mirrored by srtA depletion. The pleiotropic defect of the safA mutant is rescued by ectopic expression of safA from not only A. oris, but also Corynebacterium diphtheriae or Corynebacterium matruchotii. Importantly, the SrtA N terminus harbors a tripartite-domain feature typical of a bacterial signal peptide, including a cleavage motif AXA, mutations in which prevent SrtA cleavage mediated by the signal peptidase LepB2. Bacterial two-hybrid analysis demonstrates that SafA and SrtA directly interact. This interaction involves a conserved motif FPW within the exoplasmic face of SafA, since mutations of this motif abrogate SafA-SrtA interaction and induce SrtA cleavage and excretion as observed in the safA mutant. Evidently, SafA is a membrane-imbedded antagonist of signal peptidase that safeguards and maintains membrane homeostasis of the housekeeping sortase SrtA, a central player of cell surface assembly.

The ssgB gene is required for the early stages of sporangium formation in Actinoplanes missouriensis.

In Streptomyces, multiple paralogs of SsgA-like proteins (SALPs) are involved in spore formation from aerial hyphae. However, the functions of SALPs have not yet been elucidated in other actinobacterial genera. Here, we report the primary function of an SsgB ortholog (AmSsgB) in Actinoplanes missouriensis, which develops terminal sporangia on the substrate mycelia via short sporangiophores. Importantly, AmSsgB is the sole SALP in A. missouriensis. The transcription of AmssgB was upregulated during sporangium formation, consistent with our previous findings that AmssgB is a member of the AmBldD regulon. The AmssgB null mutant (ΔAmssgB) strain formed non-globose irregular structures on the substrate mycelium. Transmission electron microscopy revealed that the irregular structures contained abnormally septate hypha-like cells, without an intrasporangial matrix. These phenotypic changes were restored by complementation with AmssgB. Additionally, analysis of the heterologous expression of seven SALP-encoding genes from Streptomyces coelicolor A3(2) (ssgA-G) in the ΔAmssgB strain revealed that only ssgB could compensate for AmSsgB deficiency. This indicated that SsgB of S. coelicolor A3(2) and AmSsgB have comparable functions in A. missouriensis. In contrast to the ΔAmssgB strain, the ftsZ-disrupted strain showed a severe growth defect and produced small sporangium-like structures that swelled to some extent. These findings indicate that AmSsgB is crucial for the early stages of sporangium formation, not for spore septum formation in the late stages. We propose that AmSsgB is involved in sporangium formation by promoting the expansion of the "presporangium" structures formed on the tips of the substrate hyphae. IMPORTANCE: SsgB has been proposed as an archetypical SsgA-like protein with an evolutionarily conserved function in the morphological development of spore-forming actinomycetes. SsgB in Streptomyces coelicolor A3(2) is involved in spore septum formation. However, it is unclear whether this is the primary function of SsgBs in actinobacteria. This study demonstrated that the SsgB ortholog (AmSsgB) in Actinoplanes missouriensis is essential for sporangium expansion, which does not seem to be related to spore septum formation. However, the heterologous expression of ssgB from S. coelicolor A3(2) restored morphological abnormalities in the ΔAmssgB mutant. We propose that the primary function of SsgB is to initiate sporulation in differentiating cells (e.g., aerial hyphae in Streptomyces and "presporangium" cells in A. missouriensis) although its molecular mechanism remains unknown.

The global nitrogen regulator GlnR is a direct transcriptional repressor of the key gluconeogenic gene pckA in actinomycetes.

In most actinomycetes, GlnR governs both nitrogen and non-nitrogen metabolisms (e.g., carbon, phosphate, and secondary metabolisms). Although GlnR has been recognized as a global regulator, its regulatory role in central carbon metabolism [e.g., glycolysis, gluconeogenesis, and the tricarboxylic acid (TCA) cycle] is largely unknown. In this study, we characterized GlnR as a direct transcriptional repressor of the pckA gene that encodes phosphoenolpyruvate carboxykinase, catalyzing the conversion of the TCA cycle intermediate oxaloacetate to phosphoenolpyruvate, a key step in gluconeogenesis. Through the transcriptomic and quantitative real-time PCR analyses, we first showed that the pckA transcription was upregulated in the glnR null mutant of Amycolatopsis mediterranei. Next, we proved that the pckA gene was essential for A. mediterranei gluconeogenesis when the TCA cycle intermediate was used as a sole carbon source. Furthermore, with the employment of the electrophoretic mobility shift assay and DNase I footprinting assay, we revealed that GlnR was able to specifically bind to the pckA promoter region from both A. mediterranei and two other representative actinomycetes (Streptomyces coelicolor and Mycobacterium smegmatis). Therefore, our data suggest that GlnR may repress pckA transcription in actinomycetes, which highlights the global regulatory role of GlnR in both nitrogen and central carbon metabolisms in response to environmental nutrient stresses. IMPORTANCE: The GlnR regulator of actinomycetes controls nitrogen metabolism genes and many other genes involved in carbon, phosphate, and secondary metabolisms. Currently, the known GlnR-regulated genes in carbon metabolism are involved in the transport of carbon sources, the assimilation of short-chain fatty acid, and the 2-methylcitrate cycle, although little is known about the relationship between GlnR and the TCA cycle and gluconeogenesis. Here, based on the biochemical and genetic results, we identified GlnR as a direct transcriptional repressor of pckA, the gene that encodes phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, thus highlighting that GlnR plays a central and complex role for dynamic orchestration of cellular carbon, nitrogen, and phosphate fluxes and bioactive secondary metabolites in actinomycetes to adapt to changing surroundings.

Structures of the NDP-pyranose mutase belonging to glycosyltransferase family 75 reveal residues important for Mn2+ coordination and substrate binding.

Members of glycosyltransferase family 75 (GT75) not only reversibly catalyze the autoglycosylation of a conserved arginine residue with specific NDP-sugars but also exhibit NDP-pyranose mutase activity that reversibly converts specific NDP-pyranose to NDP-furanose. The latter activity provides valuable NDP-furanosyl donors for glycosyltransferases and requires a divalent cation as a cofactor instead of FAD used by UDP-D-galactopyranose mutase. However, details of the mechanism for NDP-pyranose mutase activity are not clear. Here we report the first crystal structures of GT75 family NDP-pyranose mutases. The novel structures of GT75 member MtdL in complex with Mn2+ and GDP, GDP-D-glucopyranose, GDP-L-fucopyranose, GDP-L-fucofuranose, respectively, combined with site-directed mutagenesis studies, reveal key residues involved in Mn2+ coordination, substrate binding, and catalytic reactions. We also provide a possible catalytic mechanism for this unique type of NDP-pyranose mutase. Taken together, our results highlight key elements of an enzyme family important for furanose biosynthesis.