Novel catalytic behavior of defective nanozymes with catalase-mimicking characteristics for the degradation of tetracycline.

Although nanozymes have shown significant potential in wastewater treatment, enhancing their degradation performance remains challenging. Herein, a novel catalytic behavior was revealed for defective nanozymes with catalase-mimicking characteristics that efficiently degraded tetracycline (TC) in wastewater. Hydroxyl groups adsorbed on defect sites facilitated the in-situ formation of vacancies during catalysis, thereby replenishing active sites. Additionally, electron transfer considerably enhanced the catalytic reaction. Consequently, numerous reactive oxygen species (ROS) were generated through these processes and subsequent radical reactions. The defective nanozymes, with their unique catalytic behavior, proved effective for the catalytic degradation of TC. Experimental results demonstrate that •OH, •O2-, 1O2 and e- were the primary contributors to the degradation process. In real wastewater samples, the normalized degradation rate constant for defective nanozymes reached 26.0 min-1 g-1 L, exceeding those of other catalysts. This study reveals the new catalytic behavior of defective nanozymes and provides an effective advanced oxidation process for the degradation of organic pollutants.

Metabolic and protein expression responses of Shewanella baltica in golden pomfret broths to slightly acidic electrolysed water.

Shewanella baltica is a specific spoilage organism of golden pomfret. This study aims to explore the antibacterial mechanism of slightly acidic electrolysed water (SAEW) against S. baltica (strains ABa4, ABe2 and BBe1) in golden pomfret broths by metabolomics, proteomics and bioinformatics analyses. S. baltica was decreased by at least 3.94 log CFU/mL after SAEW treatment, and strain ABa4 had the highest resistance. Under SAEW stress, amino acids and organic acids in S. baltica decreased, and nucleotide related compounds degraded. Furthermore, 100 differentially expressed proteins (DEPs) were identified. Most DEPs of strains ABe2 and BBe1 were down-regulated, while some DEPs of strain ABa4 were up-regulated, especially those oxidative stress related proteins. These results suggest that the modes of SAEW against S. baltica can be traced to the inhibition of amino acid, carbon, nucleotide and sulphur metabolisms, and the loss of functional proteins for temperature regulation, translation, motility and protein folding.

The involvement of multiple ABC transporters in daunorubicin efflux in Streptomyces coeruleorubidus.

Streptomyces genus produces a large number of antibiotics, which are always synthesized by specific biosynthetic gene clusters (BGCs). To resist autotoxicity, transporters encoded by genes located within BGC occasionally pump antibiotic along with transporter encoded by gene located outside BGC. Daunorubicin is an anthracycline antibiotic biosynthesized by Streptomyces species, playing a crucial role in the treatment of leukaemia. In existing studies, only one two-component ATP-binding cassette (ABC) transporter, encoded by drrA1-drrB1 (abbreviated as drrAB1) and located within the daunorubicin BGC, has been proven to extrude daunorubicin. In this work, two other two-component ABC transporters, encoded by drrAB2 and drrAB3 and located outside the cluster, were found to play the complementary role in daunorubicin efflux in S. coeruleorubidus. Disruption of three drrABs resulted in a 94% decrease in daunorubicin production. Furthermore, drrAB2 is regulated by the TetR family regulator DrrR1, responding to the intracellular accumulation of daunorubicin and suggesting its role in stress response and self-resistance. Although the homologues of DrrAB1 are only found in three anthracycline BGCs, the homologues of DrrAB2 and DrrAB3 are spread in many Streptomyces strains which do not contain any known anthracycline BGC. This indicates that DrrAB2 and DrrAB3 may recognize and extrude a broader range of substrates besides daunorubicin, thus playing a more extensive role in cellular detoxification.

Multi-omics profiling reveals the molecular mechanism of Bifidobacterium animalis BB04 in co-culture with Wickerhamomyces anomalus Y-5 to induce bifidocin A synthesis.

Bacteriocin is a kind of natural substance that can effectively inhibit bacteria, but its production usually limited by environment. Co-culture is a strategy to stimulate bacteriocin production. Bifidocin A produced by Bifidobacterium animalis BB04, is a novel bacteriocin with a broad-spectrum antimicrobial active of foodborne bacteria. In order to enhance bifidocin A production, bacteriocin-inducing strains were screened firstly in co-cultivation. Then, the molecular mechanism of co-cultural induction was investigated by transcriptomic and proteomic analysis. Finally, the key inducing metabolites were identified by using targeted metabolomic technology. The results showed that Wickerhamomyces anomalus Y-5 in co-cultivation could significantly enhance bifidocin A production, with a 3.00-fold increase compared to mono-culture. The induction may not depend on direct contact with cells and may instead be attributed to be continuous exposure to inducing substances at specific concentration. In co-cultivation, W. anomalus Y-5 up-regulated Hxk2 and Tap42 to activate Glucose-cAMP and Tor and HOG-MAPK pathway, stimulated the expression of the retrograde gene, produced glutamine and glycerol to maintain activity. During this process, glutamine, inosine, guanosine, adenine, uracil, fumaric acid and pyruvic acid produced by W. anomalus Y-5 could induce the synthesis of bifidocin A. In conclusion, W. anomalus Y-5 in co-cultivation induced the synthesis of bifidocin A by regulating various signaling pathways to produce inducing substances. These findings establish a foundation for high-efficient synthesis of bifidocin A and provide a new perspective into the industrial production of bacteriocin.

Antibiotic-producing plant-associated bacteria, anti-virulence therapy and microbiome engineering: Integrated approaches in sustainable agriculture.

Plant health is crucial for maintaining the well-being of humans, animals and the environment. Plant pathogens pose significant challenges to agricultural production, global food security and ecosystem biodiversity. This problem is exacerbated by the impact of climate change, which is expected to alter the emergence and evolution of plant pathogens and their interaction with their plant hosts. Traditional approaches to managing phytopathogens involved the use of chemical pesticides, but alternative strategies are needed to address their ongoing decline in performance as well as their negative impact on the environment and public health. Here, we highlight the advancement and effectiveness of biocontrol strategies based on the use of antimicrobial-producing plant-associated bacteria, anti-virulence therapy (e.g. quorum quenching) and microbiome engineering as sustainable biotechnological approaches to promote plant health and foster sustainable agriculture. Notably, Enterobacterales are emerging as important biocontrol agents and as a source of new antimicrobials for potential agricultural use. We analysed here the genomes of over 250 plant-associated enterobacteria to examine their potential to synthesize secondary metabolites. Exploration of the plant microbiome is of major interest in the search for eco-friendly alternatives for reducing the use of chemical pesticides.

Global regulator AdpA directly binds to tunicamycin gene cluster and negatively regulates tunicamycin biosynthesis in Streptomyces clavuligerus.

Since a transcriptional regulator has yet to be identified within the tunicamycin biosynthetic gene cluster in Streptomyces clavuligerus, we conducted a comprehensive investigation by focusing on the possible function of the pleiotropic regulator AdpA on tunicamycin. The genes encoding early steps of tunicamycin biosynthesis were significantly upregulated in S. clavuligerus ΔadpA. At the same time, they were downregulated in adpA overexpressed strain as shown by RNA-sequencing (RNA-seq) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis. The tunicamycin gene cluster's co-transcription pattern was understood by reverse transcriptase polymerase chain reaction (RT-PCR). Our Electrophoretic Mobility Shift Assay (EMSA) data clearly showed AdpA's binding to the upstream sequence of the tunA gene, asserting its regulatory control. In addition to its direct negative regulation of tunicamycin biosynthesis, AdpA operates at a global level by orchestrating various regulatory genes in S. clavuligerus, such as wblA, whiB, bldM, arpA, brp, and adsA involved in morphological differentiation and secondary metabolite biosynthesis as depicted in RNA-seq data. This study represents a significant milestone by unveiling the AdpA regulator's pathway-specific and global regulatory effect in S. clavuligerus.

Microbe Profile: Pseudonocardia: antibiotics for every niche.

Pseudonocardia species comprise a genus of filamentous, sporulating bacteria belonging to the phylum Actinomycetota, formerly Actinobacteria. They are found in marine and freshwater sediments and soils and associated with marine animals, insects, and plants. To date, they have mostly been studied because of their mutually beneficial symbiosis with fungus-growing ants in the tribe Attini. They have also attracted interest due to their biosynthetic capabilities, including the production of variably glycosylated polyenes and other novel antifungal compounds, and for their capacity to grow on a variety of hydrocarbons. The majority of clinically used antibiotics are derived from the specialised metabolites of filamentous actinomycete bacteria and most of these come from the genus Streptomyces. However, in the quest for novel chemistry there is increasing interest in studying other filamentous actinomycete genera, including Pseudonocardia. Here we outline the biological properties, genome size and structure and key features of the genus Pseudonocardia, namely their specialised metabolites and ecological roles.

Elucidating the bioremediation potential of laccase and peroxidase enzymes from Bacillus ligniniphilus L1 in antibiotic degradation: A computationally guided study.

This study showcased the antibiotic degradation abilities of laccase and catalase-peroxidase from Bacillus ligniniphilus L1, an extremophile, against 18 common antibiotics using computationally guided approach. Molecular docking and simulation identified six enzyme-antibiotic complexes for laccase and four for catalase-peroxidase, demonstrating significant binding affinity and stability. Enzyme activity assays corroborated computational results, indicating both enzymes could degrade all tested antibiotics with varying efficiencies. L1 laccase outperformed commercial laccase against five antibiotics, notably vancomycin, levofloxacin, tobramycin, linezolid, and rifamycin, with enhanced degradation potential upon ABTS addition. Catalase-peroxidase from L1 exhibited superior degradation efficiency over commercial peroxidase against vancomycin, linezolid, tobramycin, and clindamycin. Overall, this study underscores the computational approach's utility in understanding enzyme-mediated antibiotic degradation, offering insights into environmental contaminant remediation.

Small molecule produced by Photorhabdus interferes with ubiquinone biosynthesis in Gram-negative bacteria.

We report the identification of 3,6-dihydroxy-1,2-benzisoxazole (DHB) in a screen of Photorhabdus and Xenorhabdus, whose symbiotic relationship with eukaryotic nematodes favors secondary metabolites that meet several requirements matching those for clinically useful antibiotics. DHB is produced by Photorhabdus laumondii and is selective against the Gram-negative species Escherichia coli, Enterobacter cloacae, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and Acinetobacter baumannii. It is inactive against anaerobic gut bacteria and nontoxic to human cells. Mutants resistant to DHB map to the ubiquinone biosynthesis pathway. DHB binds to 4-hydroxybenzoate octaprenyltransferase (UbiA) and prevents the formation of 4-hydroxy-3-octaprenylbenzoate. Remarkably, DHB itself is prenylated, forming an unusable chimeric product that likely contributes to the toxic effect of this antimicrobial. DHB appears to be both a competitive enzyme inhibitor and a prodrug; this dual mode of action is unusual for an antimicrobial compound. IMPORTANCE: The spread of resistant pathogens has led to the antimicrobial resistance crisis, and the need for new compounds acting against Gram-negative pathogens is especially acute. From a screen of Photorhabdus symbionts of nematodes, we identified 3,6-dihydroxy-1,2-benzisoxazole (DHB) that acts against a range of Gram-negative bacteria, including Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, and Acinetobacter baumannii. DHB had previously been isolated from other bacterial species, but its mechanism of action remained unknown. We show that DHB is unique among antimicrobials, with dual action as an inhibitor of an important enzyme, UbiA, in the biosynthesis pathway of ubiquinone and as a prodrug. DHB is a mimic of the natural substrate, and UbiA modifies it into a toxic product, contributing to the antimicrobial action of this unusual antibiotic. We also uncover the mechanism of DHB selectivity, which depends on a particular fold of the UbiA enzyme.

Increased intracellular H2S levels enhance iron uptake in Escherichia coli.

We investigated the impact of intracellular hydrogen sulfide (H2S) hyperaccumulation on the transcriptome of Escherichia coli. The wild-type (WT) strain overexpressing mstA, encoding 3-mercaptopyruvate sulfur transferase, produced significantly higher H2S levels than the control WT strain. The mstA-overexpressing strain exhibited increased resistance to antibiotics, supporting the prior hypothesis that intracellular H2S contributes to oxidative stress responses and antibiotic resistance. RNA-seq analysis revealed that over 1,000 genes were significantly upregulated or downregulated upon mstA overexpression. The upregulated genes encompassed those associated with iron uptake, including siderophore synthesis and iron import transporters. The mstA-overexpressing strain showed increased levels of intracellular iron content, indicating that H2S hyperaccumulation affects iron availability within cells. We found that the H2S-/supersulfide-responsive transcription factor YgaV is required for the upregulated expression of iron uptake genes in the mstA-overexpression conditions. These findings indicate that the expression of iron uptake genes is regulated by intracellular H2S, which is crucial for oxidative stress responses and antibiotic resistance in E. coli. IMPORTANCE: H2S is recognized as a second messenger in bacteria, playing a vital role in diverse intracellular and extracellular activities, including oxidative stress responses and antibiotic resistance. Both H2S and iron serve as essential signaling molecules for gut bacteria. However, the intricate intracellular coordination between them, governing bacterial physiology, remains poorly understood. This study unveils a close relationship between intracellular H2S accumulation and iron uptake activity, a relationship critical for antibiotic resistance. We present additional evidence expanding the role of intracellular H2S synthesis in bacterial physiology.

Combined effect of polyphosphate kinase and lon protease in Streptomyces coelicolor A3(2) antibiotic production.

The bacterial stringent response is a global regulatory process in which polyphosphate kinase (Ppk) and lon protease are important players. Previous studies have shown that overexpression of the lon gene and deletion of the ppk gene significantly increased actinorhodin production in Streptomyces coelicolor (SCO). In this study, a recombinant SCOΔppk-lon cell, expressing the extra lon gene in Δppk cells, was simulated using a modified in silico (computational) model, ecSco-GEM, and the negative effect of Ppk on actinorhodin production was confirmed. In addition, we identified key enzymes that play a positive role in actinorhodin production. Of these, NADH dehydrogenase/complex-I, beta-ketoacyl-[acyl-carrier-protein] synthase III, glycine cleavage system, and superoxide dismutase were identified as the most significant. By confirming these results with experiments, we have shown that GEMs can be a reliable starting point for in vitro (lab-based) studies of Streptomyces..

Substrate Specificity of a Methyltransferase Involved in the Biosynthesis of the Lantibiotic Cacaoidin.

Modification of the N- and C-termini of peptides enhances their stability against degradation by exopeptidases. The biosynthetic pathways of many peptidic natural products feature enzymatic modification of their termini, and these enzymes may represent a valuable pool of biocatalysts. The lantibiotic cacaoidin carries an N,N-dimethylated N-terminal amine group. Its biosynthetic gene cluster encodes the putative methyltransferase Cao4. In this work, we present reconstitution of the activity of the enzyme, which we termed CaoSC following standardized lanthipeptide nomenclature, using a heterologously produced peptide as the model substrate. In vitro methylation of diverse lanthipeptides revealed the substrate requirements of CaoSC. The enzyme accepts peptides of varying lengths and C-terminal sequences but requires dehydroalanine or dehydrobutyrine at the second position. CaoSC-mediated dimethylation of natural lantibiotics resulted in modestly enhanced antimicrobial activity of the lantibiotic haloduracin compared to that of the native compound. Improved activity and/or metabolic stability as a result of methylation illustrates the potential future application of CaoSC in the bioengineering of therapeutic peptides.

Optimization of degradation conditions for sulfachlorpyridazine by Bacillus sp. DLY-11 and analysis of biodegradation mechanisms.

Sulfachloropyridazine (SCP) is a common sulfonamide antibiotic pollutant found in animal excreta. Finding highly efficient degrading bacterial strains is an important measure to reduce SCP antibiotic pollution. Although some strains with degradation capabilities have been screened, the degradation pathways and biotransformation mechanisms of SCP during bacterial growth are still unclear. In this study, a strain capable of efficiently degrading SCP, named Bacillus sp. DLY-11, was isolated from pig manure aerobic compost. Under optimized conditions (5 % Vaccination dose, 51.5 â„ƒ reaction temperature, pH=7.92 and 0.5 g/L MgSO4), this strain was able to degrade 97.7 % of 20 mg/L SCP within 48 h. Through the analysis of nine possible degradation products (including a new product of 1,4-benzoquinone with increased toxicity), three potential biodegradation pathways were proposed. The biodegradation reactions include S-N bond cleavage, dechlorination, hydroxylation, deamination, methylation, sulfur dioxide release, and oxidation reactions. This discovery not only provides a new efficient SCP-degrading bacterial strain but also expands our understanding of the mechanisms of bacterial degradation of SCP, filling a knowledge gap. It offers important reference for the bioremediation of antibiotic pollutants in livestock and poultry farming.

Production of hybrid macrolide antibiotics by exploiting the specific substrate recognition characteristics of multifunctional cytochrome P450 enzyme MycG.

Macrolide antibiotics are biosynthesized via enzymatic modifications, including glycosylation, methylation, and oxidation, after the core macro-lactone ring is generated by a polyketide synthase system. This study explored the diversification of macrolides by combining biosynthetic enzymes and reports an approach to produce unnatural hybrid macrolide antibiotics. The cytochrome (CYP) P450 monooxygenase MycG exhibits bifunctional activity, catalyzing late-stage hydroxylation at C-14 followed by epoxidation at C-12/13 during mycinamicin biosynthesis. The mycinose sugar of mycinamicin serves as a key molecular recognition element for binding to MycG. Thus, we subjected the hybrid macrolide antibiotic 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (IZI) to MycG, and confirmed that MycG catalyzed hydroxylation at C-22 and epoxidation at C-12/13 in IZI. In addition, the introduction of mycinose biosynthesis-related genes and mycG into rosamicin-producing Micromonospora rosaria enabled the fermentative production of 22-hydroxylated and 12,13-epoxidized forms of IZI. Interestingly, MycG catalyzed the sequential oxidation of hydroxylation and epoxidation in mycinamicin biosynthesis, but only single reactions in IZI. These findings highlight the potential for expanding the application of the multifunctional P450 monooxygenase MycG for the production of unnatural compounds.

Phylogenomic analysis uncovers an unexpected capacity for the biosynthesis of secondary metabolites in Pseudoalteromonas.

Pseudoalteromonas is a genus of marine bacteria and a promising source of natural products with antibacterial, antifungal, and antifouling bioactivities. To accelerate the exploration of new compounds from this genus, we applied the gene-first approach to study 632 public Pseudoalteromonas genomes. We identified 3968 biosynthetic gene clusters (BGCs) involved in the biosynthesis of secondary metabolites and classified them into 995 gene cluster families (GCFs). Surprisingly, only 9 GCFs (0.9 %) included an experimentally identified reference biosynthetic gene cluster from the Minimum Information about a Biosynthetic Gene cluster database (MIBiG), suggesting a striking novelty of secondary metabolites in Pseudoalteromonas. Bioinformatic analysis of the biosynthetic diversity encoded in the identified BGCs uncovered six dominant species of this genus, P. citrea, P. flavipulchra, P. luteoviolacea, P. maricaloris, P. piscicida, and P. rubra, that encoded more than 17 BGCs on average. Moreover, each species exhibited a species-specific distribution of BGC. However, a deep analysis revealed two BGCs conserved across five of the six dominant species. These BGCS encoded an unknown lanthipeptide and the siderophore myxochelin B implying an essential role of antibiotics for Pseudoalteromonas. We chemically profiled 11 strains from the 6 dominant species and identified four new antibiotics, korormicins L-O (1-4), from P. citrea WJX-3. Our results highlight the unexplored biosynthetic potential for bioactive compounds in Pseudoalteromonas and provide an important guideline for targeting exploration.

Efficient metabolic pathway modification in various strains of lactic acid bacteria using CRISPR/Cas9 system for elevated synthesis of antimicrobial compounds.

Lactic acid bacteria (LAB) are known to exhibit various beneficial roles in fermentation, serving as probiotics, and producing a plethora of valuable compounds including antimicrobial activity such as bacteriocin-like inhibitory substance (BLIS) that can be used as biopreservative to improve food safety and quality. However, the yield of BLIS is often limited, which poses a challenge to be commercially competitive with the current preservation practice. Therefore, the present work aimed to establish an optimised two-plasmid CRISPR/Cas9 system to redirect the carbon flux away from lactate towards compounds with antimicrobial activity by disrupting lactate dehydrogenase gene (ldh) on various strains of LAB. The lactic acid-deficient (ldhΔ) strains caused a metabolic shift resulting in increased inhibitory activity against selected foodborne pathogens up to 78 % than the wild-type (WT) strain. The most significant effect was depicted by Enterococcus faecalis-ldh∆ which displayed prominent bactericidal effects against all foodborne pathogens as compared to the WT that showed no antimicrobial activity. The present work provided a framework model for economically important LAB and other beneficial bacteria to synthesise and increase the yield of valuable food and industrial compounds. The present work reported for the first time that the metabolism of selected LAB can be manipulated by modifying ldh to attain metabolites with higher antimicrobial activity.

Metabolomic analysis in Amycolatopsis keratiniphila disrupted the competing ECO0501 pathway for enhancing the accumulation of vancomycin.

Vancomycin is a clinically important glycopeptide antibiotic against Gram-positive pathogenic bacteria, especially methicillin-resistant Staphylococcus aureus. In the mutant strain of Amycolatopsis keratiniphila HCCB10007 Δeco-cds4-27, the production of ECO-0501 was disrupted, but enhanced vancomycin yield by 55% was observed compared with the original strain of A. keratiniphila HCCB10007. To gain insights into the mechanism of the enhanced production of vancomycin in the mutant strain, comparative metabolomics analyses were performed between the mutant strain and the original strain, A. keratiniphila HCCB10007 via GC-TOF-MS and UPLC-HRMS. The results of PCA and OPLS-DA revealed a significant distinction of the intracellular metabolites between the two strains during the fermentation process. 64 intracellular metabolites, which involved in amino acids, fatty acids and central carbon metabolism, were identified as differential metabolites. The high-yield mutant strain maintained high levels of glucose-1-phosphate and glucose-6-phosphate and they declined with the increases of vancomycin production. Particularly, a strong association of fatty acids accumulation as well as 3,5-dihydroxyphenylacetic acid and non-proteinogenic amino acid 3,5-dihydroxyphenylglycine (Dpg) with enhancement of vancomycin production was observed in the high-yield mutant strain, indicating that the consumption of fatty acid pools might be beneficial for giving rise to 3,5-dihydroxyphenylacetic acid and Dpg which further lead to improve vancomycin production. In addition, the lower levels of glyoxylic acid and lactic acid and the higher levels of sulfur amino acids might be beneficial for improving vancomycin production. These findings proposed more advanced elucidation of metabolomic characteristics in the high-yield strain for vancomycin production and could provide potential strategies to enhance the vancomycin production.

Pseudocin 196, a novel lantibiotic produced by Bifidobacterium pseudocatenulatum elicits antimicrobial activity against clinically relevant pathogens.

Bacteriocins are broad or narrow-spectrum antimicrobial compounds that have received significant scientific attention due to their potential to treat infections caused by antibiotic-resistant pathogenic bacteria. The genome of Bifidobacterium pseudocatenulatum MM0196, an antimicrobial-producing, fecal isolate from a healthy pregnant woman, was shown to contain a gene cluster predicted to encode Pseudocin 196, a novel lantibiotic, in addition to proteins involved in its processing, transport and immunity. Following antimicrobial assessment against various indicator strains, protease-sensitive Pseudocin 196 was purified to homogeneity from cell-free supernatant. MALDI TOF mass spectrometry confirmed that the purified antimicrobial compound corresponds to a molecular mass of 2679 Da, which is consistent with that deduced from its genetic origin. Pseudocin 196 is classified as a lantibiotic based on its similarity to lacticin 481, a lanthionine ring-containing lantibiotic produced by Lactococcus lactis. Pseudocin 196, the first reported bacteriocin produced by a B. pseudocatenulatum species of human origin, was shown to inhibit clinically relevant pathogens, such as Clostridium spp. and Streptococcus spp. thereby highlighting the potential application of this strain as a probiotic to treat and prevent bacterial infections.

TetR family regulator AbrT controls lincomycin production and morphological development in Streptomyces lincolnensis.

BACKGROUND: The TetR family of transcriptional regulators (TFRs), serving as crucial regulators of diverse cellular processes, undergo conformational changes induced by small-molecule ligands, which either inhibit or activate them to modulate target gene expression. Some ligands of TFRs in actinomycetes and their regulatory effects have been identified and studied; however, regulatory mechanisms of the TetR family in the lincomycin-producing Streptomyces lincolnensis remain poorly understood. RESULTS: In this study, we found that AbrT (SLCG_1979), a TetR family regulator, plays a pivotal role in regulating lincomycin production and morphological development in S. lincolnensis. Deletion of abrT gene resulted in increased lincomycin A (Lin-A) production, but delayed mycelium formation and sporulation on solid media. AbrT directly or indirectly repressed the expression of lincomycin biosynthetic (lin) cluster genes and activated that of the morphological developmental genes amfC, whiB, and ftsZ. We demonstrated that AbrT bound to two motifs (5'-CGCGTACTCGTA-3' and 5'-CGTACGATAGCT-3') present in the bidirectional promoter between abrT and SLCG_1980 genes. This consequently repressed abrT itself and its adjacent gene SLCG_1980 that encodes an arabinose efflux permease. D-arabinose, not naturally occurring as L-arabinose, was identified as the effector molecule of AbrT, reducing its binding affinity to abrT-SLCG_1980 intergenic region. Furthermore, based on functional analysis of the AbrT homologue in Saccharopolyspora erythraea, we inferred that the TetR family regulator AbrT may play an important role in regulating secondary metabolism in actinomycetes. CONCLUSIONS: AbrT functions as a regulator for governing lincomycin production and morphological development of S. lincolnensis. Our findings demonstrated that D-arabinose acts as a ligand of AbrT to mediate the regulation of lincomycin biosynthesis in S. lincolnensis. Our findings provide novel insights into ligand-mediated regulation in antibiotic biosynthesis.

In Situ Raman Hyperspectral Analysis of Microbial Colonies for Secondary Metabolites Screening.

Since the discovery of penicillin, a vast array of microbial antibiotics has been identified and applied in the medical field. Globally, the search for drug candidates via microbial screening is ongoing. Traditional screening methods, however, are time-consuming and require labor-intensive sample processing, significantly reducing throughput. This research introduces a Raman spectroscopy-based screening system tailored to the in situ analysis of microbial colonies on solid culture media. Employing multivariate curve resolution-alternating least-squares (MCR-ALS) for spectral decomposition, our approach reveals the production of secondary metabolites at the single colony level. We enhanced the microbial culture method, enabling direct, high signal-to-noise (S/N) ratio Raman spectroscopic measurements of colonies of Escherichia coli and actinomycetes species. Through semisupervised MCR analysis using the known spectra of actinorhodin and undecylprodigiosin as references, we accurately assessed the production of these compounds by Streptomyces coelicolor A3(2). Furthermore, we herein successfully detected the production of amphotericin B by Streptomyces nodosus, even in the absence of prior spectral information. This demonstrates the potential of our technique in the discovery of secondary metabolites. In addition to enabling the detection of the above-mentioned compounds, this analysis revealed the heterogeneity of the spatial distribution of their production in each colony. Our technique makes a significant contribution to the advancement of microbial screening, offering a rapid, efficient alternative to conventional methods and opening avenues for secondary metabolites discovery.