TetR family transcriptional regulators (TFRs) are widely distributed in bacteria and archaea, and the first discovered TFR was confirmed to control the expression of tetracycline efflux pump in Escherichia coli. TFRs can bind DNAs and ligands. Small molecule ligands can induce conformational changes of TFRs, inhibiting or promoting TFRs to control target gene expression. Currently, TFRs have a wide variety of ligands, including carbohydrates, proteins, fatty acids and their derivatives, metal ions, and so on. Due to the diversity of ligands, TFRs regulate a wide range of physiological processes, from basic carbon metabolism and nitrogen metabolism to quorum sensing and antibiotic biosynthesis. On the basis of the recent studies in our laboratory and the literature, we review here the regulatory mechanism mediated by ligands of TFRs in primary and secondary metabolism, as well as the application of ligands for TFRs in the development of gene route and the activation of antibiotic biosynthesis.
Anti-Bacterial Agents ; Bacteria/metabolism* ; Bacterial Proteins/metabolism* ; Gene Expression Regulation, Bacterial ; Ligands ; Quorum Sensing

Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.
Humans ; Clostridioides/metabolism* ; Clostridioides difficile/metabolism* ; Bacterial Proteins/metabolism* ; Virulence ; Anti-Bacterial Agents/metabolism*

otrA resembles elongation factor G (EF-G) and is considered to be an oxytetracycline (OTC)-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and other aminoglycosides to Streptomyces coelicolor, the otrA gene from S. rimosus M527 was cloned under the control of the strong ermE^* promoter. The resulting plasmid, pIB139-otrA, was introduced into S. coelicolor M145 by intergeneric conjugation, yielding the recombinant strain S. coelicolor M145-OA. As expected S. coelicolor M145-OA exhibited higher resistance levels specifically to OTC and aminoglycosides gentamycin, hygromycin, streptomycin, and spectinomycin. However, unexpectedly, S. coelicolor M145-OA on solid medium showed an accelerated aerial mycelia formation, a precocious sporulation, and an enhanced actinorhodin (Act) production. Upon growth in 5-L fermentor, the amount of intra- and extracellular Act production was 6-fold and 2-fold higher, respectively, than that of the original strain. Consistently, reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the transcriptional level of pathway-specific regulatory gene actII-orf4 was significantly enhanced in S. coelicolor M145-OA compared with in S. coelicolor M145.
Aminoglycosides/pharmacology* ; Anthraquinones/metabolism* ; Anti-Bacterial Agents/pharmacology* ; Bacterial Proteins/genetics* ; Drug Resistance, Bacterial/genetics* ; Streptomyces coelicolor/metabolism*

Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10 (CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL (encoding the ribosomal protein S12) or rpoB (encoding the RNA polymerase β-subunit). Among them, L10/RpoB (H437Y) accumulated a dark pigment on a yeast extract-malt extract-glucose (YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance (NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and electrophoretic mobility shift assay (EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.
Anti-Bacterial Agents/pharmacology* ; Antioxidants/pharmacology* ; Bacterial Proteins/genetics* ; Multigene Family ; Mutagenesis, Site-Directed ; Streptomyces/metabolism*

Catalyzed by a family of enzymes called glycosyltransferases (GTases), glycosylation reactions are essential for the bioactivities of macrolide antibiotics which have been widely applied. Additionally, glycosylation is also an important strategy of microbial to get macrolide antibiotic resistance. Studies on the structure, function and application areas of macrolide GTases will lay the stable groundwork for the combinatorial biology. This paper introduced in detail the biological functions of macrolide glycosylation, and then made an in-depth discussion on the families and discoveries of macrolide GTases. The resistance mechanism with macrolide glycosyltion and the correlative GTases MGT have been reviewed afterwards. According to the flexible substrate specificity of macrolide GTases, the combinatorial biological applications on them were also seriously summarized here. At the end, the authors made a developmental prospect of macrolide GTases based on the studies of the research group.
Anti-Bacterial Agents ; metabolism ; pharmacology ; Drug Resistance, Bacterial ; Glucosyltransferases ; classification ; metabolism ; Glycosylation ; Macrolides ; chemistry ; metabolism ; Streptomyces ; enzymology ; Substrate Specificity

OBJECTIVETo investigate biological transport of tetracycline hydrochloride by human periodontal ligament fibroblasts (HPDLF) for verifying the hypothesis of delivering medicine to the periodontium and whole body through the root canal.

METHODSHPDLF and MC3T3-E1 cells were incubated in antibiotics solutions. The intracellular antibiotics contents were measured by high performance liquid chromatography (HPLC) and the cell total protein was measured by bradford protein assay.

RESULTSThe intracellular contents increased with incubation time. The extracellular medicine concentration had effect on the intracellular contents.

CONCLUSIONSTetracycline hydrochloride can be transported into HPDLF with incubation and this transport is time-dependent and concentration-dependent.

Anti-Bacterial Agents ; pharmacokinetics ; Biological Transport ; Cells, Cultured ; Fibroblasts ; metabolism ; Humans ; Periodontal Ligament ; metabolism ; Tetracycline ; pharmacokinetics

Aims: To characterize the plantaricin IIA-1A5 crude extract that biosynthesized by Lactobacillus plantarum IIA-1A5 using corn steep liquor (CSL) based medium. Methodology and results: Lactobacillus plantarum IIA-1A5 was grown in several media containing different components including corn steep liquor (CSL), molasses and MRS (de Man Rogosa Sharpe) as control medium for 24 h at 37 °C. Antibacterial activities of the cell-free supernatant were expressed as diameter of inhibition zones observed by paper disc method. The results showed that CSL medium produced cell-free supernatant of L. plantarum IIA-1A5 with significantly higher antibacterial activity againts Staphylococcus aureus ATCC 25923 (9.81 mm), Lactobacillus monocytogenes ATCC 7644 (9.61 mm), Bacillus cereus (8.97 mm) and Escherichia coli ATCC 25922 (9.23 mm) were not significantly different compared to control MRS broth media (9.59 mm). CSL medium added only with 3% yeast extract and Tween 80 produced supernatant which showed similar antibacterial activity either to 10% molasses or control medium (Medium K and B). The CSL medium was considered more efficient and low cost, therefore this medium was selected for production and characterization of plantaricin IIA-1A5 crude extract. Further characterization performed by SDS PAGE analysis showed that crude plantaricin had molecular weight of approximately 9.9 kDa, higher than that produced in control medium (8.0 kDa). However, both plantaricins were categorized under the same class for small bacteriocin (class II). This study also revealed the plantaricin IIA-1A5 produced in CSL medium was stable to heat and pH and not significantly different compared to control MRS broth media. The antibacterial activity of plantaricin IIA-1A5 crude extract against S. aureus ATCC 25923 (10.09 mm) was not significantly different with 1000 ppm sodium benzoate (9.70 mm) and 300 ppm sodium nitrite (9.82 mm). Conclusion, significance and impact of study The CSL medium produced cell-free supernatant of L. plantarum IIA 1A5 had significant antibacterial activity characterization againts S. aureus ATCC 25923, L. monocytogenes ATCC 7644, B. cereus and E. coli ATCC 25922. Comparison of the inhibition activity of plantaricin IIA-1A5 crude extract against pathogen with synthetic preservatives indicated that plantaricin IIA-1A5 crude extract have the potency to replace synthetic preservatives. CSL based medium is potential to be used for low-cost plantaricin IIA-1A5 production.
Anti-Bacterial Agents--metabolism ; Bacteriocins--metabolism ; Lactobacillus plantarum ; Microbial Viability--drug effects ; Staphylococcus aureus

Ribosomal engineering is a technique that can improve the biosynthesis of secondary metabolites in the antibiotics-resistant mutants by attacking the bacterial RNA polymerase or ribosome units using the corresponding antibiotics. Ribosomal engineering can be used to discover and increase the production of valuable bioactive secondary metabolites from almost all actinomycetes strains regardless of their genetic accessibility. As a consequence, ribosomal engineering has been widely applied to genome mining and production optimization of secondary metabolites in actinomycetes. To date, more than a dozen of new molecules were discovered and production of approximately 30 secondary metabolites were enhanced using actinomycetes mutant strains generated by ribosomal engineering. This review summarized the mechanism, development, and protocol of ribosomal engineering, highlighting the application of ribosomal engineering in actinomycetes, with the aim to facilitate future development of ribosomal engineering and discovery of actinomycetes secondary metabolites.
Actinobacteria/metabolism* ; Actinomyces/genetics* ; Anti-Bacterial Agents/metabolism* ; Multigene Family ; Ribosomes/genetics*

Iron is an essential trace element for both humans and bacteria. It plays a vital role in life, such as in redox reactions and electron transport. Strict regulatory mechanisms are necessary to maintain iron homeostasis because both excess and insufficient iron are harmful to life. Competition for iron is a war between humans and bacteria. To grow, reproduce, colonize, and successfully cause infection, pathogens have evolved various mechanisms for iron uptake from humans, principally Fe 3+ -siderophore and Fe 2+ -heme transport systems. Humans have many innate immune mechanisms that regulate the distribution of iron and inhibit bacterial iron uptake to help resist bacterial invasion and colonization. Meanwhile, researchers have invented detection test strips and coupled antibiotics with siderophores to create tools that take advantage of this battle for iron, to help eliminate pathogens. In this review, we summarize bacterial and human iron metabolism, competition for iron between humans and bacteria, siderophore sensors, antibiotics coupled with siderophores, and related phenomena. We also discuss how competition for iron can be used for diagnosis and treatment of infection in the future.
Humans ; Siderophores/metabolism* ; Iron/metabolism* ; Bacteria ; Anti-Bacterial Agents/pharmacology* ; Biological Transport

Natural cyclohexapeptide AFN A₁ fromStreptomyces alboflavus 313 has moderate antibacterial and antitumor activities. An artificial designed AFN A₁ homodimer, di-AFN A₁, is an antibiotic exhibiting 10 to 150 fold higher biological activities, compared with the monomer. Unfortunately, the yield of di-AFN A₁ is very low (0.09 ± 0.03 mg·L^-1) in the engineered strain Streptomyces alboflavus 313_hmtS (S. albo/313_hmtS), which is not friendly to be genetically engineered for titer improvement of di-AFN A₁ production. In this study, we constructed a biosynthetic gene cluster for di-AFN A₁ and increased its production through heterologous expression. During the collection of di-AFN A₁ biosynthetic genes, the afn genes were located at three sites of S. alboflavus 313 genome. The di-AFN A₁ biosynthetic gene cluster (BGC) was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24, which produced di-AFN A₁ at a titer of 0.43 ± 0.01 mg·L^-1. To further increase the yield of di-AFN A₁, the di-AFN A₁ BGC was multiplied and split to mimic the natural afn biosynthetic genes, and the production of di-AFN A₁ increased to 0.62 ± 0.11 mg·L^-1 in S. lividans TK24 by the later strategy. Finally, different Streptomyces hosts were tested and the titer of di-AFN A₁ increased to 0.81 ± 0.17 mg·L^-1, about 8.0-fold higher than that in S. albo/313_hmtS. Successful heterologous expression of di-AFN A₁ with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.
Cloning, Molecular ; Streptomyces/metabolism* ; Multigene Family ; Anti-Bacterial Agents/metabolism* ; Plasmids/genetics*