Expression of Syo_1.56 SARP Regulator Unveils Potent Elasnin Derivatives with Antibacterial Activity.

Actinomycetes are prolific producers of natural products, particularly antibiotics. However, a significant proportion of its biosynthetic gene clusters (BGCs) remain silent under typical laboratory conditions. This limits the effectiveness of conventional isolation methods for the discovery of novel natural products. Genetic interventions targeting the activation of silent gene clusters are necessary to address this challenge. Streptomyces antibiotic regulatory proteins (SARPs) act as cluster-specific activators and can be used to target silent BGCs for the discovery of new antibiotics. In this study, the expression of a previously uncharacterized SARP protein, Syo_1.56, in Streptomyces sp. RK18-A0406 significantly enhanced the production of known antimycins and led to the discovery of 12 elasnins (1-12), 10 of which were novel. The absolute stereochemistry of elasnin A1 was assigned for the first time to be 6S. Unexpectedly, Syo_1.56 seems to function as a pleiotropic rather than cluster-specific SARP regulator, with the capability of co-regulating two distinct biosynthetic pathways, simultaneously. All isolated elasnins were active against wild-type and methicillin-resistant Staphylococcus aureus with IC50 values of 0.5-20 µg/mL, some of which (elasnins A1, B2, and C1 and proelasnins A1, and C1) demonstrated moderate to strong antimalarial activities against Plasmodium falciparum 3D7. Elasnins A1, B3, and C1 also showed in vitro inhibition of the metallo-ß-lactamase responsible for the development of highly antibiotic-resistant bacterial strains.

ε-Poly-L-lysine peptide chain length regulated by the linkers connecting the transmembrane domains of ε-Poly-L-lysine synthetase.

ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL.

Mechanism of epsilon-poly-L-lysine production and accumulation revealed by identification and analysis of an epsilon-poly-L-lysine-degrading enzyme.

Epsilon-poly-L-lysine (epsilon-PL) is produced by Streptomyces albulus NBRC14147 as a secondary metabolite and can be detected only when the fermentation broth has an acidic pH during the stationary growth phase. Since strain NBRC14147 produces epsilon-PL-degrading enzymes, the original chain length of the epsilon-PL polymer product synthesized by epsilon-PL synthetase (Pls) is unclear. Here, we report on the identification of the gene encoding the epsilon-PL-degrading enzyme (PldII), which plays a central role in epsilon-PL degradation in this strain. A knockout mutant of the pldII gene was found to produce an epsilon-PL of unchanged polymer chain length, demonstrating that the length is not determined by epsilon-PL-degrading enzymes but rather by Pls itself and that the 25 to 35 L-lysine residues of epsilon-PL represent the original chain length of the polymer product synthesized by Pls in vivo. Transcriptional analysis of pls and a kinetic study of Pls further suggested that the Pls catalytic function is regulated by intracellular ATP, high levels of which are required for full enzymatic activity. Furthermore, it was found that acidic pH conditions during epsilon-PL fermentation, rather than the inhibition of the epsilon-PL-degrading enzyme, are necessary for the accumulation of intracellular ATP.

ß-carboline chemical signals induce reveromycin production through a LuxR family regulator in Streptomyces sp. SN-593.

Actinomycetes bacteria produce diverse bioactive molecules that are useful as drug seeds. To improve their yield, researchers often optimize the fermentation medium. However, exactly how the extracellular chemicals present in the medium activate secondary metabolite gene clusters remains unresolved. BR-1, a ß-carboline compound, was recently identified as a chemical signal that enhanced reveromycin A production in Streptomyces sp. SN-593. Here we show that BR-1 specifically bound to the transcriptional regulator protein RevU in the reveromycin A biosynthetic gene cluster, and enhanced RevU binding to its promoter. RevU belongs to the LuxR family regulator that is widely found in bacteria. Interestingly, BR-1 and its derivatives also enhanced the production of secondary metabolites in other Streptomyces species. Although LuxR-N-acyl homoserine lactone systems have been characterized in Gram-negative bacteria, we revealed LuxR-ß-carboline system in Streptomyces sp. SN-593 for the production of secondary metabolites. This study might aid in understanding hidden chemical communication by ß-carbolines.

miRNA Profiles of Tubular Cells: Diagnosis of Kidney Injury.

MicroRNAs (miRNAs) are small noncoding RNAs of 18-23 nucleotides that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various physiological and pathological conditions. The present study details the conserved miRNA expression profiles of tubular tissues, and discusses whether they could be used to distinguish between proximal tubule injury, diagnose acute kidney injury (AKI), and the early-stage renal tubular dysfunction. miRNA expression was assessed with miRNA array and real-time reverse transcription polymerase chain reaction using the TaqMan system. The expression profiles of miR-200a/b/c, miR-145, miR-192, miR-194, miR-216a/b, miR-217, and miR-449a in human and rat tubular tissues such as the kidneys, lung, small intestine, and various exocrine glands were adequate for discriminating tubular tissues. In the kidney, miR-192 and miR-194 were highly expressed, whereas miR-145 and miR-449a were absent. miR-145 and miR-449a were relatively specifically expressed in small intestine and lung, respectively. Therefore, the combined levels of miR-200a/b/c, miR-192, and miR-194 in plasma were very useful in diagnosing AKI induced by contact freezing in mice. Moreover, urinary miR-200a levels were useful for the diagnosis of renal tubular dysfunction in Dahl salt-sensitive rat with high salt administration. Our results indicate that miRNA expression profiles are useful as biomarkers for identification of various kidney injuries.

Induction of peroxisomes by butyrate-producing probiotics.

We previously found that peroxisomal biogenesis factor 11a (Pex11a) deficiency is associated with a reduction in peroxisome abundance and impaired fatty acid metabolism in hepatocytes, and results in steatosis. In the present study, we investigated whether butyrate induces Pex11a expression and peroxisome proliferation, and studied its effect on lipid metabolism. C57BL/6 mice fed standard chow or a high-fat diet (HFD) were treated with tributyrin, 4-phelybutyrate acid (4-PBA), or the butyrate-producing probiotics (Clostridium butyricum MIYAIRI 588 [CBM]) plus inulin (dietary fiber), and the body weight, white adipose tissue, serum triglycerides, mRNA expression, and peroxisome abundance were evaluated. Tributyrin or 4-PBA treatment significantly decreased body weight and increased hepatic mRNA expression of peroxisome proliferator-activated receptor-α (PPARα) and Pex11a. In addition, 4-PBA treatment increased peroxisome abundance and the expression of genes involved in peroxisomal fatty acid ß-oxidation (acyl-coenzyme A oxidase 1 and hydroxysteroid [17-beta] dehydrogenase 4). CBM and inulin administration reduced adipose tissue mass and serum triglycerides, induced Pex11a, acyl-coenzyme A oxidase 1, and hydroxysteroid (17-beta) dehydrogenase 4 genes, and increased peroxisome abundance in mice fed standard chow or an HFD. In conclusion, elevation of butyrate availability (directly through administration of butyrate or indirectly via administration of butyrate-producing probiotics plus fiber) induces PPARα and Pex11a and the genes involved in peroxisomal fatty acid ß-oxidation, increases peroxisome abundance, and improves lipid metabolism. These results may provide a new therapeutic strategy against hyperlipidemia and obesity.

A novel biomarker for acute kidney injury using TaqMan-based unmethylated DNA-specific polymerase chain reaction.

There has been increasing interest in the use of circulating DNA as biomarkers for various tissue injuries, cancers, and fetal conditions. DNA methylation is a well-characterized mechanism underlying the epigenetic regulation of gene expression, and many diagnostic tests based on DNA methylation patterns have been developed. We developed a novel TaqMan-based assay for the detection of acute kidney injury using a hypomethylated promoter region of Slc22a12, a urate transporter specifically expressed in proximal tubular cells. Bisulfite sequencing analysis confirmed that the CpG islands in the promoter region of mouse Slc22a12 were preferentially hypomethylated in the kidney cortex. TaqMan minor groove binder (MGB) probes reliably discriminated the DNA fragments corresponding to the unmethylated and methylated promoter regions of Slc22a12. Plasma levels of unmethylated DNA corresponding to the Slc22a12 promoter region were undetectable at baseline and were significantly elevated after acute kidney cortex necrosis. This study showed the usefulness of the TaqMan system in discriminating methylated and unmethylated DNA fragments, and the similar strategy can be applied for establishing biomarkers for various cellular injuries or pathological conditions.

MiR-216a and miR-216b as markers for acute phased pancreatic injury.

MicroRNAs (miRNAs) are endogenous small RNAs (length, 18-ss23 nucleotides) that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various diseases. In the present study, we explored cell- or tissue-specific miRNAs and assessed the applicability of miRNA profiling for identifying biomarkers of tissue injuries. miRNA analyses in various human and rat tissues identified several candidate miRNAs with possible tissue-specific expression, some of which have already been reported. In the present study, we focused on pancreas-specific miRNAs, miR-216a and miR-216b. Laser microdissection revealed that miR-216a and 216b were predominantly expressed in acinar cells of the pancreas as compared to Langerhans' islet. Plasma concentrations of miR-216a and miR-216b considerably increased in a rat model of L-arginineinduced acute pancreatitis. The current results have confirmed that miRNA expression profiling in various cells is useful for providing biomarkers for cell- or tissue-specific injuries.

Mutational analysis of the three tandem domains of ε-poly-L-lysine synthetase catalyzing the L-lysine polymerization reaction.

ε-Poly-l-lysine (ε-PL) synthetase (Pls) is a nonribosomal peptide synthetase (NRPS)-like enzyme with three tandem domains to catalyze the l-lysine polymerization reaction. Mutational analysis of the three tandem domains demonstrated that the interconnected action of all three domains is essential for the enzyme activity.

Renoprotective mechanisms of pirfenidone in hypertension-induced renal injury: through anti-fibrotic and anti-oxidative stress pathways.

Pirfenidone (PFD) is a novel anti-fibrotic agent that targets TGFß. However, the mechanisms underlying its renoprotective properties in hypertension-induced renal injury are poorly understood. We investigated the renoprotective properties of PFD and clarified its renoprotective mechanisms in a rat hypertension-induced renal injury model. Dahl salt-sensitive rats were fed a high-salt diet with or without 1% PFD for 6 weeks. During the administration period, we examined the effects of PFD on blood pressure and renal function. After the administration, the protein levels of renal TGFß, Smad2/3, TNFα, MMP9, TIMP1, and catalase were examined. In addition, total serum antioxidant activity was measured. Compared to untreated rats, PFD treatment significantly attenuated blood pressure and proteinuria. Histological study showed that PFD treatment improved renal fibrosis. PFD may exert its anti-fibrotic effects via the downregulation of TGFß-Smad2/3 signaling, improvement of MMP9/TIMP1 balance, and suppression of fibroblast proliferation. PFD treatment also increased catalase expression and total serum antioxidant activity. In contrast, PFD treatment did not affect the expression of TNFα protein, macrophage or T-cell infiltration, or plasma interleukin 1ß levels. PFD prevents renal injury via its anti-fibrotic and anti-oxidative stress mechanisms. Clarifying the renoprotective mechanisms of PFD will help improve treatment for chronic renal diseases.

Separation and purification of ε-poly-L-lysine from the culture broth based on precipitation with the tetraphenylborate anion.

A biopolymer ε-poly-L-lysine (εPL) exists as a fully protonated form (εPLH(n+)) in solutions at pH < 4. Around pH 3.5, at which the tetraphenylborate (TPB(-)) anion is not decomposed immediately, the εPLH(n+) cation associates with the TPB(-) anion to form a precipitate of 1:n stoichiometry, εPLH(TPB)(n). By the addition of NaTPB to a culture broth containing εPL and NH(4)(+) and K(+) ions, not only the polycationic εPLH(n+) but also the monovalent cations would be precipitated with the TPB(-) anion. However, εPLH(TPB)(n) was purified by washing the mixed precipitate with acetone, in which NH(4)TPB and KTPB are soluble. By mixing the εPLH(TPB)(n) precipitate and a high-concentration HCl solution, the TPB(-) anion was decomposed immediately to hydrophobic molecules. By the addition of a much larger volume of acetone to the reaction mixture, the decomposition products dissolved in the solvent. Simultaneously, εPL was precipitated as the hydrochloride salt. Thus, εPL has been separated and purified from the culture broth. Also, the method has been successfully applied to the separation of oligomeric εPL species. The present chemical separation method is rapid, simple, and easy to carry out, and can be utilized in bioengineering studies of such basic peptides or polyamines.

Development of a recombinant ε-poly-L-lysine synthetase expression system to perform mutational analysis.

ε-Poly-L-lysine (ε-PL) synthetase (Pls), which is a membrane protein with adenylation and thiolation domains characteristic of the nonribosomal peptide synthetases, catalyzes polymerization of L-lysine molecules (25-mer to 35-mer). Here, we report on the development of a recombinant Pls expression system that allowed us to perform a site-directed mutational analysis.