Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

Production of a broad spectrum streptothricin like antibiotic from halotolerant Streptomyces fimbriatus isolate G1 associated with marine sediments.

Streptomyces have been reported as a remarkable source for bioactive secondary metabolites with complex structural and functional diversity. In this study, 35 isolates of genus Streptomyces were purified from rhizospheric and marine soils collected from previously unexplored habitats and screened for antimicrobial activities. One of these isolates, G1, when tested in vitro, was found highly active against wide range of microbes including Gram-positive, Gram-negative bacteria, and different fungal pathogens. It was identified as mesophilic, alkaliphilic, and moderately halotolerant as it showed optimum growth at temperature 30 °C, pH 8.0 in casein-starch-peptone-yeast extract-malt extract medium supplemented with 5% NaCl. Sequence analysis of the 16S rRNA gene indicated 100% identity of this isolate to Streptomyces fimbriatus. Moreover, maximum antimicrobial activity was achieved in starch nitrate medium supplemented with 1% glycerol as carbon and 0.03% soy meal as nitrogen source. The antimicrobial compounds produced by this isolate were extracted in methanol. Bioassay-guided fractionation through thin layer chromatography of methanolic extract resulted in the separation of a most active fraction with an Rf value of 0.46. This active fraction was characterized by FTIR and LCMS analysis and found similar to streptothricin D like antibiotic with m/z 758.42.

Insights into the Function of the N-Acetyltransferase SatA That Detoxifies Streptothricin in Bacillus subtilis and Bacillus anthracis.

Acylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed that Bacillus subtilis and Bacillus anthracis encode the GCN5-related N-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of B. subtilis SatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of the B. anthracis Ames SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations into satA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 of BaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCE This work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.

In Bacillus subtilis, the SatA (Formerly YyaR) Acetyltransferase Detoxifies Streptothricin via Lysine Acetylation.

Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA+ restored streptothricin resistance to B. subtilissatA (BsSatA) strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetyl-coenzyme A (acetyl-CoA). A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd [dissociation constant] = 1 µM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilissatA+ in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil.IMPORTANCE Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The enzyme alluded to is a member of a family of proteins that are broadly distributed in all domains of life but poorly studied in B. subtilis and B. anthracis The initial characterization of the enzyme provides insights into its mechanism of catalysis.

[Progress in streptothricin antibiotics - A review].

Streptothricins are a group of the earliest discovered antibiotics with broad antimicrobial spectrum, and have been used for crop protection. We reviewed the studies on streptothricin resistance, biosynthesis of the three components (streptolidine, carbamoylated D-glucosamine and poly ß-lysine chain) and chemical synthesis of streptothricins. The important aspects for future streptothricin researches were also discussed.

Biosynthesis of the carbamoylated D-gulosamine moiety of streptothricins: involvement of a guanidino-N-glycosyltransferase and an N-acetyl-D-gulosamine deacetylase.

Streptothricins (STNs) are atypical aminoglycosides containing a rare carbamoylated D-gulosamine (D-GulN) moiety, and the antimicrobial activity of STNs has been exploited for crop protection. Herein, the biosynthetic pathway of the carbamoylated D-GulN moiety was delineated. An N-acetyl-D-galactosamine is first attached to the streptolidine lactam by the glycosyltransferse StnG and then epimerized to N-acetyl-D-gulosamine by the putative epimerase StnJ. After carbamoylation by the carbamoyltransferase StnQ, N-acetyl-D-GulN is deacetylated by StnI to furnish the carbamoylated D-GulN moiety. In vitro studies characterized two novel enzymes: StnG is an unprecedented GT-A fold N-glycosyltransferase that glycosylates the imine nitrogen atom of guanidine, and StnI is the first reported N-acetyl-D-GulN deacetylase.

Biosynthesis of streptolidine involved two unexpected intermediates produced by a dihydroxylase and a cyclase through unusual mechanisms.

Streptothricin-F (STT-F), one of the early-discovered antibiotics, consists of three components, a ß-lysine homopolymer, an aminosugar D-gulosamine, and an unusual bicyclic streptolidine. The biosynthesis of streptolidine is a long-lasting but unresolved puzzle. Herein, a combination of genetic/biochemical/structural approaches was used to unravel this problem. The STT gene cluster was first sequenced from a Streptomyces variant BCRC 12163, wherein two gene products OrfP and OrfR were characterized in vitro to be a dihydroxylase and a cyclase, respectively. Thirteen high-resolution crystal structures for both enzymes in different reaction intermediate states were snapshotted to help elucidate their catalytic mechanisms. OrfP catalyzes an Fe(II) -dependent double hydroxylation reaction converting L-Arg into (3R,4R)-(OH)2 -L-Arg via (3S)-OH-L-Arg, while OrfR catalyzes an unusual PLP-dependent elimination/addition reaction cyclizing (3R,4R)-(OH)2 -L-Arg to the six-membered (4R)-OH-capreomycidine. The biosynthetic mystery finally comes to light as the latter product was incorporation into STT-F by a feeding experiment.

Mining of a streptothricin gene cluster from Streptomyces sp. TP-A0356 genome via heterologous expression.

Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces sp. TP-A0356. The ST cluster from S. sp. TP-A0356 was verified by successful heterologous expression in Streptomyces coelicolor M145. Two new ST analogs were produced together with streptothricin F and streptothricin D in the heterologous host. The ST cluster was further confirmed by inactivation of gene stnO, which was proposed encoding an aminomutase supplying ß-lysines for the poly-ß-Lys chain formation. A putative biosynthetic pathway for STs is proposed based on bioinformatics analyses of the ST genes and experimental evidence.

Three new 12-carbamoylated streptothricins from Streptomyces sp. I08A 1776.

Two new streptothricins (1 and 2) and a new streptothricin acid derivative (3), all with the carbamoyl group substituted at C-12 of the gulosamine moiety, together with the known N(ß)-acetylstreptothricin D acid (4), have been isolated from the culture broth of Streptomyces sp. I08A 1776. The structures of the new compounds were determined by MS, CD, and 1D and 2D NMR spectroscopic data analysis. The isolated compounds were evaluated for antibacterial and antifungal activities. Streptothricin E (6) showed potent activity against the clinically isolated extensively drug-resistant Mycobacterium tuberculosis with MIC values of 0.25-0.5µg/mL.

Two streptothricins with a cis-streptolidine lactam moiety from Streptomyces sp. I08A 1776.

Two unique cis-fused streptothricins (1 and 2) were isolated from the culture broth of Streptomyces sp. I08A 1776. Their structures were determined by MS, CD, and 1D and 2D NMR spectroscopic data analysis. Compound 2 showed weak antibacterial activities against Bacillus subtilis and Enterococcus faecalis with MIC values of 32 and 64 µg ml(-1), respectively.

Streptothricin derivatives from Streptomyces sp. I08A 1776.

Five new streptothricin derivatives with a carbamoyl group substituted at C-12 (1-5) and three known analogues have been isolated from the culture broth of Streptomyces sp. I08A 1776 by ion exchange and hydrophilic interaction chromatographic techniques. Their structures were determined by spectroscopic and chemical methods. Compound 3 was a streptothricin derivative possessing a cis-streptolidine moiety. Its absolute configuration was defined by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. Compound 5 and streptothricin E (6) displayed antibacterial and antifungal activity with MIC values in the range 1-64 µg/mL.

Isolation, structure elucidation and antibacterial activities of streptothricin acids.

Five streptothricin acids (compounds 1-5) were isolated by ion-exchange resin chromatography and preparative RP-HPLC from the fermentation broth of Streptomyces qinlingensis. Their structures were elucidated mainly by analyses of the IR, HR-EIS-MS and NMR spectral data. They were deduced as two known compounds, streptothricin F acid (1) and streptothricin D acid (2), and three new compounds, 12-carbamoylstreptothricin E acid (3), 12-carbamoylstreptothricin D acid (4) and N-acetyl-streptothricin D acid (5), respectively. The antibacterial activities of 1-5 against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa were assayed by micro-broth dilution. Comparison of the MICs with streptothricin F and D showed that the antimicrobial activities of 1-5 were decreased significantly but varied with the structures.

Two new members of streptothricin class antibiotics from Streptomyces qinlingensis sp. nov.

Four streptothricin-group antibiotics (1~4) were isolated from the fermentation broth of Streptomyces qinlingensis sp. nov. Along with the known antibiotics streptothricins F (1) and D (3), two new members of this class (2, 4) were identified as 12-carbamoyl derivatives of 1 and 3, respectively, mainly by analysis of the IR, HR-MS and NMR spectral data. The antibacterial activities of 1~4 against Escherichia coli (MICs 3.1, 25.0, 3.1 and 12.5 microg/ml), Bacillus subtilis (MICs 6.3, 25.0, 3.1 and 50 microg/ml), Staphylococcus aureus (MICs 12.5, >100.0, 6.3, >100.0 microg/ml), Bacillus cereus (MICs 25.0, 50.0, 25.0 and 50.0 microg/ml) and Pseudomonas aeruginosa (MICs 50.0, >100.0, 50.0, >100.0 microg/ml) were assayed by micro-broth dilution. The results based on MIC data indicated that 2 and 4 exhibited significantly less potent antibacterial activities when compared to that of 1 and 3.

[A new HPLC method for determination of main constituents of the streptothricin complex. Analysis of nourseothricin, grisin and kormogrisin].

A simple and reliable HPLC method for quantitative analysis of complex antibiotics consisting of a mixture of streptothricins F, E, D and C in a biological matrix was developed. The method is based on ion-pair separation of streptothricins on the reversed-phase C18 analytical column with UV detector (210 nm). Aqueous solution of acetonitrile containing trifluoroacetic acid and octane-1-sulfonic acid sodium salt was used as eluent. Retention times of streptothricins became longer as the molecular weight increased, i.e. the component F was eluted first, then followed components E, D and C. The total time of the analysis was ca. 22 min. Composition of the standard samples of nourseothricin and grisin, as well as the streptothricin content of the commercial grisin-based kormogrisin were determined. Components F and D were found to be dominant in the streptothricin complex comprising totally 70-90%, with streptothricin F prevailing in nourseothricin (56%) and streptothricin D being the major constituent in grisin (51%), while in the kormogrisin the concentrations of components D and F were approximately the same. The portion of E varied from 5 to 20% and the concentration of streptothricin C changed within the range of 3-11%. The peaks of the admixtures present in kormogrisin did not interfere with determination of the streptothricin components. It is suggested that the method described can be applied to determination of the streptothricins in biological objects without a complex preliminary sample preparation.

A novel enzyme conferring streptothricin resistance alters the toxicity of streptothricin D from broad-spectrum to bacteria-specific.

Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics. All STs consist of a carbamoylated D-gulosamine to which the beta-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. Although many ST-resistance genes have been identified in bacteria, including clinically isolated pathogens and ST-producing Streptomyces strains, only one resistance mechanism has been identified to date. This mechanism involves the modification of the ST molecule by monoacetylation of the moiety of the beta-lysine(s). In this study, we successfully isolated a novel ST-resistance gene (sttH) from Streptomyces albulus, which is a known ST nonproducer. The in vitro analysis of SttH demonstrated that this enzyme catalyzes the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. Interestingly, the selective toxicity of ST-D possessing 3x beta-lysine moiety was altered from broad-spectrum to bacteria-specific by the hydrolysis of streptolidine lactam, although ST-F (1 x beta-lysine) was detoxified by SttH in both prokaryotes and eukaryotes (yeasts). STs have not been clinically developed due to their toxicities; however, in this study, we showed that hydrolyzed ST-D (ST-D-acid) exhibits potent antibacterial activity even when its toxicity against eukaryotic cells is reduced by SttH. This suggests that ST-D-acid is a potential candidate for clinical development or for use as a new lead compound for drug discovery.

Studies on the formation and incorporation of streptolidine in the biosynthesis of the peptidyl nucleoside antibiotic streptothricin F.

Streptothricin F (STF, 1) is a peptidyl nucleoside antibiotic produced by Streptomyces lavendulae. Studies were conducted to address the formation and timing of incorporation of the arginine-derived base streptolidine (4) during the biosynthesis of 1. [guanidino-(13)C]Streptolidine (10) was prepared by modification of an established method and used in whole-cell incorporation experiments. Analysis of the purified STF by (13)C NMR revealed a 1.9% enrichment of the guanidino carbon, confirming 4 as an advanced precursor to 1 and supporting proposals that 1 is assembled via a convergent biosynthetic pathway. To identify advanced intermediates in the conversion of L-arginine to 4, (2S,3R)-[guanidino-(13)C]capreomycidine (32) was prepared from oxazolidine aldehyde (18) via 1,1-dimethylethyl (4R,1'S)-4-(1',3'-diaminopropyl)-2,2-dimethyl-3-oxazolidinecarboxylate (30). Treatment of 30 with Br(13)CN yielded the corresponding diprotected amino alcohol, which was readily converted to 32. The STF isolated from whole-cell incorporation experiments with 32 showed no significant (13)C enrichment at the guanidino carbon. These results suggest that 32 may be an enzyme-bound intermediate, unable to enter the cell, or is not a precursor to STF.

A-53930A and B, novel N-type Ca2+ channel blockers.

A-53930A, B and C, which inhibit N-type Ca2+ channels, were isolated from the culture broth of Streptomyces vinaceusdrappus SANK 62394. A-53930A and B were new compounds which contained a carbamoyl group on the 6-hydroxyl group of the D-gulosamine part of streptothricin. A-53930C was identical to streptothricin B. A-53930A, B and C inhibited [125I]omega-conotoxin MVIIA binding to N-type Ca2+ channels (IC50= 0.17, 0.091 and 0.071 microM), but did not inhibit [3H]PN200-110 binding to L-type Ca2+ channels (IC50 > 50 microM). These compounds also inhibited [3H]norepinephrine release from chick cerebral cortex synaptosomes (IC50=91.0, 20.6 and 39.5 microM), indicating these compounds selectively block N-type Ca2+ channels which are important for neurotransmitter release. It was also revealed that although A-53930C had antimicrobial activity against gram-negative and -positive bacteria and fungi, A-53930A and B showed weak activity only against gram-negative bacteria.

Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation.

In a search for strains producing biocides with a wide spectrum of activity, a new strain was isolated. This strain was taxonomically characterized as Streptomyces rochei F20, and the chemical structure of the bioactive product extracted from its fermentation broth was determined to be a mixture of streptothricins. From a genomic library of the producer strain prepared in the heterologous host Streptomyces lividans, a 7.2-kb DNA fragment which conferred resistance to the antibiotic was isolated. DNA sequencing of 5.2 kb from the cloned fragment revealed five open reading frames (ORFs) such that ORF1, -2, -3, and -4 were transcribed in the same direction while ORF5 was convergently arranged. The deduced product of ORF1 strongly resembled those of genes involved in peptide formation by a nonribosomal mechanism; the ORF2 product strongly resembled that of mphA and mphB isolated from Escherichia coli, which determines resistance to several macrolides by a macrolide 2'-phosphotransferase activity; the ORF3 product had similarities with several hydrolases; and the ORF5 product strongly resembled streptothricin acetyltransferases from different gram-positive and gram-negative bacteria. ORF5 was shown to be responsible for acetyl coenzyme A-dependent streptothricin acetylation. No similarities in the databases for the ORF4 product were found. Unlike other peptide synthases, that for streptothricin biosynthesis was arranged as a multienzymatic system rather than a multifunctional protein. Insertional inactivation of ORF1 and ORF2 (and to a lesser degree, of ORF3) abolishes antibiotic biosynthesis, suggesting their involvement in the streptothricin biosynthetic pathway.