Identification of the streptothricin and tunicamycin biosynthetic gene clusters by genome mining in Streptomyces sp. strain fd1-xmd.

The genus Streptomyces have been highly regarded for their important source of natural products. Combined with the technology of genome sequencing and mining, we could identify the active ingredients from fermentation broth quickly. Here, we report on Streptomyces sp. strain fd1-xmd, which was isolated from a soil sample collected in Shanghai. Interestingly, the fermentation broth derived from this strain demonstrated broad-spectrum antimicrobial activity against gram-positive bacteria, gram-negative bacteria, and eukaryotes. To identify the antimicrobial substances and their biosynthetic gene clusters, we sequenced the fd1-xmd strain and obtained a genome 7,929,999 bp in length. The average GC content of the chromosome was 72.5 mol%. Knockout experiments demonstrated that out of eight biosynthetic gene clusters we could identify, two are responsible for the biosynthesis of the antibiotics streptothricin (ST) and tunicamycin (TM). The ST biosynthetic gene cluster from fd1-xmd was verified via successful heterologous expression in Streptomyces coelicolor M1146. ST production had a yield of up to 0.5 g/L after the optimization of culture conditions. This study describes a novel producer of ST and TM and outlines the complete process undertaken for Streptomyces sp. strain fd1-xmd genome mining.

Functional analysis of methyltransferases participating in streptothricin-related antibiotic biosynthesis.

Streptothricin (ST) and its related compounds produced by Streptomyces strains are broad-spectrum antibiotics that consist of carbamoylated d-gulosamine, amino-acid side chain, and streptolidine lactam moieties. BD-12, a streptothricin-related antibiotic, has a glycine-derived side chain and two N-methyl groups, whereas ST-F carrying the l-ß-lysine side chain has no methyl group. In our previous studies, we identified and characterized the BD-12 and ST biosynthetic gene clusters. Here we report the functional analysis of two methyltransferase genes (orf 6 and orf 13) in the BD-12 biosynthetic gene cluster. Combinatorial biosynthesis using these two methyltransferase genes and the ST biosynthetic gene cluster resulted in the production of three methylated forms of ST-F. Among them, N,N'-dimethyl-ST-F, a novel compound generated in the present study, showed bacteria-specific antibiotic activities, although ST-F exhibits antibiotic activities against both prokaryotes and eukaryotes. Our findings also demonstrated that the orf 6 and orf 13 genes are responsible for the N-methylations of the amide bonds in the streptolidine lactam and in the amino-acid side chain linkage, respectively, and that N-methyl modification of the streptolidine lactam confers resistance in part against an ST hydrolase, SttH.

Separation of Streptothricin Antibiotics from Culture Broth with Colorimetric Determination Using Dipicrylamine.

Our earlier method for the detection and separation of ε-poly-L-lysine using a yellow anionic dye, the dipicrylamine (DPA-) anion, was herein optimized for streptothricin antibiotics (ST), which contains the ß-lysine oligopeptides moiety, H-[NH-(CH2)3-CH(NH2)-CH2-CO]n-. We then applied this method to the detection and separation of ST in a commercially available nourseothricin, a mixture of ST species with n = 1, 2, 3, and 4. The ST species were precipitated with the DPA- anion. The precipitate was found to consist of the salts of the fully protonated ST species, STz+ (z = n + 1), with the DPA- anion. The ST(DPA)z precipitate was re-dissolved in acetonitrile. The solution was yellowish, and gave an absorption maximum at around 420 nm. Thus, the equivalent concentration of the ST species referred to the charge numbers of STz+ can be determined colorimetrically. By the addition of bis(triphenylphosphoranylidene)ammonium chloride, the ST species could be re-precipitated from the acetonitrile solution as hydrochloride salts. All of the ST species were found in the precipitate with high yields. The method was thus successfully applied to the detection and separation of ST species from the culture broth.

[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.

tRNA-Dependent Aminoacylation of an Amino Sugar Intermediate in the Biosynthesis of a Streptothricin-Related Antibiotic.

UNLABELLED: The antibiotic streptothricin (ST) possesses an amino sugar bound to an l-ß-lysine (ß-Lys) residue via a peptide bond. The peptide bond formation has been shown to be catalyzed by a nonribosomal peptide synthetase (NRPS) during ST biosynthesis. The focus of this study is the closely related ST analogue BD-12, which carries a glycine-derived side chain rather than a ß-Lys residue. Here, in Streptomyces luteocolor NBRC13826, we describe our biosynthetic studies of BD-12, which revealed that the peptide bond between the amino sugar and the glycine residue is catalyzed by a Fem-like enzyme (Orf11) in a tRNA-dependent manner rather than by an NRPS. Although there have been several reports of peptide bond-forming tRNA-dependent enzymes, to our knowledge, Orf11 is the first enzyme that can accept an amino sugar as a substrate. Our findings clearly demonstrate that the structural diversity of the side chains of ST-type compounds in nature is generated in an unusual manner via two distinct peptide bond-forming mechanisms. Moreover, the identification and functional analysis of Orf11 resulted in not only the production of new ST-related compounds, but also the provision of new insights into the structure-activity relationship of the ST-related antibiotics. IMPORTANCE: The antibiotic streptothricin (ST) possesses an amino sugar bound to an l-ß-lysine (ß-Lys) side chain via a peptide bond formed by a nonribosomal peptide synthetase (NRPS). BD-12, an analogue of ST, carries a glycine-derived side chain rather than ß-Lys, and here, we describe the BD-12-biosynthetic gene cluster from Streptomyces luteocolor NBRC13826, which contains the orf11 gene encoding a novel tRNA-dependent peptide bond-forming enzyme. The unique Fem-like enzyme (Orf11) accepts the amino sugar as a substrate and mediates the peptide formation between the amino sugar intermediate and glycine. Our studies demonstrate that the structural diversity of the side chains of ST-related compounds in nature is generated via two distinct peptide bond-forming mechanisms.

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.

A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis.

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-ß-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-ß-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-ß-lysine molecules adenylated by ORF 19 are used to elongate an L-ß-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-ß-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery.

In situ monitoring of streptothricin production by Streptomyces rochei F20 in soil and rhizosphere.

The onset of streptothricin (ST) biosynthesis in Streptomyces rochei F20 was studied by using reverse transcription-PCR (RT-PCR) to detect transcripts of ST genes during growth in liquid medium, soil, and the rhizosphere. In situ results correlated with those obtained in vitro, illustrating the growth phase-dependent manner of ST production by F20. Maximal transcription of ST resistance (sttR) and biosynthesis (sttA) genes occurred during the transition between the exponential and stationary phases of growth, when the specific growth rate (micro) started to decline. A higher level of gene expression of sttR versus sttA was observed in all experiments. In liquid culture, maximal transcript accumulation of the sttA gene was only ca. 40% that of the sttR gene. sttA and sttR mRNAs were detected in soil containing approximately 10(6) CFU of growing cells g of soil(-1). sttR mRNA was detected in sterile and nonsterile rhizosphere colonized with growing mycelium of F20 at 1.2 x 10(6) and 4.0 x 10(5) CFU g of soil(-1), respectively. However, neither sttR nor sttA transcripts were detected by RT-PCR in the rhizoplane, which supported a lower population density of F20 than the rhizosphere.

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 beta-lysine adenylating enzyme and a beta-lysine binding protein involved in poly beta-lysine chain assembly in nourseothricin synthesis in Streptomyces noursei.

Nourseothricins (syn. Streptothricins), a group of nucleoside peptides produced by several streptomycete strains, contain a poly beta-lysine chain of variable length attached in amide linkage to the amino sugar moiety gulosamine of the nucleoside portion. We show that the nourseothricin-producing Streptomyces noursei contains an enzyme (NpsA) of an apparent M(r) 56,000 that specifically activates beta-lysine by adenylation but does not bind to it as a thioester. Cloning and sequencing of npsA from S. noursei including its flanking DNA regions revealed that it is closely linked to the nourseothricin resistance gene nat1 and some other genes on the chromosome possibly involved in nourseothricin biosynthesis. The deduced amino-acid sequence revealed that NpsA is a stand-alone adenylation domain with similarity to the adenylation domains of nonribosomal peptide synthetases (NRPS). Further analysis revealed that S. noursei contains a beta-lysine binding enzyme (NpsB) of about M(r) 64,100 which can be loaded by NpsA with beta-lysine as a thioester. Analysis of the deduced amino-acid sequence from the gene (npsB) of NpsB showed that it consists of two domains. The N-terminal domain of approximately 100 amino-acid residues has high similarity to PCP domains of NRPSs whereas the 450-amino-acid C-terminal domain has a high similarity to epimerization (E)-domains of NRPSs. Remarkably, in this E-domain the conserved H-H-motif is changed to H-Q, which suggests that either the domain is nonfunctional or has a specialized function. The presence of one single adenylating beta-lysine activating enzyme in nourseothricin-producing streptomycete and a separate binding protein suggests an iteratively operating NRPS-module catalyses synthesis of the poly beta-lysine chain.

Nourseothricin (streptothricin) inactivated by a plasmid pIE636 encoded acetyl transferase of Escherichia coli: location of the acetyl group.

Escherichia coli strains harbouring the plasmid pIE636 are able to synthesize acetylcoenzyme A: streptothricin acetyltransferase (ACSAT). The (enzymatic) N-acetylation of streptothricin F is known to contribute significantly towards the loss of antibacterial activity. 13C-NMR analysis of [14C]N-acetyl-labelled streptothricin F, produced by ACSAT-catalysed acetylation of streptothricin F and subsequent purification by various chromatographical steps, unequivocally revealed streptothricin F to be acetylated at the beta-amino group (C16) (and not at the epsilon-amino group (C19)).

Metabolism of phosphate-limited cultures of Streptomyces. IV. Protein-phosphorylation of antibiotics-producing cultures.

It has been demonstrated that in the cellular proteins of Streptomyces hygroscopicus JA 6599 and Streptomyces noursei JA 3890 b, the producers of the antibiotics turimycin and nourseothricin, respectively, phosphorylated proteins are present. Numbers and concentrations of phosphorylated proteins decreased during the idiophase as characterized by phosphate limitation, antibiotic biosynthesis and phosphatase formation. Phosphoamino acids of serine, threonine and tyrosine were found in the hydrolysates of proteins. Protein tyrosyl kinase was demonstrated in the cellular extracts. The results supports the hypothesis that protein phosphorylation possesses a function in the regulation of growth and secondary product formation.

[Formation of a streptothricin-group antibiotic by a Streptomyces glaucus 1136 culture]. / Obazovanie antibiotika iz gruppy streptotritsinov kul'turoi Streptomyces glaucus 1136.

In screening of new antibiotics a streptomycete (strain 1136) was isolated from a soil sample of Armenia. It showed no antagonistic properties in streek cultures on agarized media. When grown under submerged conditions strain 1136 produced an antibiotic active against grampositive and gramnegative bacteria. By its cultural and morphological properties strain 1136 was classified as Streptomyces glaucus Agre et Preobrazhenskaya, 1983. Microbiological and chemical investigation of the antibiotic produced by strain provided its identification at the early stages of the investigation as an antibiotic of the streptothricin group. Up to now no organisms producing streptothricin antibiotics were detected among streptomycetes of the Azureus section including strain 1136.

Directed selection of differentiation mutants of Streptomyces noursei using chemostat cultivation.

A nourseothricin-producing Streptomyces noursei strain was continuously cultivated in a chemostat equipped with a stirrer for mechanical fractionation of the mycelium. Different cultivation conditions allowed the selection of six types of differentiation mutants after the culture had reached a population genetically stationary state. The mutants showed an altered control pattern of sporulation as well as altered antibiotic biosynthesis and antibiotic resistance. In addition, the stability of the recombinant plasmid pIJ385 in several differentiation type mutants as host strains was tested. The results suggest that there exists a strong correlation between the cultivation conditions employed and the type of differentiation mutants selected.

[Metabolism of phosphate-limited Streptomyces cultures. III. The ambivalent effect of phosphates in nourseothricin-producing cultures of Streptomyces noursei JA 3890b]. / Untersuchungen zum Stoffwechsel phosphatlimitierter Streptomycetenkulturen. III. Mitteilung: Uber die ambivalente Wirkung des Phosphates in Nourseothricin-bildenden Kulturen von Streptomyces noursei JA 3890b.

A common condition in the evolution of organisms and their metabolism seems to be a latent lack of available phosphate in the natural environment. Accordingly, the phosphate dependent metabolisms of the soil-living streptomycetes should be stamped by lack of phosphate, too. The biosynthesis of the streptothricin antibiotic nourseothricin by Streptomyces noursei 3890b is initiated by limitation of soluble phosphate in the fermentation medium. At the other side is shown that a certain rate of feeding of phosphate during the fermentation increases the nourseothricin biosynthesis. An ambivalente role of phosphate on the secondary metabolite biosynthesis is stated. The limitation of phosphate leads to a special physiological state of the producer, characterized by secondary product formation and dephosphorylating activities in cells. This state is temporally stabilized by the presence of a sufficient phosphate supply, realized by enzymatic hydrolysis of complex phosphate-containing substrates or by a direct feeding of inorganic phosphate to the fermentations. The occurrence of different physiological states in respect to the phosphate-dependent metabolism is described by S-shaped functions of the relationship between specific growth rate and the phosphate concentration in the medium. The special behaviour of Streptomyces noursei cells at phosphate limitation is discussed to be the result of the dephosphorylating activities in cells, hydrolyzing phosphoester-bonds of regulatory metabolites as well as energy-rich compounds.

Influence of inorganic phosphate on the lipid synthesis of a phosphate-deregulated mutant of Streptomyces noursei.

Phosphate-dependent changes of the mycelial lipid composition were studied in the streptothricin-producing parental strain Streptomyces noursei JA 3890 b/2 and its mutant RG 2. In contrast to its ancestor, the mutant was capable of producing the antibiotic nourseothricin even when large quantities of inorganic phosphate were present in the medium. The apparent insensitivity of the secondary metabolism to phosphate inhibition corresponds to a decreased level of phospholipids in the presence of excessive inorganic phosphate and, during phosphate limitation, to a much higher production of the alkaline phosphatases. A model is discussed which proposed the control by a common genetic element of both the phospholipid and antibiotic production.

Influence of nourseothricin on growth and secondary metabolism of Streptomyces noursei JA 3890b.

The nourseothricin-producing S. noursei strain JA 3890b possessed a high degree of resistance to its own antibiotic when grown in submerged cultures started from mycelium samples as inocula. In contrast, both the outgrowth of spores and the development of surface colonies from mycelium samples were severely inhibited in the presence of relatively low concentrations of nourseothricin, suggesting that the producer organism is susceptible to the autotoxic metabolite in particular stages of its development. Nourseothricin production by submerged cultures has been found to be independent of negative feedback regulation by the antibiotic.

ATP levels in phosphate-deregulated and arsenate-resistant mutants of Streptomyces noursei.

Mycelial levels of ATP and glucose-6-phosphate were investigated in mutants of streptothricin-producing S. noursei JA 3880b differing from the wild-type strain in antibiotic formation, in the control by inorganic phosphate of the secondary metabolism, and in the resistance to growth inhibition by toxic arsenate ions. As compared with the ancestral strain, mutants exhibited a lower content of ATP in the mycelium while addition of 0.1 M arsenate to growing cultures provoked only moderate changes in the level of this high-energy metabolite. The results suggest that there exists a correlation between growth resistance to arsenate and insensitivity to phosphate inhibition of the secondary metabolism, on the one hand, and the capacity to produce streptothricin-type antibiotics, on the other.

Isolation and biological properties of arsenate-resistant strains of Streptomyces noursei.

Arsenate-resistant (AsR) clones were obtained with high frequency from colony populations of streptothricin-producing strains of Streptomyces noursei by the paper strip method. Whereas in the AsR-strains obtained from both wild type and mutant NG 13 the antibiotic production was reduced to approx. 60% of the parental level, the AsR clones isolated from colony populations of mutant UV 12 displayed increased productivity (less than or equal to 150%). However, their improved capacity to produce streptothricins was lost rapidly after repeated cell propagation in submerged cultures, suggesting that unstable genetic elements were involved in enabling S. noursei to grow in the presence of toxic concentrations of arsenate.