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The crystal structure of CYP105D18 and its unique structural features for papaverine N-oxidation are presented., The bacterial CYP105 family is involved in secondary metabolite biosynthetic pathways and plays essential roles in the biotransformation of xenobiotics. This study investigates the newly identified H2O2-mediated CYP105D18 from Streptomyces laurentii as the first bacterial CYP for N-oxidation. The catalytic efficiency of CYP105D18 for papaverine N-oxidation was 1.43 s−1 µM −1. The heme oxidation rate (k) was low (

A novel bacterium, designated TRM 44457T, belonging to the genus Streptomyces , was isolated from soil sampled in cotton fields in Xinjiang, PR China. Comparative 16S rRNA gene sequence analysis indicated that strain TRM 44457T was phylogenetically most closely related to Streptomyces laurentii LMG 19959T (99.38 % sequence similarity); however, strain TRM 44457T had a relatively low DNA–DNA relatedness value with S. laurentii LMG 19959T as determined by calculating the average nucleotide identity value (84.42 %). Strain TRM 44457T possessed ll-diaminopimelic acid as the diagnostic cell-wall diamino acid, MK-9 (H6) and MK-9 (H10) as the major menaquinone. The polar lipids included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphotidylinositol, phosphatidylinositol mannosides and an unidentified phospholipid. The major fatty acids were anteiso-C15:0, iso-C16:0, anteiso-C17:0, iso-C15:0, C16:0, iso-C17:0, cyclo-C17:0 and anteiso-C17:1ω9c. The genomic DNA G+C content was 72.6 mol%. Based on the evidence from this polyphasic study, strain TRM 44457T represents a novel species of the Streptomyces , for which the name Streptomyces roseicoloratus is proposed. The type strain is TRM 44457T (=KCTC 39904T=CCTCC AA 2016040T).

Background and objective Chitinase enzymes have a various application in the field of environmental, biotechnology and medical aspects. This study aimed to the production of the chitinolytic enzymes from different species of bacteria. Materials and methods Bacterial isolation from different habitats was carried out on agar medium containing chitin as carbon and nitrogen sources. The obtained bacteria (20) were characterized and screened again in chitin broth medium. Results Out of 20 bacterial isolate, 2 new isolates, belonged to Streptomyces laurentii SN5 and Cellulosimicrobium funkei SN20, were the most active in chitin degradation compared to the other isolates. They have been characterized for the first time for their chitinase activity. They were identified using 16S rRNA gene analysis and in the liquid medium, the 2 isolates have enzyme activities of 0.533 and 0.537 U mL-1, respectively. The maximum chitinase production was obtained when those bacterial strains were grown in Luria-Bertani (LB) broth amended with 1% colloidal chitin, for 1 day and at temperature of 30°C. The optimum pH value for chitinase production was pH 7 for both S. laurentii and C. funkei. The enzyme has been purified using Sephadex G-100 and DEAE-Cellulose chromatography column and found to have a similar molecular size of ~50 kDa. Conclusion Those two bacterial species could be used in chitinase production and in the environmental recycling of disposable chitin wastes such as chitin from shrimp shell waste.

Thiostrepton (TSR), produced by Streptomyces laurentii, is a potent archetypal thiopeptide antibiotic that effectively antagonizes a broad spectrum of Gram-positive bacteria and has been used as a safe antimicrobial agent for animals. Siomycin (SIO) is a naturally produced TSR-type antibiotic that has been reported more potent than TSR. Based on a recent understanding regarding the structure–activity relationship of TSR against prokaryotic pathogens, we here constructed an ideal platform to obtain quinaldic acid (QA)-modified SIO analogs and generated a new SIO derivative, 5′-fluoro-SIO, with an unanticipated improvement in water solubility. To investigate whether oral diseases could be developed as novel indications for TSR-type antibiotics, we tested the minimum inhibitory concentrations (MICs) of these antibiotics against common oral pathogenetic microorganisms. Quantitative bioassays indicated that all of the tested TSR-type antibiotics exhibited potent antibacterial activity against the Gram-positive cariogenic microorganisms involved in the development of dental caries, as well as two major Gram-negative periodontal pathogens. Among the tested antibiotics, 5′-fluoro-SIO and SIO exhibited stronger potency than 5′-fluoro-TSR and TSR. These findings suggest that SIO may be more suitable than TSR as a lead compound to develop improved thiopeptide derivatives for clinical use and that TSR-type antibiotics have considerable potential for the prevention and treatment of dental caries and periodontitis.

Improvement of the medical textile industry has received more attention recently, especially with widespread of microbial and viral infections. Medical textiles with new properties, such as bacterial pathogens self-cleaning, have been explored with nanotechnology. In this study, an endophytic actinomycetes strain of Streptomyces laurentii R-1 was isolated from the roots of the medicinal plant Achillea fragrantissima. This is used as a catalyst for the mediated biosynthesis of silver nanoparticles (Ag-NPs) for applications in the textile industry. The biosynthesized Ag-NPs were characterized using UV-vis spectroscopy, Fourier transform infrared (FT-IR), transmission electron microscopy (TEM), and X-ray Diffraction (XRD), which confirmed the successful formation of crystalline, spherical metal nanoparticles. The biosynthesized Ag-NPs exhibited broad-spectrum antibacterial activity. Our data elucidated that the biosynthesized Ag-NPs had a highly cytotoxic effect against the cancerous caco-2 cell line. The selected safe dose of Ag-NPs for loading on cotton fabrics was 100 ppm, regarding their antibacterial activity and safe cytotoxic efficacy. Interestingly, scanning electron microscope connected with energy dispersive X-ray spectroscopy (SEM-EDX) of loaded cotton fabrics demonstrated the smooth distribution of Ag-NPs on treated fabrics. The obtained results highlighted the broad-spectrum activity of nano-finished fabrics against pathogenic bacteria, even after 5 and 10 washing cycles. This study contributes a suitable guide for the performance of green synthesized NPs for utilization in different biotechnological sectors.

Thiopeptins are highly decorated thiopeptide antibiotics similar in structure to thiostrepton A and harbor two unusual features. All thiopeptins contain a thioamide, a rare moiety among natural products, and a subset of thiopeptins present with a piperidine in the core macrocycle rather than the more oxidated dehydropiperidine or pyridine rings typically observed in the thiopeptides. Here, we report the identification of the thiopeptin biosynthetic gene (tpn) cluster in Streptomyces tateyamensis and the gene product, TpnL, which shows sequence similarity to (deaza)flavin-dependent oxidoreductases. Heterologous expression of TpnL in the thiostrepton A producer Streptomyces laurentii led to the production of a piperidine-containing analogue. Binding studies revealed that TpnL preferentially binds the deazaflavin cofactor coenzyme F(420), and in vitro reconstitution of TpnL activity confirmed that this enzyme is an F(420)H(2)-dependent dehydropiperidine reductase. The identification of TpnL and its activity establishes the basis for the piperidine-containing series a thiopeptides, one of the five main structural groups of this diverse family of antibiotics.

Solvent extracts of Mazus japonicus and Fumaria indica were investigated individually and in combinations (1:1, 1:2, or 2:1) for their antibacterial potency against Bacillus subtilis, Pseudomonas geniculata, Streptomyces laurentii, Klebsiella pneumoniae, Microbacterium oxydans, Alcaligenes faecalis, and Enterococcus faecium. Minimum inhibition concentration (MIC) and fractional inhibitory concentration (FIC) were investigated. Petroleum ether extracts showed moderate (6.06 mg.mL–1 MIC for M. japonicus and 1.65 mg.mL–1 MIC for F. indica), aqueous extract had the lowest, 1.619 mg.mL–1 MIC for M. japonicus and18.36 mg.mL–1 MIC of F. indica), and methanolic extract showed the highest (0.740 mg.mL–1 MIC for M. japonicus and 1.065 mg.mL–1 MIC for F. indica) antibacterial activities (individual analysis). When analyzing mixtures, none of the aqueous extracts showed synergistic effect against any bacteria. Methanolic extract mixtures demonstrated synergy against B. subtilis (0.082 mg.mL–1 MIC with 2:1); t...

Four different genotypes of Glycine max, viz Rawal-I, NARC-II, SA-72-60 and Ajmeri, were subjected to antimicrobial investigation in the present study. Three types of extracts, i.e. aqueous, methanol and peptide, were prepared from the seed powder of each variety. Extracts were tested for their antimicrobial ability by agar well diffusion assay against 3 g-positive strains, i.e. Streptomyces laurentii, Enterococcus faecium and Microbacterium oxydans, and four gram-negative strains, i.e. Pseudomonas geniculata, Alcaligenes faecalis, Klebsiella pneumoniae and Bacillus subtilis. Rawal-I and NARC-II exhibited maximum antibacterial activity at higher concentrations with dose-dependent relationship against Streptomyces laurentii and Pseudomonas geniculata, whereas SA-72-60 and Ajmeri were found ineffective against all tested strains. Among three extracts prepared, aqueous and peptide extracts \({(12\pm0.14\,{\rm mm},\,11\pm0.07\,{\rm mm})}\) were more effective as compared to methanolic extracts. Different combinations of Rawal-I and NARC-II in 1:2 and 2:1 ratios were tested for antibacterial efficacies which exhibited no synergistic activity with few exceptions where antagonist interaction was shown by some of the extract mixtures. Preliminary phytochemical analysis of the effective extracts revealed the presence of antimicrobial constituents like flavonoids, alkaloids and saponins.

The double-mutant strain Streptomyces laurentii ΔtsrB/T was designed and constructed based on a recent understanding regarding the structure–activity relationship of thiostrepton (TSR) against prokaryotic pathogens. Five new C-terminally methylated TSR (CmTSR) derivatives that varied in the side-ring structure were obtained via the chemical feeding of quinaldic acid (QA) analogs. These derivatives provide new insights into the tolerance of QA incorporation in TSR biosynthesis. Certain members of the tested TSR derivatives, meanwhile, exhibited much better antibacterial activities than all currently known thiopeptide antibiotics.

Among the extreme habitats, drought is most harsh abiotic stress affecting growth, development and productivity of crops. Plants also face limitations of certain nutrients such as phosphorus required for different physiological and metabolic activities. Stress-adaptive phosphorus-solubilizing microbes in rhizospheric soil can help plants to combat water scarcity and overcome the problem of phosphorus unavailability to plant systems. The present investigation deals with the isolation of drought stress adaptive and P-solubilizing microbes from rhizospheric soil of different cereals and pseudocereals and their role in mitigation of drought stress in great millet. A total of 193 rhizospheric microbes were isolated and screened for their capability to solubilize phosphorus under drought stress. Twenty isolates exhibited P-solubilizing attribute under drought stress, which were further screened for plant growth promoting (PGP) traits such as solubilization of zinc and potassium; production of Fe-chelating compounds, indole acetic acid, hydrogen cyanide and ammonia. On basis of multifunctional PGP traits, two efficient and potential microbes were evaluated for PGP in great millet in vitro under green house with different water regimes. The isolates were found to be efficient in terms of enhancing accumulation of different osmolytes such as glycine betaine, proline, sugars, increased chlorophyll content, and decreasing lipid peroxidation. The isolates were identified by 16S/18S rRNA gene sequencing as Streptomyces laurentii EU-LWT3-69 and Penicillium sp. strain EU-DSF-10. To best of our knowledge Streptomyces laurentii has been reported first time as PGP and drought adaptive bacterium. PGP drought-adaptive phosphorus solubilizers could be used as bioinoculants for crops under water scarcity ecosystems.

Eight isolates of Streptomyces sp. can be isolated from compost soil in Surabaya, Indonesia. The results of biochemical and morphological tests showed that the 8 isolates were new. Phylogenetic analysis was performed on the sequence of 16S rRNA gene. Nucleotide sequences of 16S rRNA gene Streptomyces sp. the compost soil isolates of Surabaya were analyzed and compared with the 16S rRNA gene sequence from the literature. Streptomyces sp. the compost soil of Surabaya based on the 16S rRNA gene showed the new species of Streptomyces. The result of phylogenetic tree diagram showed that Streptomyces Sp-D, Sp-Ep, Sp-G and Sp-I found in Bratang Surabaya compost house land were new isolates. Streptomyces Sp-Ep was a new type of Streptomyces closely related to Streptomyces indonesiasis and Streptomyces nashvillensis. Streptomyces Sp-Ea was Streptomyces olivoreticuli which was still related to Streptomyces yogyakartensis. Streptomyces Sp-F was Streptomyces levis strain NRRL B-24299. Streptomyces Sp-C was Streptomyces filamentosus. Streptomyces Sp-D was a new type of Streptomyces closely related to Streptomyces javensis and Streptomyces roseus. Streptomyces Sp-G was a new type of Streptomyces closely related to Streptomyces roseoviridis strain NBRC 12911 and Streptomyces thermocarboxydovorans strain AT52. Streptomyces Sp-I was a new streptomyces that was still closely related to Streptomyces cangkringensis and Streptomyces asiaticus. Streptomyces Sp-A was Streptomyces laurentii strain: LMG 19959.

Streptomyces laurentii ATCC 31255 produces thiostrepton, a thiopeptide class antibiotic. Here, we report the complete genome sequence for this strain, which contains a total of 8,032,664 bp, 7,452 predicted coding sequences, and a G+C content of 72.3%.

Plasmid pSLL of Streptomyces laurentii ATCC31255 (wild-type strain P0) is a 93-kilobase linear DNA plasmid that carries a protein bound to each 5' end of the DNA. It was self-transmitted to the pSLL-cured strain by conjugation in solid culture. The pSLL-cured strain carried a circular plasmid, pSLS, and showed a marked decrease in spore formation and thiostrepton productivity, owing to the pSLS. However, by retransmission of pSLL, these things reverted to levels seen in strain P0. Thus, plasmid pSLL suppressed the injurious effects of pSLS on the host mycelia.

Specifically 13C-labeled quinoline-2-carboxylate derivatives were synthesized from quinoline and used to study the biosynthesis of thiostrepton in a strain of Streptomyces laurentii. 13C NMR analysis of thiostrepton recovered after feeding methyl (RS)-[11-13C]-4-(1-hydroxyethyl)quinoline-2-carboxylate or methyl [11-13C]-4-acetylquinoline-2-carboxylate showed conclusively that these compounds are specifically and efficiently incorporated into thiostrepton. Both compounds were also detected in cultures of the producing organism by isotope dilution analysis. The significance of the relative endogenous concentrations of the two compounds and of the relative extent of the incorporation of exogenously added labeled material into thiostrepton are discussed in terms of the biosynthetic pathway linking tryptophan and 4-(1-hydroxyethyl)quinoline-2-carboxylate in S. laurentii. A highly specific enzyme activity was detected in cell-free extracts of S. laurentii that was capable of adenylating (12S)-4-(1-hydroxyethyl)quinoline-2-carboxylic acid. Partial purification of the enzyme was achieved. The enzyme was found to be specific for the enantiomer of the substrate which has the same absolute configuration as found in the natural antibiotic structure. The presence of one specific enzyme catalysing the adenylation process in S. laurentii was shown by photoaffinity labeling with [alpha-32P]-8-azido-ATP and subsequent SDS PAGE analysis of the labeled products. The native molecular weight of the active enzyme, determined by gel permeation chromatography, was found to be approximately 47 kDa, compared with a denatured weight of 50 kDa estimated for the photoaffinity-labeled protein. The enzyme is thus probably monomeric.

A common approach to identify and clone biosynthetic gene from an antibiotic-producing streptomycete is to clone the resistance gene for the antibiotic of interest and then use that gene to clone DNA that is linked to it. As a first step toward cloning the genes responsible for the biosynthesis of thiostrepton (Th) in Streptomyces laurentii (Sl), the Th resistance-encoding gene (tsnR) was cloned as a 1.5-kb BamHI-PvuII fragment in Escherichia coli (Ec), and shown to confer Th resistance when introduced into S. lividans TK24. The tsnR-containing DNA fragment was used as a probe to isolate clones from cosmid libraries of DNA in the Ec cosmid vector SuperCos, and pOJ446 (an Ec/streptomycete) cosmid vector. Sequence and genetic analysis of the DNA flanking the tsnR indicates that the Sl tsnR is not closely linked to biosynthetic genes. Instead it is located within a cluster of ribosomal protein operons.

A large (80 kbp) plasmid designated as pSLL was isolated from Streptomyces laurentii ATCC 31255 (strain P0). Another covalently closed circular plasmid, pSLS (16 kbp), was found in pSLL-cured PO cells (named PI), which spontaneously formed pocks on a solid medium. The frequency of the generation of pSLL-loss PI cells coordinated positively with the frequency of pock appearance. pSLS was also found as an integrated form in the chromosome of strains PO and Pl. The results indicated that strain PO harbored two plasmids, pSLL and integrated form pSLS, and pSLS seemed to be generated as a free-form plasmid with the loss of pSLL. It was also suggested that pSLL may suppress the excision of the integrated pSLS from the chromosome and that the free form of pSLS may participate in pock formation.

The biosynthesis of the thiopeptide antibiotic thiostrepton (1) has been investigated by administration of isotopically labeled precursors to cultures of Streptomyces azureus and Streptomyces laurentii. The amino acid origin of all the components of the antibiotic was demonstrated. Experiments with (S)-[1,2- 13 C 2 ]- and (S)-[2,3- 13 C 2 ]serine showed intact incorporation of serine into the thiazoline and thiazole rings as well as the dehydroalanine, alanine, and tetrahydropyridine moieties. (S)-[3- 13 C, 2 H 2 ]Serine and (2S,3S)-[3- 13 C, 2 H 1 ]serine were used to elucidate the stereochemistry of the various transformations of serine

Thiopeptide antibiotics are a group of highly modified peptide metabolites. The defining scaffold for the thiopeptides is a macrocycle containing a dehydropiperidine or pyridine ring, dehydrated amino acids, and multiple thiazole or oxazole rings. Some members of the thiopeptides, such as thiostrepton, also contain either a quinaldic acid or indolic acid substituent derived from tryptophan. Although the amino acid precursors of these metabolites are well-established, the biogenesis of these complex peptides has remained elusive. Whole-genome scanning of Streptomyces laurentii permitted identification of a thiostrepton prepeptide, TsrA, and involvement of TsrA in thiostrepton biosynthesis was confirmed by mutagenesis. A gene cluster responsible for thiostrepton biosynthesis is reported, and the encoded gene products are discussed. The disruption of a gene encoding an amidotransferase, tsrT, led to the loss of thiostrepton production and the detection of a new metabolite, contributing further support to the identification of the tsr cluster. The tsr locus also appears to possess the gene products needed to convert tryptophan to the quinaldic acid moiety, and an aminotransferase was found to catalyze an early step in this pathway. This work establishes that the thiopeptides are a type of bacteriocin, a family of genetically encoded antimicrobial peptides, and are subjected to extensive posttranslational modification during maturation of the prepeptide.

Publisher Summary This chapter reviews thiopeptides. Thiostrepton is produced in low quantities by Streptomyces azureus ATCC 14921 and Streptomyces hawaiiensis ATCC 12236, and overproduced by Streptomyces laurentii ATCC 31255; it is used as a topical veterinary antibiotic and as a selective agent in recombinant DNA experiments with streptomycetes. Nosiheptide is produced by Streptomyces actuosus ATCC 25421, Streptomyces sp. ATCC 31463, and Streptomyces glaucogriseus NRRL LL-BP189; it is used as an animal feed additive to promote weight gain. Thiostrepton inhibits protein synthesis by binding to 23S ribosomal RNA (rRNA) and ribosomal protein L-l 1 to block the GTP hydrolysis activities of the 50S ribosomal subunit. The thiopeptide antibiotics also interfere with synthesis of the stringent response factor ppGpp, and expression of the thiostrepton resistance gene prevents thiostrepton from inhibiting ppGpp formation. Thiostrepton resistance is conferred by a methylase that specifically methylates adenosine-1067 of 23S rRNA to produce 2'-0-methyladenosine; thiostrepton does not bind 23S rRNA methylated in this position. The mechanisms for nosiheptide activity and resistance appear to be the same as for thiostrepton.

l -2-Methyltryptophan was found to be an intermediate in the biosynthesis of the antibiotic thiostrepton. It was isolated from growing cultures and resting cells of Streptomyces laurentii in trapping experiments after the application of labeled l -methionine or l -tryptophan. Its formation from l -tryptophan and S-adenosylmethionine was studied in a cell-free extract of S. laurentii. Although several attempts to purify the soluble methyltransferase by standard methods failed, some of its characteristics could be determined in the crude extract. The enzyme has a sharp pH optimum at pH 7.8. The apparentKm value for S-adenosylmethionine is 120 μ m and the Ki value for S-adenosylhomocysteine is 480 μ m . The enzyme is not stereoselective with respect to d - or l -tryptophan, but the d -isomer is converted at a slower rate than the l -isomer. Indolepyruvic acid is also methylated, while indole is not a substrate. The methyl group is transferred with retention of its configuration, contrary to most other methyltransferase reactions.

Highly specific enzymes which activate 3,4-dimethylindole-2-carboxylate (DMI) and 4-(1-hydroxyethyl)quinoline-2-carboxylate (HEQ) to their acyl adenylates have been detected in cell-free extracts of the nosiheptide and thiostrepton producers, S. actuosus and S. Iaurentii, by an ATP/PPi, exchange assay.

The effect of ammonium ions in the medium on production of thiostrepton byStreptomyces laurentii was investigated. In batch cultures, the excessive initial concentration of ammonium ions inhibited thiostrepton production. Moderate feeding of ammonia accelerated, however, not only microbial growth but also thiostrepton production. Fed-batch cultures with various feed rates of ammonia and a kinetic study clarified the effect of ammonium ion consumption rate on thiostrepton production. A modified kinetic model is proposed that takes product inhibition and the influence of maximum thiostrepton content into account.

The taxonomic description of Streptomyces laurentii, a new species related to but distinguishable from the S. fradiae group, is presented. This new species produces thiostrepton but bears no taxonomic relationship to the known producers of the antibiotic: S. azureus, S. hawaiiensis, and Streptomyces X-14b.

High concentration production of an antibiotic, thiostrepton, was achieved by the fed-batch culture of Streptomyces laurentii. To produce thiostrepton efficiently, the pH of the medium had to be maintained in a very narrow range between 6.0 and 6.2. As the substrates, not only glucose but also natural nutrients such as defatted soy bean meal and corn steep liquor were demanded. All of these substrates (multi-substrate) had to be supplied during the cultivation. pH was used as the indicator to detect the deficiency of substrates. When the glucose in the medium had been exhaustively consumed, the pH increased immediately; the multi-substrate solution (MS-solution) was then supplied. The composition of the feeding solution was determined from data obtained in batch cultures. During the fed-batch culture, glucose concentration was kept lower than 5 g/l by regulating the feed amount of MS-solution with this pH-stat modal control. By this control strategy a high concentration of thiostrepton, 10.5 g/l, was obtained, while total cell mass concentration reached 157 g/l. The productivity of thiostrepton was greatly increased compared with the conventional batch culture.