Search

Rhizosphere microorganisms play important ecological roles in promoting herb growth and producing abundant secondary metabolites. Studies on the rhizosphere microbes of traditional Chinese medicines (TCMs) are limited, especially on the genomic and metabolic levels. In this study, we reported the isolation and characterization of a Steptomyces netropsis WLXQSS-4 strain from the rhizospheric soil of Clematis manshurica Rupr. Genomic sequencing revealed an impressive total of 40 predicted biosynthetic gene clusters (BGCs), whereas metabolomic profiling revealed 13 secondary metabolites under current laboratory conditions. Particularly, medium screening activated the production of alloaureothin, whereas brominated and chlorinated pimprinine derivatives were identified through precursor-directed feeding. Moreover, antiproliferative activities against Hela and A549 cancer cell lines were observed for five compounds, of which two also elicited potent growth inhibition in Enterococcus faecalis and Staphylococcus aureus , respectively. Our results demonstrated the robust secondary metabolism of S. netropsis WLXQSS-4, which may serve as a biocontrol agent upon further investigation.

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

A novel marine actinomycete strain designated ICN19 T was isolated from the subtidal sediment of Chinnamuttam coast of Kanyakumari, India and subjected to polyphasic taxonomic analysis. Neighbour-joining tree based on 16S rRNA gene sequences of validly described type strains had revealed the strain ICN19 T formed distinct cluster with Streptomyces wuyuanensis CGMCC 4.7042 T , Streptomyces tirandamycinicus HNM0039 T and Streptomyces spongiicola HNM0071 T . Morphological, physiological and chemotaxonomic characteristics were consistent with those of members of the genus Streptomyces. The strain possessed LL-diaminopimelic acid as the diagnostic diamino acid. The predominant isoprenoid quinone was identified as MK-9(H8) (70%), MK-9(H6) (20%) and MK-9(H2) (2%), with the major cellular fatty acids (>10%) being anteiso-C 15:0 , C 16:0 and iso-C 16:0 . The main polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides and three unidentified phospholipids. The dendrogram generated on the basis of MALDI-TOF mass spectra supports the strain differentiated from its neigbours. The genome sequence of strain ICN19 T was 9,010,366 bp in size with a total of 7420 protein-coding genes and 98 RNA genes. The genomic G+C content of the novel strain was 71.27 mol%. The DNA-DNA relatedness between strain ICN19 T and the reference strains with S. wuyuanensis CGMCC 4.7042 T , S. tirandamycinicus HNM0039 T and S. spongiicola HNM0071 T were 42.8%, 39.5% and 38%, respectively. Based on differences in physiological, biochemical, chemotaxonomic differences and whole-genome characteristics the isolated strain represents a novel species of the genus Streptomyces, for which the name Streptomyces marianii sp. nov. is proposed. Type strain is ICN19 T (=MCC 3599 T  = KCTC 39749 T ).

Zincphyrin IV is a potential organic photosensitizer which is of significant interest for applications in biomedicine, materials science, agriculture (as insecticide), and chemistry. Most studies on Zincphyrin are focused on Zincphyrin III while biosynthesis and application of Zincphyrin IV is comparatively less explored. In this study, we explored Zincphyrin IV production in Streptomyces venezuelae ATCC 15439 through combination of morphology engineering and "One strain many compounds" approach. The morphology engineering followed by change in culture medium led to activation of cryptic Zincphyrin IV biosynthetic pathway in S. venezuelae with subsequent detection of Zincphyrin IV. Morphology engineering applied in S. venezuelae increased the biomass from 7.17 to 10.5 mg/mL after 48 h of culture. Moreover, morphology of engineered strain examined by SEM showed reduced branching and fragmentation of mycelia. The distinct change in color of culture broth visually demonstrated the activation of the cryptic biosynthetic pathway in S. venezuelae. The production of Zincphyrin IV was found to be initiated after overexpression ssgA, resulting in the increase in titer from 4.21 to 7.54 μg/mL. Furthermore, Zincphyrin IV demonstrated photodynamic antibacterial activity against Bacillus subtilis and photodynamic anticancer activity against human ovarian carcinoma cell lines.

Streptomycetes are filamentous bacteria that differentiate by producing spore-bearing reproductive structures called aerial hyphae. The transition from vegetative to reproductive growth is controlled by the bld (bald) loci, and mutations in bld genes prevent the formation of aerial hyphae, either by blocking entry into development (typically mutations in activators) or by inducing precocious sporulation in the vegetative mycelium (typically mutations in repressors). One of the bld genes, bldC , encodes a 68-residue DNA-binding protein related to the DNA-binding domain of MerR-family transcription factors. Recent work has shown that BldC binds DNA by a novel mechanism, but there is less insight into its impact on Streptomyces development. Here we used ChIP-seq coupled with RNA-seq to define the BldC regulon in the model species Streptomyces venezuelae , showing that BldC can function both as a repressor and as an activator of transcription. Using electron microscopy and time-lapse imaging, we show that bldC mutants are bald because they initiate development prematurely, bypassing the formation of aerial hyphae. This is consistent with the premature expression of BldC target genes encoding proteins with key roles in development (e.g., whiD , whiI , sigF ), chromosome condensation and segregation (e.g., smeA-sffA , hupS ), and sporulation-specific cell division (e.g., dynAB ), suggesting that BldC-mediated repression is critical to maintain a sustained period of vegetative growth prior to sporulation. We discuss the possible significance of BldC as an evolutionary link between MerR family transcription factors and DNA architectural proteins. IMPORTANCE Understanding the mechanisms that drive bacterial morphogenesis depends on the dissection of the regulatory networks that underpin the cell biological processes involved. Recently, Streptomyces venezuelae has emerged as an attractive model system for the study of morphological differentiation in Streptomyces This has led to significant progress in identifying the genes controlled by the transcription factors that regulate aerial mycelium formation (Bld regulators) and sporulation (Whi regulators). Taking advantage of S. venezuelae , we used ChIP-seq coupled with RNA-seq to identify the genes directly under the control of BldC. Because S. venezuelae sporulates in liquid culture, the complete spore-to-spore life cycle can be examined using time-lapse microscopy, and we applied this technique to the bldC mutant. These combined approaches reveal BldC to be a member of an emerging class of Bld regulators that function principally to repress key sporulation genes, thereby extending vegetative growth and blocking the onset of morphological differentiation., (Copyright © 2019 Bush et al.)

A novel actinobacterial strain, designated S10 T , was isolated from a sand sample collected from the Qaidam Basin in Qinghai province, China. The strain S10 T exhibited antibacterial activity against MRSA. The taxonomic position of the strain S10 T was determined by a polyphasic approach. There were six copies of 16S rDNA in S10 T which were not same exactly (MH257693-MH257698). Phylogenetic analysis of 16S rRNA gene sequences indicated the strain belonging to the genus Streptomyces and it showed high sequence similarities with Streptomyces chartreusis NBRC 12753 T (99.31%), Streptomyces phaeoluteigriseus DSM 41896 T (99.24%), Streptomyces variegatus NRRL B-16380 T (99.17%) and Streptomyces flavovariabilis NRRL B-16367 T (99.17%) comparing with MH257693, MH257695, MH257696, MH257697, and MH257698. Similarities with Streptomyces kunmingensis NBRC14463 T (98.82%), Streptomyces bungoensis DSM 41781 T (98.76%), S. chartreusis NBRC 12753 T (98.69%) and S. phaeoluteigriseus DSM 41896 T (98.62%) with MH257694. Whole-genome average nucleotide identity (ANI) values between strain S10 T and S. chartreusis NBRC 12753 T , S. phaeoluteigriseus DSM 41896 T , S. variegatus NRRL B-16380T, S. flavovariabilis NRRL B-16367 T , S. kunmingensis NBRC 14463 T , S. bungoensis DSM 41781 T were 83.63%, 82.89%, 92.55%, 92.51%, 79.29, and 82.87%, respectively, suggesting that the strain S10 T represented a new species. A phylogenetic analysis comparing the S10 T genome with those of 336 other sequenced Streptomyces genomes confirmed its relatedness with Streptomyces variegatus NRRL B-16380 T and Streptomyces flavovariabilis NRRL B-16367 T . Strain S10 T contained LL-diaminopimelic acid in the cell wall. The predominant menaquinones were MK-9(H 6 ) and MK-9(H 8 ) and the major fatty acids were iso-C 15:0, anteiso-C 15:0 , iso-C 16:0 , and anteiso-C 17:0 . Phospholipids detected were diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline, three unknown phospholipids, an unknown aminophospholipid and an unknown phosphatidyl glycolipid. On the basis of these genotypic and phenotypic data, it is proposed that isolate S10 T (=JCM 31184 T =CGMCC 4.7315 T ) should be classified in the genus Streptomyces as Streptomyces qaidamensis sp. nov.

AdpA is studied and considered as a pleiotropic regulator which is involved in morphological development and secondary metabolism in many Streptomyces. In this study, AdpA sd, which was cloned from toyocamycin (TM)-producing strain Streptomyces diastatochromogenes 1628, was identified as an ortholog of AdpA and belongs to a large subfamily of the AraC/XylS family. In order to elucidate the correlation of AdpA sd with TM biosynthesis and morphological differentiation, adpA sd was placed under the control of the ermE* promoter in plasmid pIB139. By intergeneric conjugation, the resulting plasmid pIB139-adpA sd was introduced into mutant S. diastatochromogenes 1628-T62 that is defective in sporulation and had limited TM production as well as transcriptional level of gene adpA sd , yielding the recombinant strain S. diastatochromogenes 1628-T62A. As expected, due to over-expression of adpA sd , the S. diastatochromogenes 1628-T62A restored spore formation to a certain extent compared with control strain S. diastatochromogenes 1628-T62. Moreover, compared with control strain 1628-T62, the TM production of recombinant 1628-T62A was increased by 120.1% on 5 l fermenter. In addition, by using semi-quantitative reverse transcription-PCR analysis, we discovered that the transcriptional levels of gene adpA sd and the all toy genes involved in TM biosynthesis were elevated in recombinant 1628-T62A compared with S. diastatochromogenes 1628-T62. These results confirm that cloned adpA sd plays a positive role in TM biosynthesis and morphological differentiation.

Two novel Gram-stain positive, spore-forming, aerobic actinomycetes, designated NEAU-PCY-1 T and NEAU-PCY-2, were isolated from rhizosphere soil of Urtica urens L. collected from Anshan, Liaoning Province, northeast China. The 16S rRNA gene sequence analysis showed that strains NEAU-PCY-1 T and NEAU-PCY-2 exhibited 99.8% similarity with each other and are closely related to Streptomyces abietis DSM 42080 T (98.2, 98.3%) and Streptomyces fildesensis DSM 41987 T (98.0, 98.1%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the two strains formed a cluster with these two closely related species. Moreover, DNA-DNA hybridization results and some phenotypic, physiological and biochemical properties differentiated the two strains from their close relatives in the genus Streptomyces. Based on a polyphasic taxonomy study, strains NEAU-PCY-1 T and NEAU-PCY-2 are considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces urticae sp. nov. is proposed, with NEAU-PCY-1 T (= DSM 105115 T  = CCTCC AA 2017015 T ) as the type strain.

Actinobacteria are prolific producers of secondary metabolites and industrially relevant enzymes. Growth of these mycelial micro-organisms in small culture volumes is challenging due to their complex morphology. Since morphology and production are typically linked, scaling down culture volumes requires better control over morphogenesis. In larger scale platforms, ranging from shake flasks to bioreactors, the hydrodynamics play an important role in shaping the morphology and determining product formation. Here, we report on the effects of agitation on the mycelial morphology of Streptomyces lividans grown in microtitre plates. Our work shows that at the appropriate agitation rates cultures can be scaled down to volumes as small as 100 µl while maintaining the same morphology as seen in larger scale platforms. Using image analysis and principal component analysis we compared the morphologies of the cultures; when agitated at 1400-1600 rpm the mycelial morphology in micro-cultures was similar to that obtained in shake flasks, while product formation was also maintained. Our study shows that the morphology of actinobacteria in micro-cultures can be controlled in a similar manner as in larger scale cultures by carefully controlling the mixing rate. This could facilitate high-throughput screening and upscaling.

In the present study different actinomycete strains were collected and isolated from Algerian Sahara soil with the aim to select novel enzymes with promising features for biotechnological applications. The Ms1 strain was selected, amongst the others, for its capability to produce melanin in different solid media. Ms1 chromosomal DNA was sequenced and the strain assigned to Streptomyces cyaneofuscatus sp. A tyrosinase (MW∼30kD) encoding sequence was identified and the corresponding enzyme was isolated and biochemically characterized. The tyrosinase showed the highest activity and stability at neutral and alkaline pH and it was able to oxidize l-DOPA at T=55°C and pH 7. The enzyme showed variable stability in presence of various water-miscible organic solvents, while it was inactivated by reducing agents. The tyrosinase activity was unaffected by NaCl and enhanced by different cations. Furthermore, the enzyme showed a higher specificity for diphenols than monophenols showing a higher diphenolase than monophenolase activity. Finally, tyrosinase was stabilized by immobilization on nylon nanofiber membranes with a payload of 82% when 1% glutaraldeyde was used. Taken all together, these results show that the enzyme displays interesting properties for biotechnological purposes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Rubber materials have greatly contributed to human civilization. However, being a polymeric material does not decompose easily, it has caused huge environmental problems. On the other hand, only few bacteria are known to degrade rubber, with studies pertaining them being intensively focusing on the mechanism involved in microbial rubber degradation. The Streptomyces sp. strain CFMR 7, which was previously confirmed to possess rubber-degrading ability, was subjected to whole genome sequencing using the single molecule sequencing technology of the PacBio® RS II system. The genome was further analyzed and compared with previously reported rubber-degrading bacteria in order to identify the potential genes involved in rubber degradation. This led to the interesting discovery of three homologues of latex-clearing protein (Lcp) on the chromosome of this strain, which are probably responsible for rubber degrading activities. Genes encoding oxidoreductase α-subunit (oxiA) and oxidoreductase β-subunit (oxiB) were also found downstream of two lcp genes which are located adjacent to each other. In silico analysis reveals genes that have been identified to be involved in the microbial degradation of rubber in the Streptomyces sp. strain CFMR 7. This is the first whole genome sequence of a clear-zone-forming natural rubber- degrading Streptomyces sp., which harbours three Lcp homologous genes with the presence of oxiA and oxiB genes compared to the previously reported Gordonia polyisoprenivorans strain VH2 (with two Lcp homologous genes) and Nocardia nova SH22a (with only one Lcp gene)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present study the leishmanicidal effect of potential protease inhibitor producing marine actinobacterial isolate has been investigated against Leishmania donovani, the causative agent of visceral leishmaniasis. Among 89 marine actinobacteria isolated from a salt pan in Kanyakumari, only one isolate (BVK2) showed 97% of protease inhibition activity against trypsin. Moderate to high protease inhibitor activity was shown by isolate BVK2 on proteinase (30%) and chymotrypsin (85%). In optimization study for protease inhibitor production glucose as carbon source and casein as nitrogen source showed the best activity. In the in-vitro Fluorescence-activated cell sorting (FACS) assay, 100 μg/ml of BVK2 extract was active against amastigotes in infected J774A.1 macrophages and showed 87% of parasitic inhibition. The isolate BVK2 showed significant anti-parasitic activity with an IC 50 of 27.1 μg/ml after double doses were administered. The potential isolate was identified by molecular 16S rRNA gene sequencing as Streptomyces sp. VITBVK2. The results obtained suggest that the marine actinobacterial extract which have novel metabolites can be considered as a potential source for the development of drugs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Efforts to construct the Streptomyces host strain with enhanced yields of heterologous product have focussed mostly on engineering of primary metabolism and/or the deletion of endogenous biosynthetic gene clusters. However, other factors, such as chromosome compactization, have been shown to have a significant influence on gene expression levels in bacteria and fungi. The expression of genes and biosynthetic gene clusters may vary significantly depending on their location within the chromosome. Little is known about the position effect in actinomycetes, which are important producers of various industrially relevant bioactive molecules., Results: To demonstrate an impact of the chromosomal position effect on the heterologous expression of genes and gene clusters in Streptomyces albus J1074, a transposon mutant library with randomly distributed transposon that includes a β-glucuronidase reporter gene was generated. Reporter gene expression levels have been shown to depend on the position on the chromosome. Using a combination of the transposon system and a φC31-based vector, the aranciamycin biosynthetic cluster was introduced randomly into the S. albus genome. The production levels of aranciamycin varied up to eightfold depending on the location of the gene cluster within the chromosome of S. albus J1074. One of the isolated mutant strains with an artificially introduced attachment site produced approximately 50% more aranciamycin than strains with endogenous attBs., Conclusions: In this study, we demonstrate that expression of the reporter gene and aranciamycin biosynthetic cluster in Streptomyces albus J1074 varies up to eightfold depending on its position on the chromosome. The integration of the heterologous cluster into different locations on the chromosome may significantly influence the titre of the produced substance. This knowledge can be used for the more efficient engineering of Actinobacteria via the relocation of the biosynthetic gene clusters and insertion of additional copies of heterologous constructs in a suitable chromosomal position.

L-asparaginase is an important enzyme as therapeutic agents used in combination with other drugs in the treatment of acute lymphoblastic leukemia. A newly isolated actinomycetes strain, Streptomyces sp. NEAE-82, was potentially producing extracellular L-asparaginase, it was identified as Streptomyces fradiae NEAE-82, sequencing product was deposited in the GenBank database under accession number KJ467538. L-asparaginase was purified from the crude enzyme using ammonium sulfate precipitation, dialysis and ion exchange chromatography using DEAE Sepharose CL-6B. Further the kinetic studies of purified enzyme were carried out. The optimum pH, temperature and incubation time for maximum L-asparaginase activity were found to be 8.5, 40 °C and 30 min, respectively. The optimum substrate concentration was found to be 0.06 M. The Km and Vmax of the enzyme were 0.01007 M and 95.08 Uml(-1)min(-1), respectively. The half-life time (T1/2) was 184.91 min at 50 °С, while being 179.53 min at 60 °С. The molecular weight of the subunits of L-asparaginase was found to be approximately 53 kDa by SDS-PAGE analysis. The purified L-asparaginase showed a final specific activity of 30.636 U/mg protein and was purified 3.338-fold. The present work for the first time reported more information in the production, purification and characterization of L-asparaginase produced by newly isolated actinomycetes Streptomyces fradiae NEAE-82.

In this research, a microbial endophytic strain obtained from the rhizosphere of the conifer Taxus baccata and designated as Streptomyces sp. AC35 (FJ001754.1 Streptomyces, GenBank) was investigated. High 16S rDNA gene sequence similarity suggests that this strain is closely related to S. odorifer. The major fatty acid profile of intracellular lipids was also carried out to further identify this strain. Atomic force microscopy and scanning acoustic microscopy were used to image our strain. Its major excreted substances were extracted, evaluated for antimicrobial activity, purified, and identified by ultraviolet-visible spectroscopy (UV-vis), liquid chromatography-mass spectrometry (LC-MS/MS) and nuclear magnetic resonance as the bioactive isoflavone aglycones-daidzein, glycitein and genistein. Batch cultivation, performed under different pH conditions, revealed enhanced production of antimycin components when the pH was stable at 7.0. Antimycins were detected by HPLC and identified by UV-vis and LC-MS/MS combined with the multiple reaction monitoring. Our results demonstrate that Streptomyces sp. AC35 might be used as a potential source of effective, pharmaceutically active compounds.

Streptomyces lunalinharesii strain 235 produces an antimicrobial substance that is active against sulfate reducing bacteria, the major bacterial group responsible for biofilm formation and biocorrosion in petroleum reservoirs. The use of this antimicrobial substance for sulfate reducing bacteria control is therefore a promising alternative to chemical biocides. In this study the antimicrobial substance did not interfere with the biofilm stability, but the sulfate reducing bacteria biofilm formation was six-fold smaller in carbon steel coupons treated with the antimicrobial substance when compared to the untreated control. A reduction in the most probable number counts of planktonic cells of sulfate reducing bacteria was observed after treatments with the sub-minimal inhibitory concentration, minimal inhibitory concentration, and supra-minimal inhibitory concentration of the antimicrobial substance. Additionally, when the treated coupons were analyzed by scanning electron microscopy, the biofilm formation was found to be substantially reduced when the supra-minimal inhibitory concentration of the antimicrobial substance was used. The coupons used for the biofilm formation had a small weight loss after antimicrobial substance treatment, but corrosion damage was not observed by scanning electron microscopy. The absence of the dsrA gene fragment in the scraped cell suspension after treatment with the supra-minimal inhibitory concentration of the antimicrobial substance suggests that Desulfovibrio alaskensis was not able to adhere to the coupons. This is the first report on an antimicrobial substance produced by Streptomyces active against sulfate reducing bacteria biofilm formation. The application of antimicrobial substance as a potential biocide for sulfate reducing bacteria growth control could be of great interest to the petroleum industry., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Streptomycetes are filamentous bacteria that produce numerous valuable compounds, including the majority of clinically used antibiotics. At an industrial scale, most of these compounds are produced in bioreactors. Growth of streptomycetes under these conditions is characterized by the formation of complex mycelial particles, whose sizes follow a bimodal distribution. Given the correlation between specific productivity and morphology, this size heterogeneity poses a potential drawback in industry. Recent work indicates that mycelial morphology is controlled by a number of genes that encode proteins required for the synthesis of cell surface-associated glycans. Using a quantifiable system based on fluorescent markers, we here show that these glycans mediate aggregation between germlings and young mycelia, yielding mycelial particles that originate from many different individuals. We also demonstrate that at later time points aggregation between distinct particles is no longer detectable. Notably, the absence of the corresponding glycan synthases yields mycelia that are homogeneous in size, identifying mycelial aggregation as a driving factor towards size heterogeneity. Given that aggregation is widespread within streptomycetes and can also occur between different Streptomyces strains, our work paves the way to improve Streptomyces as a cell factory for the production of known metabolites, but possibly also to discover new ones.

Bacterial cell division is a highly complex process that requires tight coordination between septum formation and chromosome replication and segregation. In bacteria that divide by binary fission a single septum is formed at mid-cell, a process that is coordinated by the conserved cell division scaffold protein FtsZ. In contrast, during sporulation-specific cell division in streptomycetes, up to a hundred rings of FtsZ (Z rings) are produced almost simultaneously, dividing the multinucleoid aerial hyphae into long chains of unigenomic spores. This involves the active recruitment of FtsZ by the SsgB protein, and at the same time requires sophisticated systems to regulate chromosome dynamics. Here, we show that SepG is required for the onset of sporulation and acts by ensuring that SsgB is localized to future septum sites. Förster resonance energy transfer imaging suggests direct interaction between SepG and SsgB. The beta-lactamase reporter system showed that SepG is a transmembrane protein with its central domain oriented towards the cytoplasm. Without SepG, SsgB fails to localize properly, consistent with a crucial role for SepG in the membrane localization of the SsgB-FtsZ complex. While SsgB remains associated with FtsZ, SepG re-localizes to the (pre)spore periphery. Expanded doughnut-shaped nucleoids are formed in sepG null mutants, suggesting that SepG is required for nucleoid compaction. Taken together, our work shows that SepG, encoded by one of the last genes in the conserved dcw cluster of cell division and cell-wall-related genes in Gram-positive bacteria whose function was still largely unresolved,coordinates septum synthesis and chromosome organization in Streptomyces., (© 2016 The Authors.)

Background: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds., Results: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemia salina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus., Conclusions: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells. Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Currently, there is an ever-increasing need to develop environmentally benign processes in place of synthetic protocols. As a result, researchers in the field of nanoparticle synthesis are focusing their attention on microbes from rare biological ecosystems. One potential actinobacterium, Streptomyces minutiscleroticus M10A62 isolated from a magnesite mine had the ability to synthesize selenium nanoparticles (SeNPs), extracellularly. Actinobacteria mediated SeNP synthesis were characterized by UV-visible, Fourier transform infrared (FT-IR), X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX) and high resolution transmission electron microscopy (HR-TEM) analysis. The UV-spectral analysis of SeNPs indicated the maximum absorption at 510nm, FT-IR spectral analysis confirms the presence of capping protein, peptide, amine and amide groups. The selenium signals confirm the presence of SeNPs. All the diffraction peaks in the XRD pattern and HR-TEM confirm the size of SeNPs in the range of 10-250nm. Further, the anti-biofilm and antioxidant activity of the SeNPs increased proportionally with rise in concentration, and the test strains reduced to 75% at concentration of 3.2μg. Selenium showed significant anti-proliferative activity against HeLa and HepG2 cell lines. The wound healing activity of SeNPs reveals that 5% selenium oinment heals the excision wound of Wistar rats up to 85% within 18 days compared to the standard ointment. The biosynthesized SeNPs exhibited good antiviral activity against Dengue virus. The present study concludes that extremophilic actinobacterial strain was a novel source for SeNPs with versatile biomedical applications and larger studies are needed to quantify these observed effects of SeNPs., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Actinomycete strains OM-6519(T) and K04-0144(T) produce the bioactive compounds lactacystin and cyslabdan, respectively. Here, the taxonomic positions of these two strains were determined. The morphological and chemical features of strains OM-6519(T) and K04-0144(T) indicated that they belonged to the genus Streptomyces. Strain OM-6519(T) showed the highest 16S rRNA gene sequence similarities with Streptomyces xanthocidicus NBRC 13469(T) (99.7%), Streptomyces chrysomallus subsp. fumigatus NBRC 15394(T) (99.6%) and Streptomyces aburaviensis NRRL B-2218(T) (99.5%). However, the DNA-DNA relatedness values between strain OM-6519(T) and the three related strains were below 70%. Strain K04-0144(T) showed the highest 16S rRNA gene sequence similarities with Streptomyces corchorusii NBRC 13032(T) (99.4%), Streptomyces olivaceoviridis NBRC 15394(T) (99.4%) and Streptomyces canarius NRRL B-2218(T) (99.3%). However, the DNA-DNA relatedness values between strain K04-0144(T) and the three related strains were also below 70%. Based on morphological, cultural and physiological characteristics and DNA-DNA relatedness data, strains OM-6519(T) and K04-0144(T) should be classified as new species of the genus Streptomyces, for which the names Streptomyces lactacystinicus sp. nov. and Streptomyces cyslabdanicus sp. nov. are proposed. The type strain of S. lactacystinicus is OM-6519(T) (=NBRC 110082(T), DSM 43136(T)). The type strain of S. cyslabdanicus is K04-0144(T) (=NBRC 110081(T), DSM 42135(T)).

From an agricultural soil that had received continuous applications of organophosphorus pesticides, 30 actinobacteria strains were isolated. Two strains, identified as Streptomyces sp. AC1-6 and Streptomyces sp. ISP4, were selected because of their tolerance to diazinon and based on the relationship between diazinon removal and microbial growth. In liquid medium with diazinon at concentrations of 25 and 50 mg L(-1), both strains were able to remove approximately 40-50% and 70-90% of the initial diazinon after 24 and 96 h of incubation, respectively. This diazinon removal was accompanied by microbial growth of the strains, an initial pH decrease, and glucose consumption in the liquid medium. Evaluation of the diazinon removal achieved by the free actinobacteria and Streptomyces sp. AC1-6 immobilized on alginate beads revealed that the immobilized cells exhibited a 60% higher diazinon removal compared with the free cells. The reusability of the encapsulated biomass was confirmed, and a diazinon removal rate of more than 50% was obtained after the second batch. This work constitutes one of the few reports that describe Streptomyces strains as diazinon degraders. Given the high diazinon removal found, the streptomycetes exhibit suitable potential as diazinon-degrading actinobacteria for elimination of diazinon from liquid residues., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

ϵ-Poly-L-lysine (ϵ-PL) is an L-lysine homopolymer with strong antimicrobial activity, which is generally produced by Streptomyces strains. ϵ-PL is only produced under acidic conditions in liquid culture, and to improve the current understanding of ϵ-PL biosynthesis, the present study was undertaken to investigate the effects of ϵ-PL on its producer Streptomyces ahygroscopicus GIM8, under acidic and neutral conditions. The results indicated that a neutral pH favored ϵ-PL adsorption onto the cells, whereas minimal adsorption occurred at pH 4.0, the maximum pH for ϵ-PL production. At pH 7.0, small amounts of ϵ-PL caused considerable ATP leakage from the cells, which showed increased membrane permeability. Conversely, ATP leakage was inhibited by ϵ-PL at pH 4.0. Transmission electron microscopy investigation indicated that the cytoplasmic membrane was the primary site of ϵ-PL activity at pH 7.0, and that cell shape was maintained. Metabolic activity profiles revealed that ϵ-PL decreased cellular metabolic activity at a relatively low rate at pH 7.0. However, the toxic effect was significantly enhanced at pH 4.0. Based on these data, a mechanism for the effect of ϵ-PL on ϵ-PL-producing cells under neutral and acidic conditions is proposed. Additionally, acidic conditions may potentially be required for ϵ-PL biosynthesis in liquid culture because low pH can increase membrane permeability and prevent binding of ϵ-PL onto cells, both of which favor the secretion of the ϵ-PL produced by the cells into the broth. This research contributes to the current understanding of ϵ-PL biosynthesis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cell division is essential for spore formation but not for viability in the filamentous streptomycetes bacteria. Failure to complete cell division instead blocks spore formation, a phenotype that can be visualized by the absence of gray (in Streptomyces coelicolor) and green (in Streptomyces venezuelae) spore-associated pigmentation. Despite the lack of essentiality, the streptomycetes divisome is similar to that of other prokaryotes. Therefore, the chemical inhibitors of sporulation in model streptomycetes may interfere with the cell division in rod-shaped bacteria as well. To test this, we investigated 196 compounds that inhibit sporulation in S. coelicolor. We show that 19 of these compounds cause filamentous growth in Bacillus subtilis, consistent with impaired cell division. One of the compounds is a DNA-damaging agent and inhibits cell division by activating the SOS response. The remaining 18 act independently of known stress responses and may therefore act on the divisome or on divisome positioning and stability. Three of the compounds (Fil-1, Fil-2, and Fil-3) confer distinct cell division defects on B. subtilis. They also block B. subtilis sporulation, which is mechanistically unrelated to the sporulation pathway of streptomycetes but is also dependent on the divisome. We discuss ways in which these differing phenotypes can be used in screens for cell division inhibitors., (© 2014 Society for Laboratory Automation and Screening.)

Production of cholesterol oxidase (COD) under batch conditions through Ca-alginate immobilized cells of Streptomyces sp. was investigated. The process was studied for optimal immobilization conditions, beads operational stability and comparisons were made with the COD production via free cells. Influence of Na-alginate concentration (1-5 g L(-1) ) and initial biomass loading on enzyme production were studied. Effects of initial pH of the production medium, temperature, shaker speed, as well as reuse of beads on the COD production were also investigated. It was observed that COD production with immobilized cells (5.6 U ml(-1) ) was higher in comparison to free cells (4.5 U ml(-1) ) under optimized conditions. The maximum COD production by free cells was observed with initial pH 7.0, rpm 200 after 96 h of incubation while immobilized cells sustain a broad pH range 6.0-9.0, rpm 300 for maximum production after 72 h. The immobilized and free cells produced maximum COD in the culture incubated at 37 and 30 °C, respectively. Other parameters bead size and Na-alginate concentration found to be optimum with 1.5 mm and 4% w/v, respectively. Scanning electron microscope study of the immobilized cells indicated that the cells in Ca-alginate beads remained in normal shape with no alterations in the morphology., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described., (© 2014 Elsevier Inc. All rights reserved.)

A novel actinobacterium MBRL 251(T), isolated from a sample collected from a limestone quarry at Hundung, Manipur, India, was characterized using a polyphasic approach. The 16S ribosomal RNA gene sequence of strain MBRL 251(T) showed closest similarities with Streptomyces xanthochromogenes NRRL B-5410(T) (99.6%) and Streptomyces michiganensis NBRC 12797(T) (99.6%). The DNA relatedness between MBRL 251(T) and S. xanthochromogenes NBRC 12828(T), and S. michiganensis NBRC 12797(T) was 46.6% and 40.7%, respectively. Strain MBRL 251(T) contained LL-diaminopimelic acid as the diagnostic diamino acid, with glucose and xylose as the main cell wall sugars, whereas small amounts of galactose, mannose, rhamnose and ribose were also detected in the whole-cell wall hydrolysates. The major fatty acids identified were anteiso-C15:0 (35.1%), iso-C16:0 (21.1%) and anteiso-C17:1 (13.2%). The predominant menaquinones detected were MK-9(H6) and MK-9(H8), whereas the polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositolmannosides. The G+C content of the genomic DNA was 72.3%. The phenotypic and genotypic data showed that strain MBRL 251(T) merits the recognition as a representative of a novel species of the genus Streptomyces. It is proposed that the isolate should be classified in the genus Streptomyces as a novel species, Streptomyces hundungensis sp. nov. The type strain is MBRL 251(T) (=JCM 17577(T)=KCTC 29124(T)).

The search for the effective and safe a-glucosidase and alpha-amylase inhibitors from Actinomycetaceae being antidiabetic agents is actual problem. Twenty one Streptomyces spp. of soil samples collected from different places of China were screened for the ability to produce this kind of inhibitory activities. Fermentation broth of isolated strains had absorbance between 350-190 nm. The Streptomyces strains PW003, ZG636, and ZG731 were characterized by special absorption at 280, 275, and 400 nm, respectively. Ten of the collected actinomycete strains had the ability to inhibit alpha-glucosidase or/and alpha-amylase and the fermentation broth of the same strain had inhibitory activity varied greatly depending on the enzyme source. In the process to screen the leading compounds used as antidiabetic agents, human alpha-glucosidase and alpha-amylase were revealed as the best used in trail compared with the same enzymes from other sources. Active alpha-glucosidase inhibitor was isolated from Streptomyces strain PW638 fermentation broth and identified as acarviostatin 103 by MS and N MR spectrometry. Its IC50 value was 1.25 and 12.23 microg/mL against human intestinal N-terminal maltase-glucoamylase and human pancreatic alpha-amylase, respectively.

Streptomyces mediolani sp. AC37 was isolated from the root system of higher plant Taxus baccata and produced metabolite identified as (-)-8-O-methyltetrangomycin according to LC/MS/MS analysis. In our screening program for improvements of bioactive secondary metabolites from plant associate streptomycetes, mutation was used as a tool for the induction of genetic variations for selection of higher (-)-8-O-methyltetrangomycin producers of isolates. S. mediolani sp. AC37 was treated with UV irradiation and chemical mutagenic treatment (N-nitroso-N-methyl-urea). The radical scavenging and antioxidant capacity of (-)-8-O-methyltetrangomycin and extracts isolated from mutants were tested using EPR spin trapping technique and ABTS(·+) assay. Comparison of electron microscopic images of Streptomyces sp. AC37 and mutant strains of Streptomyces sp. AC37 revealed substantial differences in morphology and ultrastructure.

Two new isochromans, panowamycins A and B, were purified by solvent extraction, silica gel and octadecylsilyl silica gel (ODS) column chromatography followed by preparative HPLC, from a culture broth of Streptomyces sp. K07-0010, together with the known compounds NFAT-133, conglobatin, piericidin C series and dinactin. Structures of panowamycins were elucidated as new analogs of NFAT-133 by spectroscopic studies including various NMR experiments. Panowamycins A and B showed moderate antitrypanosomal activity, with IC(50) values of 0.40 and 3.30 μg ml(-1), respectively.

A new, short, and quick method was developed for preparation of specimen for observing Actinomycetes of genus Streptomyces by scanning electron microscopy. The cultures were directly grown on stubs and coated with a film of gold without using any fixative and dehydrating procedures. Using this simple preparation procedure, surface of intact sporing structures of Streptomyces was observed over a range of magnifications. As the preparation procedure is so simple and rapid, this procedure could be most useful for the routine examination and identification of Streptomyces., (Copyright © 2011 Wiley Periodicals, Inc.)

The aims of this paper were to study the biofouling and biodeterioration of photos and maps stored at Historical Archive of the Museum of La Plata (HAMP), Argentine, and two repositories of the National Archive of Cuba Republic (NARC) and to carry out the physiological characterization of the isolated fungi and bacteria. The role of the environmental microbiota in the biofouling formation was also studied. Microbial assemblages in the air were sampled by sedimentation technique while those on documents were sampled by swabbering. Biofilm formation and biofouling were monitored by scanning electron microscope (SEM). Large microbial assemblages were found at NARC archives with the prevalence of genera Aspergillus, Cladosporium and Penicillium, whereas at HAMP these values were lower, Penicillium was the only fungal genus detected. Most of the fungi degraded cellulose and produced pigments and acids, and all of the isolated bacteria had proteolytic and/or cellulolytic activity. In all cases, a higher concentration of viable bacteria than of fungi was isolated from documents. These results correlated with bacterial values detected in air at NARC repositories. However, this correlation cannot be observed at HAMP where Aspergillus, Penicillium and Talaromyces helicus (teleomorph of Penicillium) were isolated. It is the first time that the last genus is reported in documents., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The biotransformation of digitoxin and some of its derivatives extracted from Digitalis lanata by Streptomyces isolated species was investigated. Cultures of a Streptomyces strain designated EUSA2003B, isolated from an Egyptian soil sample, efficiently induced selective 12beta-hydroxylation of the steroid aglycone of digitoxin (DT) and its alpha-acetyl and beta-methyl derivatives. The transformation reaction was performed within a 5-day fermentation process, products were isolated and their aglycone moiety was obtained by acid hydrolysis and their structures were elucidated by 13C and 1H NMR. The biotransformation resulted mainly digoxin (DG, approximately 87%), meanwhile, digoxigenone (DGON, approximately 7.0%) was also afforded as a side product. The present study revealed that: 1-Streptomyces isolate EUSA2003B harbors its specific 12beta-hydroxlase and has the capability to transform DT and it's alpha-acetyl and beta-methyl derivatives into their corresponding digoxins at reasonable yields. 2-The minor structural differences in the trisaccharide side chain seemed ineffective on the transformational capability of this organism. 3-The Streptomyces might also possess a specific glycosidase that splits the saccharidic side chain beside another dehydrogenase that oxidizes C3 at the steroid nucleus into its ketone form (DGON).

During screening of microorganisms producing antifungal metabolites, Streptomyces psammoticus strain KP1404 was isolated. The culture extract of this strain showed potent disease control efficacy against Fusarium wilt on tomato plants. The antifungal metabolites ST-1 and ST-2 were isolated from the culture extract using a variety of chromatographic procedures. On the basis of MS and NMR spectrometric analysis, the structures of the antifungal active compounds ST-1 and ST-2 were determined to be the polyene antibiotics strevertene A and strevertene B, respectively. In vitro, strevertenes A and B showed inhibitory effects against the mycelial growth of Alternaria mali , Aspergillus oryzae , Cylindrocarpon destructans , Colletotrichum orbiculare , Fusarium oxysporum f.sp. lycopersici, and Sclerotinia sclerotiorum , even at concentrations of 4-16 μg/mL. Fusarium wilt development on tomato plants was strongly retarded by treatment with 1 μg/mL of these strevertenes. The disease control efficacies of strevertenes on Fusarium wilt were as remarkable as that of benomyl.

Linear chromosomes and linear plasmids of Streptomyces possess covalently bound terminal proteins (TPs) at the 5' ends of their telomeres. These TPs are proposed to act as primers for DNA synthesis that patches the single-stranded gaps at the 3' ends during replication. Most ('archetypal') Streptomyces TPs (designated Tpg) are highly conserved in size and sequence. In addition, there are a number of atypical TPs with heterologous sequences and sizes, one of which is Tpc that caps SCP1 plasmid of Streptomyces coelicolor. Interactions between the TPs on the linear Streptomyces replicons have been suggested by electrophoretic behaviors of TP-capped DNA and circular genetic maps of Streptomyces chromosomes. Using chemical cross-linking, we demonstrated intramolecular and intermolecular interactions in vivo between Tpgs, between Tpcs and between Tpg and Tpc. Interactions between the chromosomal and plasmid telomeres were also detected in vivo. The intramolecular telomere interactions produced negative superhelicity in the linear DNA, which was relaxed by topoisomerase I. Such intramolecular association between the TPs poses a post-replicational complication in the formation of a pseudo-dimeric structure that requires resolution by exchanging TPs or DNA.

A filamentous soil bacterium, strain K202, was isolated from soil where an edible mushroom (Boletopsis leucomelas) was growing and identified as belonging to the genus Streptomyces on the basis of its morphological characteristics and the presence of LL-2, 6-diaminopimelic acid. We studied the existence states of Cs and its migration from extracellular to intracellular fluid in the mycelia of Streptomyces sp. K202. The results indicated that Cs accumulated in the cells through at least 2 steps: in the first step, Cs(+) was immediately and non-specifically adsorbed on the negatively charged cell surface, and in the second step, this adsorbed Cs(+) was taken up into the cytoplasm, and a part of the Cs entering the cytoplasm was taken up by an energy-dependent transport system(s). Further, we confirmed that a part of the Cs(+) was taken up into the mycelia competitively with K(+), because K(+) uptake into the intact mycelia of the strain was significantly inhibited by the presence of Cs(+) in the culture media. This suggested that part of the Cs is transported by the potassium transport system. Moreover, (133)Cs-NMR spectra and SEM-EDX spectra of the mycelia that accumulated Cs showed the presence of at least 2 intracellular Cs states: Cs(+) trapped by intercellular materials such as polyphosphate and Cs(+) present in a cytoplasmic pool., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Diamagnetic levitation is a technique that uses a strong, spatially varying magnetic field to simulate an altered gravity environment, as in space. In this study, using Streptomyces avermitilis as the test organism, we investigate whether changes in magnetic field and altered gravity induce changes in morphology and secondary metabolism. We find that a strong magnetic field (12T) inhibit the morphological development of S. avermitilis in solid culture, and increase the production of secondary metabolites., Methodology/principal Findings: S. avermitilis on solid medium was levitated at 0 g*, 1 g* and 2 g* in an altered gravity environment simulated by diamagnetic levitation and under a strong magnetic field, denoted by the asterix. The morphology was obtained by electromicroscopy. The production of the secondary metabolite, avermectin, was determined by OD(245 nm). The results showed that diamagnetic levitation could induce a physiological response in S. avermitilis. The difference between 1 g* and the control group grown without the strong magnetic field (1 g), showed that the magnetic field was a more dominant factor influencing changes in morphology and secondary metabolite production, than altered gravity., Conclusion/significance: We have discovered that magnetic field, rather than altered gravity, is the dominant factor in altered gravity simulated by diamagnetic levitation, therefore care should to be taken in the interpretation of results when using diamagnetic levitation as a technique to simulate altered gravity. Hence, these results are significant, and timely to researchers considering the use of diamagnetic levitation to explore effects of weightlessness on living organisms and on physical phenomena.

Surugapyrone A, a novel 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging 2-pyrone, was isolated from the culture filtrate of an actinomycete strain USF-6280. This strain was assigned to the genus Streptomyces owing to its morphological and chemotaxonomic characteristics. Phylogenetic analysis based on 16S ribosomal RNA gene sequences supported the classification of the producing strain as a member of the genus Streptomyces and indicated that it was closely related to Streptomyces coelicoflavus. As the morphological, physiological and biochemical characteristics of the producing strain were in agreement with those of the type strain of S. coelicoflavus, we concluded that strain USF-6280 should be identified as a member of S. coelicoflavus. The structure of surugapyrone A was determined to be 4-hydroxy-6-isopropyl-3-methyl-2-pyrone on the basis of the spectroscopic data. The results of feeding experiments with (13)C-labeled compounds indicated that surugapyrone A was biosynthesized through a polyketide pathway involving isobutyrate, acetate and propionate.

To investigate the function of nsdA in Streptomyces bingchengensis, it was cloned and sequenced, which presented an 89.89% identity with that of S. coelicolor. The lambdaRED-mediated PCR-targeting technique was used to create nsdA replacement in the S. bingchengensis_226541 chromosome. The nsdA disruption mutant, BC29, was obtained, which produced more pigment and spores than did the ancestral strain. HPLC analysis revealed that the disruption of nsdA efficiently increased milbemycin A(4) production and nanchangmycin production by 1.5-fold and 9-fold, respectively. Complementation of the nsdA mutation restored the phenotype and antibiotic production. These results showed that nsdA negatively affected sporulation and antibiotic production in S. bingchengensis.

The antibiotic meroparamycin was produced in the free culture system of Streptomyces sp. strain MAR01. Five solid substrates (rice, wheat bran, Quaker, bread, and ground corn) were screened for their ability to support meroparamycin production in solid-state fermentation. In batch culture, wheat bran recorded the highest antibacterial activity with the lowest residual substrate values. The highest residual substrate values were recorded for both ground corn and Quaker. On the other hand, no antibacterial activity was detected for rice as a solid substrate. The use of the original strength of starch-nitrate medium in the solid-state fermentation gave a lower antibacterial activity compared with the free culture system. Doubling the strength of this medium resulted in the increase in the activity to be equivalent to the free culture. The initial pH (7.0) of the culture medium and 2 ml of spore suspension (1 ml contains 5x10(9) spores/ml) were the optima for antibiotic production. The water was the best eluent for the extraction of the antibiotic from the solid-state culture. Ten min was enough time to extract the antibiotic using a mixer, whereas, 60 min was required when shaking was applied. Semicontinuous production of meroparamycin using a percolation method demonstrated a more or less constant antibacterial activity over 4 runs (450-480 microg/ml). The semicontinuous production of the antibiotic was monitored in a fixed-bed bioreactor and the maximum activity was attained after the fourth run (510 microg/ml) and the overall process continued for 85 days.

A novel nucleoside phytotoxin, albucidin (1), was isolated from the culture broth of Streptomyces albus subsp. chlorinus NRRL B-24108 using bioassay directed fractionation. The structure of the new natural product, albucidin, was determined by NMR and MS; however, the compound has been reported earlier in the literature following synthetic modification of oxetanocin. This is the first report of herbicidal activity for compounds of this structural type. Albucidin shows high levels of broad spectrum activity following post-emergence applications as well as moderate levels of pre-emergence activity. Accordingly, albucidin could be an important new lead for herbicide discovery.

A novel salt-tolerant strain DUT_AHX, which was capable of utilizing nitrobenzene (NB) as the sole carbon source, was isolated from NB-contaminated soil. Furthermore, it was identified as Streptomyces albidoflavus on the basis of physiological and biochemical tests and 16S ribosomal DNA (rDNA) sequence analysis. It can grow in the presence of NaCl up to 12% (w/v) or NB up to 900 mg/l in mineral salts basal (MSB) medium. The exogenously added osmoprotectants such as glycin, glutamic acid, proline, betaine and ectoine can improve growth of strain DUT_AHX in the presence of 10% (w/v) NaCl. NB-grown cells of strain DUT_AHX in modified MSB medium can degrade NB with the concomitant release of ammonia. Moreover, crude extracts of NB-grown strain DUT_AHX mainly contained 2-aminophenol 1,6-dioxygenase activity. These indicate that NB degradation by strain DUT_AHX might involve a partial reductive pathway. The proteins induced by salinity stress or NB were analyzed by native-gradient polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. In NB-induced proteins de novo, 141 kDa protein on the native-gradient PAGE gel was excised and electroeluted. Furthermore, enzyme tests exhibit the 2-aminophenol 1,6-dioxygenase activity of purified 141 kDa protein is 11-fold that of the cell-free extracts. The exploitation of strain DUT_AHX in salinity stress will be a remarkable improvement in NB bioremediation and wastewater treatment in high salinity.

The beta-lactamase inhibitory protein, BLIP-II, found in the culture supernatant of Streptomyces exfoliatus SMF19, shows no discernible sequence identity with other beta-lactamase inhibitory proteins identified in Streptomyces spp. A null mutant of the gene encoding BLIP-II (bliB::hygr) showed a bald appearance on solid media. Although BLIP-II was initially isolated from the supernatant of submerged cultures, sites of BLIP-II accumulation were seen in the cell envelope. The mutation of bliB was also associated with changes in the formation of septa and condensation of the chromosomal DNA associated with sporulation. The bliB mutant exhibited infrequent septa, showing dispersed chromosomal DNA throughout the mycelium, whereas the condensed chromosomes of the wild-type were separated by regularly spaced septa giving the appearance of a string of beads. Therefore, on the basis of these results, it is suggested that BLIP-II is a regulator of morphological differentiation in S. exfoliatus SMF19.

Chitin from Streptomyces lunalinharesii spores, detected on its outermost surface layer, was isolated and characterized by chemical and spectroscopic methods, transmission electron microscopy and flow cytometry analysis. Gold-chitinase- and gold-lectin (Lycopersicum esculentum agglutinin, LEA)-conjugated labels were used in microscopy experiments, whereas a fluorescence-lectin (LEA) conjugate was used in flow cytometry analysis. Chitin isolation consisted of several steps of hot alkali and nitrous acid treatment, and the final material was obtained in the colloidal form. The infrared and the 13C CP/MAS NMR spectra of Streptomyces sp. colloidal chitin and colloidal chitin obtained from commercial crab shell chitin were very similar. Incubation of the spores with gold-labeled lectin, or gold-labeled recombinant chitinase, showed the presence of gold particles around the spore surface, indicating the specific binding of the lectin or the recombinant chitinase with the chitin present on the outermost surface. Flow cytometry analysis, using the fluorescence-lectin conjugate, confirmed these results. According to scanning electron microscopy, S. lunalinharesii presented spore surface ornamentation belonging to the spiny group. This is the first detailed characterization of chitin on the spore's outermost layer from a Streptomyces species.

Immobilized bacteria are being assessed by industry for drug delivery, novel fermentation systems and the protection of organisms in harsh environments. Alginate bioreactors containing Streptomyces nodosus were examined for community structure, cell viability and amphotericin production under different growth conditions. When cell proliferation was encouraged, substrate hyphae were found inside the alginate matrix and within multicellular projections on the surface of the capsule. The periphery of these projections had erect and branched hyphae, morphologically identical to aerial hyphae. Antibiotic production from immobilized organisms was assessed using conditioned culture medium to eliminate the emergence of a free-dwelling population. These organisms sporulated with reduced antibiotic production compared with free-dwelling cultures. The commitment to sporulate was independent of a surface but dependent on community size and nutritional status. This is the first report of the sporulation of S. nodosus in liquid cultures and description of the multicellular community the organism adopts at a solid-liquid interface., (© 2008 The Authors. Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Spontaneous white mutants from sectors of Streptomyces hygroscopicus 111-81 were isolated. The comparison of morphological, cultural, and biochemical properties of the mutants and ancestor showed the differences in colors of aerial, substrate mycelia, and sporulation. Changes in resistance to antibiotics and sensitivity to lysozyme indicated alterations in cell walls and cell membranes of the mutants. They showed antifungal activity close to that of the parent strain on fermentation medium FM2, with unchanged component composition of the AK-111-81 antibiotic complex. The cells of spontaneous white mutants are characterized with electron-transparent structures, vacuoles, aggregation of ribosomes, intrahyphal growth, and lack of multiple cell septa, which was established by transmission electron microscopy. The appearance of white sectored-mutants in S. hygroscopicus 111-81 is connected with exhausting of nutrients causing the substrate limitation and is a stress response to starvation.

Cyslabdan, a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus, was isolated from the culture broth of Streptomyces sp. K04-0144 by Diaion HP-20 and ODS column chromatographies and preparative HPLC. The structure of cyslabdan was elucidated by spectroscopic analyses including NMR. The compound has a labdane-type diterpene skeleton connecting with an N-acetylcysteine via thioether linkage.

An afsA homologue (srrX) and three gamma-butyrolactone receptor gene homologues (srrA, srrB and srrC) are coded on the giant linear plasmid pSLA2-L in Streptomyces rochei 7434AN4, a producer of two polyketide antibiotics, lankacidin and lankamycin. Construction of gene disruptants and their phenotypic study revealed that srrX and srrA make a gamma-butyrolactone receptor system in this strain. Addition of a gamma-butyrolactone fraction to an srrX-deficient mutant restored the production of lankacidin and lankamycin, indicating that the SrrX protein is not necessary for this event. In addition to a positive effect on antibiotic production, srrX showed a negative effect on morphological differentiation. The receptor gene srrA reversed both effects of srrX, while the second receptor gene homologue srrC had only a positive function in spore formation. Furthermore, disruption of the third homologue srrB greatly increased the production of lankacidin and lankamycin. Electron microscopic analysis showed that aerial mycelium formation stopped at a different stage in the srrA and srrC mutants. Overall, these results indicated that srrX, srrA, srrB and srrC constitute a complex regulatory system for antibiotic production and morphological differentiation in S. rochei.

During the course of screening for industrially important microorganisms, an alkali-tolerant and thermotolerant actinomycete, strain DAS 131T, was isolated from a soil sample collected from the Gulbarga region, Karnataka province, India. The strain was characterized by a polyphasic approach that showed that it belonged to the genus Streptomyces. Growth was observed over a wide pH range (pH 6-12) and at 45 degrees C. The 16S rRNA gene sequence of strain DAS 131T was deposited in the GenBank database under the accession number DQ317411. 16S rRNA gene sequence analysis revealed that strain DAS 131T was most closely related to Streptomyces venezuelae ISP 5230T (AY999739) with a sequence similarity of 99.5% (8 nucleotide differences out of 1,477). Despite this very high sequence similarity, strain DAS 131T was phenetically distinct from S. venezuelae. The DNA relatedness between these strains was 54%, indicating that strain DAS 131T is a distinct genomic species. On the basis of phenetic and genetic analyses, strain DAS 131T is classified as a new species in the genus Streptomyces, for which we propose the name Streptomyces gulbargensis sp. nov.