Genomic and Metabolomic Investigation of a Rhizosphere Isolate Streptomyces netropsis WLXQSS-4 Associated with a Traditional Chinese Medicine.

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.

Exploration of cryptic organic photosensitive compound as Zincphyrin IV in Streptomyces venezuelae ATCC 15439.

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.

Morphology-driven downscaling of Streptomyces lividans to micro-cultivation.

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.

Streptomyces urticae sp. nov., isolated from rhizosphere soil of Urtica urens L.

Two novel Gram-stain positive, spore-forming, aerobic actinomycetes, designated NEAU-PCY-1T 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-1T and NEAU-PCY-2 exhibited 99.8% similarity with each other and are closely related to Streptomyces abietis DSM 42080T (98.2, 98.3%) and Streptomyces fildesensis DSM 41987T (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-1T 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-1T (= DSM 105115T = CCTCC AA 2017015T) as the type strain.

AdpAsd, a Positive Regulator for Morphological Development and Toyocamycin Biosynthesis in Streptomyces diastatochromogenes 1628.

AdpA is studied and considered as a pleiotropic regulator which is involved in morphological development and secondary metabolism in many Streptomyces. In this study, AdpAsd, 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 AdpAsd with TM biosynthesis and morphological differentiation, adpAsd was placed under the control of the ermE* promoter in plasmid pIB139. By intergeneric conjugation, the resulting plasmid pIB139-adpAsd 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 adpAsd, yielding the recombinant strain S. diastatochromogenes 1628-T62A. As expected, due to over-expression of adpAsd, 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 adpAsd 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 adpAsd plays a positive role in TM biosynthesis and morphological differentiation.

Chromosomal position effect influences the heterologous expression of genes and biosynthetic gene clusters in Streptomyces albus J1074.

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 ß-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.

Efficacy of protease inhibitor from marine Streptomyces sp. VITBVK2 against Leishmania donovani - An in vitro study.

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 IC50 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.

Endophytic Streptomyces sp. AC35, a producer of bioactive isoflavone aglycones and antimycins.

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.

Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan.

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.

Investigation on the effects of ϵ-poly-L-lysine on a producing strain Streptomyces ahygroscopicus GIM8, for better understanding its biosynthesis.

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.

Removal of the insecticide diazinon from liquid media by free and immobilized Streptomyces sp. isolated from agricultural soil.

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.

Cholesterol oxidase production from entrapped cells of Streptomyces sp.

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.

Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins.

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.

Inhibitory effect of components from Streptomyces species on alpha-glucosidase and alpha-amilase of different origin.

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.

Biotransformation of extracted digitoxin from Digitalis lanata by Streptomyces.

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).

Streptomyces marianii sp. nov., a novel marine actinomycete from southern coast of India.

A novel marine actinomycete strain designated ICN19T 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 ICN19T formed distinct cluster with Streptomyces wuyuanensis CGMCC 4.7042T, Streptomyces tirandamycinicus HNM0039T and Streptomyces spongiicola HNM0071T. 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-C15:0, C16:0 and iso-C16:0. 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 ICN19T 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 ICN19T and the reference strains with S. wuyuanensis CGMCC 4.7042T, S. tirandamycinicus HNM0039T and S. spongiicola HNM0071T 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 ICN19T (=MCC 3599T = KCTC 39749T).

Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.

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.

Genetics of Streptomyces rimosus, the oxytetracycline producer.

From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.

Responses of a novel salt-tolerant Streptomyces albidoflavus DUT_AHX capable of degrading nitrobenzene to salinity stress.

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.

Solid-state fermentation for the production of meroparamycin by streptomyces sp. strain MAR01.

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.