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ABSTRACT: In Streptomyces griseus, AdpA, the key transcriptional activator in the A-factor regulatory cascade, switches on the transcription of multiple genes
required for secondary metabolism and morphological differentiation. Streptomyces avermitilis also contains an ortholog of adpA, which is named adpA-a. To clarify the in vivo function of adpA-a, an adpA-a-disrupted strain was constructed by double crossover recombination. No difference in avermectin production was found between
the adpA-a-disruptant and the wild-type strain. However, this disruptant neither formed spores nor produced melanin and its phenotype
was restored to the original wild-type by a single copy of the adpA-a gene integrated into the chromosome. This report shows that adpA-a is involved in regulation of morphological differentiation and melanin production in S. avermitilis.
Chinese Science Bulletin 04/2012; 52(5):623-630. · 1.32 Impact Factor
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ABSTRACT: Streptomyces avermitilis is an industrially important soil bacterium known for production of avermectins, which are antiparasitic agents useful in animal health care, agriculture, and treatment of human infections. ku genes play a key role in the non-homologous end-joining pathway for repair of DNA double strand breaks. We identified homologs of eukaryotic ku70 and ku80 genes, termed ku1 and ku2, in S. avermitilis. Mutants with deletion of ku1, ku2, and both genes were constructed and their phenotypic changes were characterized. Deletion of ku genes had no apparent adverse effects on growth, spore formation, or avermectin production. The ku mutants, in comparison to wild-type strain, were slightly more sensitive to the DNA-damaging agent ethyl methanesulfonate, but not to UV exposure or to bleomycin. Gene targeting frequencies by homologous recombination were higher in the ku mutants than in wild-type strain. We conclude that ku-deleted strains will be useful hosts for efficient gene targeting and will facilitate functional analysis of genes in S. avermitilis and other industrially important bacterial strains.
Journal of Industrial Microbiology 02/2012; 39(6):917-25. · 1.80 Impact Factor
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ABSTRACT: Two Gram-positive, rod-shaped, non-spore-forming bacteria (strains RHLT1-17T and RHLT2-1T) were isolated from Hailuogou glacier in Szechwan province, P. R. China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains belonged to the genus Nocardioides and shared 97.8 % sequence similarity to each other. The highest level of sequence similarity (97.6 %, 98.4 %, respectively) were found with Nocardioides kribbensis. Strain RHLT1-17T grow at 0-35°C and strain RHLT2-1T grow at 0-25°C. The major cellular fatty acids of strain RHLT1-17T were C17:1ω8c (32.69 %) and iso-C16:0 (21.74 %). The major cellular fatty acids of strain RHLT2-1T were C18:1 ω9c (28.72%), summed feature 3 (17.14 %; comprising C16:1ω7c and/or C16:1ω6c), iso-C16:0 (14.35 %), C16:0 (9.96 %) and iso-C14:0 (8.34 %). Both strains contained LL-2, 6-diaminopimelic acid in the cell-wall peptidoglycan, MK-8(H4) as the predominant menaquinone. On the basis of a polyphasic approach, two novel species Nocardioides szechwanensis sp. nov. (type strain = RHLT1-17T =CGMCC 1.11147T =NBRC 108562T) and Nocardioides psychrotolerans sp. nov. (type strain = RHLT2-1T =CGMCC 1.11156T =NBRC 108563T) are proposed.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 02/2012; · 2.11 Impact Factor
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ABSTRACT: Gram-positive, rod-shaped bacteria, strains Hh8(T), Hh15(T) and Hh40-2, were isolated from the No. 1 glacier in Xinjiang, north-west China. Colonies of strain Hh8(T) were orange-yellow, convex and round on PYG plates. Strain Hh8(T) grew at 0-19 °C and pH 5.5-10.5. Colonies of strain Hh15(T), which was able to grow at 0-20 °C and pH 5.5-12, were lemon yellow, convex and round on PYG plates. Phylogenetic analysis based on 16S rRNA gene sequences showed that these three strains were related to members of the genus Cryobacterium. The major cellular fatty acids of the novel strains were anteiso-C(15:0), iso-C(16:0), iso-C(15:0) and anteiso-C(15:1) A. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, two novel species, Cryobacterium flavum sp. nov. (type strain Hh8(T) = CGMCC 1.11215(T) = NBRC 107879(T)) and Cryobacterium luteum sp. nov. (type strain Hh15(T) = CGMCC 1.11210(T) = NBRC 107880(T)), are proposed.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 07/2011; 62(Pt 6):1296-9. · 2.11 Impact Factor
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ABSTRACT: The role of the extracytoplasmic function (ECF) σ factor Sig6 (SAV663) in avermectin production by Streptomyces avermitilis was investigated by gene-deletion, complementation and over-expression experiments. Inactivation of Sig6 had no major effect on growth, stress responses, or morphology. Avermectin yield was increased 2- to 2.7-fold (~680 μg/ml) relative to the wild-type strain by deletion of the sig6 gene, and was restored to the wild-type level by introduction of a single copy of sig6. Introduction of extra multi-copy or integrative sig6 vectors into the wild-type decreased avermectin yield by 56-63%. Taken together, these findings indicate that Sig6 plays a negative regulatory role in avermectin production in S. avermitilis. RT-PCR analysis demonstrated that this role of Sig6 is mediated by the pathway-specific activator gene aveR.
Biotechnology Letters 06/2011; 33(10):1955-61. · 1.68 Impact Factor
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ABSTRACT: We investigated the function of maltose ABC transporter system encoded by malEFG-a and the effect of its overexpression on antibiotic production in Streptomyces avermitilis. A malEFG-a deletion mutant was unable to grow in a minimal medium with maltose as sole carbon source and produce avermectin. Maltose utilization and avermectin production were restored by introduction of a single copy of malEFG-a. RT-PCR analysis showed that the expression of malE-a was induced by maltose, and was strongly repressed by glucose. When multi-copy, integrative malEFG-a gene expression vectors were introduced into wild-type strain ATCC31267 and ivermectin-producer OI-31, antibiotic production increased by 2.6- to 3.3-fold and the time required for fermentation decreased by about 10%. The overexpression of malEFG-a improved the utilization rate of starch, and thereby enhanced avermectin production. Such an approach would be useful for the improvement of commercial antibiotic production using starch as the main carbon source in the fermentation process.
Bioresource technology 12/2010; 101(23):9228-35. · 4.25 Impact Factor
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ABSTRACT: The bacteriophage phiSASD1, isolated from a failed industrial avermectin fermentation, belongs to the Siphoviridae family. Its four predominant structural proteins, which include the major capsid, portal and two tail-related proteins, were separated and identified by SDS-PAGE and N-terminal sequence analysis. The entire double-stranded DNA genome of phiSASD1 consists of 37,068 bp, with 3'-protruding cohesive ends of nine nucleotides. Putative biological functions have been assigned to 24 of the 43 potential open reading frames. Comparative analysis shows perfect assembly of three "core" gene modules: the morphogenesis and head module, the tail module and the right arm gene module, which displays obvious similarity to the right arm genes of Streptomyces phage phiC31 in function and arrangement. Meanwhile, structural module flexibility within phiSASD1 suggests that assignment of phage taxonomy based on comparative genomics of structural genes will be more complex than expected due to the exchangeability of functional genetic elements.
Virology 07/2010; 403(1):78-84. · 3.35 Impact Factor
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ABSTRACT: Ribosome recycling factor (RRF), encoded by frr gene, is involved in the release of ribosomes from the translational post-termination complex for a new round of initiation. In this study, the frr gene with either its own promoter or with ermE p was cloned into a multi-copy vector, pKC1139, and a single-site integrative vector, pSET152, respectively. The resulting plasmids were transformed into Streptomyces avermitilis wild-type strain ATCC31267, avermectin high-producing mutant strain 76-02-e, and the engineered strain GB-165 that produces only avermectin B. The results showed that overexpression of frr increased avermectin yield (by 3- to 3.7-fold in the wild-type strain) and revealed an frr gene "copy number effect"; i.e., multiple copies of frr had a greater promoting effect on avermectin production than a single copy in each of the three transformed S. avermitilis strains. Comparison of the growth and expression of the ave genes in an frr-overexpressing strain and wild-type ATCC31267 indicated that frr overexpression promoted cell growth as well as the expression of ave genes (including pathway-specific positive regulatory gene aveR for avermectin biosynthesis and ave structural genes), leading in turn to avermectin overproduction. These findings provide an effective approach for the improvement of antibiotic production in Streptomyces.
Journal of Industrial Microbiology 03/2010; 37(7):673-9. · 1.80 Impact Factor
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Canadian Journal of Microbiology 01/2010; 56(1):88. · 1.36 Impact Factor
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ABSTRACT: The chromosome of Streptomyces has been shown to be unstable, frequently undergoing gross chromosomal rearrangements. However, the mechanisms underlying this phenomenon remain unclear, with previous studies focused on two chromosomal ends as targets for rearrangements. Here we investigated chromosomal instability of Streptomyces avermitilis, an important producer of avermectins, and characterized four gross chromosomal rearrangement events, including a major deletion in the central region. The present findings provide a valuable contribution to the mechanistic study of genetic instability in Streptomyces.
Thirty randomly-selected "bald" mutants derived from the wild-type strain all contained gross chromosomal rearrangements of various types. One of the bald mutants, SA1-8, had the same linear chromosomal structure as the high avermectin-producing mutant 76-9. Chromosomes of both strains displayed at least three independent chromosomal rearrangements, including chromosomal arm replacement to form new 88-kb terminal inverted repeats (TIRs), and two major deletions. One of the deletions eliminated the 36-kb central region of the chromosome, but surprisingly did not affect viability of the cells. The other deletion (74-kb) was internal to the right chromosomal arm. The chromosome of another bald mutant, SA1-6, was circularized with deletions at both ends. No obvious homology was found in all fusion sequences. Generational stability analysis showed that the chromosomal structure of SA1-8 and SA1-6 was stable.
Various chromosomal rearrangements, including chromosomal arm replacement, interstitial deletions and chromosomal circularization, occurred in S. avermitilis by non-homologous recombination. The finding of an inner deletion involving in the central region of S. avermitilis chromosome suggests that the entire Streptomyces chromosome may be the target for rearrangements, which are not limited, as previously reported, to the two chromosomal ends.
BMC Microbiology 01/2010; 10:198. · 3.04 Impact Factor
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ABSTRACT: The function of the regulatory protein AveR in Streptomyces avermitilis was examined. An aveR deletion mutant abolished avermectin production and produced more oligomycin, and its phenotype was complemented by a single copy of the aveR gene. Removal of the C-terminal HTH domain of AveR abolished avermectin biosynthesis, indicating the importance of HTH domain for AveR function. Promoter titration and promoter probe assays suggested that the transcription of aveA1, encoding polypeptide AVES1 of avermectin PKS, was activated by AveR. Chromatin immunoprecipitation (ChIP) assay showed that the predicted promoter regions of both the ave cluster and the olm cluster were target sites of AveR, and the DNA-binding activity of AveR was dependent on its HTH domain. RT-PCR analysis revealed that the transcriptions of ave structural genes were dependent on AveR, but that of olm structural genes and putative pathway-specific regulatory genes increased in the aveR mutants. Consistent with these observations, overexpression of aveR successfully increased avermectin production. These results indicated that aveR encodes a pathway-specific activator essential for avermectin biosynthesis and it also negatively affects oligomycin biosynthesis.
MGG Molecular & General Genetics 12/2009; 283(2):123-33. · 2.58 Impact Factor
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ABSTRACT: The avermectin analogue doramectin (CHC-B1), which is produced in mutants that have an altered biosynthesis pathway of avermectin, is one of the most effective agricultural pesticides and antiparasitics. We report here the construction of a bkdF olmA double-deletion mutant lacking one of the branched-chain alpha-keto acid dehydrogenase encoding genes (bkdF) and the oligomycin PKS encoding gene cluster (olmA) in Streptomyces avermitilis 76-05. We then characterized the production of various antibiotics in cultures of the deletion mutant. In a fermentation medium supplemented with cyclohexanecarboxylic acid, this double mutant produced doramectin and its analogues but no oligomycin. The mutant proved to be genetically stable, without any antibiotic resistance markers inserted into its chromosome, and could potentially become an industrial doramectin-producing strain after further improvement.
Canadian Journal of Microbiology 12/2009; 55(12):1355-63. · 1.36 Impact Factor
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ABSTRACT: In eukaryotes, Ca2+ is an important second messenger and regulates diverse cellular activities ranging from muscle contraction to fertilization. In bacteria, growing evidence suggests that Ca2+ also plays important regulatory roles in various physiological processes. Here we review current understanding of calcium regulation in bacteria from the following aspects: 1) the concept of bacterial [Ca2+]i and its determination; 2) cellular processes affected by [Ca2+]i changes; 3) transportation of Ca2+ across bacterial membrane; 4)eukaryotic calcium binding proteins in bacteria and their functions.
ACTA MICROBIOLOGICA SINICA 12/2009; 49(12):1564-70.
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ABSTRACT: Genetic instability, such as morphological variation, is a common occurrence in Streptomyces. It causes lots of trouble in fermentation industry and research, such as strain degeneration and reduction of antibiotic productivity. The relationship between genetic instability and chromosome instability in Streptomyces has been well studied. It is very important to reveal the molecular mechanism of genetic instability for strain improvement and for construction of stable overproducing strains. In this review, the present status and prospect on the study of the genetic instability in Streptomyces are presented.
ACTA MICROBIOLOGICA SINICA 10/2009; 49(10):1271-6.
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ABSTRACT: Spinosad is a new class of insecticides produced by Saccharopolyspora spinosa. The aim of this study was to construct a starch-utilizing strain that overproduced spinosad by intergeneric fusion between S. spinosa and Streptomyces avermitilis. Protoplast fusion is an important technique for engineering microbial strains, especially for microorganisms with few available molecular genetic tools. Protoplast fusion was conducted with UV-irradiated protoplasts of S. spinosa and S. avermitilis. Among 76 recombinants screened by ESI-MS and HPLC, a starch-utilizing strain F17, identified as S. spinosa, was obtained. The yield of spinosad in F17 was increased by 447.22%, compared with the yield of the wild-type strain. This is the first report of intergeneric protoplast fusion between S. spinosa and S. avermilitis, which shows great potential for industrial applications.
Canadian Journal of Microbiology 09/2009; 55(9):1070-5. · 1.36 Impact Factor
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ABSTRACT: Two new recombinants of avermectin polyketide synthases were constructed by domain and module swapping in Streptomyces avermitilis 73-12. However, only the strain, S. avermitilis OI-31, formed by domain substitution could produce ivermectin. Analysis of the ivermectin synthesized gene cluster showed that decreased amount of aveC transcripts was one of the factors causing low yield of ivermectin. Overexpression of aveC could improve ivermectin yield.
Bioorganic & medicinal chemistry letters 10/2008; 18(20):5359-63. · 2.65 Impact Factor
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ABSTRACT: A new actinomycete strain, isolated from soil in China, strongly inhibited in vitro proliferation of human hepatoma, chronic myelogenous leukemia, and colonic carcinoma cell lines. The strain, designated L033, was identified as a strain of Streptomyces avermitilis based on cultural property, morphology, carbon source utilization, 16s rRNA gene analysis, and DNA-DNA relatedness studies. The anticancer component from L033 was purified to homogeneity by preparative positive-phase high-performance liquid chromatography and crystallization. Nuclear magnetic resonance and mass spectrometric analysis showed that this compound had the same structure as oligomycin A. Different with other reported naturally occurring strains of S. avermitilis, L033 produced high quantity of oligomycin A (maximal 1,461 microg/ml). Therefore, L033 was considered of great potential as an industrial oligomycin-A-producing strain.
Applied Microbiology and Biotechnology 10/2008; 81(5):839-45. · 3.42 Impact Factor
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ABSTRACT: Among eight components of avermectin, B1 fractions have the most effective antiparasitic activities and the lowest level of
toxic side-effects and are used widely in veterinary and agricultural fields. Intraspecific protoplast fusion between two
strains of Streptomyces avermitilis, one an avermectin high producer (strain 76-05) and the other a genetically engineered strain containing the mutations aveD
− and olmA
− (strain 73-12) was performed for enhancement and selective production of avermectin B in the absence of oligomycin. Two recombinant
strains (F23 and F29) were isolated and characterized with regards to the parental merits. F23 and F29 produced only the four
avermectin B components with high yield and produced no oligomycin. The avermectin production of F23 and F29 was about 84.20%
and 103.45% of the parental strain 76-05, respectively, and increased about 2.66-fold and 3.50-fold, respectively, compared
to that of parental strain 73-12. F23 and F29 were genetically stable prototrophic recombinants and F29 was quite tolerant
of fermentation conditions compared to avermectin high producer parental strain 76-05. The ability to produce avermectin B
with high yield without the production of other avermectin components and oligomycin will make F23 and F29 useful strains
for avermectin production. Strain F29’s tolerance of fermentation conditions will also make it suitable for industrial applications.
Chinese Science Bulletin 02/2007; 52(5):616-622. · 1.32 Impact Factor
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ABSTRACT: Ivermectin, 22, 23-dihydroavermectin B1, is commercially important in human, veterinary medicine, and pesticides. It is currently synthesized by chemical reduction of the double bond between C22 and C23 of avermectins B1, which are a mixture of B1a (>80%) and B1b (<20%) produced by fermentation of Streptomyces avermitilis. The cost of ivermectin is much higher than that of avermectins B1 owing to the necessity of region-specific hydrogenation at C22-C23 of avermectins B1 with rhodium chloride as the catalyst for producing ivermectin. Here we report that ivermectin can be produced directly by fermentation of recombinant strains constructed through targeted genetic engineering of the avermectin polyketide synthase (PKS) in S. avermitilis Olm73-12, which produces only avermectins B and not avermectins A and oligomycin. The DNA region encoding the dehydratase (DH) and ketoreductase (KR) domains of module 2 from the avermectin PKS in S. avermitilis Olm73-12 was replaced by the DNA fragment encoding the DH, enoylreductase, and KR domains from module 4 of the pikromycin PKS of Streptomyces venezuelae ATCC 15439 using a gene replacement vector pXL211. Twenty-seven of mutants were found to produce a small amount of 22, 23-dihydroavermectin B1a and avermectin B1a and B2a by high performance liquid chromatography and liquid chromatography mass spectrometry analysis. This study might provide a route to the low-cost production of ivermectin by fermentation.
Applied Microbiology and Biotechnology 10/2006; 72(5):986-94. · 3.42 Impact Factor
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ABSTRACT: Gene deletion vector pXL05(pKC1139::ΔolmA1 +ΔolmA4) was used to disrupt oligomycin PKS encoding genes(olmA) inStreptomyces avermitilis CZ8-73, the producer of anthelmintic avermectins B and the cell growth inhibitor oligomycin.olmA gene cluster in the chromosome was displaced by deletion allele on the plasmid via double crossover. Four of disruptants
were confirmed by Southern blotting. Shaking flask experiments and HPLC analyses showed that the four mutants no longer produced
the toxic oligomycin, but only made four components of avermectins B, which were avermectin B1a, B1b, B2a, B2b. The yields
of avermectins B in these mutants were separately equal to those in CZ8-73. This revealed thatolmA genes deletion did not affect the biosynthesis of avermectins. The deletion mutants were proved to be genetically stable,
and thus might be promising strains in industrial production of avermectins B.
Chinese Science Bulletin 01/2004; 49(4):350-354. · 1.32 Impact Factor
