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1.
Streptomyces venezuelae ISP5230 produces a group of jadomycin congeners with cytotoxic activities. To improve jadomycin fermentation process, a genetic
engineering strategy was designed to replace a 3.4-kb regulatory region of jad gene cluster that contains four regulatory
genes (3′ end 272 bp of jadW2, jadW3, jadR2, and jadR1) and the native promoter upstream of jadJ (PJ) with the ermEp* promoter sequence so that ermEp* drives the expression of the jadomycin biosynthetic genes from jadJ in the engineered strain. As expected, the mutant strain produced jadomycin B without ethanol treatment, and the yield increased
to about twofold that of the stressed wild-type. These results indicated that manipulation of the regulation of a biosynthetic
gene cluster is an effective strategy to increase product yield. 相似文献
2.
A method to type the potential angucycline producers in actinomycetes isolated from marine sponges 总被引:1,自引:0,他引:1
Angucyclines are aromatic polyketides with antimicrobial, antitumor, antiviral and enzyme inhibition activities. In this study,
a new pair of degenerate primers targeting the cyclase genes that are involved in the aromatization of the first and/or second
ring of angucycline, were designed and evaluated in a PCR protocol targeting the jadomycin cyclase gene of Streptomyces venezuelae ISP5230. The identity of the target amplicon was confirmed by sequencing. After validation, the primers were used to screen
49 actinomycete isolates from three different marine sponges to identify putative angucycline producers. Seven isolates were
positively identified using this method. Sequence analysis of the positive amplicons confirmed their identity as putative
angucycline cyclases with sequence highly similar to known angucycline cyclases. Phylogenetic analysis clustered these positives
into the angucycline group of cyclases. Furthermore, amplifications of the seven isolates using ketosynthase-specific primers
were positive, backing the results using the cyclase primers. Together these results provided strong support for the presence
of angucycline biosynthetic genes in these isolates. The specific primer set targeting the cyclase can be used to identify
putative angucycline producers among marine actinobacteria, and aid in the discovery of novel angucyclines. 相似文献
3.
Jakeman DL Graham CL Young W Vining LC 《Journal of industrial microbiology & biotechnology》2006,33(9):767-772
The jadomycins are a unique family of benzoxazolophenanthridine antibiotics produced by Streptomyces venezuelae ISP5230 following heat or ethanol shock or phage infection. We have modified the culture conditions by altering the carbon source, buffer, inoculum size, and timing of ethanol shock, thereby reducing growing times and improving jadomycin B production. Our optimized conditions use glucose as the carbon source, MOPS as buffer, low concentrations of phosphate, a defined inoculum concentration and an immediate ethanol shock to induce jadomycin B production; results that contrast previous studies. The altered media will facilitate the isolation of related jadomycin B congeners. 相似文献
4.
Janice L. Doull Amrit K. Singh Melanie Hoare Stephen W. Ayer 《Journal of industrial microbiology & biotechnology》1994,13(2):120-125
Summary The novel benzoxazolophenanthridine antibiotic, jadomycin B, is produced byStreptomyces venezuelae ISP5230 following a 42 °C heat shock or exposure to ethanol. To further characterize these unusual culture conditions, studies were carried out using different media, varying nutrient concentrations, initial pH, and time of application of heat or ethanol stress. Highest titers of jadomycin B accumulated 48 h afterS. venezuelae ISP5230 was inoculated into ad-galactose-l-isoleucine production medium (pH 7.5) which was supplemented with ethanol (6%, v/v) between 6 and 13 h. Cultures supplemented with ethanol later than 17 h post inoculation into the production medium produced little or no jadomycin B. Among other heat-shock inducing treatments examined, infection with phage SV1 was associated with increased jadomycin B production. Although jadomycin B titers showed little change with variations in the concentration of phosphate in the production medium, the nature of the nitrogen source was found to be important. Different colored pigments, presumed to be jadomycin B analogs, were formed when other amino acids replacedl-isoleucine in the medium as the sole nitrogen source. Increased jadomycin B titers accompanied increasedl-isoleucine andd-galactose concentrations in the production medium. 相似文献
5.
A new phycoerythrin, SCH-phycoerythrin, was purified from Synechococcus sp. ECS-18 by DEAE-Sephacel anion exchange chromatography and Sephacryl S-300 gel filtration. The protein pigment had an
absorbance maximum at 542 nm and a fluorescence maximum at 565 nm. The native molecular mass was approximately 219 kDa as
determined by gel filtration, and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of two
subunits, with molecular mass of 19 and 17.9 kDa. These observations are consistent with the (αβ)6 subunit composition that is characteristic of phycoerythrins. The α- and β-subunits showed immunological identity by Ouchterlony
double immunodiffusion with an anti-phycoerythrin antiserum. The DNA sequence of the SCH-phycoerythrin gene was determined
by PCR amplification using primers based on the conserved N-terminal amino acid sequence of the α- and β-subunits of phycoerythrins. 相似文献
6.
To determine the species of the yeast-like symbionts (YLS) in the brown planthoppers (BPH), Nilaparvata lugens, YLS were first isolated and purified by ultracentrifugation from the fat bodies of BPH, and then 18S rDNA and internal transcribed
spacer (ITS)–5.8S rDNA sequences of YLS were amplified with the different general primers for fungi. The results showed that
the two different 18S and ITS–5.8S rDNA sequences of YLS were obtained. One 2291-bp DNA sequence, which contained 18S and
ITS–5.8S rDNA, showed the high similarity to Cryptococcus and was named Cryp-Like symbiotes. Another 1248-bp DNA sequence, which contained a part of 18S and ITS–5.8S rDNA, showed the high similarity
to Pichia guilliermondii and was named Pichia-Like symbiotes. It was further proved that Cryp- and Pichia-Like symbiotes existed in BPH through nested PCR with specific primers for two symbiotes and in situ hybridization analysis
using digoxigenin-labeled probes. Our results showed that BPH harbored more than one species of eukaryotic YLS, which suggested
that diversity of fungal endosymbiotes may be occurred in planthoppers, just like bacterial endosymbiotes. 相似文献
7.
“Candidatus Liberobacter,” the uncultured bacterium associated with citrus Huanglongbing (HLB) disease, is an α-Proteobacteria, and two species, “Candidatus L. africanum” and “Candidatus L. asiaticum,” have been characterized by sequence analysis of the 16S rDNA and β operon (rplKAJL-rpoBC) genes. These genes were isolated by PCR and random cloning of DNA from infected plants. However, this strategy is laborious
and allowed selection of only three Liberobacter DNA fragments. In this paper, we described isolation of additional genes
using Random Amplified Polymorphic DNA (RAPD). In total, 102 random 10-mer primers were used in PCR reactions on healthy and
Liberobacter-infected plant DNA. Eight DNA bands amplified from infected plant DNA were cloned and analyzed. Six of them were
found to be part of the Liberobacter genome by sequence and hybridization experiments. On these DNA fragments, four genes
were identified: nusG, pgm, omp, and a hypothetical protein gene. These results indicate that RAPD can be used to clone DNA of uncultured organisms.
Received: 14 September 1998 / Accepted: 6 October 1998 相似文献
8.
A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis 总被引:2,自引:0,他引:2
A simple, two-step efficient method to perform multiple-site mutagenesis of a gene from bacterial genome was developed. The method was named polyacrylamide gel electrophoresis (PAGE)-mediated overlap extension polymerase chain reaction (PCR) (POEP). The first step involves synthesis of individual fragments containing mutant sites with 15- to 25-bp overlap between two adjacent fragments. Mutations were introduced into the overlapping oligonucleotide primers which ensured the particular primer-template annealing. PAGE was used to remove contaminating parental templates, mispriming fragments, and leftover primers. The second step involves synthesis of the mutant full-length fragment. All purified PCR products from the first step were combined and used as the template for a second PCR using high-fidelity DNA polymerase, with the two outermost flanking oligonucleotides as primers. Using the POEP method, we have successfully introduced eight EcoRI sites into the Escherichia coli β-galactosidase (Lac Z) gene. The overall rate of obtaining the multiple mutant sites was 100%. The POEP method is simple, involving only two steps, and reliable for multiple-site mutagenesis and is promising to be widely used in gene modification. 相似文献
9.
Streptomyces luteogriseus strain 099, producing a new type of macrolide antibiotic with anti-coxB6 virus and anti-HIV protease activities, was isolated
from soil. PCR was optimized to amplify β-ketoacyl-ACP synthase (KS) genes. The system was optimized around the use of higher concentrations of DMSO (15% vs. 10% v/v)
and dNTP (500 μM vs. 50–200 μM) and a lower annealing temperature (55 °C vs. 60–70 °C) than the normal PCR method used to amplify high GC content DNA. 相似文献
10.
11.
Thapa LP Oh TJ Lee HC Liou K Park JW Yoon YJ Sohng JK 《Applied microbiology and biotechnology》2007,76(6):1357-1364
The pSKC2 cosmid, which has 32 kb and 28 open-reading frames, was isolated from Streptomyces kanamyceticus ATCC12853 as the gene cluster of kanamycin. This gene cluster includes the minimal biosynthetic genes of kanamycin with the
resistance and regulatory genes. It was heterologously expressed in Streptomyces venezuelae YJ003, which has the advantage of fast growth, good efficiency of the transformation host, and rapid production of the aminoglycosides
antibiotic. The isolated compound was analyzed by electrospray ionization–mass spectrometry, liquid chromatography–mass spectrometry,
high-performance liquid chromatography, and tandem mass spectrometry and shows a molecular weight of 485 as kanamycin A. 相似文献
12.
In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the
3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses.
Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline,
streptomycin or spectinomycin, and were recovered at frequencies in the range of 10−5 to 10−8 per recipient. A total of 12 distinct plasmids, designated pB1–pB12, was identified. Their sizes ranged between 41 to 69 kb
and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated
resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids, displaying extremely
high transfer frequencies in filter crosses, ranging from 10−1 to 10−2 per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP
group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged
to the IncPβ subgroup, whereas two plasmids were identified as IncPα plasmids. Analysis of the IncP-specific PCR products
revealed considerable differences among the IncPβ plasmids at the DNA sequence level. In order to characterize the gene “load”
of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative
proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two
putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide
dismutase, parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified.
Received: 11 October 1999 / Accepted: 11 January 2000 相似文献
13.
Geldanamycin belongs to benzoquinone ansamycin antibiotic and has potent antitumor activities. In this study, a bacterial
artificial chromosome (BAC) library with an average insert size of up to 150 kb was constructed from genomic DNA of Streptomyces autolyticus JX-47. A genetic-screening strategy was established using BAC end-sequencing and three pairs of primers designed to target
the remote regions, gdmA1, gdmA3 and gdmRI, of the geldanamycin gene cluster. Three clones covering geldanamycin biosynthesis
gene cluster were obtained, which together spanned a 250-kb genomic region, and a 150227-bp insert in the clone p4E9 was sequenced.
Comparison with the reported geldanamycin gene cluster sequences from S. hygroscopicus revealed that it had the same gene arrangement and high gene homology in the polyketide synthase (PKS) region and its downstream
with 84–100% DNA identity and 81–100% amino acid (AA) identity. Its DNA homology with the whole gene cluster sequence from
S. hygroscopicus strain 17997 reached 99% identity. However, upstream of the PKS region exhibited great diversity, where only ORF16 was conserved,
and the other genes including gdmL and gdmX were displaced. 相似文献
14.
Jung WS Han AR Hong JS Park SR Choi CY Park JW Yoon YJ 《Applied microbiology and biotechnology》2007,76(6):1373-1381
To develop a system for combinatorial biosynthesis of glycosylated macrolides, Streptomyces venezuelae was genetically manipulated to be deficient in the production of its macrolide antibiotics by deletion of the entire biosynthetic
gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes. Two engineered deoxysugar biosynthetic
pathways for the biosynthesis of thymidine diphosphate (TDP)-d-quinovose or TDP-d-olivose in conjunction with the glycosyltransferase–auxiliary protein pair DesVII/DesVIII derived from S. venezuelae were expressed in the mutant strain. Feeding the representative 12-, 14-, and 16-membered ring macrolactones including 10-deoxymethynolide,
narbonolide, and tylactone, respectively, to each mutant strain capable of producing TDP-d-quinovose or TDP-d-olivose resulted in the successful production of the corresponding quinovose- and olivose-glycosylated macrolides. In mutant
strains where the DesVII/DesVIII glycosyltransferase–auxiliary protein pair was replaced by TylMII/TylMIII derived from Streptomyces fradiae, quinovosyl and olivosyl tylactone were produced; however, neither glycosylated 10-deoxymethynolide nor narbonolide were
generated, suggesting that the glycosyltransferase TylMII has more stringent substrate specificity toward its aglycones than
DesVII. These results demonstrate successful generation of structurally diverse hybrid macrolides using a S. venezuelae in vivo system and provide further insight into the substrate flexibility of glycosyltransferases.
Won Seok Jung and Ah Reum Han contributed equally to this work. 相似文献
15.
Properties of normal and mutant polypeptide fragments from the dimer self-association sites of human red cell spectrin 总被引:2,自引:0,他引:2
Lecomte MC Nicolas G Dhermy D Pinder JC Gratzer WB 《European biophysics journal : EBJ》1999,28(3):208-215
We have examined the properties and interactions of expressed polypeptide fragments from the N-terminus of the α-chain and the C-terminus of the β-chain of human erythroid spectrin. Each polypeptide comprises one complete structural repeating unit, together with the incomplete
repeat that interacts with its partner when spectrin tetramers are formed. The shared repeat thus generated is made up of
two helices from the C-terminal part of the β-chain and one helix from the N-terminus of the α-chain. Three mutant β-chain fragments with amino acid substitutions in the incomplete terminal repeat were also studied. The α- and β-chain fragments were both substantially monomeric, as shown by sedimentation equilibrium. Circular dichroism analysis and
thermal denaturation profiles revealed that the complete repeat present in each fragment had entered the stable tertiary fold.
Unexpectedly, the conformational stability of the folded β-chain repeat was found to be grossly perturbed by the mutations, all of them well beyond its C-terminal boundary; possible
explanations for this phemomenon are considered. Sedimentation equilibrium showed that in equimolar mixtures the wild-type
α- and β-chain peptides formed a 1:1 complex. Mixing curves, observed by circular dichroism, revealed that association was accompanied
by an increase in α-helicity. From continuous-variation profiles an association constant in the range 1–2×106 M–1 was inferred. The association was unaffected by the apparently unstructured anionic tail of 54 residues, found at the C-terminus
of the spectrin β-chain. Of the three mutations in the β-chain fragment, one (an Ala→Val replacement in the A helix segment of the incomplete repeat) had a relatively small effect on the association with the α-chain fragment, whereas Trp→Arg mutations in the A and in the remote B helix segments were much more deleterious. These observations are consistent with the relative severities of the haemolytic
conditions associated with the mutations.
Received: 10 August 1998 / Revised version: 13 October 1998 / Accepted: 13 October 1998 相似文献
16.
A novel simple and rapid PCR-based site-directed mutagenesis method 总被引:11,自引:0,他引:11
Rabhi I Guedel N Chouk I Zerria K Barbouche MR Dellagi K Fathallah DM 《Molecular biotechnology》2004,26(1):27-34
Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted
to specific purposes are still being developed. Herein we describe a straightforward and efficient method with versatile applications
for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector.
In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing
the sequence of interest. The primers are designed so that the desired modifications are introduced at the 5′ end of one of
the primers, whereas the other primer starts with the nucleotide at position (−1) of the one to be modified. The PCR is carried
out using Pfu DNA polymerase. The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli. Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation
is confirmed by direct DNA sequencing. This procedure was used efficiently to introduce substitutions, deletions, and insertions
in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat α integrin
CD11b A-domain and the human CD8β cloned in pPICZαB, pGEX-2T, and CDM8 expression vectors, respectively. 相似文献
17.
18.
Subunit E of the vacuolar ATPase (V-ATPase) contains an N-terminal extended α helix (Rishikesan et al. J Bioenerg Biomembr
43:187–193, 2011) and a globular C-terminal part that is predicted to consist of a mixture of α-helices and β-sheets (Grüber et al. Biochem
Biophys Res Comm 298:383–391, 2002). Here we describe the production, purification and 2D structure of the C-terminal segment E133-222 of subunit E from Saccharamyces cerevisiae V-ATPase in solution based on the secondary structure calculation from NMR spectroscopy studies. E133-222 consists of four β-strands, formed by the amino acids from K136-V139, E170-V173, G186-V189, D195-E198 and two α-helices,
composed of the residues from R144-A164 and T202-I218. The sheets and helices are arranged as β1:α1:β2:β3:β4:α2, which are
connected by flexible loop regions. These new structural details of subunit E are discussed in the light of the structural
arrangements of this subunit inside the V1- and V1VO ATPase. 相似文献
19.
H. Kobayashi Hiroko Oyamada Naoko Iwadate Hiromi Suzuki Hideko Mitobe Kaori Takahashi Nobuyuki Shibata Shigeo Suzuki Yoshio Okawa 《Archives of microbiology》1998,169(3):188-194
A mannan of Candida glabrata IFO 0622 digested by Arthrobacter exo-α-mannosidase and a β-1,2-linked mannobiose obtained from the parent mannan by acid treatment was analyzed using 13C nuclear magnetic resonance spectroscopy. The results show that the β-1,2-linked mannobiosyl residue is esterified to a phosphate
group through position C-1 in the α-configuration, Manβ1– 2Manα1–HPO3–. The results of immunochemical assays of these mannans using the commercial antigenic factor sera of the genus Candida (Candida Check, Iatron) indicate that the main recognition site of serum no. 6 in this kit is the mannotetraosyl side-chain
Manβ1–2Manα1– 2Manα1–2Man in C. glabrata mannan and also suggest that the phosphate-containing unit (such as Manβ1– 2Manα1–HPO3– in this mannan) behaves as one of the antigenic determinants of serum no. 6, but not of serum no. 5. Therefore, the present
and previous findings indicate that serum no. 5 recognizes relatively longer β-1,2-linked oligomannosyl side-chains, Manβ1–[2Manβ1–]n 2Man (n = 1–6), attached to the phosphate groups previously observed in the cell wall mannans of Candida albicans, Candida stellatoidea, and Candida tropicalis.
Received: 18 March 1997 / Accepted: 16 September 1997 相似文献