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ABSTRACT: The whole-genome sequence of Thermoanaerobacter tengcongensis, an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China, was completed in 2002. However, in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system. In order to establish such a system, the plasmid pBOL01 containing the replication origin of the T. tengcongensis chromosome and a kanamycin resistance cassette, in which kanamycin resistance gene expression was controlled by the tte1482 promoter from T. tengcongensis, was constructed and introduced into T. tengcongensis via electroporation. Subsequently, the high transformation efficiency occurred when using freshly cultured T. tengcongensis cells without electroporation treatment, suggesting that T. tengcongensis is naturally competent under appropriate growth stage. A genetic transformation system for this strain was then established based on these important components, and this system was proved to be available for studying physiological characters of T. tengcongensis in vivo by means of hisG gene disruption and complementation.
Journal of Genetics and Genomics 10/2012; 39(10):561-70. · 1.88 Impact Factor
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ABSTRACT: Nitroalkane oxidase (NAO) catalyzes neutral nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His179, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel nitroalkane oxidase with TIM barrel structure.
Biochemical and Biophysical Research Communications 02/2011; 405(3):344-8. · 2.48 Impact Factor
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ABSTRACT: HpdR, an IclR-family regulator in Streptomyces coelicolor, is a substrate-dependent repressor for the tyrosine catabolic gene hppD. In this study, S1 nuclease protection assays revealed that hpdR is subject to a negative autoregulation. Purified HpdR showed specific DNA-binding activity for the promoter region of hpdR, indicating that the autoregulation of hpdR is performed directly. The disruption of hpdR led to reduced production of CDA by S. coelicolor J1501, suggesting a positive effect of hpdR on CDA biosynthesis. Electrophoretic mobility shift assays showed that HpdR specifically bound to the promoter region of hmaS (SCO3229 in the CDA gene cluster), encoding 4-hydroxymandelic acid synthase. Disruption of hmaS in J1501 abolished CDA production. It is possible that hpdR regulates CDA biosynthesis by controlling the transcription of hmaS.
Microbiology 09/2010; 156(Pt 9):2641-8. · 3.06 Impact Factor
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ABSTRACT: In actinomycetes, the onset of secondary metabolite biosynthesis is often triggered by the quorum-sensing signal gamma-butyrolactones (GBLs) via specific binding to their cognate receptors. However, the presence of multiple putative GBL receptor homologues in the genome suggests the existence of an alternative regulatory mechanism. Here, in the model streptomycete Streptomyces coelicolor, ScbR2 (SCO6286, a homologue of GBL receptor) is shown not to bind the endogenous GBL molecule SCB1, hence designated "pseudo" GBL receptor. Intriguingly, it could bind the endogenous antibiotics actinorhodin and undecylprodigiosin as ligands, leading to the derepression of KasO, an activator of a cryptic type I polyketide synthase gene cluster. Likewise, JadR2 is also a putative GBL receptor homologue in Streptomyces venezuelae, the producer of chloramphenicol and cryptic antibiotic jadomycin. It is shown to coordinate their biosynthesis via direct repression of JadR1, which activates jadomycin biosynthesis while repressing chloramphenicol biosynthesis directly. Like ScbR2, JadR2 could also bind these two disparate antibiotics, and the interactions lead to the derepression of jadR1. The antibiotic responding activities of these pseudo GBL receptors were further demonstrated in vivo using the lux reporter system. Overall, these results suggest that pseudo GBL receptors play a novel role to coordinate antibiotic biosynthesis by binding and responding to antibiotics signals. Such an antibiotic-mediated regulatory mechanism could be a general strategy to coordinate antibiotic biosynthesis in the producing bacteria.
Journal of Biological Chemistry 08/2010; 285(35):27440-8. · 4.77 Impact Factor
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ABSTRACT: EndoTT encoded by tte0829 of Thermoanaerobacter tengcongensis binds and cleaves single-stranded (ss) and damaged double-stranded (ds) DNA in vitro as well as binding dsDNA. In the presence of a low concentration of NaCl, EndoTT cleaved ss regions of damaged dsDNA efficiently but did not cleave DNA that was entirely ss or ds. At high concentrations of NaCl or MgCl(2) or ATP, there was also specific cleavage of ssDNA. This suggested a preference for ss/ds junctions to stimulate cleavage of the DNA substrates. EndoTT has six specific sites (a-f) in the oriC region (1-70 nt) of T. tengcongensis. Substitutions of nucleotides around site c prevented cleavage by EndoTT of both sites c and d, implying that the cleavage specificity may depend on both the nucleotide sequence and the secondary structure of the ssDNA. A C-terminal sub-fragment of EndoTT (residues 107-216) had both endonucleolytic and DNA-binding activity, whereas an N-terminal sub-fragment (residues 1-110) displayed only ssDNA-binding activity. Site-directed mutations showed that G(170), R(172) and G(177) are required for the endonuclease activity of EndoTT, but not for DNA-binding, whereas D(171), R(178) and G(189) are partially required for the DNA-binding activity.
Nucleic Acids Research 02/2010; 38(11):3709-20. · 8.03 Impact Factor
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ABSTRACT: Nikkomycins are a group of peptidyl nucleoside antibiotics produced by Streptomyces ansochromogenes. They are competitive inhibitors of chitin synthase and show potent fungicidal, insecticidal, and acaricidal activities. Nikkomycin X and Z are the main components produced by S. ansochromogenes. Generation of a high-producing strain is crucial to scale up nikkomycins production for further clinical trials.
To increase the yields of nikkomycins, an additional copy of nikkomycin biosynthetic gene cluster (35 kb) was introduced into nikkomycin producing strain, S. ansochromogenes 7100. The gene cluster was first reassembled into an integrative plasmid by Red/ET technology combining with classic cloning methods and then the resulting plasmid(pNIK)was introduced into S. ansochromogenes by conjugal transfer. Introduction of pNIK led to enhanced production of nikkomycins (880 mg L(-1), 4 -fold nikkomycin X and 210 mg L(-1), 1.8-fold nikkomycin Z) in the resulting exconjugants comparing with the parent strain (220 mg L(-1) nikkomycin X and 120 mg L(-1) nikkomycin Z). The exconjugants are genetically stable in the absence of antibiotic resistance selection pressure.
A high nikkomycins producing strain (1100 mg L(-1) nikkomycins) was obtained by introduction of an extra nikkomycin biosynthetic gene cluster into the genome of S. ansochromogenes. The strategies presented here could be applicable to other bacteria to improve the yields of secondary metabolites.
Microbial Cell Factories 01/2010; 9:6. · 3.55 Impact Factor
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ABSTRACT: Nikkomycins are a group of peptidyl nucleoside antibiotics and act as potent inhibitors of chitin synthases in fungi and insects. Nikkomycin X and Z are the main components produced by Streptomyces ansochromogenes. Of them, nikkomycin Z is a promising antifungal agent with clinical significance. Since highly structural similarities between nikkomycin Z and X, separation of nikkomycin Z from the culture medium of S. ansochromogenes is difficult. Thus, generating a nikkomycin Z selectively producing strain is vital to scale up the nikkomycin Z yields for clinical trials.
A nikkomycin Z producing strain (sanPDM) was constructed by blocking the imidazolone biosynthetic pathway of nikkomycin X via genetic manipulation and yielded 300 mg/L nikkomycin Z and abolished the nikkomycin X production. To further increase the yield of nikkomycin Z, the effects of different precursors on its production were investigated. Precursors of nucleoside moiety (uracil or uridine) had a stimulatory effect on nikkomycin Z production while precursors of peptidyl moiety (L-lysine and L-glutamate) had no effect. sanPDM produced the maximum yields of nikkomycin Z (800 mg/L) in the presence of uracil at the concentration of 2 g/L and it was approximately 2.6-fold higher than that of the parent strain.
A high nikkomycin Z selectively producing was obtained by genetic manipulation combined with precursors feeding. The strategy presented here might be applicable in other bacteria to selectively produce targeted antibiotics.
Microbial Cell Factories 11/2009; 8:61. · 3.55 Impact Factor
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ABSTRACT: In bacteria, many "atypical" response regulators (ARRs) lack the conserved residues important for phosphorylation by which typical response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. However, such mechanisms have not been established. JadR1, an OmpR-type ARR of Streptomyces venezuelae, appears to activate the transcription of jadomycin B (JdB) biosynthetic genes while repressing its own gene. JadR1 activities were inhibited in cells induced to produce JdB, which was found to bind directly to the N-terminal receiver domain of JadR1, causing JadR1 to dissociate from target promoters. The activity of a NarL-type ARR, RedZ, that regulates production of another antibiotic was likewise modulated by the end product (undecylprodigisines), implying that end-product-mediated control of antibiotic pathway-specific ARRs may be widespread. These results could prove relevant to knowledge-based improvements in yield of commercially important antibiotics.
Proceedings of the National Academy of Sciences 06/2009; 106(21):8617-22. · 9.68 Impact Factor
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ABSTRACT: SSB2 and SSB3 are ssDNA-binding proteins of Thermoanaerobacter tengcongensis. This work aimed to disclose novel properties of both proteins.
We performed electrophoretic mobility shift assays (EMSAs) using oligonucleotides spanning the replication origin of T. tengcongensis and non-denaturing polyacrylamide gels. Western blotting assays were used to study the expression patterns of both proteins.
SSB2 bound to 35-nt, 59-nt and 70-nt ssDNA spanning the replication origin and formed one,two or three DNA-protein complexes. The number of the SSB2-DNA complexes was determined by both the length of the ssDNA and the concentration of SSB2. SSB3 formed one more DNA-protein complex with 59-nt or 70-nt ssDNA in comparison with SSB2. Storage of the proteins at -70 degrees C led to the disappearance of one SSB2-(70-nt) complex, or two SSB3-(59-nt) complexes or three SSB3-(70-nt) complexes in the EMSA, indicating the distinct loss of the SSBs's conformations. Moreover,SSB2 and SSB3 displayed different expression patterns at variable incubation temperatures in vivo.
SSB2 and SSB3 could bind ssDNA with various conformations that were determined by the length of ssDNA, the concentration of the proteins, as well as the temperature of treatment. To our knowledge, this is the first disclosure of the characteristics of SSB2 and SSB3 on 35-70 nt oligonucleotides.
ACTA MICROBIOLOGICA SINICA 05/2009; 49(4):453-9.
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ABSTRACT: The nikkomycin-producing strain Streptomyces ansochromogenes has a homologue (adpA-L) of the key pleiotropic Streptomyces regulatory gene adpA. Gene disruption and genetic complementation revealed that adpA-L was required for both nikkomycin biosynthesis and morphological differentiation. Transcriptional analysis suggested that the transcription of sanG, the specific activator gene for nikkomycin biosynthesis, was dependent on AdpA-L. In gel-shift and DNase 1 footprinting assays, the purified His(6)-tagged recombinant AdpA-L protein bound the upstream region of sanG at five sites, which are spread over more than one kilobase of DNA and most of which is inside the transcribed region. A consensus AdpA-L-binding sequence, 5'-TGGCNNVWHN-3' (V: C, A or G; W: A or T; H: A, T or C; N: any nucleotide) was found in these binding sites. Transcriptional analysis of sanG carrying mutated AdpA-L binding sites showed that transcription of sanG was eliminated when site I was mutated and its trascription was decreased when site V was mutated, whereas it was increased when the binding sites II, III or IV were mutated. Meanwhile, nikkomycin production of the mutated site III strain was enhanced comparing with the wild-type strain as control. This work highlights a new level of complexity in the regulation of nikkomycin biosynthesis.
Molecular Microbiology 04/2009; 72(3):710-23. · 5.01 Impact Factor
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ABSTRACT: Homogentisate 1,2-dioxygenase (HmgA) converts homogentisate to maleylacetoacetate in the pathway of tyrosine catabolism in living organisms. Studies published to date have not elucidated the transcriptional regulation of hmgA and its biological function in Streptomyces spp. Here it is shown that the transcription of hmgA in Streptomyces coelicolor was activated by HpdA, an AsnC-type activator, in the presence of tyrosine. Disruption of hmgA led to enhanced production of diffusible brown pigment, suggesting that hmgA is required for tyrosine catabolism in Streptomyces coelicolor. The production of actinorhodin in the hmgA disruption mutant was several fold higher than that in the wild-type strain (M145), indicating that hmgA of tyrosine catabolism correlates with actinorhodin biosynthesis. Furthermore, S1 mapping showed that the transcription of actII-ORF4, a pathway-specific activator gene of actinorhodin biosynthesis, was increased in the hmgA disruption mutant. These results reveal the transcriptional regulation of hmgA and the positive contribution of hmgA disruption to the actinorhodin biosynthesis in Streptomyces coelicolor.
FEMS Microbiology Letters 04/2008; 280(2):219-25. · 2.04 Impact Factor
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ABSTRACT: Streptomyces coelicolor produces a brown pigment on nutrient-limited agar medium (Tyr-PM) using l-tyrosine as the sole nitrogen and carbon source. The pigment production is associated with the second step of l-tyrosine catabolism catalysed by 4-hydroxyphenylpyruvate dioxygenase (HppD), which converts 4-hydroxyphenylpyruvate (4HPP) to 2, 5-dihydroxyphenylacetate (homogentisate) to provide the carbon and energy substrates for the growth of S. coelicolor on Tyr-PM. An hppD mutant did not produce brown pigment, and its normal growth was impaired on Tyr-PM. hpdA and hpdR, located close to hppD, were identified as activator and repressor genes for hppD transcription in the presence of tyrosine. hpdA, divergently transcribed from hppD, is negatively autoregulated in the absence of tyrosine, whereas it is repressed by both its own protein and HpdR in the presence of tyrosine. Electrophoretic mobility shift assays and footprinting experiments showed that HpdA and HpdR each bind to an overlapping region spanning the promoters of both hppD and hpdA, and that 4HPP, instead of tyrosine, is the specific ligand modulating the binding patterns and footprints of HpdA and HpdR on the hppD-hpdA promoter region. These results suggested that the transcription of hppD is subject to coarse and fine control by a complex regulatory system.
Molecular Microbiology 09/2007; 65(4):1064-77. · 5.01 Impact Factor
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ABSTRACT: Spore cortex-lytic enzymes are essential for germination in Bacilli. A gene-encoding spore cortex-lytic enzyme designated sleB was cloned from Bacillus thuringiensis. Disruption of sleB did not affect vegetative growth of B. thuringiensis, but the fall in optical density at 600 nm in the mutant spores was much slower than in the wild type strain during spore germination induced by L-alanine. Moreover, the mutant spores did not become completely dark, as compared with the wild type strain. These showed that sleB is required for normal spore germination in B. thuringiensis. Reverse transcription polymerase chain reaction analysis indicated that sleB is transcribed during sporulation. Western blot experiment also proved that SleB accumulated in sporulating cells as a precursor protein, and in spores as a mature processed form.
Current Microbiology 05/2007; 54(4):292-5. · 1.82 Impact Factor
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ABSTRACT: One polysaccharide deacetylase gene was cloned from Bacillus thuringiensis and designated pdaA. Disruption of pdaA did not affect vegetative growth and sporulation but obviously affected spore germination. When L-alanine was added into the spore suspension, the spores of the pdaA disruption mutant showed a slow and partial reduction in absorbance at OD600 and became phase pale gray compared with phase dark of the wild-type strain. In contrast with the outgrowing of wild-type spores after germination, the pdaA mutant spores were blocked at the stage of spore germination. Transmission electron micrographs revealed a significant difference between the pdaA mutant and the wild-type strain in the spore cortex. Introduction of the pdaA gene into the pdaA disruption mutant complemented the germination-negative phenotype. Reverse transcription--polymerase chain reaction showed that pdaA was transcribed after incubation for 10 h in CCY medium.
Canadian Journal of Microbiology 11/2006; 52(10):935-41. · 1.36 Impact Factor
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ABSTRACT: DNA sequence analysis of a 7.5 kb XhoI DNA fragment from the region flanking the nikkomycin biosynthesis gene cluster in Streptomyces ansochromogenes revealed one 3.3 kb open reading frame (ORF), designated sanG. The deduced product of sanG (1061 amino acids), which is similar to PimR of Streptomyces natalensis, contains an OmpR-like DNA binding domain in its N-terminal portion and A- and B-type nucleotide binding motifs in the middle of the protein. Disruption of sanG abolished nikkomycin biosynthesis, reduced sporulation and led to brown pigment accumulation. All aspects of this complex phenotype were complemented by a single copy sanG which was integrated into the chromosome. The introduction of multiple copies of sanG resulted in increased nikkomycin production. S1 mapping results indicated that sanG is transcribed from at least three promoters (P1, P2 and P3), P1 being strongly upregulated when production of nikkomycins starts. Two putative transcription units for nikkomycin biosynthesis, starting from sanN and sanO, are dependent on the expression of sanG, whereas a putative transcription unit starting from sanF was not regulated by sanG. These results suggested that sanG encodes a transcriptional activator important for nikkomycin biosynthesis that, unusually, also has pleiotropic effects on secondary metabolism and development.
Molecular Microbiology 04/2005; 55(6):1855-66. · 5.01 Impact Factor
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ABSTRACT: A 1.4-kb DNA fragment from Streptomyces ansochromogenes accelerated mycelium formation of S. ansochromogenes when present on a multicopy plasmid. The DNA fragment contains one complete open reading frame, designated samR, encoding a protein with 213 amino acids that contains a likely DNA-binding helix-turn-helix motif close to its N-terminus. The deduced SamR protein resembles the product of the hppR gene, which is involved in the regulation of catabolism of 3-(3-hydroxyphenyl) propionate in Rhodococcus globerulus. A samR disruption mutant was constructed that presented a bald phenotype and failed to form aerial hyphae and spores. We suggest that samR plays an important role in the emergence of aerial hyphae from substrate mycelium. An almost identical gene of Streptomyces coelicolor was also subjected to gene disruption. Surprisingly, the mutant was able to develop an aerial mycelium, but it remained white and deficient in sporulation instead of forming gray spores.
Archives of Microbiology 04/2002; 177(3):274-8. · 1.43 Impact Factor
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ABSTRACT: A new gene,scrX fromStreptomyces coelicolor was cloned and sequenced. It consists of 660 base pair, encoding a peptide of 220 amino acids. There are three rare codons
inscrX which are AAA, AAA and ATA.scrX gene may be a typical differentiation regulator which was strictly controlled at translational level. The comparison of amino
acids also revealed that ScrX belonged to Ic1R family which acted in transcriptional regulation of prokaryote. Studies on
gene function by gene disruption and complementation indicated thatscrX may play a positive regulation role in spore formation ofStreptomyces coelicolor.
Science in China Series C Life Sciences 03/2000; 43(2):157-168. · 1.61 Impact Factor
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ABSTRACT: The promoters, PTH4 and PTH270 involved in the regulation ofStreptomyces coelicolor differentiation were subcloned intoStreptomyces promoter, i.e. probe plasmid pIJ4083, and the recombinant plasmids, pIJ4470 and pIJ4471, were constructed. Two promoters
could drive the expression of reporter gene encoding catechol dioxygenase when pIJ4470 and pIJ4471 were introduced into some
white mutants (C85, C70, C71, C17 and C119). The total RNA was isolated from these strains containing recombinant plasmid.
Probes were prepared by labelling 5′-ends of PTH4 and PTH270 DNA fragments using radioisotope. DNA -RNA hybridization was carried out with the probes and RNAs isolated from different
strains. The S1 mapping result showed that all RNAs from strains of C85/pIJ4470, C85/4471, C70/pIJ4470, C70/pIJ4471 and C17/pIJ4470
as well as C17/pIJ4471 gave rise to strong positive hybridization signal, whereas RNAs from C71/pIJ4470 and C71/pIJ4471 did
not give any positive signal. RNAs from C119/pIJ4470 and C119/pIJ4471 gave weak hybridization signal. The result indicated
that the transcription of both PTH4 and PTH270 promoters might depend onwhiG, an essential gene inStreptomyces differentiation, and partially depend on whiH, but they did not depend on other differentiation genes (whiA, whiB andurhiI).
Science in China Series C Life Sciences 04/1997; 40(3):246-250. · 1.61 Impact Factor
