A new GntR family regulator Ste1 in Streptomyces sp. 139

APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY

A new GntR family regulator Ste1in Streptomyces sp.139

Liping Bai &Xiaoqiang Qi &Yang Zhang &Chen Yao &Lianhong Guo &Rong Jiang &Ren Zhang &Yuan Li

Received:26March 2013/Revised:17June 2013/Accepted:18June 2013/Published online:5July 2013#Springer-Verlag Berlin Heidelberg 2013

Abstract The novel exopolysaccharide Ebosin produced by Streptomyces sp.139has remarkable in vivo antirheumatic arthritis activity,and its biosynthesis gene cluster (ste )consisting of 27ORFs has been identified.The present inves-tigation focused on the function of ste1gene.Database searching revealed that Ste1is homologous to the GntR family regulator originated from microbes.To confirm its function in Ebosin biosynthesis,the gene was disrupted.The mutant strain Streptomyces sp.139D1was found to have a much higher Ebosin production than that of the wild-type strain,whilst the complementation strain Streptomyces sp.139C1showed a decrease in the exopolysaccharide produced.Real-time qPCR analysis indicated that in the mutant strain Streptomyces sp.139D1,transcription levels of gene ste5,ste8,and ste17increased significantly compared with those in the wild-type strain.The electrophoretic mobility shift assay demonstrated that Ste1binds with higher affinity to the pro-moter 1and 3regions in the ste gene cluster.It is concluded that ste1plays the negative regulation as a transcription re-pressor during Ebosin biosynthesis.Growing on minimal agar medium supplemented with glucose and R2YE agar medium,the mutant strain Streptomyces sp.139D1exhibited a Bld phenotype.

Keywords Gene ste1.GntR family regulator .Ebosin production .Real-time qPCR .The electrophoretic mobility shift assay .Phenotype of the mutant strain

Introduction

Exopolysaccharides (EPSs)are long-chain polysaccharides consisting of branched,repeating units of sugars or sugar derivatives (Welman and Maddox 2003)which are not at-tached permanently to the surface of microbial cells but se-creted into their surroundings during growth (Laws 2001).The EPSs produced by bacteria are involved in many protec-tive cellular functions that improve bacterial competition in different habitats (Fanning et al.2012;Carter et al.2012).The biosynthesis of EPSs includes their assembly on a lipid carrier by sequential transfer of monosaccharides from nucleotide sugars by glycosyltransferases and the subsequent polymeri-zation and export of the repeating units (Van Kranenburg et al.1999).EPSs have been found recently to possess some health benefits such as cholesterol-lowering properties (Jin et al.2012;Lima et al.2008),antitumor activity (Fernandes et al.2011),anti-inflammatory effect (Joo and Yun 2005),and antidiabetic activity (Hwang et al.2008).

Ebosin,a novel exopolysaccharide,produced by Strepto-myces sp.139has remarkable antirheumatic arthritis activity in vivo,and its biosynthesis gene cluster (ste )consisting of 27ORFs has been identified (Wang et al.2003).Some of the ste genes have been functionally characterized (Bai et al.2011;Li et al.2010;Zhang et al.2012)and this study focused on the function of ste1gene.

According to database searching,the product of ste1is homologous to the GntR family of transcriptional regulators which play roles in the reversal of the Bld phenotype and antibiotic production of bacteria (Seo et al.2002;Rigali et al.2002).The Streptomyces coelicolor genome contains 57putative GntR-like proteins (Brandan and Janet 2006).DasR regulates the sugar phosphotransferase system and WhiH plays a crucial role in the septation of aerial hyphae in S.coelicolor (Brandan and Janet 2006;Rigali et al.2006).DasA and DasR are responsible for aerial mycelium forma-tion in Streptomyces griseus (Seo et al.2002).This paper reports the GntR-like ste1gene involved in the biosynthesis of Ebosin.

Liping Bai and Xiaoqiang Qi contributed equally to this work.L.Bai :X.Qi :Y .Zhang :C.Yao :L.Guo :R.Jiang :Y .Li (*)Key Laboratory of Biotechnology of Antibiotics,Ministry of

Health,Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences &Peking Union Medical College,Beijing,China e-mail:yuanwli08@https://www.wendangku.net/doc/8f16051329.html,

R.Zhang

School of Biological Sciences,University of Wollongong,Wollongong,Australia

Appl Microbiol Biotechnol (2013)97:8673–8682DOI 10.1007/s00253-013-5076-6

Materials and methods

Bacterial strains and culture conditions

All strains used in this study were presented in Table1. Streptomyces sp.139was isolated from a soil sample in China and kept in the China General Microbiology Cul-ture Collection Center(no.0405).The strain was cul-tured at28°C with shaking(250rpm)in ether TSB medium supplemented with5mM MgCI2and0.5% glycine or fermentation medium(1%glucose,starch, 2%soybean extract,0.2%tryptone,0.2%beef extract, 0.4%yeast extract,0.05%K2HPO4,0.3%CaCO3,pH 7.3).For morphogenesis study,the ste1gene disruption mutant strain Streptomyces sp.139D1was grown on the minimal medium supplemented with glucose and R2YE agar plates(Van Wezel et al.2005).Escherichia coli DH5αwas grown at37°C in Luria–Bertani(LB) medium.

DNA preparation and Southern blot analysis

Isolation of E.coli plasmid DNA and standard recombinant DNA techniques were performed as described by Sambrook and Russell(2001).Streptomyces plasmid and genomic DNA were isolated as mentioned by Kieser et al.(2000). For Southern blot analysis,a DIG high prime DNA labeling and detection starter kit II obtained from Roche(USA)were used following the instructions of the manufacturer.Disruption of the gene ste1in Streptomyces sp.139

Using Streptomyces sp.139chromosomal DNA as the template,a817-bp fragment(F1)upstream of ste1was PCR amplified with primers P1and P2(Table2).An-other1,019bp fragment(F2)downstream of ste1was also PCR amplified using primers P3and P4(Table2). The PCR amplification was performed under the follow-ing conditions:an initial denaturation at98°C for3min, then30cycles of20s at98°C,30s at62°C,1min at 72°C,and finally10min at72°C.A1.2-kb fragment F3carrying the kanamycin resistance(Km r)gene was digested with Xba I from plasmid pFD666(Denis and Brzezinski1992).The fragment containing F1,F3,and F2was cloned into pKC1139(Bierman et al.1992)to construct plasmid pKC1D.After propagation in E.coli ET12567(MacNeil et al.1992),pKC1D was introduced into Streptomyces sp.139by polyethylene glycol-mediated proto-plast transformation(Kieser et al.2000).Incubated at28°C for 16–20h,the plates were overlaid with soft R2YE(0.7%agar) containing kanamycin(40μg/ml).Plasmid pKC1D bears a temperature-sensitive Streptomyces replication origin (Bierman et al.1992)that is unable to replicate at temperatures above34°C.Therefore,the transformants were first incubated at28°C for2days until pinpoint size colonies became visible and then shifted to37°C for further incubation.Mutants resulted from a double crossover via homologous recombina-tion grew out of the original pinpoint-sized colonies in several days.

Table1Strains and plasmids used in this study

Strains or plasmids Relevant characteristics Source or reference

Strains

Streptomyces sp.139Ebosin-producing strain CGMCC0405 Strain D1ste1knockout mutant of Streptomyces sp.139This study

Strain C1ste1-complemented strain This study

E.coli DH5αF?recA1endA1hsdR17deoR thi-1supE44gyrA96relA1Δ

(lacZYA-argF)U169λ?(φ80dlacZΔM15)

Sambrook et al.2001 E.coli ET12567Methylation-deficient E.coli;dam?dcm?hsdM MacNeil et al.(1992) E.coli BL21(DE3)F?ompT hsdSB(r B?m B?)dcm galλ(DE3)Novagen

Plasmids

pKC1139Shuttle plasmid(E.coli–Streptomyces);pSG5,pBR322;

aac(3)IV lacZa oriT RK2;Am r

Bierman et al.(1992) pKC1D pKC1139-derived plasmid carrying F1,F2,and Km r fragments;Km r Am r This study

pKC1C pKC1139-derived plasmid-carrying0.45-kb ErmE*promoter

fragment and ste1;Am r

This study

pGEM-3Zf-ErmE*Resource of ErmE*promoter;Ap r Zhang et al.(2012) pET30a T7promoter,His-tag;Km r Novagen

pET30a-ste1pET30a-derived plasmid carrying ste1;Km r This study

Ap r ampicillin resistance,Am r apramycin resistance,Km r kanamycin resistance

Complementation of gene ste1disruption mutant

A765-bp ste1fragment was amplified by PCR with the primers P5and P6(Table2)using the genomic DNA of Streptomyces sp.139as template.The0.45-kb fragment of erm E*promoter was isolated from the plasmid pGEM-3zf-erm E*(Zhang et al.2006)digested by EcoR I and BamH I.The erm E*fragment and the765-bp ste1fragment were jointed together by ligation and then inserted into plasmid pKC1139 digested by EcoR I and Hind III to create pKC1C,which was then transformed into E.coli ET12567.After cultivating the recombinant strain with apramycin resistance(Am r),the plas-mid pKC1C was isolated and transformed into the protoplasts of Streptomyces sp.139D1.The complementing strain was named as Streptomyces sp.139C1.

Isolation of Ebosin

Ebosin was isolated from the supernatants of fermentation cultures of Streptomyces sp.139,Streptomyces sp.139D1, and Streptomyces sp.139C1(at28°C for96h),respectively, as described before(Jing et al.2003).

Assay for Ebosin activity

An enzyme-linked immunospecific assay(ELISA)method was used to analyze the competitive binding activity of isolated Ebosin with interleukin-1(IL-1)for IL-1R(14). IL-1in100μl(0.01μg,PeproTECH)was coated onto a 96-well immunoplate(Nunc)at4°C overnight.To each well,250μl of3%BSA in phosphate-buffered saline (PBS)buffer(KH2PO40.024%,Na2HPO40.363%,KCl 0.02%,and NaCl0.8%,pH7.4)was added and the plate was kept at4°C for4h,followed by washing three times with PBS and PBS+0.05%Tween20(PBST),respectively, and blotted dry.The EPSs were then diluted in PBS and added50μl to each well,at mean time;50μl of IL-1R (1:100,R&D)was also added to each well.The plate was incubated at4°C for3h.After binding,the plate was washed by PBS and PBST three times separately,then100μl of diluted solution(1:1,000)of goat polyclonal anti-human IL-1R antibody(R&D)was transferred into each well and the plate was allowed to stand at4°C for1h.The plate was washed again as above,and100μl of diluted solution (1:1,000)of second antibody(rabbit polyclonal anti-goat IgG antibody conjugated with SA-HRP,Promega)was added to each well and the plate was kept at4°C for1h. After final washes as above,100μl of3,3′,5,5-tetramethyl benzidine dihydrochloride solution was trans-ferred into each well,and reaction took place at room temperature for1h(solution turned blue in color)before being stopped by the addition of100μl of2N HCl. The absorbance at450nm was recorded as a measure-ment of the reaction.

Table2Primers used in

this study Sequence(5′→3′)Primer name DNA fragments

CTGGAATTCCACCATCGTCGCCACCT P1(Eco RI)F1

GCATCTAGAGGCTCCCGCTACAAGTTC P2(Xba I)F1

GCATCTAGACGCTCGTCGGTCATCTGC P3(Xba I)F2

AGCAAGCTTGTCTTGGGCGTGTCCTTG P4(Hin dIII)F2

CGCGGATCCATGAGTACGGACGTCAGCAG P5(Bam HI)ste1

CCCAAGCTTTCACCCCCGCGGTCGCCGCAGT P6(Hin dIII)ste1

ATCGGCCACGTCACTTG P7Promoter1

CCTGGCGCCGCGGAATGT P8Promoter1

CGCAAGGCATTCCTGAGG P9Promoter2

GGATGCCGTTCCACTGCT P10Promoter2

TGCCTTCCGGCTGCGGTGTC P11Promoter3

CGCGAACGGCTGGACAAC P12Promoter3

Real-time qPCR

GCTGATCCTGCTGGTGGTGC

CCATCGTGCGGAACTTGAGG ste5F ste5

CTCGGCAAGCTCAGCCAGAC ste5R ste5

CGAGCAGCAGGAACAGCACC ste8F ste8

CTGGACGGCGACGAGAT ste8R ste8

CGACGCAGTGGAACGAG ste17F ste17

TGGTCGAGGTCATCAACAAG ste17R ste17

TGGACCTCGATGACCTTCTC hrdB F hrdB

hrdB R hrdB

Cloning and expression of the ste1gene in E.coli

The765-bp ste1gene fragment was cloned into the plasmid pET30a(Invitrogen)digested with BamH I and Hind III to construct pET30a-ste1.Then,the plasmid was transformed into E.coli BL21(DE3).An overnight cul-ture of E.coli BL21(DE3)/pET30a-ste1was diluted 1:100with LB broth and subjected to further incubation at37°C until the absorbance at600nm reached～0.6. Isopropyl-β-thiogalactoside was added to the culture at a final concentration of0.6mM.After further incubation at 37°C for10h,the bacterial cells were harvested by centrifugation(3,000×g,10min)and suspended in the binding buffer(5mM imidazole,0.5M NaCl,20mM Tris–Cl,pH8.0).The cells were sonicated and the cell debris was removed by centrifugation(14,000×g,10min) at4°C.

Purification of the recombinant Ste1

After centrifugation,the supernatant was collected and load-ed on a2.0-ml Ni-NTA His-Bind resin column(Novagen) pre-equilibrated with a binding buffer.The column was then eluted with5ml of the binding buffer,10ml of the washing buffer(60mM imidazole,0.5M NaCI,20mM Tris–HCl,pH 8.0),and5ml of the eluting buffer(250mM imidazole, 0.5M NaCI,20mM Tris–HCl,pH8.0)successively.The fractions containing the recombinant protein Ste1were col-lected and His-tagged Ste1was dialyzed with H2O at4°C and stored at4°C for further use.

Real-time qPCR analysis

After the mutant strain Streptomyces sp.139D1and the complementing strain Streptomyces sp.139C1were cul-tured at28°C for36h,the mycelia of the two strains were collected with centrifugation(3,000×g,10min)and washed by PBS buffer individually(NaCl137mM,KCl 2.7mM,Na2HPO410mM,KH2PO42mM,pH7.4).Total RNAs of the mycelia from the strains were isolated respec-tively with Pure Yield RNA Midiprep System(Promega, Madison,USA)according to the manufacturer’s instruc-tion.Contaminating DNA was removed using DNAse (TaKaRa)digestion.With Superscript III First-Strain syn-thesis system kit for RT-PCR(Invitrogen),cDNA was syn-thesized based on the manufacturer’s protocols.Amplifica-tion and detection of cDNA in qPCR were performed with FastStart Universal SYBR Green Master kit(Roche)fol-lowing the manufacturer’s instructions using50ng cDNA per reaction.All transcripts were normalized relative to HrdB(RNA polymerase principal sigma factor)transcript quantities.The primers used in these real-time qPCR re-actions are listed in Table2.Electrophoretic mobility gel shift assay

To evaluate the ability of the Ste1binding to the promoter regions in the ste gene cluster,the electrophoretic mobility gel shift assay(EMSA)was performed.A Biotin3′End DNA Labeling Kit and a LightShift Chemiluminescent EMSA Kit(Pierce)were used following the instructions of manufacturer.In this assay,the protein Ste1was purified as mentioned above.The promoters1,2,and3in the gene cluster predicted by the BDGP Neural Network Promoter Prediction(https://www.wendangku.net/doc/8f16051329.html,/seq_tools/promoter.html) were used in the EMSA assay,which were PCR amplified with primers P7–P12(Table2),respectively,using the ge-nomic DNA of Streptomyces sp.139as a template.

Morphogenesis analysis of the strains by scanning electron microscopy

Morphological studies of surface-grown aerial hyphae and spores of Streptomyces sp.139,Streptomyces sp.139D1, and Streptomyces sp.139C1were performed with a FEI Quanta200scanning electron microscope(Li et al.2007). The strains were grown on the minimal medium with glucose and R2YE agar plates(Sambrook et al.2001)at28°C for 4days and the agar blocks containing spores and hyphae were cut.For preparation of the specimens,the agar blocks were fixed with2.5%glutaraldehyde(in0.1M phosphate buffer,pH7.0)at4°C overnight,then with1%osmium tetroxide(in0.1M phosphate)for2–4h.Afterwards,each specimen was dehydrated successively by30,50,70,85,95, and100%of ethanol for15–20min each before air drying for1h.The specimens were finally sputter coated with platinum–gold and examined using the microscope. Statistical analysis

Data were showed as the mean±SD from at least three independent experiments.The significance of differences between groups was evaluated by Student’s t test.P values less than0.05were considered significant.

Results

Homology analysis between Ste1and GntR family transcriptional repressors

The DNA sequence of ste1presented in this study was depos-ited in GenBank under accession number AY131229.The protein sequence alignment of Ste1and two members of the GntR family transcriptional repressors were shown in Fig.1. Ste1bears75%identity and82%similarity over253-aa region with the transcriptional repressor DasR in S.griseus

(Seo et al.2002).To SCO5231of S.coelicolor A3(2)(Bentley et al.2002;Hsiao and Kirby 2008),identity and similarity were respectively 73and 82%over the 253-aa region.Construction of gene ste1disruption mutant and gene complementation

To examine the function of ste1relating to Ebosin biosyn-thesis,the gene was disrupted with a double crossover gene knockout process (Fig.2a ).The expected mutant colonies (Km r Am s )were selected randomly,and genomic DNA of the mutant strain Streptomyces sp.139D1and wild-type strain Streptomyces sp.139were digested,respectively,with BamH I and then subjected to agarose gel electrophoresis.With the 817-bp fragment (F1)upstream of ste1as a probe,southern hybridization was carried out.As shown in Fig.2b ,a distinctive hybridization band of 6.5kb was detected in the mutant strain and a 5.8-kb band appeared in the wild-type strain as predicted.These confirmed that the kanamycin resistance cassette had been integrated into ste1in the strains with Km r Am s ,which therefore lost the gene function of ste1.

Gene complementation of the knockout mutant was performed by transforming the mutant strain Streptomyces sp.139D1with pKC1C.It was evidenced by the transformants with resistances to both apramycin and kanamycin (Am r Km r )and correct restriction digestion mapping of the isolated plas-mid by BamH I and Hind III (not shown).The complementing strain was named Streptomyces sp.139C1.

Ebosin production of the mutant strain Streptomyces sp.139D1and the complementing strain Streptomyces sp.139C1Using the method described by Jing et al.(2003),Ebosin was isolated from the supernatants of fermentation cultures of Streptomyces sp.139,Streptomyces sp.139D1,and Strep-tomyces sp.139C1(28°C for 96h).As shown in Fig.3a ,the crude extract of Ebosin in the mutant strain was 6.435±0.295g/l (P <0.05),remarkably higher than that of the wild-type strain (4.47±0.02g/l).Complementation of the mutant strain resulted in a reduction of the EPS production (5.325±0.045g/l)comparing with the wild-type strain.The-se imply that the ste1gene is a negative regulator for the Ebosin biosynthesis gene cluster.

During fermentation of the strains,the mycelium weights and pH values were checked at 28°C for 24,48,72,and 96h,respectively.The results showed that the mycelium weight of the wild-type strains was higher than that of Streptomyces sp.139D1and Streptomyces sp.139C1(Fig.3c ),but the changes in pH value were similar for the three strains during fermentation (Fig.3d ).

Bioactivities of Ebosin subsisted in the fermentation culture of Streptomyces sp.139D1and Streptomyces sp.139C1The ELISA method mentioned before (14)was used to analyze the competitive binding activity of Ebosin with IL-1for IL-1R.After cultivation at 28°C for 96h,an aliquot of the supernatant from each fermentation

culture

Fig.1Amino acid alignment of Ste1(AAN04228)with the transcriptional repressors in the GntR family.DasR from S.

griseus (Q8VV01)and SCO5231(DasR)from S.coelicolor A3(2)(NP_629378)are shown

Fig.2Functional deletion of ste1.a Homologous recombination scheme for deletion of ste1gene in

Streptomyces sp.139.Restriction maps of the wild-type

Streptomyces sp.139and Strain D1(ste1-deleted mutant)show the predicted fragment sizes upon BamH I digestion.b Southern blotting analysis of Streptomyces sp.139D1and the wild-type Streptomyces sp.139.Chromosome DNA digested with BamH I,F1fragment as a hybridization probe.1Streptomyces sp.139D1;2Streptomyces sp.

139

Fig.3Analysis for fermentation of Streptomyces sp.139,Streptomyces sp.139D1,and Streptomyces sp.139C1.a The production of Ebosin from Streptomyces sp.139,Streptomyces sp.139D1,and Streptomyces sp.139C1.b Mycelium wet weight of the fermentation process at different times.c pH of the fermentation process at different times.d The competitive binding activities of the fermentation supernatant with IL-1for IL-1R.An asterisk indicated a statistically significant difference compared to the wild type (P <0.001)

of Streptomyces sp.139D1,Streptomyces sp.139C1,and the wild-type strain was diluted 100-fold with PBS and 1ml was used for the assay.The binding activity of Ebosin from the mutant strain was 48.4%(P <0.001),which is significantly higher than that of the wild-type strain (17.5%).Comparing with the mutant strain,the binding activity of Ebosin of the gene-complementing strain decreased to 24.53%(Fig.3b ).Such results proved again that the ste1gene may function as a neg-ative regulator during Ebosin biosynthesis.

Transcription levels of the genes ste5,ste8,and ste17

involved in Ebosin biosynthesis were regulated by gene ste1To investigate the effects of gene ste1on transcription levels of the genes involved in Ebosin biosynthesis,real-time qPCR was used to quantify the amount of transcripts of the genes ste5,ste8,and ste17encoding galactosyltransferase (Wang et al.2003),chain length determinants (Wzz)(Wang et al.2003),and α-D -glucose-1-phosphate cytidylyltransferase (Qi et al.2009),respectively,at 36h culture.

Results showed (Fig.4)that in the mutant strain Streptomyces sp.139D1,a dramatic increase in the transcription levels of ste5,ste8,and ste17was seen at 36h,which were 3.3-(P <0.01),2.3-(P <0.05),and 6.1-(P <0.001)folds,respectively,higher than those of wild-type strain Streptomyces sp.139.Whilst,in the complementing strain Streptomyces sp.139C1,the tran-scription levels of these three genes were 46.3,76.7,and 43.1%,respectively,lower than those of the mutant strain Streptomyces sp.139D1.These indicated that the ste1gene played negative regulation in transcription levels during Ebosin biosynthesis.

Ste1binds with high affinity to the promoter region of the gene cluster in Streptomyces sp.139

The predicted promoters 1,2,and 3located upstreams of ste5,ste2,and ste1(Fig.5a )were amplified by PCR with primers P7–P12(Table 2),respectively,and used in the EMSA assay together with the recombinant Ste1protein expressed in E .coli (Fig.5b ).As shown in Fig.5c ,Ste1binds to the pro-moters 1and 3with higher affinity in the gene cluster (ste )resulting in the retardations of the two biotin-labeled DNA fragments,respectively.However,the labeled promoter 2fragment remained un-retardid at the same Ste1protein con-centration indicating that Ste1did not bind to the promoter 2region in the gene cluster (ste ).

Ste1is required for morphogenesis of Streptomyces sp.139Repeated plating showed that the ste1mutant Streptomyces sp.139D1made no spores on either the minimum medium supplemented with glucose or R2YE agar medium.It was observed by cryo-scanning electron microscopy that the mutant strain grown on the two media for 4days exhibits a strictly vegetative growth (Bld phenotype).When the gene-complementing strain Streptomyces SP.139C1was cultivat-ed for 4days,abundant aerial hyphae were produced,which were similar as the wild-type strain Streptomyces sp.139growing on the same media (Fig.6a,b ).

Discussion

Many bacteria are known to produce polysaccharides,which can either be secreted into the environment as exopolysaccharides (Laws 2001),form a capsule around the cell as capsular polysaccharides,or attach to the cell membrane as the O-antigens of lipopolysaccharides (Rob-erts 1996).Different classes of EPSs can be distinguished on the basis of their biosynthesis mechanisms and precursors required (Alan and Maddox 2003).The processes of EPS synthesis are very complex and are regulated at both tran-scriptional and posttranscriptional levels,with multiple reg-ulatory systems (Janczarek 2011).In Sinorhizobium meliloti ,several regulatory genes of EPS1and EPS2syn-thesis have been identified.Among them,the exoR ,exoS ,exoX ,and exsB genes negatively affect EPS1synthesis,and mucR negatively regulates EPS2synthesis (Janczarek 2011).

On the other hand,the SyrM and PhoB proteins are positive regulators of EPS1and EPS2production (Bahlawane et al.2008).A phosphorylated form of PhoB activates the transcrip-tion of target genes by binding to the PHO box sequence located in the promoters of phosphate-regulated genes (Y uan et al.2006).In Rhizobium leguminosarum ,up to now,only

a

Fig.4Effects of ste1gene on transcription levels of ste5,ste8,and ste17in the ste gene cluster.qRT-PCR was used to quantify the amounts of transcripts produced at 36h liquid culture.All transcripts were normalized relative to HrdB (RNA polymerase principal sigma factor)transcript quantities.Asterisks indicated statistically significant differ-ence:ste5(P <0.01),ste8(P <0.05),and ste17(P <0.001)compared to the wild type

few regulatory genes involved in this process have been described (Janczarek 2011).psiA (a polysaccharide inhibition gene)and psrA (a polysaccharide restoration gene)were the first to be identified among these genes and have been found on the symbiotic megaplasmid pSym of R .leguminosarum .For proper EPS production,a balanced number of psiA ,pssA ,and psrA copies is required.A psrA mutant produces a de-creased amount of EPS in comparison to the wild-type strain,showing a positive role of this gene in EPS production.In contrast,the gene exoR negatively regulates EPS production.Streptomyces ,a group of Gram-positive bacteria,are well known as important industrial microorganisms for their pro-duction of naturally derived antibiotics.As producers of around two thirds of all known antibiotics,the soil-dwelling filamentous streptomycetes are a paradigm of secondary metabolite-producing microorganisms (Hopwood 1999).Genes for the biosynthesis of secondary metabolites are com-monly grouped together in clusters on the chromosome in-cluding their pathway-specific regulatory genes.Pathway-specific regulators can affect positively (activators)or nega-tively (repressors)on the expression of gene cluster elements (Bibb 2005;Olano et al.2008).S .coelicolor ,for example,produces several antibiotics (actinorhodin calcium-dependent antibiotic,undecylprodigiosin,and methylenomycin)and the onset of their biosynthesis is controlled by specific regulators (actII-orf4,cdaR ,redD ,and redZ ).At mean time,there

are

Fig.5a Gene organization around ste1234and dasRABC from S.griseus and S.coelicolor A3(2).b SDS-PAGE image of recombinant Ste1protein.M protein molecular weight markers;lane 1supernatant of cell lysate after sonication;lane 2pellet of cell lysate after sonication;lane 3after affinity chromatography.c Electrophoretic mobility analysis

(EMSA)of His-tagged Ste1binding to different putative promoter re-gions.Biotin-labeled promoters 1,2,and 3were incubated with (+)or without (?)purified His-tagged Ste1.The arrows indicated the DNA –protein

complexes

Fig.6Phenotype of Streptomyces sp.139,Streptomyces sp.139D1,and Streptomyces sp.139C1on different media.Scanning electron micrographs of Streptomyces sp.139,Streptomyces sp.139D1,and

Streptomyces sp.139C1grown on a minimal agar medium and b R2YE agar medium

several pleiotropic genes(afs,abs,and bld)which affect not only the antibiotic production,but also the morphological development of the bacterium(Huang et al.2005).One intu-itive approach for the improvement of antibiotics production seems to be deregulation of the expression of secondary metabolite pathways with overexpression of pathway-specific positive regulators or by inactivation of pathway re-pressors(38).

Although the Ebosin biosynthesis gene cluster in Strepto-myces sp.139has been identified and the function of several ste genes characterized,nothing is known of the regulation at molecular level.The ste1gene is predicted to specify a transcriptional regulator,and for understanding its role in the biosynthesis of Ebosin,the gene knockout approach was taken in this study.The remarkable increase in Ebosin pro-duction by the ste1mutant Streptomyces sp.139D1com-pared to the wild-type strain indicates the possible negative role of the gene product as a transcription repressor.This was also supported by gene complementation experiment and Ebosin bioactivity assays.The transcription repressor role was confirmed by real-time qPCR analysis and electropho-retic mobility gel shift assay,which demonstrated that the Ste1protein binds to promoters1and3of the ste cluster.

The scanning electron microscopy revealed that the mu-tant strain Streptomyces sp.139D1growing on both of the minimum medium with glucose and R2YE agar medium appeared Bld phenotype typical of bld mutant,suggesting another role of Ste1in the formation of aerial hyphae and spores.Many genes,especially those bld genes,have been identified functional in this developmental process of Strep-tomyces(Bibb2005)and two gntR family genes dasA and dasR found playing essential roles in S.griseus(Seo et al. 2002).Our results here also demonstrated that ste1,as a gntR-like gene,is responsible for aerial mycelium formation of Streptomyces sp.139.

Acknowledgments This research has been supported by a grant from the Natural Science Foundation of China(31070086)and by the Fun-damental Research Funds for the Central Universities in China (2012N09).A grant from the Natural Science Foundation of Beijing, China(5092020)also supported this project.

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感受现代科技

感受现代科技 【学习目标】 1、知识：感受现代科技给人类生活带来的新变化，认识科技与生活，科技发展与社会发展的关系，懂得“科学技术是第一生产力”的道理。 2、能力与情感：感悟现代科技的神奇与力量，理解科技是社会发展的强大推力，激发学生 对科技重要性的认识，增强学生对科学的兴趣，培养学生热爱科学的精神。 3、过程与方法：依据教学内容和学生的认识规律设置了“课前预习”、“课堂助学”、“课堂巩固”、“课后拓学”、“教学反思”五个模块的教学整合，运用多媒体等教学手段，采用自主体验、 探究活动、案例情境等方法来完成教学目标。 【学习重点、难点】 领略现代科技的神奇与力量，理解“科技是第一生产力”。 【学习过程】 一、预习初探： （一）快快行动，书外的知识真有趣： 1、生活体验：观察生活，请你说说我们身边有哪些科技产品？例举实例说说这些科技产品给我们的生活带来哪些新变化？ 2、想象天地：展现你的想象天份，想象你准备发明一样科技产品，使你的未来生活更美好。 3、图片收集：上网收集有关科技产品的图片，准备创办科技小展览，领略现代科技的神 奇与力量。 （二）阅读课本，书本的知识真寻味： 4、我们现在的生活与科技________________。丰足的衣食，舒适的住行，千百年来一直是人类_________________。 5、科学技术是________________的强大推力，是________生产力。______________已成为当代经济发展的火车头。 6、________________是人类文明的标志。科学技术的进步为人类创造了巨大的 ______________和_________________。

《交通规划》课程教学大纲

《交通规划》课程教学大纲 课程编号：E13D3330 课程中文名称：交通规划 课程英文名称：Transportation Planning 开课学期：秋季 学分/学时：2学分/32学时 先修课程：管理运筹学，概率与数理统计，交通工程学 建议后续课程：城市规划，交通管理与控制 适用专业/开课对象：交通运输类专业/3年级本科生 团队负责人：唐铁桥责任教授：执笔人：唐铁桥核准院长： 一、课程的性质、目的和任务 本课程授课对象为交通工程专业本科生，是该专业学生的必修专业课。通过本课程的学习，应该掌握交通规划的基础知识、常用方法与模型。课程具体内容包括：交通规划问题分析的一般方法，建模理论，交通规划过程与发展历史，交通调查、出行产生、分布、方式划分与交通分配的理论与技术实践，交通网络平衡与网络设计理论等，从而在交通规划与政策方面掌握宽广的知识和实际的操作技能。 本课程是一间理论和实践意义均很强的课程，课堂讲授要尽量做到理论联系实际，模型及其求解尽量结合实例，深入浅出，使学生掌握将交通规划模型应用于实际的基本方法。此外，考虑到西方在该领域内的研究水平，讲授时要多参考国外相关研究成果，多介绍专业术语的英文表达方法以及相关外文刊物。课程主要培养学生交通规划的基本知识、能力和技能。 二、课程内容、基本要求及学时分配 各章内容、要点、学时分配。适当详细，每章有一段描述。 第一章绪论（2学时） 1. 交通规划的基本概念、分类、内容、过程、发展历史、及研究展望。 2. 交通规划的基本概念、重要性、内容、过程、发展历史以及交通规划中存在的问题等。

第二章交通调查与数据分析（4学时） 1. 交通调查的概要、目的、作用和内容等；流量、密度和速度调查；交通延误和OD调查；交通调查抽样；交通调查新技术。 2. 交通中的基本概念，交通流量、速度和密度的调查方法，调查问卷设计与实施，调查抽样，调查结果的统计处理等。 第三章交通需求预测（4学时） 1. 交通发生与吸引的概念；出行率调查；发生与吸引交通量的预测；生成交通量预测、发生与吸引交通量预测。 2. 掌握交通分布的概念；分布交通量预测；分布交通量的概念，增长系数法及其算法。 3. 交通方式划分的概念；交通方式划分过程；交通方式划分模型。 第四章道路交通网络分析（4学时） 1. 交通网络计算机表示方法、邻接矩阵等 2. 交通阻抗函数、交叉口延误等。 第五章城市综合交通规划（2学时） 1. 综合交通规划的任务、内容；城市发展战略规划的基本内容和步骤 2. 城市中长期交通体系规划的内容、目标以及城市近期治理规划的目标与内容 第六章城市道路网规划（2学时） 城市路网、交叉口、横断面规划及评价方法。 第七章城市公共交通规划（2学时） 城市公共交通规划目标任务、规划方法、原则及技术指标。 第八章停车设施规划（2学时） 停车差设施规划目标、流程、方法和原则。 第九章城市交通管理规划（2学时） 城市交通管理规划目标、管理模式和管理策略。 第十章公路网规划（2学时） 公路网交通调查与需求预测、方案设计与优化。 第十一章交通规划的综合评价方法（2学时） 1. 交通综合评价的地位、作用及评价流程和指标。 2. 几种常见的评价方法。 第十二章案例教学（2学时）