Patent Application: US-22405007-A

Abstract:
described are methods and means for metabolic engineering and improved product formation by a filamentous micro - organism or a low g + c gram - positive bacterium . disclosed is that dasr and dasr binding sites play an important and universal role in the control of gene expression in micro - organisms . based on this finding , provided are multiple useful applications , such as a method for regulating the expression of a gene of interest , a method for controlling metabolism , a method for decreasing undesired expression and many more . moreover , provided are means that can be used to establish said methods : for example a micro - organism in which the dasr binding site in operable linkage with a particular gene has been modified to obtain increased or decreased expression of a protein encoded by said gene .

Description:
e . coli dh5α , and bl21 ( de3 ) were used for subcloning and dasr overexpression experiments . s . coelicolor m145 , m510 ( m145 δredd ), m511 ( m145 δactii - iv ) and m512 ( m145 δactii - iv δredd ) ( floriano and bibb , 1996 ) and streptomzyces lividans 1326 were all obtained from the john innes centre strain collection , streptomyces avermitilis nrrl 8165 ( ma - 4680 ), streptonzyces hygroscopicus atcc27438 , streptomyces limosus atcc 19778 , streptomyces rinzosus atcc 10970 , streptonzyces roseosporus atcc 31568 and streptonzyces venezuelae atcc15439 were obtained from the atcc strain collection and streptomyces acrinzycini dsm 40540 , streptomyces cinnanzonensis dsm 40467 , streptomyces clavuligerus nrrl 3585 , streptomyces collinus dsm 40733 and streptomyces griseus nrrl b2682 from the dsmz strain collection . the dasr mutant bap29 ( δdasr :: accc4 ) was created by replacing the coding region of the gene by the apramycin resistance gene cassette , using plasmid pwhm3 , according to a routine procedure ( nothaft et al ., 2003 ). the same strategy was used to create the knock - out mutants for ptsh ( bap1 ), crr ( bap2 ), ptsi ( bap3 ), nage1 ( bap4 ), nage2 ( bap5 ), and nage1 / e2 ( bap6 ). bap1 - 3 were published previously ( nothaft et al ., 2003 ). s . coelicolor strains were grown at 28 ° c . using tryptic soy broth without dextrose as complex medium ( tsb , difco ) or minimal medium ( van wezel et al ., 2005 ). e . coli cultures were grown in luria - bertani broth ( lb ) at 37 ° c . phenotypic characterization of mutants was done on minimal medium agar plates with various carbon sources as indicated in the text ( kieser et al ., 2000 ). quantification of act and red was performed as described previously ( martinez - costa et al ., 1996 ). a 222 - bp dna fragment corresponding to the − 202 /+ 8 region relative to the start of s . coelicolor crr gene ( sco1390 ) was chosen for dnase i footprinting . the dna fragment was amplified from chromosomal dna by pcr . 50 fmoles of 32 p end - labelled probe were incubated with the relevant proteins ( dasr -( his ) 6 and / or bsa ) and dnasei ( 0 . 4 μg / ml ) as described ( sambrook et al ., 1989 ). computational prediction . multiple alignments and position weight matrices were generated as described previously ( rigali et al ., 2004 ) by the target explorer automated tool on the world wide web at trantor . bioc . columbia . edu / target explorer !) ( sosinsky et al ., 2003 ). the weight matrix was deposited as “ dasr4 ”. the minimum score obtained by a sequence scanned by matrix dasr4 is − 38 . 55 and the maximum score is 17 . 25 . according to the current experimental validations , a dasr - binding site could be defined as a sequence of 16 nucleotides that , when scanned by the dasr4 matrix , obtains a score comprised between higher than 6 ( and up to 17 . 25 ). illustratively , a truly and experimentally validated dasr - binding site with a score of only − 2 . 97 has been found upstream of gdha encoding a nadp - specific glutamate dehydrogenase . transmission electron microscopy ( tem ) for the analysis of thin sections of hyphae and spores was performed with a philips em410 transmission electron microscope ( mahr et al ., 2000 ). phase contrast micrographs were produced using a zeiss standard 25 phase - contrast microscope , and a 5 megapixel digital camera . uptake assays with 20 μm n -[ 14 c ] acetyl - d - glucosamine ( 6 . 2 mci mmol − 1 ) into mycelia were performed as described ( nothaft et al ., 2003 ). purification of recombinant histidine - tagged dasr ( rigali et al ., 2004 ) and western blot analysis with antibodies raised against hpr and iia crr have been described elsewhere ( nothaft et al ., 2003 ). rna was isolated from mycelium of s . coelicolor m145 and bap29 . minimal medium cultures containing 50 mm glycerol were inoculated with spores and grown until od 550 of 0 . 6 ( exponential growth ). n - acetylglucosamine was added at 0 . 5 % and samples were taken after 0 , 15 , 30 and 60 minutes . rt - pcr analyses were conducted with the superscript iii one - step rt - pcr kit ( invitrogen ). rt - pcrs without reverse transcription were used as control for absence of residual dna . for semi - quantitative analysis , samples were taken at three - cycle intervals between cycles 18 to 35 to compare non - saturated pcr product formation ( van wezel et al ., 2005 ). data were verified in three independent experiments . oligonucleotides used for the rt - pcr experiments described in fig2 b were : for redz ( 5 ′- cgacatgaaagtgcaggtgg - 3 ′ ( seq id no : 518 ) and 5 ′- tcgggctfggtcagcaaaagc - 3 ′ ( seq id no : 519 )), for actii - orf4 ( 5 ′- gctgcagacgtacgtgtaccacac - 3 ′ ( seq id no : 520 ) and 5 ′- gcgtcgatacggagctgcattcc - 3 ′ ( seq id no : 521 )), for redd ( 5 ′- tcatgggagtgcggagaacgcg - 3 ′ ( seq id no : 522 ) and 5 ′- cgccccacagttcgtccaccag - 3 ′ ( seq id no : 523 )), sco6273 ( 5 ′- cgggggcgaactcgtcaaggtc - 3 ′ ( seq id no : 524 ) and 5 ′- gccgagatgtcgatgaggacgcgg - 3 ′ ( seq id no : 525 )), for kaso ( 5 ′- gcgggatgctcagtgagcacgg - 3 ′ ( seq id no : 526 ) and 5 ′- gacgaggtcggcgaggacggg - 3 ′ ( seq id no : 527 )) and for rpsi ( 5 ′- gagaccactcccgagcagccgc - 3 ′ ( seq id no : 528 ) and 5 ′- gtagcggttgtccagctcgagca - 3 ′( seq id no : 529 )). mycelia of s . coelicolor m145 and bap29 were grown in minimal medium with 50 mm glycerol , harvested at different time points within exponential phase , washed , resuspended in 20 mm hepes , ph7 . 5 and 50 mm mgso 4 and sonicated ; cell debris was removed after centrifugation . dna and rna were eliminated by dnase and rnase treatment . the proteins extracts were dialysed twice at 4 ° c . against water , followed by addition of 6 m solid urea and 2m thiourea , tritonx - 100 ( 2 . 5 % ( v / v )) ipg buffer ( 0 . 5 % ( v / v )), dtt ( 25 mm ) and bromophenolblue . membrane proteins were removed by ultracentrifugation for 1 h at 65 . 000 g . 1 . 5 mg of the cytoplasmic protein fraction was applied on 24 - cm ipg strips ( ph range 4 - 7 ) on an ipgphor unit ( amersham / pharmacia ). the ipg strips were subjected to 12 . 5 % polyacrylamide gels that were run on the ettan dalt ii system ( amersham / pharmacia ). the gels were stained with phastgel blue r and scanned . proteome patterns were compared using two gel sets derived from independent experiments . protein intensities were analysed by densitometric gray scale analysis with tina software ( raytest ). protein spots were excised , subjected to in - gel digestion with trypsin and analysed by liquid chromatography tandem mass spectrometry ( lc - ms / ms ) ( marvin - guy et al ., 2005 ). emsas were performed with fluorescent probes ( 10 nm ) with an alf express sequencer ( filee et al ., 2001 ). purified dasr ( 3 μm ) and 1000 - fold excess of non - specific dna were used in the reaction mixture . predicted cis - acting elements were taken from the promoter regions of actii - orf4 ( sco5085 ; 5 ′- cacattgaaatc tgttgagtaggcctgt tattgtcgcccc - 3 ′ ( seq id no : 530 )), and redz ( sco5881 ; 5 ′- acaagatcttct tgaggtggaaaccact ttcgtatcagtct - 3 ′ ( seq id no : 531 )). known cis - acting elements upstream of crr ( sco1390 ; ccgtgaggag tgtggtctagacctct aatcggaaca - 3 ′ ( seq id no : 532 )), and crp ( sco3571 ; 5 ′- tgcggcatccttgtgacagatcacactgtttggact - 3 ′ ( seq id no : 533 )) were used as positive and negative controls , respectively . the 16 nt dre sites are underlined . chitinase activity was determined as described previously ( zhang et al ., 2002 ) using a colorimetric assay with carboxymethylchitin - remazol brilliant violet 5r ( loewe biochemica gmbh , germany ) as substrate . d - ala - d - ala aminopeptidase measurements were performed with d - ala - paranitroanilide as substrate ( cheggour et al ., 2000 ). bca protein assay ( pierce ) was used for determining protein concentrations . samples ( 1 μl ) of diluted spore suspensions were spotted on minimal medium plates containing 0 . 5 % mannitol with or without 1 % glcnac and incubated three days at 28 ° c . for the bioassay , we inoculated 10 ml of molten soft nutrient agar ( sna ) with 500 μl of a bacillus subtilis overnight culture ( 0d 600 ˜ 1 ), and poured the mixture into square 12 - cm - side petri dishes . plates were kept 2 h at 4 ° c . to solidify sna and to allow diffusion of antibiotics produced , then incubated overnight at 30 ° c . to allow the s . coelicolor genome to be scrutinized for the occurrence of the dasr operator site , we performed dnasei footprinting on the dre of the crr - ptsi operon , encoding the pts enzyme iia ( iia crr ) and enzyme i ( ei ) ( fig1 a ). the protected sequence ( tgtggtctagacctct ( seq id no : 10 )) corresponded to positions − 130 to − 115 relative to the start of crr , and had a 13 out of 16 by match to the derived dasr binding site consensus sequence ( see below ). this information was used to determine the dre sites of target genes that we had already validated ( table 1 and ( rigali et al ., 2004 )). using this training set , we built a refined position weight matrix (“ dasr4 ”; see materials and methods ), resulting in an alignment matrix that was used to scan the complete s . coelicolor genome . a genome scan revealed 160 dre sites for 131 transcription units , representing over 200 candidate genes . about 40 % of the target genes are related to sugar or aminosugar metabolism , with n - acetylglucosamine ( glcnac ) as the central saccharidic component ( fig1 b and table 2 ). the relationship with glcnac also connects to the identification of cell wall - associated peptidases . the predicted dasr regulon further includes genes for nitrogen metabolism including genes related to glutamine / glutamate amino acid metabolism . the list of targets also includes 16 transcription factors (# 27 , 31 , 46 , 51 , 56 , 62 , 67 , 71 , 77 , 81 , 84 , 105 , 119 , 129 , 130 , and 131 in table 2 ), suggesting an extensive level of indirect transcriptional control by dasr . a dasr null mutant ( bap29 ) was constructed by replacing almost the complete coding region ( nt 14 - 635 out of 765 ) by the apramycin resistance cassette ( aacc4 ), to study the role of dasr in vivo . the dasr mutant of s . coelicolor showed medium - dependent development : on some media development was enhanced , while on others it was completely abolished . this is summarised in fig1 . this strongly suggests that the function of dasr depends on the carbon and nitrogen sources used . surprisingly , the dasr mutant showed strongly enhanced antibiotic production ( fig2 a ). overproduction of dasr in strain m145 ( pft241 dasr + ) resulted in a reversed , non - sporulating bald ( bld ) phenotype ( fig2 b ). closer inspection of the dasr mutant by cryo - scanning electron microscopy ( cryo - sem ) showed that spores were almost completely absent in the dasr mutant , and aerial hyphae collapsed readily during sample preparation ( fig2 c ). analysis at high resolution by transmission em of cross - sections from s . coelicolor m145 and bap29 revealed that while m145 produced normal spores , the dasr mutant produced many spores ( approximately 30 %) with smaller or larger voids close to the spore wall ( fig2 d ; voids indicated by arrows ), suggesting extensive detachment of the cytoplasmic membrane from the spore wall . additionally , spore morphologies were significantly more heterogeneous ; while wild - type spores typically have a size of 0 . 6 by 0 . 8 μm , the dasr mutant showed an unusually strong variation in spore sizes ( 0 . 5 - 1 . 4 μm in length , but with the same width of 0 . 8 μm ). in addition , many mutant spores had a wall with a thickness similar to that of aerial hyphae , failing to create the typical thick spore wall . these observations connect well to our in silico predictions ( table 2 ) that dasr controls genes involved in the fate of peptidoglycan , including the genes for the metabolism of the precursor n - acetyglucosamine . how does dasr control the switch to development ? inclusion of pts genes in the dasr regulon allowed us to propose that at least one link is through the dasr - mediated control of the pts . in a previous publication ( nothaft et al ., 2003 ), we described normal but retarded development for the individual pts knock - out mutants , namely bap1 ( δptsh , the gene for hpr ), bap2 ( δcrr , the gene for iia crr ), and bap3 ( δptsi , the gene for enzyme i ( ei )). more detailed phenotypic analysis of the pts mutants revealed that while eventually all of the pts mutants were on some media able to produce spores , morphogenesis was significantly delayed on diverse complex and minimal media agar plates with the strongest differences when grown in the presence of mannitol and arabinose . strains were grown for 5 days on sfm or r2ye agar plates . as shown in fig3 a , on sfm the crr mutant bap2 produces a white aerial mycelium but failed to produce spores under these conditions , while deletion of ptsh ( bap1 ) or ptsi ( bap3 ) allowed the production of some grey - pigmented spores . interestingly , on r2ye agar plates all three pts mutants show vegetative arrest ( so - called bald or bld phenotype ). we recently discovered in a proteomics screen of these mutants that the expression patterns ( in bap2 and bap3 ) or the modification patterns ( in bap1 ) of the whig protein , a key developmental σ factor for early aerial growth ( chater et al ., 1989 ), strongly differed from those in the parental strain m145 . to establish the expression of whig , its transcription was analysed in all three mutants and in m145 . interestingly , whig transcription was strongly reduced in the bap2 and bap3 mutants ( fig3 b ), providing a likely explanation for their failure to complete sporulation , since whig mutants have a characteristic non - sporulating phenotype . hence , we propose that dasr acts as the nutrient sensor , and translates this through control of the pts , which in turn controls whig and — in view of the developmental arrest of the pts mutants — most likely at least one or more other early developmental genes . it might be noteworthy that the phosphotransferases ei , hpr , and iia crr provide a perfect signalling system through reversible metabolite - dependent phosphorylation , which in other bacteria is used for diverse but always carbon - related responses ( brückner and titgemeyer , 2002 ). to obtain an assessment of the effect of dasr , we compared the protein profiles of bap29 and its parent m145 . protein extracts were prepared from mycelia grown in the presence of glycerol ( a neutral carbon source ) and analyzed by two - dimensional gel electrophoresis . about 4 % of the protein spots on the coomassie - brilliant - blue - stained gels were altered in intensity by more than two - fold and eight were identified by mass spectrometry ( fig4 a ). 11 of the most spectacular differences between m145 and bap29 were analyzed , and of these we could positively identify nine proteins by mass spectrometry ( fig4 a ). two of the proteins were predicted in our in silico screen , namely the multiple sugar import protein msik and naga ( n - acetylglucosamine - 6 - p deacetylase ). only naga and msik were included in table 2 and contained predicted dre sites . the others could be related to central and to secondary metabolism ( see below and fig5 ). binding of dasr to the dre in the msik promoter region was demonstrated by emsa ( see below ). according to a role of msik in the uptake of inducers of polysaccharides - degrading systems , the induction of these enzymatic arsenals ( about a hundred of genes ) should be also affected due to the dasr deletion ( see discussion ). the lack of a dre upstream of the other seven genes suggests indirect control of these genes by dasr . visualising the metabolic pathways related to proteins identified by proteome analysis revealed that most gravitate around glcnac and glutamate metabolism , fitting well with the in silico and in vitro data presented above ( fig5 ). interestingly , two of the targets identified in our proteomics screen , namely actva4 and gpsi , which are both up - regulated in the dasr mutant , are involved in the production of the antibiotic γ - actinorhodin ( bibb , 2005 ) which correlated well with the early an activated production of blue γ - actinorhodin in the dasr mutant ( fig2 a ). gpsi is the guanosine pentaphosphate ( pppgpp ) synthetase that synthesizes the ppgpp precursor . it has been established that the stringent factor ppgpp has a causal role in activating actii - orf4 transcription ( hesketh et al ., 2001 ). the high amount of gpsi in bap29 suggests an increased pool of ppgpp precursors and therefore early and enhanced production of actinorhodin . the function of actva4 is unknown but the gene is included in the cluster responsible for actinorhodin production ( 20 genes ) and depending on the transcriptional activator actii - orf4 ( arias et al ., 1999 ). as follows from table 2 , actii - orf4 features among the predicted dasr target genes . direct binding of purified dasr to the dre upstream of this gene is substantiated by our observation that dasr protein directly binds to a double - stranded oligonucleotide containing the dre element found in the actii - orf4 promoter region ( fig9 ). this proves that indeed dasr controls actinorhodin production by binding to the pathway - specific activator gene for the synthesis of this exciting compound , suggesting that dasr plays a crucial role in the control of antibiotic production in actinomycetes . excitingly , comparison of the secreted proteins in abstracts of the dasr mutant and its parent s . coelicolor m145 by one - dimensional gel electrophoresis showed that one single protein was extraordinarily highly over - expressed in the dasr mutant ( fig4 b ). this protein was identified by mass spectrometry as sco5074 . this protein was recently shown to be part of the actinorhodin biosynthesis cluster , and is a secreted dehydratase that is most likely responsible for tailoring of the secreted antibiotic ( hesketh & amp ; chater 2003 ; taguchi et al , 2000 ). the gene product most likely assists cyclisation - dehydration of the alcohol in the actinorhodin precursor to give the pyran ring , a reaction that can proceed spontaneously but far less efficiently without it . as described in taguchi et al . ( 2000 ), the actvi - orf3 disruption mutant produces less ( about half as much ) actinorhodin as the parent . this is in line with our observation that while the wild - type strain produced a blue pigment , the dasr mutant produced a purple / violet pigment , most likely a variant of actinorhodin due to the extreme over - expression of sco5074 . also highly interesting is that using the novel bioinformatics techniques described above , we identified dre sites upstream of many more genes involved in the regulation and / or production of antibiotics . these targets are summarised in table 3 . with 16 predicted genes , chitin - related ( chi ) genes constitute a large subset of potential dasr targets , including chitinases , chitin binding proteins , extracellular β - n - acetylglucosaminidases ( convert chito - oligosaccharides into glcnac and chitobiose ), and intracellular β - n - acetylglucosaminidases ( hydrolyse chitobiose to glcnac ). to substantiate this , we determined the overall chitinolytic activity of bap29 and m145 grown under inducing ( chitin ) or repressing ( glycerol , glycerol plus glcnac , and glucose plus chitin ) conditions . as depicted in fig6 a , we observed a strongly reduced chitinolytic activity in bap29 , when cells were grown on chitin in the presence or absence of glucose . similar observations were made when total β - n - acetylglucosaminidase activities were assayed fig6 a ). seven examples were selected to validate the predicted cis - trans relationship between dasr and chi genes , for the chitinolytic system that is required for the utilization of chitin , a polymer of n - acetylglucosamine that is the one but most abundant carbon source on earth . positive dna - dasr interactions were observed for all tested promoters ( fig9 ), although some had low binding efficiency . the transcription of three chitinase genes was monitored by rt - pcr on rna isolated from cultures grown under conditions inducing ( glycerol and glcnac ) or not inducing ( glycerol ) the uptake of glcnac ( fig6 b ). the genes analysed were chii ( sco1444 ), chif ( sco7263 ), and sco6300 , encoding a putative secreted β - n - acetylglucosaminidase . as deduced from the global chitinolytic activity , a basal expression was observed for all three genes in cultures grown solely on glycerol . for chii and sco6300 , there was no significant difference in transcript levels between m145 and bap29 . excitingly , chif transcription was fully dependent on dasr : while we failed to detect any transcript in rna preparation from bap29 , there was strong chif transcription in the rna samples of the wild type . these data suggest that dasr positively controls the chitinolytic system , in contrast to its repressing function towards genes for glcnac transport and its subsequent intracellular catabolism ( see below ). other extracellular enzymes are also controlled by dasr ( fig7 ). indeed , we found that besides the chitinolytic system also the activity of mannanases , α - amylases , xylanases depend on dasr ( fig7 ). all of these polysaccharide - degrading systems were in fact affected in both substrate induction and glucose control by dasr , underlining its crucial position in the control mechanisms for enzyme secretion . since many of the predicted dasr targets were involved in the fate of carbon sources ( table 2 ), we analyzed the effects of the dasr mutation on sugar import . transport assays revealed that pts - mediated internalization of glcnac had become constitutive in bap29 , while in the parent m145 uptake was induced by glcnac ( fig8 a ). this correlated to constitutive protein levels of the universal pts phosphotransferases hpr and iia crr ( fig8 b ), and was supported by rt - pcr of the respective genes ( malx2 , nage2 , crr - ptsi and ptsh ; fig8 c ) that encode the pts permease complex ( iib glcnac , iic glcnac , iia crr , ei , hpr ). as shown above , in silico prediction and proteome analysis identified msik ( sco4240 ), encoding the universal atpase msik , as a target for dasr ( fig4 ). dna binding experiments showed that dasr directly binds to the dre present in the msik promoter region ( fig9 ), showing that dasr is involved in the regulation of msik - dependent abc - type ( atp - binding cassette ) transporters , which include those for uptake of cellobiose , trehalose , maltose , xylobiose , chitobiose , and probably another further 20 to 30 carbohydrates ( bertram et al ., 2004 ). an obvious consequence of dasr - dependent control of msik is that dasr indirectly controls the availability of sugar operon inducers , thus affecting the expression of all extracellular sugar hydrolases . this corresponds well to our discovery that besides the chitinolytic system also the expression of mannanases , α - amylases , xylanases depends on dasr ( fig7 ). the observed cell - wall anomalies in the dasr mutant ( fig2 d ) are at least in part explained by the finding that several genes encoding peptidoglycan - associated peptidases are included in the list of potential dasr targets ( table 2 ). in fact , a site has been predicted 71 bp upstream a five - membered dppa operon ( sco6486 - 6490 ). dppa itself encodes a putative binuclear zinc - dependent , d - specific aminopeptidase ( pfam 04951 ), 30 % identical and 50 % similar to dppa of bacillus subtilis ( dppa bsu ) ( cheggour et al ., 2000 ); dppa bsu is only active on d - ala - d - ala and d - ala - gly - gly substrates . the physiological role of dppa bsu , is probably an adaptation to nutrient deficiency by hydrolysing the d - ala - d - ala dipeptide required in peptidoglycan biosynthesis ( cheggour et al ., 2000 ). an other orf of the dppa operon ( sco6489 ) is also involved in peptidoglycan precursors or peptidoglycan degradation products catabolism . the predicted gene product of sco6489 is 32 % identical and 47 % similar to ldca ( l , d - carboxypeptidase a ) from e . coli that hydrolyses the peptide bond between the di - basic amino acid and the c - terminal d - alanine in the tetrapeptide moiety in peptidoglycan ( templin et al ., 1999 ). the inactivation of ldca in e . coli results in a dramatic decrease in the overall cross - linkage of peptidoglycan . to assess whether dasr controls the expression of the dppa operon , we performed dna binding studies with purified his - tagged dasr and a fragment corresponding to 193 by upstream of dppa . analysis using emsas established a weak but significant interaction of dasr with the dppa promoter ( fig9 ). to further substantiate a regulatory role for dasr on the expression of dppa , the intracellular d - ala - d - ala ( seq id no :) aminopeptidase activity was measured in mutant bap29 and compared to the parental strain s . coelicolor m145 . both strains were grown for 24 hours in mm supplemented with various carbon sources ( fig1 ). we failed to detect substantial variation in the total d - ala - d - ala ( seq id no :) aminopeptidase activity between m145 grown in chitin . however , in glucose + chitin we measured an average 85 % of loss of activity in mutant bap29 compared to m145 . in glycerol and glycerol + glcnac the dasr mutant had about 70 % and 33 % increased activity , respectively , thus revealing an opposite effect . these experiments show that dasr controls dppa activity according to the culture conditions and therefore modulates the d - ala - d - ala pool required for peptidoglycan precursors biosynthesis . considering the large number of n - acetylglucosamine - related genes in the list of predicted dre sites , we investigated the impact of dasr on the regulation of the nag metabolic genes . emsas were conducted using purified dasr protein and dna fragments encompassing the predicted dre sites for nagb ( glucosamine - 6 - p isomerase ) and the nagka operon ( glcnac kinase , and glcnac - 6 - p deacetylase ). in both cases a dasr - dre complex could be demonstrated ( fig9 ). this is consistent with our proteome analysis on naga ( fig4 ), and with rt - pcr analysis of nagb , which is constitutively expressed in the dasr mutant ( fig8 c ). the dasr regulon further focuses on the fate of n - acetylglucosamine through control of genes for nitrogen metabolism , including aminosugar and glutamine / glutamate metabolism . our proteome analysis revealed glutamate dehydrogenase ( gdha ; completely dependent on dasr ) and phosphoserine aminotransferase ( sco4366 , repressed ) as targets , which catalyse opposite reactions ( altermann and klaenhammer , 2005 ) ( fig4 & amp ; 5 ). acetate that is liberated from n - acetylglucosamine by naga is converted by acyl - coa synthetase ( strong dre site upstream of sco3563 and confirmed by emsa ) to acetyl - coa , the precursor of the tca cycle . acetyl - coa is alternatively converted by a thiolase ( thil ; a target detected by proteomics , fig4 ) to acetoacetyl - coa to enter fatty acid metabolism . this may well extend to an unusual type of control at the translational level , as the last two genes in the operon containing all major trnas for gln ( anticodon cug ) and glu ( anticodon cuc )— in the order trna gln - trna glu - trna glu - trna gln - trna glu — are predicted to be regulated by dasr , while the first three are not , suggesting fine - tuning of trna availability by dasr . supporting evidence for such control at the level at trna abundance comes from the presence of a predicted dre site upstream of glu - trna gln amidotransferase ( table 2 ). hence , a picture emerges of a hyper - controlled core network of the dasr regulon , crucial for the cell &# 39 ; s energy balance and revolving around the triangle glcnac - gln / glu - acetyl - coa , with almost complete control of all metabolic steps involved . thus , dasr plays a particularly prominent role in the control of central metabolism and is a very attractive target for metabolic engineering . all targets relating to n - acetylglucosamine and glutamate metabolism are highlighted in table 4 . amazingly , our analysis of the thermobifido fusca genome showed that in fact every single step of glycolysis is controlled by dasr , with highly reliable dre sites located upstream of the respective enzyme - encoding genes ( table 9 and fig1 ). the implications of this are truly daunting , as it means that in this industrially relevant actinomycete the flux through glycolysis can be easily controlled by the enhanced or reduced expression ( or inactivation ) of dasr . a pivotal question is what is the effector molecule that modulates dasr ? as shown in here , many of the targets for dasr relate to the generation ( chitinolytic system ), transport ( pts glcnac ), and metabolism ( glycolysis via fructose 6 - p ) of n - acetylglucosamine . we therefore looked for the inducer among the intermediate molecules that gravitate around aminosugar metabolism . a binding interference experiment was set up where the ability of compounds to interfere with binding of dasr to the nagb and crr promoters was tested . these compounds were : n - acetylglucosamine , n - acetylglucosamine - 6 - p , glucosamine - 6 - p , glutamate , glutamine , acetyl - coa , and fructose 6 - p . these binding interference experiments identified glucosamine - 6 - p as the inducer / effector molecule , as it was the only of the compounds tested that prevented the formation of a complex of dasr with the nagb ( fig1 ) and crr promoter regions ( not shown ). the finding that glucosamine - 6 - p serves as an effector of dasr is explained by its central position at the metabolic crossroads between ( glcnac ) n extracellular degradation , n - acetylglucosamine transport and intracellular metabolism , lipid and nitrogen metabolism , glycolysis , and peptidoglycan synthesis ( fig1 ). two possible transporters for n - acetylglucosamine were identified on the s . coelicolor genome , namely the adjacent genes nage1 ( sco2906 ) and nage2 ( sco2907 ). mutants were created for both genes by replacing the entire gene by the apramycin resistance cassette aacc4 . a double mutant was also produced ( bap6 ). the method used to do this was by using pwhm3 , as described previously ( nothaft et al ., 2003 ). mutants deleted for the transport genes nage1 ( bap4 ), nage2 ( bap5 ), or both ( bap6 ), were plated on r2ye agar plates with or without n - acetylglucosamine ( 1 % w / v ). other strains on the agar plates are : s . coelicolor m145 ( parent of all mutants ), the dasr mutant bap29 and the pts mutants ptsh ( bap1 ), crr ( bap2 ), and ptsi ( bap3 ). for phenotypes of the pts mutants see also fig3 a ). excitingly , in the absence of nage2 ( or nage2 and nage1 ) addition of n - acetylglucosamine has no effect on development , while the nage1 mutant and the parental strain s . coelicolor m145 become arrested in the vegetative state . this proves that indeed nage2 is the transporter of n - acetylglucosamine and is essential for import of the inducer molecule for the dasr control system . clearly , influencing the activity of nage2 ( positively or negatively ) will have a strong effect on the amount of inducer molecules introduced into the streptomyces cell , and therefore will strongly effect the dasr regulatory system . while we describe here around 200 targets ( table 2 ), the true number is without doubt much larger ; for example , we used a highly restrictive dre position weight matrix to avoid false positives , but we have evidence that by doing so many true dre sites have been obscured . additionally , we identified at least eight transcription factor genes in the list of predicted dasr targets . dasr controls many sensing / transport elements and the expression of glu - and gln - trnas . this suggests that dasr may be receptive to diverse environmental changes and governs many other regulons , and most likely at both the transcriptional and at the translational level . this multi - level control by dasr is summarized in fig1 . besides the absolute size of the regulon , a prerequisite for a global - acting regulator is further that it should act in concert with single - acting transcription factors ( moreno et al ., 2001 ). indeed , dasr controls chi - related genes , which are also regulated by the chis / chir two - component system ( kormanec et al ., 2000 ) and by a third unknown dna - binding protein identified recently ( fujii et al ., 2005 ), suggesting a multi - partner control of the chitinolytic system . another example arises from our studies on the regulation of the pts , where we observed that besides dasr also sco6008 , encoding a rok - family regulator ( titgemeyer et al ., 1994 ), is required for activation of pts genes . the wide - spread dasr regulon is a target for novel screening procedures excitingly , the dasr regulatory network is highly conserved in s . avermitilis and s . scabies , with more than 75 % of the dre sites predicted in s . coelicolor also found upstream of the orthologous genes in s . avermitilis , providing a strong phylogenetic argument for the presented predictions . the strong conservation of the dasr regulon in other actinomycetes also suggests that dasr may control many genes for natural products and enzymes in this class of bacteria . the conservation of the dasr regulon is underlined by the high conservation of dasr proteins ( fig1 ). considering the predicted control of clavulanic acid production in s . clavuligerus ( table 3 ), we cloned the dasr gene by pcr using oligonucleotides matching the − 50 /− 30 and + 900 /+ 920 regions of s . coelicolor dasr , with nt positions relative to the start of the gene . the clone was sequenced , and the predicted gene product differed in a single amino acid position , namely an asn55 in s . clavuligerus dasr and asp55 in s . coelicolor dasr . on this basis it is obvious that the dasr binding site in s . clavuligerus is highly similar to that in s . coelicolor . the corresponding nucleic acid and amino acid sequence are disclosed in fig1 . surprisingly , we recently discovered that the core gene cluster naga - nagb - dasr ( and the dre elements ) is also widespread among low g + c gram - positives , including bacillus , lactococcus , listeria and streptococcus . not only the organization is conserved , but also also the sequence of the dre sites , even though the g + c content of the dna of bacillus ( 43 %) is around 30 % lower than that of streptomycetes ( 72 - 73 %). the dre sites of bacillus subtilis and bacillus halodurans are summarised in table 5 , those of streptococcus species in table 7 , of lactococcus lactis in table 6 , and of listeria innocua and listeria monocytogenes in table 8 . the derived consensus sequences for dasr binding sites in these species are summarised in cartoons in fig1 . from this we conclude that the dasr core regulon is a very important concept , as its presence in such divergent micro - organisms means that the dasr control system has survived at least half a billion years of evolution . finding a tool to manipulate the activity of dasr is therefore very important , as it will allow controlling the expression of many industrially and medically relevant compounds ( enzymes , antibiotics , anti - tumor agents , agricultural compounds , and other secondary metabolites ) from the outside rather than by genetic engineering . this is for example a prerequisite for setting up novel screening strategies , as individual strain manipulation is not an option . addition of inducer ( notably n - acetylglucosamine and derivatives ) will trigger or at least enhance the expression of a wide range of natural products , allowing more ready screening . an obvious example is the control of cryptic clusters , which are silenced and therefore cannot be identified by activity - based screening assays . we show that antibiotic biosynthesis clusters are activated by the removal or reduced activity of dasr , and we anticipate that addition of inducer will relieve these clusters and thus boost the potential of novel screening procedures . as shown above , on media that do allow development ( e . g . mannitol - containing solid media ) the dasr mutant showed enhanced production of the pigmented antibiotics actinorhodin ( act ) and undecylprodigiosin ( red ). the relative increase in antibiotic production in the dasr mutant was quantified by determining act and red concentrations in the spent agar of solid - grown cultures ( mm without any added carbon source ). under these conditions s . coelicolor grows solely on agar , enabled by induction of the daga agarase ( buttner et al ., 1987 ). spectroscopic measurements showed that act and red production were consistently enhanced in bap29 by factors of 3 . 2 (± 0 . 2 ) and 3 . 9 (± 0 . 3 ), respectively ( averages of three independent experiments ). as shown in fig4 , in further support of enhanced act production by the dasr mutant , preliminary proteome analysis of extracellular fractions of m145 and bap29 identified two proteins encoded by genes in the act cluster that were strongly up - regulated in the dasr mutant , namely actvi - orf3 , encoded by sco5074 , a secreted protein involved in stereospecific pyran ring formation of actinorhodin ( hesketh and chater , 2003 ; ichinose et al ., 1999 ), and actva - orf4 , the product of sco5079 ( caballero et al ., 1991 )), a conserved hypothetical cytoplasmic protein . we observed putative dre sites upstream of actii - orf4 and redz encoding transcriptional activators of the act and red gene clusters , respectively ( for dre sites see table 2 ). the dre site upstream of actii - orf4 ( nt positions − 59 /− 44 relative to the translational start site ) lies precisely between the canonical − 35 and − 10 sequences of the promoter , a position strongly suggesting that dasr should function as a transcriptional repressor . the dre site upstream of redz ( nt positions − 201 / 186 relative to the translational start site ) lies around 50 bp upstream of the − 35 sequence of the redz promoter . electrophoretic mobility gel shift assays ( emsas ) with purified his 6 - tagged dasr and double - stranded oligonucleotide probes showed direct binding to the predicted dre sites of redz and actii - orf4 and to the positive control ( dre site of crr - ptsi ), while dasr did not bind to the cis - acting element of crp , which lacks similarity to the dre element and was therefore used as the negative control ( fig2 a ). no free template was found when dasr was bound to the crr fragment , while over 50 % of the redz probe was bound , and only around 10 % of the probe containing the actii - orf4 dre site . hence , we established direct binding of the dasr protein to the predicted dre sites , with binding efficiencies corresponding to their ‘ statistical strength ’. the role of dasr in the control of actii - orf4 , redz and redd was further assessed by semi - quantitative rt - pcr on rna samples collected from the parental strain ( m145 ) and the dasr mutant ( bap29 ) grown on mm mannitol agar plates for 30 h ( vegetative growth ), 42 h ( initiation of aerial growth ), and 72 h ( aerial growth and spores ). rt - pcr analysis revealed strongly enhanced transcription of actii - orf4 at all time points in the dasr mutant and a discrete but significantly enhanced transcription of redz ( fig2 b ). apparently , not only does the degree of dasr - dependent transcriptional repression relate to the strength of the dna - protein interactions , but the relative positioning of the dre site with respect to the promoter consensus sequence is also an important factor . it was shown previously that enhanced expression of redz strongly induces redd transcription , even by - passing the block in antibiotic production in blda mutants ( guthrie et al ., 1998 ). indeed , the enhanced expression of redz was reflected in a clearly enhanced expression of the red pathway - specific activator gene redd . in conclusion , the known activator genes of the red cluster are negatively controlled by dasr , explaining the enhanced production of red in a dasr mutant . for the act cluster , dasr competes with the transcriptional activator atra for binding to the promoter region of actii - orf4 , and inactivation of dasr — either through modulation of its in vivo activity or through gene inactivation — then results in enhanced act production . a signalling cascade from initial detection of the nutritional status of the environment to the onset of physiological and chemical differentiation should contain at least the following steps : ( 1 ) availability and sensing of an extracellular signal ; ( 2 ) transport of ‘ signalling nutrients ’ into the cell ; ( 3 ) their intracellular modification into an inducer molecule ; ( 4 ) its binding to a global regulator , which is the checkpoint for ( 5 ) signalling the information to pathway - specific activators and ( 6 ) the switch to development and antibiotic production . our experiments suggest that the glcnac sensory cascade controlled by dasr is a global system that triggers antibiotic production in direct response to nutrients ( fig2 ). the steps are : ( 1 ) sensing of glcnac ; ( 2 ) transport via the pts glcnac ; ( 3 ) conversion by naga to glucosamine - 6 - p ; ( 4 ) binding of the signalling molecule to dasr , thus inhibiting its repressing activity on actii - orf4 and redz and activating the pathways for biosynthesis of actinorhodin and undecylprodigiosin . arguing from the regulatory pathway deduced from the newly extended characterization of the dasr regulon ( fig2 ), we anticipated that dasr - dependent transport and phosphorylation of glcnac via the pts could be a decisive signal to trigger actinorhodin and undecylprodigiosin biosynthesis in s . coelicolor . this hypothesis was tested by plating s . coelicolor m510 ( δredd ), m511 ( δactii - orf4 ) and m512 ( δredd , δactii - orf4 ) on mm agar plates with or without glcnac . as a consequence of the deletion of the respective pathway - specific activators , s . coelicolor m510 cannot produce the red - pigmented undecylprodigiosin and m511 fails to produce the blue - pigmented actinorhodin , while m512 produces neither antibiotic . these strains allowed us to specifically monitor each of the antibiotics , which is a necessary control because their pigmentation is ph - dependent , and biosynthetic derivatives show varying colours ( bystrykh et al ., 1996 ; ichinose et al ., 1999 ). neither strain produced significant amounts of actinorhodin or undecylprodigiosin when grown for five days on mm with agar as the sole carbon source . in the presence of glcnac ( 10 mm ), production of act or red was induced in s . coelicolor m510 and m511 , respectively ( fig2 ). no pigmented antibiotic was observed for m512 , as expected ( not shown ). thus , under starvation conditions production of act and red is induced both in response to increased levels of glcnac and by the absence of dasr . is the glcnac - mediated control of antibiotic production a more widespread phenomenon in streptomycetes ? to assess this , we evaluated the effect of glcnac on total antimicrobial activity ( bactericidal and bacteristatic ) of several streptomycetes , using bacillus subtilis as the indicator strain . the tested strains were spotted on minimal medium containing mannitol ( 0 . 5 %) with or without glcnac ( 1 %). excitingly , growth inhibition zones indicative of antibiotic production were much larger for streptomyces clavuligerus ( a producer of cephamycin ), streptomyces collinus ( produces kirromycin ), streptomyces griseus ( streptomycin producer ), streptomyces hygroscopicus ( produces hygromycin ), streptomyces rimosus ( produces oxytetracycline ), and streptomyces venezuelae ( chloramphenicol , methymycin ) ( fig2 ). n - acetylglucosamine did not seem to affect antibiotic activity against b . subtilis in streptomyces acrimycini , streptomyces avermitilis streptomyces cinnamonensis , streptomyces limosus , and streptomyces lividans . interestingly , we observed a repressing effect in streptomyces roseosporus . these results suggest that the relief of antibiotic production by glcnac ( and through dasr ) is a common control mechanism in streptomycetes . in order to assess if dasr plays a role in silencing cryptic secondary metabolite clusters in streptomycetes , we analysed the expression level of a putative antibiotic biosynthetic cluster for a hypothetical type i polyketide ( sco6273 - 6288 ), the only cryptic cluster studied so far . induction of this biosynthetic pathway depends on a pathway - specific activator , kaso ( sco6280 ), which is in turn repressed by the γ - butyrolactone ( scb1 ) binding protein scbr ( takano et al ., 2005 ). repression of kaso is relieved by production of scb1 . to test the possible “ awakening ” of this cluster by the absence of dasr we performed semi - quantitative rt - pcr on rna samples collected from the parental strain ( m145 ) and the dasr mutant ( bap29 ) grown on mm mannitol agar plates for 30 h ( vegetative growth ), 42 h ( initiation of aerial growth ), and 72 h ( aerial growth and spores ). excitingly , kaso transcripts were detected in the 30 - h and 72 - h rna samples of bap29 , but were not seen in m145 in any of the samples ( fig2 ). most likely as a result of the induction of kaso , transcription of sco6273 , the last orf of the biosynthetic cluster and encoding a putative type i polyketide synthase , was dramatically increased ( fig2 ). the enhanced expression of sco6273 was observed only during vegetative growth . no dre site was predicted upstream of kaso , and the cis - trans relationship between dasr and this cryptic cluster is under investigation . chitin is the main form of storage of glcnac and the second most abundant polymer on earth , and as such is of immense importance for soil - dwelling bacteria . glcnac is a rich n - and c - source and , with its metabolic products acetate , ammonia and fructose - 6 - p , stands at the crossroads of the major primary metabolic pathways . this underlines the selective advantage of being able to colonise different types of chitin - containing substrates ( saito et al ., 2003 ; schrempf , 2001 ). our experiments suggest that glcnac can provide opposite signals , namely expansion ( growth and developmental block ) under nutrient - rich conditions and growth cessation followed by development ( antibiotic production ; sporulation ) under nutrient - limited conditions . there are two major sources of glcnac : chitin and the bacterium &# 39 ; s own cell wall , and they may trigger opposite responses . bacterial chitinases mainly generate chito - oligosaccharides and n , n ′- diacetylchitobiose ( glcnac ) 2 from chitin , and little glcnac . also , dasr mutants have a five - fold lower chitinolytic activity than the parental strain ( colson et al ., 2007 ), but overproduce antibiotics , suggesting that chitinases do not produce the signal . the ‘ glcnac effect ’ was observed only at higher concentrations (& gt ; 5 mm ). perhaps the most likely natural source would be autolysis of the bacterial cell wall . large amounts of glcnac were found to accumulate locally after programmed cell lysis , when general nutrient limitation necessitates development of an aerial mycelium at the expense of the vegetative hyphae ( miguelez et al ., 2000 ). since we show that nutrient sensing , cell wall lysis and proteolysis and secondary metabolism ( in particular antibiotic production ) are all linked directly to the function of dasr , there is a highly suggestive clustering within a single regulon of genes involved in the catabolism of peptidoglycan precursors , together with antibiotic pathway - specific activators . the contrast between the large number of secondary metabolites produced by streptomycetes and the relatively limited knowledge on the global regulatory mechanisms that trigger their production implies that much is to be gained in terms of drug discovery by learning from the organism itself . we propose a signalling cascade from nutrient stress to antibiotic production . our deduced pathway proposes glcnac as an important signalling molecule for streptomycetes , allowing them to determine the nutritional status of the habitat . the signal that is transported by the pts glcnac is metabolized to glucosamine - 6 - p , inactivating dasr , which in turn is responsible for suppression of antibiotic production and development under nutrient - rich conditions . besides the pts glcnac , dasr controls many more abc sugar transporters and the functions of several of these are currently under investigation . the observation that antibiotic production can be awakened and / or enhanced by interfering with the dasr - 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