Patent Document:

the bacterial strains used in this application are listed in table 1 . e . coli k - 12 strains jm109 was used for propagating plasmids , and was grown and transformed by standard procedures ( sambrook et al ., 1989 ); transformants were selected in l broth containing 1 % ( w / v ) glucose , and ampicillin at a final concentration of 200 μg ml − 1 . l broth with 1 % glucose and 30 μg ml − 1 chloramphenicol was used to grow et12567 . streptomyces coelicolor a3 ( 2 ) m145 and streptomyces lividans 1326 were obtained from the john innes centre strain collection , and were used for transformation and propagation of streptomyces plasmids . protoplast preparation and transformation were performed as described by kieser et al . ( 2000 ). sfm medium ( mannitol , 20 gl − 1 ; soya flour , 20 gl − 1 ; agar , 20 gl − 1 , dissolved in tap water ) was used to make spore suspensions . minimal medium ( mm ) and r2ye agar plates ( kieser et al ., 2000 ) were used for promoter - probing experiments ; r2ye was also used for regenerating protoplasts and , after addition of the appropriate antibiotic , for selecting recombinants . for standard cultivation of streptomyces , and for plasmid isolation , yeme ( kieser et al ., 2000 ) or tryptone soy broth ( difco ) containing 10 % ( w / v ) sucrose ( designated tsbs ), were used . growth curves were performed in nmmp ( liquid minimal medium ), with 1 % mannitol as the carbon source . for this purpose , strains were grown on mmd medium , a solid minimal medium with mannitol ( 1 % w / v ) and 100 mm 2 - deoxyglucose , which is lethal for glk −+ strains . therefore colonies that develop on this medium have to be glk − . for each mutant , three independent colonies that were able to grow on mmd were selected , and tested for glucose kinase activity . strains that lack glucose kinase activity are glucose kinase deficient ( δglka ). strains were checked by pcr , which showed that the nature of the mutations varied from large deletions to point mutations . those harboring very large deletions ( beyond the glka gene ) were discarded . pij2925 ( janssen and bibb , 1993 ) is a puc - derived plasmid used for routine subdloning . for cloning in streptomyces we used phjl401 ( larson and herschberger , 1986 ), and pwhm3 ( vara et al .). phjl401 is a shuttle vector containing the e . coli puc19 and streptomyces scp2 * ( lydiate et al ., 1985 ) origins of replication , giving approximately 10 copies per chromosome in streptomyces ; pwhm3 is a shuttle vector containing the e . coli puc19 and streptomyces pij101 origins of replication ( approximately 50 copies per chromosome ). e . coli plasmid dna was isolated from s . lividans prior to transformation to s . coelicolor . pij2587 ( van wezel et al ., 2000a ) was used for promoter probing experiments . it is an e . coli - streptomyces shuttle vector derived from phjl401 with puc19 and scp2 * origins of replication ; it possesses a copy number of approximately 10 per chromosome in streptomycetes . fragments harboring upstream sections of the respective ssga - like genes , as well as of ssgr , were amplified by pcr , using 30 - mer oligonucleotides . the oligonucleotides were designed such , that restriction sites were added so as to clone the upstream sequences as ecori - bamhi fragments , with the bamhi site proximal to the start of the genes . in this way , possible promoter sequences were positioned in the desired orientation and immediately upstream of the promoterless redd gene in pij2587 . red production by the transformants on r2ye plates was assessed visually . the exact inserts of the constructs are shown in table 2 . exact location of the dna fragments harboring upstream sequences of the various ssga - like genes , as well as ssgr , are shown . the inserts were cloned as ecori - bamhi fragments into pij2587 , to allow transcription of the promoterless redd gene . fragments were amplified by pcr , using the relevant oligonucleotides . 3 . constructs for the expression of ssga and ssga - like genes in streptomyces for the overexpression of ssga in actinomycetes , pgws4 - sd was used , an integrative expression construct based on pset152 ( bierman et al ., 1992 ), containing ssga behind the strong and constitutive erme promoter . construction of this construct was described in european patent application 99929959 . 7 ( publication number 1090121 ). for complementation experiments , dna fragments harboring ssga - like genes and upstream sequences were inserted into phjl401 . these constructs for the expression of ssgb , ssgc , ssgd , ssge , ssgf , and ssgg , were designated pgwb1 , pgwc1 , pgwd1 , pgwe1 , pgwf1 , and pgwg1 , respectively . presence of active promoters was confirmed by promoter probing ( see below ). restriction maps of ssgb - g and flanking regions , and exact inserts of the expression constructs are shown in fig8 a - 8f . as an alternative to expression constructs harboring complete copies of any of the ssga - like genes ssgb - g , hybrid constructs were produced containing the ssgra operon with the part of ssga following the bamhi site replaced by comparable parts of any of the ssga - like genes . the architecture of these constructs is shown in fig9 a . in this way , timing of expression of the hybrid ssgax genes is similar to that of ssga . the phjl401 - based constructs contain the bglii - bamhi part of ssgra operon ( i . e ., the − 440 /+ 1000 region relative to the ssgr translational start codon ; cf fig4 a ), fused to an approximately 600 bp pcr - generated bamhi - hindiii dna fragment , with the bamhi site created at a position corresponding to the fully conserved proline residue ( aa residue 24 in the ssga ( seq id no : 11 ) sequence ) shown in the alignment ( fig7 ). the regions − 1000 /+ 3 and + 410 /+ 1400 relative to the start of the ssgb gene were amplified by pcr , using 30 - mer oligonucleotides . these were designed such as to introduce a bamhi site in the middle of the insert , allowing introduction of the apramycin resistance cassette . a thiostrepton resistance cassette on the vector part of pgwb2 allowed distinction between single and double recombination events . transformants were selected for apra r and subsequently screened for thiostrepton sensitivity , indicative of loss of the plasmid . the region deleted in the mutant and replaced by the apramycin resistance cassette is shown in fig8 a . to create a construct for the deletion of ssgr , the 1400 bp bglii - bamhi fragment containing ssgr and part of ssga ( fig4 a ) was inserted into bamhi - digested pij2925 , and the ncoi - sphi segment of ssgr was removed , to create an in - frame deletion in the ssgr gene on the plasmid . subsequently , the apramycin resistance cassette aacc ( 4 ) ( kieser et al ., 2000 ) was inserted into the ecori site of the construct ( outside the ssgra insert ), producing pgwr2 . this construct allows production of an in - frame deletion mutant of ssgr . after transformation of the non - replicating construct to s . coelicolor m145 , initial integrants ( apramycin resistant ) were selected , allowed to sporulate on sfm plates without antibiotics , and replicated non - selectively to allow a second recombination event to take place , and plated for single colonies . the latter were replicated to sfm containing apramycin , to screen for double recombinants , which should have lost the plasmid and hence have become sensitive to apramycin . about 30 % of all apramycin sensitive colonies were sporulation mutants . testing of four sporulating and four non - sporulating double recombinants by pcr revealed that all sporulation mutants carried the expected 300 bp in - frame deletion , while sporulating colonies had a wild - type ssgr gene . one of the mutant colonies was selected , and designated gsr1 . location of the deletion is shown in fig4 a . for complementation experiments plasmid pgwr1 was designated . this is a low - copy number phjl401 - based vector that harbors a 1 . 3 kb pcr - generated insert including the entire ssgr gene , and approximately 300 bp of upstream sequences , including the ssgr promoter region . for analysis of the effect of enhanced expression of ssga on the growth rate and on productivity of actinomycetes , growth curves were performed in small scale fermentations using a bioflo 3000 5l bench top fermenter ( new brunswick biosciences ). 4 . 5 liters of tryptone soy broth with 10 % sucrose ( ts medium ) containing 50 mm cucl 2 and the relevant antibiotics , were inoculated with a 100 ml preculture , grown for 30 hours at 30 ° c . in a spring - coiled flask . the fermentation inoculum was 0 . 2 g / l . fermentations were performed at 30 ° c ., and the ph was fixed at 6 . 5 , by the addition of 2n phosphoric acid or 2n naoh . dissolved oxygen tension was set at 80 % and maintained by changing the stirrer speed . the specific enzymatic activity of tyrosinase secreted by transformants of s . lividans 1326 harboring pij703 ( katz et al ., 1983 ) was determined as described previously , following the conversion of 1 mm dopa spectrophotometrically at 475 nm ( lerch and ettinger , 1972 ). activity ( expressed as δa 475 / sec . ml ) was corrected for biomass content of the samples . to assess the total antimicrobial activity present in the culture fluid of s . roseosporus fermentations , 40 ml samples were taken at regular intervals , biomass was harvested by centrifugation ( 20 minutes at 10 , 000 rpm ), and residual debris removed by filtration using a 0 . 22 μm bacterial filter ( millipore ). a lawn of streptomyces avermitilis atcc31267 was streaked on minimal medium agar plates containing 0 . 5 m cacl 2 , with mannitol as the sole carbon source ( 1 % w / v ). sterile 0 . 6 cm antibiotic assay filter paper discs ( whatman grade aa ) were placed onto the plates and 10 μl of filtered supernatant was spotted on these filters . plates were allowed to grow for four days and photographed . zones of clearing ( dark halos ) represent growth inhibition due to antibiotics present in the culture fluid of the s . roseosporus fermentations . polymerase chain reactions ( pcrs ) were performed in a minicycler ( mj research , watertown , mass . ), using pfu polymerase ( stratagene , la jolla , calif . ), and the buffer provided by the supplier , in the presence of 5 % ( v / v ) dmso and 200 μm dntp . no additional mg ++ was added to the reaction mixture . the following pcr program was used for 30 cycles : 45 seconds melting at 94 ° c ., 1 minute annealing at 54 ° c ., and 90 seconds extension at 72 ° c . the reaction was completed by an additional ten minutes incubation at 72 ° c . rna was purified from mm agar plates with mannitol as the carbon source , as described by kieser et al . ( 2000 ), except that dnase i treatment was used in addition to salt precipitation to eliminate dna from the nucleic acid preparations . for each nuclease s1 protection assay , about 0 . 02 pmol ( about 10 4 cerenkov counts minute − 1 ) of labeled probe was hybridized to 30 μg of rna in natca buffer at 45 ° c . overnight after denaturation at 70 ° c . for 15 minutes . all subsequent steps were carried out as described previously ( kieser et al ., 2000 ), using an excess of probe . the probes used for mapping ssgr and ssga transcripts were amplified from dna of s . coelicolor cosmid q11 . the following probes were amplified ; for mapping ssgr transcripts , a 393 nt probe covering the − 320 /+ 72 region relative to the ssgr translational start codon , and for ssga a 233 nt probe covering the − 192 /+ 41 region relative to the translational start codon of ssga . s . coelicolor strains were grown in 500 ml minimal medium ( smm ) supplemented with 1 % ( w / v ) glucose or mannitol , under vigorous shaking at 28 ° c . cells were harvested at 30 minute intervals , washed twice and then resuspended in cold standard buffer ( 50 mm tris ph 7 . 4 ; 5 mm mgcl 2 , 40 mm nh 4 ac , 50 mm nacl , 1 mm dtt ). crude extracts were prepared by sonication at 50 w ( labsonic u ( braun ); five times for 30 seconds each time ) and subsequent removal of cell debris by centrifugation . glucose kinase activity in cell extracts was assayed using 50 μg of total protein , in a reaction mixture containing 50 mm tris - cl ( ph 7 . 0 ), 20 mm glucose , 25 mm mgcl 2 , 0 . 5 mm nadp , 1 mm atp , and 0 . 7 u glucose - 6 - p dehydrogenase ( skarlatos and dahl , 1998 ). s . coelicolor strains were grown as described for the glucose kinase activity assay . proteins of cell extracts were separated by sds - polyacrylamide gel electrophoresis on a 7 . 5 % polyacrylamide gel and transferred to a hybond - c super ( amersham ) by electroblotting . glk was detected with a rabbit polyclonal antiserum raised against glk ( his 6 ) of s . coelicolor . glk antibodies ( mahr et al ., 2000 ) were visualized using the ecl western blot analysis system ( amersham ). to analyze what the effect is of enhanced expression of ssga ( seq id no : 11 ) on growth rate and on product formation by actinomycetes , we used streptomyces coelicolor m145 , genetically the most well - characterized actinomycete , whose genome sequence was published recently ( bentley et al ., nature 417 : 141 ( 2002 )), and the related but industrially more relevant strain streptomyces lividans 1326 as initial test systems . s . coelicolor forms large clumps during fermentation , and grow slowly ( doubling time approximately three hours in defined media ). mainly due to these growth problems , s . coelicolor has never been used in industrial fermentations . the ssga gene was introduced in the wild - type strain m145 , which dramatically altered its morphology ( van wezel et al ., 2000c ). recent tests in 5l fermentations showed strong reduction of the adaptation ( lag ) phase , and doubling of specific growth rate on introduction of m145 + ssga . such an improvement of growth rate strongly reduces fermentation time , making the production process much more cost effective , as more fermentations can be run in the same time span . while the growth effects were very promising , we tested what effect this seemingly improved morphology had on the yield of secreted enzymes in small - scale fermentations . for this purpose , pij703 , a plasmid expressing the tyrosinase gene ( melc ) ( katz et al ., 1983 ) was introduced into s . lividans 1326 , a strain often used for the commercial production of enzymes that require streptomyces as the production host . excitingly , expression of ssga had a very positive effect on both growth rate and enzyme production ( fig1 ). specific growth rate of the enzyme - producing s . lividans during fermentation had approximately doubled . another important observation is the reduced so - called lag phase , or the time the culture requires entering exponential growth . precultures of s . lividans harboring pgws4 - sd entered exponential growth significantly earlier than the control strain . clearly , the smaller mycelial clumps of the ssga transformant were much better suited for the production of the secreted enzyme tyrosinase , as shown by the spectacular increase in tyrosinase activity of the ssga transformant (“ 1326 ssga ” in fig1 ). this strain reached a peak of 0 . 94 ( arbitrary units ) around 20 hours after start of the fermentation , while the control strain harboring the control plasmid pset 152 produced 0 . 55 arbitrary units after almost 35 hours . for the analysis of the effect of growth improvement on antibiotic production , we introduced pgws4 - sd into streptomyces roseosporus , producer of the glycopeptide antibiotic daptomycin . control transformants harbored pset152 . similarly to the experiments described above for s . coelicolor and s . lividans , growth behavior was altered , with enhanced fragmentation and therefore reduced pellet formation . an example of growth curves is shown in fig2 a . 40 ml samples were taken at regular intervals from both fermentations , and analyzed for antimicrobial activity using a standard antibiotic assay ( see materials and methods section ). as indicator strain we used streptomyces avermitilis . the size of the zone of clearing around the filter disc is a measure for the antibiotic concentration in the samples . also in the case of s . roseosporus the effect of enhanced ssga expression on growth rate was positive , although not as strong as in the case of s . coelicolor and s . lividans , probably because the latter two strains produce larger pellets than s . roseosporus . however , the effect of the introduction of pgws4 - sd on antibiotic production by this strain was spectacular . while early samples taken from exponentially growing cells contained little antibiotic , as was apparent from the small zones of clearing in fig2 b ( control strain , samples 1 - 3 ) and fig2 c ( s . roseosporus expressing ssga , samples 1 - 4 ), samples taken from stationary phase cultures ( fig2 b , samples 5 - 6 ; fig2 c , samples 5 - 8 ) showed a strong increase of the zones growth inhibition around the filter discs . excitingly , the much larger clearing zones around filter discs containing supernatants from pgws4 - sd transformants as compared to when supernatants from control pset152 transformants were applied to the discs , clearly show that enhanced expression of ssga has a very positive effect on the antibiotic production of s . roseosporus . in summary , the experiments described above convincingly show that ssga has a strongly positive effect on enzyme production and on antibiotic production of several actinomycetes . an ssgr insertional knock - out mutant of s . griseus was shown to have a whi ( non - sporulating ) phenotype ( jiang and kendrick , 2000 ). however , since this mutant was created by insertion of a resistance cassette , this effectively blocks transcription from the ssgr promoter into ssga . to rule out this possibility , and especially to study the role of ssgr in regulating ssga transcription in s . coelicolor , an in - frame - deletion mutant of s . coelicolor ssgr was created , as described in the materials and methods section . this effectively removed the approximately 300 bp ncoi - sphi section of ssgr , resulting in an in - frame deletion rendering ssgr effectively inactive ( fig4 a ). this mutant was designated gsr1 . gsr1 had a phenotype very similar to that of the ssga mutant gsa3 , forming aerial hyphae , but few spores , and only after prolonged incubation on sfm plates ( fig3 ). apparently , the ssgra gene cluster has a very similar role in s . coelicolor and s . griseus . to analyze the growth - phase - dependent transcription of ssgr , nuclease s1 mapping experiments were performed on rna isolated from solid cultures of s . coelicolor m145 . rna was isolated at 12 to 24 - hour intervals during five days , so as to provide representative samples to analyze development - dependent transcription . a 393 bp dna fragment encompassing the − 320 /+ 72 region relative to the translational start site of ssgr , was amplified by pcr , with the relevant 20 - mer oligonucleotides , and used as probe . two transcripts were observed with a length of approximately 210 and 72 nt . the 5 ′ ends of these transcripts correspond to nt positions − 135 and + 1 , relative to the translational start site of ssgr , respectively . thus , the latter transcript lacks a leader sequence . transcription is developmentally regulated , and is strongly enhanced after approximately 64 hours , corresponding to the onset of sporulation . since ssgr and ssga are transcriptionally linked ( van wezel et al ., 2000c ), and both are essential for correct sporulation , a functional relationship between the two genes was investigated . ssgr encodes a member of the family of iclr - type regulators , including several repressors and activators . to analyze the possible dependence of ssga transcription on ssgr , nuclease s1 mapping experiments were performed on rna isolated from solid cultures of s . coelicolor m145 , and its congenic ssgr in - frame deletion mutant gsr1 . a 233 bp dna fragment encompassing the − 192 /+ 41 region relative to the ssga translational start site was amplified by pcr , with oligonucleotides t7 - af ( 32 p - labeled at its 5 ′ end ) and t7 - ar ( fig4 b ), and used as probe in the mapping experiments . rna was isolated at 12 to 24 - hour intervals for five days , so as to provide representative samples to analyze development - dependent transcription . in wild - type s . coelicolor , two transcripts were observed of approximately 115 nt and 100 nt ( bands a1 and a2 in fig6 , respectively ). promoter probe data indicated that ssga is transcribed from the ssgr promoter as well as from its own promoter . however , it is unclear if both bands a1 and a2 represent de novo transcription . if so , the promoter sequences overlap . the abundance of both ssga transcripts increased , reaching a maximum after approximately 80 hours , corresponding to sporulation . interestingly , ssgr transcripts reached a maximum already after 64 hours ( using the same rna samples ), corresponding to the onset of sporulation . this is in accordance with our complementation data , and suggests that ssgr activates transcription of ssga . to test this hypothesis , transcription of ssga in the ssgr mutant was analyzed . excitingly , no ssga transcripts could be detected in the ssgr mutant ( fig6 , right panel ). therefore , we conclude that ssgr is a likely transcriptional activator of ssga . expression of ssga ( seq id no : 11 ) restores sporulation to an ssgr mutant if the non - sporulating phenotype of the ssgr mutant is solely due to the absence of ssga transcripts , it follows that expression of ssga ( seq id no : 11 ) in such a mutant would counteract the mutation . therefore , two constructs were introduced into gsr1 , one with ssga preceded by its own ( ssgr - activated ) regulatory sequences , and one with ssga positioned behind the ssgr - independent and constitutive erme promoter . in a control experiment , we also introduced ssgr expression constructs in the ssga mutant . in this case , no effect was expected . in another control experiment , it was tested if the ssgr and ssga mutants could be complemented by wild - type copies of ssgr and ssga , respectively . the results are shown in fig3 . expectedly , the ssga and ssgr mutants could be complemented by the introduction of wild - type ssga ( using plasmid pgws4 ; van wezel et al ., 2000c ) and ssgr ( using plasmid pgwr1 , see materials and methods section ), respectively . this underlines that the non - sporulating phenotype of the ssgr and ssga mutants is solely due to the absence in these mutants of ssgr or ssga , respectively . as shown in fig3 , the data clearly show that expression of ssgr has no effect on the development of the ssga mutant . interestingly , expression of ssga using the ssgr - independent erme promoter fully restored development , while introduction of multiple copies of ssga behind its own promoter did not complement the ssgr mutant ( fig3 , gsr3 and gsr4 , respectively ). again , this strongly suggests that ssgr activates ssga transcription . apparently , positioning of an ssgr - independent promoter in front of ssga relieves its dependence on an active copy of ssgr . this allows altering the regulation of ssga expression , offering possibilities for growth improvement of streptomyces . dna sequences required for , and mode of , ssgr binding to the ssga promoter region interestingly , a clone harboring 233 bp upstream of the translational start codon of ssga showed no detectable promoter activity , even though s1 nuclease mapping experiments revealed two transcription starts in this region ( see previous section ). the clone was sequenced , and shown to contain the published dna sequence . this is apparently confirmed by promoter - probe experiments using a clone with a larger upstream region ( fragment 2 in fig1 a ), which did stimulate red production . the possibility that the dna sequence added to fragment 1 to give fragment 2 ( fig1 a ) one or more promoters , was ruled out by nuclease s1 mapping experiments , which failed to reveal a transcriptional start site inside the ssgr gene ( not shown ). it is most likely that the dna sequence between nt positions − 600 /− 50 , relative to the ssga translational start site , contains all the necessary elements for activation of ssga transcription by ssgr . members of the superfamily of ic 1 r - like regulators bind as homodimers to two well - separated imperfect inverted repeats , so that in total four proteins must bind for activity ( see , for example , zhang et al ., 2002 ). these inverted repeats are separated by at least 100 bp , and on binding , the spacer dna is folded away . it is likely that the region around the stop codon of the ssgr gene constitutes the downstream one of these two elements ; it is highly conserved among s . coelicolor and s . griseus ( fig4 c ), and harbors an a - rich stretch similar to that found for other ic 1 r - type binding sites . activity of ic 1 r - like regulators is lost on binding of a substrate . while this substrate is not yet known , we anticipate that this may be a high - energy metabolic intermediate such as acetyl - coa , phosphoenolpyruvate ( pep ) or citrate , which signal a nutrient - rich state . alteration of the substrate - binding domain should allow the use of unique and non - metabolizable inducers , for improved control of growth and morphology of streptomyces in liquid - grown cultures . ssgr has a similar effect on morphology and fragmentation of liquid - grown cultures of s . coelicolor as ssga ( seq id no : 11 ) since we here show that ssgr transactivates ssga , it is logical that overexpression of ssgr stimulates ssga , and thus fragmentation of the mycelium . indeed , introduction of low - and high - copy number vectors into s . coelicolor m145 results in similar fragmentation as observed for ssga overexpression , with increased fragmentation and reduced branching , with a stronger effect when multi - copy constructs were used ( not shown ). summarizing the regulatory role of ssgr , it is a key regulator of ssga expression , and provides a very useful tool to fine tune or modulate the expression pattern of ssga , and hence control mycelial morphology of streptomycetes and other actinomycetes in submerged culture , and especially in industrial fermentations . ssga ( seq id no : 11 ) belongs to a family of developmentally active proteins the recently completed genome sequences of s . avermitilis and s . coelicolor revealed six and seven genes with relevant homology to ssga , respectively ( bentley et al ., 2002 ; ikeda et al ., 2003 ). the genes encode relatively small ( 130 - 140 aa ) proteins , which share between 30 - 50 % amino acid identity ( keijser et al ., 2003 ; van wezel et al ., 2000c ). the herein described and used ssg proteins have been renamed to bring their names into conformity with keijser et al . ( ssge ( seq id no : 14 ) and ssgg ( seq id no : 10 )). proteins with relevant similarity to ssga ( seq id no : 11 ) are designated salps ( s sg a - like p roteins ). homologues of s . coelicolor ssga ( sco3926 ), ssgb ( sco1541 ), ssgd ( sco7622 ), and ssge ( sco3158 ), are found on the s . avermitilis genome ( sav3926 , 6810 , 1687 , and 3605 , respectively ), with high conservation in these otherwise distantly related species : the ssgb gene products differ in only one amino acid residue . the highest conservation is found in two sections of the proteins , corresponding to amino acid residues 13 - 30 and 40 - 65 of ssga ( seq id no : 11 ). in total 20 amino acid residues ( about 15 % of the protein ) are fully conserved among all 19 salp proteins identified so far . however , there are no sequences in these proteins that resemble known functional motifs . the amino acid sequences of the salp - family proteins from s . coelicolor were retrieved from the entrez protein databases at ncbi ( world wide web 3 ( www3 ). ncbi . nlm . nih . gov / entrez / index . htlm ). the multalin program ( corpet , 1988 ) was used to create a multiple alignment of these sequences . the output file was loaded into the software boxshade ( world wide web ( www ). ch . embnet . org / software / box form . html . the resulting alignment of the s . coelicolor salps is shown in fig7 , with identical and similar amino acids shaded black and grey , respectively . short amino acid stretches showing high homology among all orthologues were selected and used to search the swiss - prot and trembl databases with the scanprosite program for matching protein sequences ( gattiker et al ., 2002 ). the identity of the hits with these searches determined whether the signature sequence had to be adjusted . the sequence was made more specific when too many hits were obtained , while one or more degenerations were allowed in case not all known ssga - like amino acid sequences were detected by the search pattern . the process was reiterated until enough specificity was achieved . eventually , the following signatures for ssga - like proteins were distilled : where [ iv ] represents i or v in a particular position , x represents any amino acid , and x ( n ) means a number ( n ) of ambiguous amino acids . signatures a ( seq id no : 1 ) and c ( seq id no : 3 ) exclusively recognized all ssga - like sequences , while signature b ( seq id no : 2 ) ( a shorter and therefore less limiting version of signature c ( seq id no : 3 )) also detected a few other protein sequences , including putative morpho - proteins . to establish if any of the ssga - like genes constitutes a functional homologue of ssga , phjl401 - derived low - copy number vectors ( 10 copies per chromosome ) harboring the respective genes ssgb - g ( see materials and methods section ) were introduced in the ssga mutant . none of these constructs restored full development ( particularly sporulation ) to the ssga mutant , illustrating that increasing the copy number and expression level of these genes does not fully compensate for the absence of ssga ( seq id no : 11 ). since in this particular experiment the ssga - like genes are expressed from their own promoters , we also used hybrid constructs in which the ssga - like genes were regulated in the same way as the ssgra operon . tn this way , effects due to differences in timing of gene expression or in the highly heterologous n - terminal sections of the proteins , were ruled out . the constructs are described in the materials and methods section , and represented schematically in fig9 a . surprisingly , despite the low degree of homology ( less than 40 % aa identity for the predicted gene products ), introduction of multiple copies of the ssgrc hybrid gene restored sporulation to the ssga mutant , producing plenty viable spores . therefore , ssgc ( seq id no : 15 ) is most likely a functional homologue of ssga ( seq id no : 11 ). no visible complementation of the ssga mutant was observed when any of the other genes ssgb , ssgd , ssge , ssgf , or ssgg ( the latter not shown ) were introduced , suggesting these morphogenes are not functionally related to ssga . to further study the role of ssgb in the development of s . coelicolor m145 , the corresponding gene was disrupted . the gene organization around ssgb is shown in fig8 a . ssgb was replaced by the apramycin resistance cassette aacc4 , using suicide vector pgwb2 ( see materials and methods section ). the knock - out strategy resulted in replacement of the complete coding sequence of ssgb by the apramycin cassette in a created bamhi site . integrity of the resulting ssgb mutants ( designated gsb1 ) was confirmed by southern hybridization ( not shown ). the ssgb disruption mutants were arrested in the aerial growth phase , failing to produce spores ( fig1 ). phase contrast microscopy revealed long , undifferentiated aerial hyphae and confirmed the absence of spores ( not shown ). thus , ssgb is a so - called whi ( sporulation ) gene . introduction of ssgb into gsb1 on the low - copy number vector phjl401 restored sporulation ( confirmed by phase - contrast microscopy ). surprisingly , gsb1 colonies were significantly larger than those of the parental m145 . in e . coli , such large colonies are induced by the mlc ( making large colonies ) phenotype , most likely due to pleiotropic and favorable effects on glucose uptake and / or glycolytic activity . we propose that ssgb directly or indirectly acts as a repressor of an mlc system in streptomyces . the larger colonies reflect enhanced growth rates . much to our surprise , gsb1 transformants produced increased levels of actinorhodin . thus , deletion of ssgb results in pleiotropic effects on morphology and antibiotic production . the morphology of surface - grown colonies of s . coelicolor gsb1 and its parental strain m145 were analyzed by cryo scanning electron microscopy . while m145 produced long and regular spore chains , gsb1 formed very smooth and non - coiling aerial hyphae , failing to sporulate ( fig1 ). the mutants appear to be blocked in an early stage of aerial growth , although the morphology of whi mutants does not necessarily correspond to a frozen developmental stage . few irregularly shaped , branched spore chains could be identified . in these pseudo - sporulating hyphae , septum distance varied greatly , as transpired from study of the mutant by transmission electron microscopy ( not shown ). to test the effect of enhanced expression of ssgb on the morphology of s . coelicolor , this strain was transformed with a multi - copy plasmid containing the ssgb gene and 500 bp of upstream sequence , containing the putative ssgb promoter . these transformants produced significantly smaller pellets ( fig1 ). furthermore , fragmentation was enhanced by ssgb , although not as severe as that observed for ssga . considering the effect of ssgb ( seq id no : 9 ) on the morphology of liquid - grown mycelium , and the significantly enlarged colonies formed by the ssgb deletion mutant , it is clear that ssgb plays a role in determining mycelial morphology . effect of in creased expression of other ssga - like genes on the morphology of s . coelicolor to analyze possible effects of ssgc , ssgd , ssge , ssgf , and ssgg on mycelial morphology in liquid culture , phjl401 - and pwhm3 - derived constructs harboring these genes and their promoters ( see m & amp ; m section ) were introduced into s . coelicolor , and the resulting transformants were subsequently cultivated in yeme or in tsbs medium . the mycelial morphology was checked by phase - contrast microscopy . while no noticeable effect was observed for increased expression of ssge and ssgg , exciting morphological effects were observed in transformants over - expressing ssgc , ssgd or ssgf . introduction of pwhm3 / ssgc in s . coelicolor resulted in a phenotype reminiscent of the same strain harboring ssga , only with a far less pronounced effect ; it resulted in more open mycelial structures , and a slight degree of fragmentation . unexpectedly , introduction of pwmh3 / ssgd results in extremely small colonies which can hardly grow , and phjl401 / ssgd transformants form strongly condensed mycelial clumps . close analysis of these clumps suggested a strong degree of branching , probably as the result of over - expression of ssgd . interestingly , overexpression of the vegetatively expressed ssgd has an almost opposite effect as overexpression of the developmentally regulated genes ssga , ssgb , and ssgc , which have different effects on growth and morphology , but all result in open and / or fragmented mycelial structures , with reduced branching . finally , overexpression of ssgf using pwhm3 / ssgf completely blocked sporulation of s . coelicolor . furthermore , antibiotic production was strongly enhanced , an effect observed in both solid - and liquid - grown cultures . no significant changes in mycelial morphology of liquid - grown cultures were observed . the exciting observation that overexpression of salp - family proteins has diverse and very different effects on the morphology and on antibiotic production of streptomyces in submerged culture as well as on plates , suggests that together , these four genes can be exploited to finely control the morphology and antibiotic production of streptomycetes and other actinomycetes . this discovery is expected to have great impact on the control of industrial fermentations . the observations for the function and expression of salps are summarized in table 3 . the experiments described above show that transcription of ssgr is initiated at a time point corresponding temporally to the onset of sporulation in solid cultures , and thus approximately to the phase that marks the transition from exponential to stationary phase in submerged cultures . this is immediately followed by the onset of ssga transcription , as the result of its activation by ssgr . to analyze the timing of transcription of the ssga - like genes ssgb - g , their respective promoter regions were cloned into pij2587 , thereby using the production of the red - pigmented antibiotic undecylprodigiosin as a visible marker . the exact nature of the dna inserts of pij2587 are shown in table 2 , and experimental details are given in the materials and methods section . the results are shown in fig1 . additional experiments were performed where each of the promoters was tested throughout the life cycle , by streaking transformants every morning and evening for a period of seven days . in this way , the onset of promoter activity could be accurately determined ( data not illustrated ). timing of the expression of most ssga - like genes was developmentally controlled , and additional experiments showed that ssga , ssgb , ssgc , ssge , and ssgf were expressed during aerial growth and sporulation ( table 3 ). surprisingly , the ssgd promoter region already stimulated red production very early during growth , even before colonies were visible , showing that it is expressed during early vegetative growth , and possibly as early as spore germination . this experiment again shows that ssgd is very different from the other salps , in terms of the timing of its expression as well as its effect on streptomyces morphology . the insert harboring the upstream region of the ssgg gene hardly stimulated red production , and if a promoter is located on this dna sequence , it was too weak to establish its timing . as described above , the dna fragment harboring the ssga promoter fails to stimulate red production due to the lack of the complete ssgr target sequence . the data presented here are summarized in table 3 and fig1 . to analyze the transcriptional regulation of the members of the family of ssga - like morphogenes , the ability of the ssgra operon promoters , and of the promoters of the individual genes ssgb - ssgg , to transcribe redd in the presence of mannitol or glucose as carbon sources was tested . the plasmids were introduced into s . coelicolor m512 , and the resulting transformants assayed for red production . as described above , on the complex medium r2ye , all transformants produced significant amounts of red , except the control strain m512 / pij2587 , which remained colorless . the ssgd promoter strongly stimulated red production during early vegetative growth , as soon as colonies were visible . in contrast , transcription from the promoters of all ssga - like genes , except that of the vegetative ssgd promoter , showed differentiation - dependent expression , with a maximum when aerial mycelium was produced . this shows that these morphogenes are developmentally regulated . to analyze the relationship between feed and morphogenes , the influence of ccr on promoter activity of the various promoters was tested . surprisingly , promoters of all ssga - like genes , except that of the vegetatively expressed ssgd , were repressed specifically by glucose . for this , transformants of m512 harboring the relevant promoter - probe constructs ( see table 2 ) were grown on mm plates containing either 1 % glucose or 1 % mannitol as carbon source . as typical examples we show the effect of glucose on the ssgra promoter regions ( various fragments shown in fig1 a ; plate shown in fig1 b ), and on the ssgc and ssgd promoters ( pij2587 - ssgcp or pij2587 - ssgdp , fig1 ). while the vegetative ssgdp invariably stimulated red production , independent of the carbon source , glucose had a strong repressive effect on the activity of the developmental ssgr , ssga , and ssgc promoters , as shown in fig1 and 16 , respectively . this strongly suggests that the genes are under ccr . introduction of the same promoter - probe vectors into a glucose kinase mutant derivative of m512 , designated m512 δglka , revealed that glucose had no repressive effect on the promoters in this strain ( fig1 ). this proves that glucose repression of these promoters occurs in a ccr - dependent manner . in biotechnological fermentations , it is of course profitable to use cheap carbon sources , such as molasses and other less well - defined sugar extracts . however , we noticed a severe dependence of antibiotic production on the carbon source used . to assess the nature of these effects , we streaked s . coelicolor m145 ( wild - type ) and seven congenic mutant derivatives of this strain on nmmp plates with various carbon sources . we analyzed mutants of the act biosynthesis pathway ( m511 , to study red production ), of the red biosynthesis pathway ( m510 , m550 ; to study act production ), of the pleiotropic regulatory gene afsr ( disturbed in regulation of the red and act pathways ) and an afsr suppressor , of the maltose repressor gene malr , and of the glka mutant j1915 . the latter is a control to establish if the effects can be related to glucose repression , which is absent in this mutant . the results are shown in fig1 . while galactose , xylose , and sucrose failed to stimulate antibiotic production in many of the strains used except the act - and red - overproducing afsrsup , arabinose and rhamnose strongly stimulated pigment production in all strains used . for example , the redd mutant ( m510 ) produces no visible antibiotics on most of the carbon sources used , but large amounts of act on arabinose and on rhamnose . interestingly , the sugars have different effects on different strains : while pigment production is stimulated in some strains , it is repressed in others . unexpectedly , ccr has no direct effect on antibiotic production : while strains grown on glucose generally show reduced levels of antibiotic production , glucose has the same effect on the wild - type ( m145 ) as on its congenic glka mutant ( j1915 ), which lacks glucose repression . also , we observed no difference in antibiotic production by strains grown on arabinose alone or on a combination of glucose and arabinose . surprisingly , arabinose and rhamnose strongly stimulated antibiotic production in all strains , while production is very low on sugars such as sucrose and xylose . therefore , it is clear that the effect of carbon utilization should be carefully checked for each individual mutant and antibiotic . arabinose and rhamnose are metabolized via the pentose phosphate pathway . affecting this route has a stimulatory effect on carbon fluxes feeding secondary metabolism . this is a very important observation , as glucose is a major constituent of large - scale fermentations , and repression of ssgra , ssgb , and ssgc would have a dramatic influence on mycelial morphology , resulting in enhanced branching and reduced fragmentation , and therefore — undesirably — in large mycelial clumps . these negative effects are counteracted by using non - repressing carbon sources , although these are typically pure and therefore more expensive , or by the enhanced expression of ssga , ssgb , ssgc , and / or ssgr ( van wezel et al ., 2000 bcd ). glucose kinase is expressed constitutively in submerged cultures , independent of the carbon source used ( mahr et al ., 2000 ). this is logical , since glucose kinase is known to be involved in ccr exerted by glucose , but also by carbohydrates that do not require the presence of a catalytically active glucose kinase . furthermore , glucose kinase activity was similar in stationary phase cultures of s . coelicolor a3 ( 2 ) m145 wild - type cells grown in liquid minimal medium under repressing and non - repressing conditions , using glucose , fructose , glycerol , or mannitol as the sole carbon source , respectively . to assess the growth - phase dependence of glucose kinase activity , s . coelicolor m145 was grown in the phosphate - rich minimal medium nmmp , with casaminoacids ( cas ) and glucose or mannitol as the carbon source . western analysis showed that glucose kinase was produced constitutively in both cultures ( fig2 ). surprisingly , two minor bands appeared , migrating slightly faster than the main glk band . these bands were particularly strong during mid - and late exponential growth in the presence of glucose ( 17 to 24 hours ), but not in the mannitol - grown cultures . to assess the relationship between the appearance of these bands and glk activity , protein extracts prepared from the same growth curves were analyzed using a glucose kinase activity assay . in obvious conflict with the significant amount of glk present in the protein extracts , hardly any activity was observed in the mannitol - grown cultures during exponential growth , but increased when stationary phase was reached . even more surprisingly , we observed a sharp rise in glk activity ( up to approximately 500 nmol / min . mg ) during mid - exponential phase in the glucose - grown cultures ( fig1 ), coinciding with the appearance of the two faster migrating protein bands after approximately 17 hours . on transition to stationary phase , activity dropped to a significantly lower level , comparable to that of mannitol - grown cultures . s . coelicolor has a second gene encoding a protein with glucose kinase activity , designated glkii ( genbank accession number sco6260 ), which is inactive in normal cells , and whose expression can be induced at high frequency in glka mutants grown for a prolonged period in the presence of glucose ( angell et al ., 1994 ). however , the protein is significantly larger than glk ( 355 instead of 318 residues ), and can therefore not correspond to the faster migrating bands . several more proteins with similarity to glk occur in s . coelicolor , but their homology to glk ( far less than 40 % amino acid identity ) is most likely too low for cross - reactivity of the antibodies . glucose kinase is probably activated by post - translational modification ( van wezel , unpublished results ). extensive studies of morphological characteristics of actinomycetes under different culture conditions showed that in non - buffered submerged cultures , fragmentation strongly increased towards stationary phase . this surprising observation prompted analysis of ph effects on morphology of actinomycetes . for this purpose , the actinomycetes saccharopolyspora erythraea , streptomyces coelicolor , streptomyces clavuligerus , and streptomyces lividans were grown in 100 ml ts cultures buffered with 100 mm mops at ph 4 . 5 , 5 . 0 , 5 . 5 , 6 . 0 , 7 . 0 or 8 . 0 . interestingly , all actinomycetes analyzed showed reduced pellet formation and reduced branching as soon as ph dropped to between 5 . 5 and 6 . 0 . the effect was even more pronounced when transformants harboring ssga - expression plasmid pgws4 - sd were analyzed . a typical example of such an experiment is shown in fig2 , showing s . coelicolor with pgws4 - sd after 30 hours of growth in buffered ts medium . while strong fragmentation typical of ssga overexpression was observed at a ph 5 . 5 or lower , larger mycelial structures were formed in cultures buffered at ph of 6 . 0 and higher , with a gradual increase of mycelium size on increasing ph . apparently , an important physiological change is effected by alteration of ph , which can be exploited to increase or reduce fragmentation of liquid - grown actinomyces mycelium , depending on what is desirable at a certain stage of the production process . this is a very important new observation , which allows the control of mycelial morphology as well as the fine tuning of ssga - ( seq id no : 11 -) induced fragmentation by fluctuation of the ph . ideally , precultures should contain fragmented mycelium , with average mycelium size 10 - 50 μm . in that way , the preculture contains a maximal number of growth nuclei , which show optimal transfer of nutrients and oxygen due to the small mycelium size , which strongly reduces the start - up ( lag ) phase ( see also fig1 ). in our 5l fermentation experiments ( such as shown in fig1 and 2 ) this lag phase varied between four hours ( fragmented preculture ) and 12 hours ( large pellets in preculture ). however , in the production phase , typically after completion of the exponential growth phase , larger mycelial structures are required , with an optimal average pellet size between 80 - 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Classification Label: 2