Patent Application: US-12296705-A

Abstract:
the invention relates to industrial microbiology , in particular to fermentation technology and especially to fermentation methods for filamentous microorganisms , in particular , filamentous microorganisms such as actinomycetes . the present invention discloses a method for improving the production of a product of interest in a liquid culture of filamentous microorganisms comprising providing the filamentous microorganisms with an agent for altering the morphology of microstructures of the filamentous microorganisms . the invention also relates to a method for improving the production of a product of interest in a liquid culture of filamentous microorganisms comprising altering in the filamentous microorganisms the expression level or copy number of an endogenous agent for altering the morphology of microstructures of the filamentous microorganisms . the invention further provides multiple methods for obtaining a filamentous microorganism with altered , preferably enhanced , fragmentation characteristics . however , a filamentous microorganism with reduced fragmentation characteristics is also included .

Description:
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 ( http :// 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 ( http :// 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 : signature a = ( seq id no : 1 ) [ iv ] [ pl ] [ av ] x [ fl ] xy [ deh ] x ( 2 , 3 )[ dh ] p signature b = ( seq id no : 2 ) wx [ fvl ] xr [ ed ][ lm ][ lv ] xxg signature c = ( seq id no : 3 ) wx [ fvl ] xr [ ed ][ lm ] [ lv ] xxgx ( 5 ) gxg [ de ] v 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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