Journal of Bacteriology, March 2004, p . 1345-1354, Vol . 186, No . 5

In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis

Olga N . Sekurova,¹ Trygve Brautaset,¹ Håvard Sletta,² Sven E . F . Borgos,¹ Øyvind M . Jakobsen,¹ Trond E . Ellingsen,² Arne R . Strøm,¹ Svein Valla,¹ and Sergey B . Zotchev^1*

Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim,¹ SINTEF Industrial Biotechnology, SINTEF, N-7034 Trondheim, Norway²

Received 10 September 2003/ Accepted 21 November 2003

 
Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455 . Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficientnystatin production, whereas no significant roles could be demonstratedfor the other two regulatory genes . To determine the in vivotargets for the NysR regulators, chromosomal integration vectorswith the xylE reporter gene under the control of seven putativepromoter regions upstream of the nystatin structural and regulatorygenes were constructed . Expression analyses of the resultingvectors in the S . noursei wild-type strain and regulatory mutantsrevealed that the four regulators differentially affect certainpromoters . According to these analyses, genes responsible forinitiation of nystatin biosynthesis and antibiotic transportwere the major targets for regulation . Data from cross-complementationexperiments showed that nysR genes could in some cases substitutefor each other, suggesting a functional hierarchy of the regulatorsand implying a cascade-like mechanism of regulation of nystatinbiosynthesis.

 
Antibiotic production by Streptomyces bacteria has received much attention in recent years due to the problems associatedwith a constantly increasing incidence of multiresistant microbial pathogens . Considerable efforts are directed toward understandingthe antibiotic biosynthetic pathways in Streptomyces spp . and manipulating the corresponding gene clusters in order to produce novel compounds with improved properties [17] . In addition, remarkable progress is being made in dissecting the functions of the genes that regulate antibiotic production in streptomycetes[11] . Coupling these two fields of research is of great importanceboth for a fundamental understanding of the antibiotic biosynthesisprocesses and for the rational engineering of novel antibioticproducers.

Biosynthesis of secondary metabolites, and in particular antibiotics, by Streptomyces bacteria is a complex process involving several levels of regulation . Many pleiotropic regulatory genes have been isolated from streptomycetes; in most cases, these genesaffect antibiotic biosynthesis by influencing the expressionof the pathway-specific regulatory genes [reviewed in reference 11] . The latter genes are usually found physically linked to the structural antibiotic biosynthesis genes on the chromosomes of streptomycetes . Both positive and negative regulators directly affecting the expression of structural genes via binding totheir promoter regions have been identified in antibiotic biosyntheticgene clusters . In some cases, it has been shown that expressionof the pathway-specific regulatory genes is controlled by signaling molecules, such as A-factor, through the action of other regulators encoded by genes located outside of the biosynthetic gene clusters[22] . Since antibiotic biosynthesis in Streptomyces spp . islinked to the process of cell differentiation [10], it is likely that expression of most of the pathway-specific regulators depends on some sort of signal transmitted via a complex network . Analysis of the regulatory genes in the antibiotic biosynthetic gene clusters is crucial for understanding the mechanisms of regulation, as well as for designing strategies for the construction ofstrains with enhanced antibiotic production.

Most of the detailed analyses of pathway-specific regulators described in the literature are concerned with the biosynthesisof nonmacrolide antibiotics such as actinorhodin, undecylprodigiosin, and daunorubicin [1, 32, 39] . However, several studies whereregulatory genes for macrolide antibiotic biosynthesis wereanalyzed have also been reported . At least some of these regulatorsmust be rather special, since they control the expression ofvery large polyketide synthase [PKS]-encoding genes, which impliessynthesis of unusually long mRNAs . The transcriptional activatorSrmR encoded within the spiramycin biosynthetic gene clusterof Streptomyces ambofaciens has been shown to be required forthe transcription of at least one of the PKS genes involvedin assembly of the spiramycin macrolactone ring [14] . The acyB2-encodedregulator of Streptomyces thermotolerans has been shown to activatethe expression of the acyltransferase gene involved in biosynthesisof the macrolide antibiotic carbomycin [2] . Five regulatory genes associated with the tylosin biosynthetic gene clusterof Streptomyces fradiae have been found [3] . Gene inactivationexperiments have confirmed differential roles for two regulatorsof the SARP [Streptomyces antibiotic regulatory protein] family,TylS and TylT, in controlling tylosin production [4] . TylS wasshown to control the expression of a global regulator [TylR] for the tylosin cluster, while TylT appeared not to be essential for antibiotic biosynthesis . In a separate report, the transcriptional repressor TylQ was found to play a central role in controlling tylosin biosynthesis in S . fradiae [31].

A detailed genetic analysis of PikD, the positive regulatorfor the pikromycin biosynthetic gene cluster in Streptomyces venezuelae, has recently been reported [40] . PikD belongs toa LAL family of transcriptional regulators containing nucleotide triphosphate [NTP] binding motifs and a C-terminally located helix-turn-helix [HTH] motif of the LuxR type [12] . Presumably,these functional features are responsible for the ability ofLAL regulators to bind DNA and activate the transcription of target genes upon NTP hydrolysis . It was shown that PikD is required for pikromycin biosynthesis, and the ability of thisprotein to act as a transcriptional activator depends on thepresence of functional NTP binding motifs.

The polyene macrolide antibiotic nystatin produced by Streptomyces noursei ATCC 11455 is widely used in treatments of fungal infections. Brautaset et al . have previously cloned and sequenced the entire nystatin biosynthetic gene cluster and located six putative regulatory genes within its flanking region [7] . In the presentwork, we describe a comprehensive in vivo analysis of these genes by means of their inactivation in S . noursei, determination of targets by use of the xylE reporter system, and cross-complementationexperiments.

 
Bacterial strains, media, and growth conditions. Bacterial strains, plasmids, and recombinant phages used inthis study are listed in Table 1 . Some of the plasmids are described below . S . noursei strains were maintained on ISP2 agar medium [Difco, Detroit, Mich.] and grown in liquid Trypticase soy broth [TSB] medium [Oxoid] for DNA isolation . Escherichia coli strains were handled by standard techniques [26] . Conjugation from E.coli ET12567[pUZ8002] to S . noursei and gene replacement wereperformed as described previously [13, 28] . Nystatin productionwas assessed by high-performance liquid chromatography [25]of the dimethylformamide extracts of cultures from 500-ml shakeflask fermentations in 100 ml of semidefined SAO-23 medium [28].

 
DNA manipulation and sequence analysis. General techniques for DNA manipulation were used as describedelsewhere [16, 26] . DNA fragments were isolated from agarosegels with the QIAEX kit [QIAGEN, Hilden, Germany] . Southernblot analysis was performed with a DIG High Prime labeling kit[Roche Biochemicals, Mannheim, Germany] according to the manufacturer'smanual . Oligonucleotide primers were purchased from AmershamPharmacia Biotech [Little Chalfont, Buckinghamshire, UnitedKingdom] . Analyses of the amino acid sequences were performedby using the PSORT Prediction [http://psort.nibb.ac.jp/form.html], MEME [http://meme.sdsc.edu/meme/website/meme.html], Pfam [http://www.sanger.ac.uk/Software/Pfam/search.shtml], and MOTIF [http://motif.genome.ad.jp/] search engines.

Construction of plasmids for gene inactivation . [i] nysRI in-frame deletion vector. A 1.37-kb DNA fragment designated SR1, encompassing the regionupstream of nysRI and some of its coding region, was amplifiedfrom the phage N58 template by using primers SOS1 [5'-GCAATGAATTCCGTGGCTCG-3']and SOS2 [5'-GGCTCTAGAGTCAG TAAGCCGGAAGAAC-3'] [restrictionenzyme sites are underlined] . A 1.50-kb DNA fragment designatedSR2, encompassing the 3' end of nysRI and the downstream region,was amplified from the N58 template by using primers SOS3 [5'-GCCTCTAGAGACCAGGACCGCCACCTCC-3'] and SOS4 [5'-GACAAGCTTCGGTGCTG CGGACGAGTTC-3'] . The SR1 and SR2 PCR products were digested with the EcoRI/XbaI and XbaI/HindIIIendonucleases, respectively, and ligated together with the 3.0-kbEcoRI-HindIII fragment from pSOK201, yielding the nysRI replacementvector pSR12 . The in-frame deletion affecting the nysRI genewithin the pSR12 plasmid eliminated the coding sequence foramino acids [aa] 13 to 943 in the NysRI protein, thus affectingall functional features predicted for this polypeptide.

[ii] nysRII in-frame deletion vector. A 1.43-kb DNA fragment designated SR3, encompassing the regionupstream of nysRII and some of its coding region, was amplifiedfrom the phage N58 template by using primers SOS5 [5'-GCAGAATTCGAGTCCGTGCTGCTCATCG-3'] and SOS6 [5'-GCACTGCAGGTGGTCGGTTGGTTCC-3'] . A 1.52-kb DNA fragment designated SR4, encompassing the 3' end of nysRII and the downstreamregion, was amplified from the N58 template by using primersSOS7 [5'-GGCCTGCAGAGCTGTACCTGCTCCTGG-3'] and SOS8 [5'-GACAAGCTTCCTGCCGCACCAACTCGAC-3'].The SR3 and SR4 PCR products were digested with the EcoRI/PstIand PstI/HindIII endonucleases, respectively, and ligated togetherwith the 3.0-kb EcoRI-HindIII fragment from pSOK201, yieldingthe nysRII replacement vector pSR34 . The in-frame deletion affecting the nysRII gene within the pSR34 construct eliminated the coding sequence for aa 14 to 936 in the NysRII protein, thus affecting most of this polypeptide, including the C-terminal HTH domain.

[iii] nysRIII in-frame deletion vector. A 1.42-kb DNA fragment designated SR5, encompassing the regionupstream of nysRIII and some of its coding region, was amplifiedfrom the phage N58 template by using primers SOS9 [5'-GACGAATTCAACTGGTCGCGCTGTTCTG-3'] and SOS10 [5'-GACCTGCAGTCAGGAGGAGCGAGGAGTC-3'] . A 1.50-kb DNA fragment designated SR6, encompassing the 3' end of nysRIII and the downstream region, was amplified from the N58 template by using primers SOS11 [5'-GCACTGCAGTGGAGAAGCACCTCACCAG-3'] and SOS12 [5'-GAGAAGCTTGAGTATTCGGAGGCCGCTC-3'] . The SR5 and SR6 PCR products were digested with the EcoRI/PstI and PstI/HindIII endonucleases, respectively, and ligated together with the 3.0-kb EcoRI-HindIII fragment from pSOK201, yielding the nysRIII replacementvector pSR56 . The in-frame deletion affecting the nysRIII genewithin the pSR56 construct eliminated the coding sequence foraa 29 to 899 in the NysRIII protein, thus affecting all functionalfeatures predicted for this polypeptide.

[iv] nysRIV and orf2 insertional inactivation. The plasmids constructed for insertional inactivation of nysRIV and orf2 were designated pNR4K and pLRD6K, respectively [see Table 1 for details].

[v] orf3 "frameshift" deletion. A 1.3-kb DNA fragment from the S . noursei genome encompassingthe 3' ends of orf3 and orf2 was amplified by PCR with primersNR5D1 [5'-GCGAGCGGCCGCTTCACCCCGCAACTCA-3'] and NR5D2 [5'-CGCGAAGCTTGGCCGACTGCTCGACGTC-3'].The PCR product was digested with NotI and HindIII and then ligated with a 1.7-kb EcoRI-NotI DNA fragment from phage N58 [encompassing nysRIV and the N-terminal part of orf3] and a 3.0-kb EcoRI-HindIII fragment from pSOK201 . The resulting plasmid,pNR5D, contained the S . noursei DNA fragment with a 43-bp deletionin the coding region of orf3 . This deletion creates a frameshiftmutation within the ORF3 coding region, subsequently leadingto truncation of its product . As a result of this truncation,165 C-terminal amino acid residues of orf3 were eliminated andreplaced with 14 aa encoded by another reading frame [and thusunrelated to orf3].

Construction of plasmids for expression of regulatory genes from the ermE*p promoter . [i] nysRI expression vector. A 0.6-kb DNA fragment representing a promoterless 5' end ofnysRI was PCR amplified from the phage N1 template by usingprimers NR1.1 [5'-CGCCGCATGCTGTTCTCACCCCACGT-3'] and NR1.2 [5'-GGCGCGACCGGTTCGGCCT-3']. The PCR product was digested with SphI/AgeI and then ligated together with a 2.8-kb AgeI-EcoRI DNA fragment from phage N1 into the pGEM7Zf[-] vector digested with SphI/EcoRI . From theresulting construct, a 3.4-kb SphI-HindIII fragment was isolatedand ligated together with a 0.3-kb EcoRI-SphI fragment frompGEM7ZfErmE*li, containing the ermE*p promoter, into the EcoRI/HindIII-digestedpSOK804 vector [for details, see Results, Table 1, and Fig. 2], resulting in the pNRE2 construct.

 
[ii] nysRII expression vector. A 2.2-kb SalI-BclI fragment from phage N58 [representing the3' end of nysRII] was cloned into SalI/BamHI-digested pGEM11Zf[-].A 0.8-kb fragment representing the 5' end of the nysRII genewas PCR amplified from the phage N58 template with primers NSR2.1 [5'-GCCGGCATGCGACGAACAGGACGAGAGGT-3'] and NSR2.3 [5'-GCCGTGGTCGACGAAGG-3']. The PCR fragment was digested with SphI/SalI and then ligated,together with a 2.2-kb SalI-HindIII fragment from the pGEM11Zf[-]-basedconstruct, into the SphI-HindIII-digested pGEM3Zf[-] vector.From the latter, a 3.0-kb SphI-HindIII fragment was isolatedand ligated, together with the 0.3-kb EcoRI-SphI ermE*p promoterfragment, into the EcoRI/HindIII-digested pSOK804 vector, resultingin the pC3A1 construct.

[iii] nysRIII expression vector. A 2.8-kb SacI-NruI fragment from phage N58, encompassing 89nucleotides [nt] upstream of the nysRIII start codon and a largeportion of the coding region, was ligated together with a 0.5-kbNruI-EcoRI fragment from the same phage, representing the 3'end of this gene, into pGEM3Zf[-] . The nysRIII gene was excisedfrom this construct as a 3.2-kb SphI-EcoRI fragment and ligated into pGEM7Zf[-] . From the pGEM7Zf[-]-based construct the nysRIII gene was excised as a 3.2-kb SphI-HindIII fragment and ligatedtogether with the 0.3-kb EcoRI-SphI ermE*p promoter fragmentinto the EcoRI/HindIII-digested pSOK804 vector, resulting inthe pNTE3 construct.

[iv] nysRIV expression vectors. The long [L] and short [S] versions of the nysRIV gene werePCR amplified from N58 recombinant phage DNA with primers NR4P3[5'-CTCAGCATGCCGAAAGGATGGCG-3'] and NR4P5 [5'-AGGCAAGCTTCGGCGACACGGGCGT-3']or primers NR4P4 [5'-CTCAGCATGCGTACGACCGGCGGG-3'] and NR4P5,respectively . The corresponding PCR products of 0.78 [NR4L]and 0.73 [NR4S] kb were digested with SphI and HindIII and thenligated, together with the 0.3-kb EcoRI-SphI fragment containing the ermE*p promoter, with the EcoRI-HindIII-digested pSOK804vector, yielding vectors pNR4EL and pNR4ES, respectively.

PCR amplification of putative promoter regions. Seven intergenic regions from the nystatin biosynthetic clusterthat might contain promoters have been amplified by PCR [seeFig . 4] . A 315-bp DNA fragment designated nysHp and containing the region between the nysH and nysDIII genes was amplified from the N40 template by using primers NHP1 [5'-GCAGTCTAGAGAGGAACACCCCGGTTGAC-3'] and NHP2 [5'-GCAGAAGCTTGGCAAACCCTTCTCGAACAC-3'] . In PCR a 315-bp intergenic fragment designated nysDIIIp was amplified from the N40 template by using primers ND31 [5'-GCAGTCTAGAGGCAA ACCCTTC TCGAACAC-3'] and ND32 [5'-GCAGAAGCTTGAGGAACACCCCGGTTGAC-3']. A 202-bp fragment encompassing the region between the nysDIII and nysI genes and designated nysIp was amplified from the sametemplate with the help of primers NIP1 [5'-GCCAACTGGTAG CAGTTCTCAAGCTTTCG-3'] and NIP2 [5'-GCGGTCTAGACTCAACTCAACCCATCTCG-3'] . The primers for nysAp, the intergenic region upstream of the nysA gene, were NSAP1 [5'-GCAGAAGCTTCGGTTACTTGGTCTCATGC-3'] and NSAP2 [5'-GCAGTCTAGAGCCTTGCTCACCCCTGCGG-3']; the 212-bp PCR product was amplified from the N76 template.A 212-bp fragment encompassing the region upstream of the nysDI gene and designated nysDIp was amplified from the N76 template by using primers ND11 [5'-GCAGTCTAGACGGTTACTTGGTCTCA TGC-3'] and ND12 [5'-GCAGAAGCTTGCCTTGCTCACCCCTGCGG-3'] . The 351-bp nysRIp and nysRIVp DNA fragments upstream of the nysRI and nysRIV genes,respectively, were amplified from the N58 template by usingprimers NR11 [5'-GCAGAAGCTTGAGACGGCACCATGCCAC-3'] and NR12 [5'-GCAGTCTAGACACGCGTTCCTCCACGTG-3']for the nysRIp fragment and primers NR41 [5'-GCAGAAGCTTGTCGTACGCCCGTCCGG-3'] and NR42 [5'-GCAGTCCAGAGAGACGCGCATCCTTTCGG-3'] for the nysRIVp fragment.

 
Construction of xylE-based promoter probe vectors. PCR-amplified fragments of intergenic regions were digestedwith the HindIII and XbaI endonucleases and ligated into the pGEM3Zf[-] vector . The 1.5-kb XbaI-BglII fragment with the promoterlessxylE gene was excised from the pIJ4081 vector and subclonedinto XbaI/BamHI-digested pGEM3Zf[-], resulting in pGEM-XylE1.The HindIII-XbaI promoter-containing fragments from constructsbased on pGEM3Zf[-] were ligated, together with the 1.5-kb XbaI-EcoRI fragment containing the reporter gene xylE from pGEM-XylE1, into the HindIII/EcoRI-digested integrative vector pSOK804, resulting in seven constructs [Table 1] . Each of the pSOK804-basedconstructs contains one of the seven intergenic regions upstreamof the reporter gene xylE.

The resulting promoter-probe constructs were introduced by conjugation into the nysRI, nysRII, nysRIII, and nysRIV mutants for theXylE assay experiments.

Assay for XylE activity. For quantitative enzymatic assays, protein extracts from S.noursei cultures were prepared . For the precultures, 20 ml ofliquid TSB medium in 250-ml shake-flasks containing 3 g of 3-mm-diameterglass beads was inoculated with spore suspensions and incubatedovernight at 30°C with shaking at 250 rpm . On the next day,50 ml of MP5 medium [containing, per liter, 25 g of glycerol,3 g of yeast extract, 2 g of NaCl, 0.2 g of K₂HPO₄, 0.2 g ofMgSO₄, and 0.02 g of FeSO₄ [pH 7.2]] was inoculated with 1.5ml of precultures and incubated at 30°C for 24 or 48 h withshaking at 250 rpm . Cells were harvested by centrifugation for10 min at 5,000 rpm [Sorvall], washed with 10 ml of 20 mM phosphatebuffer [pH 7.2], and resuspended in 10 ml of sample buffer [100mM phosphate buffer [pH 7.5]-10% acetone [vol/vol]-20 mM EDTA[pH 8.0]] . A 3-ml volume of cell suspension was sonicated for2 min, and 10 µl of 10% Triton-100 was added per ml of extract . Extracts were placed on ice for 15 min and then centrifuged for 10 min at 15,000 rpm, and cell supernatants were used for XylE assays.

The reaction mixture for measurement of catechol dioxygenase activity consisted of 1.9 ml of assay buffer [100 mM phosphatebuffer [pH 7.5], 0.2 mM catechol] preincubated at 37°C for1 min and 100 ml of cell extract . The optical density at 375nm was measured over 6 min.

Protein concentrations in extracts were measured according tothe Bio-Rad Protein Assay method, by using bovine serum albuminas the standard . The catechol dioxygenase activity was calculatedas the rate of change in optical density at 375 nm per minuteper milligram of protein.

 
In silico analysis of putative regulatory genes and their deduced gene products. Three genes, designated nysRI, nysRII, and nysRIII, are locateddownstream of the nysE gene, encoding putative thioesterase,in the nystatin biosynthetic gene cluster of S . noursei ATCC11455 [7] [Fig. 1A] . The nysRIII gene's putative start codon overlaps by 11 nt with the 3' end of the nysRII gene, suggesting that these two genes might be translationally coupled . Analysis of the deduced primary sequences of the proteins encoded bythe nysRI, nysRII, and nysRIII genes revealed their significant similarity to each other and to a number of putative transcriptional activators of the LAL family [12] [data not shown] . Severalfunctional features were predicted for the NysRI, NysRII, and NysRIII polypeptides [Fig . 1B] . Those include HTH DNA bindingmotifs of the LuxR type [15] located at the C termini of allthree proteins [Fig . 2B] and Walker A and B NTP binding motifs[38] at the N termini of NysRI and NysRIII [Fig . 2A] . In addition, two putative transmembrane regions were predicted in the central part of NysRIII, while tetratricopeptide repeats [TPR] [20] were detected in both the NysRI and NysRIII polypeptides [see Discussion].

 
The nysRIV gene, previously designated orf4 [7], is located404 nt downstream of nysRIII . The start codon for nysRIV hasbeen reassigned, according to a better match of an upstreamsequence [AGGA] to the consensus Shine-Dalgarno sequence [30], and is likely to be located 48 nt upstream of the start codon originally proposed [7] . Thus, nysRIV presumably encodes a 226-aarather than a 210-aa protein [see below] . A database searchshowed a high degree of NysRIV sequence identity [63%] to the regulatory protein PteR encoded within the pentaene macrolide antibiotic gene cluster of Streptomyces avermitilis [23] . Detailedsequence analysis revealed the presence of a PAS-like domain at the N terminus of NysRIV [33] and a putative C-terminal HTHmotif of the LuxR type [Fig . 1B and 2B].

orf3, located downstream of nysRIV, encodes a protein of 253 aa similar to transcriptional repressors of the DeoR family[36]. orf2, which is transcribed in the direction opposite thatof all the other putative regulatory genes, encodes a 354-aapolypeptide similar to transcriptional regulators of the AsnCtype [19].

Inactivations of the regulatory genes and their effects on nystatin biosynthesis. Disruption of the nysRI gene, described previously, has ledto complete elimination of nystatin biosynthesis in the S . nourseimutant NRD2 [7] . However, judging from the operon-like organizationof the nysRI, nysRII, and nysRIII genes [Fig . 1A], this mutation very likely had a polar effect . In order to determine the individual roles of these three genes in the regulation of nystatin biosynthesis, we constructed in-frame deletion mutants . The deletions were generated via selection of a second crossover event after integration of the pSR12, pSR34, and pSR56 gene replacement vectors [see Materials and Methods] into the genome of the S . noursei wild-type [WT] strain . The resulting mutant strains, SR12, SR34, and SR56 [see Fig . 1A for genotypes], were analyzed for nystatin production.

Mutant SR12 [nysRI] produced nystatin at a severely reducedlevel, i.e., 0.5% of that in the WT [Table 2] . This result confirmedthe assumed polar effect of the nysRI disruption in the NDR2mutant, since not even traces of nystatin could be detectedupon fermentation of the latter [7] . Nystatin production inmutants SR34 [nysRII] and SR56 [nysRIII] was reduced by 93 and91%, respectively, compared to that in the WT strain [Table2].

 
The nysRIV gene was inactivated by insertion of the Km^r gene into its coding sequence via a gene replacement procedure using plasmid pNR4D [Table 1; Fig . 1A] . The resulting NR4K mutantproduced nystatin at a level of ca . 2% of WT production [Table2].

orf3 and orf2 were inactivated by deletion and disruption with the Km^r cassette by using plasmids pNR5D and pLDR6K, respectively,creating frameshift mutations [see Materials and Methods] [Table1] . Neither the orf3 nor the orf2 mutation had a significanteffect on nystatin biosynthesis, suggesting that these genesare not directly involved in the regulation of nystatin biosynthesis,at least under the conditions tested [data not shown].

Complementation of the nysRI, nysRII, nysRIII, and nysRIV mutants by expression of the regulatory genes from the ermE*p promoter. The pSOK804 plasmid vector, containing an integration function[an integrase gene and AttP] from the streptomycete temperatephage VWB [37] and part of the pSET152 vector [6], was constructed[Table 1] . Plasmid pSOK804 was able to integrate site-specificallyinto one site in the genome of S . noursei [data not shown],at a frequency about 2 orders of magnitude higher than thatfor the pSET152 vector previously used for gene expression inS . noursei [41] . pSOK804-based integration vectors were assembledfor the expression of the nysRI, nysRII, nysRIII, and nysRIV genes in S . noursei [see Materials and Methods and Table 1]. To circumvent potential problems related to self-regulationof these genes' endogenous promoters, we chose to use the constitutive ermE*p promoter [5] for their expression . Five integrative expressionvectors were constructed [see Materials and Methods and Table1] and used for complementation of the corresponding S . nourseimutants . The results of these experiments are summarized inTable 2 . Nystatin synthesis was either partly or fully restoredin the SR12, SR34, SR56, and NR4K mutants upon introductionof the vectors expressing the respective regulatory genes, suggestingthat the mutations did not have polar effects . Only vector pNR4EL,expressing the longer, 226-aa version of NysRIV, was able tocomplement NR4K, thus corroborating the new assignment of thenysRIV start codon [see above].

The vectors used in complementation experiments were also introduced into WT S . noursei in order to test whether potential overexpressionof the regulators might increase nystatin production . Interestingly,while no effect was observed with nysRI and nysRIII, additionalexpression of nysRII from ermE*p provided for a 21% increasein nystatin production [Table 2] . Expression of nysRIV fromthe pNR4EL vector in the WT S . noursei strain had the strongestpositive effect on nystatin synthesis: the resulting recombinantstrain produced nystatin at a level 36% above that of the WT[pSOK804] [Table 2].

Promoter activity studies with the regulatory mutants. Although definitive roles in controlling nystatin biosynthesiswere established for LAL-family regulators and NysRIV by theexperiments described above, their individual contributionsto the process, as well as the target genes, remained unknown.To address these questions, seven putative promoter regionsfor the structural and regulatory genes from the nystatin cluster[Fig . 3] were cloned upstream of a promoterless xylE reportergene [see Materials and Methods] . Since we deduced that thenysH-nysDIII and nysDI-nysA intergenic regions contain divergentpromoters, these regions were cloned in two alternative orientationsto allow the assessment of both promoters . The reporter cassetteswere cloned into the pSOK804 integrative vector, and the resultingplasmids were introduced into the S . noursei WT strain and theregulatory mutants . XylE activity assays of crude extracts preparedfrom the recombinant strains were used to monitor relative expressionlevels from the various promoters [Fig . 4].

 
In the WT background, XylE activity could be detected for all promoter-probe vectors . Data from the XylE assay [Fig . 4] showedthat expression from the promoter for the putative mycosamine transferase gene nysDI [and probably for the cotranscribed nysDIIand nysN genes] was 2 times higher in the SR56 and NR4K mutantsthan in the WT background at the 24-h time point . Interestingly,this pattern was changed after 48 h: XylE activity in the SR56mutant was reduced to ca . 30% of that in the WT, while that in the NR4K mutant continued to increase . nysDIIIp, the promoter for a putative GDP-mannose dehydratase gene, depended only weakly on the regulators: the XylE activity measured for the nysDIIIp::xylE construct in the regulatory mutants was ca . 20 to 50% lower than that in the WT . This difference was most visible at the24-h time point and was less profound after 48 h.

Expression from the nysAp promoter for the PKS loading module gene nysA [and presumably for the cotranscribed nysB and nysCgenes] was very strongly dependent on all four regulators . Essentiallyno XylE activity was observed in the corresponding protein extractsexcept for the SR34 mutant, where only very low XylE activity[ca . 3% of the WT level] was detected . Compared to the promotersfor mycosamine biosynthesis and attachment genes, nysAp providedfor ca . 30-times-higher XylE expression in the WT.

nysIp, the promoter presumably driving the expression of the NysI, NysJ, and NysK PKS proteins, responsible for further elongation and termination of synthesis of the nystatin polyketide chain, showed limited dependence on the presence of the regulators.The strongest effects observed were those in the SR34 and NR4Kmutants at 24 h, where XylE activity due to expression fromnysIp was ca . 60 to 70% lower than that in the WT background.Interestingly, nysIp seemed to be dependent on the NysRIII regulatorat 48 h, while no such trend could be observed when XylE activityin the protein extract from the 24-h culture was measured [Fig. 4] . Also, the XylE activity in the NR4K mutant almost reached the level of that in the WT at 48 h.

According to the XylE assay, the promoter for the transportergene nysH [and presumably for the cotranscribed nysG gene] was essentially independent of NysRI, while its activity was greatly diminished in the nysRII, nysRIII, and nysRIV mutants [see Discussionand Fig . 4].

nysRIp [the promoter region upstream of the regulatory gene nysRI] showed only moderate dependence on the NysRIII and NysRIV regulators, since the XylE activities in the corresponding mutants were diminished by ca . 50 to 60% from that in the WT . At the same time, nysRIp was strongly dependent on NysRI and NysRII [Fig . 4] . This result suggested that NysRI regulates its own expression and that NysRII is involved in this process as well. The nysRIp promoter seemed to be very strong and was superseded only by nysRIVp, which provided the highest level of XylE expressiondemonstrated in these experiments . The activity of the nysRIVppromoter was greatly affected in all three LAL regulatory mutants,while NysRIV seemed to be moderately autoregulating its own expression, as XylE activity in the NR4K mutant was diminished by ca . 60%.

In order to gain more insight into the results of the complementation experiments described in the preceding section, a control experiment designed to assess the efficiency of the ermE*p promoter in S . noursei was performed . In this experiment, XylE activity was measured in protein extracts from the WT strain expressing xylE from the nysAp and ermE*p promoters . Apparently, ermE*pprovided for a much more efficient [ca . 12-times-higher] expressionof xylE than nysAp [Fig . 4]. ermE*p appears to be the strongestof the promoters investigated in this study.

Cross-complementation experiments. The xylE promoter fusion experiments provided important clueson the target genes controlled by the four NysR regulators inthe nystatin gene cluster . However, the possible hierarchy ofthe regulators remained obscure . In order to gain deeper insightinto the mechanism of regulation of nystatin biosynthesis, cross-complementationexperiments were carried out . The idea behind these studieswas to test which of the regulatory genes, when expressed fromthe heterologous ermE*p promoter, could substitute for eachother in the regulatory mutants.

Accordingly, pSOK804-based expression vectors containing four pathway-specific regulatory genes were introduced into the SR12, SR34, SR56, and NR4K mutants, and nystatin production by recombinant strains was assessed [Table 3] . Nystatin production in the SR12mutant could be restored to the same extent [ca . 60% of the WT level] by introduction of any of the four regulatory genes. Interestingly, both nysRII and nysRIII were able to complement the SR12 mutant, while no cross-complementation was observed between the nysRII and nysRIII genes, suggesting that these regulatory genes can be placed on the same hierarchy level [see Discussion] . nysRIV was able to restore nystatin biosynthesis to a significant level [60 to 87% of the WT] in all regulatory mutants.

 
The results obtained in the gene inactivation experiments clearly show that at least four regulatory genes control nystatin production in S . noursei . The nysRI, nysRII, nysRIII, and nysRIV genesare required for efficient nystatin biosynthesis and probablyrepresent transcriptional activators for the gene cluster . Clearly,nysRII and nysRIII are not as important in this respect as nysRI,since their inactivation has less profound effects on antibioticproduction . This notion is exemplified by the fact that, accordingto the xylE fusion experiments, NysRI regulates an endogenouspromoter for its own gene, which is also likely to drive theexpression of nysRII and nysRIII . The operon-like structureof nysRI-nysRII-nysRIII and the apparent polar effect of nysRIdisruption imply that these three genes might be transcribedfrom the same promoter located upstream of nysRI . nysRIp isthe second strongest [after nysRIVp] of the nys promoters andremains at least partially active in all regulatory mutants.Both nysRIp and nysRIVp seem to be weaker than the ermeE*p promoterwhen used for the expression of xylE in WT S . noursei [Fig. 4] . This fact, along with the results from the complementation experiments [Table 2], suggests that a high level of expressionof the regulatory genes alone does not ensure a high level ofnystatin production, since only partial complementation was observed when nysRI and nysRIV were expressed from ermE*p . Itseems plausible, therefore, that the mechanism governing gene expression in the nystatin biosynthetic cluster requires coordinated expression of the regulatory genes, which is provided through the intrinsic regulatory genes' promoters.

Remarkably, XylE expression from the nysRIp promoter is strongly reduced only in nysRI and nysRII mutants, suggesting that transcriptionof the nysRI-nysRII-nysRIII genes is autoregulated . Taking theabove into consideration, it is conceivable that in both ofthese mutants, neither of the regulators is efficiently expressed.It seems, however, that the basal level of expression of NysRIIand NysRIII in the nysRI mutant is sufficient to activate thenysHp promoter [Fig. 4] . The latter appears to be strongly dependenton the availability of the NysRII, NysRIII, and NysRIV regulators.

The NysRI, NysRII, and NysRIII proteins seem to derive froma common ancestor, since they contain homologous regions andtheir domain organization, especially for NysRI and NysRIII,is similar [Fig . 1B] . Proteins with significant similarity to NysRI, NysRII, and NysRIII are found mostly in actinomycetesand are proposed to be transcriptional activators for antibiotic biosynthesis, lipase, and cholesterol oxidase genes [18, 21,27, 29] . Genes encoding proteins similar to NysRI to -III arealso located in the biosynthetic gene cluster for the polyeneantibiotic candicidin [9] . Most of these proteins might be consideredmembers of the LAL subfamily of transcriptional regulators proposedby De Schrijver and De Mot [12], on the basis of their sizeand the presence of the N-terminal NTP binding and C-terminalLuxR HTH motifs . The NTP binding motifs in the transcriptionalregulator PikD have been shown to be required for its activity[40] . Therefore, it seems likely that NysRI and NysRIII alsorequire NTP binding and hydrolysis for their function . The presenceof the TPRs, which are implicated in protein-protein interactions[35], in NysRI and NysRIII suggests that these proteins mightinteract with other proteins . It is well documented that protein-protein interactions play an important role in transcriptional controlin bacteria [34].

All experimental data obtained for the nysRIV gene [see below] point to its central role in controlling nystatin biosynthesis. Indeed, very little nystatin is produced by the nysRIV disruption mutant, while expression of nysRIV from the ermE*p promoter results in significant stimulation of nystatin production in the WT strain . Also, nysRIV can complement all nysR regulatory mutants . Detection of a PAS-like domain within NysRIV suggests that this protein might respond to the energy levels in thecell . PAS domains are found in many signaling proteins, wherethey serve as signal sensor domains [24] . Promoter probe studies clearly demonstrate that nysRIVp is strongly downregulated in all three LAL regulator mutants . However, the nysRIVp promoter appears to be the strongest of the seven nys promoters studied, and even in the absence of LAL regulators, its activity is at the level of the nysDIIIp and nysIp promoters . The latter fact suggests that a relatively high level of nysRIV transcription is required for this gene's product to exert a positive effect on nystatin biosynthesis.

The xylE fusion experiments with the promoters for the nystatin structural genes provided the first clues on the regulatory mechanism controlling nystatin biosynthesis in S . noursei . Apparently, promoters driving the expression of nysDIII and nysDI-nysDII-nysN[presumably cotranscribed] are only weakly dependent on theNysR regulators.

Regulation of the promoters driving the expression of PKS genes, nysIp and nysAp, is strikingly different . First, the level of XylE expression from the nysAp promoter in the WT strain seems to be at least 35 times higher than that from nysIp . This is not surprising, since nysA encodes the loading module of the nystatin PKS, expression of which is pivotal for initiationof biosynthesis [8] . The mutations in the nysR regulatory geneshave much stronger effects on nysAp than on nysIp [see Fig.4], suggesting that initiation of nystatin biosynthesis promotedby the nysA gene product is the primary target for the regulators.

Cross-complementation experiments helped to establish a hierarchy among the four NysR regulators of the nystatin gene cluster. Differences in the degree of complementation observed in these experiments could most probably be attributed to the expressionof the genes in trans from the heterologous promoter . The ability of nysRII and nysRIII to complement the nysRI mutant impliesthat NysRII and NysRIII can each substitute for NysRI and thatexpression of these proteins in the SR12 mutant is severely affected . It is not clear, however, why both nysRII and nysRIII can complement the nysRI mutant, since deletion of either ofthese genes has a detrimental effect on nystatin biosynthesis,and these genes cannot substitute for each other in cross-complementationexperiments [Table 3] . The answer to this question probablylies in the plausibility of concerted action of the regulatorsor might be that such complementation is due to the use of anonnatural constitutive promoter, ermE*p, in the complementation experiments.

Based on the data from promoter analysis and cross-complementation experiments, the following tentative model can be suggested. Expression of the LAL regulatory operon would start with NysRI,which positively regulates its own promoter . However, it seemsthat NysRII is required for efficient transcription from nysRIp,while NysRIII is not essential [Fig . 4] . It is thus logical to assume that NysRI and NysRII function in concert as autoregulators of the LAL operon, ensuring its efficient transcription . NysRII seems to play a pivotal role here, as expression of this proteincan alleviate the effect of nysRI mutation . Since no cross-complementationis observed in the case of nysRII and nysRIII, it seems likelythat their products are both required for efficient nysRIV expression.Since nysRIV can complement all regulatory mutants, but noneof the other regulators can complement NR4K, nysRIV most probably directly controls the expression of nystatin biosynthetic genes.

 
We thank A . M . Lian for conducting initial promoter studiesand R . Aune for performing the fermentations . We are also gratefulto L . Van Mellaert, C . R . Hutchinson, and J . White for providingvectors pKTO2, pGEM7ZfErmE*Li, and pIJ4081, respectively.

This work was supported by the Research Council of Norway, SINTEF, and Alpharma AS.

 
* Corresponding author . Mailing address: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway . Phone: 47 73 59 86 79 . Fax: 47 73 59 12 83 . E-mail: sergey.zotchev@biotech.ntnu.no.

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