Carboxyethylarginine synthase, encoded by the paralogous ceaS1 and ceaS2 genes, catalyzes the first reaction in the shared biosynthetic pathway leading to clavulanic acid and the otherclavam metabolites in Streptomyces clavuligerus . The nutritional regulation of ceaS1 and ceaS2 expression was analyzed by reverse transcriptase PCR and by the use of the enhanced green fluorescent protein-encoding gene [egfp] as a reporter . ceaS1 was transcribedin complex soy medium only, whereas ceaS2 was transcribed inboth soy and defined starch-asparagine [SA] media . The transcriptionalstart points of the two genes were also mapped to a C residue98 bp upstream of ceaS1 and a G residue 51 bp upstream of theceaS2 start codon by S1 nuclease protection and primer extensionanalyses . Furthermore, transcriptional mapping of the genes encoding the beta-lactam synthetase [bls1] and proclavaminate amidinohydrolase [pah1] isoenzymes from the paralogue gene cluster indicated that a single polycistronic transcript of 4.9 kb includesceaS1, bls1, and pah1 . The expression of ceaS1 and ceaS2 ina mutant strain defective in the regulatory protein CcaR wasalso examined . ceaS1 transcription was not affected in the ccaRmutant, whereas that of ceaS2 was greatly reduced compared tothe wild-type strain . Overall, our results suggest that differentmechanisms are involved in regulating the expression of ceaS1and ceaS2, and presumably also of other paralogous genes thatencode proteins involved in the early stages of clavulanic acidand clavam metabolite biosynthesis.

 
Members of the genus Streptomyces exhibit a complex life cycle that involves a hierarchy of regulatory genes controlling and coordinating antibiotic production and sporulation [14, 20].Streptomyces clavuligerus produces a number of ß-lactamcompounds, including cephamycin C, clavulanic acid, and at leastfour other known clavam metabolites [8, 12] . Clavulanic acidand the other clavams differ from cephamycin C in that theirbicyclic nucleus contains an oxygen atom instead of the sulfuratom found in the more conventional cephamycin-type antibiotics[6] . Clavulanic acid is a clinically important inhibitor ofß-lactamases, whereas the other clavam metabolitesproduced by S . clavuligerus show weak antibacterial and antifungalactivities [35] . The four clavam metabolites produced by S.clavuligerus are commonly referred to as the 5S clavams dueto their stereochemistry, which differs from the 5R stereochemistryof clavulanic acid . The ß-lactamase-inhibitory activityof clavulanic acid has been associated with this 5R stereochemistry[6] . Since clavulanic acid is produced industrially by fermentationusing S . clavuligerus, the regulation of clavulanic acid and5S clavam biosynthesis is a point of great interest.

The biosynthetic pathway leading to clavulanic acid and the5S clavams is partially shared, at least to the level of clavaminicacid [Fig . 1B] [10] . However, in S . clavuligerus, the genesinvolved in the biosynthesis of clavulanic acid and the 5S clavamsreside in three distinct gene clusters that are not physicallylinked [K . Tahlan, H . U . Park, and S . E . Jensen, unpublisheddata] . The clavulanic acid gene cluster is situated immediatelydownstream of the cephamycin gene cluster, and together theyform a larger gene cluster often referred to as the ß-lactamsupercluster [1, 13, 44] . This clavulanic acid gene clustercontains genes encoding enzymes involved in the early sharedstages of the clavulanic acid and 5S clavam pathway [the earlygenes], as well as genes encoding proteins involved in the laterstages of clavulanic acid biosynthesis only [the late genes][15, 17, 21, 24] . The clavam gene cluster encompasses the geneencoding clavaminate synthase 1 [cas1], a paralogue of the cas2gene from the clavulanic acid gene cluster and one of the genesinvolved in the biosynthesis of both clavulanic acid and the5S clavams . In addition to cas1, the clavam cluster includesother genes involved exclusively in the biosynthesis of the5S clavams and some genes of unknown function [26] . The paraloguegene cluster contains paralogues of additional genes found inthe clavulanic acid gene cluster that encode enzymes involvedin the early stages of both clavulanic acid and 5S clavam biosynthesis[18, 42].

 
The biosynthesis of both clavulanic acid and the 5S clavams initiates with the condensation of glyceraldehyde-3-phosphateand L-arginine to give N²-[2-carboxyethyl]arginine, catalyzedby the enzyme carboxyethylarginine synthase [CEAS] [Fig. 1B][19] . There are two copies of the gene encoding CEAS presentin S . clavuligerus; ceaS2 is located in the clavulanic acidgene cluster, and ceaS1 is found in the paralogue gene cluster[Fig . 1A] [15, 33, 42] . The next reaction in the pathway iscatalyzed by the enzyme ß-lactam synthetase [BLS][3, 23], which is also encoded by two separate genes [bls1 andbls2] [15, 42] . BLS activity closes the ß-lactam ringto give deoxyguanidino proclavaminic acid [3, 23], which is then converted to guanidino proclavaminic acid by clavaminate synthase [CAS] [5] . Two genes encode CAS in S . clavuligerus [22], cas1 from the clavam gene cluster and cas2 from the clavulanicgene cluster [22, 26] . Guanidino proclavaminic acid is hydrolyzedto give proclavaminic acid by the action of proclavaminate amidinohydrolase [PAH] [46], which is encoded by pah1 and pah2 [1, 15, 18, 46].Next, in a series of two sequential reactions, CAS convertsproclavaminic acid to clavaminic acid [4, 36] . Clavaminic acidis thought to be the branch point of the pathway leading toclavulanic acid and the 5S clavams [10] . The only other stepknown in the pathway beyond clavaminic acid is the reductionof clavaldehyde to clavulanic acid by the action of the enzymeclavulanic acid dehydrogenase [27], which is encoded by cad, a late gene from the clavulanic acid gene cluster [15] . It isstill not known how clavaminic acid is converted to clavaldehyde,nor have any of the steps specifically leading from clavaminicacid to the 5S clavams been elucidated [16].

The transcriptional activators CcaR and ClaR are known to regulate the expression of the clavulanic acid biosynthetic genes [16]. The ccaR gene lies within the cephamycin biosynthetic gene cluster, and CcaR is the pathway-specific transcriptional regulator for cephamycin biosynthesis, as well as controlling expression ofthe claR gene from the clavulanic acid gene cluster [2, 31,32] . ClaR is the pathway-specific transcriptional regulatorfor clavulanic acid biosynthetic genes, but it affects onlyexpression of the late genes for clavulanic acid biosynthesis[29] . The early genes responsible for the steps shared withthe 5S clavams are not regulated by ClaR [29] . In this manner,CcaR coordinates the biosynthesis of cephamycin C, a ß-lactamantibiotic, with clavulanic acid, a ß-lactamase inhibitor[16].

The cas1 and cas2 paralogues can functionally replace each otherand are known to be nutritionally regulated . The cas1 paralogueis expressed only when S . clavuligerus is grown on complex soymedium, whereas the cas2 paralogue is expressed during growthon both complex soy and defined SA media [30] . Thus, a cas1mutant can still produce clavulanic acid in both soy and SAmedia, because cas2 is expressed in both media . In contrast,a cas2 mutant produces clavulanic acid only when grown on soymedium, because cas1 is not expressed in SA medium [30] . Similarphenotypes were also observed when mutants defective in eachof the ceaS, bls, and pah paralogues were prepared and tested[15, 18, 42].

Since ceaS1 and ceaS2 encode proteins catalyzing the first reactionin the shared clavulanic acid and 5S clavam biosynthetic pathways,and since they are both located at the boundaries of their respectivegene clusters [Fig . 1A], they seemed to be likely candidatesfor points of regulation . With this in mind, the nutritionalregulation of ceaS1 and ceaS2 was examined at the transcriptionallevel to determine if they are regulated in a manner similarto cas1 and cas2 . Previous studies have suggested that ClaRdoes not regulate ceaS2 transcription [29], but the effect ofCcaR on ceaS1 and ceaS2 transcription is still not known . These effects were also examined and are discussed in the presentstudy . Lastly, the transcriptional start points [TSPs] of ceaS1, bls1, pah1, and ceaS2 were also mapped.

 
Bacterial strains, plasmids, media, and culture conditions. The bacterial strains and plasmids used in this study are describedin Table 1 . Escherichia coli cultures were grown as describedearlier [38], and cultures containing plasmids were supplementedwith ampicillin [100 µg/ml], apramycin [50 µg/ml],chloramphenicol [25 µg/ml], kanamycin [50 µg/ml], or spectinomycin [100 µg/ml], as appropriate . S . clavuligerus cultures were maintained on either MYM [40] or ISP 4 medium, as described previously [42], and cultures containing plasmidswere supplemented with apramycin [25 µg/ml] or thiostrepton [5 µg/ml] . S . clavuligerus cultures for the isolationof chromosomal DNA were grown in trypticase soy broth supplementedwith 1% starch [TSBS], and cultures for the isolation of exconjugantswere grown in AS-1 medium supplemented with 10 mM MgCl₂, as described earlier [42] . To prepare RNA from S . clavuligerus,spores were pregerminated for 4 h at 28°C in 2YT medium[38] and then used to inoculate SA or soy culture medium [30].RNA was isolated after 96 and 120 h of growth in soy mediumand after 72 and 96 h of growth in SA medium . All Streptomycesliquid cultures were grown at 28°C on a rotary shaker at250 rpm.

 
DNA isolation, manipulation, and Southern analysis. Routine manipulation of plasmid DNA isolated from E . coli cultures, including labeling of double-stranded DNA probes with [-³²P]dCTP by nick translation and end labeling using [-³²P]dATP, was performedusing standard procedures [38] . DNA sequencing was carried outusing the DYEnamic ET Terminator Cycle Sequencing kit [AmershamPharmacia, Baie d'Urfe, Quebec, Canada] by the Molecular BiologyService Unit, University of Alberta . Manual sequencing of DNAwas performed using the Thermo Sequenase Radiolabeled TerminatorCycle Sequencing kit [U.S . Biochemical] according to the manufacturer'sdirections . DNA fragments fractionated by agarose and polyacrylamidegel electrophoresis [PAGE] were isolated using the QIAquickGel Extraction kit [Qiagen Inc.] and the crush-and-soak method[38], respectively . PCRs were carried out using the Expand high-fidelityPCR system [Roche] according to the manufacturer's instructions.Plasmid DNA was introduced into S . clavuligerus by intergenericconjugation as described previously [42] . Southern analysisof S . clavuligerus DNA was also carried out as described elsewhere[38], using the following wash conditions . After overnight incubationwith labeled probes, the nylon membranes were washed twice for15 min each time with 2x SSC [1x SSC is 0.15 M NaCl plus 0.015M sodium citrate]-0.1% sodium dodecyl sulfate [SDS] [38] atroom temperature, once for 20 min with 1.0x SSC-0.1% SDS, and once for 20 min with 0.1x SSC-0.1% SDS . All incubations andwashes were carried out at 65°C unless otherwise indicated.

RNA isolation, RT-PCR, S1 nuclease mapping, primer extension, and Northern blot analysis. RNAs from wild-type S . clavuligerus and S . clavuligerus ccaR::tsrA were isolated using the modified Kirby procedure [20] . Reversetranscriptase [RT]-PCR analysis of RNA was carried out using C . therm polymerase [Roche] for reverse transcription in two-step RT-PCR . All RT reactions were carried out at 62°C for 30min according to the manufacturer's instructions with the following changes . The reactions were set up in a final volume of 10 µlusing 0.5 µg of total RNA per reaction and 15.8 U of RNAguardRNase Inhibitor [Amersham] . The reverse primers ceaS1-RT-Rev,ceaS2-RT-Rev, and CAN 122 [Table 2] were used to synthesizecDNA corresponding to the ceaS1, ceaS2, and hrdB transcripts, respectively . The PCRs were performed using 5 µl of theRT product from the reaction described above in a final volumeof 50 µl, employing the Expand high-fidelity PCR systemwith buffer 2 . The primer pairs ceaS1-RT-For plus ceaS1-RT-Revand ceaS2-RT-For plus ceaS2-RT-Rev were used for PCR amplificationof the ceaS1 and the ceaS2 RT products, respectively . Dimethylsulfoxide [DMSO; 5% [vol/vol] final concentration] was usedin these reactions with the following program: 94°C for2 min, followed by 25 cycles of 94°C for 1 min, 62°Cfor 1 min, and 72°C for 1 min . The primers CAN 123 and CAN122 were used to amplify the hrdB RT product by PCR using 10%[vol/vol] DMSO and the following program: 94°C for 2 min, followed by 25 cycles of 94°C for 1 min, 66°C for 1min, and 72°C for 1 min . The RT-PCR products were analyzedby fractionation on 1.5% agarose Tris-borate-EDTA gels . Theidentities of the RT-PCR products were also verified by restrictionanalysis, followed by PAGE and by sequencing of both DNA strands[data not shown].

 
High-resolution S1 nuclease mapping was carried out using thesodium trichloroacetate method [20] . All double-stranded DNA probes were prepared by PCR using custom primers . DMSO [5% [vol/vol]] was included in the reactions, with the following program: 94°C for 2 min, followed by 10 cycles of 94°C for 45 s, 60°Cfor 45 s, and 72°C for 45 s, and finally 15 cycles of 94°Cfor 45 s, 65°C for 45 s, and 72°C for 45 s . The primersceaS1-S1-For and ceaS1-PR-EX, along with p2.8-18 as a template,were used to prepare the probe to map the ceaS1 TSP . The primersceaS2-S1-For and ceaS2-PR-EX, along with the template plasmidpBB5.3A, were used to prepare the probe for mapping the TSPof ceaS2 . The primers bls1-S1-For and bls1-S1-Rev, and pah1-S1-Forand pah1-UP-rev, along with p5.7 as a template, were used toprepare probes for S1 protection analysis of bls1 and pah1,respectively.

Primer extension analysis was performed using C . therm polymerase for reverse transcription in two-step RT-PCR [Roche] according to the manufacturer's instructions with the following changes. Twenty-microliter reaction mixtures were set up using 5 pmolof the end-labeled reverse primers ceaS1-PR-EX and ceaS2-PR-EX[Table 2] and 40 U of RNaseOUT Recombinant RNase Inhibitor [Invitrogen],with the following program: extension at 60°C for 60 minand termination at 80°C for 10 min.

DNA sequencing ladders were prepared for size estimation usingthe reverse primers and the template plasmids used in the preparationof the S1 probes or in the primer extension reactions . Sampleswere separated on 6% denaturing polyacrylamide sequencing gelsfor analysis as described earlier [38].

Northern blot analysis was carried out using established techniques [30] with 40 µg of RNA isolated from wild-type S . clavuligerusgrown on soy medium for 96 and 120 h . Molecular Weight MarkerIII [Roche] was run along with the RNA samples for size estimation.The primers pah1-S1-For and pah1-S1-Rev were used to generatea 288-bp probe by PCR using p5.7 as a template . This probe wasused for both S1 nuclease protection analysis [data not shown] and Northern hybridization . Probe annealing and subsequent washes were carried out under the same high-stringency conditions used in Southern blot analysis.

Preparation of enhanced green fluorescent protein [EGFP] reporter constructs. A 781-bp DNA fragment spanning the ceaS1 promoter region wasamplified by PCR using p2.8-18 as a template and the primerpair KTA-ceaS1-For and KTA-ceaS1-Rev . Similarly, a 721-bp DNA fragment encompassing the ceaS2 promoter region was amplified by PCR using pBB5.3A as the template and the primers KTA-ceaS2-For and KTA-ceaS2-Rev . The PCR products were treated with Taq DNA polymerase before ligation to pCR2.1TOPO [Invitrogen] according to the manufacturer's instructions . This gave pTOPO-ceaS1-4and pTOPO-ceaS2-8, which contain the ceaS1 and ceaS2 promoter regions in pCR2.1TOPO, respectively . The double-stranded DNA sequence of the inserts was obtained using universal primersto ensure that no mutations were introduced.

The ceaS2 promoter region from pTOPO-ceaS2-8 was isolated as a BamHI/KpnI fragment and ligated into the corresponding sites of pIJ8660 to give pIJ8660-ceaS2 . pIJ8660-ceaS2 contains theceaS2 promoter region fused to a promoterless egfp gene foruse as a reporter of expression driven by the ceaS2 promoter.

For unexplained reasons, we were unable to subclone the ceaS1 promoter region into pIJ8660 and therefore used an alternative approach . The ceaS1 promoter region from pTOPO-ceaS1-4 was isolated as a BamHI/KpnI fragment and ligated into the corresponding sites of pTO6 to give pTO6-ceaS1 . The entire cassette encompassing the ceaS1 promoter region fused to the egfp gene from pTO6-ceaS1 was isolated as a 2.37-kb BamHI/EcoRI fragment and introduced into the corresponding sites of pSET152 to give pSET-ceaS1,which served as the ceaS1 reporter construct.

The plasmids pSET-ceaS1 and pIJ8660-ceaS2 were introduced into wild-type S . clavuligerus by conjugation . Strains that had the plasmids integrated at the C31 attB site in the chromosome wereisolated based on apramycin resistance and were verified bySouthern hybridization [data not shown].

Confocal microscopy. S . clavuligerus cultures harboring egfp reporter constructswere grown in TSBS for 36 h . Five-hundred-microliter amountsof the TSBS cultures were used to inoculate 25 ml of eitherSA or soy medium . After 72 h of growth, 1-ml amounts of thecultures were harvested and washed once in acetonitrile andthen twice in sterile distilled water . The washed mycelia weremounted in 40% [vol/vol] glycerol before observations were madeunder the microscope.

Confocal microscopy was carried out using a Leica DM IRB inverted microscope . An argon laser [50 to 52% attenuation] provided excitation at 488 nm . Fluorescence due to EGFP excitation was detected between 505 and 520 nm, and corresponding differential interference contrast images were also obtained.

Western analysis. Five-milliliter amounts of S . clavuligerus cultures grown insoy and SA media were harvested and resuspended in 1 ml of lysingbuffer [100 mM HEPES [pH 7.2], 0.5 mg of lysozyme/ml, 2x Complete EDTA free protease inhibitor cocktail [Roche]] . The suspensionswere incubated at 37°C for 30 min, the cell membranes werebroken by ultrasonic disruption, and then the cell debris wasremoved by centrifugation . Samples of cell extracts [CFEs] containing50 µg of total protein were separated by SDS-PAGE [12%gels] as described earlier [38] . The proteins were transferredonto polyvinylidene difluoride membranes [Immobilon-P; Millipore]using a Bio-Rad Transblot apparatus . The BM ChemiluminescenceWestern Blotting Kit [Mouse/Rabbit] [Roche] was used to detectproteins in accordance with the manufacturer's instructions.The primary antibody used to detect EGFP was the commerciallyavailable BD Living Colors A.v . Peptide Antibody [BD Biosciences]and was used at 1/400 dilution.

HPLC analyses of culture filtrates, bioassays, and growth determination. High-performance liquid chromatographic [HPLC] analysis of culturesupernatants after imidazole derivatization was performed underpreviously described conditions [30] . Bioassays were used todetect clavulanic acid and alanyl clavam production using Klebsiellapneumoniae ATCC 15380 and Bacillus sp . strain ATCC 27860, respectively,as the indicator organisms [18, 30] . Growth of S . clavuligerusin fermentation medium was estimated by measuring the opticaldensity of broken mycelia at 495 nm as described earlier [7].

 
Nutritional regulation of ceaS1 and ceaS2. In previous studies, when ceaS1 and ceaS2 mutant strains were prepared individually, both mutant strains still retained some ability to produce both clavulanic acid and the 5S clavams, depending on the fermentation medium used [15, 33, 42] . TheceaS1 mutant produced both clavulanic acid and the 5S clavamsin soy medium but only clavulanic acid in SA medium . In contrast,the ceaS2 mutant produced small amounts of clavulanic acid andthe 5S clavam metabolites in soy medium only, while no clavulanicacid or 5S clavam production was observed in cultures grownin SA medium [15, 42] . Based on the observed phenotypes, ceaS1 was postulated to be expressed in soy medium only, whereas ceaS2 was expressed in both soy and SA media . To verify this hypothesis, we examined the effects of growth in soy and SA media on both ceaS1 and ceaS2 expression at the transcriptional level.

RT-PCR was used to detect ceaS1 and ceaS2 transcripts, using RNA isolated from wild-type S . clavuligerus grown on SA medium for 72 and 96 h and on soy medium for 96 and 120 h . When RNA isolated from SA cultures was subjected to analysis, ceaS1 transcripts were not detected, whereas the same samples showed the presence of ceaS2 transcripts [Fig . 2A] . In similar analyses of RNA isolatedfrom soy-grown cultures, both ceaS1 and ceaS2 transcripts weredetected by RT-PCR [Fig . 2A] . The expression of hrdB, whichencodes a constitutively expressed sigma factor in Streptomyces,was monitored as a control, and hrdB transcripts were detectedat similar levels in all samples tested [Fig . 2] . HPLC analysisof culture supernatants showed the expected levels of clavulanicacid and 5S clavams in cultures grown in both media used forRNA isolation and analysis [data not shown].

 
The promoter regions of ceaS1 and ceaS2 [–501 to +215 and –551 to +95 bp relative to the putative start codonsof ceaS1 and ceaS2, respectively] were subcloned in front ofa promoterless egfp gene to give plasmids pSET-ceaS1 and pIJ8660-ceaS2, respectively . Fluorescence arising due to EGFP expression was used as a reporter to monitor transcription driven from the promoters . These promoter constructs were introduced into wild-type S . clavuligerus, where they integrated into the chromosome via the C31 attB site . The integration of the reporter plasmidsinto the S . clavuligerus chromosome was confirmed by Southern hybridization using DNA probes specific for both egfp and the respective promoters [data not shown].

The S . clavuligerus reporter strains C1G and C2G [Table 1] weregrown on soy and SA media for 72 h, after which mycelia wereharvested and analyzed by confocal microscopy to examine fluorescencearising due to EGFP expression and excitation . Fluorescencewas observed in all samples except in the C1G strain grown onSA medium [Fig . 3A] . Cell extracts [CFEs] were also preparedfrom the same samples that were subjected to confocal microscopy,and the presence of EGFP in the CFEs was analyzed by Westernblotting . A 27-kDa band corresponding to EGFP was observed inall of the samples except for the lane containing CFE from theC1G strain grown on SA medium [Fig . 3B].

 
Bioassays indicated that all of the strains used in microscopicand Western analyses produced the expected levels of both clavulanicacid and alanyl clavam, and growth assays indicated that allthe strains grew at similar rates [data not shown].

Transcriptional mapping of ceaS1 and ceaS2 promoters. S1 nuclease protection and primer extension analyses were usedto map the TSPs of both ceaS1 and ceaS2 . RNA for the analysis was isolated from wild-type S . clavuligerus grown on soy medium [for 96 and 120 h] . S1 protection analysis, using a probe extending from –261 to +26 bp relative to the putative ceaS1 start codon, indicated that the ceaS1 TSP was located 98 to 99 bp upstream of the ceaS1 start codon [Fig . 4A] . Primer extensionanalysis was also conducted using the reverse primer ceaS1-PR-EX[Fig . 4B], and the ceaS1 TSP was mapped to a C residue located98 bp upstream of the ceaS1 ATG start codon [Fig . 4C].

 
A probe extending from –204 to +22 bp relative to theceaS2 start codon was used in an S1 protection assay to mapthe ceaS2 TSP, which was located 51 to 52 bp upstream of theceaS2 start codon [Fig . 5A] . Primer extension analysis using the reverse primer ceaS2-PR-EX [Fig . 5B] confirmed that the ceaS2 transcript originated from a G residue located 51 bp upstream of the ceaS2 ATG start codon [Fig . 5C].

 
Mapping of the bls1 and pah1 transcripts. Since bls2 and pah2 are transcribed as part of a larger polycistronic transcript that also includes ceaS2 and cas2 [30], the regionsupstream of bls1 and pah1 were examined using S1 nuclease protectionassays to determine if they were also expressed as part of apolycistronic message . The bls1 transcript was mapped usingRNA isolated from wild-type S . clavuligerus grown on soy mediumfor 96 h, together with a probe extending from –137 to+16 bp relative to the proposed bls1 ATG start codon . Only full-lengthprotection of the probe was observed, indicating that therewas no individual TSP located in the 23-bp intergenic regionbetween ceaS1 and bls1 [Fig. 6A].

 
The intergenic region between bls1 and pah1 was examined by S1 nuclease protection assays using a probe extending from –257 to +21 bp relative to the proposed pah1 ATG codon, but once again, only full-length protection of the probe was observed[data not shown] . Because the DNA sequence ladder was unclearin the region of the full-length protected probe, a second 116-bpprobe [–257 to –151 bp upstream of the proposedpah1 start codon] was also used in an S1 protection assay . Againonly full-length protection of the probe was observed [Fig.6B], indicating that there was no promoter immediately upstreamof pah1 . Since the probes used for the S1 nuclease protectionstudies did not cover the entire 317-bp intergenic region betweenbls1 and pah1, it was still possible that a TSP might be foundfurther upstream of pah1 . Northern analysis of RNA isolatedfrom wild-type S . clavuligerus grown on soy medium for 96 and120 h was conducted to investigate this possibility . The 288-bpprobe prepared for the first pah1 S1 protection assay was usedas the pah1 Northern probe . Only a single large band of 4.9 kb hybridized to the probe [Fig . 7], indicating that pah1 doesnot have an individual promoter and that it is transcribed aspart of a tricistronic operon together with bls1 and ceaS1.

 
Effects of CcaR on ceaS1 and ceaS2 transcription. RNAs isolated from wild-type and ccaR::tsrA strains of S . clavuligerusgrown on soy medium for 96 and 120 h were analyzed by RT-PCRto monitor the effect of CcaR status on ceaS1 and ceaS2 expression.The transcription of ceaS1 was comparable in both the wild-typeand the ccaR::tsrA strains [Fig . 2B] . When ceaS2 transcription was examined in the same RNA samples, almost no transcriptswere detectable in the ccaR::tsrA mutant compared to the wild-typestrain [Fig . 2B] . The expression of hrdB was also monitoredas a control and was found to be constant in all of the samples[Fig . 2B].

The levels of clavulanic acid and 5S clavam production in the wild-type and the ccaR::tsrA mutant cultures used to isolateRNA were also determined by HPLC analysis of culture supernatants.The wild-type strain produced the expected levels of clavulanicacid and the 5S clavams after 96 and 120 h of growth on soymedium . Under the same conditions, the ccaR::tsrA strain didnot produce any detectable levels of clavulanic acid, whereasnormal 5S clavam production was observed [data not shown].

 
Previous studies have shown that paralogous pairs of genes encodethe enzymes involved in the early stages of the shared biosynthetic pathway leading to clavulanic acid and the 5S clavams [15, 18,22, 42] . In order to understand to what extent each of thesesets of genes contributes to the production of clavulanic acidand the 5S clavams, the transcriptional regulation of the geneswas examined.

The ceaS1 transcript originated from a single TSP located at a C residue 98 bp upstream from the start codon, but the ceaS1 promoter region showed little similarity to known Streptomyces promoters [Fig . 4C], a reflection of the large diversity foundin Streptomyces promoter sequences [39] . When the bls1 and thepah1 transcripts were analyzed by S1 nuclease protection assays,no individual TSP was detected for either of these genes andonly full-length protection of the probes was observed . Thissuggested that neither bls1 nor pah1 has its own dedicated promoterand that the upstream ceaS1 promoter drives the transcriptionof these genes.

The large intergenic region of 317 bp that separates bls1 and pah1 was not fully covered by the probes used for S1 nuclease protection studies . Therefore, Northern analysis was used to ensure that any pah1 transcript originating from a promoter located further upstream was not missed . Since pah1 and pah2 show 72% end-to-end identity at the nucleotide level [18], theprobe used in Northern analysis was carefully chosen to be specificfor pah1 . The only hybridization seen was to a band 4.9 kb insize, again indicating that pah1 was transcribed as part ofa large polycistronic transcript [Fig . 7] . This 4.9-kb transcriptis postulated to include ceaS1, bls1, and pah1, as the predictedlength of a transcript extending from the ceaS1 TSP to the stopcodon of pah1 would be 4.7 kb . The next gene downstream of pah1is oat1, which is oriented in the direction opposite to pah1transcription, and therefore its presence on the 4.9-kb polycistronictranscript can be ruled out . In addition, computational analysisof the 126-bp intergenic region between pah1 and oat1 predictedthe presence of considerable secondary structure, consistingof multiple stem-loops with a cumulative G of –101.4,which could function as a transcriptional terminator [data notshown].

The transcriptional arrangement of ceaS1, bls1, and pah1 issimilar to that of their paralogous counterparts from the clavulanicacid gene cluster [Fig . 1A] . The ceaS2, bls2, pah2, and cas2genes from the clavulanic acid gene cluster are transcribedas a 5.3-kb polycistronic transcript . In addition, cas2 is also transcribed as a 1.2-kb monocistronic transcript [30] . The mostsignificant difference between the 5.3-kb transcript arising from the clavulanic acid gene cluster and the 4.9-kb transcript arising from the paralogue gene cluster is the absence of thecas1 coding sequence in the paralogue gene cluster . cas1 islocated elsewhere on the S . clavuligerus chromosome and is expressed as a 1.4-kb monocistronic transcript [30].

The transcript comprising ceaS2 was also mapped and was also found to arise from a single TSP located 51 bp upstream of the ceaS2 start codon . As was the case for ceaS1, the proposed ceaS2promoter region did not show any significant similarity to anyknown Streptomyces promoters . Since S1 nuclease and primer extensionanalyses were used to identify all of the TSPs described in this study, it should be noted that both of the analysis methods employed are predictive of the TSP, provided the mRNA is not processed.

The nutritional regulation of ceaS1 and ceaS2 transcription was examined using RT-PCR, which demonstrated that ceaS1 was transcribed in soy medium only and not in SA medium . Under the same conditions and using the same RNA preparations, ceaS2 was transcribed in both soy and SA media at comparable levels . The ceaS1 and ceaS2 promoter regions were also subcloned in front of a promoterless egfp gene, and EGFP expression was used as a reporter to monitor transcription driven by the respective promoters . Confocal microscopy was used to detect fluorescencedue to EGFP expression and excitation, and the results confirmedthat ceaS1 was expressed in soy medium only and not in SA medium, whereas ceaS2 was expressed in both media tested . The results obtained from confocal microscopy were confirmed by Westernanalysis, which indicated that the fluorescence observed inthe samples was due to true EGFP expression . Since ceaS1, bls1,and pah1 are expressed only as a 4.9-kb polycistronic message,it can be inferred that bls1 and pah1 will show the same general trend of nutritional regulation as ceaS1 . In combination, our results indicate that ceaS1, bls1, and pah1 are expressed insoy medium but not in SA medium, whereas ceaS2, bls2, pah2,and cas2 are expressed in both soy and SA media . This explainsthe clavulanic acid- and 5S clavam-producing phenotypes observedwhen mutants defective in these genes were prepared and testedin previous studies [15, 18, 42].

The ccaR gene from the cephamycin gene cluster encodes a pathway-specifictranscriptional regulator that coordinates the production ofboth cephamycin C and clavulanic acid [2, 31] . Its effect onclavulanic acid biosynthesis is exerted, at least in part, throughactivation of the expression of a second pathway-specific transcriptionalregulator, ClaR, from the clavulanic acid gene cluster . Mutantsdefective in CcaR do not produce any cephamycin C, and clavulanicacid production is also knocked out [2, 31], as the transcription of claR is reduced to near zero in these mutants [32] . ClaRpositively regulates genes involved exclusively in the biosynthesisof clavulanic acid [29] . Previous studies have shown that ceaS2expression is not under the control of claR [29], but the detailed effects of CcaR on ceaS1 and ceaS2 expression are not known.

Our results indicate that ceaS1 transcription is unaffected in the ccaR mutant compared to the wild-type strain, whereas the transcription of ceaS2 is almost eliminated in the ccaR mutant . Therefore, CcaR controls the production of clavulanic acid through at least two routes, an indirect route mediatedby ClaR and a second route that may involve CcaR directly regulatingceaS2 promoter activity.

CcaR belongs to a family of transcriptional regulators calledthe Streptomyces antibiotic regulatory proteins [SARPs], which bind to specific heptameric repeats and promote transcription[45] . Imperfect heptameric repeats can be identified in theregion upstream of ceaS2 [Fig . 5C], consistent with the idea that CcaR may bind directly to the ceaS2 promoter region to regulate transcription . Such a notion, however, has not yetbeen demonstrated experimentally, and it is also possible thatCcaR exerts its effect indirectly through additional proteins[20a, 38a] . Since claR is not expressed in the ccaR mutant,it can also be inferred that claR has no effect on ceaS1 transcription,which was unaffected in the ccaR mutant . Similarly, due to thepolycistronic nature of the transcript including ceaS1, bls1,and pah1, it follows that neither bls1 nor pah1 is affectedby CcaR or ClaR.

The reason why S . clavuligerus possesses two sets of genes encoding enzymes involved in the early stages of clavulanic acid and5S clavam biosynthesis is still unclear . One suggestion is thatthis could be a strategy to increase precursor and metaboliteflux through the shared part of the pathway by increasing thegene dosage . This is consistent with the observation that bothsets of the paralogous genes are expressed in complex soy medium,where precursor availability and growth would support greatermetabolite production levels than are possible on defined SAmedium, where only the ceaS2-oat2 set of paralogues is expressed.In addition, the increased production of these secondary metabolitesmay be of greater advantage in complex medium to ward off competition,especially that posed by faster-growing organisms . In definedmedia, such as SA, the expression of only one set of paralogousgenes may suffice to provide an adequate supply of precursors,given the lower levels of clavulanic acid and 5S clavam production.

Another explanation put forth is that the two sets of paralogous genes may belong to two separate pathways, with one leadingto clavulanic acid and the other to the 5S clavams . Since thetwo pathways proceed through common early steps, a sharing of biosynthetic intermediates results, but the two pathways maybe regulated differently . This is consistent with our observationthat, although clavulanic acid production was knocked out inthe ccaR mutant, the 5S clavams were still produced during growthon soy medium . The ccaR mutant shows ceaS1 transcription equivalent to that seen in the wild-type strain and still produces wild-type levels of 5S clavams, whereas ceaS2 transcription is almost absent and clavulanic acid production is lost . This suggests that ceaS1, bls1, and pah1 may be more closely associated withthe production of 5S clavams via a CcaR-independent pathway,whereas ceaS2 and its related paralogues are associated withclavulanic acid production and are regulated by CcaR . This isan attractive hypothesis from the point of view that the producerorganisms would be best served by coordinating production of a ß-lactam antibiotic [cephamycin C] with productionof a ß-lactamase inhibitor [clavulanic acid] throughthe action of a common regulator [CcaR] . In contrast, no apparentadvantage would be gained by coordinating production of the5S clavams with production of cephamycin C . Further investigationis required before any firm conclusions can be drawn . In previousstudies [2], it was reported that production of both 5S clavamand clavulanic acid was lost in a ccaR mutant, whereas in our hands, mutation of ccaR had no effect on 5S clavam production. This inconsistency may be attributed to differences in the methodologies and growth media used for culture propagation in the two studies or to the extensive variability that has been observed in 5S clavam production profiles within this species [43].

The functional holoenzyme forms of CEAS2 [9], BLS2 [25], andPAH2 [11] have all been characterized structurally and shownto be oligomers . Since these proteins were overexpressed andpurified from E . coli, only homo-oligomers were observed . Itis reasonable to expect that when the corresponding homo-oligomericforms of CEAS1, BLS1, and PAH1 are expressed and purified, theymay have somewhat different activities or kinetic properties,just as was seen for the CAS1 and CAS2 monomers [37] . Thesedifferences in activities may be important under the specific nutritional conditions in which each of these isoenzymes is expressed . It is also possible that within S . clavuligerus the two isozymic forms of each protein can form hetero-oligomers,which could provide another mechanism to modulate enzyme activitybased on nutritional and precursor availability.

Studies are under way to investigate the interactions amongthe different isoenzymes to gain greater understanding of theroles of the paralogous genes involved in clavulanic acid and5S clavam biosynthesis.

 
This work was supported by a grant from the Canadian Institutesof Health Research . K.T . was supported by a studentship fromthe Alberta Heritage Foundation for Medical Research.

We thank B . K . Leskiw and her laboratory members for help withRNA analysis . We also thank R . Bhatnagar and J . Scott from theMicroscopy Unit at the Department of Biological Sciences, Universityof Alberta, for contributing their expertise on confocal microscopy.

 
* Corresponding author . Mailing address: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 . Phone: [780] 492-4434 . Fax: [780] 492-9234 . E-mail: ktahlan@ualberta.ca .

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