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Antimicrobial Agents and Chemotherapy, April 2005, p. 1529-1541, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1529-1541.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Expression of ccaR, Encoding the Positive Activator of Cephamycin C and Clavulanic Acid Production in Streptomyces clavuligerus, Is Dependent on bldG

Dawn R. D. Bignell ,{dagger},{ddagger} Kapil Tahlan,{dagger} Kimberley R. Colvin, Susan E. Jensen, and Brenda K. Leskiw*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada

Received 10 August 2004/ Returned for modification 24 October 2004/ Accepted 28 November 2004


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ABSTRACT
 
In Streptomyces coelicolor, bldG encodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment from Streptomyces clavuligerus contained an open reading frame that encoded a protein showing 92% identity to the S. coelicolor BldG protein and 91% identity to the BldG ortholog in Streptomyces avermitilis. Sequencing of the region downstream of bldG in S. clavuligerus revealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins in S. coelicolor and S. avermitilis, respectively. Northern analysis indicated that, as in S. coelicolor, the S. clavuligerus bldG gene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstream orf3 gene. High-resolution S1 nuclease mapping of S. clavuligerus bldG transcripts revealed the presence of three bldG-specific promoters, and analysis of expression of a bldGp-egfp reporter indicated that the bldG promoter is active at various stages of development and in both substrate and aerial hyphae. A bldG null mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as cephamycin C, clavulanic acid, and the 5S clavams. This inability to produce cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makes bldG the first regulatory protein identified in S. clavuligerus that functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production.


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INTRODUCTION
 
Streptomyces spp. are unique among prokaryotic organisms because their life cycle involves filamentous, vegetative mycelia and a series of complex morphological changes resulting in the formation of aerial hyphae and chains of unigenomic spores.

In Streptomyces coelicolor, the genetically best-characterized streptomycete, a number of genes have been characterized that function in the global regulation of morphological differentiation. Such genes are referred to as bld (bald) genes, since mutants lack the characteristic fuzzy coating of aerial hyphae seen in the wild-type strain. Interestingly, some of the bld mutants that have been isolated are also defective in the production of secondary metabolites, such as antibiotics, suggesting that the corresponding bld genes are involved in the regulation of both differentiation processes.

To date, 14 S. coelicolor bld mutants have been isolated and eight of the bld genes have been cloned and characterized. The bld gene products are diverse in nature (7, 16, 28, 33, 35, 36, 43, 53) and include a putative anti-anti-sigma factor, BldG (11) that is the subject of this study. Characterization of the bldG locus (11) revealed both a bldG-complementing gene (SCO3549) and a second, transcriptionally linked open reading frame designated orf3 (SCO3548) (6). bldG and orf3 encode proteins showing similarity to the SpoIIAA and RsbV anti-anti-sigma factors and the SpoIIAB and RsbW anti-sigma factors of Bacillus subtilis, respectively. Anti-sigma factor proteins in Bacillus function to regulate negatively the activity of alternative sigma factors, such as the sporulation-specific ({sigma}F) and stress response-specific ({sigma}B) sigma factors, by binding to them and preventing them from associating with core RNA polymerase (5, 15, 32). This inhibition is reversed, however, when the anti-anti-sigma factor binds to the anti-sigma factor, allowing the sigma factor to direct transcription (2, 3, 13, 14).

Of the eight characterized bld genes, only bldA, bldH, and bldN have been studied in other Streptomyces spp. besides S. coelicolor. bldN and its ortholog in Streptomyces griseus, adsA, were found to influence only morphological differentiation in both organisms (7, 59), while bldH and its S. griseus ortholog adpA were shown to affect both morphological differentiation and antibiotic production (37, 53). bldA has been studied in four different species and, while it was demonstrated to control both aerial hypha formation and secondary metabolite production in S. coelicolor, Streptomyces lividans, and S. griseus (26, 28, 29), it was found to be required only for morphological differentiation in Streptomyces clavuligerus (54). Thus, although the study of bld genes in S. coelicolor is, for the most part, useful for ascertaining the role of these genes in other Streptomyces spp., some differences are likely to be observed, and comparative analysis of gene organization and expression could prove informative.

S. clavuligerus is known to produce a variety of secondary metabolites, including cephamycin C, clavulanic acid, and additional compounds with the clavam structure, which are herein referred to as the 5S clavams (reviewed in reference 30). Clavulanic acid is an important commercial metabolite because of its potent ß-lactamase inhibitory activity and is routinely used along with ß-lactam antibiotics for the treatment of infections caused by ß-lactamase-producing microorganisms (22). The production of cephamycin C and clavulanic acid is controlled by the CcaR regulatory protein, which belongs to the SARP (for Streptomyces antibiotic regulatory proteins) family of transcriptional activators (57). Disruption of ccaR has been shown to abolish cephamycin C and clavulanic acid production (1, 41), while the overexpression of ccaR results in a two- to threefold increase in metabolite production (41). A second regulatory gene, claR, encodes a protein showing similarity to members of the LysR family of transcriptional activators. ClaR is required only for clavulanic acid production (38), and transcription of this gene is abolished in a ccaR mutant (42), indicating that expression of claR is dependent on CcaR.

Given the industrial importance of clavulanic acid production and given the potential for orthologs of S. coelicolor bld genes, other than bldA, to be involved in the regulation of secondary metabolite production in S. clavuligerus, we set out to identify and characterize the ortholog of the S. coelicolor bldG gene to determine its function in S. clavuligerus.


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MATERIALS AND METHODS
 
Bacterial strains, culture conditions, and plasmids. S. clavuligerus and Escherichia coli strains used in this study are listed in Table 1. S. clavuligerus was maintained on either MYM agar (49) or ISP-4 agar (Difco, Detroit, Mich.). Plasmid-containing S. clavuligerus cultures were supplemented as required with thiostrepton (5 µg/ml), apramycin (25 µg/ml), or kanamycin (50 µg/ml). Cultures for isolation of chromosomal DNA were grown in Trypticase soy broth supplemented with 1% (wt/vol) soluble starch (TSB-S). Cultures for the extraction of total RNA were grown in either soy (40) using pregerminated spores (~3 x 109) as the inoculum or in TSB-S medium using 24-h TSB-S seed cultures (1% [vol/vol]) as the inoculum. Spore pregermination was carried out in 2xYT medium as described by Kieser et al. (25), except that the spores were not sonicated after pregermination. Liquid cultures for the analysis of clavulanic acid, cephamycin C, and 5S clavam production were prepared by growing strains in both soy medium and starch asparagine (SA) medium as described previously (40), except that mycelial fragments, rather than spores, were used to inoculate seed cultures in the case of the bldG mutants. All S. clavuligerus cultures were grown at 28°C on a rotary shaker at 250 rpm. Growth conditions and media for E. coli cultures were as described previously (46). Plasmid-containing cultures were supplemented as required with ampicillin (100 µg/ml), apramycin (50 µg/ml), kanamycin (50 µg/ml), or chloramphenicol (25 µg/ml). All cosmids, plasmid vectors, and recombinant plasmids used are listed in Table 1.


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  		TABLE 1. Bacterial strains and plasmids used in this study

Introduction of DNA into Streptomyces strains. The preparation of S. clavuligerus protoplasts and their subsequent transformation were as described previously (39) except that cultures for protoplast preparations were grown in TSB-S medium. Plasmid DNA that was transformed into protoplasts was first passaged through E. coli ET12567 to reduce cleavage of the DNA by the highly active restriction-modification system of S. clavuligerus. Transformants were selected as described by Kieser et al. (25). Conjugative transfer of DNA from E. coli to S. clavuligerus was performed as described previously (52).

DNA isolation, manipulation, and PCR. Standard methods for DNA manipulation, such as restriction endonuclease digestion, ligation, generation of blunt ends, random primer labeling, end labeling, Southern analysis, E. coli transformation, and isolation of plasmid DNA from E. coli cultures, were performed as described previously (46). Isolation of chromosomal DNA from Streptomyces strains was performed using procedure 3 described by Hopwood et al. (20). PCRs for amplification of DNA were routinely performed in 50-µl volumes using EXPAND Hi-Fidelity polymerase (Roche) according to the manufacturer's instruction. Oligonucleotide primers used for PCRs are listed in Table 2.


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  		TABLE 2. Oligonucleotide primers used in this study

Cloning and sequencing of bldG and surrounding genes from S. clavuligerus. To isolate the bldG coding region from S. clavuligerus, a library of S. clavuligerus NRRL 3585 chromosomal fragments in the cosmid vector pWE15 (Stratagene) was screened by colony hybridization using an internal region of the S. coelicolor bldG gene as probe. The probe was generated by PCR using pAU64 as the template and the primers BKL63 and BKL64 (Tables 1 and 2). Cosmid DNA from 10 hybridizing clones was isolated, digested with ApaI, and screened by Southern analysis using the same S. coelicolor bldG probe. A single hybridizing, 0.6-kb ApaI fragment from cosmid 3B12 (Table 1) was purified using the QIAquick gel extraction kit (QIAGEN) and ligated into pBluescript SK(+) in both orientations to generate pAU328 and pAU329 (Table 1). Sequencing of the insert in both plasmids was performed by the Molecular Biology Service Unit (Department of Biological Sciences, University of Alberta). Since the 0.6-kb DNA fragment cloned into pAU328 and pAU329 was found to consist of the bldG coding sequence with little sequence information either upstream or downstream of the gene, further sequencing was performed using sequence-specific primers and various bldG-hybridizing cosmid clones (Table 1) that had been digested with either NcoI or EcoRI-BamHI. As well, a ca. 3.6-kb EcoRI-BamHI fragment from cosmid 1E7 shown to hybridize to the S. coelicolor bldG probe was purified by the trough purification method (61) and ligated into pBluescript SK(+) to generate pAU330 (Table 1), which was subsequently used as a template in sequencing reactions.

RNA isolation. Total RNA was extracted from S. clavuligerus liquid cultures as described elsewhere (25). RNA was extracted from soy cultures grown for 72, 96, and 120 h and from TSB-S cultures grown for 24, 36, 48, and 72 h.

Northern analysis. Northern analysis was performed according to the methods of Williams and Mason (58) as described previously (11). The probe used to detect bldGScl transcripts was a labeled PCR product generated using pAU328 as the template and the primers DBG54 and DBG55. A labeled PCR product generated using the bldG-hybridizing cosmid 7D8 as template and the primers DBG53 and DBG57 was used to detect transcripts of orf3Scl. ccaR transcripts were detected using a gel-purified, ca. 1,200-bp fragment generated by digestion of pDA1102 (Table 1) with BamHI. Hybridizations in all cases were performed overnight at 44°C in a solution containing 50% formamide. Estimation of transcript sizes and controls for RNA loading were described previously (11). All signals were detected using a PhosphorImager (model 445 SI; Molecular Dynamics).

High-resolution S1 nuclease protection assays. High-resolution S1 mapping of the bldGScl promoters was performed using the sodium-trichloroacetate procedure described by Kieser et al. (25). The probe used to map promoters 1 and 2 (P1 and P2) was generated by PCR using the bldG-hybridizing cosmid 7D8 as the template and primers DBG50 and DBG51 (Table 2). The resulting 273-bp product was cloned into pCR2.1TOPO to generate pAU334 and sequenced to determine the orientation of the insert in the vector. Next, PCR was carried out using pAU334 as the template and M13 reverse and DBG51 as primers. The DBG51 oligonucleotide was end labeled using T4 polynucleotide kinase and [{gamma}-32P]ATP prior to PCR so that only the desired strand of the PCR product would be labeled for S1 mapping. The resulting 32P-labeled PCR product, which consisted of 263 bp of S. clavuligerus sequence and 100 bp of a nonhomologous extension to distinguish between full-length protection of the probe and probe-probe reannealing, was then purified by crushing and soaking (46). The probe used to map promoter 3 (P3) was generated by PCR using cosmid 7D8 as the template and the primers DBG45 and DBG48. After ligation into pCR2.1TOPO and sequencing to determine the orientation of the insert, the plasmid, designated pAU335, was used as template for a second round of PCR using the M13 reverse and DBG45 primers. In this case, DBG45 was end labeled prior to PCR. The resulting gel-purified probe consisted of 379 bp of S. clavuligerus sequence and 90 bp of a nonhomologous extension.

S1 mapping reactions were set up using 40 µg of total RNA from S. clavuligerus and 60 fmol of end-labeled probe. The resulting S1 products were separated by denaturing gel electrophoresis on a urea-6% polyacrylamide gel along with sequencing ladders generated by the dideoxy chain termination method (47) using the ThermoSequenase radiolabeled terminator cycle sequencing kit (U.S. Biochemicals, Cleveland, Ohio). For mapping the location of P1 and P2, the sequencing ladder was produced using pAU334 as the template and DBG51 as the primer, while the ladder used to map P3 was generated using pAU335 as the template and DBG45 as the primer. Signals were then detected using a PhosphorImager (model 445 SI; Molecular Dynamics).

Preparation of bldGp-egfp reporter construct. The S. clavuligerus bldG promoter region was PCR amplified using cosmid 3B12 as the template and the primers DBG48 and DG51. The resulting 557-bp fragment was cloned into pCR2.1TOPO to give pAU336, and after sequencing to ensure that no PCR-induced mutations were present the fragment was released from pAU336 by digestion with BamHI and XbaI. The resulting 655-bp fragment was gel purified (46) and ligated into BamHI-XbaI-digested pIJ8660 to give pAU337. pAU337 and the parent vector pIJ8660 were introduced into wild-type S. clavuligerus by conjugation. Apramycin-resistant strains containing the integrated plasmids were verified by PCR and sequencing using the DBG54 and KTA-GFP-Rev primers (data not shown).

Confocal microscopy. S. clavuligerus strains containing the egfp reporter constructs were grown on ISP-4 agar (Difco), which supports both aerial hypha formation and sporulation. Microscope coverslips (no. 0; CANEMCO Supplies, St. Laurent, Quebec, Canada) were sterilized in ethanol and were inserted into the medium at a 45° angle. Five-microliter amounts of the appropriate S. clavuligerus spore stocks were used as inocula along the base of the coverslips. The coverslips were carefully pulled out of the medium after 2, 4, and 8 days of growth, which coincided with different stages in the life cycle of S. clavuligerus. They were then mounted in 40% glycerol, and the ends were sealed with nail polish. Confocal microscopy was carried out using a Leica DM IRB inverted microscope as described by Tahlan et al. (51). Both fluorescence and differential interference contrast images were obtained and were processed using Adobe Photoshop 7.0.

Construction of an S. clavuligerus bldG null mutant. The S. clavuligerus bldG null mutant was constructed using the REDIRECT PCR targeting system described by Gust et al. (19). Briefly, a disruption cassette consisting of oriT and the aac(3)IV apramycin resistance gene was created by PCR amplification of a gel-purified 1,384-bp EcoRI-HindIII fragment from pIJ773 (Table 1), using DBG43 and DBG44 as primers (Table 2). The oligonucleotide primers contained 39-nucleotide (nt) nonhomologous extensions corresponding to sequence downstream of the S. clavuligerus bldG GTG start codon in the case of DBG43 and upstream of the TGA stop codon in the case of DBG44. The resulting PCR product was electroporated into E. coli BW25113 that contained both the bldG cosmid 5D4 and the pIJ790 plasmid. After sequencing of the mutant cosmid using the Red-Up and Red-Down primers (Table 2) to confirm the presence of the disruption cassette, the cosmid, designated pAU331, was transformed into E. coli ET12567/pUZ8002 and then moved by conjugation into S. clavuligerus. Exconjugants were screened for apramycin resistance (aprar) and kanamycin sensitivity (kans), indicating that a double crossover event had occurred, and protoplasts from putative mutant exconjugants were prepared and plated in serial dilution onto modified R5B medium (4) in order to isolate colonies arising from single genomes. Two aprar kans colonies were then examined by Southern analysis to confirm that they contained the desired bldG gene disruption.

Construction of a bldG complementation vector. A bldG complementation vector was constructed as follows. First, a blunted 1,072-bp XbaI-SmaI thiostrepton resistance gene (tsr) fragment from pAU5 was cloned into the blunted NheI site in pSET152 to generate pAU3-45. The S. clavuligerus bldG coding sequence and promoter were amplified by PCR using the bldG-hybridizing cosmid 3C2 as template and the primers DBG48 and DBG60 (Table 2). The resulting 1,194-bp product was ligated into the pCR2.1TOPO vector to give pAU332 and was sequenced to ensure no PCR-induced mutations were present. Next, pAU332 was digested with EcoRI to release a 1,216-bp bldG-containing fragment that was subsequently purified (61) and ligated into EcoRI-digested pAU3-45 to give pAU333.

Analysis of culture supernatants by HPLC. The production of clavulanic acid and 5S clavam metabolites was followed by high-performance liquid chromatography (HPLC) of culture supernatants after imidazole derivatization as described previously (39), except that a Phenomenex Bondclone 10-µ C18 column was used in the analysis. Culture supernatants from wild-type S. clavuligerus grown in soy and SA media were diluted 1 in 5 and 1 in 2, respectively, before derivatization, while undiluted supernatants were used in the case of the bldGScl mutant strains.

Bioassays. The production of clavulanic acid, cephamycin C, and alanylclavam was followed using bioassays as described previously (52).

Preparation of S. clavuligerus cell extracts. Crude extracts from wild-type S. clavuligerus and the bldG mutant strains were prepared as described previously (9), except that the extracts were prepared from liquid cultures grown in soy or TSB-S medium.

Western analysis. Western analysis was performed as described previously (9) using 30 to 50 µg of S. clavuligerus cell extract. The BldG primary antibody (9) was used at a dilution of 1 in 10,000, while the CcaR-specific antibody (1) was used at a dilution of 1 in 5,000. Extracts isolated from wild-type S. coelicolor M145 and from the S. coelicolor bldG mutant {Delta}bldG 1DB (Table 1) were used as positive and negative controls for BldG detection, respectively, while purified CcaR (56) and extract isolated from a ccaR mutant strain (Table 1) were used as positive and negative controls, respectively, for CcaR detection.

Nucleotide sequence accession number. A 2,525-bp sequence consisting of the complete bldG and orf3 open reading frames as well as partial sequence of the putative RNA helicase gene upstream of bldG and of the putative pyrophosphate synthase gene downstream of orf3 in S. clavuligerus has been deposited in GenBank under the accession number AY821553.


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RESULTS
 
Cloning and sequence analysis of bldG and surrounding genes from S. clavuligerus. The bldG gene from S. clavuligerus (herein referred to as bldGScl) was isolated from a cosmid library of S. clavuligerus chromosomal DNA after hybridization with a DNA fragment corresponding to the S. coelicolor bldG gene (bldGSco). A total of 10 hybridizing cosmid clones were identified in this screen, and the presence of bldG in these clones was verified by digestion with ApaI and probing with the same bldGSco-specific fragment. A ca. 0.6-kb ApaI fragment from each clone was found to hybridize to the bldG-specific probe, and this fragment was gel purified from one cosmid clone (3B12) and ligated in both orientations into pBluescript SK(+) to give pAU328 and pAU329. Sequencing of the 0.6-kb fragment in these plasmid clones revealed the presence of a 354-bp open reading frame (Fig. 1A) preceded by a putative ribosome binding site (GGAGG). The deduced protein sequence encoded by this open reading frame was found to be 92% identical and 94% similar to the S. coelicolor BldG protein and 91% identical and 94% similar to the BldG ortholog (SAV4615) from Streptomyces avermitilis. Alignment of the three protein sequences (Fig. 1B; for alignment of BldGSco with the B. subtilis SpoIIAA and RsbV proteins, see reference 11) revealed that the serine residue, previously shown to be phosphorylated in S. coelicolor and in the Bacillus proteins (9, 34, 60) and which is known or believed to be important for the regulation of protein activity (2, 3, 9, 12, 13), is conserved in both the S. avermitilis and S. clavuligerus BldG proteins, suggesting that they too are posttranslationally modified by phosphorylation. Interestingly, the S. clavuligerus BldG was found to contain a four-amino-acid (TGPA) insertion (Fig. 1B) that is absent from the S. avermitilis and S. coelicolor proteins. The significance of this, however, is not yet known.



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  		FIG. 1. (A) Schematic diagram of the sequenced 2,525-bp region of the S. clavuligerus chromosome containing bldGScl. Block arrows represent the coding regions indicated, with the direction of the arrow indicating the direction of transcription, while the thin line represents the flanking S. clavuligerus chromosomal DNA. The curved lines through the RNA helicase and pyrophosphate synthase open reading frames indicate that the sequence of these open reading frames in the 2,525-bp region is not complete. The locations of the ApaI sites used to clone the 0.6-kb bldGScl-containing fragment are shown, as are the distances between the different coding regions. (B) Alignment of the BldG protein sequences from S. coelicolor, S. avermitilis, and S. clavuligerus. The alignment was generated using PepTool version 2.0 (BioTools Inc., Edmonton, Alberta, Canada). Black shading indicates mismatched amino acids in at least one of the sequences. The serine residue that is known to be phosphorylated in the S. coelicolor BldG protein is indicated by the arrow.

Since the ca. 0.6-kb ApaI fragment contained little sequence information for open reading frames surrounding bldGScl, a ca. 3.6-kb BamHI-EcoRI, bldGSco-hybridizing fragment from cosmid 1E7 was gel purified and ligated into pBluescript SK(+) to give pAU330. This plasmid clone, as well as various bldGSco-hybridizing cosmid clones that had been digested with either NcoI or BamHI-EcoRI, were used as templates for DNA sequencing. Analysis of the sequence data revealed that cosmid 1E7 had resulted from ligation of noncontiguous Sau3A fragments. Furthermore, 33 bp at the 3' end of the 3.6-kb insert in pAU330 was noncontiguous DNA and contained the EcoRI site used for the subcloning. Figure 1A shows that a 432-bp open reading frame was found downstream of bldGScl, which was preceded by a putative ribosome binding site (GAGGGGGA) and which encoded a protein showing 72% identity and 83% similarity to the ORF3 putative anti-sigma factor protein of S. coelicolor and 69% identity and 80% similarity to the S. avermitilis SAV4614 protein encoded downstream of SAV4615. Farther downstream of the S. clavuligerus orf3 ortholog (herein referred to as orf3Scl) was a partial open reading frame showing similarity to the putative pyrophosphate synthase genes found in the same regions of the S. coelicolor and S. avermitilis chromosomes. Analysis of the nucleotide sequence upstream of bldGScl revealed the presence of a partial, divergent open reading frame encoding a protein showing similarity to RNA helicases, including those encoded upstream of bldGSco and SAV4615. At least five putative in-frame start codons were identified using FramePlot (21), none of which were preceded by a convincing ribosome binding site and two of which corresponded to the start codons predicted for the S. coelicolor and S. avermitilis putative RNA helicases. The putative start codon closest to the bldGScl coding region would result in a 79-bp helicase-bldGScl intergenic region, while the start codon farthest away from the bldGScl coding region would give a 160-bp intergenic region.

Northern analysis revealed the presence of both bldG monocistronic and polycistronic transcripts in S. clavuligerus. Previous analysis indicated that bldGSco is transcribed both as a monocistronic and a polycistronic transcript, the latter including the downstream orf3Sco gene (11). Sequence analysis of the bldG locus in S. clavuligerus revealed a larger intergenic region between bldGScl and orf3Scl compared to that in either S. coelicolor or S. avermitilis (296 bp in S. clavuligerus versus 109 bp in S. coelicolor and 129 bp in S. avermitilis), which made us wonder whether orf3Scl might be transcribed separately from bldGScl. To investigate this, Northern analysis was performed using RNA isolated from liquid cultures grown in duplicate in soy medium for 72, 96, and 120 h. The probe used to detect bldGScl transcripts was a 232-bp PCR product corresponding to sequence internal to bldGScl, while a 248-bp PCR product was used to detect orf3Scl transcripts. The results (Fig. 2) showed that the bldGScl-specific probe hybridized to two transcripts of about 800 and 1,500 nt, which correlated well with the expected sizes of a bldGScl monocistronic transcript and a bldGScl-orf3Scl polycistronic transcript, respectively. Analysis of the bldGScl-orf3Scl intergenic region using the Mfold program (http://bioweb.pasteur.fr/seqanal/interfaces/mfold.html) revealed the presence of multiple potential hairpin structures, any of which might serve as a rho-independent terminator for generation of the bldGScl monocistronic transcript. Alternatively, the monocistronic transcript might result from degradation of the larger bldGScl-orf3Scl polycistronic transcript, and these hairpin structures might serve as stabilization factors to prevent further mRNA degradation. Both the mono- and polycistronic bldGScl transcripts were detected at all time points examined. The time points were chosen because they correspond to the times when secondary metabolite production by S. clavuligerus is typically observed in soy medium (52). The 248-bp orf3Scl-specific probe hybridized only to a ~1,500-nt transcript, suggesting that expression of this gene occurs only from a promoter upstream of bldGScl. This was confirmed by S1 nuclease mapping, which showed that an orf3Scl-specific promoter is not present within the intergenic region between bldGScl and orf3Scl (data not shown).



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  		FIG. 2. Northern analysis of bldGScl and orf3Scl transcripts. Total RNA (40 µg) was isolated from liquid cultures of S. clavuligerus NRRL 3585 grown in soy medium for 72, 96, and 120 h as indicated. The probe for bldGScl was a 232-bp random primer-labeled PCR product corresponding to sequence internal to bldGScl, while a 248-bp random primer-labeled PCR product was used to detect orf3Scl transcripts. The marker bands (Marker III; Roche) used for estimating transcript sizes were detected after hybridizing with random primer-labeled Marker III DNA. To control for RNA loading levels, the same membranes were hybridized with an end-labeled oligonucleotide probe (BKL54) specific for 16S rRNA.

Expression of bldG in S. clavuligerus and S. coelicolor occurs from three separate promoters. High-resolution S1 nuclease mapping and primer extension analysis had previously demonstrated that the S. coelicolor bldG gene is transcribed from two promoters located 82 and 123 nt upstream of the translation start codon (11). To determine the transcription initiation site(s) for the S. clavuligerus bldG gene, S1 nuclease mapping was performed using a 363-bp end-labeled probe that included a 100-bp nonhomologous extension to distinguish between full-length probe protection and probe-probe reannealing (Fig. 3A). The probe was uniquely labeled at one end so that only those S1 products resulting from protection by bldGScl mRNA would be detected. The experiment was performed at least twice using duplicate RNA samples, and representative results are shown in Fig. 3B. Two major S1 nuclease-protected products were observed which corresponded to transcription start sites located 79 to 80 and 120 nt upstream of the translation start codon (designated P1 and P2, respectively). These transcription start sites corresponded to the same two start sites previously observed for the S. coelicolor bldG gene. Unexpectedly, however, full-length protection of the probe was also detected, which suggested that a third bldG promoter existed in S. clavuligerus farther upstream of the probe sequence. A second probe was therefore designed to map the location of this third promoter, and it consisted of 379 bp of Streptomyces sequence and 90 bp of nonhomologous sequence to distinguish between full-length protection and probe-probe reannealing. The results (Fig. 3C) indicated that the third bldGScl promoter (P3) is located 277 nt upstream of the translation start codon. Similar S1 mapping experiments performed using RNA isolated from wild-type S. coelicolor and a newly designed bldGSco-specific probe revealed that a third, previously unidentified, promoter for bldGSco located 280 bp upstream of the translation start codon also exists (data not shown).



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  		FIG. 3. High-resolution S1 nuclease mapping of bldGScl transcripts. (A) The probe used to map P1 and P2 (M13R-DBG51) was an end-labeled 363-bp PCR product consisting of 263 bp of S. clavuligerus sequence and 100 bp of nonhomologous sequence. The probe used to detect P3 (M13R-DBG45) was an end-labeled 469-bp PCR product consisting of 379 bp of S. clavuligerus sequence and 90 bp of nonhomologous sequence. In both cases, the nonhomologous sequence (indicated by the curved line) was used to distinguish between full-length protection and probe-probe reannealing. The end of the probe that was labeled with 32P is indicated by the circle. (B and C) Total RNA (40 µg) isolated from wild-type S. clavuligerus grown in soy medium for 72, 96, and 120 h as indicated was hybridized with either end-labeled M13R-DBG51 (B) or end-labeled M13R-DBG45 (C) and was then treated with S1 nuclease. The sequencing ladder used to map P1 and P2 (B) was generated with the DBG51 oligonucleotide, while the ladder used to map P3 (C) was generated using the oligonucleotide DBG45. P+S1, control lane containing probe that went through the S1 procedure; P-S1, control lane containing probe that did not go through the S1 procedure; *, the most probable transcription start site(s).

Analysis of the sequence upstream of bldGScl P1 revealed the presence of a potential –10 sequence (TTGAAT) and –35 sequence (GTGCAT) with a spacer region of 16 nt. A putative –10 sequence was also observed upstream of P2 (GACAAT); however, an appropriately spaced –35 sequence could not be identified. In the case of P3, neither –10 nor –35 sequences of appropriate spacing could be found.

Localization of bldG expression in S. clavuligerus. We were next interested in determining whether expression of bldGScl is confined to a specific region of the S. clavuligerus colony. Previous studies of bldG expression in S. coelicolor revealed that both bldG transcripts and BldG protein are present throughout growth on solid medium (9, 11); however, the spatial localization of bldG promoter activity has never been investigated. We cloned a 655-bp fragment spanning P1 to P3 of the bldGScl promoter region into the egfp reporter vector pIJ8660, which integrates into the {phi}C31 site in the Streptomyces chromosome. The resulting construct, pAU337, was moved by conjugation from E. coli into wild-type S. clavuligerus. As a negative control, pIJ8660 was independently transferred to S. clavuligerus. Enhanced green fluorescent protein (EGFP) production was then monitored in both the test and the control strains by confocal microscopy after growth on ISP-4 solid medium for 2, 4, and 8 days, which coincided with different stages in the life cycle of S. clavuligerus. The results (Fig. 4) showed that a progressive increase in EGFP, reflecting bldGScl promoter activity, was observed over time and that while bldGScl promoter activity was localized primarily within the substrate mycelia during the early stages of growth, when aerial hyphae first began to appear (day 2) (Fig. 4A), by day 4 it was evident that expression occurred strongly in both substrate and aerial mycelia (Fig. 4B). This was also true in the later stages of growth (day 8), when chains of spores could be observed (Fig. 4C), and a close-up image of individual spores (Fig. 4D) indicated the presence of bldGScl promoter activity within the spores themselves. Under the same conditions, no fluorescence was observed at any time with the control strain (data not shown). Thus, it appears as though bldGScl expression takes place throughout the developing colony.



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  		FIG. 4. Localization of bldG promoter activity. Wild-type S. clavuligerus containing the bldGp-egfp reporter construct (pAU337) was grown on ISP-4 solid medium for 2 (A-1 to A-3), 4 (B-1 to B-3), and 8 (C-1 to C-3 and D-1 to D-3) days before being analyzed by confocal microscopy. Differential interference contrast images are shown on the left (A-1 to D-1), while fluorescence images are shown in the middle (A-2 to D-2) and overlays of the two images are shown on the right (A-3 to D-3). The location of the substrate mycelia (SM) and aerial hyphae (AH) in panels A to C are indicated, while D panels show a close-up image of individual spores observed after 8 days of growth. Bars, 40 µm (A to C) and 8 µm (D).

A constructed bldGScl null mutant is defective in both aerial hypha formation and secondary metabolite production. To determine whether the bldG ortholog in S. clavuligerus is involved in morphological differentiation and secondary metabolite production, as observed in S. coelicolor, a bldGScl null mutant was constructed using the REDIRECT PCR targeting system described by Gust et al. (19). The first step of this protocol involved the generation of a mutant cosmid (see Materials and Methods) in which 276 bp of internal bldGScl sequence was replaced with a cassette containing the aac(3)IV apramycin resistance gene and oriT, the latter allowing conjugative transfer of the mutant cosmid from E. coli to Streptomyces. After introduction of the mutant cosmid into wild-type S. clavuligerus, two single aprar kans exconjugants were selected and were screened by Southern analysis to confirm that replacement of the wild-type gene with the disruption cassette had taken place. Figure 5A shows that ApaI digestion of chromosomal DNA from the wild-type S. clavuligerus strain was expected to generate a 622-bp bldGScl-containing fragment, while a 1,717-bp fragment would result from replacement of bldGScl with the aac(3)IV+oriT cassette. Hybridization of the bldGScl-specific probe with the expected 622-bp ApaI fragment in the wild-type S. clavuligerus strain, and the absence of such a band in the mutant lanes, confirmed that gene replacement had taken place in the mutant strains (Fig. 5B). As expected, the 1,717-bp ApaI fragment was detected in the mutant lanes after hybridization with the aac(3)IV+oriT probe. In addition, the 1,717-bp fragment was also detected with the bldGScl-specific probe, as there was a small amount of sequence overlapping the bldGScl-specific probe present in the mutant DNA.



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  		FIG. 5. Southern analysis of S. clavuligerus bldG mutants. (A) Schematic diagram showing the bldGScl locus in the wild-type and bldG mutant strains. The location of the ApaI sites and the sizes of the resulting ApaI digestion products are indicated, and arrows show the direction of transcription of the bldGScl and aac(3)IV open reading frames. The thick lines represent flanking chromosomal DNA, while the thin line represents S. clavuligerus DNA contained within the pAU331 cosmid used to create the bldG mutant strains. FRT, flip recombinase target; aac(3)IV, apramycin resistance gene; oriT, origin of transfer. (B) Chromosomal DNA (4 µg) from the wild-type and the 4-44 and 4-52b bldG mutant strains was digested with ApaI and was separated on a 1% agarose gel. Similarly digested cosmid 5D4 and plasmid pAU331 were included as controls. After transfer to nylon membrane, the DNA was hybridized with a 232-bp random primer-labeled PCR product corresponding to bldGScl. The same membrane was then stripped and hybridized with a gel-purified, random primer-labeled 1,384-bp EcoRI-HindIII fragment containing aac(3)IV and oriT (isolated from pIJ773). {lambda} phage DNA digested with PstI was used as the molecular weight marker, and marker band sizes are indicated along with the sizes of the ApaI fragments that hybridized with the bldGScl- and aac(3)IV+oriT-specific probes (bold arrows).

The two bldGScl mutant strains, designated 4-44 and 4-52b, produced very little or no aerial hyphae when grown on either MYM medium or ISP-4 medium (data not shown), suggesting that, as in S. coelicolor, bldGScl is necessary for proper morphological development. To determine the effect of the mutation on secondary metabolite production, the two mutant strains, as well as the wild-type strain, were fermented in soy and SA medium in single shake flask cultures, and culture supernatants from 72-, 96-, and 120-h cultures were then analyzed by bioassays and HPLC. The mutants were completely blocked for clavulanic acid production in both soy and SA medium and were unable to produce either of the 5S clavam metabolites clavam-2-carboxylate or alanylclavam in soy medium as determined by HPLC and bioassays, respectively (data not shown). This lack of production was not attributed to a reduced growth rate of the mutants compared to the wild-type strain, as analysis of protein levels at each time point suggested that the growth rates of all strains were similar throughout the fermentation (data not shown). Bioassays also indicated that the production of cephamycin C was abolished in both mutant strains. Western analysis of crude extracts isolated at each time point, using polyclonal antibodies raised against purified S. coelicolor BldG protein fused to a maltose binding protein (MBP) tag, confirmed that the BldGScl protein was present in the wild-type strain but was absent from the 4-44 mutant strain (Fig. 6A).



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  		FIG. 6. Analysis of BldG, CcaR, and ccaR transcripts in the S. clavuligerus bldG 4-44 mutant. (A) Western analysis of BldGScl in the S. clavuligerus wild-type and bldG 4-44 mutant strains. Crude extracts (50 µg) isolated from soy cultures grown for 72, 96, and 120 h were separated on an SDS-15% PAGE and were transferred to a PVDF membrane. The proteins were then probed with antibodies raised against an MBP-BldGSco fusion protein at a dilution of 1 in 10,000. Extracts from wild-type S. coelicolor M145 and from the S. coelicolor bldG mutant {Delta}bldG 1DB were used as positive and negative controls for BldG detection, respectively. (B) Western analysis of CcaR in the S. clavuligerus wild-type and bldG 4-44 mutant strains. Crude extracts (30 µg) were isolated from TSB-S cultures grown for 24, 36, and 48 h and were separated on an SDS-10% PAGE. After transfer to PVDF membrane, the proteins were probed with MBP-CcaR-specific antibodies at a dilution of 1 in 5,000. Purified CcaR and extract from a ccaR disruption mutant (ccaR::apr) were included as positive and negative controls, respectively. (C) Northern analysis of ccaR transcripts. Total RNA (40 µg) isolated from the wild-type and bldG 4-44 mutant strains grown in TSB-S medium for 24, 36, 48, and 72 h was separated on a 1.25% agarose gel and transferred to nylon membrane. The probe used to detect ccaR transcripts was a ca. 1,200-bp random primer-labeled fragment containing the entire ccaR coding region. The molecular weight marker bands (M) were detected after hybridizing with random primer-labeled Marker III DNA (Roche). To control for RNA loading levels, the same membrane was hybridized with an end-labeled oligonucleotide probe (BKL54) specific for 16S rRNA.

Complementation of the bldGScl mutant phenotype. To verify that the observed phenotype of the mutant strains was solely due to the replacement of bldGScl, a copy of the wild-type bldGScl gene cloned into the integrative pSET152 derivative pAU3-45 (Table 1) (see Materials and Methods) was introduced into the S. clavuligerus 4-44 and 4-52b mutant strains by protoplast transformation. As expected, aerial hypha formation was restored to the 4-44 and 4-52b mutant strains by the presence of the wild-type bldGScl gene, whereas introduction of the plasmid vector alone had no effect (data not shown). Bioassays of supernatants from cultures grown in soy medium for 72 and 96 h indicated that production of clavulanic acid, cephamycin C, and 5S clavams was also restored in the bldG 4-44 mutant complemented with bldGScl (data not shown). Similar results were observed with the bldG 4-52b mutant strain.

Expression of ccaR is abolished in a bldGScl mutant background. Since the biosynthesis of cephamycin C and clavulanic acid is known to be controlled by the transcriptional activator CcaR, we next looked at whether the lack of secondary metabolite production observed in the bldGScl mutant strains was due to the absence of CcaR. Western analysis was performed using crude extracts isolated from the wild-type strain and the bldG 4-44 mutant strain grown in TSB-S medium for 24, 36, and 48 h. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to polyvinylidene difluoride (PVDF) membrane, the extracts were probed with polyclonal antibodies raised against an MBP-CcaR fusion protein. Figure 6B shows that the CcaR protein could be readily detected in the wild-type extracts but was absent from the mutant extracts, and similar results were observed with extracts isolated from the bldG 4-52b mutant strain (data not shown). To determine if this was due to a loss of ccaR gene expression, Northern analysis was performed using RNA isolated from the wild-type and bldG 4-44 strains and a random primer-labeled ccaR-specific probe. Although it had been previously demonstrated by another group that ccaR is expressed as a ~0.9-kb monocistronic transcript only prior to the appearance of cephamycin C and clavulanic acid (41), we have shown here that the ccaR-specific probe used could hybridize to two transcripts of ~1,000 to 1,100 nt and ~1,400 to 1,500 nt (Fig. 6C). These transcripts were detected in the wild-type samples at all time points examined, including time points when cephamycin C production was detected by bioassays (data not shown). Similar results were observed with RNA samples isolated from cultures grown in soy medium (data not shown), and increasing the stringency of the membrane washes had no effect on binding of the probe to either transcript, suggesting that both transcripts were hybridizing specifically to the probe. Moreover, neither transcript could be detected in any of the bldG 4-44 mutant lanes, indicating that the observed absence of CcaR protein in the mutant strain was due to loss of ccaR gene expression.


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DISCUSSION
 
This study describes the cloning and sequencing of the S. coelicolor bldG ortholog from S. clavuligerus and demonstrates that, as in S. coelicolor, bldGScl is necessary for both morphological differentiation and secondary metabolite production in S. clavuligerus. This is the first classical bld gene to be identified that affects both processes in S. clavuligerus, as previous studies of the S. clavuligerus bldA gene revealed that a bldA mutant was defective in aerial hypha formation but was still able to produce clavulanic acid and cephamycin C (54).

The bldGScl protein product shows 92% identity and 94% similarity to the S. coelicolor BldG protein and 91% identity and 94% similarity to the BldG ortholog in S. avermitilis. All three proteins show similarity to anti-anti-sigma factor proteins of Bacillus spp. and of other organisms, which typically function along with a cognate anti-sigma factor protein to regulate the activity of a target sigma factor. For example, in B. subtilis the SpoIIAB anti-sigma factor regulates the activity of the sporulation-specific sigma factor {sigma}F by binding to it and preventing association of the sigma factor with core RNA polymerase (15, 34). Once a specific stage in the sporulation process is reached, however, this inhibition is reversed by the SpoIIAA anti-anti-sigma factor, which binds to SpoIIAB, allowing {sigma}F to direct transcription of genes involved in sporulation (3, 14). An important feature of this system of sigma factor regulation is the regulation of the anti-anti-sigma factor by phosphorylation, since only the unphosphorylated form of the protein is able to bind to the anti-sigma factor (3, 12). Analysis of the BldGSco protein sequence revealed that the serine residue known to be phosphorylated in SpoIIAA as well as in other anti-anti-sigma factors is conserved in BldGSco (11), and phosphorylation studies have confirmed that this residue is phosphorylated in S. coelicolor by an unknown kinase (9). Given that this residue is also conserved in both the S. avermitilis and S. clavuligerus BldG orthologs, it is likely that the activity of these proteins is also controlled by reversible phosphorylation. In the case of S. clavuligerus, this idea is further supported by the EGFP results obtained in this study. bldGScl promoter activity was detected throughout the developing colony and at all stages of growth examined, suggesting that regulation of bldGScl activity occurs posttranslationally.

Despite the sequence similarity of the BldG proteins with other known anti-anti-sigma factors, it is not yet clear whether these proteins function in a similar manner to regulate sigma factors in Streptomyces spp. In all three Streptomyces spp. where the bldG locus has been sequenced, an open reading frame has been found downstream of bldG that encodes a protein showing similarity to anti-sigma factor proteins. This genetic organization is similar to that observed in B. subtilis, with cognate anti-sigma factor/anti-anti-sigma factor pairs (17, 24). Furthermore, results from a previous study and from this study demonstrated that in S. coelicolor and S. clavuligerus, this downstream open reading frame (orf3) is expressed as a polycistronic transcript that includes bldG, suggesting that the two protein products might work together to regulate a sigma factor. However, whether the orf3-encoded anti-sigma factor is involved along with BldG in the regulation of morphological differentiation and secondary metabolite production in S. coelicolor has not yet been elucidated. Deletion of the orf3Sco open reading frame resulted in a variety of phenotypes that could not be complemented by orf3Sco, suggesting that one or more secondary site mutations may have been acquired (10). Also not known is whether a sigma factor is the target of BldG regulation, since a sigma factor gene was not found in the vicinity of bldG in S. coelicolor, S. avermitilis, or S. clavuligerus. This is in contrast to the situation in Bacillus spp., where the target sigma factor gene is normally encoded along with its anti-sigma and anti-anti-sigma factor regulators (17, 24). The absence of a sigma factor gene within the bldG locus may imply that more than one sigma factor is being regulated by BldG and its cognate anti-sigma factor, or that these regulatory proteins play a different role in Streptomyces spp. than in Bacillus.

In S. clavuligerus, the production of cephamycin C and clavulanic acid is controlled by the pathway-specific regulator CcaR, which is encoded within the cephamycin C gene cluster (1, 41, 55). Disruption of ccaR results in a loss of cephamycin C and clavulanic acid biosynthesis, indicating that CcaR is essential for the production of these compounds. Previously it had been demonstrated that a single ccaR transcript, which approximately corresponds with the smaller of the two transcripts we observed, was produced in S. clavuligerus prior to the appearance of cephamycin C and clavulanic acid, while no ccaR transcripts were detected once secondary metabolite production had begun (41). We have shown here that in wild-type S. clavuligerus, ccaR is expressed as two transcripts of ~1 and ~1.4 kb. Although the intensity of the transcript bands decreased in RNA samples from the later time points, both were detectable throughout growth. Since these results differed from those of Perez-Llarena et al. (41), and because it wasn't clear how those authors staged their cultures, we took care to grow the cultures from both germinated spores (soy medium) and from seed cultures (TSB medium), and we consistently observed the appearance of the two transcripts regardless of the culture conditions used. While we are not sure why our results differ from those observed by Perez-Llarena et al., one possibility is that the larger of the two transcripts is more faintly visible on the Northern blot, as are the transcripts from the later time points, and may have been below detection in their experiments.

DNA binding studies have indicated that CcaR interacts with the bidirectional cefD-cmcI promoter region of the cephamycin gene cluster to control expression of the middle (cefDE) and late (cmcI) stages of the cephamycin C biosynthetic pathway (48). It has also been demonstrated by Kyung et al. (27) that CcaR binds to the promoter region of the lat gene in the cephamycin gene cluster, although this needs to be further examined, as a similar study performed by Santamarta et al. (48) was unable to show binding of CcaR to the lat promoter. In the case of the clavulanic acid gene cluster, CcaR acts indirectly through the ClaR transcriptional regulator, which is required only for expression of the late genes for clavulanic acid biosynthesis (38, 42). More recently, it has been demonstrated that CcaR also controls (directly or indirectly) expression of the ceaS2 promoter, leading to expression of a polycistronic transcript encompassing the early (ceaS2-bls2-pah2-cas2) clavulanic acid biosynthetic genes (51). In this study we report that expression of ccaR itself is dependent upon bldGScl, making bldGScl the first gene identified in S. clavuligerus that functions in a regulatory cascade above ccaR and claR to control cephamycin C and clavulanic acid production. Production of the 5S clavams was also shown to be dependent on bldGScl; however, since recent findings suggest that production of the 5S clavams occurs independently of CcaR (51), it is likely that bldGScl mediates its effect through the control of additional regulatory proteins that are involved specifically in the biosynthesis of these compounds. The identity of such additional regulatory proteins is currently under investigation.

The results of this study support the notion of functional conservation of bld genes among Streptomyces spp. and reinforce the need to further characterize such genes in S. coelicolor so that the knowledge gained can be applied to industrially important organisms, such as S. clavuligerus. In the case of bldG, it still needs to be determined exactly how the protein product functions to control secondary metabolite production, whether it is through the direct regulation of one or more sigma factors or through some other unknown mechanism. Also, the involvement of anti-sigma factor proteins such as that putatively encoded by orf3 remains to be elucidated.


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ACKNOWLEDGMENTS
 
This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Alberta Heritage Foundation for Medical Research, and the Alberta Ingenuity Fund.

We thank K. Gislason for technical help with the Northern analysis. We also thank R. Bhatnagar and J. Scott from the Microscopy Unit at the Department of Biological Sciences, University of Alberta, for helping with microscopic analysis.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, CW405 Biological Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. Phone: (780) 492-1868. Fax: (780) 492-9234. E-mail: brenda.leskiw{at}ualberta.ca. Back

{dagger} D.R.D.B. and K.T. contributed equally to this work. Back

{ddagger} Present address: Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom. Back


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Antimicrobial Agents and Chemotherapy, April 2005, p. 1529-1541, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1529-1541.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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