Expression of ccaR, encoding the positive activator of cephamycin C and clavulanic acid production in Streptomyces clavuligerus, is dependent on bldG

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.

FIG. 1.

(A) Schematic diagram of the sequenced 2,525-bp region of the S. clavuligerus chromosome containing bldG_Scl. 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 bldG_Scl-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.

FIG. 2.

Northern analysis of bldG_Scl and orf3_Scl 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 bldG_Scl was a 232-bp random primer-labeled PCR product corresponding to sequence internal to bldG_Scl, while a 248-bp random primer-labeled PCR product was used to detect orf3_Scl 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.

FIG. 3.

High-resolution S1 nuclease mapping of bldG_Scl 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).

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).

FIG. 5.

Southern analysis of S. clavuligerus bldG mutants. (A) Schematic diagram showing the bldG_Scl 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 bldG_Scl 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 bldG_Scl. 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). λ 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 bldG_Scl- and aac(3)IV+oriT-specific probes (bold arrows).

FIG. 6.

Analysis of BldG, CcaR, and ccaR transcripts in the S. clavuligerus bldG 4-44 mutant. (A) Western analysis of BldG_Scl 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-BldG_Sco fusion protein at a dilution of 1 in 10,000. Extracts from wild-type S. coelicolor M145 and from the S. coelicolor bldG mutant ΔbldG 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.