Fig 1.
Diagrammatic representation of the partial clavulanic acid biosynthetic pathway from Streptomyces clavuligerus.
Genes encoding enzymes known to be involved in the “early” shared stages of 5S clavam and clavulanic acid production, and those predicted to encode proteins involved exclusively in the biosynthesis of clavulanic acid (“late” steps) are indicated. In addition, genes encoding enzymes with known biosynthetic functions are shown next to arrows representing the respective reactions catalyzed by them, and the question mark indicates the unknown protein(s) responsible for the 5S to 5R epimerization and side chain modification of clavam intermediates during clavulanic acid biosynthesis. The two 5S clavam intermediates related to clavaminic acid (R1 = N-glycyl and R2 = N-acetyl-glycyl, respectively), which accumulate in the orf15 and orf16 gene mutants are shown in the dashed box.
Fig 2.
Preparation and analysis of S. clavuligerus cpe deletion mutants.
(A and B) The thick arrows depict genes with arrowheads indicating the direction of transcription. (A) The relative arrangement of genes from the chromosomal locus surrounding cpe in S. clavuligerus is shown, and the bent arrows represent the known promoters for the different transcriptional units. The DNA sequences of the overlapping regions between cpe-orf13 (bottom) and orf13-orf14 (top) are indicated in open boxes, whereas the respective start and stop codons are shown in filled boxes. (B) Diagrammatic representation of the Δcpe::apra and Δcpe-INF mutants, which were prepared such that the 5' and 3' ends of cpe were retained and intervening DNA sequences were replaced by an apramycin resistance cassette or an in-frame 81-bp sequence in the respective mutants. (C and D) HPLC analysis of 96 hour wt S. clavuligerus and different cpe mutant SA culture supernatants for assessing clavulanic acid production using the phosphate buffer system [32]. Peaks corresponding to imidazole-derivatized clavulanic acid are indicated by the star symbol, which were detected at 311nm.
Table 1.
Bacterial strains used in the current study.
Table 2.
Plasmids and cosmids used in the current study.
Fig 3.
Cellular localization of Cpe in S. clavuligerus.
C-terminal FLAG-tagged copies of Cpe, secreted Blip and cytoplasmic CcaR were expressed in wt S. clavuligerus separately for western blot analysis. Cultures were used for isolating secreted (left panel) and cell/cytoplasmic (right panel) fractions, which were probed using anti-FLAG antibodies. The analysis of protein fractions from S. clavuligerus strains containing plasmids pHM:blipFLAG (expressing BlipFLAG), pHM:cpeFLAG (expressing CpeFLAG), pHM:ccaRFLAG (expressing CcaRFLAG) or pHM11a (Cont, empty vector) is shown. Lane M contains the PageRuler Plus Prestained Protein Ladder, which functioned as the molecular weight marker for resolving protein samples during 12% SDS-PAGE.
Fig 4.
Transcriptional analysis of genes from the clavulanic acid biosynthetic gene cluster (BGC) in wt S. clavuligerus and the Δcpe-INF mutant.
(A) The overall architecture of the BGC is shown with each hollow arrow representing a gene and the arrowhead its orientation. The known transcriptional units are also indicated, and the broken lines represent transcripts (B) The first gene from each transcriptional unit in (A) was selected for analysis to determine its comparative expression level in the two respective strains. RNA isolated from wt S. clavuligerus and the Δcpe-INF mutant after 48 hours of growth in SA medium was used for RT-PCR (+) analysis. As controls, treated RNA samples were used directly in PCR without RT or cDNA synthesis (-). The expression of the constitutively expressed hrdB gene (extreme right boxed panel) was used as internal control to normalize expression levels between different samples/strains.
Fig 5.
Functional analysis of different domains of CpeSc and its homologues during clavulanic acid production in S. clavuligerus.
(A and B) LC-MS analysis of 96 hour SA culture supernatants after imidazole derivatization using the ammonium bicarbonate buffer system [38]. Cultures of wt S. clavuligerus or the Δcpe-INF mutant expressing Cpe from S. clavuligerus (pHM:cpeSc), S. flavogriseus (pHM:cpeSf), S. jumonjinensis/S. katsurahamanus (pHM:cpeSj/Sk) or the C-terminal domain of CpeSc (pHM:cpeCt) were used in the analysis. (A) Liquid chromatography profiles showing the elution of the peaks corresponding to imidazole-derivatized clavulanic acid (indicated by the star symbol). (B) Mass spectra of the major peaks corresponding imidazole-derivatized clavulanic acid [M+H]+ (m/z = 224) and the fragmented product [M-imidazole]+ (m/z = 156), which were only detected in supernatants from clavulanic acid producing strains shown in (A).
Table 3.
Clavulanic acid production in wild type (wt) S. clavuligerus and the Δcpe-INF mutant expressing different variants of CpeSc.