Figure 1.
S. marcescens Db10 is able to inhibit growth of Gram-positive bacteria.
A. Activity assays using B. subtilis NCIB3610, S. aureus 113 and M. luteus ATCC4698 as indicator strains, with S. marcescens Db10 or S. marcescens Db10 SMA2290::Tn5 (NRS2992) as the producer strains. B. Culture supernatant assays (described in experimental procedures) indicate the diffusible molecule is produced by S. marcescens Db10 in stationary phase. Clearance halo sizes (radius of cleared area) are averages of three replicates; error bars represent standard error of the mean. A representative growth curve is shown for reference.
Figure 2.
Transcriptional analysis of the alb operon.
A. Map of the alb operon. Genes belonging to the alb operon are indicated as light grey arrows, genes that do not belong to the alb operon are indicated as dark grey arrows. Each gene is shown to scale. The region upstream of the alb1 gene is highlighted by a broken box. The transcription start site is shown as a black arrow. The site of the transposon (Tn) insertion (at nucleotide 2745) in alb4 is indicated with a grey triangle. B. RT-PCR analysis of the biosynthetic gene cluster. The template used in the PCR reaction is indicated above the gels as Db10 (wild type S. marcescens Db10 cDNA) or transposon (Tn5 mutant cDNA). Reactions were performed in the presence (+) and absence (−) of reverse transcriptase. Con. represents S. marcescens Db10 genomic DNA as a positive control (+) and water as a negative control (−). The region of the chromosome amplified is indicated to the left of each gel. Primers were designed to amplify a product internal to a single gene, or spanning the intergenic region between two genes. Twenty five cycles of PCR amplification were used with the exception of SMA2294 and SMA2294-alb1 where 30 cycles were used. C. 5′ RACE analysis to identify the transcriptional start site of the alb operon. The region upstream of the alb1 gene is shown and the methionine start codon is boxed. Asterisks indicate the number of times a particular base was identified as the transcriptional start site. Putative −10 and −35 regions are highlighted by a broken box. The −41 bp and −42 bp start sites are highlighted together as it is not possible to distinguish between these sites more specifically (see text).
Figure 3.
Proposed pathway for althiomycin biosynthesis in S. marcescens Db10.
Alb4 and Alb5 form a hybrid NRPS-PKS assembly line, which consists of six modules as indicated. Domains within the NRPS-PKS are as follows. A: adenylation; PCP: peptidyl carrier protein; C: condensation; Cy: condensation/heterocyclisation; Ox: flavin-dependent oxidase; E: epimerisation; KS: ketosynthase; AT: acyl transferase; ACP: acyl carrier protein; TE: thioesterase. Alb6 (not shown) likely functions as an external ‘editing’ thioesterase. Alb3 is similar to known S-adenosylmethionine (SAM)-dependent methyltransferases and Alb2 is similar to known oxidases. The fact that no althiomycin-related compounds accumulate in alb2/alb3 mutants suggests that N-oxidation and O-methylation take place in trans during chain assembly on the hybrid NRPS-PKS. However, the precise timing of the N-oxidation reaction is currently unclear.
Figure 4.
Spectroscopic analysis of althiomycin produced by S. marcescens Db10.
A. Comparison of the extracted ion chromatogram at m/z 440.0600 for wild type S. marcescens Db10 (dashed line) and Db10 Δalb6 (solid line). B. Top panel: high resolution mass spectrum of althiomycin detected in culture supernatant of S. marcescens Db10. Bottom panel: simulated mass spectrum for the C16H18N5O6S2+ ion. C. Summary of COSY and HBMC NMR correlations observed for althiomycin isolated from S. marcescens Db10.
Figure 5.
The tailoring enzymes are required for althiomycin production.
A. Assay to assess antimicrobial activity of S. marcescens Db10 and the alb4–5 mutant strain (SAN5) against B. subtilis 3610. B. Visualisation of proteins corresponding to the predicted size of Alb4 and Alb5. Total cellular protein from Db10 (wild type S. marcescens Db10), Δalb3 (SAN4) or Δalb4–5 (SAN5) mutants was isolated from cultures grown to stationary phase and separated by SDS-PAGE, followed by staining with Coomassie blue. C–E. Activity assays were performed using Bacillus subtilis NRS1473 as the indicator lawn. The producer strains used are indicated above as: Db10+VC (S. marcescens Db10 pSUPROM); Db10+alb2 (S. marcescens Db10 pSAN2); Δalb2+VC (SAN3 pSUPROM); Δalb2+alb2 (SAN3 pSAN2); Db10+alb3 (S. marcescens Db10 pSAN3);Δalb351–262+VC (SAN88 pSUPROM); Δalb351–262+alb3 (SAN88 pSAN3); Db10+alb6 (S. marcescens Db10 pSAN38); Δalb6+VC (SAN60 pSUPROM); Δalb6+alb6 (SAN60 pSAN38). ‘VC’ represents the empty vector control.
Figure 6.
The PPTase encoded by SMA2452 is required for althiomycin biosynthesis.
A. Assay to assess the antimicrobial activity of S. marcescens Db10, the alb4–5 mutant strain (SAN5), the Δ4147 mutant strain (SAN96) and the Δ2452::Cml mutant strain (SAN112) against B. subtilis 3610. B. Activity assays were performed using Bacillus subtilis NRS1473 as the indicator lawn. The producer strains are indicated above as: Db10+VC (S. marcescens Db10 pSUPROM); Db10+2452 (S. marcescens Db10 pSAN46); Δ2452::Cml+VC (SAN112 pSUPROM); Δ2452::Cml+2452 (SAN112 pSAN46). ‘VC’ represents the empty vector control.
Figure 7.
The alb1 gene encodes an althiomycin resistance determinant.
A. Activity assays using B. subtilis NRS1473 as the indicator lawn for the producer strains: Db10+VC (S. marcescens Db10 pSUPROM); Db10+alb1 (S. marcescens Db10 pSAN1); Δalb1+VC (SAN2 pSUPROM); Δalb1+alb1 (SAN2 pSAN1) B. RT-PCR analysis of alb1–alb6 transcript levels in each of the alb mutant strains. The template used in the PCR reaction is indicated above the gels as: Db10 (wild type S. marcescens Db10 cDNA); Δ1 (SAN2(Δalb1) cDNA); Δ2 (SAN2 (Δalb2) cDNA); Δ3 (SAN4 (Δalb3) cDNA); Δ4–5 (SAN5 (Δalb4–5) cDNA); Δ6 (SAN60 (Δalb6) cDNA). Reactions were performed in the presence (+) or absence (−) of reverse transcriptase. Con. represents S. marcescens Db10 genomic DNA as a positive control (+) and water as a negative control (−). The primer pairs used to amplify a product internal to a particular gene are indicated to the right of each gel. Twenty five cycles of PCR amplification were used. C. Activity assays using B. subtilis 3610 as the indicator lawn and S. marcescens ATCC274 as the producer strain. D. Activity assays using Db10 (S. marcescens Db10 pSUPROM), Db10 Δalb4–5 (SAN5 pSUPROM) and Db10 PT5-alb1–6 (SAN100 pSUPROM) as the producer strain and using ATCC274+VC (S. marcescens 274 pSUPROM) and ATCC274+alb1 (S. marcescens 274 pSAN1) as the indicator lawn. ‘VC’ represents the empty vector control.
Table 1.
Comparative Analysis of the Althiomycin Biosynthetic Proteins.
Figure 8.
The 4-methoxy-3-pyrrolin-2-one moiety of althiomycin is shared by other bioactive natural products.
The position of the 4-methoxy-3-pyrrolin-2-one moiety in althiomycin is highlighted by a broken box and this moiety is depicted in grey within the other molecules.
Table 2.
Selected Bacterial Strains and Plasmids Used in This Study.