Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Aug;160(Pt 8):1609-1617.
doi: 10.1099/mic.0.078576-0. Epub 2014 May 20.

Role of the phosphopantetheinyltransferase enzyme, PswP, in the biosynthesis of antimicrobial secondary metabolites by Serratia marcescens Db10

Amy J Gerc  1 , Nicola R Stanley-Wall  1 , Sarah J Coulthurst  1
Affiliations

Role of the phosphopantetheinyltransferase enzyme, PswP, in the biosynthesis of antimicrobial secondary metabolites by Serratia marcescens Db10

Amy J Gerc et al. Microbiology (Reading). 2014 Aug.

Abstract

Phosphopantetheinyltransferase (PPTase) enzymes fulfil essential roles in primary and secondary metabolism in prokaryotes, archaea and eukaryotes. PPTase enzymes catalyse the essential modification of the carrier protein domain of fatty acid synthases, polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs). In bacteria and fungi, NRPS and PKS enzymes are often responsible for the biosynthesis of secondary metabolites with clinically relevant properties; these secondary metabolites include a variety of antimicrobial peptides. We have previously shown that in the Gram-negative bacterium Serratia marcescens Db10, the PPTase enzyme PswP is essential for the biosynthesis of an NRPS-PKS dependent antibiotic called althiomycin. In this work we utilize bioinformatic analyses to classify PswP as belonging to the F/KES subfamily of Sfp type PPTases and to putatively identify additional NRPS substrates of PswP, in addition to the althiomycin NRPS-PKS, in Ser. marcescens Db10. We show that PswP is required for the production of three diffusible metabolites by this organism, each possessing antimicrobial activity against Staphylococcus aureus. Genetic analyses identify the three metabolites as althiomycin, serrawettin W2 and an as-yet-uncharacterized siderophore, which may be related to enterobactin. Our results highlight the use of an individual PPTase enzyme in multiple biosynthetic pathways, each contributing to the ability of Ser. marcescens to inhibit competitor bacteria by the production of antimicrobial secondary metabolites.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
The PPTase PswP is required for the biosynthesis of three secondary metabolites with antimicrobial activity against Sta. aureus. (a) Antimicrobial activity assays using Sta. aureus 113 as the indicator lawn. The producer strains are indicated above: Db10 (wild-type Ser. marcescens Db10), Δalb4-5 (SAN5), ΔpswP (SAN112), swrA : : Tn5 (JESM267), Δalb4-5 swrA : : Tn5 (SAN124) and Δalb4-5 swrA : : Tn5 ΔentB (SAN181). (b) Antimicrobial activity assays show complementation of the pswP deletion by expression of the pswP gene in trans, using kanamycin-resistant Sta. aureus Newman spa : : tet sbi : : kan as the indicator lawn. The producer strains are indicated above: Db10+VC (Ser. marcescens Db10 pSUPROM, vector control), Db10+pswP (Ser. marcescens Db10 pSAN46), ΔpswP+VC (SAN112 pSUPROM) and ΔpswP+pswP (SAN112 pSAN46).
Fig. 2.
Fig. 2.
Identification of conserved motifs of the F/KES subfamily of Sfp-type PPTases within PswP of Ser. marcescens Db10. Conserved PPT motifs 1A, 1, 2 and 3 are denoted with a dashed boxed. Residues previously identified as being highly conserved across the F/KES subfamily are indicated below the amino acid sequence; residues of PswP that match the consensus shown are shaded black. Analysis is based on the motif alignment analysis performed by Copp & Neilan (2006).
Fig. 3.
Fig. 3.
Comparison of the SMA2450–2452 and SMA4408–4415 gene clusters of Ser. marcescens Db10 with the enterobactin biosynthetic gene cluster of E. coli CFT073 (c0668–c0683). Genes are drawn approximately to scale and protein length is indicated below the encoding gene (as the number of amino acids). Homologous genes are indicated by the same colour, the level of identity between homologous proteins is shown below the corresponding genes as an absolute value and as a percentage.
Fig. 4.
Fig. 4.
An althiomycin and serrawettin W2 mutant of Ser. marcescens Db10 is unable to kill Sta. aureus in the presence of additional iron. Antimicrobial activity assays in the absence (a) or presence (b) of 50 μM FeCl3, using Sta. aureus as the indicator lawn, with a schematic of the location of each producer strain shown (c). The producing strains were: 1, Ser. marcescens Db10 (wild-type); 2, SAN5 (Δalb4-5); 3, JESM267 (swrA : : Tn5); 4, SAN124 (Δalb4-5 swrA : : Tn5); 5, SAN112 (ΔpswP). The insets show a magnified image of producer strain 4 SAN124 in both (a) and (b).
Fig. 5.
Fig. 5.
The PPTase PswP and SMA4415 (EntB) are required for the biosynthesis of a siderophore in Ser. marcescens Db10. CAS assay for the detection of siderophore biosynthesis, as indicated by the presence of an orange halo. (a) The producer strains are indicated above: Db10 (wild-type Ser. marcescens Db10), Δalb4-5 (SAN5), swrA : : Tn5 (JESM267), Δalb4-5 swrA : : Tn5 (SAN124), ΔentB (SAN176), ΔpswP (SAN112) and Δalb4-5 swrA : : Tn5 ΔentB (SAN181). (b) Complementation of the pswP deletion by expression of the pswP gene in trans. The producer strains are indicated above: Db10+VC (Ser. marcescens Db10 pSUPROM, vector control), Db10+pswP (Ser. marcescens Db10 pSAN46), ΔpswP+VC (SAN112 pSUPROM) and ΔpswP+pswP (SAN112 pSAN46).
See this image and copyright information in PMC

References

    1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. (1997). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402. 10.1093/nar/25.17.3389 - DOI - PMC - PubMed
    1. Beld J., Sonnenschein E. C., Vickery C. R., Noel J. P., Burkart M. D. (2014). The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life. Nat Prod Rep 31, 61–108. 10.1039/c3np70054b - DOI - PMC - PubMed
    1. Blin K., Medema M. H., Kazempour D., Fischbach M. A., Breitling R., Takano E., Weber T. (2013). antiSMASH 2.0–a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res 41 (Web Server issue), W204-W212. 10.1093/nar/gkt449 - DOI - PMC - PubMed
    1. Chirgadze N. Y., Briggs S. L., McAllister K. A., Fischl A. S., Zhao G. (2000). Crystal structure of Streptococcus pneumoniae acyl carrier protein synthase: an essential enzyme in bacterial fatty acid biosynthesis. EMBO J 19, 5281–5287. 10.1093/emboj/19.20.5281 - DOI - PMC - PubMed
    1. Copp J. N., Neilan B. A. (2006). The phosphopantetheinyl transferase superfamily: phylogenetic analysis and functional implications in cyanobacteria. Appl Environ Microbiol 72, 2298–2305. 10.1128/AEM.72.4.2298-2305.2006 - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources