prodigiosin and serratamolide

Serratia marcescens is a Gram-negative bacterium of the Enterobacteriaceae family that can produce numbers of biologically active secondary metabolites. However, our understanding of the regulatory mechanisms behind secondary metabolites biosynthesis in S. marcescens remains limited. In this study, we identified an uncharacterized LysR family transcriptional regulator, encoding gene BVG90_12635, here we named psrA, that positively controlled prodigiosin synthesis in S. marcescens. This phenotype corresponded to PsrA positive control of transcriptional of the prodigiosin-associated pig operon by directly binding to a regulatory binding site (RBS) and an activating binding site (ABS) in the promoter region of the pig operon. We demonstrated that L-proline is an effector for the PsrA, which enhances the binding affinity of PsrA to its target promoters. Using transcriptomics and further experiments, we show that PsrA indirectly regulates pleiotropic phenotypes, including serrawettin W1 biosynthesis, extracellular polysaccharide production, biofilm formation, swarming motility and T6SS-mediated antibacterial activity in S. marcescens. Collectively, this study proposes that PsrA is a novel regulator that contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in S. marcescens and provides important clues for future studies exploring the function of the PsrA and PsrA-like proteins which are widely present in many other bacteria.

Topics: Bacterial Proteins; Biofilms; Depsipeptides; Movement; Operon; Polysaccharides, Bacterial; Prodigiosin; Promoter Regions, Genetic; Serratia marcescens; Transcription Factors

The genus Serratia is a predominantly unexplored source of antimicrobial secondary metabolites. The aim of the current study was thus to isolate and evaluate the antimicrobial properties of biosurfactants produced by Serratia species. Forty-nine (n = 34 pigmented; n = 15 non-pigmented) biosurfactant producing Serratia strains were isolated from environmental sources and selected isolates (n = 11 pigmented; n = 11 non-pigmented) were identified as Serratia marcescens using molecular typing. The swrW gene (serrawettin W1 synthetase) was detected in all the screened pigmented strains and one non-pigmented strain and primers were designed for the detection of the swrA gene (non-ribosomal serrawettin W2 synthetase), which was detected in nine non-pigmented strains. Crude extracts obtained from S. marcescens P1, NP1 and NP2 were chemically characterised using ultra-performance liquid chromatography coupled to electrospray ionisation mass spectrometry (UPLC-ESI-MS), which revealed that P1 produced serrawettin W1 homologues and prodigiosin, while NP1 produced serrawettin W1 homologues and glucosamine derivative A. In contrast, serrawettin W2 analogues were predominantly identified in the crude extract obtained from S. marcescens NP2. Both P1 and NP1 crude extracts displayed broad-spectrum antimicrobial activity against clinical, food and environmental pathogens, such as multidrug-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Cryptococcus neoformans. In contrast, the NP2 crude extract displayed antibacterial activity against a limited range of pathogenic and opportunistic pathogens. The serrawettin W1 homologues, in combination with prodigiosin and glucosamine derivatives, produced by pigmented and non-pigmented S. marcescens strains, could thus potentially be employed as broad-spectrum therapeutic agents against multidrug-resistant bacterial and fungal pathogens.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Depsipeptides; Lipoproteins; Methicillin-Resistant Staphylococcus aureus; Peptides, Cyclic; Prodigiosin; Pseudomonas aeruginosa; Secondary Metabolism; Serratia marcescens; Surface-Active Agents

Topics: Allelopathy; Biosynthetic Pathways; Chromatography, High Pressure Liquid; Depsipeptides; Endophytes; Maytenus; Metabolome; Microbial Interactions; Prodigiosin; Serratia marcescens; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Bacterial secondary metabolites are naturally produced to prevail amongst competitors in a shared habitat and thus represent a valuable source for antibiotic discovery. The transformation of newly discovered antibiotic compounds into effective drugs often requires additional surfactant components for drug formulation. Nature may also provide blueprints in this respect: A cocktail of two compounds consisting of the antibacterial red pigment prodigiosin and the biosurfactant serrawettin W1 is naturally produced by the bacterium Serratia marcescens, which occurs in highly competitive habitats including soil. We show here a combinatorial antibacterial effect of these compounds, but also of prodigiosin mixed with other (bio)surfactants, against the soil-dwelling bacterium Corynebacterium glutamicum taken as a model target bacterium. Prodigiosin exerted a combinatorial inhibitory effect with all tested surfactants in a disk diffusion assay which was especially pronounced in combination with N-myristoyltyrosine. Minimal inhibitory and bactericidal concentrations (MIC and MBC) of the individual compounds were 2.56 μg/mL prodigiosin and 32 μg/mL N-myristoyltyrosine, and the MIC of prodigiosin was decreased by 3 orders of magnitude to 0.005 μg/mL in the presence of 16 μg/mL N-myristoyltyrosine, indicative of synergistic interaction. Investigation of bacterial survival revealed similar combinatorial effects; moreover, antagonistic effects were observed at higher compound concentrations. Finally, the investigation of microcolony formation under combined application of concentrations just below the MBC revealed heterogeneity of responses with cell death or delayed growth. In summary, this study describes the combinatorial antibacterial effects of microbial biomolecules, which may have ecological relevance by inhibiting cohabiting species, but shall furthermore inspire drug development in the combat of infectious disease.

Topics: Anti-Bacterial Agents; Corynebacterium glutamicum; Depsipeptides; Disinfectants; Drug Combinations; Microbial Sensitivity Tests; Prodigiosin; Serratia marcescens; Surface-Active Agents

The EepR transcription factor positively regulates secondary metabolites and tissue-damaging metalloproteases. To gain insight into mechanisms by which EepR regulates pigment and co-regulated factors, genetic suppressor analysis was performed. Suppressor mutations that restored pigment to the non-pigmented ∆eepR mutant mapped to the hexS ORF. Mutation of hexS also restored haemolysis, swarming motility and protease production to the eepR mutant. HexS is a known direct and negative regulator of secondary metabolites in Serratia marcescens and is a LysR family regulator and an orthologue of LrhA. Here, we demonstrate that HexS directly controls eepR and the serralysin gene prtS. EepR was shown to directly regulate eepR expression but indirectly regulate hexS expression. Together, these data indicate that EepR and HexS oppose each other in controlling stationary phase-associated molecules and enzymes.

Topics: Bacterial Proteins; Depsipeptides; DNA Transposable Elements; Electrophoretic Mobility Shift Assay; Escherichia coli; Flagella; Gene Expression Regulation, Bacterial; Genes, Regulator; Mass Spectrometry; Metalloendopeptidases; Prodigiosin; Secondary Metabolism; Serratia marcescens; Transcription Factors

Swarming motility and hemolysis are virulence-associated determinants for a wide array of pathogenic bacteria. The broad host-range opportunistic pathogen Serratia marcescens produces serratamolide, a small cyclic amino-lipid, that promotes swarming motility and hemolysis. Serratamolide is negatively regulated by the transcription factors HexS and CRP. Positive regulators of serratamolide production are unknown. Similar to serratamolide, the antibiotic pigment, prodigiosin, is regulated by temperature, growth phase, HexS, and CRP. Because of this co-regulation, we tested the hypothesis that a homolog of the PigP transcription factor of the atypical Serratia species ATCC 39006, which positively regulates prodigiosin biosynthesis, is also a positive regulator of serratamolide production in S. marcescens. Mutation of pigP in clinical, environmental, and laboratory strains of S. marcescens conferred pleiotropic phenotypes including the loss of swarming motility, hemolysis, and severely reduced prodigiosin and serratamolide synthesis. Transcriptional analysis and electrophoretic mobility shift assays place PigP in a regulatory pathway with upstream regulators CRP and HexS. The data from this study identifies a positive regulator of serratamolide production, describes novel roles for the PigP transcription factor, shows for the first time that PigP directly regulates the pigment biosynthetic operon, and identifies upstream regulators of pigP. This study suggests that PigP is important for the ability of S. marcescens to compete in the environment.

Topics: Bacterial Proteins; Depsipeptides; Erythrocytes; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Hemolysis; Hexosyltransferases; Humans; Membrane Proteins; Movement; Mutation; Operon; Prodigiosin; Sequence Homology, Amino Acid; Serratia marcescens; Signal Transduction; Transcription Factors

Serratia marcescens has been known as a temperature-dependent producer of two chemically different exolipids (red pigment prodigiosin and biosurfactant serrawettin W1) in parallel. During genetic investigation of such control mechanisms, mini-Tn 5 insertional mutant Tan1 overproducing these exolipids was isolated. The gene concerning such disregulation was identified as hexS by DNA cloning followed by sequencing and homology analysis of the presumed product with 314 amino-acids. The product HexS was the homologue of HexA of Erwinia carotovora ssp. carotovora and classified as a transcriptional regulator belonging to LysR family. By RT-PCR analysis, the hexS mutant was shown to over-transcribe the pigA gene (the first gene of the pig cluster involved in prodigiosin synthesis) and the swrW gene encoding serrawettin W1 synthetase belonging to the nonribosomal peptide synthetase family. In contrast, transcription of the pswP gene encoding phosphopantetheinyl transferase in Tan1 was in the level of parent strain 274. Purified protein encoded in his(6)-hexS demonstrated binding activity to DNA fragments of the upstream region of pigA and swrW genes and not to that of the pswP gene. S. marcescens strain 274 transformed with a low-copy plasmid carrying hexS demonstrated reduced production of prodigiosin and serrawettin W1, and reduced activity of exoenzymes (protease, chitinase, and DNase) except phospholipase C. Possible generation of virulent S. marcescens by derepression or mutation of the hexS gene in infected tissues or ex vivo environments was suggested.

Topics: Amino Acid Sequence; Base Sequence; Depsipeptides; Down-Regulation; Molecular Sequence Data; Mutagenesis; Prodigiosin; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Serratia marcescens; Transcription Factors

Serratia marcescens mutants defective in production of the red pigment prodigiosin and the biosurfactant serrawettin W1 in parallel were isolated by transposon mutagenesis of strain 274. Cloning of the DNA fragment required for production of these secondary metabolites with different chemical structures pointed out a novel open reading frame (ORF) named pswP. The putative product PswP (230 aa) has the distinct signature sequence consensus among members of phosphopantetheinyl transferase (PPTase) which phosphopantetheinylates peptidyl carrier protein (PCP) mostly integrated in the nonribosomal peptide synthetases (NRPSs) system. Since serrawettin W1 belongs to the cyclodepsipeptides, which are biosynthesized through the NRPSs system, and one pyrrole ring in prodigiosin has been reported as a derivative of L -proline tethered to phosphopantetheinylated PCP, the mutation in the single gene pswP seems responsible for parallel failure in production of prodigiosin and serrawettin W1.

Topics: Bacterial Proteins; Depsipeptides; Peptides, Cyclic; Prodigiosin; Serratia marcescens; Transferases (Other Substituted Phosphate Groups)