The Kiss of Death: Serratia marcescens Antibacterial Activities against Staphylococcus aureus Requires Both de novo Prodigiosin Synthesis and Direct Contact.
ABSTRACT: Prodigiosin possesses antibacterial activities, but as a highly hydrophobic compound, it raised the question about how Serratia marcescens introduce this compound to other microbes. Here, we demonstrate that the production of prodigiosin by newly isolated S. marcescens RH10 correlates with its antibacterial activity against a multidrug-resistant strain of S. aureus, with this pathogen's viability decreasing 6-log over 24 h. While S. marcescens RH10 does secrete membrane vesicles that carry prodigiosin, this antibiotic was not active in this form, with 5 mg/L prodigiosin leading to only a 1.22-fold reduction in the S. aureus viability while the same quantity of purified prodigiosin led to a 2800-fold reduction. Contact assays, however, showed increased activity, with a 3-log loss in the S. aureus viabilities in only 6 h as long as de novo production of prodigiosin occurred. The role of prodigiosin was confirmed further by generating an isogenic ΔpigA mutant in S. marcescens RH10, based on the draft genome sequence reported here, to inhibit the synthesis of prodigiosin. In all experiments performed, this mutant was unable to kill S. aureus. Finally, the possibility that the type VI secretion system present in S. marcescens may also be important was also explored as it is known to be used by this strain to kill other microbes. The results here, however, found no obvious activity against S. aureus. In conclusion, the results presented here show prodigiosin requires both cell-to-cell contact and de novo synthesis for it to be effective as an antibiotic for its native host. IMPORTANCE The antibacterial activities of prodigiosin are well-established but, as a hydrophobic molecule, the mechanisms used to introduce it to susceptible microbes has never been studied. We found here, in contrast to violacein, another hydrophobic antibiotic that can be transferred using membrane vesicles (MVs), prodigiosin is also carried from Serratia marcescens in MVs released but its resulting activities were severely mitigated compared to the freely added compound, suggesting it is more tightly bound to the MVs than violacein. This led us to hypothesize that cell-to-cell contact is needed, which we demonstrate here. As well, we show de novo synthesis of prodigiosin is needed for it to be effective. As violacein- and prodigiosin-producing bacterial strains are both beneficial to amphibians, where they help protect the skin against pathogens, the findings presented here provide an important ecological perspective as they show the mechanisms used differ according to the antibacterial produced.
Project description:<i>Serratia marcescens</i> is a bacterial species widely found in the environment, which very efficiently colonizes mosquitoes. In this study, we isolated a red-pigmented <i>S. marcescens</i> strain from our mosquito colony (called <i>S. marcescens</i> VA). This red pigmentation is caused by the production of prodigiosin, a molecule with antibacterial properties. To investigate the role of prodigiosin on mosquito-<i>S. marcescens</i> interactions, we produced two white mutants of <i>S. marcescens</i> VA by random mutagenesis. Whole genome sequencing and chemical analyses suggest that one mutant has a nonsense mutation in the gene encoding prodigiosin synthase, while the other one is deficient in the production of several types of secondary metabolites including prodigiosin and serratamolide. We used our mutants to investigate how <i>S. marcescens</i> secondary metabolites affect the mosquito and its microbiota. Our <i>in vitro</i> tests indicated that <i>S. marcescens</i> VA inhibits the growth of several mosquito microbiota isolates using a combination of prodigiosin and other secondary metabolites, corroborating published data. This strain requires secondary metabolites other than prodigiosin for its proteolytic and hemolytic activities. In the mosquito, we observed that <i>S. marcescens</i> VA is highly virulent to larvae in a prodigiosin-dependent manner, while its virulence on adults is lower and largely depends on other metabolites.
Project description:Prodigiosin, a red linear tripyrrole pigment, has long been recognised for its antimicrobial property. However, the physiological contribution of prodigiosin to the survival of its producing hosts still remains undefined. Hence, the aim of this study was to investigate the biological role of prodigiosin from Serratia marcescens, particularly in microbial competition through its antimicrobial activity, towards the growth and secreted virulence factors of four clinical pathogenic bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa) as well as Staphylococcus aureus and Escherichia coli. Prodigiosin was first extracted from S. marcescens and its purity confirmed by absorption spectrum, high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). The extracted prodigiosin was antagonistic towards all the tested bacteria. A disc-diffusion assay showed that prodigiosin is more selective towards Gram-positive bacteria and inhibited the growth of MRSA, S. aureus and E. faecalis and Gram-negative E. coli. A minimum inhibitory concentration of 10 μg/μL of prodigiosin was required to inhibit the growth of S. aureus, E. coli and E. faecalis whereas > 10 μg/μL was required to inhibit MRSA growth. We further assessed the effect of prodigiosin towards bacterial virulence factors such as haemolysin and production of protease as well as on biofilm formation. Prodigiosin did not inhibit haemolysis activity of clinically associated bacteria but was able to reduce protease activity for MRSA, E. coli and E. faecalis as well as decrease E. faecalis, Salmonella Typhimurium and E. coli biofilm formation. Results of this study show that in addition to its role in inhibiting bacterial growth, prodigiosin also inhibits the bacterial virulence factor protease production and biofilm formation, two strategies employed by bacteria in response to microbial competition. As clinical pathogens were more resistant to prodigiosin, we propose that prodigiosin is physiologically important for S. marcescens to compete against other bacteria in its natural soil and surface water environments.
Project description:The focal intent of this study was to find out an alternative strategy for the antibiotic usage against bacterial infections. The quorum sensing inhibitory (QSI) activity of marine sponges collected from Palk Bay, India was evaluated against acyl homoserine lactone (AHL) mediated violacein production in Chromobacterium violaceum (ATCC 12472), CV026 and virulence gene expressions in clinical isolate Serratia marcescens PS1. Out of 29 marine sponges tested, the methanol extracts of Aphrocallistes bocagei (TS 8), Haliclona (Gellius) megastoma (TS 25) and Clathria atrasanguinea (TS 27) inhibited the AHL mediated violacein production in C. violaceum (ATCC 12472) and CV026. Further, these sponge extracts inhibited the AHL dependent prodigiosin pigment, virulence enzymes such as protease, hemolysin production and biofilm formation in S. marcescens PS1. However, these sponge extracts were not inhibitory to bacterial growth, which reveals the fact that the QSI activity of these extracts was not related to static or killing effects on bacteria. Based on the obtained results, it is envisaged that the marine sponges could pave the way to prevent quorum sensing (QS) mediated bacterial infections.
Project description:Prodigiosin is a red pigment produced by <i>Serratia marcescens</i> with anticancer, antimalarial, and antibacterial effects. In this study, we extracted and identified a red pigment from a culture of <i>S. marcescens</i> strain ZPG19 and investigated its effect on the growth performance and intestinal microbiota of Kunming mice. High-performance liquid chromatography/mass spectrometry revealed that the pigment had a mass-to-charge ratio (<i>m</i>/<i>z</i>) of 324.2160, and thus it was identified as prodigiosin. To investigate the effect of prodigiosin on the intestinal microbiota, mice (n = 5) were administered 150 μg/kg/d prodigiosin (crude extract, 95% purity) via the drinking water for 18 days. Administration of prodigiosin did not cause toxicity in mice. High-throughput sequencing analysis revealed that prodigiosin altered the cecum microbiota abundance and diversity; the relative abundance of <i>Desulfovibrio</i> significantly decreased, whereas <i>Lactobacillus reuteri</i> significantly increased. This finding indicates that oral administration of prodigiosin has a beneficial effect on the intestinal microbiota of mice. As prodigiosin is non-toxic to mouse internal organs and improves the mouse intestinal microbiota, we suggest that it is a promising candidate drug to treat intestinal inflammation.
Project description: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.
Project description:Prodigiosin, a secondary metabolite produced by Serratia marcescens, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, information on the regulatory mechanism behind prodigiosin biosynthesis in S. marcescens remains limited. In this work, a prodigiosin-hyperproducing strain with the BVG90_22495 gene disrupted (ZK66) was selected from a collection of Tn5G transposon insertion mutants. Using real-time quantitative PCR (RT-qPCR) analysis, ?-galactosidase assays, transcriptomics analysis, and electrophoretic mobility shift assays (EMSAs), the LysR-type regulator MetR encoded by the BVG90_22495 gene was found to affect prodigiosin synthesis, and this correlated with MetR directly binding to the promoter region of the prodigiosin-synthesis positive regulator PigP and hence negatively regulated the expression of the prodigiosin-associated pig operon. More analyses revealed that MetR regulated some other important cellular processes, including methionine biosynthesis, cell motility, H2O2 tolerance, heat tolerance, exopolysaccharide synthesis, and biofilm formation in S. marcescens Although MetR protein is highly conserved in many bacteria, we report here on the LysR-type regulator MetR exhibiting novel roles in negatively regulating prodigiosin synthesis and positively regulating heat tolerance, exopolysaccharide synthesis, and biofilm formation.IMPORTANCE Serratia marcescens, a Gram-negative bacterium, is found in a wide range of ecological niches and can produce several secondary metabolites, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin shows diverse functions as an immunosuppressant, antimicrobial, and anticancer agent. However, the regulatory mechanisms behind prodigiosin synthesis in S. marcescens are not completely understood. Here, we adapted a transposon mutant library to identify the genes related to prodigiosin synthesis, and the BVG90_22495 gene encoding the LysR-type regulator MetR was found to negatively regulate prodigiosin synthesis. The molecular mechanism of the metR mutant hyperproducing prodigiosin was investigated. Additionally, we provided evidence supporting new roles for MetR in regulating methionine biosynthesis, cell motility, heat tolerance, H2O2 tolerance, and exopolysaccharide synthesis in S. marcescens Collectively, this work provides novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers novel roles for the highly conserved MetR protein in regulating prodigiosin synthesis, heat tolerance, exopolysaccharide (EPS) synthesis, and biofilm formation.
Project description:Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for natural compound discovery and combinatorial biosynthesis.
Project description:Serratia marcescens, a gram-negative bacterium, found in a wide range of ecological niches can produce several high-value products, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, the regulatory mechanisms behind prodigiosin synthesis in Serratia marcescens remains limited. Here, a transposon mutant library was constructed to identify the genes related to prodigiosin synthesis, and BVG90_02415 gene encoding a peptidoglycan synthesizing enzyme D-Ala-D-Ala carboxypeptidase DacA was found to negatively regulates prodigiosin synthesis. Quantitative measurements revealed that disruption of dacA increased prodigiosin production 1.46-fold that of the wild-type strain JNB5-1 in fermentation medium. By comparing differences in cell growth, pigA gene expression level, cell morphology, membrane permeability, and intracellular prodigiosin concentration between wild-type strain JNB5-1 and dacA mutant SK4-72, results revealed that the mechanism for hyper-producing of prodigiosin by the dacA mutant was probably that dacA disruption enhanced prodigiosin leakage, which in turn alleviated feedback inhibition of prodigiosin and increased expression of pig gene cluster. Collectively, this work provides a novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers new roles of DacA protein in regulating cell growth, cell morphology, and membrane permeability in Serratia marcescens. Finally, this study offers a new strategy for improving production of high-value compounds in Serratia marcescens.
Project description:The emergence of bacterial strains resistant to different antibiotics has prompted the search for new sources of antimicrobial compounds. Studies have shown that jambolan [Syzygium cumini (L.) Skeels], a tropical fruit from the Mirtaceae family, contains a great variety of phytochemical compounds with high antioxidant and antimicrobial activity. This study aimed to determine the centesimal composition and physicochemical characteristics of the pulp and seed of S. cumini (L.) Skeels, as well as the content of total phenolic compounds and the antioxidant, antibacterial, antibiofilm and anti-quorum sensing (QS) activities of the phenolic extracts obtained from the pulp and the seeds of this fruit. The in vitro antibacterial and anti-QS activities of active films incorporating phenolic extracts were also evaluated. Additionally, we performed molecular docking of phenolic compounds present in jambolan with the CviR QS regulator of Chromobacterium violaceum. The composition and physicochemical characteristics of the samples presented similar values to those found for the species. However, the seed phenolic extract had a higher content of phenolic compounds and antioxidant activity than the pulp. Both phenolic extracts presented antibacterial activity against Aeromonas hydrophila, C. violaceum, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium, Serratia marcescens, Listeria monocytogenes, and Staphylococcus aureus. The seed phenolic extract was particularly inhibitory against S. aureus. The pulp phenolic extract inhibited swarming motility and biofilm formation of A. hydrophila, E. coli, and S. marcescens in sub-MIC concentrations. The pulp and seed phenolic extracts inhibited violacein production in C. violaceum. Films incorporating both phenolic extracts inhibited the growth of bacteria, particularly Pseudomonas fluorescens, L. monocytogenes, and S. aureus, as well as QS in C. violaceum. Molecular docking showed that a variety of compounds found in pulp and seed extracts of jambolan, particularly chlorogenic acid and dihydroquercetin, potentially bind CviR protein and may interfere with QS. Our results indicate that pulp and seed of jambolan are good sources of antibacterial, antibiofilm, and anti-QS compounds that can be used in the development of natural preservatives and for application in antibacterial active films.
Project description:Serratia marcescens is a Gram-negative bacterium with both environmental and host-associated strains. Pigmentation is reportedly inversely correlated with infection frequency, and prodigiosin is one of Serratia pigments that has medical and industrial applications. Here, we report the draft genome sequence of prodigiosin-hyperproducing Serratia marcescens strain N2, isolated from Cairo, Egypt. The sequence is assembled into 142 contigs, with a combined size of 5,570,793 bp. The assembled genome carries typical S. marcescens genes, with potential prodigiosin-biosynthesizing genes detected.