LysR-Type Transcriptional Regulator MetR Controls Prodigiosin Production, Methionine Biosynthesis, Cell Motility, H2O2 Tolerance, Heat Tolerance, and Exopolysaccharide Synthesis in Serratia marcescens.
ABSTRACT: 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: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.
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 (PG), a red linear tripyrrole pigment normally secreted by <i>Serratia marcescens</i>, has received attention for its reported immunosuppressive, antimicrobial, and anticancer properties. Although several genes have been shown to be important for prodigiosin synthesis, information on the regulatory mechanisms behind this cellular process remains limited. In this work, we identified that the transcriptional regulator RcsB encoding gene <i>BVG90_13250</i> (<i>rcsB</i>) negatively controlled prodigiosin biosynthesis in <i>S. marcescens</i> Disruption of <i>rcsB</i> conferred a remarkably increased production of prodigiosin. This phenotype corresponded to negative control of transcription of the prodigiosin-associated <i>pig</i> operon by RcsB, probably by binding to the promoter region of the prodigiosin synthesis positive regulator FlhDC. Moreover, using transcriptomics and further experiments, we revealed that RcsB also controlled some other important cellular processes, including swimming and swarming motilities, capsular polysaccharide production, biofilm formation, and acid resistance (AR), in <i>S. marcescens</i> Collectively, this work proposes that RcsB is a prodigiosin synthesis repressor in <i>S. marcescens</i> and provides insight into the regulatory mechanism of RcsB in cell motility, capsular polysaccharide production, and acid resistance in <i>S. marcescens</i> <b>IMPORTANCE</b> RcsB is a two-component response regulator in the Rcs phosphorelay system, and it plays versatile regulatory functions in <i>Enterobacteriaceae</i> However, information on the function of the RcsB protein in bacteria, especially in <i>S. marcescens</i>, remains limited. In this work, we illustrated experimentally that the RcsB protein was involved in diverse cellular processes in <i>S. marcescens</i>, including prodigiosin synthesis, cell motility, capsular polysaccharide production, biofilm formation, and acid resistance. Additionally, the regulatory mechanism of the RcsB protein in these cellular processes was investigated. In conclusion, this work indicated that RcsB could be a regulator for prodigiosin synthesis and provides insight into the function of the RcsB protein in <i>S. marcescens</i>.
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: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.
Project description:The genome of Streptococcus mutans encodes 4 LysR-type transcriptional regulators (LTTRs), three of which, MetR, CysR (cysteine synthesis regulator), and HomR (homocysteine synthesis regulator), are phylogenetically related. MetR was previously shown to control methionine metabolic gene expression. Functional analysis of CysR and HomR was carried out by phenotypical studies and transcriptional analysis. CysR is required to activate the transcription of cysK encoding the cysteine biosynthesis enzyme, tcyABC and gshT genes encoding cysteine and glutathione transporter systems, and homR. HomR activates the transcription of metBC encoding methionine biosynthesis enzymes, tcyDEFGH involved in cysteine transport, and still uncharacterized thiosulfate assimilation genes. Control of HomR by CysR provides evidence of a cascade regulation for sulfur amino acid metabolism in S. mutans. Two conserved motifs were found in the promoter regions of CysR and HomR target genes, suggesting their role in the regulator binding recognition site. Both CysR and HomR require O-acetylserine to activate transcription. A global sulfur amino acid supply gene regulatory pathway is proposed for S. mutans, including the cascade regulation consequent to transcriptional activation of HomR by CysR. Phylogenetic study of MetR, CysR, and HomR homologues and comparison of their potential regulatory patterns among the Streptococcaceae suggest their rapid evolution.
Project description:<i>Serratia marcescens</i> is a common bacterium well-known for the red secondary metabolite prodigiosin. However, color mutants have long been described. Non-pigmented strains can be found to exist both naturally and under laboratory conditions. It is unclear why <i>S. marcescens</i> loses prodigiosin synthesis capacity in certain conditions. In the present study, we find that the spontaneous color mutants arise within a few generations (about five passages) and rapidly replace the wild-type parent cells (about 24 passages), which indicates a growth advantage of the former. Although, the loss of prodigiosin synthesis genes (<i>pigA-N</i>) is frequently reported as the major reason for pigment deficiency, it was unexpected that the whole gene cluster is completely preserved in the different color morphotypes. Comparative transcriptomic analysis indicates a dramatic variation at the transcriptional level. Most of the <i>pig</i> genes are significantly downregulated in the color morphotypes which directly lead to prodigiosin dyssynthesis. Besides, the transcriptional changes of several other genes have been noticed, of which transcriptional regulators, membrane proteins, and nearly all type VI secretion system (T6SS) components are generally downregulated, while both amino acid metabolite and transport systems are activated. In addition, we delete the transcription regulator <i>slyA</i> to generate a non-pigmented mutant. The Δ<i>slyA</i> strain loses prodigiosin synthesis capacity, but has a higher cell density, and surprisingly enhances the virulence as an entomopathogen. These data indicate that <i>S. marcescens</i> shuts down several high-cost systems and activates the amino acid degradation and transport pathways at the transcriptional level to obtain extra resources, which provides new insights into the competitive growth advantage of bacterial spontaneous color mutants.
Project description:RNAseq analysis was performed to evaluate gene expression differences between strains JNB5-1 and ZK66. S. marcescens JNB5-1 and ZK66 cells were grown in LB medium for 12 h before harvesting. The collected cells were then treated with RNAprep pure Kit (TIANGEN) to extract total bacterial RNA and delivered to GENEWIZ in dry ice for transcriptome resequencing analysis. For annotation, the genome of S. marcescens WW4 (NC_020211.1) was used as reference. A total of 24048918 reads matched to the referenced genome in the sample of JNB5-1, and 24569696 reads in the sample of ZK66. The differentially expressed genes (DEGs) were determined between strains JNB5-1 and ZK66 with the standards of false discovery rate (FDR) ≤ 0.05, fold change |log2Ratio|≥1. Transcriptome data showed that expression of 641 genes were upregulated while 784 genes were down regulated by at least 2-fold when comparing the metR mutant strain ZK66 to its parent strain JNB5-1. Based on the annotation of KEGG_B_class, the up-regulated and down-regulated genes were classified into 30 and 27 major cellular processes, respectively. The transcriptome data indicated that MetR may regulate a variety of cellular processes in S. marcescens, including prodigiosin synthesis, and cell motility. Overall design: Gene expression of strains JNB5-1 and ZK66 were generated by deep sequencing, in triplicate, using Illumina HiSeq X10.
Project description:Prodigiosin is an important secondary metabolite produced by Serratia marcescens. It can help strains resist stresses from other microorganisms and environmental factors to achieve self-preservation. Prodigiosin is also a promising secondary metabolite due to its pharmacological characteristics. However, pigmentless S. marcescens mutants always emerge after prolonged starvation, which might be a way for the bacteria to adapt to starvation conditions, but it could be a major problem in the industrial application of S. marcescens. To identify the molecular mechanisms of loss of prodigiosin production, two mutants were isolated after 16 days of prolonged incubation of wild-type (WT) S. marcescens 1912768R; one mutant (named 1912768WR) exhibited reduced production of prodigiosin, and a second mutant (named 1912768W) was totally defective. Comparative genomic analysis revealed that the two mutants had either mutations or deletions in rpoS. Knockout of rpoS in S. marcescens 1912768R had pleiotropic effects. Complementation of rpoS in the ?rpoS mutant further confirmed that RpoS was a positive regulator of prodigiosin production and that its regulatory role in prodigiosin biosynthesis was opposite that in Serratia sp. ATCC 39006, which had a different type of pig cluster; further, rpoS from Serratia sp. ATCC 39006 and other strains complemented the prodigiosin defect of the ?rpoS mutant, suggesting that the pig promoters are more important than the genes in the regulation of prodigiosin production. Deletion of rpoS strongly impaired the resistance of S. marcescens to stresses but increased membrane permeability for nutritional competence; competition assays in rich and minimum media showed that the ?rpoS mutant outcompeted its isogenic WT strain. All these data support the idea that RpoS is pleiotropic and that the loss of prodigiosin biosynthesis in S. marcescens 1912768R during prolonged incubation is due to a mutation in rpoS, which appears to be a self-preservation and nutritional competence (SPANC) trade-off.
Project description: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 <i>de novo</i> production of prodigiosin occurred. The role of prodigiosin was confirmed further by generating an isogenic Δ<i>pigA</i> 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 <i>de novo</i> synthesis for it to be effective as an antibiotic for its native host. <b>IMPORTANCE</b> 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 <i>de novo</i> 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.