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, β-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.
Our understanding of the molecular mechanisms behind bacteria-phage interactions remains limited. Here we report that a small protein, SrpA, controls core cellular processes in response to phage infection and environmental signals in Pseudomonas aeruginosa. We show that SrpA is essential for efficient genome replication of phage K5, and controls transcription by binding to a palindromic sequence upstream of the phage RNA polymerase gene. We identify potential SrpA-binding sites in 66 promoter regions across the P. aeruginosa genome, and experimentally validate direct binding of SrpA to some of these sites. Using transcriptomics and further experiments, we show that SrpA, directly or indirectly, regulates many cellular processes including cell motility, chemotaxis, biofilm formation, pyocyanin synthesis and protein secretion, as well as virulence in a Caenorhabditis elegans model of infection. Further research on SrpA and similar proteins, which are widely present in many other bacteria, is warranted.
Prodigiosin, a red linear tripyrrole pigment normally secreted by Serratia marcescens, 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 BVG90_13250 (rcsB) negatively controlled prodigiosin biosynthesis in S. marcescens. Disruption of rcsB conferred a remarkably increased production of prodigiosin. This phenotype corresponded to RcsB negative control of transcription of the prodigiosin-associated pig operon 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 in S. marcescens. Collectively, this work proposes that RcsB is a prodigiosin synthesis repressor in S. marcescens and provides insight into the regulatory mechanism of RcsB on cell motility, capsular polysaccharide production, and acid resistance in S. marcescens. Importance RcsB is a two-component response regulator in the Rcs phosphorelay system, and plays versatile regulatory functions in Enterobacteriaceae. However, information on the function of the RcsB protein in bacteria, especially in S. marcescens remains limited. In this work, we illustrated experimentally that the RcsB protein was involved in diverse cellular processes in S. marcescens, including prodigiosin synthesis, cell motility, capsular polysaccharide production, biofilm formation, and acid resistance. Additionally, the regulatory mechanism of the RcsB protein on 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 RcsB protein in S. marcescens.
The underlying mechanisms of phage-host interactions largely remained to be elucidated. In this work, Pseudomonas aeruginosa phage C11 was first characterized as a Myoviridae virus having a linear dsDNA molecule of 94109 bp with 1173 bp identical terminal direct repeats (TDR). Then the mutants resistant to phage C11 were screened in a Tn5G transposon mutant library of P. aeruginosa PAK, including two mutants with decreased adsorption rates (DAR) and five mutants with wild-type adsorption rates (WAR). When the WAR mutants were incubated with phage C11, their growth rates were significantly inhibited; the replication of the phage genomic DNA was detected in all the WAR mutants with the real-time quantitative PCR analysis; and the synthesized phage genomic DNA was processed into monomers for packaging evidenced by the southern blot analysis. Moreover, with strain PAK as indicator, small quantities of phage C11 were synthesized in the WAR mutants. Taken together, these data suggested the identified genes of the WAR mutants are necessary for efficient synthesis of the infectious phage particles. Finally, the WAR mutants were detected sensitive to two other Pseudomonas phages closely related with C11, further implying the evolved diversity and complexity of the phage-host interactions in both sides.
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