Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites

Abstract

Serratia marcescens generates secondary metabolites and secreted enzymes, and it causes hospital infections and community-acquired ocular infections. Previous studies identified cyclic AMP (cAMP) receptor protein (CRP) as an indirect inhibitor of antimicrobial secondary metabolites. Here, we identified a putative two-component regulator that suppressed crp mutant phenotypes. Evidence supports that the putative response regulator eepR was directly transcriptionally inhibited by cAMP-CRP. EepR and the putative sensor kinase EepS were necessary for the biosynthesis of secondary metabolites, including prodigiosin- and serratamolide-dependent phenotypes, swarming motility, and hemolysis. Recombinant EepR bound to the prodigiosin and serratamolide promoters in vitro. Together, these data introduce a novel regulator of secondary metabolites that directly connects the broadly conserved metabolism regulator CRP with biosynthetic genes that may contribute to competition with other microbes.

Importance: This study identifies a new transcription factor that is directly controlled by a broadly conserved transcription factor, CRP. CRP is well studied in its role to help bacteria respond to the amount of nutrients in their environment. The new transcription factor EepR is essential for the bacterium Serratia marcescens to produce two biologically active compounds, prodigiosin and serratamolide. These two compounds are antimicrobial and may allow S. marcescens to compete for limited nutrients with other microorganisms. Results from this study tie together the CRP environmental nutrient sensor with a new regulator of antimicrobial compounds. Beyond microbial ecology, prodigiosin and serratamolide have therapeutic potential; therefore, understanding their regulation is important for both applied and basic science.

FIG 1

Hyperhemolysis phenotype of crp mutants and genetic analysis. (A) Photograph demonstrating suppression of the crp hemolysis (left) and pigment (right) phenotypes by mutation of eepR and eepS. The eepR and eepS colonies appear red because of the blood agar (left), but their severe lack of pigment phenotype is clear when grown on LB agar (right). The crp pigB mutant is included as a control and only has a defect in pigment production. The crp mutant is strain number CMS613, the crp eepR mutant is CMS795, the crp eepS mutant is CMS1075, and the crp pigB mutant is CMS794. (B) Map of the genetic context of eepR and eepS; ORF numbers are from the Db11 genome, and “SMDB11_” was removed from each ORF number to save space. The asterisk indicates a predicted CRP binding site. (C) Predicted protein domains and amino acid length of EepR. The amino acid location of the predicted phosphorylation site is boxed. (D) Predicted protein domains of EepS. The amino acid locations of predicted phosphorylation sites are boxed.

FIG 2

Direct regulation of eepR by cAMP-CRP. (A) Expression of the eepR promoter measured from a chromosomal lacZ reporter integrated at eepR in a WT and crp mutant background. The WT strain is CMS376, and the crp mutant is CMS786. (B) As described for panel A but at an OD₆₀₀ of 4.0. (C) qPCR analysis of eepR expression from the WT and crp mutant measured at an OD₆₀₀ of 1.5. The WT strain is CMS376, and the crp mutant is CMS1687. (D) EMSA of His₈-CRP interaction with the biotin-labeled eepR predicted promoter (P_eepR; 2 ng) in vitro. His₈-CRP produced a gel shift of labeled P_eepR that could be inhibited by an excess of unlabeled P_eepR (P_eepR-UL) but not by a nonspecific unlabeled amplicon (Nonspecific-UL), a 360-bp internal region of eepR. The gel shift required cAMP; whereas 0 and 0.1 μM did not support binding, 10 μM was sufficient. (E) Chromatin affinity purification of His₈-CRP suggests binding of the eepR promoter in vivo. PCR amplification of the eepR promoter was elevated from ChAP purification of CRP-bound DNA in the WT strain containing a plasmid expressing His₈-CRP (+CRP) compared to the WT strain with the empty vector (−CRP). The flhDC promoter was included as a positive control and the oxyR promoter as a negative control.

FIG 3

Growth and complementation analysis of eepR mutant pigment phenotypes. (A) Prodigiosin extracted and measured from stationary-phase bacteria. WT, CMS376; ΔeepR strain, CMS2097; ΔeepR repaired strain, CMS2921; ΔeepS strain, CMS2701; eepS::Tn strain, CMS1076; eepS::Tn repaired strain, CMS2032; Nima ΔeepR strain, CMS2089; Nima ΔeepS strain, CMS2091; Nima ΔeepR ΔeepS strain, CMS2093; K904 ΔeepR strain, CMS2094; K904 ΔeepS strain, CMS2924. Means and standard deviations are shown (n = 8). An asterisk indicates P < 0.05 by ANOVA with Tukey's posttest. (B) Complementation of the ΔeepR mutant phenotype in K904 and K904 ΔeepR strains using plasmid pMQ369 (peepR) and a vector negative control (pMQ132). (C) Culture pigmentation in the ΔeepR strain (CMS2097) without (left) and with (right) l-arabinose (L-Arab.)-induced expression of the prodigiosin biosynthetic operon.

FIG 4

Regulation of pigA by EepR and epistasis analysis. (A) β-Galactosidase-based expression from the pigA promoter at an OD₆₀₀ of 4. WT, CMS376; crp strain, CMS1687; crp eepR strain, CMS2157; eepR strain, CMS2097; eepR repaired strain, CMS2921. (B) qPCR analysis of the pigA promoter at an OD₆₀₀ of 2. WT, CMS376; crp strain, CMS1687; crp eepR strain, CMS795; eepR strain, CMS2097. (C) EMSA analysis of MBP-EepR interaction with biotin-labeled pigA promoter (P_pigA; 2 ng) in vitro. MBP-EepR produced a gel shift of labeled P_pigA that could be inhibited by an excess of unlabeled P_pigA (P_pigA-UL). Recombinant MBP was not sufficient to produce a gel shift of P_pigA. An asterisk indicates significant difference from the WT by ANOVA with Tukey's posttest.

FIG 5

EepR is necessary for serratamolide and serratamolide-dependent phenotypes. (A) Hemolysis and swarming are EepR dependent in strain K904, but swimming is not. K904 ΔeepR mutant, CMS2904. (B) Swarming motility is defective in the ΔeepR (CMS2097) mutant and could be restored when the chromosomal eepR deletion allele was replaced by the wild-type eepR gene (CMS2921). (C) Hemolysis and swarming phenotypes of the ΔeepR mutant (CMS2097) can be rescued by induced expression of swrW on a plasmid (pswrW/pMQ367) but not by the vector control (pMQ125). (D) Means and standard deviations from surfactant radii around colonies on a swarming agar plate (n ≥ 5 independent isolates). WT, CMS376; swrW strain, CMS635; crp strain, CMS1687; crp swrW strain, CMS2281; eepR strain, CMS2097; crp eepR strain, CMS2701; crp eepS strain, CMS2395. (E) HPLC-MS analysis of serratamolide levels in supernatants from stationary-phase cultures. An asterisk indicates the serratamolide peak. WT, CMS376; pigP strain, CMS2096; eepR strain, CMS2097; swrW strain, CMS635).

FIG 6

Positive regulation of serratamolide biosynthetic gene, swrW, by EepR. (A) β-Galactosidase analysis of swrW expression from cultures at an OD₆₀₀ of 4. Means and standard deviations are shown. An asterisk indicates significant difference from results for the WT by ANOVA with Tukey's posttest. WT, CMS376; eepR strain, CMS2097; eepS strain, CMS1076. (B) Quantitative RT-PCR analysis of swrW expression from cultures at an OD₆₀₀ of 3. Means and standard deviations are shown. An asterisk indicates significant differences by Student's t test. WT, CMS376; eepR strain, CMS2097. (C) EMSA analysis of MBP-EepR interaction with biotin-labeled swrW promoter (P_swrW; 2 ng) in vitro. MBP-EepR produced a gel shift of labeled P_swrW that could be inhibited by an excess of unlabeled P_swrW (P_swrW-UL). Recombinant MBP was not sufficient to produce a gel shift of P_swrW.