Suppressor analysis of eepR mutant defects reveals coordinate regulation of secondary metabolites and serralysin biosynthesis by EepR and HexS

Abstract

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.

Fig. 1.

Genetic screen for eepR suppressor mutations. Transposon mutations were introduced into the CMS2097 strain (∆eepR) to find pigmented suppressor mutants. (a) A portion of one plate is shown with one red suppressor (reep) mutant (black arrow) visible among the pigmentless ∆eepR colonies. (b) Location of ∆eepR suppressor mutations (vertical arrows) in the hexS gene (horizontal bar). Of the 12 insertions, 8 are shown, the other 4 are in the hexS ORF but not mapped. (c) Prodigiosin pigmentation of strains grown on LB agar for 20 h at 30 °C. WT refers to parental strain PIC3611; eepR, to CMS2097; hexS, to CMS2922; eepR hexS, to CMS2204; Nima, to parental strain CMS1787; Nima eepR, to CMS2089; and Nima eepR hexS, to CMS2320.

Fig. 2.

Genetic evidence suggests coordinated swarming and haemolysis regulation by EepR and HexS. (a) Swarming motility after incubation for 20–48 h on LB medium with 0.6 % agar. (b) Haemolysis phenotype after 4 days of growth on tryptic soy agar +5 % sheep erythrocytes.

Fig. 3.

Growth and MS analysis of serratamolide production by the eepR hexS double mutant. (a) Growth curve analysis of the wild-type strain PIC3611, ∆eepR, ∆hexS and ∆eepR ∆hexS in LB medium. (b) MS analysis of serratamolide biosynthesis of cultures grown for 24 h and normalized to OD₆₀₀ 2.0. Means and standard deviations are shown, n=3 independent samples. PIC3611-derived strains were used.

Fig. 4.

Genetic evidence suggests coordinated protease regulation by EepR and HexS. Protease activity in supernatants from stationary-phase bacterial cultures normalized to OD₆₀₀ 2.0. Azocasein was used as a colorimetric protease substrate. Means and standard deviations are shown, n=3 independent experiments, each with three biological replicates. (a) Protease activity from the PIC3611 background. (b) Protease activity from the Nima strain background.

Fig. 5.

Genes regulated by HexS and EepR. (a–d) qRT-PCR analysis of gene expression using RNA from strains grown to OD₆₀₀ 3. (a, b) Analysis of eepR and prtS expression in WT compared to the ∆hexS mutant; (c, d) analysis of hexS and eepR expression in WT compared to the ∆eepR mutant. Gene expression was determined using PIC3611-derived strains. The phexS plasmid refers to pMQ294. Means and standard deviations are shown. At least three independent replicates were used for each experiment.

Fig. 6.

EepR and HexS promoter binding analyses. EMSA analysis with biotinylated promoter DNA. Labelled promoter DNA was used in each reaction at 2 ng per reaction. Specific promoters are noted in the upper left-hand corner of each panel. MBP and MBP–HexS were used at 38 µM and 28 µM, respectively. MBP–EepR was used at 17.5 µM (*) or 35 µM (+); 500 ng of poly-dIdC was added per reaction to prevent non-specific protein–DNA interactions, and unlabelled promoters were used at 500 ng. –, indicates no addition of a particular reagent. (a) Representative EMSA using recombinant MBP or MBP–HexS. (b) Representative EMSA using recombinant MBP or MBP–EepR. Each EMSA experiment had a consistent result in at least three independent experiments.

Fig. 7.

Multicopy expressions of lhrA and hexS inhibit prodigiosin biosynthesis and flhDC expression in the hexS mutant. (a) Prodigiosin pigmentation of strains grown on LB agar with kanamycin for 20 h at 30 °C. Plasmids with the hexS or lhrA genes under control of the P_lac promoter inhibit pigmentation in both the wild-type and the ∆hexS mutant. Experiments were performed with the PIC3611 strain background and vector indicates pMQ131. (b) β-Galactosidase activity produced by WT and ∆hexS strains bearing a plasmid-borne flhDC-lacZ transcriptional reporter after growth in LB medium to OD₆₀₀ 3. A representative experiment with three independent biological replicates is shown. Mean and standard deviation is shown. (c) Model, described in Discussion, for coordinated regulation of secondary metabolite biosynthetic genes (pigA-N and swrW) and the prtS protease gene by EepR and HexS. The line between EepR and HexS indicates that each inhibits transcription of the other.