Cyclic AMP negatively regulates prodigiosin production by Serratia marcescens

Abstract

Many Serratia marcescens strains produce the red pigment prodigiosin, which has antimicrobial and anti-tumor properties. Previous reports suggest that cyclic AMP (cAMP) is a positive regulator of prodigiosin production. Supporting this model, the addition of glucose to growth medium inhibited pigment production in rich and minimal media. Unexpectedly, we observed highly elevated levels of prodigiosin production in isogenic strains with mutations in genes involved in cAMP production (cyaA and crr) and in cAMP-dependent transcriptional signaling (crp). Multicopy expression of the Escherichia coli cAMP-phosphodiesterase gene, cpdA, also conferred a striking increase in prodigiosin production. Exogenous cAMP decreased both pigment production and pigA-lacZ transcription in the wild-type (WT) strain, and pigA-lacZ transcription was significantly increased in a crp mutant relative to WT. Suppressor and epistasis analysis indicate that the hyperpigment phenotype was dependent upon pigment biosynthetic genes (pigA, pigB, pigC, pigD and pigM). These experiments establish cAMP as a negative regulator of prodigiosin production in S. marcescens.

Fig. 1

Mutation of crp confers a hyperpigment phenotype and prodigiosin production is inhibited by glucose. (Top) Growth and prodigiosin production by WT (fimC-4) and an isogenic crp (crp-1 fimC-2) mutant strain in LB medium. The fimC mutation is used to maintain a homogenous culture, as crp mutants aggregate and form robust biofilms on test tubes that could complicate growth analysis. The average of 4 independent replicates per strain are shown. (Bottom) Growth and prodigiosin production by WT (fimC-4) and an isogenic crp (crp-1 fimC-2) mutant strain in LB medium supplemented with 2% glucose. The averages of 4 independent replicates per strain are shown. These experiments were performed on different days with the same trend.

Fig. 2

Exogenous cAMP inhibits prodigiosin production, whereas multicopy expression of a bona fide cAMP-PDE gene confers a hyperpigment phenotype. (A) Exogenous cAMP in LB medium elicits a dose-dependent reduction in prodigiosin production from the WT strain. Prodigiosin levels normalized by culture density are shown as a percentage of WT without exogenous cAMP. This experiment shows the average of 11-12 independent replicates per cAMP concentration from three experiments performed on different days. (B) Prodigiosin levels with respect to culture density in the presence or absence of cAMP (10 mM) in LB medium. Prodigiosin production by the cyaA, but not crp mutant is sensitive to exogenous cAMP. This experiment shows the average of 6 independent replicates per genotype; the experiment was performed two times on different days. (C) Phosphodiesterase (PDE) activity from crude lysates of a cpdA mutant of E. coli with an empty pBBR1-based vector, the vector expressing WT cpdA from E. coli (pcpdA), or a mutant version (pcpdA-1) using bis-pNPP as a substrate. The WT cpdA gene confers PDE activity, whereas the pcpdA-1 mutant confers an intermediate phenotype, indicating partial function. The data are from 10-12 independent replicates from two separate experiments performed on different days. Experiments were performed in LB medium. (D) Prodigiosin production by WT S. marcescens bearing an empty pBBR1-based vector, the vector expressing WT cpdA from the E. coli P_lac promoter (pcpdA), or a partial-function mutant version (pcpdA-1). The cpdA gene confers a hyperpigment phenotype to the WT, and the pcpdA-1 mutant confers an intermediate phenotype. The data are from n≥10 independent replicates from two separate experiments performed on different days. Experiments were performed in LB medium. Asterisk = statistically significant difference from prodigiosin levels achieved without cAMP (p<0.05) by one-way ANOVA with the Tukey post-test. Error bars = one standard deviation.

Fig. 3

A chromosomal pigA-lacZ fusion is regulated by CRP and exogenous cAMP. (A) Exogenous cAMP in LB medium elicits a dose-dependent reduction in chromosomal pigA-lacZ ß-galactosidase activity at A₆₀₀=5.5 in a WT background. This experiment shows the average of 12 independent replicates per cAMP concentration, performed on two different days. (B) ß-galactosidase activity as expressed from the chromosomal pigA promoter with respect to culture density in LB medium. This experiment shows the average of 3 independent replicates per genotype, the experiment was performed two times on different days with similar results. Experiments were performed in LB medium. (C) Competitive EMSA to determine the ability of unlabeled DNA to compete with His₈-CRP-flhD interactions. Biotin-labeled flhD promoter DNA (1 ng) was incubated with His₈-CRP at 0 ng (lane 1) or 50 ng (lanes 2-7) per reaction. Unlabeled promoter fragments of flhD (lanes 3-4) and pigA (lanes 5-7) were incubated in the specified binding reactions at 0 ng (lanes 1-2), 50 ng (lane 3), 200 ng (lanes 4 - 5), 400 ng (lane 6) or 600 ng (lane 7). The asterisk indicates the migration of unbound labeled-flhD promoter DNA, signifying successful competitive inhibition of His₈-CRP-flhD promoter interactions (lanes 3-4). (D) EMSA analysis with crude lysates. Biotin-labeled pigA promoter DNA (2 ng, lanes 1-6) was incubated with crude lysates from WT (lanes 2-3, 10 μg of protein) or crp-23 cultures (lanes 5-6, 8 μg of protein). Lanes 1 and 4 have no protein added. Unlabeled pigA promoter DNA (1100 ng) was added (lanes 3 and 6) to compete for binding. Asterisk = statistically significant difference from prodigiosin levels achieved without cAMP (p<0.05) by one-way ANOVA with the Tukey post-test. Error bars = one standard deviation.