Mutation of crp mediates Serratia marcescens serralysin and global secreted protein production.

The bacterial species Serratia marcescens secretes both beneficial and cytotoxic proteins. Here we report that a crp mutant exhibited elevated secreted protease activity. A genetic screen revealed that the gene coding for the metalloprotease serralysin was necessary for the elevated proteolysis, and this was confirmed by western blot analysis. Proteomic analysis of secreted proteins corroborated increased secretion of serralysin protease by crp mutants compared to the wild type. The crp-mutant-secreted fractions also contained less chitinase and chitin binding protein. These data support the hypothesis that cAMP-CRP is an upstream indirect regulator of serralysin production and they provide novel insight into the S. marcescens secretome.

Serratia marcescens quinoprotein glucose dehydrogenase activity mediates medium acidification and inhibition of prodigiosin production by glucose.

Serratia marcescens is a model organism for the study of secondary metabolites. The biologically active pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), like many other secondary metabolites, is inhibited by growth in glucose-rich medium. Whereas previous studies indicated that this inhibitory effect was pH dependent and did not require cyclic AMP (cAMP), there is no information on the genes involved in mediating this phenomenon. Here we used transposon mutagenesis to identify genes involved in the inhibition of prodigiosin by glucose. Multiple genetic loci involved in quinoprotein glucose dehydrogenase (GDH) activity were found to be required for glucose inhibition of prodigiosin production, including pyrroloquinoline quinone and ubiquinone biosynthetic genes. Upon assessing whether the enzymatic products of GDH activity were involved in the inhibitory effect, we observed that d-glucono-1,5-lactone and d-gluconic acid, but not d-gluconate, were able to inhibit prodigiosin production. These data support a model in which the oxidation of d-glucose by quinoprotein GDH initiates a reduction in pH that inhibits prodigiosin production through transcriptional control of the prodigiosin biosynthetic operon, providing new insight into the genetic pathways that control prodigiosin production. Strains generated in this report may be useful in large-scale production of secondary metabolites.

The LysR Transcription Factor, HexS, Is Required for Glucose Inhibition of Prodigiosin Production by Serratia marcescens.

Generation of many useful microbe-derived secondary metabolites, including the red pigment prodigiosin of the bacterium Serratia marcescens, is inhibited by glucose. In a previous report, a genetic approach was used to determine that glucose dehydrogenase activity (GDH) is required for inhibiting prodigiosin production and transcription of the prodigiosin biosynthetic operon (pigA-N). However, the transcription factor(s) that regulate this process were not characterized. Here we tested the hypothesis that HexS, a LysR-family transcription factor similar to LrhA of Escherichia coli, is required for inhibition of prodigiosin by growth in glucose. We observed that mutation of the hexS gene in S. marcescens allowed the precocious production of prodigiosin in glucose-rich medium conditions that completely inhibited prodigiosin production by the wild type. Unlike previously described mutants able to generate prodigiosin in glucose-rich medium, hexS mutants exhibited GDH activity and medium acidification similar to the wild type. Glucose inhibittion of pigA expression was shown to be dependent upon HexS, suggesting that HexS is a key transcription factor in secondary metabolite regulation in response to medium pH. These data give insight into the prodigiosin regulatory pathway and could be used to enhance the production of secondary metabolites.

Cyclic AMP negatively regulates prodigiosin production by Serratia marcescens.

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.