A molecular genetic investigation into 2-methyl-3-amyl-6-methoxyprodigiosene (prodigiosin) biosynthesis in Serratia marcescens

Abstract

Serratia marcescens is a Gram-negative enteric bacterium. The distinguishing feature of this species is the production of a bright red, non-diffusible pigment: 2-methyl-3-amyl-6- methoxyprodigiosene (prodigiosin). Prodigiosin is a classical secondary metabolite, produced in late-log to stationary phases of growth. It belongs to a family of structurally related tripyrrolic compounds, produced by a number of prokaryotic genera, which possess anti-bacterial, anti-fungal, anti-protozoal and immunosuppressive properties. The biological function(s) of prodigiosin in Serratia marcescens is unknown. Prodigiosin is synthesised from proline, alanine, serine, methionine, glycine and acetate. Little is know about the biosynthetic pathway, except that it is bifurcated, and terminated in the condensation of a bipyrrole and a monopyrrole to form prodigiosin. Virtually no published information exists on the pathway precursors, the pathway enzymes or the genes encoding them. The aim of this study was to investigate prodigiosin biosynthesis at the genetic level. Thomson (1996), isolated the prodigiosin biosynthetic (pig) gene cluster from a S. marcescens chromosomal DNA library. After subcloning, approximately half of the pig cluster (11.5 Kb) was sequenced in this study. The remainder was sequenced in a parallel study in this laboratory. From both studies, 16 putative open reading frames (ORFs) were identified, which are arranged unidirectionally, with the exception of orf76 at the extreme 3' end of the cluster. In the 5' half of the cluster, sequenced in this study, homologues of bacterial acyl-CoA dehydrogenase, phosphoenolpyruvate synthase and ornithine aminotransferase were identified by similarity. A homologue of a hypothetical protein mapping to the red (undecylprodigiosn) locus of Streptomyces coelicolor A3(2) was also identified by similarity; another putative ORF does not have any database homologues. The pig cluster was randomly mutagenised by using TnphoA'-2, which simultaneously generated some lacZ gene fusions strains. Non-pigmented, hyperpigmented and orange-pigmented mutants were isolated. Cloning and sequencing of transposon insertions from non-pigmented TnphoA'-2 mutated strains revealed that gene fusions to the first and fourth putative ORFs had been obtained. Additionally, it was found that in one non-pigmented mutant strain, a transposon insertion is located in an ORF encoding a putative homologue of the Escherichia coli integral inner-membrane histidine sensor kinase EnvZ; sequence data suggest that a putative homologue of the corresponding response-regulator OmpR is present upstream of the transposon insertion site. Another non-pigmented mutant was found to have a transposon insertion in a putative homologue of Escherichia coli hscA, which encodes a "cold-shock" induced molecular chaperonin. Southern blot analyses showed that insertions in these latter coding regions are external to the pig cluster. Two hitherto unknown loci, which are essential to pigment biosynthesis, were therefore identified in this study. Strains carrying gene fusions in orf1 and orf4 of the pig cluster showed differential LacZ expression under prodiogiosin biosynthesis-permissive conditions. LacZ expression was not abolished by growing these strains at a temperature at which prodigiosin biosynthesis does not occur, suggesting that transcription of orf1 and orf4 is not temperature-sensitive. Other work done in this study included strain construction by the use of a Serratia marcescens generalised transducing phage (ΦOT8), and the construction of a Lac- strain of this species in which lacZ fusions were generated by TnphoA'-2 mutagenesis, and transduced into from an isogenic Lac+ strain