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A molecular genetic investigation into 2-methyl-3-amyl-6-methoxyprodigiosene (prodigiosin) biosynthesis in Serratia marcescens
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Abbasi, Sophia Y. (1998) A molecular genetic investigation into 2-methyl-3-amyl-6-methoxyprodigiosene (prodigiosin) biosynthesis in Serratia marcescens. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b1362916~S15
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.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QR Microbiology | ||||
Library of Congress Subject Headings (LCSH): | Serratia marcescens -- Genetics, Microbial metabolites -- Synthesis | ||||
Official Date: | September 1998 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Biological Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Salmond, George | ||||
Sponsors: | Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC) | ||||
Extent: | [11], xiii, 218 p. | ||||
Language: | eng |
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