The enteric bacterium Serratia marcescens is an opportunistic human pathogen. The strain studied here makes the red pigment prodigiosin (Pig) and the ß-lactam antibiotic (5R)-carbapen-2-em-3 carboxylic acid. Mutants were isolated which were affected for pigment production. Approximately 20% of these mutants were also concomitantly deficient for the production of antibiotic. These mutants were presumed to be defective in the rap (regulation of antibiotic and pigment) gene. This study set out to investigate the rap gene which had been cloned by direct cosmid complementation of a Rap mutant from a cosmid library (pNRT300). Sequence analysis of the rap gene revealed a predicted product showing strong homology to S1yA, classified by Libby et al., (1994) as a virulence determinant in Salmonella. Homologues of the rap gene were detected in several genera including Salmonella, Yersinia, Enterobacter and species of the enteric plant pathogen Erwinia. The Erwinia horEc gene (homologue of rap) was cloned and encoded a product which was highly homologous to both the SlyA and Rap proteins. The gene arrangement around the rap locus in Erwinia was identical to that in Serratia in that rap and horEc were both situated upstream of two genes encoding homologues of the lipoprotein Pcp and a gene encoding a protein of unknown function from Yersinia enterocolitica. This observation led to the search for the Yersinia homologue of rap (horte) which was subsequently cloned and sequenced. This gene too encoded a protein highly homologous to Rap and HorE. Data base searches revealed that these proteins shared a significant level of homology with a number of bacterial protein regulators involved in exoenzyme production, virulence in plant and human pathogens, multiple antibiotic resistance and xenobiotic catabolism. The findings of this study cast serious doubt on the conclusions of Libby et al., (1994) and in a recent report which was published whilst this thesis was being compiled, Ludwig et al., (1995) reclassified S1yA as a regulatory protein capable of activating cryptic haemolysin genes in Escherichia coll. Marker exchange mutants (horEc:k: anR) of the Envinia carotovora subspecies carotovora were found to be affected in the production of a carbapenem antibiotic and showed decreased levels of production of multiple exoenzyme virulence factors. Transcriptional fusion data revealed that the horEc mutation affected the transcription of carA a carbapenem biosynthetic gene. Antibiotic and exoenzymes are known to be regulated by a small molecule dependent regulatory system analogous to the Lux system controlling bioluminescence in Photobacterium fischeri. The results of regulatory studies in which autoinducer was added exogenously, or carR was added in trans imply a role for HorE in this pheromone-signalling system. The functional expression of prodigiosin in a Erwinia carotovora subspecies carotovora was found to be dependent on autoinducer and the gene product of horEc. Some interesting observations were also made regarding differential patterns of prodigiosin gene expression within bacterial colonies. These patterning effects were strikingly strain-specific.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:338673 |
| Date | January 1996 |
| Creators | Thomson, Nicholas Robert |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/4194/ |
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