1 CHAPTER 1 Introduction Serratia marcescens is a species of Gram-negative, short rod-shaped, enteric bacterium in the family Enterobacteriaceae that is able

to inhabit a wide variety of ecological niches and causes disease in plant, vertebrate and invertebrate hosts (Grimont & Grimont, 1978). It is classified as an opportunistic pathogen that causes nosocomial infections that are clinically problematic because multi-drug resistance is widespread within the species (Stock et al., 2003; Traub, 2000). One of the characteristic features of many S. marcescens strains is the production of cell-associated red color pigment called prodigiosin (2-methyl-3-pentyl-6-

methoxyprodigiosin). Prodigiosin is a linear tripyrrole typical secondary metabolite, appearing only in the later stages of bacterial growth (Williams et al., 1971). Currently, prodigiosins are of great interest because they have been shown to possess numerous biological activities including antimicrobial, antifungal, anti-protozoal, anti-malarial, immunosuppressive and anticancer properties (Williamson et al., 2007). The complex and exquisite regulation of prodigiosin production has been widely studied in Serratia 39006 strain, where it has been revealed to involve a complex regulatory network, integrating information from a variety of physiological and environmental cues, including quorum sensing (Slater et al., 2003; Fineran et al., 2005). Quorum sensing (QS) is a mechanism of intercellular communication which enables bacteria to detect their cell population density and use this information to coordinately regulate gene expression and is a widespread phenomenon in gram-negative bacteria (Williams et al., 2000). One type of quorum signaling system that has been described is the LuxS/AI-2 (autoinducer-2) that also regulates prodigiosin biosynthesis in

2 some Serratia strains (Coulthurst et al., 2004). In S. marcescens 274, LuxS synthesizes the AI-2 signal, which is required for maximum pigmentation (Coulthurst, et. al., 2004). The LuxS mutant of Sma 274 exhibits reduced prodigiosin production, haemolysis and virulence, and LuxS regulation of pigment production does indeed occur via an extracellular signal, most likely AI-2 (Coulthurst et al., 2004). This present study shall deal with the cloning and characterization of the LuxS particularly making use of the local strain of Serratia marcescens to validate results of previous studies on the effect of LuxS mutant in the phenotype. The end view is to contribute in whatever little step to a macroscopic study of averting cancer.

Objectives of the Study: The main objective of this study is to clone and characterize the LuxS gene of the bacteria Serratia marcescens that involves in the biosynthesis of prodigiosin. Specifically, it aims to: 1. To determine the sequence of LuxS gene in local strains of S. marcescens; 2. To determine the copy number of the LuxS homolog in the local strains of S. marcescens genome; and 3. To characterize the gene sequence through bioinformatics.

Scope and Limitation of the Study This study focuses on the amplification of the LuxS gene of the local strains of S. marcescens. This gene will be cloned by PCR and will be analyzed through agarose gel electrophoresis and bio-informatics.

3 Significance of the Study Research into the biology of pigment production will stimulate interest in the bioengineering of strains to synthesize useful prodiginine derivatives. An understanding of the biosynthesis prodiginines will allow engineering of bacterial strains capable of synthesizing novel pordiginines through rational design and mutasynthesis experiments. Bacterial prodiginines and synthetic derivatives are effective proapoptotic agents with multiple cellular targets, and they are active against numerous cancer cell lines, including multidrug-resistant cells, (Williamson, et.al, 2007) Characterization of the gene sequence through bio-informatics enables early detection of mis-breeding, genetic drift and enables correlation of genetic information with phenotype. This set of information could serve as a guide in genetic engineering. Characterized genes also make attractive teaching tools.