[The identification of genes involved in Exo-polysaccarides production in Rhodobacter sphaeroides 2.4.1 regulated by the response regulator RSP0087]

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Acknowledgement

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Abstract

The photosynthetic bacterium Rhodobacter sphaeroides 2.4.1T has two chromosomes, CI (C3<0 Mb) and CII (C0<9 Mb). In this study a low-redundancy sequencing strategy was adopted to analyse 23 out of 47 cosmids from an ordered CII library. The sum of the lengths of these 23 cosmid inserts was C495 kb, which comprised C417 kb of unique DNA. A total of 1145 sequencing runs was carried out, with each run generating 559O268 bases of sequence to give C640 kb of total sequence. After editing, C2<8% bases per run were estimated to be ambiguous. After the removal of vector and Escherichia coli sequences, the remaining C565 kb of R. sphaeroides sequences were assembled, generating C291 kb of unique sequences. BLASTX analysis of these unique sequences suggested that C131 kb (45% of the unique sequence) had matches to either known genes, or database ORFs of hypothetical or unknown function (dORFs). A total of 144 strong matches to the database was found; 101 of these matches represented genes encoding a wide variety of functions, e.g. amino acid biosynthesis, photosynthesis, nutrient transport, and various regulatory functions. Two rRNA operons (rrnB and rrnC) and five tRNAs were also identified. The remaining 160 kb of DNA sequence which did not yield database matches was then analysed using CODONPREFERENCE from the GCG package. This analysis suggested that 122 kb (42% of the total unique DNA sequence) could encode putative ORFs (pORFs), with the remaining 38 kb (13%) possibly representing non-coding intergenic DNA. From the data so far obtained, CII does not appear to be specialized for encoding any particular metabolic function, physiological state or growth condition. These data suggest that CII contains genes which are functionally as diverse as those found on any other bacterial chromosome and also contains sequences (pORFs) which may prove to be unique to this organism.

Table of Contents

ACKNOWLEDGEMENT2

DECLARATION3

ABSTRACT4

TABLE OF CONTENTS6

CHAPTER 1: INTRODUCTION13

CHAPTER 2: LITERATURE REVIEW15

CHAPTER 3: METHODOLOGY29

CHAPTER 4: DISCUSSION AND ANALYSIS35

Sequence strategies and genome analysis35

Sequence 'skimming' of the small chromosome37

DNA composition and codon usage37

Analysis of the sequencing strategy41

A variety of gene functions map to CII47

Comparison to other bacterial genomes49

CHAPTER 5: CONCLUSION53

REFERENCES59

Chapter 1: Introduction

Rhodobacter sphaeroides 2.4.1T is a facultative photoheterotroph belonging to the a-3 subdivision of the Proteobacteria (Woese et al., 1984). The metabolic diversity of this group of organisms is unparalleled (Gest, 1972; Kiley & Kaplan, 1988). The structure and function of the photosynthetic membrane protein complexes and their regulation, photolithotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis and tetrapyrrole ...