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Abstract:
The genetic code is degenerate – 20 proteinogenic amino acids are coded for by 61
functional codons. One might then expect all the codons for an amino acid, called
synonymous codons, to be used equally. However, across all domains of life, preferential use
of synonymous codons has been observed. This phenomenon is called codon bias.
Synonymous substitutions, nucleotide level changes that do not modify the amino acid
sequence, were widely viewed to be selectively neutral. Mounting evidence, however,
indicates otherwise – synonymous substitutions can induce non-neutral and measurable
fitness effects.
Experimental studies with randomised codon usage in genes from both prokaryotes
and eukaryotes reveal large-scale fitness effects, ranging from altered growth, global changes
in both transcription and translation and protein output and function. The effects of
synonymous substitutions become particularly prominent in bacteria, where growth rate is
often limited by the speed of translation. However, these observations mainly arise from
selectively enriching certain codons, a pattern that is rare in naturally evolved genomes. To
investigate the functional implications of naturally diverged patterns of codon usage we have
identified an essential and highly expressed gene that exclusively exhibits synonymous
differences across three genomes of Pseudomonas fluorescens bacteria. These variants were
swapped between genomes to test for effects on gene expression and fitness. To swap the
synonymous alleles between genomes, I have optimised a scar-free genetic engineering
technique that is routinely used for SBW25, to A506 and Pf0-1. The resulting mutants varied
from the corresponding wild type strains at only one locus – the locus of the gene that was
swapped. I proceeded to test for the effects synonymous substitutions on gene expression and
fitness and demonstrated that changing established codon usage patterns of a gene in one
strain to that of another strain has considerable effects on gene expression (transcription).
Having optimised a technique for manipulating genomes of P. fluorescens strains
(besides SBW25), I proceeded to examine the genetic basis of one of the most studied and
extensively characterised phenotype of P. fluorescens – the ability to form mats at the airliquid
interface. First, I compared mat formation phenotype in A506, Pf0-1 and PICF7 to the
model strain SBW25; while, SBW25, A506 and PICF7 formed mats, Pf0-1 did not. To
identify the genetic routes to mat formation in PICF7, I developed tools for transposon
suppressor analysis, which revealed that unlike SBW25, which utilises the cellulose
biosynthetic machinery encoded by the wss genes to form mats, PICF7 makes use of the pel
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operon to synthesise the Pel exopolysaccharide. Pel is an exopolymer known to form biofilms
(mats) in P. aeruginosa. Using genetic engineering techniques I identified that A506 does not
use wss genes either, but the pga operon to produce mats, a pathway seldom utilised in
SBW25. The lack of mat forming ability in liquid medium by Pf0-1 is intriguing as it
possesses some of the structural genes known from P. aeruginosa and is reported to form
mats on solid surfaces. While different structural genes are exploited by the three mat forming
strains (SBW25, A506 and PICF7), the central regulatory pathways that fine-tune the
expression of the operons have remained the same. This is suggestive of a modular
mechanism, wherein multiple structural genes can substitute for one another across genomes.
So far, our understanding of P. fluorescens genome evolution has been limited to
SBW25. With the development of genetic tools within the scope of this thesis, we now have
the opportunity to expand on the comparative study of P. fluorescens and the efficacy of these
tools for the same has been amply demonstrated through the examination of the effects of
changing naturally evolved synonymous codons as well as the variation in molecular routes
exploited to colonise the air-liquid interface.
