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 randomized 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 optimized 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 optimized 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 characterized phenotype of P. fluorescens – the ability to form mats at the air liquid 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 utilizes the cellulose biosynthetic machinery encoded by the wss genes to form mats, PICF7 makes use of the Pel vii operon to synthesize 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 utilized 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 colonize the air-liquid interface.
