Genotypic and phenotypic analysis of plant-associated Pseudomonas : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Environmental Microbiology at Massey University, Auckland, New Zealand

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The ecological success of Pseudomonas in plant environments is largely determined by the phenotypes that it expresses: efficient utilization of plantderived nutritional substrates is fundamentally important for bacterial competitive growth. Not surprisingly then, Pseudomonas up-regulates the expression of many genes involved in nutrient scavenging when colonizing the plant surfaces. A typical example is the hut genes dedicated to the utilization of histidine and urocanate (the first intermediate of the histidine degradation pathway) in the model organism of P. fluorescens SBW25. Previous work has defined the genes involved in the histidine/urocanate uptake, degradation and regulation. This study aims to extend our understanding of histidine/urocanate utilization to the population level. A total of 230 Pseudomonas strains were isolated from the phyllosphere of sugar beets grown in Oxford (UK) and Auckland (New Zealand) and their ability to grow on histidine and urocanate was tested. The results revealed considerable variation of phenotypes, for example, strains were capable of growing on histidine but not on urocanate (His+, Uro-, 11%) and vice versa (His-, Uro+, 13%). Interestingly, His+, Uro- strains were commonly found in the Auckland population, whereas His-, Uro+ strains were more prevalent in the Oxford population. Introduction of cloned copies of the histidine- and urocanatespecific transporter genes (hutTh and hutTu) from P. fluorescens SBW25 restored the ability of many naturally His- and Uro- strains to utilize histidine and urocanate, respectively. Together, the data indicate that Pseudomonas populations are polymorphic with respect to the transporters. The genetic relatedness of the two Pseudomonas populations from Oxford and Auckland was estimated using multi-locus sequence analysis (MLSA) of three genes (gapA, gltA and acnB). For each of the three genes, oligonucleotide primers were designed to amply the DNA fragment (~600 nt) which was subjected to subsequent DNA sequencing. The DNA sequences of three genes (615 nt for gltA, 303 nt for gapA, 273 nt for acnB) were concatenated and used for phylogenetic analysis. Results showed that the Pseudomonas population from Auckland is phylogenetically distinct from that of Oxford; there is a clear correlation between the MLSA genotypes and the phenotypes (i.e., utilization of histidine vs. urocanate). Taken together, my data show that the two Pseudomonas populations colonizing the phyllosphere of sugar beets in Oxford and Auckland are genetically diverse and display distinct phenotypes in terms of their ability to grow on histidine and urocanate as the sole source of carbon and nitrogen. Furthermore, the observed phenotypic diversity is attributable to variation in histidine- and urocanate-specific transports, not genes for histidine catabolism. [Of note, the results reported in this thesis on the polymorphism of histidine and urocanate utilization in plant-associated Pseudomonas has been published in the journal of Environmental Microbiology, wherein I am the second author (see Appendix B).]
Pseudomonas, Genetics, Genotypic analysis, Phenotypic analysis