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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Date
2011
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Massey University
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Abstract
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).]
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Keywords
Pseudomonas, Genetics, Genotypic analysis, Phenotypic analysis