Molecular mechanism of xylose utilization in a plant growth-promoting bacterium Pseudomonas fluorescens SBW25 : a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Auckland, New Zealand
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2015
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Massey University
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Abstract
Pseudomonas fluorescens SBW25 is a plant growth-promoting bacterium that was
originally isolated from the phyllosphere of field-grown sugar beet. It is capable of
aggressively colonizing sugar beet and a number of other crops such as wheat, maize
and peas, and inhibiting the damping-off disease caused by Pythium ultimum. P.
fluorescens SBW25 has become an important model organism for studying the
molecular interactions between bacteria and plants. Previous promoter-trapping
analysis showed that SBW25 elevates expression of over 100 genes in its genome
when colonizing sugar beet seedlings. These include a candidate gene for xylose
utilization, suggesting that SBW25 colonization may be critically dependent on the
presence and catabolism of plant-derived xylose. The overall aim of this project is to
unravel the molecular basis of xylose utilization by P. fluorescens SBW25 and
demonstrate its ecological significance for bacterial survival in complex plant
experiments.
Bacterial degradation of xylose is sequentially mediated by two enzymes - an
isomerase (XutA) and a xylulokinase (XutB) - with xylulose as an intermediate. P.
fluorescens SBW25, though capable of growth on xylose as a sole carbon source,
encodes only one degradative enzyme XutA in the xylose utilization (xut) locus. Here,
using site-directed mutagenesis and transcriptional assays I identified two functional
xylulokinase-encoding genes (xutB1 and xutB2), and further showed that expression
of xutB1 is specifically induced by xylose. Surprisingly, the xylose-induced xutB1
expression is mediated by the mannitol-responsive regulator MtlR, using xylulose
rather than xylose as the direct inducer. In contrast, expression of the xutA operon is
regulated by XutR in a xylose- and xylulose-dependent manner. Moreover, the data
indicate a complex overlapping cellular responses to xylose and other structurally
similar sugars such as mannitol, sorbitol, fructose as well as ribose.
Both XutR and MtlR are transcriptional activators of the AraC family, members of
which typically use DNA-looping to modulate levels of gene expression. The
functionality of XutR has been subjected to detailed genetic and biochemical
analyses, including promoter mapping, electrophoretic mobility shift assay (EMSA)
and DNase I footprinting assay. My data leads to a XutR regulatory model that does
not involve DNA-looping. XutR functions as a dimer, which recognizes two inverted
repeat sequences; but binding to one half site is very weak requiring inducer
molecules such as xylose for activation.
To determine the ecological significance of xylose utilization for bacterial
colonization in planta, a Xut- mutant (?xutA) was subjected to competitive
colonization on sugar beet seedlings together with a neutrally marked wild-type
strain. Results showed that the ?xutA mutant was significant less competitive than the
wild-type strain both in the shoot and the rhizosphere. Together, the data show that
xylose utilization is an important trait for P. fluorescens SBW25 to colonize surfaces
of plants.
It should be noted that xylose can only support slow bacterial growth of P.
fluorescens SBW25, and thus it is not the sugar of choice in the presence of other
preferred carbon and energy sources such as succinate, glucose and arabinose. The
underlying mechanism is called carbon catabolite repression (CCR). CCR has been
well studied in E. coli, where it is mainly mediated by the catabolite-activation protein
CAP charged with cAMP. However, CCR still remains elusive for non-enteric
bacteria such as Pseudomonas. Previous studies in other Pseudomonas species
indicate that CCR in Pseudomonas occurs at post-transcriptional levels and involves
specific binding of the Crc protein to mRNAs of respective catabolic genes. However,
genetic tools suitable for studying post-transcriptional gene expression are lacking,
particularly vectors derived from mini-Tn7. Mini-Tn7 vectors possess the advantage
of delivering reporter fusions into the chromosome in a site- and orientation-specific
manner.
To facilitate the study of CCR in Pseudomonas, I have successfully constructed and
validated a panel of five mini-Tn7 vectors for analysis of post-transcriptional gene
expression in Pseudomonas. Four vectors allow construction of translational fusions
to ß-galactosidase (lacZ), while the fifth is designed for functional analysis of
noncoding RNA genes. Translational fusions can be constructed without a functional
promoter in the vector or from an inducible promoter of either Ptac or PdctA. I show
that promoterless fusions have value for determining levels of translation, whereas
fusions to inducible promoters have utility in the analysis of mRNA-binding factors.
Next, a combination of site-directed mutagenesis and gene expression assays were
used to identify regulators that are involved in the succinate-mediated repression of
the xut operon. Succinate is an intermediate of the tricarboxylic acid (TCA) cycle and
it is preferentially used by P. fluorescens SBW25 as a source of carbon and energy. In
this work, I have successfully identified the major regulatory components of CCR in
P. fluorescens SBW25. These include a two-component signal transduction system
CbrAB, two small non-coding RNAs CrcY and CrcZ, and a two-protein complex
composed of Crc and Hfq. Results showed that when succinate is present, the Crc/Hfq
complex inhibits expression of xut genes via binding of the mRNA transcript; when
succinate disappears, CbrAB activates the expression of CrcY and CrcZ, which in
turn sequesters the Crc/Hfq complex and relieves repression of the xut operon.
Taken together, data presented in this thesis indicate novel mechanisms of xylose
utilization in terms of not only the catabolic genes but also the mode of their
regulation, and reveal complexity and redundancy of regulators involved in the
succinate-mediated repression of xylose utilization genes.
Description
Appendix I removed from digital copy for copyright reasons:
Liu, J., Rainey, P. B., & Zhang, X.-X. (2014).
Mini-Tn7 vectors for studying post-transcriptional
gene expression in Pseudomonas. Journal of
Microbiological Methods, 107, 182-185. doi.org/10.1016/j.mimet.2014.10.015
Keywords
Pseudomonas fluorescens, Molecular aspects, Research Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology::Genetics