Regulation of histidine catabolism in Pseudomonas fluorescens SBW25 : a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Auckland, New Zealand
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2019
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Massey University
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Abstract
The pathway of histidine utilization (hut) has been a model for studying bacterial gene
expression, particularly the coordination between cellular carbon and nitrogen
metabolisms. Early studies in enteric bacteria led to the concept of catabolite
repression, which explains the inhibitory effects of glucose on the utilization of
alternative carbon sources such as histidine and lactose. Briefly, transcription of hut
genes is activated by the catabolite-activating protein (CAP) charged with cAMP and
the NtrBC/NAC cascade when histidine is used as a source of carbon and nitrogen,
respectively. However, this well-defined paradigm does not hold for many non-enteric
bacteria, including the closely related Pseudomonas.
This work aims to define the molecular basis of hut gene expression in Pseudomonas,
using the plant growth-promoting bacterium P. fluorescens SBW25 as a model.
Previous work identified all hut genes involved in histidine uptake and subsequent
enzymatic breakdown, which are organized in three transcriptional units in the hut
locus: hutF, hutCD and hutU-G. Like in enteric bacteria, histidine-induced expression
of hut operons is mediated by the HutC repressor with urocanate, the first intermediate
of the histidine degradation pathway, as the effector molecule. However, the precise
interactions between HutC and its hut operator sites remain elusive; more importantly,
recent progress suggests a new role of HutC in global gene regulation beyond histidine
catabolism. Moreover, two two-component systems CbrAB and NtrBC are involved in
hut activation, but it remains unknown whether they act in a direct or indirect manner.
In this study, I first examined the molecular interactions between His6-tagged HutC
protein and probe DNAs of the PhutU and PhutF promoters. Results of electrophoretic
mobility shift assay (EMSA) and DNase I footprinting indicate that HutC binds to a
consensus sequence of TGTA-N2-TACA (named Phut site), and involves complex
oligomerization in response to varying concentrations of urocanate. A novel weak
HutC binding sequence (termed Pntr site) was identified in the PhutF promoter, which
may help strengthen the repression of hutF. Significantly, this Pntr site shows no
sequence similarity to the previously recognized Phut site, instead it is homologous to
the NtrC-binding consensus sequence (GCACCA-N3-TGGTGC).
Next, the Phut consensus sequence was used to predict HutC target genes in the
genome of P. fluorescens SBW25. This led to the identification of 88 candidate
promoters, eight of which were subject to experimental verification by EMSA and
DNase I footprinting. Phenotypic analysis of the hutC deletion mutant showed that
hutC is involved in cell motilities. The data is consistent with the predicted global
regulatory role of HutC.
Histidine utilization poses a significant challenge as it produces excess nitrogen over
carbon. The rate of histidine utilization (hut) thus must be carefully regulated. Here we
show, for the first time, that expression of hut genes is positively regulated by two
global regulators CbrAB and NtrBC in a direct manner, while subjecting to histidine
concentration-dependent negative control of the HutC repressor. hut expression is
further regulated at the post-transcriptional level by the CbrAB-CrcYZ-Crc/Hfq cascade
in response to the presence of succinate (the most preferred carbon source for
Pseudomonas). When growing in nutrient-complex conditions such as a minimal salts
medium supplemented with succinate and histidine wherein histidine is the sole
nitrogen but less-preferred carbon source, CbrAB is involved in directly activating hut
transcription but indirectly repressing hut translation. Under this condition, NtrBC plays
the dominant role in transcriptional activation of hut genes, but it requires assistance
from the HutC repressor. A combination of genetic and biochemical analyses show that
HutC acts as a governor to monitor and control the histidine catabolic rate, preventing
production of excess ammonium and consequent inactivation of the NtrBC system.
HutC additionally recognizes the NtrC binding site responsible for ntrBC expression,
which provides a negative feedback for NtrBC autoregulation.
Together, data presented in this thesis extend our understanding of carbon catabolite
repression to the cellular nitrogen catabolism of Pseudomonas: carbon/nitrogen
metabolic balance is maintained by the interplay of CbrAB and NtrBC at the hut
operator site, and it requires the local regulator HutC to prevent hut expression from
exceeding a critical upper limit. The finding that the HutC regulator is capable of
recognizing two distinct DNA binding motifs (Phut and Pntr) has broader implications
in gene regulation. Further biochemical analysis is required to unravel the molecular
basis of the observed dual site recognition.
Description
Listed in 2019 Dean's List of Exceptional Theses
Keywords
Pseudomonas fluorescens, Metabolism, Regulation, Genetic regulation, Bacterial genetics, Dean's List of Exceptional Theses