Mechanisms of carbon catabolite repression in Streptococcus pneumoniae.
Fleming, Eleanor.
2016
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Abstract:
Streptococcus pneumoniae (the pneumococcus) is a Gram positive asymptomatic colonizer of
the human nasopharynx, capable of spreading to disparate sites throughout the body of a
susceptible host. Previous work from multiple laboratories collectively supports the
model in which pneumococcus 'grazes' on glycosylated host mucosal surfaces during
colonization. Pneumococcus encodes an ... read moreaverage of ten surface-associated glycosidases
that can release a variety of mono-, di-, and trisaccharides from these glycosylated
surfaces. It also encodes up to thirty transport systems for subsequent uptake of these
carbohydrates and breakdown by one of their many encoded carbohydrate catabolism
pathways. Expression of these glycosidases, transporters and downstream metabolic
enzymes is likely regulated in response to carbohydrate availability. Carbon catabolite
repression (CCR) is a regulatory process used by both Gram positive and negative
bacteria to achieve maximum growth in mixed carbohydrate environments, such as the host
sites occupied by pneumococcus. CCR organizes available carbohydrates into a hierarchy;
the most easily metabolizable carbohydrate is the first to be transported and
metabolized by a bacterium before it resorts to less preferred carbohydrates. This
hierarchy is created through repression of metabolic enzymes and transporters for less
easily metabolized carbohydrates when a preferred carbohydrate is available. Given the
carbohydrate diversity at the sites occupied by the pneumococcus and the large fraction
of their genome dedicated to carbohydrate metabolism, we hypothesize that CCR is of
utmost importance to this pathogen's fitness. To gain support for this hypothesis and to
extend our understanding of carbohydrate gene regulation in this pathogen, the goal of
my thesis was to determine how the pneumococcus accomplishes CCR. The first half of my
thesis describes my investigation into transcriptional and non-transcriptional
mechanisms of CCR that are conserved in related species. We determined that the two key
aspects of serine-phosphorylated histidine phospho-carrier protein
(HPr-Ser∼P)-mediated CCR are active in the pneumococcus; it restricts
carbohydrate transport thereby enabling transcriptional repression of non-preferred
carbohydrate metabolism genes independently of and in conjunction with the global CCR
transcriptional regulator, carbon catabolite protein A (CcpA). We identified three
examples of carbohydrate-binding transcriptional regulators whose regulation activity
profiles are consistent with them responding to intracellular carbohydrate inducers
which are excluded by HPr-Ser∼P restriction of transport. We also demonstrate the
influence of HPr-Ser∼P on CcpA-dependent transcriptional regulation, suggesting
these proteins co-repress in the pneumococcus. Finally, we obtained results strongly
suggesting that all or some of these CCR activities of HPr-Ser~P are uniquely essential
to the pneumococcus. In the second half of my thesis I investigate the role of the
phosphoenolpyruvate-dependent transport system (PTS) transporter ManLMN to pneumococcal
carbohydrate metabolism and CCR. ManLMN plays a predominant role in carbohydrate
metabolism and metabolic regulation in many Gram positive species, in particular low G+C
species, but its role in the pneumococcus had not been explored. Despite the
preponderance of carbohydrate transporters encoded by the pneumococcus, we found that
ManLMN is required for growth on five non-preferred carbohydrates, partially required
for growth on an additional two non-preferred carbohydrates, while growth on four
different preferred carbohydrates did not require ManLMN. We determined that ManLMN is
essential in these conditions because it is required for induction of other PTS
transporters and downstream metabolic enzymes. Constitutively expressing a lactose PTS
in place of ManLMN resulted in abrogation of CCR of a lactose metabolism gene,
suggesting ManLMN has a unique role in CCR as a multi-substrate transporter. To explore
this idea further we performed a selection for suppressor mutations that allow the
ΔmanLMN strain to grow on specific carbohydrates. Through this analysis we
identified the virulence regulator SmrC as well as two uncharacterized small proteins as
potential carbohydrate regulators in the
pneumococcus.
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Andrew Camilli.
Committee: Carol Kumamoto, Abraham Sonenshein, John Leong, and Michael Malamy.
Keyword: Microbiology.read less - ID:
- 4f16cf04n
- Component ID:
- tufts:20332
- To Cite:
- TARC Citation Guide EndNote
