title: Characterisation of the ATP-dependent ligase, Lig E:  An investigation into its significance in Neisseria gonorrhoeae

authors: Pan, Jolyn

abstract: The rapid rise in multidrug-resistant Neisseria gonorrhoeae isolates has caused major public concern for the treatment of its sexually transmitted disease, gonorrhoea. Previously easily treatable, gonorrhoea is now spreading at alarming rates due to the bacterium’s ability to take up DNA from its environment without regulation. With approximately 106 million new cases annually, thorough investigations into novel gonococcal pathways that can be targeted to limit its survival and spread are crucial. One possible target is the disruption of its DNA repair systems, which include ligases that seal breaks in DNA, and would greatly discourage bacterial growth. Interestingly, in addition to its replicative ligase, N. gonorrhoeae expresses a minimal ATP-dependent ligase, Ligase E (Lig E), which contains a signal peptide that indicates a likely periplasmic location as opposed to the cytosolic location of genomic DNA. Hence, I hypothesise that Lig E is working on repairing fragmented extracellular DNA which has a role in both biofilm formation and bacterial competence, both of which would be greatly beneficial for the persistence and acquisition of antibiotic resistance genes in N. gonorrhoeae.

In this thesis, I focussed on characterising Lig E from N. gonorrhoeae (Ngo-Lig E) to determine its contribution to gonococcal phenotype, as well as its structure, function and cellular location. To analyse its biological role, several ngo-lig E mutants were created in vivo and their growth, stress response, biofilm formation as well as infection and adhesion to a human cell line were studied. I showed that Ngo-Lig E is not essential for normal planktonic N. gonorrhoeae growth and survival; however, it is important for its biofilm formation and subsequent adhesion onto human cells. Following this, I further investigated the potential role of Ngo-Lig E on biofilm formation by looking at its morphology via scanning electron microscopy and confocal laser scanning microscopy after growing them under constant shear forces with a continual supply of fresh media. Data from this supported earlier indications that Ngo-Lig E is important for N. gonorrhoeae in biofilm formation, as well as the extent of damage the bacterium can cause to human tissue. Taking its natural competence into account, I also investigated the potential role of Ngo-Lig E in the uptake of fragmented DNA during transformation and found a decreased ability of N. gonorrhoeae to repair and take up nicked DNA when ngo-lig E was absent. Investigations into the potential location of Ngo-Lig E showed that it is likely transported into both the periplasmic and extracellular space in N. gonorrhoeae, where it may perform the aforementioned dual-role in biofilm formation and DNA uptake. Finally, I solved the three-dimensional structure of Ngo-Lig E, highlighting its minimal architecture and lack of complete encirclement around its substrate DNA. 

The work presented in this thesis provides the first report of a solved structure of Lig E from a human pathogen, the first report of in vivo characterisation of this enzyme, as well as the first report of attempting to characterise its role and location. Collectively, this thesis has allowed for the establishment of important characteristics of Ngo-Lig E that were previously unknown. Although further research into this enzyme is necessary, the results presented here bring to light a potential novel pathway that may be targeted by future drug developments to tackle the emerging threat of multidrug-resistant N. gonorrhoeae in our community.

text: Characterisation of the ATP-dependent ligase, Lig E:  An investigation into its significance in Neisseria gonorrhoeae The rapid rise in multidrug-resistant Neisseria gonorrhoeae isolates has caused major public concern for the treatment of its sexually transmitted disease, gonorrhoea. Previously easily treatable, gonorrhoea is now spreading at alarming rates due to the bacterium’s ability to take up DNA from its environment without regulation. With approximately 106 million new cases annually, thorough investigations into novel gonococcal pathways that can be targeted to limit its survival and spread are crucial. One possible target is the disruption of its DNA repair systems, which include ligases that seal breaks in DNA, and would greatly discourage bacterial growth. Interestingly, in addition to its replicative ligase, N. gonorrhoeae expresses a minimal ATP-dependent ligase, Ligase E (Lig E), which contains a signal peptide that indicates a likely periplasmic location as opposed to the cytosolic location of genomic DNA. Hence, I hypothesise that Lig E is working on repairing fragmented extracellular DNA which has a role in both biofilm formation and bacterial competence, both of which would be greatly beneficial for the persistence and acquisition of antibiotic resistance genes in N. gonorrhoeae.

In this thesis, I focussed on characterising Lig E from N. gonorrhoeae (Ngo-Lig E) to determine its contribution to gonococcal phenotype, as well as its structure, function and cellular location. To analyse its biological role, several ngo-lig E mutants were created in vivo and their growth, stress response, biofilm formation as well as infection and adhesion to a human cell line were studied. I showed that Ngo-Lig E is not essential for normal planktonic N. gonorrhoeae growth and survival; however, it is important for its biofilm formation and subsequent adhesion onto human cells. Following this, I further investigated the potential role of Ngo-Lig E on biofilm formation by looking at its morphology via scanning electron microscopy and confocal laser scanning microscopy after growing them under constant shear forces with a continual supply of fresh media. Data from this supported earlier indications that Ngo-Lig E is important for N. gonorrhoeae in biofilm formation, as well as the extent of damage the bacterium can cause to human tissue. Taking its natural competence into account, I also investigated the potential role of Ngo-Lig E in the uptake of fragmented DNA during transformation and found a decreased ability of N. gonorrhoeae to repair and take up nicked DNA when ngo-lig E was absent. Investigations into the potential location of Ngo-Lig E showed that it is likely transported into both the periplasmic and extracellular space in N. gonorrhoeae, where it may perform the aforementioned dual-role in biofilm formation and DNA uptake. Finally, I solved the three-dimensional structure of Ngo-Lig E, highlighting its minimal architecture and lack of complete encirclement around its substrate DNA. 

The work presented in this thesis provides the first report of a solved structure of Lig E from a human pathogen, the first report of in vivo characterisation of this enzyme, as well as the first report of attempting to characterise its role and location. Collectively, this thesis has allowed for the establishment of important characteristics of Ngo-Lig E that were previously unknown. Although further research into this enzyme is necessary, the results presented here bring to light a potential novel pathway that may be targeted by future drug developments to tackle the emerging threat of multidrug-resistant N. gonorrhoeae in our community.

year: 2025

title: Review: The effect of temperature on enzyme activity: new insights and their implications

authors: Daniel, Roy M.

subjects: Equilibrium Model

abstract: The two established thermal properties of enzymes are their activation energy and their thermal stability. Arising from careful measurements of the thermal behaviour of enzymes, a new model, the Equilibrium Model, has been developed to explain more fully the effects of temperature on enzymes. The model describes the effect of temperature on enzyme activity in terms of a rapidly reversible active-inactive transition, in addition to an irreversible thermal inactivation. Two new thermal parameters, T eq and ΔH eq, describe the active–inactive transition, and enable a complete description of the effect of temperature on enzyme activity. We review here the Model itself, methods for the determination of T eq and ΔH eq, and the implications of the Model for the environmental adaptation and evolution of enzymes, and for biotechnology.

text: Review: The effect of temperature on enzyme activity: new insights and their implications The two established thermal properties of enzymes are their activation energy and their thermal stability. Arising from careful measurements of the thermal behaviour of enzymes, a new model, the Equilibrium Model, has been developed to explain more fully the effects of temperature on enzymes. The model describes the effect of temperature on enzyme activity in terms of a rapidly reversible active-inactive transition, in addition to an irreversible thermal inactivation. Two new thermal parameters, T eq and ΔH eq, describe the active–inactive transition, and enable a complete description of the effect of temperature on enzyme activity. We review here the Model itself, methods for the determination of T eq and ΔH eq, and the implications of the Model for the environmental adaptation and evolution of enzymes, and for biotechnology.

year: 2007

title: Te Whakaruruhau Transition and Wellbeing programme: An implementation evaluation

authors: Robins, Kathryn

subjects: Maori

abstract: Based on the recognition that many women who come into refuge have few options
but to return to the sort of environment in which they have been abused, the
Transition and Wellbeing programme aims to provide medium to long term housing
for families as they re‐establish themselves in the community.
Data for the evaluation of the programme was collected from two main sources; the
women on the programme and key informants at Te Whakaruruhau. Five women,
two of whom are housed in transitional accommodation were interviewed
individually, while focus groups were conducted with middle and senior management
teams.
The completion of the interviews enabled the development of a programme logic,
which describes the ‘theory’ of the programme. The model details the activities or
what the programme does. These activities include linking women to programmes
and resources, providing practical support, addressing specific cultural needs and the
availability of quality advocates. These activities are built on foundational values, such
as, whanaungatanga, manaakitanga and wairuatanga. The programme is only made
possible with internal and external inputs. Of the external inputs, funding
contributions are considered vital to the functioning of the programme.
The women’s perspectives identified varying outcomes from their participation in the
programme. The logic model details the intended outcomes in the short, medium and
long‐term, reflecting personal, relational and community wellbeing. Short term
outcomes include improved communication, improved self‐esteem, improved
confidence and personal growth. Medium outcomes saw (re)engagement in training,
education and for some, (re)entry into the workforce. Long term outcomes related to
the establishment of a sustainable life style free from violence.
The participants did not see a need to modify the programme in any significant way.
Some did think that it could be usefully extended by adding to the existing activities a
hands on, artistic approach. Programme developers may consider more creative ways
in which to assess and measure the impact of the programme. Finally, it is suggested
that the programme could benefit from a more systemic assessment process to
determine whether women are “ready” to enter it.

text: Te Whakaruruhau Transition and Wellbeing programme: An implementation evaluation Based on the recognition that many women who come into refuge have few options
but to return to the sort of environment in which they have been abused, the
Transition and Wellbeing programme aims to provide medium to long term housing
for families as they re‐establish themselves in the community.
Data for the evaluation of the programme was collected from two main sources; the
women on the programme and key informants at Te Whakaruruhau. Five women,
two of whom are housed in transitional accommodation were interviewed
individually, while focus groups were conducted with middle and senior management
teams.
The completion of the interviews enabled the development of a programme logic,
which describes the ‘theory’ of the programme. The model details the activities or
what the programme does. These activities include linking women to programmes
and resources, providing practical support, addressing specific cultural needs and the
availability of quality advocates. These activities are built on foundational values, such
as, whanaungatanga, manaakitanga and wairuatanga. The programme is only made
possible with internal and external inputs. Of the external inputs, funding
contributions are considered vital to the functioning of the programme.
The women’s perspectives identified varying outcomes from their participation in the
programme. The logic model details the intended outcomes in the short, medium and
long‐term, reflecting personal, relational and community wellbeing. Short term
outcomes include improved communication, improved self‐esteem, improved
confidence and personal growth. Medium outcomes saw (re)engagement in training,
education and for some, (re)entry into the workforce. Long term outcomes related to
the establishment of a sustainable life style free from violence.
The participants did not see a need to modify the programme in any significant way.
Some did think that it could be usefully extended by adding to the existing activities a
hands on, artistic approach. Programme developers may consider more creative ways
in which to assess and measure the impact of the programme. Finally, it is suggested
that the programme could benefit from a more systemic assessment process to
determine whether women are “ready” to enter it.

year: 2008-06-26