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Decoding the dual target antiactivator QseM
No full textRegulation of transfer of the symbiosis island of Mesorhizobium japonicum strain R7A, ICEMlSymR7A, is tightly controlled by a complex multi-component regulatory system. The master antiactivator of this system is QseM, which robustly inactivates two proteins, FseA and TraR, with both FseA and QseM containing a DUF2285 domain. Through these interactions QseM blocks both quorum sensing and Integrative and Conjugative Element (ICE) transfer, effectively quashing biological noise that could otherwise unleash the transfer mechanism population-wide with deleterious effects. Quorum sensing (QS) systems are widely utilised by bacteria to time critical changes in cellular physiology and have been increasingly implemented into the development of synthetic circuits to take advantage of this timed population-wide response to improve circuit function. However, these QS systems are prone to inappropriate activation by biological noise and crosstalk, leading to inefficient operation and signal collapse.
There is a lack of components that can be utilised in combination to control cross-talk and enable complex circuit construction, especially in the cases where integration of multiple quorum sensing pathways is desired. Identification and adaption of naturally evolved components is one way in which this lack of components can be overcome. Some QS systems have evolved antiactivators, which dampen the effects of biological noise and improve the function of natural QS circuits. The role of QseM in regulating ICEMlSymR7A and its imperviousness to biological noise makes it an ideal candidate for use in synthetic applications. However, the mechanisms QseM utilises to exert antiactivation are unknown. The aims of this work were to identify critical features in QseM that are involved in the interaction and inactivation of FseA and TraR with the view of adapting it to control biological noise and crosstalk in synthetic circuits.
To identify critical residues and motifs within QseM, alanine scanning mutagenesis was performed in combination with previous work to create 33 QseM mutants. These mutants were assessed for function against FseA and TraR and two critical regions in QseM were identified, one in the first predicted α-helix and one shared between the end of the third, and fourth α- helices. The majority of the residues critical for FseA inactivation were also critical for TraR inactivation; however, a four-residue motif, RLLD, was specific for FseA with mutation of an arginine at position 28 abolishing inactivation but not interaction in Bacterial-two-Hybrid assays.
The NMR structure of QseM except for its N- and C-terminal regions was determined by our collaborators and the structure of the whole QseM protein was reinforced by the development of co-evolution models of QseM (this work) and FseA (William Jowsey, this lab, unpublished data). Overlay of mutations critical for FseA inactivation on the structure showed that these clustered on one side of QseM, with helix one and the end of helix three to four forming a broad interaction interface on the surface of the protein. The structure of QseM identified it as an uncharacterised tetra-helical HTH domain protein with the typical DNA- binding HTH features, but the mutational analysis showed that critical DNA-binding HTH residues were not important for QseM function. The conservation of the HTH structure may be due to its suggested evolutionary history as a duplication of the DUF2285 domain of FseA.
FseA inactivation is at the core of QseM function with homologues down to 43% identity able to completely inactivate FseA despite their own FseA homologues showing as little as 25% identify. However only one of five QseM homologues tested, found on an ICE in the marine bacterium Stappia indica, was able to inactivate ICEMlSymR7A TraR. Substitution of regions within QseM with a homologue that was unable to inactivate TraR indicated that the C-terminus of QseM is involved in TraR inactivation. Incorporation of TraR into the regulatory system of QseM appears to a be a recent evolutionary event, with very few homologues outside of mesorhizobia containing linked qseM and traR genes that were close in the genome.
The mutational and structural analyses provided evidence that QseM acts as a competitive inhibitor of FseA dimerisation, obscuring the FseA dimerisation domain in a cleft between the RLLD and GY motifs of QseM, similar to the mechanism that FseA may use to dimerise. QseM also interacts with the AHL-binding domain of TraR, blocking its function likely through the formation of inactive heterodimers.
Finally, a chimeric LuxR family regulator was created using TraR and LasR. The 170 amino acid AHL-binding domain of TraR was fused to the DNA-binding domain of LasR as a ‘QseM-tag’. This chimera responded to the native TraR AHL 3-oxo-C6-HSL and had a similar AHL response profile despite having reduced function. Upon addition of AHL, the chimera activated transcription of the LasR target promoter, lasIp indicating that a successful fusion had been made. Importantly, it was shown that the chimeric protein was susceptible to QseM- mediated inactivation while the donor LasR was not. The construction of this chimera highlighted the modular chimeric QS regulators can be developed to improve specificity and regulation
Determining the ability of zeolitic imidazolate framework nanoparticles to remove copper from industrial wastewater
Get PDFVarious valuable metal elements (e.g., gold, silver, platinum, copper, and aluminium) are
used in electronic devices. Recycling of electronic waste can extract valuable metal
elements, which can be sold as raw materials for the manufacture of new products. Mint
Innovation New Zealand is a company that has developed a novel process using
microorganisms to recycle electronic waste. Although the technology is useful in
generating value from electronic waste, it produces highly acidic wastewater streams
containing highly concentrated dissolved heavy metal ions, which must be treated before
safe disposal into the environment. Copper is one such dissolved heavy metal ion present
in Mint Innovation’s wastewater streams, which must be reduced to below 10 mg/L in
order to comply with Auckland Council Trade-Waste agreements.
Suitable methods for selectively removing high concentrations of heavy metals from
wastewater with low pH conditions are yet to be discovered. ZIF-8 nanoparticles were
selected as a potential adsorbent material for removing copper from Mint Innovation’s
wastewater due to their large surface area, high pore density, narrow pore sizes, good
stability and abundance of possible binding sites. ZIF-8 was combined with chitosan and
PSS separately to produce ZIF-8@chitosan and ZIF-8@PSS composites, respectively.
Various reactant combinations were investigated for preparing ZIFs (ZIF-8, ZIF8@chitosan and ZIF-8@PSS).
During adsorption in idealised solutions (pH 6.5, [Cu2+] = 60 mg/L), copper removal to
below 10 mg/L limits was achieved by all ZIFs within 15 minutes. ZIFs also selectively
adsorbed copper in idealised solutions containing co-existing calcium or sodium ions.
However, ZIFs released large quantities of zinc (> 100 mg/L) ions into solution upon
binding to copper ions. The release of Zn2+ from ZIFs was enhanced in idealised solutions
with initial Cu2+ concentrations above 100 mg/L or initial pH less than 3.
During adsorption in Mint Innovation’s wastewater, ZIFs were rapidly degraded by the
low pH and strongly oxidising conditions. Upon raising the pH from 0.7 to 4 in one waste-stream, ZIF-8 successfully removed nearly all copper (89.8%), aluminium (93%)
and calcium (89.3%) ions. However, the reliability of this experiment as a measure of
metal removal by ZIF-8 was low because only a small number of wastewater samples (n
= 3) were collected, which was due to both time constraints and having limited access to
the analytical equipment required for detecting metal ions in wastewater solutions.
Binding of Cu2+ to ZIFs was thought to have destabilised the framework structure,
resulting in Zn2+ being released into solution. In attempt to stabilise ZIF particles and
explore their compatibility with industry standard water filtration equipment, ZIF8@PSS was coated onto nylon ultrafiltration membranes via direct deposition or in situ
self-assembly. The ZIF-8@PSS coated membranes were referred to as Mixed Matrix
Membranes (MMMs). The chemical composition and physical characteristics of ZIFs and
ZIF-8@PSS coated MMMs were analysed by Fourier transform infrared spectroscopy
(FTIR), dynamic light scattering (DLS), scanning electron microscopy (SEM). and water
contact angle (CA) measurements.
Direct deposition or in situ self-assembly both produced ZIF-8@PSS coated MMMs with
non-uniform surfaces, resulting from ZIF-8@PSS aggregation in some regions of the
membrane surface, but failing to attach to others (based on FTIR, SEM and CA analysis).
The filtration performance of ZIF-8@PSS coated MMMs was tested using solutions of
Congo red (CR) dye. ZIF-8@PSS coated MMMs failed to remove CR dye from solution
during continuous filtration. It was believed that nanosized CR molecules could simply
pass-through openings in the MMMs surface where ZIF-8@PSS failed to attach. Because
copper ions are smaller than CR molecules, it was predicted that the ZIF-8@PSS coated
MMMs will not remove copper ions during filtration.
ZIFs successfully removed copper ions to below 10 mg/L in both idealised solutions and
wastewater produced by Mint Innovation. However, Zn2+ release from ZIFs during ion
exchange with copper means that one pollutant is simply replaced with another during
wastewater treatment. Future research should focus on ZIF modifications that inhibit the
ion-exchange mechanism of ZIFs without reducing the ability of ZIF to bind to copper
ions
Planning for Street Art in Aotearoa-New Zealand Cities
Get PDFThe relation between street art, the city, and urban planning is complex. Unlike typical infrastructure, street art is temporary in nature, a kind of soft infrastructure, limiting the representation in national and regional policies. There is also negative stereotyping about the perception of street art as forms of resistance, rebellion, and vandalism which create NIMBY syndrome making certain spaces undesirable. However, street art is increasingly being recognised as an important urban design element to beautify cities. It is also an expression of public participation regarding how citizens consume public space. Planning is an important tool to mobilise street art, as it holds the ability to influence how cities define their local narrative. Street art is underpinned by a network of localised planning regulations that are inconsistent from region to region, subjecting street art to planning disputes.
Using qualitative methods, the research aims to identify how the current planning legislative frameworks have impacted the production of street art in Aotearoa-New Zealand’s urban context. Utilising Aotearoa-New Zealand urban centres Christchurch, Dunedin, and Wellington as case studies, illustrates the discrepancies in strategies.
The research found that there are positive and negative aspects that impact the street art production. Perceptions emphasised the need for more consistent directive and leadership across Aotearoa-New Zealand, but still ensures innovation, creativity, and narratives of space are reflected throughout art in urban spaces. It was identified that localised planning approaches stem from the creative city movements where the aspirations of the creative class are aspired to through tailored strategies—removing homogeneity between urban ideals. Recognising the relevance of the creative cities model in localised provisions is fundamental in the production of street art across Aotearoa-New Zealand’s urban realms. Privatisation, ownership, and sense of space are debates that increase the complexity of this research.
Creating a more enabling approach to street art allows both communities to have a say, and street artists to maintain greater self-expression. However, the lack of leadership from national government is reflected in the planning frameworks. Recommendations for improving street arts impact within the urban realm include:
- National government to implement a consistent definition of street art to provide a consistent understanding
- Centralise greater advocacy for street art helping fragmentation
- A policy shift to provide permissive planning regulations
- Provide greater education opportunities surrounding awareness of the role street art has on urban development
Adopting these recommendations may help street art become a more enabling process and positively impact the production of street art in urban areas. Implementing these recommendations involves greater collaboration and the need for a sufficient balancing act between national and local government, key stakeholders involved in the public art discourse, and the local community
More than a trickle, not yet a flood: Māori Employment and Urban Migration during World War Two
Get PDFThis thesis explores the connection between Māori urban migration and the Māori home
front war effort during World War Two. It argues that Māori urban migration was
occurring on a meaningful and notable scale before the end of World War Two, and this
can be seen in the impact urban migration had on the war effort. The relationship
between war work and migration is examined through case studies of three different
industries: Market Gardens, Freezing Works, and the Public Service. This framework
was chosen because it allows for the examination of various employment patterns, as
they relate to gender, kin, and different forms of migration.
The historiography for the Māori home front in World War Two has largely been
limited to analyses of the Māori War Effort Organization and its legislative legacy.
Other areas of research usually limit the Māori home front experience to being the
prologue for events in the 1950s and beyond, in terms of urban migration and industrial
developments. The period of Māori urban migration after 1950 has often been described
as a ‘flood’, and the period before that as a ‘drift’. This thesis offers an examination of
the 1939-1945 period which places the war at the forefront of the story of Māori urban
migration, and challenges the assumption that Māori urban migration only becomes
notable after the war. Making use of primary sources ranging from minute books, to
official correspondence, housing surveys, and census data, this thesis offers a new
perspective on the Māori home front. It contributes to the scholarship by exploring the
link between employment, migration, and the war effort
New homoleptic and heteroleptic [Pd2L4] helicates.
Get PDFThis thesis describes the synthesis and characterisation of a series of new homoleptic and heteroleptic [Pd2L4] helicates, synthesised from a series of new ligands based on the previously reported 1,4-bis(3-pyridyloxy)benzene ligand. The anion exchange process on these homoleptic and heteroleptic helicates was also explored.
Chapter 1 gives a brief introduction into supramolecular chemistry, and the ideas behind the self-assembly of homoleptic and heteroleptic architectures with some key examples from the literature. Considerations for the binding of ions inside a supramolecular architecture are also addressed.
Chapter 2 discusses the synthesis and characterisation of ligands 1 – 6, where the ligands vary in the difference in the sterics of the central group. Chapter 2 then moves into detailing the synthesis and characterisation of the homoleptic helicates [Pd2(1)4], [Pd2(3)4] and [Pd2(5)4]. The work done shows that varying the sterics of the central group of the ligand leads to a preference for some homoleptic cages over others. The anion exchange process on the homoleptic helicates [Pd2(1)4], [Pd2(4)4] and [Pd2(5)4] with the tetrabutylammonium salts of ClO4-, ReO4- and NO3-, is also discussed.
Chapter 3 details the synthesis and characterisation of the dynamic libraries of the heteroleptic helicates [Pd2(1)x(3)4-x], [Pd2(1)x(5)4-x] and [Pd2(3)x(5)4-x]. The anion exchange process on the heteroleptic helicates [Pd2(1)x(3)4-x], [Pd2(1)x(4)4-x] and [Pd2(1)x(5)4-x] with the tetrabutylammonium salts of ClO4- and ReO4- is also discussed.
Chapter 4 details the synthesis and characterisation of the dynamic libraries of the heteroleptic tri-ligand helicates [Pd2(1)x(2)y(3)z], [Pd2(1)x(3)y(6)z], [Pd2(1)x(4)y(5)z], [Pd2(2)x(3)y(6)z] and [Pd2(3)x(5)y(6)z].
Appendix 2 and 3 contain details on the synthesis and characterisation, and anion exchange processes for the other homoleptic and heteroleptic helicates synthesised from ligands 1 – 6. The experimental information for chapters 2, 3 and 4 are in appendix 2, 3 and 4, respectively
Regulation of the Pho Regulon and Reconstruction of the Photosynthetic Apparatus under Phosphate Limitation in Synechocystis sp. PCC 6803
Get PDFFor millennia, cyanobacteria have evolved strategies for acclimatization to dynamic environments – this requires a molecular response system. Phosphate is essential for cellular integrity and metabolism, yet inorganic phosphate is often the limiting nutrient for most aquatic environments. This study characterizes two periplasmic phosphate-binding proteins, namely SphX and SphZ, which are essential for the functional response of the SphS-SphR signal transduction system in Synechocystis sp. PCC 6803 and incorporates both into a model. SphX offers a competitive role so as to ensure inorganic phosphate is not depleted from the environment at a faster rate than is necessary for metabolism and, therefore, hinders luxury uptake; whereas, SphZ, encoded by sll0540, is the auxiliary sensor necessary for the SphS response under phosphate limitation in tandem with a protein complex association with the phosphate-specific transport 2 system and the negative regulator, SphU. Cross-regulation between the pho regulon and the photosynthetic apparatus is presented as well as a parameterized analysis for low-temperature fluorescence emission spectroscopy
Investigating the effect of marine reserves on coastal sharks in southern New Zealand
Get PDFWith the rapid increase in the use of marine protected areas, there is a need to better understand the value they offer top predators. However, in Aotearoa (New Zealand), no research has previously investigated the effects of marine reserves (i.e. Type 1 Marine Protected Areas) on such species. Research on marine reserves in New Zealand has generally focused on commercially important species, such as blue cod (Parapercis colias), snapper (Pagrus auratus), and spiny lobster (Jasus edwardsii). Considering there are 44 marine reserves of varying characteristics that overlap with the distribution of several coastal shark species, New Zealand presents an ideal location to research the effects of marine reserves on top predators. This study used a non-invasive methodology to investigate the effect of six marine reserves on the distribution of coastal shark species. Baited remote underwater video (BRUV) systems were deployed in 317 sites over two distinct regions: 192 sites in Ata Whenua (Fiordland) from July 2018 to December 2019, and 125 sites in Whaka ā Te Wera (Paterson Inlet), Rakiura (Stewart Island), from February 2018 to November 2019. A generalized linear modelling (GLM) approach was used to assess the effect of the marine reserves on two trophic levels of sharks in Fiordland. Firstly, GLM was used to assess the effect of protection on the combined relative abundance of the most common mesopredatory sharks detected. These species included the spiny dogfish (Squalus acanthias), school shark (Galeorhinus galeus), and carpet shark (Cephaloscyllium isabellum). A significant effect of the marine reserves was evident for mesopredators, suggesting marine reserves have led to an increase in their abundance or a change in distribution. Secondly, GLM was used to assess the effect of protection on the presence of broadnose sevengill sharks (Notorynchus cepedianus) and by contrast, there was no significant effect detected. For the Stewart Island data, a species distribution modelling approach was used to assess the fine scale effects of the Te Wharawhara (Ulva Island) Marine Reserve on the relative abundance of sevengill sharks. Again, no significant effect was evident. Given the marine reserves sampled are relatively small (< 40 km2), they appear to provide minimal conservation benefits for the large and mobile sevengill shark. In comparison, mesopredatory sharks with smaller home ranges may be completely protected in marine reserves, possibly allowing their populations to increase. These findings suggest that to be effective for shark conservation, marine reserves need to be appropriately sized for the ranging behaviour of the species intended to be protected. This study is the first of its kind to assess the effect of marine reserves on coastal sharks in New Zealand, and the first attempt to quantify the distribution of sevengill sharks, as well as other coastal shark species, in Fiordland
Science communication to support dissemination and implementation: a case study and conceptual framework
Get PDFSuccessful dissemination and implementation of evidence-based healthcare practices requires effective communication across traditional disciplinary and professional boundaries. This thesis presents a project-based inquiry into how such communication can be improved and facilitated through a theoretically grounded approach that draws from science communication, theories of learning, and dissemination and implementation science. The project involved collaboration between researchers from the School of Physiotherapy and the Centre for Science Communication at the University of Otago, with the aim to produce theoretically grounded communication resources for a case study, WellPro. The case study WellPro is an evidence-based wellbeing programme developed in the School of Physiotherapy for people with long-term conditions and multimorbidity.
Methods
The approach of this thesis project has been pragmatic and based on reading of scholarly literature involving theories from science communication, learning, and dissemination and implementation sciences, consultation with key stakeholders and development of communication resources. As the creative component of this Master of Science Communication thesis, the communication resources have been developed to promote WellPro and to support newly formed interprofessional teams to implement WellPro in healthcare districts across Aotearoa New Zealand. Through an iterative process of critical examination of theoretical frameworks and creative practice, a set of theory-based assertions were compiled into an original communication framework. The AIMS framework can be used to support the development of future communication resources for dissemination and implementation practices.
Results
The set of communication resources produced for WellPro comprise an infographic, a short promotional video, an orientation guide and outlines for team planning workshops. The development process of the communication resources involved the production and editing of creative materials including audio, film, written copy and group activities.
The creative process necessitated and guided assembly of the AIMS framework, an original conceptual framework that integrates perspectives from disparate disciplines. Drawing on theories of science communication, theories of learning and other disciplines has great potential to improve dissemination and implementation of evidence-based healthcare practices and ultimately lead to equitable healthcare outcomes. The AIMS communication framework provides a foundation for further exploration and evaluation of this approach and future development of communication resources. Thus, in addition to being an interdisciplinary collaboration in its own right, the project utilises an interdisciplinary approach to facilitating interprofessional communication in dissemination and implementation activities.
Conclusion
The WellPro communication resources presented here provide a solid foundation for further development of communication resources to support the implementation of WellPro. Of particular value in future will be the evaluation of both the programme and the communication resources used to support its implementation. The AIMS communication framework is a tool that can be used in both development of future resources and in guiding evaluation of existing resources
Rock Advertising v MWB in the UK Supreme Court: “no oral variation” clauses can be enforceable
Get PDFIn Rock Advertising Ltd v MWB Business Exchange Centres [2018] UKSC 24, the United Kingdom Supreme Court found that an informal oral variation of a contract that featured a “no oral variation” clause was not enforceable, overturning a Court of Appeal finding that it was the “no oral variation” clause that had no force (MWB v Rock Advertising [2016] EWCA Civ 553). This resolved an ongoing lack of clarity in English law, with previous Court of Appeal decisions suggesting that “no oral variation” clauses could (United Bank v Asif, CA, 11 February 2000, unreported) and could not (World Online Telecom v I-Way [2002] EWCA Civ 413, Globe Motors v TRW [2016]) be enforced. The facts of Rock Advertising provide a useful illustration of how “no oral variation” issues can arise, so we will start there, before examining the arguments for and against enforceability made in the case, and finally commenting on what the case means for the position in New Zealand.Peer Reviewe
