title: Identification of genes regulated by cold temperature - a study of mdtJI in E. coli O157:H7

authors: Urlich, Shelley

subjects: E. coli O157:H7

abstract: E. coli O157:H7, a serious food-borne pathogen, is capable of adapting to distinctly different environments, ranging from the ruminant, to soil and water.  Critical to the success of this pathogen, is the ability to adapt rapidly to changes in the environment. These changes rarely occur in isolation and bacteria, through regulatory networks, can respond to multiple challenges simultaneously, often through master regulators. Understanding the adaptive process of E. coli O157:H7, particularly in response to cold temperatures, is vital for elucidating the pathogens ability to persist during food processing. Of major concern to the meat industry, is the ability of E. coli O157:H7 to survive multiple hurdle intervention strategies that include both chilling and freezing. 

The aim of this project was to identify genes involved in the cold shock response of E. coli O157:H7 when exposed to refrigeration temperatures (4, 0, -1.5 C). We hypothesized that E. coli O157:H7 is able to withstand chill temperatures by up-regulating genes that allow survival in unfavorable conditions, for example, when the cell is expelled from the ruminant host, into soil or water environments. It is likely that E. coli O157:H7, utilizing similar adaptive mechanisms can withstand prolonged periods at refrigeration temperatures. Furthermore, we speculated that quorum sensing (QS) has overtime become integrated into these adaptive pathways, potentially forming an integrated component of the E. coli O157:H7 adaptive stress response, including the cold shock response.  

A number of genes were identified as being up-regulated in E. coli O157:H7 during incubation at 4 C on meat. Of these, four were of particular interest, as they had been previously linked to cell survival processes: slp (carbon starvation lipoprotein), hslJ (heat inducible protein), mdtI (multidrug efflux pump) and mdtJ (multidrug efflux pump). RT-PCR data showed that slp and the mdtJI complex are expressed more at refrigeration temperatures than at 37 C while hslJ expression was greatest at 37 C.  mdtJI was selected for further analysis, because mdtJI was the only gene that was not expressed at 37 C when grown on BHI media, plus these genes had, at the initiation of this project, not been annotated or assigned function. Using a luxCDABE promoter reporter, real time analysis of the effect of temperature downshift on mdtJI expression was confirmed. Furthermore, expression was demonstrated to increase at high cell density at 37 C, suggesting a regulatory connection to quorum sensing. This coupled with the finding that 400nt upstream of the mdtJI promoter was a gene encoding a transporter of AI-2, a QS autoinducer, suggested a link to the LuxS/AI-2 QS regulatory network. Data presented here was unable to confirm a regulatory link to AI-2 itself but it did reveal a link to LuxS.    

In conclusion, data presented in this thesis has confirmed that mdtJI is involved in the adaptive response, specifically adaptation to cold temperatures in E. coli O157:H7, and possibly, to growth cessation. The influence of LuxS on mdtJI expression in E. coli O157:H7 is most likely to be through metabolic activity, rather than via a QS mechanism.

text: Identification of genes regulated by cold temperature - a study of mdtJI in E. coli O157:H7 E. coli O157:H7, a serious food-borne pathogen, is capable of adapting to distinctly different environments, ranging from the ruminant, to soil and water.  Critical to the success of this pathogen, is the ability to adapt rapidly to changes in the environment. These changes rarely occur in isolation and bacteria, through regulatory networks, can respond to multiple challenges simultaneously, often through master regulators. Understanding the adaptive process of E. coli O157:H7, particularly in response to cold temperatures, is vital for elucidating the pathogens ability to persist during food processing. Of major concern to the meat industry, is the ability of E. coli O157:H7 to survive multiple hurdle intervention strategies that include both chilling and freezing. 

The aim of this project was to identify genes involved in the cold shock response of E. coli O157:H7 when exposed to refrigeration temperatures (4, 0, -1.5 C). We hypothesized that E. coli O157:H7 is able to withstand chill temperatures by up-regulating genes that allow survival in unfavorable conditions, for example, when the cell is expelled from the ruminant host, into soil or water environments. It is likely that E. coli O157:H7, utilizing similar adaptive mechanisms can withstand prolonged periods at refrigeration temperatures. Furthermore, we speculated that quorum sensing (QS) has overtime become integrated into these adaptive pathways, potentially forming an integrated component of the E. coli O157:H7 adaptive stress response, including the cold shock response.  

A number of genes were identified as being up-regulated in E. coli O157:H7 during incubation at 4 C on meat. Of these, four were of particular interest, as they had been previously linked to cell survival processes: slp (carbon starvation lipoprotein), hslJ (heat inducible protein), mdtI (multidrug efflux pump) and mdtJ (multidrug efflux pump). RT-PCR data showed that slp and the mdtJI complex are expressed more at refrigeration temperatures than at 37 C while hslJ expression was greatest at 37 C.  mdtJI was selected for further analysis, because mdtJI was the only gene that was not expressed at 37 C when grown on BHI media, plus these genes had, at the initiation of this project, not been annotated or assigned function. Using a luxCDABE promoter reporter, real time analysis of the effect of temperature downshift on mdtJI expression was confirmed. Furthermore, expression was demonstrated to increase at high cell density at 37 C, suggesting a regulatory connection to quorum sensing. This coupled with the finding that 400nt upstream of the mdtJI promoter was a gene encoding a transporter of AI-2, a QS autoinducer, suggested a link to the LuxS/AI-2 QS regulatory network. Data presented here was unable to confirm a regulatory link to AI-2 itself but it did reveal a link to LuxS.    

In conclusion, data presented in this thesis has confirmed that mdtJI is involved in the adaptive response, specifically adaptation to cold temperatures in E. coli O157:H7, and possibly, to growth cessation. The influence of LuxS on mdtJI expression in E. coli O157:H7 is most likely to be through metabolic activity, rather than via a QS mechanism.

year: 2009

title: Population Genetics and Autecology of the Endemic Shrub Epiphyte Pittosporum cornifolium

authors: Clarkson, Fiona Marie

subjects: autecology

abstract: A comprehensive investigation of the population genetics and autecology of the endemic New Zealand shrub epiphyte Pittosporum cornifolium (Pittosporaceae) is presented. Pittosporum cornifolium has a wide geographic range and is well adapted to a variety of lifestyles namely terrestrial, rupestral, and more commonly, epiphytic. The primary habitats of P. cornifolium are lowland and coastal ecosystems which, in recent times (<200 years) have been subjected to widespread clearance and fragmentation resulting in major reductions to the species potential population range. The study focused on five populations in the North Island from Coromandel to Taranaki where habitat loss of lowland and coastal ecosystems has been significant. Population-level genetic analysis using Inter-Simple Sequence Repeat (ISSR) markers revealed that while P. cornifolium exhibited high genetic diversity at the species-level, genetic diversity was relatively low at the population-level. The outcrossing dioecious breeding system and unique evolutionary history of P. cornifolium are likely to be key factors influencing the observed high intra-specific diversity whereas reduced genetic diversity at population-level is probably due to geographic isolation caused by recent habitat fragmentation. Ecological parameters were investigated to determine the current ecological status of the five populations and results did not reveal any substantial ecological impediments to regeneration and dispersal modes. Ecological data were incorporated with information from national data sets to provide a more comprehensive overview of P. cornifolium autecology and to develop a predicted environmental distribution map. Key findings indicate P. cornifolium is typically affiliated with old growth forest systems and well drained low nutrient substrates, while low mean daily temperatures (<0.6°C) restrict environmental distribution. Both genetic and autecological research was applied to determine levels of intra-specific divergence in cultivated P. cornifolium individuals from the Poor Knights Islands (outer Hauraki Gulf), which are morphologically distinct from mainland forms. The Poor Knights Islands individuals were the most genetically distinct as revealed by ISSR analysis, having higher pairwise levels of genetic distance than iii mainland populations as well as more unique loci. A single mutation in the sequence of the Internal Transcribed Spacer (ITS) region was revealed in the Poor Knights Islands individuals, distinguishing them from mainland P. cornifolium and additional members of a monophyletic clade which have shared ITS sequences. Furthermore, P. cornifolium from the Poor Knights Islands have significant morphological and anatomical differences such as larger leaves and leaf tissue depths. Long term isolation on the offshore islands is likely to have had the most significant effect on this population divergence. The differences in the Poor Knights Islands individuals may warrant the delineation of a new subspecies or even species. However, a more comprehensive examination of the taxon across its mainland range, the Poor Knights Island group, and other northern offshore islands where the species is present is recommended to clarify current inferences. The results of this research have provided a framework for the development of species specific conservation and restoration strategies for P. cornifolium and reveal the importance of provenance and microhabitat (lifestyle) when sourcing seed for reintroduction projects.

text: Population Genetics and Autecology of the Endemic Shrub Epiphyte Pittosporum cornifolium A comprehensive investigation of the population genetics and autecology of the endemic New Zealand shrub epiphyte Pittosporum cornifolium (Pittosporaceae) is presented. Pittosporum cornifolium has a wide geographic range and is well adapted to a variety of lifestyles namely terrestrial, rupestral, and more commonly, epiphytic. The primary habitats of P. cornifolium are lowland and coastal ecosystems which, in recent times (<200 years) have been subjected to widespread clearance and fragmentation resulting in major reductions to the species potential population range. The study focused on five populations in the North Island from Coromandel to Taranaki where habitat loss of lowland and coastal ecosystems has been significant. Population-level genetic analysis using Inter-Simple Sequence Repeat (ISSR) markers revealed that while P. cornifolium exhibited high genetic diversity at the species-level, genetic diversity was relatively low at the population-level. The outcrossing dioecious breeding system and unique evolutionary history of P. cornifolium are likely to be key factors influencing the observed high intra-specific diversity whereas reduced genetic diversity at population-level is probably due to geographic isolation caused by recent habitat fragmentation. Ecological parameters were investigated to determine the current ecological status of the five populations and results did not reveal any substantial ecological impediments to regeneration and dispersal modes. Ecological data were incorporated with information from national data sets to provide a more comprehensive overview of P. cornifolium autecology and to develop a predicted environmental distribution map. Key findings indicate P. cornifolium is typically affiliated with old growth forest systems and well drained low nutrient substrates, while low mean daily temperatures (<0.6°C) restrict environmental distribution. Both genetic and autecological research was applied to determine levels of intra-specific divergence in cultivated P. cornifolium individuals from the Poor Knights Islands (outer Hauraki Gulf), which are morphologically distinct from mainland forms. The Poor Knights Islands individuals were the most genetically distinct as revealed by ISSR analysis, having higher pairwise levels of genetic distance than iii mainland populations as well as more unique loci. A single mutation in the sequence of the Internal Transcribed Spacer (ITS) region was revealed in the Poor Knights Islands individuals, distinguishing them from mainland P. cornifolium and additional members of a monophyletic clade which have shared ITS sequences. Furthermore, P. cornifolium from the Poor Knights Islands have significant morphological and anatomical differences such as larger leaves and leaf tissue depths. Long term isolation on the offshore islands is likely to have had the most significant effect on this population divergence. The differences in the Poor Knights Islands individuals may warrant the delineation of a new subspecies or even species. However, a more comprehensive examination of the taxon across its mainland range, the Poor Knights Island group, and other northern offshore islands where the species is present is recommended to clarify current inferences. The results of this research have provided a framework for the development of species specific conservation and restoration strategies for P. cornifolium and reveal the importance of provenance and microhabitat (lifestyle) when sourcing seed for reintroduction projects.

year: 2011

title: Ascending the Poutama: Culturally responsive diabetes care for Māori communities

authors: Crosswell, Rebekah Jane

subjects: culturally responsive

abstract: Introduction
Type 2 diabetes (T2D) significantly impacts Māori populations in Aotearoa New Zealand (NZ) who experience health disparities including suboptimal disease management.

Aim
To explore culturally responsive approaches to supporting Māori living with T2D from the perspective of healthcare providers.

Methods
Nine semi-structured interviews were conducted with clinicians from various disciplines (general practitioner, podiatrist, exercise physiologist, three kaiāwhina, dietitian, two pharmacists); five clinicians identify as Māori, four as non-Māori. Kaupapa Māori inductive thematic analysis was used.

Results
Four key themes emerged: empowering patient-centred communication, whānau involvement, interprofessional collaboration, and culturally appropriate analogies.

Discussion
The findings underscore the need for holistic, culturally responsive T2D care that prioritises culturally appropriate communication to improve patient engagement, health outcomes and address Māori health disparities.

text: Ascending the Poutama: Culturally responsive diabetes care for Māori communities Introduction
Type 2 diabetes (T2D) significantly impacts Māori populations in Aotearoa New Zealand (NZ) who experience health disparities including suboptimal disease management.

Aim
To explore culturally responsive approaches to supporting Māori living with T2D from the perspective of healthcare providers.

Methods
Nine semi-structured interviews were conducted with clinicians from various disciplines (general practitioner, podiatrist, exercise physiologist, three kaiāwhina, dietitian, two pharmacists); five clinicians identify as Māori, four as non-Māori. Kaupapa Māori inductive thematic analysis was used.

Results
Four key themes emerged: empowering patient-centred communication, whānau involvement, interprofessional collaboration, and culturally appropriate analogies.

Discussion
The findings underscore the need for holistic, culturally responsive T2D care that prioritises culturally appropriate communication to improve patient engagement, health outcomes and address Māori health disparities.

year: 2025