title: Cultivating microbial dark matter - A combination of traditional and molecular techniques

authors: King, Charlotte

abstract: The services bacteria offer the world are immense and impact people's lives daily. They were first described by Antoni van Leeuwenhoek in 1676, but bacteria have been utilised in the medical, agricultural, and food and beverage industries even before they were formally identified. Microbes’ roles in the environment for cycling of nutrients, climate regulation and pollution are becoming more well understood, and they are also utilised to answer a wide variety of research questions in disparate fields. Traditional cultivation techniques were initially developed to study bacteria and understand their functions as isolates. Such cultivation techniques have become rarer as new molecular and bioinformatic tools have been developed. These cultivation- independent techniques have allowed the study of microbial life genetic data which led to the finding that 99% of all microbial species were yet to be cultured and studied in a laboratory. The applications of these unstudied organisms could be unprecedented, though their cultivation remains challenging due to the requirement of specific nutrients that are hard to identify, very slow growth rates, environmental conditions that are difficult to replicate in a laboratory setting, live in symbiosis with other organisms, or live in extreme climates. Therefore, much of the information may be overlooked and hidden in this microbial dark matter. Methods such as metagenomics, metagenome-assembled genomes (MAGs), and single amplified genomes (SAGs) have provided us with genomic data for previously uncharacterised microbes from diverse environments. However, such data is still limited. Omics tools can provide insights into what is living there and what it can potentially do. However, this data is regularly incomplete due to the processes involved in sequencing, and conclusions are often drawn from associations relating to cultured organisms. This can lead to potentially spurious conclusions as a gene may behave differently in different species, and the absence of genes may depend on how the data was analysed. Therefore, cultured isolates of the organism are vital. To combat this issue of an uncultivated majority, it is important to have a targeted approach to culture, what is wanted, not just what is easy.

This thesis aimed to provide an easy-to-follow, straightforward workflow to cultivate target environmental organisms using standard molecular tools and methods readily accessible in any standard microbiological laboratory. The idea is to isolate and culture what is specifically wanted or needed, not just what is easy. Specifically, it combined classical cultivation and molecular techniques to target a sulphur-reducing, obligate anaerobe from an environmental sample— namely a Desulfurella sp. from a geothermal pool in Rotorua, New Zealand.
In order to provide the desired targeted approach, specific primers needed to be designed and assessed to first find the right sampling site for the initial inoculum and second to ensure the target is not lost throughout the culturing process. I designed and evaluated primers for targeting specific taxa at different stringency levels. Initially, I tested published primers (EPS_F/EPS_R, Gittel et al., 2012) aimed at Epsilonproteobacteria, which were reclassified into the Campylobacterota phylum (including Desulfurella sp.). These primers did not target Desulfurella sp., so I modified them (EPS_FM/EPS_RL) to produce a longer amplicon for broader Campylobacterota detection. However, these also amplified non-target species (e.g., Desulfobacterota). Finally, I designed a third set of primers (DS_F/DS_R) specific to Desulfurella sp., confirmed by in silico testing. These primers allow assessment of Desulfurella sp. and broader Campylobacterota before sequencing.
The second experimental chapter focuses on enriching the target using the previously described primers, classical cultivation techniques, and molecular tools. Site selection was based on the 1000 Springs Project (Power et al., 2018). Culturing was performed originally with DSMZ Desulfurella medium and later modified, investigating pH (4 and 6) and temperature (50°C and 30°C) variations and adapting it for solid media by replacing elemental sulphur with sodium polysulfide. I used a vacuum food sealer to cultivate anaerobes, an accessible and portable alternative to expensive anaerobic chambers. Automated Ribosomal Intergenic Spacer Analysis (ARISA) was used to assess axenicity before sending samples for 16S rRNA gene and whole genome sequencing. Desulfurella was detected in each culture using species-specific primers (DS_F/DS_R), enabling its phylogenetic placement. However, despite Desulfurella presence in the 16S rRNA gene amplicon sequencing, a metagenome-assembled genome (MAG) could not be produced. This thesis discusses lessons learned and suggests improvements for future studies.

text: Cultivating microbial dark matter - A combination of traditional and molecular techniques The services bacteria offer the world are immense and impact people's lives daily. They were first described by Antoni van Leeuwenhoek in 1676, but bacteria have been utilised in the medical, agricultural, and food and beverage industries even before they were formally identified. Microbes’ roles in the environment for cycling of nutrients, climate regulation and pollution are becoming more well understood, and they are also utilised to answer a wide variety of research questions in disparate fields. Traditional cultivation techniques were initially developed to study bacteria and understand their functions as isolates. Such cultivation techniques have become rarer as new molecular and bioinformatic tools have been developed. These cultivation- independent techniques have allowed the study of microbial life genetic data which led to the finding that 99% of all microbial species were yet to be cultured and studied in a laboratory. The applications of these unstudied organisms could be unprecedented, though their cultivation remains challenging due to the requirement of specific nutrients that are hard to identify, very slow growth rates, environmental conditions that are difficult to replicate in a laboratory setting, live in symbiosis with other organisms, or live in extreme climates. Therefore, much of the information may be overlooked and hidden in this microbial dark matter. Methods such as metagenomics, metagenome-assembled genomes (MAGs), and single amplified genomes (SAGs) have provided us with genomic data for previously uncharacterised microbes from diverse environments. However, such data is still limited. Omics tools can provide insights into what is living there and what it can potentially do. However, this data is regularly incomplete due to the processes involved in sequencing, and conclusions are often drawn from associations relating to cultured organisms. This can lead to potentially spurious conclusions as a gene may behave differently in different species, and the absence of genes may depend on how the data was analysed. Therefore, cultured isolates of the organism are vital. To combat this issue of an uncultivated majority, it is important to have a targeted approach to culture, what is wanted, not just what is easy.

This thesis aimed to provide an easy-to-follow, straightforward workflow to cultivate target environmental organisms using standard molecular tools and methods readily accessible in any standard microbiological laboratory. The idea is to isolate and culture what is specifically wanted or needed, not just what is easy. Specifically, it combined classical cultivation and molecular techniques to target a sulphur-reducing, obligate anaerobe from an environmental sample— namely a Desulfurella sp. from a geothermal pool in Rotorua, New Zealand.
In order to provide the desired targeted approach, specific primers needed to be designed and assessed to first find the right sampling site for the initial inoculum and second to ensure the target is not lost throughout the culturing process. I designed and evaluated primers for targeting specific taxa at different stringency levels. Initially, I tested published primers (EPS_F/EPS_R, Gittel et al., 2012) aimed at Epsilonproteobacteria, which were reclassified into the Campylobacterota phylum (including Desulfurella sp.). These primers did not target Desulfurella sp., so I modified them (EPS_FM/EPS_RL) to produce a longer amplicon for broader Campylobacterota detection. However, these also amplified non-target species (e.g., Desulfobacterota). Finally, I designed a third set of primers (DS_F/DS_R) specific to Desulfurella sp., confirmed by in silico testing. These primers allow assessment of Desulfurella sp. and broader Campylobacterota before sequencing.
The second experimental chapter focuses on enriching the target using the previously described primers, classical cultivation techniques, and molecular tools. Site selection was based on the 1000 Springs Project (Power et al., 2018). Culturing was performed originally with DSMZ Desulfurella medium and later modified, investigating pH (4 and 6) and temperature (50°C and 30°C) variations and adapting it for solid media by replacing elemental sulphur with sodium polysulfide. I used a vacuum food sealer to cultivate anaerobes, an accessible and portable alternative to expensive anaerobic chambers. Automated Ribosomal Intergenic Spacer Analysis (ARISA) was used to assess axenicity before sending samples for 16S rRNA gene and whole genome sequencing. Desulfurella was detected in each culture using species-specific primers (DS_F/DS_R), enabling its phylogenetic placement. However, despite Desulfurella presence in the 16S rRNA gene amplicon sequencing, a metagenome-assembled genome (MAG) could not be produced. This thesis discusses lessons learned and suggests improvements for future studies.

year: 2024

title: NATO's strategic communication as international public relations: The PR practitioner and the challenge of culture in the case of Kosovo

authors: Schoenberger-Orgad, Michèle Anne

subjects: circuit of culture

abstract: The role of supranational organizations is changing as they are becoming more involved in, responding to, and finding solutions for, many different events occurring in our world. This article discusses one such organization, the North Atlantic Treaty Organization (NATO) and its public relations strategies during its military campaign in Kosovo in 1999. The circuit of culture model provides the basis for a discussion of the public relations practitioner as cultural intermediary, and aligns the model with the method of critical discourse analysis to examine the public relations strategies used by NATO during this campaign. The article contends that the charismatic skill of the NATO spokesperson played an important role in establishing the organization as a humanitarian military powerhouse through discursive positioning of the organization acting in the interests of the international community. By doing so and, at the same time, NATO legitimized its own continuation as a viable supranational organization for the 21st century.

text: NATO's strategic communication as international public relations: The PR practitioner and the challenge of culture in the case of Kosovo The role of supranational organizations is changing as they are becoming more involved in, responding to, and finding solutions for, many different events occurring in our world. This article discusses one such organization, the North Atlantic Treaty Organization (NATO) and its public relations strategies during its military campaign in Kosovo in 1999. The circuit of culture model provides the basis for a discussion of the public relations practitioner as cultural intermediary, and aligns the model with the method of critical discourse analysis to examine the public relations strategies used by NATO during this campaign. The article contends that the charismatic skill of the NATO spokesperson played an important role in establishing the organization as a humanitarian military powerhouse through discursive positioning of the organization acting in the interests of the international community. By doing so and, at the same time, NATO legitimized its own continuation as a viable supranational organization for the 21st century.

year: 2011

title: Hōmai te Waiora ki Ahau: te ara whakamua - towards the establishment of construct validity

authors: Palmer, Stephanie

abstract: Hōmai te Waiora ki Ahau: te ara
whakamua, is about the development of a
tool to measure psychological wellbeing
among Māori. Why is it relevant? Because
a quick look at the June 2002 edition of
New Zealand’s Journal of Psychology will
show you that the wellbeing measures
being used in this country are not responsive to the needs of
Māori, are not based on Māori concepts or
Constructs, do not facilitate Māori participation in
te ao Māori and do not provide pathways through
which Māori can develop a positive Māori
identity.
It is highly unlikely that the tools
which psychologists use to measure
wellbeing among Māori will help Māori to
experience whānau ora and that, as we all
know, is the paramount health objective for
Māori (Ministry of Health, 2002). In this
regard, it would seem that the powers that
be in psychology are failing to meet their
Treaty obligations to Māori. Therefore,
Hōmai te Waiora ki Ahau simply aims to assist change. This presentation will briefly
describe the context of the development of
this measure, the methodologies used to
develop this tool, the outcomes of a small
pilot-study, and current challenges and
future directions for Hōmai te Waiora ki
Ahau.

text: Hōmai te Waiora ki Ahau: te ara whakamua - towards the establishment of construct validity Hōmai te Waiora ki Ahau: te ara
whakamua, is about the development of a
tool to measure psychological wellbeing
among Māori. Why is it relevant? Because
a quick look at the June 2002 edition of
New Zealand’s Journal of Psychology will
show you that the wellbeing measures
being used in this country are not responsive to the needs of
Māori, are not based on Māori concepts or
Constructs, do not facilitate Māori participation in
te ao Māori and do not provide pathways through
which Māori can develop a positive Māori
identity.
It is highly unlikely that the tools
which psychologists use to measure
wellbeing among Māori will help Māori to
experience whānau ora and that, as we all
know, is the paramount health objective for
Māori (Ministry of Health, 2002). In this
regard, it would seem that the powers that
be in psychology are failing to meet their
Treaty obligations to Māori. Therefore,
Hōmai te Waiora ki Ahau simply aims to assist change. This presentation will briefly
describe the context of the development of
this measure, the methodologies used to
develop this tool, the outcomes of a small
pilot-study, and current challenges and
future directions for Hōmai te Waiora ki
Ahau.

year: 2003