title: The DNA Binding Protein Lsr2 from Mycobacterium tuberculosis

authors: Summers, Emma Louise

subjects: DNA binding protein

abstract: Lsr2 is a small, basic DNA binding protein that is highly conserved in mycobacteria and related actinomycetes. Lsr2 is essential for growth in Mycobacterium tuberculosis and previous studies have shown that Lsr2 is involved in down-regulating a wide range of genes involved in cell wall synthesis and metabolic functions. This regulatory function is likely to influence bacterial growth and survival.

This research investigated the biochemistry and 3D structure of Lsr2 from M. tuberculosis. Transmission electron microscopy (TEM) analysis of Lsr2 in complex with DNA revealed a regular fibril-like arrangement of protein coating double-stranded DNA. In addition, it was shown that Lsr2 physically protected DNA from DNase activity. The structure of the C-terminal DNA binding domain of Lsr2, determined by others part way through this research, prompted site directed mutagenesis of residues proposed to interact with DNA. Modification of arginine residues significantly reduced the binding of Lsr2 to DNA and fibril-like structures were not observed using TEM, for arginine mutants.

The first crystal structure of the N-terminal domain of Lsr2 is reported here. Two high resolution structures in monoclinic and hexagonal space groups were solved using X-ray crystallography and ab initio phasing. Proteolytic processing of the N-terminus of Lsr2 was revealed by the structure in P2₁ and this process was recreated using the protease trypsin which resulted in crystal formation in a P3₁21 space group. Both structures show linear chains of dimeric N-terminal Lsr2, as shown by crystallographic symmetry, linked by overlapping anti-parallel β-sheets, revealing a mechanism of protein oligomerisation. Oligomerisation only occurs after the removal of the first three residues M1, A2 and K3. In solution, protein oligomerisation was recreated with trypsin, resulting in the formation of large protein complexes. A change in DNA topology after the addition of trypsin to full-length Lsr2/DNA complexes was observed using TEM. This mechanism is likely to be important to M. tuberculosis under “stress” conditions where proteases are known to be upregulated and where cross-linking and condensation of DNA is critical.

text: The DNA Binding Protein Lsr2 from Mycobacterium tuberculosis Lsr2 is a small, basic DNA binding protein that is highly conserved in mycobacteria and related actinomycetes. Lsr2 is essential for growth in Mycobacterium tuberculosis and previous studies have shown that Lsr2 is involved in down-regulating a wide range of genes involved in cell wall synthesis and metabolic functions. This regulatory function is likely to influence bacterial growth and survival.

This research investigated the biochemistry and 3D structure of Lsr2 from M. tuberculosis. Transmission electron microscopy (TEM) analysis of Lsr2 in complex with DNA revealed a regular fibril-like arrangement of protein coating double-stranded DNA. In addition, it was shown that Lsr2 physically protected DNA from DNase activity. The structure of the C-terminal DNA binding domain of Lsr2, determined by others part way through this research, prompted site directed mutagenesis of residues proposed to interact with DNA. Modification of arginine residues significantly reduced the binding of Lsr2 to DNA and fibril-like structures were not observed using TEM, for arginine mutants.

The first crystal structure of the N-terminal domain of Lsr2 is reported here. Two high resolution structures in monoclinic and hexagonal space groups were solved using X-ray crystallography and ab initio phasing. Proteolytic processing of the N-terminus of Lsr2 was revealed by the structure in P2₁ and this process was recreated using the protease trypsin which resulted in crystal formation in a P3₁21 space group. Both structures show linear chains of dimeric N-terminal Lsr2, as shown by crystallographic symmetry, linked by overlapping anti-parallel β-sheets, revealing a mechanism of protein oligomerisation. Oligomerisation only occurs after the removal of the first three residues M1, A2 and K3. In solution, protein oligomerisation was recreated with trypsin, resulting in the formation of large protein complexes. A change in DNA topology after the addition of trypsin to full-length Lsr2/DNA complexes was observed using TEM. This mechanism is likely to be important to M. tuberculosis under “stress” conditions where proteases are known to be upregulated and where cross-linking and condensation of DNA is critical.

year: 2012

title: Cost-effective policies for improving water quality by reducing nitrate emissions from diverse dairy farms: An abatement–cost perspective

authors: Doole, Graeme J.

subjects: agricultural production

abstract: A flexible and replicable approach for the appraisal of the cost-effectiveness of various policy instruments for the regulation of nonpoint pollution from heterogeneous farms is presented. The framework is employed to evaluate three regulatory instruments for achieving reductions in nitrate leaching among a large population of dairy farms in the Waikato region of New Zealand. These instruments are (1) a differentiated policy that allows individual abatement effort to vary among farms to satisfy a catchment-wide reduction in leaching load, (2) a uniform policy that requires all farms to reduce emissions by a constant proportion, and (3) a threshold policy that requires all farms to emit beneath a given threshold leaching load. A 30% reduction in leaching will cost around 1.4, 1.96, and 4.41 million dollars across the catchment with a differentiated, uniform, and threshold policy, respectively. In comparison, a 50% reduction in leaching will cost around 7.77, 8.58, and 11.24 million dollars across the catchment with a differentiated, uniform, and threshold policy, respectively. Thus, a differentiated policy achieves required environmental outcomes at least cost. This result highlights the value of modelling heterogeneous farms in policy evaluation for nonpoint pollution control.

text: Cost-effective policies for improving water quality by reducing nitrate emissions from diverse dairy farms: An abatement–cost perspective A flexible and replicable approach for the appraisal of the cost-effectiveness of various policy instruments for the regulation of nonpoint pollution from heterogeneous farms is presented. The framework is employed to evaluate three regulatory instruments for achieving reductions in nitrate leaching among a large population of dairy farms in the Waikato region of New Zealand. These instruments are (1) a differentiated policy that allows individual abatement effort to vary among farms to satisfy a catchment-wide reduction in leaching load, (2) a uniform policy that requires all farms to reduce emissions by a constant proportion, and (3) a threshold policy that requires all farms to emit beneath a given threshold leaching load. A 30% reduction in leaching will cost around 1.4, 1.96, and 4.41 million dollars across the catchment with a differentiated, uniform, and threshold policy, respectively. In comparison, a 50% reduction in leaching will cost around 7.77, 8.58, and 11.24 million dollars across the catchment with a differentiated, uniform, and threshold policy, respectively. Thus, a differentiated policy achieves required environmental outcomes at least cost. This result highlights the value of modelling heterogeneous farms in policy evaluation for nonpoint pollution control.

year: 2011

title: Observations by and conversations with health workers and hospital personnel involved in transferring Maori patients and whānau to Waikato Hospital in Aotearoa New Zealand

authors: Masters-Awatere, Bridgette

subjects: Science & Technology

abstract: The predominant focus of Aotearoa New Zealand’s public health system on biomedical models of health has left little room for meaningful engagement with holistic indigenous approaches. Culturally appropriate provision and support are recognized for their relevance and importance during hospital transferals. Hospital staff involved in transfers to one of New Zealand’s trauma centers share their observations of whānau Māori engagement during an admission away from their home base. Sixteen key informants share their experiences, which are presented as strategies and challenges to whānau engagement. Three main themes highlight challenges within the health system that make it difficult for hospital staff to engage whānau in the desired ways and as often as both parties would like. Key informants described services and practices that are not designed with patients and their whānau in mind; instead they are designed by clinicians around the needs of administrative systems. As employees within the public health system, key informants felt powerless to challenge dominant settings. Nevertheless, employees managed to circumnavigate processes. Our findings highlight the need for continued decolonization and anti-racism work within public health settings.

text: Observations by and conversations with health workers and hospital personnel involved in transferring Maori patients and whānau to Waikato Hospital in Aotearoa New Zealand The predominant focus of Aotearoa New Zealand’s public health system on biomedical models of health has left little room for meaningful engagement with holistic indigenous approaches. Culturally appropriate provision and support are recognized for their relevance and importance during hospital transferals. Hospital staff involved in transfers to one of New Zealand’s trauma centers share their observations of whānau Māori engagement during an admission away from their home base. Sixteen key informants share their experiences, which are presented as strategies and challenges to whānau engagement. Three main themes highlight challenges within the health system that make it difficult for hospital staff to engage whānau in the desired ways and as often as both parties would like. Key informants described services and practices that are not designed with patients and their whānau in mind; instead they are designed by clinicians around the needs of administrative systems. As employees within the public health system, key informants felt powerless to challenge dominant settings. Nevertheless, employees managed to circumnavigate processes. Our findings highlight the need for continued decolonization and anti-racism work within public health settings.

year: 2020