Eukaryotic microorganisms for producing lipids and antioxidants

Disclosed are compositions and methods related to eukaryotic microorganisms that can produce unsaturated fatty acids which can be purified and used.

Optimisation and analysis of three polymerase chain reaction (PCR) systems for the detection of environmentally significant microorganisms

Conventional assessments of water quality are based on the determination of bacterial indicator numbers. The detection of an indicator organism only provides presumptive evidence of the presence of harmful organisms By using polymerase chain reaction (PCR) to monitor water quality, indicators and pathogenic organisms can be specifically detected with a high sensitivity. The research illustrates the importance of optimisation and the maintenance of reaction efficiency in the accuracy and precision of the PCR.

Isolation and development of oil producing microorganisms including Thraustochytrids from Indian and Australian marine biodiversity

 The author isolated and characterized several Thraustochytrid strains from Indian and Australian marine biodiversity and compared these strains for their application in biodiesel and DHA production depend on their fatty acid profile. Strain having best productivity was further optimized for the coproduction of DHA and biodiesel.

Fermentation of omega-3 and carotenoid producing marine microorganisms

 The objective of the thesis was to understand and develop the process of fermentation in marine microorganisms for the production of omega-3 fatty acids and carotenoids. Among marine microorganisms, thraustochytrids that belong to phytoplankton group was identified as sources of omega-3 fatty acids and other valuable co-products. In this research, more efficient and cost-effective production of omega-3 oils and other value added products was discussed by addressing the below key objectives. Fermentation strategy using lower cost raw materials, particularly carbon. Screening for new strains that can naturally produce high levels of PUFAs together with useful co-products that can be harvested along with the omega-3 oil.

Identifying and integrating patient and caregiver perspectives for clinical practice guidelines on the screening and management of infectious microorganisms in hemodialysis units

Introduction: The integration of patient and caregiver input into guideline development can help to ensure that clinical care addresses patient expectations, priorities, and needs. We aimed to identify topics and outcomes salient to patients and caregivers for inclusion in the Kidney Health Australia Caring for Australasians with Renal Impairment (KHA-CARI) clinical practice guideline on the screening and management of infectious microorganisms in hemodialysis units.

Methods: A facilitated workshop was conducted with 11 participants (patients [n = 8], caregivers [n = 3]). Participants identified and discussed potential topics for inclusion in the guidelines, which were compared to those developed by the guideline working group. The workshop transcript was thematically analyzed to identify and describe the reasons underpinning their priorities.

Findings: Patients and caregivers identified a range of topics already covered by the scope of the proposed guidelines and also suggested additional topics: privacy and confidentiality, psychosocial care during/after disease notification, quality of transportation, psychosocial treatment of patients in isolation, patient/caregiver education and engagement, and patient advocacy. Five themes characterized discussion and underpinned their choices: shock and vulnerability, burden of isolation, fear of infection, respect for privacy and confidentiality, and confusion over procedural inconsistencies.

Discussion: Patients and caregivers emphasized the need for guidelines to address patient education and engagement, and the psychosocial implications of communication and provision of care in the context of infectious microorganisms in hemodialysis units. Integrating patient and caregiver perspectives can help to improve the relevance of guidelines to enhance quality of care, patient experiences, and health and psychosocial outcomes.

Recent advances in chloroplast and mitochondrial division

Stomatin, originally identified as a major protein of the human erythrocyte membrane, is widely expressed in various tissues. Orthologues are found in vertebrates, invertebrates, plants, and microorganisms. Related proteins exhibit a common core structure, termed the prohibitin (PHB) domain, with varying extensions. Stomatin has an unusual topology, similar to caveolin-1, with a hydrophobic domain embedded at the cytoplasmic side of the membrane. Additional anchoring is provided by palmitoylation and the membrane affinity of the PHB domain. Stomatin associates with cholesterol-rich microdomains (lipid rafts), forms oligomers, and thereby displays a scaffolding function by generating large protein-lipid complexes. It regulates the activity of various membrane proteins by reversibly recruiting them to lipid rafts. This mechanism of regulation has been shown for GLUT-1 and may also apply for ion channels. Stomatin is located at the plasma membrane, particularly in microvilli, in endocytic and exocytic vesicles, and cytoplasmic granules. Stomatin-carrying endosomes are highly dynamic and interact with lipid droplets suggesting a role in intracellular lipid transport. This subcellular distribution and the caveolin-like protein structure suggest important membrane organizing functions for stomatin. A general picture emerges now that cell membranes contain cholesterol-rich domains that are generated and regulated by scaffolding proteins like caveolins, stomatins, and flotillin/reggie proteins.

Biodegradation of benzo[a]pyrene in soil by Mucor sp. SF06 and Bacillus sp. SB02 co-immobilized on vermiculite

Two indigenous microorganisms, Bacillus sp. SB02 and Mucor sp. SF06, capable of degrading polycyclic aromatic hydrocarbons (PAHs) were co-immobilized on vermiculite by physical adsorption and used to degrade benzo[a] pyrene (BaP). The characteristics of BaP degradation by both free and co-immobilized microorganism were then investigated and compared. The removal rate using the immobilized bacterial-fungal mixed consortium was higher than that of the freely mobile mixed consortium. 95.3% of BaP was degraded using the co-immobilized system within 42 d, which was remarkably higher than the removal rate of that by the free strains. The optimal amount of inoculated co-immobilized system for BaP degradation was 2%. The immobilized bacterial-fungal mixed consortium also showed better water stability than the free strains. Kinetics of BaP biodegradation by co-immobilized SF06 and SB02 were also studied. The results demonstrated that BaP degradation could be well described by a zero-order reaction rate equation when the initial BaP concentration was in the range of 10—200 mg/kg. The scanning electronic microscope (SEM) analysis showed that the co-immobilized microstructure was suitable for the growth of SF06 and SB02. The mass transmission process of co-immobilized system in soil is discussed. The results demonstrate the potential for employing the bacterial-fungal mixed consortium, co-immobilized on vermiculite, for in situ bioremediation of BaP.

Disease fingerprinting with cDNA microarrays reveals distinct gene expression profiles in lethal type 1 and type 2 cytokine-mediated inflammatory reactions

Development of polarized immune responses controls resistance and susceptibility to many microorganisms. However, studies of several infectious, allergic, and autoimmune diseases have shown that chronic type-1 and type-2 cytokine responses can also cause significant morbidity and mortality if left unchecked. We used mouse cDNA microarrays to molecularly phenotype the gene expression patterns that characterize two disparate but equally lethal forms of liver pathology that develop in Schistosoma mansoni infected mice polarized for type-1 and type-2 cytokine responses. Hierarchical clustering analysis identified at least three groups of genes associated with a polarized type-2 response and two linked with an extreme type-1 cytokine phenotype. Predictions about liver fibrosis,  apoptosis, and granulocyte recruitment and activation generated by the microarray studies were confirmed later by traditional biological assays. The data show that cDNA microarrays are useful not only for determining  coordinated gene expression profiles but are also highly effective for molecularly “fingerprinting” diseased tissues. Moreover, they illustrate the potential of genome-wide approaches for generating comprehensive views on the molecular and biochemical mechanisms regulating infectious  disease pathogenesis.

UV-induced DNA damage promotes resistance to the biotrophic pathogen Hyaloperonospora parasitica in Arabidopsis

Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280–320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response.

Aspects of the production of viscous capsular material by the yoghurt starter, streptococcus thermophilus

The technology of modern fermented milk production is not complicated and relies largely on the characteristics of the microorganisms used in its manufacture. Biochemical substances excreted by the starter cultures contribute to the chemical, physical and organoleptic properties of cultured milks. Chemical and organoleptic properties of yoghurt starter cultures have been widely studied over several decades. Conversely the biosynthetic processes and genetic control of the production of viscous extracellular material (slime) by selected thermophillic streptococci is still insufficiently understood. This study attempted to elucidate physiological aspects and the genetic control of slime production. An attempt to chemically induce ropiness was also preformed. Twenty strains of Gram positive, thermo-tolerant, milk dotting, catalase negative cocci were collected from a variety of sources. All strains were identified as Streptococcus thermophilus. Four of the isolates were identified as capable of producing an extracellular, ‘ropy’ capsular material. A negative staining method for highlighting capsular material under light microscopy was described. Ropy isolates displayed thick capsular zones of between 6-8 μm. The isolates graded as non-ropy produced only small capsular zones (less than 2 μm); two variants displayed no capsular material. Instability of the ropy phenotype during subculture and prolonged storage was described for all four ropy isolates at varied temperatures. Instability during transfer was reported as moderate with a loss of no more than 45% of ropy colonies after 15 subcultures at 48°C A significant increase in instability, during transfer, associated with an increase in incubation temperature (37-48°C) was also reported. Prolonged storage of ropy variants over ten days resulted in a drop in the number of ropy colonies. The loss was minimal when cultures were stored at 8°C, but excessive (approaching 100%) at 37°C This suggested the presence of capsular degradative substances. Analysis of the plasmid profiles of 20 strains identified only two strains harboured plasmid DNA. All plasmids were small, less than 23kilobases, and each strain possessed a single plasmid species. Only one ropy strain contained plasmid DNA that was shown, with the aid of curing experiments, not to be linked to production of the ropy phenotype. The amino acid analogue p-fluoro-DL-phenylalanine was unsuccessful in generating ropy colonies from non-ropy variants of Streptococcus thermophilus at low concentrations. Some technological considerations for the use of ropy variants of Streptococcus thermophilus in yoghurt starter cultures were made.

Effects of phytophthora cinnamomi on heathland flora and fauna of the Eastern Otway Ranges

The plant pathogen, Phytophthora dnnamomi, is a cause of dieback disease observed in sclerophyll vegetation in Australia, The effects of P. dnnamomi on flora and fauna were studied at two locations in heathland vegetation near the coastal town of Anglesea, Victoria. The pathogen was isolated from soils beneath diseased heathland plants. The extent of diseased vegetation was assessed by the presence and absence of highly sensitive indicator species, Xanthorrhoea australis and hopogon ceratophyllus. The characteristics of heathland vegetation exhibiting dieback disease associated with the presence of P. dnnamomi were investigated. Plant species richness was similar between diseased and non-diseased areas however diseased areas were characterised by significant declines in the cover and frequency of susceptible species, increases in resistant species and increases in percent cover of open ground. Compared to non-diseased areas, diseased areas exhibited fewer shrub species and decreased shrub cover. The percentage cover and number of species of sedges, lilies and grasses were higher in diseased areas. Structural differences were significant between 0-0.6 m with decreased cover of vegetation in diseased areas. Differences in structure between diseased and non-diseased areas were not as great as expected due to increases in the cover of resistant species. A number of regenerating X australis were observed in post-disease areas. Cluster analysis of floristic data could clearly separate diseased and non-diseased trap stations. The population dynamics and habitat use of eight small mammal species present were compared in diseased and non-diseased areas using trapping and radio-tracking techniques. The number of small mammal species captured in post-disease areas was significantly lower than non-diseased areas. Mean captures of Antechinus stuartii and Rattus fiisdpes were significantly lower in diseased areas on Grid B. Mean captures of Rattus lutreolus were significantly lower in diseased areas on both study grids. Significant differences were not observed in every season over the two year study period. Radio tracking revealed more observations of Sminthopsis leucopus in non-diseased vegetation than in diseased. Cercartetus nanus was frequently observed to utilise the disease susceptible X. australis for nesting. At one location, the recovery of vegetation and small mammal communities in non-diseased and diseased vegetation after fuel reduction burning was monitored for three years post-fire. Return of plant species after fire in both disease classes were similar, reaching 75% of pre-fire richness after three years. Vegetation cover was slower to return after fire in diseased areas. Of the seven small mammal species captured pre-fire, five were regularly captured in the three years after fire. General linear model analysis revealed a significant influence of disease on capture rates for total small mammals before fire and a significant influence of fire on capture rates for total small mammals after fire. After three years, the influence of fire on capture rates was reduced no significant difference was detected between disease classes. Measurements of microclimate indicate that diseased, burnt heathland was likely to experience greater extremes of temperature and wind speed. Seeding of diseased heathland with X. australis resulted in the establishment of seedlings of this sensitive species. The reported distributions of the mamma] species in Victoria were analysed to determine which species were associated with the reported distribution of dieback disease. Twenty-two species have more than 20% of their known distribution in diseased areas. Five of these species, Pseudomys novaehollandiae, Pseudomys fumeust Pseudomys shortridgei, Potorous longipes and Petrogale pencillata are rare or endangered in Victoria. Four of the twenty-two species, Sminthopsis leucopus, Isoodon obesulus, Cercartetus nanus and Rottus lutreolus am observed in Victorian heathlands. Phytophthora cinnamomi changes both the structure and floristics of heathland vegetation in the eastern Qtway Ranges. Small mammals respond to these changes through decreased utilisation of diseased heathland. The pathogen threatens the diversity of species present and future research efforts should be directed towards limiting its spread and rehabilitating diseased areas.

Use of phosphite as a control for Phytophthora cinnamomi in southeastern Victorian vegetation communities

One of the major aims of the research presented in this thesis was to assist managers of native vegetation communities in southeastern Australia in understanding the dynamics of P. cinnamomi with an important ecological species, Xanthorrhoea australis. It trialed the use of phosphite in large-scale field applications to establish the usefulness of this management option for the first time on Victorian flora. This thesis describes the process of disease development within mature X. Australia plants. For the first time it was shown that within X. australis plants, secondary disease symptoms are related to the percentage of stem that has been infested by the disease. It was evident that after initial invasion the pathogen moves via root xylem and throughout the plant within vascular to the stem, especially within the desmium. The research shows that the pathogen could not be isolated consistently even though it was considered to be responsible for disease symptoms. Trials of a control fungicide (Foli-R-fos 200) shows that protection occurs in many susceptible plants when 2 and 6g a.i./L phosphite is applied. Phytotoxicity occurred in native plants at Anglesea and within controlled environment trials when using ≥ 6g a.i./L. It will be shown that 2g a.i./L phosphite controls disease in sprayed plots within heathlands at Anglesea and a recently burnt coastal woodland community at Wilson’s Promontory. The proportion of healthy X. australis plants treated with phosphite was significantly higher than the proportion in control plots without phosphite. The research shows that phosphite was recovered from leaves of three species treated with Foli-R-fos 200 in the field. For the first time it has been shown that seed germination was reduced in two species when high concentrations of phosphite were applied. The first documentation of the effect that phosphite has on soil properties showed that nitrogen and oxidised organic carbon were the only parameters to alter significantly. This thesis provides answers to some important questions, answers that can now be used by managers in formulating better policies and actions at an operational level. There has been a dire need in Victoria to address many issues regarding P. cinnamomi and this thesis provides relevant and informative approaches to disease control, and a better understanding of the disease progress.

Aspects of the interaction between Xanthorrhoea australis and Phytophthora cinnamomi in south-western Victoria, Australia

Diseases in natural ecosystems are often assumed to be less severe than those observed in domestic cropping systems due to the extensive biodiversity exhibited in wild vegetation communities. In Australia, it is this natural biodiversity that is now under threat from Phytophthora cinnamomi. The soilborne Oomycete causes severe decline of native vegetation communities in south-western Victoria, Australia, disrupting the ecological balance of native forest and heathland communities. While the effect of disease caused by P. cinnamomi on native vegetation communities in Victoria has been extensively investigated, little work has focused on the Anglesea healthlands in south-western Victoria. Nothing is known about the population structure of P. cinnamomi at Anglesea. This project was divided into two main components to investigate fundamental issues affecting the management of P. cinnamomi in the Anglesea heathlands. The first component examined the phenotypic characteristics of P. cinnamomi isolates sampled from the population at Anglesea, and compared these with isolates from other regions in Victoria, and also from Western Australia. The second component of the project investigated the effect of the fungicide phosphonate on the host response following infection by P. cinnamomi. Following soil sampling in the Anglesea heathlands, a collection of P, cinnamomi isolates was established. Morphological and physiological traits of each isolate were examined. All isolates were found to be of the A2 mating type. Variation was demonstrated among isolates in the following characteristics: radial growth rate on various nutrient media, sporangial production, and sporangial dimensions. Oogonial dimensions did not differ significantly between isolates. Morphological and physiological variation was rarely dependant on isolate origin. To examine the genetic diversity among isolates and to determine whether phenotypic variation observed was genetically based, Random Amplified Polymorphic DNA (RAPD) analyses were conducted. No significant variation was observed among isolates based on an analysis of molecular variance (AMQVA). The results are discussed in relation to population biology, and the effect of genetic variation on population structure and population dynamics. X australis, an arborescent monocotyledon indigenous to Australia, is highly susceptible to infection by P. cinnamomi. It forms an important component of the heathland vegetation community, providing habitat for native flora and fauna, A cell suspension culture system was developed to investigate the effect of the fungicide phosphonate on the host-pathogen interaction between X. australis and P. cinnamomi. This allowed the interaction between the host and the pathogen to be examined at a cellular level. Subsequently, histological studies using X. australis seedlings were undertaken to support the cellular study. Observations in the cell culture system correlated well with those in the plant. The anatomical structure of X australis roots was examined to assist in the interpretation of results of histopathological studies. The infection of single cells and roots of X. australis, and the effect of phosphonate on the interaction are described. Phosphonate application prior to inoculation with P. cinnamomi reduced the infection of cells in culture and of cells in planta. In particular, phosphonate was found to stimulate the production of phenolic material in roots of X australis seedlings and in cells in suspension cultures. In phosphonate-treated roots of X australis seedlings, the deposition of electron dense material, possibly lignin or cellulose, was observed following infection with P. cinnamomi. It is proposed that this is a significant consequence of the stimulation of plant defence pathways by the fungicide. Results of the study are discussed in terms of the implications of the findings on management of the Anglesea heathlands in Victoria, taking into account variation in pathogen morphology, pathogenicity and genotype. The mode of action of phosphonate in the plant is discussed in relation to plant physiology and biochemistry.