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  • (2012) Tebben, Jan
    Thesis
    In the marine environment chemical signals play critical roles at every organisational level. One emerging area of interest in the field of Marine Chemical Ecology is the role of bacterially derived chemical signals, in particular for the induction of larval settlement of marine invertebrates. Although bacteria have long been recognised to induce larval settlement, to date no inducer of bacterial origin has been fully characterised. In Chapter 2, I describe the isolation of the bacterium Pseudoalteromonas J010 from the surface of crustose coralline algae, which trigger the larval settlement of acroporid corals. In a bioassay-guided fashion, I characterised the metamorphic inducer in this bacterium as tetrabromopyrrole (TBP). TBP rapidly induces larval metamorphosis, however the larvae fail to attach to the substratum when exposed to TBP. The toxic nature of this compound suggests that larval metamorphosis to TBP is a stress response, rather than an evolved response to a habitat specific cue. The production of TBP may provide Pseudoalteromonas J010 with an advantage to persist in the highly competitive biofilm environment. To further explore this hypothesis, I screened the allelochemical profile of this strain and characterised further bioactive metabolites, including novel korormicin derivatives and a polybrominated pyrrole with anti-larval, anti-bacterial, anti-fungal and anti-protozoal properties (Chapter 3). Because, TBP did not explain the inductive properties of CCA on coral larval settlement, I addressed the origin and characteristics of inductive cues from CCA (Chapter 4). This resulted in the isolation of purified fractions that readily induced complete larval settlement, including i) low molecular weight organic-soluble compounds identified as glycoglycerolipids and ii) high molecular weight polymeric aqueous-soluble cue(s). These common algal metabolites may explain the highly inductive properties of CCA on acroporid coral larval settlement. I further demonstrated that these settlement inducers can be immobilised, enabling controlled settlement of coral larvae on target surfaces. Targeted larval settlement on substrata is common practise in reef rehabilitation, particularly for techniques based on sexual reproduction of corals. To improve current reef rehabilitation techniques, I explored methods to enhance post settlement survival of settled coral spat (Chapter 5).

  • (2016) Ye, Jun
    Thesis
    Organic farming relies on the activities of bacterial communities for optimal soil productivity. Understanding the responses of bacterial communities to different soil amendments, including organic fertilizers and biochars, can provide information for soil management. This thesis firstly describes the bacterial diversity and functions that are central to soil processes of organic soils and their responses to fertilizers with different C/N ratios. Secondly, this thesis analyzes the effect of a mineral-enhanced biochar (MEB) on the bacterial community of organically amended soil. Finally, the thesis investigates the direct interactions between biochar and bacteria that underpin metabolic processes in the soil. Bacterial taxa that are resilient to different fertilizers were identified and defined as the core community of organic soil. The phyla Bacteroidetes and Planctomycetes, the family Cytophagaceae and the class Acidobacteria-5 were thus found in organic soil regardless of the type of fertilizer being applied. These core bacterial taxa were further linked to the functional potential of organic soil. The C/N ratio of fertilizer was also found to have a positive correlation with microbial N assimilation in organic soil. MEB was applied in combination with compost to soil and this resulted in synergistic effects on soil properties. Specifically, the soil nitrate content was increased, which correlated with an enrichment of bacterial nitrifiers due to the MEB addition. As a consequence, plants produced larger leaves, which demonstrates that MEB could be used to manipulate specific agricultural outcomes in organic farming. To understand the detailed mechanism that supports the beneficial effects of biochar and MEB, a novel method was developed to visualize the in situ interactions between bacteria and surfaces on a single-cell level. Distinct bacterial communities were found to exist on the surface of biochar and MEBs compared to surrounding soils and surface-associated bacteria were found to have the capacities to fix carbon dioxide using chemolithotrophic processes. This provides a bacterial mechanism on how biochar and MEBs can drive carbon sequestration into the soil environment. Together, the discoveries and models presented in this thesis provide new insights into the functions of soil microbiomes in organically amended soil.

  • (2013) Ling, Gee Chong
    Thesis
    In all ecosystems bacteria predominantly live on surfaces, encapsulated within an extracellular matrix, and readily establish a multicellular and most often a multispecies organisation known as a biofilm. Microbial biofilm formation is sequential, involving multiple developmental stages and resulting in dynamic communities with cooperative traits resembling those of multicellular organisms. Such traits enable biofilms to survive in harsh environments and increase bacterial tolerance to antimicrobials, leading to a range of negative impacts such as fouling on industrial surfaces and bacterial infections. This study investigates the impact of micro-fabricated poly-dimethyl siloxane (PDMS) surfaces on initial biofilm formation as well as subsequent development, to explore the utility of surface modification and patterning for biofilm control. Bacterial populations establishing biofilms on different micro-fabricated PDMS surfaces, with micro-scale trenches from 1 to 10 μm, and smooth control surfaces, were first studied using the model biofilm forming bacterium Pseudomonas aeruginosa. Surface micro-fabrication had significant effects on several important biofilm traits, such as reduced microcolony formation, biofilm height, and biovolumes. Similar outcomes were observed for P. aeruginosa grown in other culture conditions, including the use of different culture media such as Luria Broth (LB), Vaatanen Nine Salt Solution (VNSS) and M9 Minimal Media (M9), which displayed broader impacts of different physiological conditions for biofilm development, as well as in studies using two model marine surface colonising organisms, Phaeobacter gallaeciensis and Pseudoalteromonas tunicata. Altered biofilm formation of P. aeruginosa biofilms on micro-fabricated PDMS surfaces also showed decreased chemical resistance of their biofilm populations, compared to biofilms established on control surfaces. Marine field experiments allowing diverse biofilm community formation similarly showed a marked impact on biofilm development by the modified surfaces. In addition to altered biofilm traits similar to those observed for biofilm populations, the biofilm community composition analysed using terminal restriction fragments (T-RF) for phylogenetic fingerprinting, was significantly changed at different times during the long term exposure, as revealed by the changes in T-RF profiles, and hence biofilm development. The effects of micro-fabricated PDMS surfaces on microbial biofilm formation and development are pronounced, both in laboratory settings and in marine field trials. The findings contribute to ongoing and future research for the delivery of novel biofilm control tools.

  • (2013) Ballestriero, Francesco
    Thesis
    Marine bacteria produce a wide array of biologically active metabolites involved in defence strategies and pathogenesis against a range of metazoans target organisms. In return bacteriovorous metazoans, including nematodes, have developed sophisticated strategies to neutralise or avoid such bioactives. The overall objective of this study was to investigate the antagonistic activities of bacteria-eukaryote interactions. The first aim of this thesis was to develop a novel functional (meta) genomic screen for the identification of inhibitors that target the nematode Caenorhabditis elegans. Environmental DNA from marine habitats was expressed in Escherichia coli and the resulting functional (meta) genomic libraries were screened by selective grazing of the nematode C. elegans, in a simple, rapid, high-throughput manner. Next, this project aimed at characterizing antagonistic activities that target C. elegans and study their toxic effect in the nematode. Genetic and microscopic analysis uncovered known and novel bioactive compounds including the small molecules tambjamine and violacein, which appear to facilitate bacterial colonisation and induce apoptosis in the nematode. An array of genes and gene products involved in antinematode activities was also identified including a gene encoding for a novel protein involved in a fast killing activity. Finally, this study investigated the sophisticated strategies that the nematode mounts to neutralize or avoid bacterial toxic compounds. The role of both C. elegans behavioural and immune system strategies in mediating the nematode defence in response to toxic bacterial compounds were investigated. In the nematode, a complex behavioural strategy known as aversive learning is employed to avoid violacein toxicity. When noxious violacein cannot be avoided, the toxic compound appears to activate the insulin-like signaling cascade, an evolutionary conserved innate immunity pathway. In summary, this work combines functional (meta) genomics and C. elegans genetics to identify bioactive compounds from marine bacteria and uncover animal defence strategies in response to bacterial secondary metabolites.

  • (2013) Hui, Janice Gee Kay
    Thesis
    Prophage have been identified in most sequenced bacterial genomes, however the effects of prophage genes on the host are poorly understood. Bacteriophage have been shown to play a role in the cell death phenomenon observed during biofilm development of Pseudomonas aeruginosa PAO1. The filamentous Pf4 prophage is important for mediating cell death within microcolonies, dispersal events and variant formation during biofilm development. Further, these effects were shown to result from the establishment of a superinfective phage phenotype. These observations have demonstrated the importance of phage activity in bacterial biofilms and their effects leading to biofilm variant formation and bacterial adaptation. However, little is known about the genetic mechanisms and triggers that lead to the conversion from lysogenic to the superinfective Pf4 phage. The aim of this study was to determine the factors and genes that induce the conversion of the lysogenic Pf4 phage into its lytic, superinfective form during P. aeruginosa PAO1 biofilm development. Here, the mechanisms in the induction into the superinfective phage and the gene responsible for variant formation in the biofilm were demonstrated.Two morphotypic variantswere isolated during the cell death and dispersal phase of the biofilm. These variants, the small colony variant SCV2 and spreader variant S4, carrya superinfective Pf4 phage and exhibited changes in motility and biofilm formation. Genetic analysis of these variants identified mutations within the immunity region of the Pf4 prophage genome. The mutations were identified to lie within the putative repressor c gene of the prophage and the putative promoter of the gene. Moreover, meta-genomic sequencing of pre- and post- dispersal biofilm populations identified the same mutations. The majority of the mutations were within the prophage genome at a frequency of up to 79% in contrast to mutations atless than 7% within the PAO1 genome. Collectively, these results suggest a role of the repressor C in superinfective phage conversion and strong selection for mutations within the immunity region of the phage to facilitate adaptation to biofilm lifecycle in the presence of phage infection. Variant formation is a common trait during biofilm development, as a result of genetic changes in the biofilm community. Furthermore, it has been observed that the appearance of variants from the biofilm correlated with the occurrence of the conversion into the superinfective phage. Environmental stresses have previously shown to cause phenotypic variants and were here tested for induction of the superinfective phage of PAO1 biofilm. Reactive oxygen and nitrogen species have been shown to accumulate within the biofilm microcolonies. Biofilms exposed to oxidative stress induced the conversion of the superinfective phage. Phenotypic variants are a commonly isolated cystic fibrosis patients suffering from lung infection, and have shown to exhibit mutator phenotypes with lost of mut genes of the mismatch repair system. DNA damaging agent mitomycin C and mutS mutant biofilms have also shown to induce superinfective phage. These results have indicated that the oxidative stress and DNA damage are triggers to induce mutations within the biofilm resulting in the conversion into the superinfective form. Interestingly, the OxyR oxidative stress response regulator binds within the repressor c gene, suggesting a potential role of OxyR in the conversion of the superinfective phage. Biofilms have been estimated to be associated with 80% of chronic bacterial infections and have been a challenge for treatment and therapy. While the mechanisms contributing to the conversion into the superinfective Pf4 phage remains elusive, this study has demonstrated the complexity involved in the study of phage infection in biofilms. Phage genes play a major role in the ability of the phage to cause infection against the host and provide resistance for host against infection. The proposed modelinvolves oxidative stress induced mutations within the repressor C region of the prophage andlead to selection for variants that are resistant against superinfective phage. Thus, biofilm variants carrying the superinfective Pf4 phage persist within the biofilm.As P. aeruginosa is known to be a pathogenic bacterium and is involved in several biofilm-related diseases such as chronic infections and cystic fibrosis, it is important to understand the mechanism of biofilm development and to further improve the current treatments conducted.

  • (2014) Nielsen, Shaun
    Thesis
    Most benthic marine invertebrates have a biphasic life cycle, in which a planktonic larval stage alternates with a benthic adult stage. The transition between the larval and adult stage is typically guided by habitat-derived settlement cues and thus understanding the nature and distribution of settlement cues is a central theme in larval ecology. Both coralline algae and their epiphytic bacterial biofilms can be important settlement cues for marine invertebrate larvae, but the relationship between settlement and specific communities of bacteria is largely unknown. I investigated bacterial mediated settlement for larvae of the Australian sea urchins Heliocidaris erythrogramma and Holopneustes purpurascens and compared this to the community ecology of bacteria on coralline algae. I conducted a meta-analysis of putative larval cues from macroalgae to test the importance of coralline algae as settlement cues for invertebrate larvae generally and sea urchin larvae specifically. The meta-analysis revealed that coralline algae were the most inductive macroalgae for a variety of larval groups, but epiphytic bacteria only enhanced larval settlement for a few larval groups including sea urchins. Using larvae of H. erythrogramma and H. purpurascens, I next showed in larval settlement assays that bacteria on coralline algae enhanced settlement for both species but only larvae of H. erythrogramma responded to specific variation in the bacterial community composition. This specificity of response was then tested by isolating bacteria from the surface of coralline algae, and testing these against settlement by both sea urchins. One bacterium, Pseudoalteromonas luteoviolacea, was isolated from different species of corallines and induced larval settlement of both sea urchins, suggesting a common settlement cue across coralline algae. 16S rRNA tag sequencing surveys of the relative abundance of bacteria on these algae in the field indicated a high abundance of bacterial groups not examined in larval assays and an extremely low number of the genus Pseudoalteromonas, suggesting a very low abundance of P. luteoviolacea in natural communities. In summary, this thesis presents a systematic overview of larval settlement studies using meta-analysis, a statistical framework for correlating larval settlement and bacterial communities and provides an understanding of dominant bacterial members that have yet to be examined in larval settlement studies.

  • (2014) Lutz, Carla Maree
    Thesis
    Bacteria are dominant organisms in the marine environment, providing the foundation for food webs that are essential for ecosystem structure and function. The ubiquity of bacteria in the marine environment is a result of their capacity to survive in many different niches. This is largely due to the evolution of a range of adaptive responses that allow bacteria to survive stressors such as nutrient deprivation, fluctuations in salinity and temperature and predation by bacterivorous bacteria and heterotrophic protists. The environmental persistence of marine bacteria can be attributed to multiple intra- and inter-specific strategies such as biofilm formation on biotic and abiotic surfaces, as well as interactions with other organisms, e.g. algae and heterotrophic protists. This thesis examines factors that influence resistance to protozoan grazing, specifically, the role of habitat of origin, such as association with algal surfaces, plays in resistance to predation by the ciliate Tetrahymena pyriformis. Results from this study found that bacteria derived from algal surfaces had superior biofilm growth performance when compared to seawater-derived bacterial isolates. However, seawater-derived bacterial isolates demonstrated higher resistance to grazing than surface-attached bacteria. A variety of protozoan grazing resistance mechanisms were detected, including toxicity, biofilm formation and intracellular survival. Intracellular survival was employed by Shewanella spp. as T. pyriformis feeding on these organisms led to expulsion of Shewanella-filled vacuoles into the external milieu. The role of intracellular survival as an environmental persistence mechanism for Shewanella sp. cp20 was investigated. Bottom-up environmental controls, specifically phosphorus starvation, played a role in the production of bacteria-filled expelled vacuoles. Long-term survival of Shewanella sp. cp20 within expelled vacuoles suggests that residence within vacuoles may provide a vehicle for environmental dispersal. Shewanella sp. cp20 genome analysis identified genes required for tolerance of the intracellular niche and subversion of T. pyriformis processes, e.g. secretion systems, detoxification enzymes, effector proteins and metal efflux systems, all of which have been shown to play a role in intracellular survival. This was the first study to describe intracellular survival for a member of the Shewanella genus and to identify virulence traits within this common aquatic organism.

  • (2011) Huynh, Tran Trieu
    Thesis
    Bacteria in the environment live predominantly as surface-attached communities, called biofilms, and are different to their free-living planktonic counterparts [1-3]. Biofilms are responsible for 65% of infections in humans [4] and cause problems in industrial settings. Carbon starvation has been shown to induce dispersal of biofilms of the opportunistic pathogen, Pseudomonas aeruginosa [5-6]; however, the molecular pathway controlling dispersal is unknown. This dissertation aimed to characterise the molecular pathway regulating dispersal of P. aeruginosa PAO1 biofilms during glucose starvation. An improved online biofilm monitoring system was used to continuously quantify biofilm formation and dispersal. Glucose starvation was applied to 4 day-old biofilms, resulting in dispersal. Biofilms of strains mutated in genes that are involved in biofilm formation and/or dispersal (i.e. nirS, vfr, bdlA, rpoS, lasRrhlR and Pf4 bacteriophage) behaved similarly to the wild type strain when starved, suggesting that these genes are not associated with glucose starvation-induced dispersal. Thus, the molecular pathway regulating starvation-induced dispersal may be distinct from dispersal or biofilm development mediated by those regulators (e.g. nitric oxide mediated dispersal). In contrast, cAMP was found to be important as biofilms of the cyaA mutant strain were unable to disperse. Results showed that biofilms treated with a proton ionophore were defective for dispersal, suggesting that energy derived from oxidative phosphorylation is essential for dispersal. In contrast, induction of the stringent response or translation inhibition did not inhibit dispersal of glucose-starved biofilms, indicating that neither pathway was required. Proteomic analysis revealed that more than 100 proteins were differentially expressed in starved vs. unstarved cells of both biofilm and planktonic cells. These proteins belonged to various functional classes including motility, energy and carbon metabolism. Further, a non-dispersing mutant was identified that carried a transposon insertion in a probable non-ribosomal synthase, but the product of this gene is still being characterised. Overall, the results showed that carbon starvation-induced dispersal of P. aeruginosa biofilms is cAMP and energy dependent and may also require flagellar motility. The online biofilm monitoring system is an advanced, reliable, inexpensive biofilm growth system that continuously quantifies biomass accumulation biofilms or dispersal cells in the effluent.

  • (2011) Low, Min Hui
    Thesis
    Biotic and abiotic surfaces submerged in aquatic systems are prone to rapid colonisation by marine organisms in a process called biofouling. Biofouling starts with the formation of an organic conditioning layer, which promotes adhesion of single celled microorganisms and the development of surface attached biofilm communities. Once established, biofilms provide a suitable environment for the settlement of macrofoulers, and this biofouling process results in major economic losses to various maritime industries. This study investigates the use of polydimethylsiloxane (PDMS) surfaces with specific micro-scaled surface topographies as a novel, nontoxic, alternative antifouling solution. The surfaces were placed in environmental chambers enclosed in 1.2 µm filters, and exposed to the marine environment. The effects of surface topographies on the architecture of natural established attached communities were assessed. Further, the impact of enhanced protozoan predation pressure by the addition of a heterotrophic flagellate, Rhynchomonas nasuta on biofilm architecture and composition was investigated. Results show that some micro-fabricated PDMS surfaces had microcolonies that grew along the textured grooves on the surface. Surprisingly, there was a high number of natural flagellates (2 - 5 µm) on the pre-established biofilms. Enhanced grazing by R. nasuta did not result in significant effects on biofilm biovolumes on micro-fabricated surfaces but the attached biovolumes were significantly affected by surface topographies. Four and 10 µm micro-scaled surfaces supported the lowest biovolumes compared to the flat PDMS control surfaces, indicating that these surfaces limited microbial attachment. The 10 µm surface also indicated that the enhanced grazing pressure reduced the attached community. Depending on seasonal variation, enhanced grazing pressure by R. nasuta induced shifts in microbial community composition. No correlation was observed between the micro-fabricated surfaces and community composition. This in situ study provides insights into the effects of PDMS micro-fabricated surfaces on attachment of microorganisms and the stability of the attached communities under grazing pressure. The 4 and 10 µm micro-fabricated surfaces may be promising nontoxic anti-microfouling surfaces for use in broad biomedical and industrial applications.

  • (2011) Koenig, Joanna Caroline
    Thesis
    Soil and groundwater pollution by chlorinated aliphatic hydrocarbons (CAHs) poses a threat to environmental and human health. A large number of sites around the world are contaminated with the CAHs carbon tetrachloride (CT) and perchloroethene (PCE). The employment of bacteria to remediate pollution events, termed in situ bioremediation, is currently gaining momentum as an effective and environmentally friendly treatment technology. A class of anaerobic bacteria named organohalide respiring bacteria (ORB) are able to remove PCE directly by utilizing it as an electron acceptor for growth. Unfortunately, no such bacteria are known which can transform CT in the same manner. Furthermore, CT and its partly dechlorinated product chloroform (CF) negatively impact the enzymatic degradation of PCE by ORB, creating major challenges for the bioremediation of sites co-contaminated with CT and PCE. While it cannot be used as a growth substrate, CT can be transformed by certain reactive metabolites of anaerobic bacteria. In particular, sulphide (HS-) and ferrous iron (Fe(II)) generated by sulphate- and iron-reducing bacteria (SRB and IRB) can transform CT. In this work, experimental results are presented which support the exploitation of this CT transformation pathway combined with the activity of organohalide respiring bacteria in view of bioremediating mixed CT and PCE plumes. Firstly, experiments were carried out to investigate the solvent tolerance of anaerobic bacteria in general, and revealed that fermentative strains are more tolerant to CAHs than SRB and IRB. Avenues to take advantage of this finding are proposed. Secondly, IRB and SRB enriched from several environmental sources displayed the ability to survive and produce Fe(II) and HS- respectively in the presence of CT and PCE. Thirdly, the potential of naturally-occuring electron-shuttling compounds was tested for their enhancing effects on CT transformation by Fe(II) and HS-. Phenolic-rich solutions including tea and wine were found to significantly decrease the proportion of CF formed in CT dechlorination and in some cases also increased dechlorination rates. Finally, a proof-of-concept study incorporating HS- production by Desulfovibrio vulgaris and organohalide respiration by ORB pointed to the feasibility of combining these two biogeochemical processes to remediate CT and PCE mixtures.