April 2012

Event Date: 
Wednesday, May 30, 2012 - 18:00 - 18:15
Institution: 
UNSW
Title: 

Deep sequencing of evolving populations in bacterial biofilms

Abstract: 

 

Bacterial communities growing as biofilms are subject to a distinct lifecyle, featuring initial surface attachment, microcolony formation and dispersal of cells. Bacterial biofilms are sometimes characterised by high levels of heritable phenotypic variants, presumably resulting from genetic diversification during the biofilm lifecyle. As biofilms are a favoured lifestyle of many environmental and pathogenic bacteria, identifying the evolutionary processes responsible for this diversification has important implications, both for our understanding of ecological processes, such as niche adaptation, and to clinically relevant questions, such as the evolution of antibiotic resistance.
I've used longitudinal genome-wide deep sequencing to reveal the underlying genetic structure of bacterial populations growing as biofilms, for the model organisms Phaeobacter gallaeciensis 2.10 (an abundant marine bacterium) and Pseudomonas aeruginosa 18A (a clinical Cystic Fibrosis isolate). Biofilms were grown under defined laboratory conditions known to generate reproducible phenotypic diversification. Samples from different stages of biofilm development were then sequenced to very high coverage (>800x). By accounting for sequencing errors using a matched-sample approach, variants with population frequencies as low as 0.5% could be accurately identified.
In general, the extent and nature of genetic variation was comparable for biofilms of both model organisms, being driven by selection for a small number of non-synonymous variants within key genes involved in biofilm- and competition-related pathways. These results also demonstrate that genome-wide deep sequencing can rapidly, accurately and comprehensively describe genetic variation within evolving populations.

 

Event Date: 
Wednesday, May 30, 2012 - 18:15 - 18:30
Institution: 
University of Sydney
Title: 

A proteomic interrogation of flagellum function

Abstract: 

The eukaryotic flagellum is involved in an array of biological processes and is implicated in an ever-growing spectrum of inherited human disease. Trypanosoma brucei, the causative agent of sleeping sickness in humans, produces a single flagellum that is essential in many critical aspects of cell biology and pathogenicity. Here we employ a combined approach utilising reverse genetics and comparative proteomics to interrogate the protein composition of the paraflagellar rod, an accessory structure of the trypanosome flagellum that is required for correct flagellum function. We have identified more than twenty novel components and confirmed the localisation of a large test set by epitope tagging. This discovery process was iterated to identify at least two co-dependent sub-groups of proteins. The protein domain characteristics of the sub-groups suggest a link between calcium sensing and adenine nucleotide homeostasis in the paraflagellar rod which has wider implications for the regulation of flagellum function generally.

Event Date: 
Wednesday, May 30, 2012 - 19:15 - 20:00
Institution: 
Mark Wainwright Analytical Centre, UNSW
Title: 

Pushing temporal and spatial resolution limits- Light and Optical Microscopy in 2012.

Abstract: 

 

The last 10 years has seen major changes in the way light and fluorescence microscopy is used as a tool by researchers from most scientific disciplines. These advances broach specimen preparation, hardware and the analysis of the images. In particular microscopes have become increasingly more sophisticated; they image faster, longer, at lower light levels, and at greater spatial resolution than ever anticipated. The scope of this talk is to familiarize the audience with some of the latest techniques in light and optical microscopy including; live cell imaging, confocal microscopy and the superresolution techniques such as STED, SIM and PALM. The focus will be on understanding what the temporal and spatial resolution that in practice can be obtained and whether a technique can be used to obtain qualitative AND quantitative data for your experiment. Lastly, the main challenges facing researchers using light and optical microscopy will be presented.

 

Event Date: 
Wednesday, June 27, 2012 - 18:00 - 18:15
Institution: 
Macquarie University
Title: 

Nullarbor Cave Slime: Looking at Life in the Dark

Abstract: 

 
Beneath Australia’s large, dry Nullarbor desert lies an extensive underwater cave system, where microbial communities known as ‘slime curtains’ exist in complete darkness. In the absence of photosynthesis and other nutrient inputs from above, these microbial cave communities may derive their energy from the oxidation of inorganic compounds, such as ammonia, sulfate, nitrate and nitrite, which are relatively abundant in cave waters. We have carried out metagenomic sequencing to explore the diversity and metabolic potential of Nullarbor ‘cave slime’ from Weebubbie cave. Of particular interest was the finding that the dominant organism in this community was an archaea related to Nitrosopumilus maritimus. N. maritimus derives energy by oxidising ammonia to nitrite via the enzyme ammonia monooxygenase and is capable of growing at the very low concentrations of ammonia found in the open ocean. Putative ammonia monooxygenase encoding genes were recovered from this environment using metagenomic sequencing and PCR.  Other genes involved in biological nitrogen cycling, including archaeal nitrite oxidoreductase were also observed in the metagenome. 16S ribosomal RNA surveys conducted to compare multiple bacterial communities from two cave systems, Warbla and Weebubbie, indicate that communities from different caves are distinct and harbor a wide range of microorganisms. We are presently carrying out Scanning Electron Microscopy and Fluorescence In Situ Hybridization to gain further insight into the structure of this unusual microbial slime community. 

Event Date: 
Wednesday, June 27, 2012 - 18:15 - 18:30
Institution: 
University of Western Sydney
Title: 

The Taguchi methods, or how to quickly and efficiently optimise PCR conditions.

Abstract: 

Originally, the Taguchi methods were formulated for the optimisation of industrial processes, where several factors (3 to 50) of complex multifactorial experiments were tested at different levels (Taguchi, 1986). The Taguchi methods use orthogonal arrays to organise the ‘control’ parameters/factors affecting a process and the levels at which they should vary. A particular algorithm (quadratic loss function) is then applied in order to predict the optimum conditions of a process, whilst accounting for performance variations due to ‘noise’ factors beyond the control of the design. In a normal factorial strategy, every parameter should be individually tested at several levels, thus becoming extremely time-consuming, labour-intensive and expensive. The Taguchi methodology allows for testing only a few combinations, therefore dramatically decreasing the total number of experiments and simultaneously identifying the optimum condition of several factors.
Because some functional genes are present only in small fractions of microbial communities, and only few copies can be present in each genome, their detection by classical PCR methods can be challenging. Optimisation of the experimental conditions of a PCR includes the different components of the reaction mix (concentrations of salt, primers, enzyme, DNA template, etc.) as well as the cycling features (time and temperature of the denaturation, annealing and extension steps, number of cycles, etc.). We used this approach for the optimisation of the detection by PCR of functional genes of non-cultivable microorganisms present in environmental samples. In particular, we tested the different parameters involved in a (touchdown/nested) PCR and estimated the optimum settings for the detection of the functional gene pmoA, coding for the putative active site of the particulate methane monooxygenase, involved in the oxidation of methane by methanotrophic bacteria. The application of the Taguchi method allowed the suppression of a nesting step and thus a significant reduction in the amplification time, as well as reagent cost.
 

Event Date: 
Wednesday, June 27, 2012 - 19:15 - 20:00
Institution: 
Faculty of Agriculture & Environment, University of Sydney, Sydney, NSW.
Title: 

Sulfur cycling in the rhizosphere: the role of sulfatase and sulfonatase diversity.

Abstract: 

Growth of healthy, high-yielding crop plants requires a stable input not only of nitrogen and phosphorus, but also of sulfur (S). Although S is naturally present in soils, it is usually bound in organic form as sulfate esters or sulfonates, which are not directly bioavailable to plants. Sulfur can be supplemented by addition of inorganic fertilizer, but most sulfate for plant nutrition is provided by microbial turnover of organically-bound sulfur. To identify the rhizosphere organisms responsible for this turnover, we focused on the key genes atsA, which encodes arylsulfatase, and asfA, which is required for aryldesulfonation. Functional T-RFLP analysis was used to analyse atsA diversity in a range of agricultural and natural soils, and clear atsA community differences associated with land use and soil/bedrock types were observed, which were mirrored in the arylsulfatase activity of the cultivable fraction of the population. Soil arylsulfatase activity is routinely assayed as a measure of soil health, but these data highlight the need for detailed studies on arylsulfatase gene diversity in the soil. Sulfonatase diversity was measured in rhizospheres of field-grown wheat plants and in a sulfate-limited Agrostis-dominated grassland, and the effect of adding sulfate in long-term or short-term treatments was tested. Functional asfA community analysis showed that desulfonation genes from both wheat and Agrostis rhizospheres were dominated by Variovorax and Polaromonas species. This distribution of taxa was also found in a cultivation-dependent analysis, and these genera appear to be key players in rhizosphere sulfonate transformations in several environments. Increasing our understanding of the rhizosphere microbes that catalyse soil organosulfur turnover will allow us to develop management practices to maximize soil sulfur availability, and minimize the costs associated with fertilization.

JAMS attendees started off the night well lubricated thanks to the free beers courtesy of some happy financial planning in our favour. The evening started with Anna Simonin from the University of Sydney discussing Neurospora crassa, a filamentous fungus that forms extensive networks by fusion of the hyphae. Anna presented some amazing live imaging of the heady flow of cytoplasm between the fungal filaments. This clever architecture is thought to influence how nutrients are distributed around the colony. To explore how these streams of nutrient traffic may be contributing to Neurospora’s substrate utilisation, the movement of stable isotope labelled amino acids was tracked within a mutant unable to fuse filaments, a mutant that had lowered fusion ability and the wild type.
 

Report by Jeff Powell

On 12 April, the 'cowboys' at the Hawkesbury Institute for the Environment played host to the 'aliens' from the Sydney region for JAMS Goes West. The mood was both enthusiastic and informative and approximately 30 people participated. The morning consisted of five short talks by representatives of five Sydney-based institutions.