THE UNIVERSITY OF SYDNEY
Rm 564, Building G08, The University of Sydney
NSW, AUSTRALIA 2006
The role of quorum sensing in chitin biodegradation
The 1011 ton global annual turnover of chitin has generated extensive interest in the regulation of chitin processing enzyme production in bacteria. Some bacteria regulate chitinase production by N-Acyl-L-homoserine lactone (AHL) mediated quorum sensing. In this study, a description of bacterial community succession during chitin particle colonisation and depolymerisation in activated sludge is presented. It was discovered that Betaproteobacteria and Bacteroidetes lineages dominate chitin colonisation in sludge and that AHLs bind to chitin at concentrations that upregulate AHL dependent transcription in bacterial cells associated with the chitin surface. There was no requirement for high cell density (a quorum) at the chitin surface. Further, N-Acetyl glucosamine (GlcNAc), the monomer of the chitin polymer, is shown to inhibit AHL dependent gene transcription representing a previously unrecognised mechanism by which the chitinase reaction product negatively regulates chitinase production. Evidence is presented supporting a role for both competitive inhibition at the AHL binding site of LuxR type transcriptional regulators and catabolite repression. The quorum sensing inhibitor activity of GlcNAc adds to its list of possible therapeutic benefits.
Microbial life in Movile Cave – an unusual cave ecosystem
Movile Cave (Mangalia, Romania) is a unique cave ecosystem sustained by in situ chemoautotrophic primary production, analogous to deep-sea hydrothermal vents. The cave has been cut-off from the surface for the past 5.5 million years with the primary energy source coming mainly from hydrogen sulfide and methane released from the thermal fluids. Invertebrates, many of which are endemic to Movile Cave, are isotopically lighter in both carbon and nitrogen than surface organisms, indicating that chemoautotrophic primary production, primarily driven by methane and sulfur oxidizing microorganisms, occurs in the cave. In this talk, I will present our recent work on the microbiology of the Movile Cave ecosystem, with particular emphasis on functional diversity of Bacteria involved in aerobic one-carbon (methane and methylated amine) metabolism. Insights from metagenomic and genomic sequence analyses of the microbial community and isolates, respectively, will be discussed in detail.
Structure, diel functional cycling and viral ecological filtering in the microbiome of a pristine coral atoll in the Indian Ocean
Given the role of microbes as both indicators and drivers of ecosystem health, establishing baselines in pristine environments is crucial to predicting the response of marine habitats to environmental change. Here we describe a survey of microbial community composition and metatranscriptomic gene expression across the Indian Ocean, encompassing the first samples from the pristine Salomon Atoll in the Chagos Archipeligo. We observed strong patterns in beta-diversty which reflected Longhurst biogeographical provinces established using primary productivity and thermohaline properties of ocean currents. Samples from within Salomon Atoll showed a highly unique community which was remarkably different even from adjacent samples despite constant water exchange. This pattern was driven by the dominance of the photosynthetic cyanobacterium Synechococcus within the lagoon, the diel activity of which was responsible for driving shifts in the transcriptional profile of samples. Inside the lagoon, increases in the expression of genes related to photosynthesis and nutrient cycling associated with the bottom-up control of bacterial populations, however the expression of viral proteins increased five-fold within the lagoon during the day, indicating a concomitant top-down control of bacterial dynamics byphage. Indeed, genome recruitment against Synechococcus reference genomes suggested viruses provide an ecological filter for determining the diversity patterns in this system. This study also represented a proof of concept for using a ‘citizen oceanography’ approach utilzing tools that may easily be adapted to deployment on any ocean going yacht, greatly expanding the scale and outreach of marine microbiology studies.
Understanding the roles of non-coding RNAs in Enterohaemorhaggic E. coli pathogenesis
Expression of virulence genes in pathogenic bacteria is tightly regulated in response to environmental cues at both the transcriptional and post-transcriptional level. RNAs that do not encode proteins (non-coding RNAs) are now appreciated to play important roles in post-transcriptional gene regulation by interacting with mRNAs and modulating translation and stability. High throughput sequencing studies are now uncovering hundreds of non-coding RNAs in pathogenic bacteria and the challenge now is to understand the function of these RNA species.
A major subclass of bacterial non-coding RNA, termed small RNAs (sRNAs), requires the RNA chaperone Hfq to anneal to mRNA targets and effect regulation. Using UV-crosslinking and NextGen sequencing techniques (CRAC or CLIP-Seq) we have generated high resolution maps of Hfq-RNA interactions in the human pathogen Enterohaemorhaggic E. coli (EHEC). Within this dataset of Hfq binding sites we have identified 55 new sRNAs (Tree et al Molecular Cell) and we are now looking to identify the mRNA targets of these sRNAs and understand their role in pathogenesis.
Recently it has been demonstrated that RNA-RNA interactions can be extracted from CLIP-Seq data allowing ncRNAs to be sequencing in complex with their mRNA targets (a technique termed CLASH). This analysis gives insights into the function of ncRNAs in vivo. Small RNAs have been shown to recruit the RNA endonuclease, RNase E, when duplexed with an mRNA target and we have recently demonstrated that sRNA-mRNA interactions can be sequenced from RNaseE CLIP-Seq data. We have confirmed a subset of these interactions using translational GFP fusions. Using this dataset we have identified mRNA targets for our newly identified EHEC sRNAs and have begun assigning functions to some of these novel RNA species. We have found that the EHEC specific sRNA, Esr41, represses translation of select iron uptake receptors indicating a role in modulating iron availability.
Bridging the gap between functional genes and biogeochemistry: a DMSP case study
A large fraction of the surface ocean food web is active in producing and cycling both dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS). In addition to the potential climatic significance of DMS production, the role that these compounds play in mediating ecosystem dynamics remains unknown. An interdisciplinary dataset of biological, chemical and physical measurements was used to test current hypotheses of the role of light and carbon supply in regulating upper-ocean sulfur cycling in oligotrophic regions. Our results suggest that UV-A radiation dose plays an important role in both phytoplankton DMS production and bacterial DMSP degradation. We suggest a modified ‘bacterial switch’ hypothesis where the prevalence of different bacterial DMSP degradation pathways is regulated by a complex set of factors including carbon supply, temperature, and UV-A dose. Finally, numerical models of varying complexity were used to link genetic and enzyme data to biogeochemical rates.
Developing MicroPIE and a Microbial Ontology
The study of the evolution of microbial traits requires both phylogenetic as well as phenotypic trait information (also called phenomics). Next generation sequencing has enable high throughput (meta)genomic analyses, but collecting phenotypic information, either de novo or from published taxonomic literature, to create character matrices is still tedious and time-consuming. I am part of a team of researchers developing tools to provide faster collection of microbial phenomic information from published literature. We have created a natural language processing tool, Microbial Phenomics Information Extractor, or MicroPIE, that uses existing parsers, machine-learning tools, and a library of microbial-specific terms derived from ~1000 taxonomic descriptions from the Archaea, Bacteroidetes, Cyanobacteria, and Mollicutes. We have also developed an ontology of terms found in prokaryotic taxonomic descriptions, that is organized using a formal logical framework. This ontology will be used to assist MicroPIE in character identification and extraction, facilitate the identification of trait synonyms used in prokaryotic taxonomic descriptions, and to populate character matrices with higher-level character states. The taxon-character matrices extracted using MicroPIE can be combined with phylogenomic trees and analyzed using the Arbor software package, which is a scalable, web-services based platform for conducting phylogenetic comparative analyses to test evolutionary hypotheses. I’ll show some preliminary results from an analysis of trait evolution in cyanobacteria.
Bacterial secondary metabolite prodigiosin inhibit biofilm development by cleaving extracellular DNA
Prodigiosin a bacterial secondary metabolite is a heterocyclic compound belongs to the class of tripyrrole, synthesized by various strains of bacteria includes Serratia species. Research on prodigiosin is under limelight for past 10 years from clinical and pharmacological aspects in relevance to its potential to be drug for cancer therapy by inducing apoptosis in several cancer cell lines. Reports suggest that prodigiosin promotes oxidative damage to DNA in presence of copper ion and consequently lead to inhibition of cell-cycle progression and inducing cell death. However, prodigiosin has not been previously implicated in biofilm inhibition. We performed experiments to reveal any link between prodigiosin and biofilm inhibition through degradation of extracellular DNA which plays a major role in biofilm establishment. Our study showed that prodigiosin (extracted from Serratia culture) has strong DNA cleaving property but does not intercalate with nitrogenous bases of DNA. Using P. aeruginosa PA14 wild-type strain as a model organism we showed that bacterial cells treated with prodigiosin showed significant reduction in its cells surface hydrophobicity and consequently affecting surface energies and physico-chemical property essential for bacterial adhesion and aggregation. We also found that prodigiosin did not influence planktonic growth of P. aeruginosa however, was successful in inhibiting the establishment of biofilms includes decrease in biofilm thickness, adhesion to substratum, decrease in biovolume, microcolony formation and also significantly dispersed pre-established biofilm of P. aeruginosa. This novel function on the biofilm inhibition of prodigiosin could be used to interfere with risks associated with bacterial biofilms.