Microbial community structure and photoheterotrophic activity across different Australian oceanic environments oceanic environments Investigator PhD Candidate Jaume Bibiloni Isaksson Supervisors Dr. Justin Seymour – C3 Plant Functional Biology and Climate Change Cluster, UTS Dr. Mark Brown School of Biotechnology and Biomolecular Sciences, UNSW

D. J. Des Marais Science, 8 September 2000

Classification of Phototrophic Organisms

http://www. seagrant. umn http://www.seagrant.umn.edu/newsletter/2005/10/all_plankton_great_and_small.html

Photoheterotrophic bacteria Proteorhodopsin bacteria (PRB) contains proteorhodopsin Aerobic anoxygenic phototrophic bacteria (AAnPB) contains bacteriochlorophyll a

Proteorhodopsin Proteorhodopsin (PR) are retinal-binding integral membrane proteins belonging to the rhodopsin superfamily Fig. 2 Secondary structure of proteorhodopsin. Béjà et al. (2000)

Proteorhodopsin PR is a light-driven proton pump that may enhance ATP production by heterotrophic bacteria that otherwise rely on organic material oxidation for energy. A single amino acid substitution in PR protein sequence bacteria absorb different wavelengths of light: 490 nm (blue) to 520 nm (green) Fig. 3 Proteorhodopsin and possible relation to proton pumping (Walter et al., 2007)

Bacteriochlorophyll a

Aerobic Anoxygenic Photoheterotrophs

Why are photoheterotrophs important?

PR genes have been identified in a wide variety of cultured and uncultured bacterioplankton, such as α and γ-Proteobacteria, Flavobacteria, Actinobacteria, and Archaea. PR in Pelagibacter ubique, a cultured representative, the most abundant bacterial group inhabiting the upper ocean marine, clades SAR11 up to 70% of the bacteria in the Sargasso Sea 7 to 57% North Atlantic Ocean Contain PR 13% of the bacteria in the Mediterranean Sea

AAnPB have been identified in a wide variety of cultured and uncultured bacterioplankton, such as α, β, and γ-Proteobacteria. Roseobacter group belong to AAnPB, a cultured representative, 1 in 5 bacteria cells inhabiting coastal waters. Also important in DMSP degradation (cleavage/demethylation). Results liberation of climate-influencing dimethyl sulfide, thus key in marine sulfur cycle. (not all Roseobacter synthesize Bchla). ~ 10 to 20 in mesotrophic estuaries positively correlate with Chla in variety of environments AAnPB up to 25% in hyperoligotrophic South Pacific Ocean

Both AAnBP and PRB are incapable of photoautotrophy (do not have enzyme RubisCO) and rely on heterotrophy of their cellular energetics Sunlight provide additional energy to cell, reducing the need for respiratory oxidation of organic substrates The ability to use light energy seems to allow more economical utilization of dissolved organic matter, thus photoheterotrophic bacteria may play a unique role in the microbial carbon pump (MCP).

Objectives i) Identify major bacteria and archaea groups including PR and Bchla groups  ii) Estimate the ratio of PR and Bchla genes expression and abundance to bacterial populations  iii) Examine how the abundance, diversity and activity of different PR and Bchla types varies temporally in different oceanographic coastal region, which are suffering from the impacts of climate change iv) Perform laboratory growth experiments to test the dynamics of photoheterotrophy under different light and nutrient condition

Hypotheses H1: PR and AAnBP diversity, abundance and activity will vary throughout the sample period/location, since significant physicochemical changes in seawater will provide more suitable conditions for different types of bacteria H2: PR-bearing bacteria abundance will become more abundant during summer (Austral) period, since PR bearing bacteria may help to provide energetic and physiological advantages under in more oligotrophic conditions

Experimental procedures Sampling and environmental parameters Monthly samples (surface samples) Port Hacking Mooring (IMOS platform) (2011-2012) Temporally. Maria Island, Port Hacking, Stradbroke Mooring stations (2012) Spatially/temporally Data is available via the IMOS portal http://imos.aodn.org.au/webportal/ Future sampling (different depths) North Australia research voyage (July 2013) Spatially East Australian current research voyage (April 2014) Spatially South Australia research voyage (Sept 2014) Spatially Environmental data will be collected in situ.

Broome Stradbroke Brisbane Freemantle Sydney Port Hacking Tasmania Maria Island

Experimental procedures Molecular experiments DNA/RNA extraction (Standards Kit extraction) PCR amplification of 16S rRNA, PR and pufM genes 454/Illumina pyrosequencing of 16S rRNA, PR and pufM genes to assess diversity Determination of ratios of 16S rRNA (all bacteria, SAR11 clade) and PR (SAR11 clade, Flavobacteria) and pufM gene abundance/expression via real-time quantitative PCR Screen for PR and Bchla genes in seawater isolates

Data Analysis 454/Illumina pyrosequencing data with QIIME Relationship between abundance, diversity and physicochemical parameters using PERMANOVA in PRIMER+ PR/pufM sequence compilations and analysis using ARB software Network analysis Data Analysis

Two major questions to be answer in this project How do Australian marine microbial communities (i.e. photoheterotrophs) and their biogeochemical function vary in space and time? Will shifting ocean circulation patterns alter microbial community structure and function in ways that will have potential feed-back effects on ecosystem stability?

People in our Lab Dr Justin Seymour - Leader and supervisor Dr Mark Brown – Co-supervisor (UNSW) Dr Tom Jeffries - Post-doc Lauren Messer - PhD student Project is funding by an ARC grant from Australia government