NASA Astrobiology Institute (NAI)

1. Microbial diversity and population structure studies in the Rio Tinto

   Project Investigators: Linda Amaral Zettler

   Other Project Members

   Maria Aguilera (Collaborator)

   Ricardo Amils (Collaborator)

   Carmen Palacios (Collaborator)

   Susanna Theroux (Doctoral Student)

   Erik Zettler (Collaborator)

   Virginia Souza-Egipsy (Collaborator)

   Donald Nuzzio (Collaborator)

   Nicholas Scalfone (Research Staff)

   Erika del Castillo (Research Staff)

   Summary

   As part of our Microbial diversity and population structure studies in the Rio Tinto, we hope to better understand how environmental conditions such as pH and metal concentrations help shape the underlying microbial community structures in extreme environments that serve as terrestrial analogs for Mars. The iron-based mineralogy found in the Rio Tinto coupled with low pH are two characteristics that tie this extreme environment to Mars. To this end, we have been sampling stations along the river that differ in the concentration and oxidation state of iron and other metals (see http://amarallab.mbl.edu/rt_main/rt.html for more detailed information and photographs of the study locations) and using molecular techniques coupled with physicochemical measurements to investigate microbial diversity in the water column at both spatial and temporal scales. When possible, determination of as many in situ physico-chemical parameters are made on biofilms as well, using microelectrodes available for field measurements. This allows for the correlation of biological diversity information with physicochemical parameters of the river. The outcome of this study will provide a comprehensive view of the microbial ecology of the system, a first step towards establishing an ecological genomics project for the Rio Tinto.

   Astrobiology Roadmap Objectives:

   • Objective 3.3: Origins of energy transduction
   • Objective 5.1: Environment-dependent, molecular evolution in microorganisms
   • Objective 5.2: Co-evolution of microbial communities

   Project Progress

   The experimental phase of our project is now complete and we are currently analyzing data and writing up our results for publication in peer reviewed journals. Our completed study includes three large data sets that are at different stages of analysis and publication. The first of these is a Serial Analysis of Ribosomal Sequence Tags of the V6 hypervariable region (SARST-V6) dataset that targets bacterial diversity at all three of our stations (22 samples, >10,000 tags total (~60 bp in length) conducted during October 2002 and is part of a manuscript entitled “Contrasting Microbial Community Assembly Hypotheses: a Reconciling Tale from the Rio Tinto” under revision for publication. A second dataset is a comprehensive analysis of microbial diversity at the 3-domain level (Bacteria, Archaea and Eukarya) and includes all three stations (27 samples and >8,000 sequences of 800-1000 bp in length spanning the V4-V8 regions) also from our October 2002 sampling campaign. A manuscript summarizing these data entitled “Environmentally Structured Community Assembly of the Three Domains of Life in an Extreme Ecosystem” is currently in preparation.

   
   

   Three domain tree of life in the Rio Tinto based on sequences from the V4-V8 rRNA gene hypervariable region based on a RAxML analysis with 8002 sequences.

   shows an example of novel higher order taxonomic groups (taxa with stars) within the Bacteria and Archaea that this approach has helped identify. The final dataset is a seasonal comparison (dry vs. rainy season) across all three domains using massively parallel tag pyrosequencing of the V6 (Bacterial and Archaeal) and V9 (Eukaryal) hypervariable regions of rRNA genes (2 seasons, 3 stations – 50 samples in total >400,000 tags).

   
   

   Pie chart bacterial V6 tag taxonomic breakdowns of three replicate samples from Berrocal Station Site 2 from September 2003. The 6 most abundant taxonomic groups are indicated in the legend.

   shows a pie chart representation of the taxonomic breakdown of bacterial tags for three replicate samples from the Berrocal site. These data are currently in the analysis phase.

   Our combined approach of using clone library construction, SARST-V6 and massively-parallel tag sequencing continues to reveal novel phylotypes in the Rio Tinto. Many of these taxa exist only at low frequency at certain times of the year but then increase in abundance during other times of the year. An example of this is illustrated in

   
   

   Pie chart taxonomic comparisons of V6 tags from Berrocal Station site 2 in September 2003 (BE2_3B03) and January 2004 (BE2_3B04).

   that contrasts a dry (BE2_3B03- Sept03) versus rainy (BE2_3B04 – Jan04) season sample from Berrocal. In this example, tags related to Gallionella are abundant in January but rare in September while the opposite is true for tags related to Acidiphilium.

   Cross-Team Collaborations

   Collaboration continues with Dr. Ricardo Amils and lab members Angeles Aguilera, Virginia Souza-Egipsy and Elena González-Toril. As part of this collaboration Dr. Aguilera submitted and was awarded the grant entitled: “Heavy metal toxicity in the eukaryotic microbial community of Rio Tinto (Huelva); Implications for Bioremediation (BIOTIN)” for which collaborative field work was conducted in March of 2006. A review paper and a research paper co-authored by Dr. Aguilera and members of the CAB and MBL published in 2007 were products of this collaboration. Dr. Virginia Souza-Egipsy recently submitted the following manuscript to FEMS Microbiology: “Prokaryotic community structure in photosynthetic biofilms from extreme acidic streams, Río Tinto (SW, Spain)” Virginia Souza-Egipsy, Elena González-Toril , Erik Zettler, Linda Amaral-Zettler, Ricardo Amils and Angeles Aguilera.

Publications

Aguilera, A., Amaral-Zettler, L., Souza-Egipsy, V., Zettler, E. & Amils, A.  (2007).  Eukaryotic Community Structure from the Rio Tinto (SW, Spain), a Highly Acidic River;.  .  Netherlands: Springer.

Aguilera, A., Zettler, E., Gomez, F., Amaral-Zettler, L., Rodriguez, N. & Amils, R.  (2007).  Distribution and seasonal variability in the benthic eukaryotic community of Rio Tinto (SW, Spain), an acidic, high metal extreme environment.  Syst Appl Microbiol, 30(7):531-46  [Online].