Starting Month: M12
Ending Month: M36
The main aim of this objective is to establish a well-defined collection of prokaryotic isolates associated with the anoxic sediment of the Etoliko Lagoon, enhancing the worldwide culture collection of anaerobes and generating a UniquELT microbial collection that can be further exploited for bioprospecting purposes.
Task 4.1 – Culture-dependent anaerobic isolation of microbial strains M13-M29.
Task 4.2 – Genomic sequencing and in silico analysis of metabolic properties M24-M36.
Task Leader: Assoc. Prof. George Tsiamis, University of Patras
Task Leader: Assoc. Prof. George Tsiamis, University of Patras
Microcosms will be prepared in the anaerobic chamber (Coy Laboratories) of the Laboratory of Molecular Genetics and Microbiology, University of Patras, in an atmosphere consisting of 95 to 97% N2 and up to 5% H2. A minimum set of five (5) samples will be sieved to remove debris and then combined with anoxic sterile ultrapure H2O to form a slurry (60% wet sediment, 40% water). Thirty-milliliter aliquots of the slurry will be dispensed into 60-ml serum bottles. Different final concentrations of acetate (20 mM), lactate (40 mM), or formate (80 mM) will be used as electron donors. These energy sources will be added separately to each bottle. Controls will also be prepared without added energy sources. The enrichments will be transferred onto sterile sediment slurries three times and incubated under a nitrogen headspace.
DNA will be extracted from triplicate cultures that had been transferred eight times. We will be using a BIO 101 Fast DNA Spin Kit for Soil (Q-Biogene), which has been providing excellent results for DNA extraction from sediment slurries and from sediment-free cultures. The DNA will be diluted 1:10 with sterile PCR-quality water, and 1 μl will be used as a template for PCR amplification in a 25-μl reaction mixture with eubacterial and archaeal primers. We will be using the Topo TA Cloning Kit (Invitrogen) to clone PCR products. Competent cells will be plated on Luria-Bertani (LB) plates with ampicillin (100 μg/ml) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) (50 μg/ml). White colonies will be picked onto fresh LB plates containing ampicillin and X-Gal, and clones will be checked for inserts by colony PCR. Clones will be sequenced using plasmid specific primers. Sequences will be checked for chimeras by uchime (Edgar, Haas, Clemente, Quince, & Knight, 2011) and then analyzed by the Basic Local Alignment Search Tool (BLAST) and by the Sequence Match tool of Ribosomal Database Project II. Phylogenetic trees will be constructed using PAUP* 4.0b, with maximum-likelihood trees being constructed using a heuristic search.
The genomes of the selected isolates (at least 20 genomes) will be sequenced to study their metabolic profile and their genomes will be used as reference material in order to improve metagenomic and metatranscriptomic assembly. Genomic DNA will be prepared from pure cultures and will be sequenced using Illumina's MiSeq and PacBio technology. Gene predictions and annotations will be performed by Rapid Annotation using Subsystem Technology (RAST) server and Integrated Microbial Genomes/Expert Review (IMG/ER). Furthermore, a Bioconductor pipeline, based on R, will be developed based on the recently established protocols in order to identify the availability and abundance of different types of bacteria on exposure to various substances. Comparative genomic analysis will help us to identify genes involved in nitrogen fixation, P-solubilization, production of indole compounds, biofilm formation, stress tolerance, cell wall degrading enzymes, chitinases, glucanase and siderophores.
Task 3.4 – Communication / Dissemination activities M24-M36.
Task Leader: Assoc. Prof. George Tsiamis, University of Patras
Project newsletter: a final UniqELT e-newsletter will by produced (M36) to inform all potential stakeholders on the project results. It will be published on the project website by an online integration to the news blog, accessible through the project website, to inform about the project latest events and relevant news items.
Scientific publications: open access to all the scientific publications will be ensured.
Milestones 4.1 Characterization of isolated strains (M27) - 4.2 Genome sequence (M36)
Deliverables
4.1 Anaerobic pure cultures deposited to National and International Microbial Collections (T4.1; M29) - 4.2 Report on the phylogenetic analysis of the isolated bacteria (T4.1; M29) - 4.3 Genomic sequences of isolated strains and accession numbers (T4.2; M36) - 4.4 Metabolic profile of the isolated strains (T4.2; M36) - 4.5 One publication to peer review journal (T4.3; M36) – 4.6 Final project e-newsletter (T4.3; M36)