Computational protocol: Impact of spatial organization on a novel auxotrophic interaction among soil microbes

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Protocol publication

[…] Time-lapse microscopy was performed on agar medium cultures that were prepared using the same basic medium described above. A 6-well tissue culture plate (Ref: 353046, Falcon) was used and 1 μl of S. indica, B. subtilis or mock solution prepared as described above were inoculated on each well accordingly to experiment design. An Olympus IX83 microscope, UPlanFLN ×4 objective and cellSens software were used for recording the growth. Okolab stage top incubator (H301-T-UNIT-BL-Plus system, and H301-EC chamber) were used for incubation, with a temperature sensor and lens heater set to 30°C and stabilized for at least 2 h prior to the experiment. Different fields of view were chosen at interior and periphery of each colony and images from those fields were recorded using the automated microscope stage and Olympus cellSens software. Images were taken in 1 h intervals and put together as image series. ImageJ (Fiji) [] was used for measuring the mean intensity on each field of view over time, normalized against the intensity value of the first frame from each view point (as shown in Fig. ). [...] In order to understand the loss of thiamine biosynthesis function in S. indica, we performed a phylogenetic analysis of 162 fungi species including S. indica and its close relative S. vermifera, and focusing on thiamine biosynthesis genes. This focus meant that we can only use species with fully sequenced and well-annotated genomes. To collate fungi species fulfilling these criteria, we first used the KEGG database [] in which there are 102 species with annotated genomes that are mapped to metabolic pathways. This dataset, however, contains only 16 species in the Agaricomycetes class, in which the species S. indica and S. vermifera belong. To get more close relatives of S. indica, we used the NCBI Taxonomy [] and SILVA rRNA [] databases and collated 60 species that are from the Agaricomycetes class and that have full genome assembly information. For the resulting 162 species, we downloaded SSU-rRNA sequences from SILVA rRNA database []. Where multiple sequences were present for a given species, we used the longest sequence with highest quality. We then aligned the resulting 162 rRNA sequences using the MUSCLE tool [], and built a maximum-likelihood phylogenetic tree using PhyML []. Onto the resulting phylogenetic tree, we mapped the presence and absence information of the thiamine biosynthesis genes THI6, THI20, THI5, THI4, and THI80 (see Fig. ). This information was already available for the 102 species that are contained in the KEGG database. To obtain it for the additional 60 species from the Agaricomycetes class (and including the species S. indica and S. vermifera), we ran a BLAST analysis, in which we queried each of these species genomes against the THI6, THI20, THI5, THI4, and THI80 gene sequences from S. cerevisiae. A gene was considered present in a given genome if there was a match of homologous sequences with an e-value smaller than 1e−6 []. […]

Pipeline specifications

Software tools cellSens, ImageJ
Application Microscopic phenotype analysis
Organisms Bacillus subtilis, Sillago indica, Bacillus subtilis BEST7003
Chemicals Carbon, Thiamine