Generated November 10, 2021
Xiaoxuan Ge, Michael P. Thorgersen, Farris L. Poole II, Adam M. Deutschbauer, John-Marc Chandonia, Pavel S. Novichkov, Paul D. Adams, Adam P. Arkin, Terry C. Hazen, Michael W. W. Adams
Submitted to Microbiology Resource Announcements
This Narrative contains data for isolate EB106-08-02-XG196, also referred to as XG196, which is described in the above manuscript. Data for another isolate from ORR EB106 sediment , XG77, is in another narrative, here.
An 8-meter-deep borehole of 8.9 cm diameter (designated EB-106) located 21.1 meters downstream from the S-3 ponds area was drilled at ORR. The sediment was collected and cut into 22 cm segments all under anaerobic conditions, as reported elsewhere (Ge et al., 2019)</li>. For microbial enrichment, sediment samples (1 g) were incubated anaerobically in 5 ml of a defined medium containing 1.3 mM KCl, 2 mM MgSO4, 0.1 mM CaCl2, 0.3 mM NaCl, 30 mM NaHCO3, 5 mM NaH2PO4 and 20 mM NaNO3, with added vitamins and minerals as described (Widdel and Bak, 1992)</li>. A mixture of organic compounds (2 mM of formate , acetate, ethanol, lactate, succinate and glucose together with 0.1 g/L yeast extract) was used as carbon source. A mixture of metals (MM) containing 5 µM cadmium acetate (Cd(CH3COO)2·2H2O), 100 µM manganous chloride (MnCl2·2H2O), 30 µM cobalt chloride (CoCl2·6H2O), 100 µM nickel chloride (NiCl2·6H2O), 10 µM cupric chloride (CuCl2·2H2O), 10 µM ferrous ammonium sulfate (Fe(NH4)2(SO4)2 ·6H2O) and 100 µM uranyl acetate (UO2(CH3COO)2·2H2O) was used to mimic the metal contamination in the groundwater near the ORR S-3 ponds (Table S1).
| Metal (1 ×) | Compound added | Final Conc.(µM) |
|---|---|---|
| Mn2+ | MnCl2·2H2O | 100 |
| Fe6+ | Fe(NH4)2(SO4)2 ·6H2O | 10 |
| Co2+ | CoCl2·6H2O | 30 |
| Ni2+ | NiCl2·6H2O | 150 |
| Cu2+ | CuCl2·2H2O | 10 |
| Cd2+ | Cd(CH3COO)2·2H2O | 5 |
| U6+ | UO2(CH3COO)2·2H2O | 100 |
The ZymoBead Genomic DNA kit was used to extract genomic DNA. More than 1 µg of purified genomic DNA was sent out to the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) for Illumina sequencing.
This pipeline was performed in another KBase Narrative, which contains other unpublished data. Relevant objects from that Narrative have been copied to this one.
A summary of the methods follows and the provenance of each object can be found by opening up the "Data explorer" window (click on the binoculars icon under each object in the data panel).
Genome quality control was perfomred using CheckM using default parameters. CheckM provides robust estimates of genome completeness and contamination by using collocated sets of genes that are ubiquitous and single-copy within a phylogenetic lineage. More documentation describing CheckM is here.
The genome was then annotated using the Annotate Domains in a Genome App using all domain libraries. This app annotates domains from COGs, CDD, NCBI-curated domains, SMART, PRK, Pfam, and TIGRFAMs databases. More detail on annotating domains in KBase is here. Note that the 4966 genes listed in the Annotate Domains output include only the protein-coding genes with annotated domains; in total, the genome contains 5750 protein-encoding genes.
| Created Object Name | Type | Description |
|---|---|---|
| EB106-08-02-XG196.domains | DomainAnnotation | Domain Annotations |
Finally, we classified the genome. As discussed in the manuscript, our initial classification was done by 16S rRNA alignment. We also built species trees for XG196 using two more KBase apps that rely on phylogenetic marker genes other than the 16S rRNA:
GTDB-Tk was run on the genome with default parameters. This app assigns objective taxonomic classifications to bacterial and archaeal genomes, using a set of domain-specific phylogenetic marker genes. More info about the app is here.
We used the Insert Genome into Species Tree App, using default parameters, to make a species tree called "EB106-08-02-XG196.tree" using 49 marker genes. More info about this app is here.
All classification methods produced consistent results: the most similar genome to XG196 that has been previously described is Bacillus niacini.
| EB106-08-02-XG196.genome |
We imported the final genome into Genbank. Due to compatibility issues, we had to re-run the annotation pipeline in Genbank instead of using the same annotations created in KBase.
| Created Object Name | Type | Description |
|---|---|---|
| EB106-08-02-XG196-genbank-RAST.genome | Genome | Annotated genome |
Some RAST tools will not run unless the taxonomic domain is Archaea, Bacteria, or Virus. These tools include: call selenoproteins, call pyrroysoproteins, call crisprs, and call prophage phispy features. You may not get the results you were expecting with your current domain of Unknown. The RAST algorithm was applied to annotating an existing genome: Bacillus sp. EB106-08-02-XG196. The sequence for this genome is comprised of 55 contigs containing 6010169 nucleotides. The input genome has 5721 existing coding features and 234 existing non-coding features. Input genome has the following feature types: Non-coding assembly_gap 12 Non-coding gene 78 Non-coding misc_binding 20 Non-coding misc_feature 4 Non-coding ncRNA 6 Non-coding rRNA 14 Non-coding regulatory 42 Non-coding tRNA 57 Non-coding tmRNA 1 gene 5721 The genome features were functionally annotated using the following algorithm(s): Kmers V2; Kmers V1; protein similarity. In addition to the remaining original 5721 coding features and 234 non-coding features, 0 new features were called, of which 0 are non-coding. Output genome has the following feature types: Coding gene 5721 Non-coding assembly_gap 12 Non-coding gene 78 Non-coding misc_binding 20 Non-coding misc_feature 4 Non-coding ncRNA 6 Non-coding rRNA 14 Non-coding regulatory 42 Non-coding tRNA 57 Non-coding tmRNA 1 Overall, the genes have 2881 distinct functions. The genes include 3670 genes with a SEED annotation ontology across 1414 distinct SEED functions. The number of distinct functions can exceed the number of genes because some genes have multiple functions.
