Hybrid genome de novo assembly with methylome analysis of the anaerobic thermophilic subsurface bacterium Thermanaerosceptrum fracticalcis strain DRI-13T

- Thermanaerosceptrum fracticalcis strain DRI-13 T.
- Using a de novo hybrid assembly of Illumina and Oxford Nanopore sequences we produced a circular genome with corresponding methylome profile of the recently characterized thermophilic, anaerobic, and fumarate-respiring subsurface bacterium, Thermanaerosceptrum fracticalcis, strain DRI-13 T to understand how this microorganism survives the deep subsurface..
- Conclusions: The de novo hybrid assembly of strain DRI-13 T genome has provided a more contiguous and accurate view of the subsurface bacterium T.
- fracticalcis, strain DRI-13 T .
- BMC Genomics https://doi.org/10.1186/s z.
- The recently characterized thermophilic, anaerobic, fumarate-respiring bacterium, Thermanaerosceptrum fracticalcis, strain DRI-13 T was isolated from a ~ 876 m deep borehole intersecting the Death Valley Regional Flow system in the United States Nevada National Se- curity Site [1].
- fractical- cis strain DRI-13 T .
- Strain DRI-13 T is a novel genus in the Peptococcaceae family with an average nucleotide identity of 66% with its two closest relatives (Pelotoma- culum propionicicum GCA and Pelotoma- culum thermopropionicum GCA .
- DRI-13 T genome is available from JGI and NCBI GCA_.
- fracticalcis DRI-13 T Hybrid genome assembly comparison.
- fracticalcis DRI-13 T.
- GCA Locus: ELL41697) was compared to strain DRI-13 T by BLAST checking for mis-annotation, but yielded no result.
- Two gene clusters encoding bifurcating hy- drogenases in the DRI-13 T genome having a range of 38–66% AASI to Thermotoga maritima (GCA_.
- Strain DRI-13 T possesses genes for mismatch repair but it is notably missing the gene for mutH.
- Strain DRI-13 T encodes only tatA and tatC genes and.
- Nitrogenase genes were present in all of the genomes used for DRI-13 T comparisons..
- Results of this analysis did not find that any of the matching spacers came from organisms that would exist in a simi- lar environment as DRI-13 T .
- In addition to a CRISPR system a putative Bacteriophage Exclusion (BREX) sys- tem was identified in strain DRI-13 T genome.
- Strain DRI-13 T methylation distribution plot showed that the microorganism ’ s methylation level varies be- tween different contexts (Fig.
- Plotted methylation signals in 100 k bp bins across DRI-13 T ’s genome revealed mCpG and Dcm levels were below 0.5% across genome, while Dam levels are around.
- 4 DNA methylation context distribution and whole genome plot of strain DRI-13 T .
- a Methylation level distribution for strain DRI-13 T .
- b Strain DRI-13 T ’ s genome is partitioned into 100kbp non-overlapping bins.
- The number of annotated genes in 1 million bp bins across all 3 Mbp of the DRI-13 T genome for both strands show an unequal distribution (Table 4 and Fig.
- 638,577 bp) in DRI-13 T .
- Strain DRI-13 T was characterized to utilize fumarate as the major (if not only) carbon and energy source.
- All genes necessary to carry out glycolysis via Embden-Meyerhof pathway were found to be present, although strain DRI-13 T showed only limited growth in the presence of glucose..
- None of which when appear to stimulate growth of strain DRI-13 T .
- A gene present in the genome for Pyruvate orthophosphate diki- nase, which has been shown to catalyze the formation of PEP in other organisms, may allow for gluconeogenesis pathway to function in strain DRI-13 T [50, 51].
- This should lead to an inability for acetyl-coA to enter the TCA cycle, thus restricting the usefulness of these biochemical reactions in strain DRI-13 T .
- Close relatives of DRI-13 T P.
- Strain DRI-13 T has multiple methods to oxidize elec- tron carriers and remove electrons via H 2 production.
- Hydrogen production has been observed in cultures of DRI-13 T when utilizing fumarate.
- a Gene counts distribution based on specific strands of strain DRI-13 T in 1Mbp non-overlapping pins.
- b Methylation profile of one of strain DRI- 13 T ’ s CRISPR array.
- Flagellar motility in DRI-13 T has been observed previ- ously.
- If aerotaxis is not conducted, it remains unclear if DRI -13 T responds to chemical attractants or repel- lents.
- A type IV pilus was annotated in DRI-13 T , but no twitching motility nor biofilm formation has been ob- served.
- Comparatively, the pilus in DRI-13 T was between two identical transposon sequences, and this was not the case for T.
- As such, this suggests that DRI- 13 T may have acquired its pilus by horizontal gene transfer.
- While biofilms come at an energetic cost, they have been found in the subsurface with greater cell density, diversity and in fractured mineral ecosystems similar to where DRI-13 T was isolated [58, 59].
- Embedded within the DRI-13 T genome are four pre- dicted prophages (Fig.
- The impact of these prophages upon strain DRI-13 T fitness is unknown..
- Strain DRI-13 T ’s genome contains multiple DNA methylation contexts, including low and consistent amounts of mCpG across the entire genome, and even lower levels of Dcm (Fig.
- What was observed in terms of Dcm methylation may be contributed by DRI-13 T ’s own unique methylation motif similar to mCHG con- text.
- DRI-13 T shows a high level of Dam relative to the only few Dam sites found in the genome (Table 3).
- Strain DRI-13 T had a relatively high amount of mCHH on the forward strand at 1Mbp - 2Mbp seg- ments, while on the reverse strand showed mCHH de- pletion (Fig.
- For strain DRI-13 T we see clear depletion of DNA methylation of all context near tran- scription start sites as well as transcription termination sites (TSS).
- Promoter sequences are essential in regulat- ing gene expression, and the depletion of DNA methyla- tion near strain DRI-13 T promoters also agrees with previous publications characterizing bacteria DNA methylation [67].
- Even though much of strain DRI-13 T ’s protein encoding genes lack DNA methylation at TSS, there is a small cluster of 45 genes, named ‘cluster 1’, that have enriched DNA mCpG proximal toward TSS..
- Strain DRI-13 T may thus use DNA methylation to regulate gene expression by controlling promoter methylation level..
- We analyzed DRI-13 T ’s CRISPR array spacer methyla- tion contexts located at 600kbp and found them to be completely devoid of mCpG, Dcm, and Dam.
- On the other hand, DRI- 13 T ’s CRISPR array showed periodicity of mCHH signal across the array.
- It is possible that RNAPol traveling through DRI-13 T ’s CRIS PR array generates repeat RNAs and causes mCHH methylation on adjacent CRISPR spacers [69].
- No spacer reads mapped to any of the prophage elements in DRI-13 T or any other location in the genome aside from their positions in the CRISPR array.
- DRI-13 T genome has three putative BREX genes (brxHI, brxD, pglX) in a 36kbp gene cluster that matches the core BREX system description (Supplemental Table S1).
- The presence of a BREX system in DRI-13 T ’s gen- ome may explain the observed integration of phage DNA in the genome.
- None of the close relatives to DRI-13 T possessed any genes to a BREX system.
- The presence of this system may explain why DRI-13 T possesses these prophage elements while the other genomes lack prophage sequences.
- In this study, we used a hybrid genome assembly of Thermanaerosceptrum fracticalcis strain DRI-13 T we could increase the detail of metabolism, respiration, DNA repair, ABC transporters, and cellular defense and introduce the first view of the methylome of this subsur- face bacterium.
- The DRI-13 T methylated mC and mA profile seem quite different from surface microbes, like laboratory strain E.
- fracti- calcis strain DRI-13 T by cultivating the microorganism in one-liter batch cultures grown on 10 mM fumarate.
- fracticalcis strain DRI-13 T was prepared using the procedure outlined in Jain, et al.
- fracticalcis strain DRI-13 T culture (>.
- The DRI-13 T genome is available from JGI and NCBI GCA .
- Strain DRI-13 T metaplot of all genes for given DNA methylation context.
- A) Alignment of strain DRI-13 T ’ s DNA adenine methyltransferase protein sequence versus well studied 6mA methyltransferase in selected prokaryote and eukaryote species.
- Alignment of strain DRI-13 T ’ s DNA cytosine methyltransferase protein sequence versus well studied 5mC methyltransferase in selected prokaryote and eukaryote species..
- A sub cluster of strain DRI-13 T ’ s CDS shows enriched mCpG.
- Heatmapping of all strain DRI-13 T CDS.
- DRI-13 T genome is avail- able from JGI and NCBI GCA .
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