ABSTRACT
We report complete genomes of four strains of Ochrobactrum pituitosum and three strains of Pseudochrobactrum saccharolyticum isolated from gut microbiomes of Caenorhabditis elegans colonized with microbial communities extracted from garden compost. Genomes were assembled from Oxford Nanopore long read data and polished with Illumina short read data.
KEYWORDS: Ochrobactrum, Pseudochrobactrum, assembly, annotation, environmental sample, C. elegans, compost
ANNOUNCEMENT
We announce closed genome assemblies and annotations of four Ochrobactrum pituitosum strains (NCBI: txid571256) and three Pseudochrobactrum saccharolyticum strains (NCBI: txid354352). Both taxa are part of the Family Brucellaceae (NCBI: txid118882).
Strains were isolated from the gut microbiome of Caenorhabditis elegans strain N2 (Caenorhabditis Genomic Center) (1). Three microbial communities were extracted from garden compost, frozen at −80°C, re-thawed, and acclimatized on plates (M9 buffer, 1.5% agar) containing 4 cm2 cellulose paper at 20°C. After 2 weeks, community-degraded paper was suspended in buffer, and 100 µL of the slurry was transferred to an identical piece of paper on fresh agar. Approximately 100 nematodes were added to the paper, with nematodes feeding leading to the colonization of the gut by bacteria.
After three 2-week transfers, nematodes were collected from the paper, surface-sterilized, and pulverized for 5 min using a tissue lyser (TissueLyser II, Qiagen) for microbiome extraction. Extracted samples were serially diluted and plated on TSA agar plates and incubated at 30°C for 24–48 h. Single colonies were purified by re-streaking colonies on TSA plates and stored in 70% glycerol saline aliquots at −80°C. Samples were thawed and re-grown (overnight) in shaking 6 mL LB broth at 28°C. Genomic DNA was extracted following the method described in a previous study (2) and purified using the Genomic DNA Clean & Concentrate kit–10 (Zymo Research). DNA was quantified using the Invitrogen Qubit dsDNA Broad Range (BR) assay kit.
Long-read sequencing (Oxford Nanopore Technology) was performed by Eurofins (Germany). Short-read sequencing was performed on a NextSeq500 platform (MPI for Evolutionary Biology) at 150 bp read length, paired-end sequencing, and 60-fold coverage. In-house library preparation used a Tn5 transposase enzyme library (3). Short-read raw data were quality-controlled using MultiQC v1.11 (4). Here, and in the following, default parameters were applied unless otherwise noted.
Raw nanopore sequencing (5) was filtered for low quality and short reads using Filtlong v0.2.1 (6) before being assembled using Flye v2.9.3 (7); parameters were optimized for bacterial genomes (7). Contigs were polished using Medaka v1.8 (8). Assembled genomes were annotated using Bakta v.1.8.2 (9). Quality of the assembled genomes was assessed using QUAST v5.2 (10), CheckM2 v1.0.1 (11), and Mash v2.3 (12). As a final QC step, sequence-cleaned reads were mapped onto the assembly using minimap2 v2.24 (13). Finally, long-read assemblies were polished with our Illumina short reads using nextpolish v1.4.1 (14) and postprocessed with samtools v1.2.1 (15).
Assembled genomes were circularized with DNApler (16) (default parameters) such that the start site coincides with the dnaA gene start codon. Average genome coverage was 84.0× with a GC content of 57.8%.
Genera were identified by 16S metabarcode sequencing (using primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC) to amplify the hypervariable V3-V4 region) and analyzed using phylos v1.52.0 (17). Isolates were inferred from a gyrA neighbor-joining phylogeny (Tamura-Nei distance) using ClustalΩ v1.2.4 (1, 18).
Strains taxonomy was determined by ANI analysis (19, 20), including type strains Ochrobactrum pituitosum CCUG:50899 and Pseudochrobactrum saccharolyticum DSM:25620, using FastANI v0.1.3 (21) (Fig. 1). All samples,raw reads, assemblies, and annotations are deposited on the ENA, see Table 1.
Fig 1.
Clustered heatmap of ANI similarity values for seven announced strains and type strains P. saccharolyticum DSM25260 and O. pituitosum CCUG50899.
TABLE 1.
Summary table
| Sample | Property | Value |
|---|---|---|
| SAMEA118333800 | Organism | O. pituitosum |
| Taxon ID | 71256 | |
| Isolate/strain | MPB25535 | |
| Short read exp | ERX14406277 | |
| Short read run | ERR15002230 | |
| Short read count | 1,727,681 | |
| Short read GC% | 53.3 | |
| Short read mean length (Rl, R2) | 150,150 | |
| Long read exp | ERX14512245 | |
| Long read run | ERR15107547 | |
| Long read count | 87,517 | |
| Long read mean length | 6113 | |
| Long read GC% | 55.0 | |
| Assembly | GCA_965577835 | |
| Total size | 4,963,799 | |
| Contigs | 3 | |
| GC% | 53.4 | |
| N50 | 13,839,640 | |
| ANI (O. pituitosum CCUG50899) | 98.06 | |
| Annotation | ERZ26904280 | |
| Annotated CDSs | 4662 | |
| SAMEA118333801 | Organism | O. pituitosum |
| Taxon ID | 71256 | |
| Isolate/Strain | MPB25451 | |
| Short read exp | ERX14406278 | |
| Short read run | ERR15002231 | |
| Short read count | 1,790,809 | |
| Short read mean length (R1, R2) | 150,150 | |
| Short read GC% | 53.5 | |
| Long read exp | ERX14512247 | |
| Long read run | ERR15107549 | |
| Long read count | 50,100 | |
| Long read mean length | 4595 | |
| Long read GC% | 51.8 | |
| Assembly | GCA_965579405 | |
| Total size | 5,097,185 | |
| Contigs | 3 | |
| GC% | 53.5 | |
| N50 | 6,950,440 | |
| ANI (O. pituitosum CCUG50899) | 98.27 | |
| Annotation | ERZ26904286 | |
| Annotated CDSs | 4825 | |
| SAMEA118333802 | Organism | O. pituitosum |
| Taxon ID | 71256 | |
| Isolate/Strain | MPB25440 | |
| Short read exp | ERX14406279 | |
| Short read run | ERR15002232 | |
| Short read count | 1,595,120 | |
| Short read mean length (Rl, R2) | 150,150 | |
| Short read GC% | 53.5 | |
| Long read exp | ERX14512249 | |
| Long read run | ERR15107551 | |
| Long read count | 44,497 | |
| Long read mean length | 6424 | |
| Long read GC% | 53.5 | |
| Assembly | GCA_965579335 | |
| Total size | 4,550,329 | |
| Contigs | 4 | |
| GC% | 53.5 | |
| N50 | 7959060 | |
| ANI (O. pituitosum CCUG50899) | 98.40 | |
| Annotation | ERZ26904287 | |
| Annotated CDSs | 4285 | |
| SAMEA118333803 | Organism | O. pituitosum |
| Taxon ID | 71256 | |
| Isolate/Strain | MPB25478 | |
| Short read exp | ERX14512100 | |
| Short read run | ERR15107402 | |
| Short read count | 1,630,005 | |
| Short read mean length (R1, R2) | 150,150 | |
| Short read GC% | 53.3 | |
| Long read exp | ERX14512251 | |
| Long read run | ERR15107553 | |
| Long read count | 74,814 | |
| Long read mean length | 3862 | |
| Long read GC% | 53.3 | |
| Assembly | GCA_965579345 | |
| Total size | 4,852,914 | |
| Contigs | 4 | |
| GC% | 53.3 | |
| N50 | 39,450,584 | |
| ANI (O. pituitosum CCUG50899) | 98.52 | |
| Annotation | ERZ26904288 | |
| Annotated CDSs | 4552 | |
| SAMEA118333804 | Organism | P. saccharolyticum |
| Taxon ID | 54352 | |
| Isolate/Strain | MPB25528 | |
| Short read exp | ERX14406336 | |
| Short read run | ERR15002289 | |
| Short read count | 440,692 | |
| Short read mean length (Rl, R2) | 150,150 | |
| Short read GC% | 51.5 | |
| Long read exp | ERX14512252 | |
| Long read run | ERR15107554 | |
| Long read count | 48,318 | |
| Long read mean length | 5292 | |
| Long read GC% | 56.3 | |
| Assembly | GCA_965579315 | |
| Total size | 3,733,418 | |
| Contigs | 3 | |
| GC% | 51.5 | |
| N50 | 12184706 | |
| ANI (P. saccharolyticum DSM25260) | 96.29 | |
| Annotation | ERZ26904290 | |
| Annotated CDSs | 3348 | |
| SAMEA118333805 | Organism | P. saccharolyticum |
| Taxon ID | 54352 | |
| Isolate/Strain | MPB25488 | |
| Short read exp | ERX14406304 | |
| Short read run | ERR15002257 | |
| Short read count | 419,074 | |
| Short read mean length (Rl, R2) | 150,150 | |
| Short read GC% | 51.4 | |
| Long read exp | ERX14512253 | |
| Long read run | ERR15107555 | |
| Long read count | 33,095 | |
| Long read mean length | 6,247 | |
| Long read GC% | 55.1 | |
| Assembly | GCA_965579355 | |
| Total size | 3,753,639 | |
| Contigs | 2 | |
| GC% | 51.5 | |
| N50 | 10,475,352 | |
| ANI (P. saccharolyticum DSM25260) | 96.35 | |
| Annotation | ERZ26904289 | |
| Annotated CDSs | 3365 | |
| SAMEA118333806 | Organism | P. saccharolyticum |
| Taxon ID | 54352 | |
| Isolate/Strain | MPB25441 | |
| Short read exp | ERX14406305 | |
| Short read run | ERR15002258 | |
| Short read count | 478,435 | |
| Short read mean length (R1, R2) | 150,150 | |
| Short read GC% | 51.6 | |
| Long read exp | ERX14512255 | |
| Long read run | ERR15107557 | |
| Long read count | 149,774 | |
| Long read mean length | 5140 | |
| Long read GC% | 54.7 | |
| Assembly | GCA_965579305 | |
| Total size | 3,839,354 | |
| Contigs | 2 | |
| GC% | 51.6 | |
| N50 | 5,324,660 | |
| ANI (P. saccharolyticum CCUG33852) | 96.20 | |
| Annotation | ERZ26904291 | |
| Annotated CDSs | 3473 |
ACKNOWLEDGMENTS
We thank the Max Planck Society for generous core support and acknowledge funding from the Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Center 1182 "Origin and Function of Metaorganisms" (grant no. SFB1182). We are also grateful to Pauline Buffard for short read sequencing data, and Ishan Bhatt for contributions to bioinformatic processing.
Contributor Information
Carsten Fortmann-Grote, Email: grotec@evolbio.mpg.de.
Zhenjiang Zech Xu, Nanchang University, Nanchang, Jiangxi, China.
DATA AVAILABILITY
Samples and data were submitted to the ENA BioProject PRJEB87311, see Table 1 for accessions. Code for gyrA and ANI analysis is deposited in https://archive.softwareheritage.org/swh:1:dir:067262a26b6b51b384fe7955736135216cbbca5d.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Samples and data were submitted to the ENA BioProject PRJEB87311, see Table 1 for accessions. Code for gyrA and ANI analysis is deposited in https://archive.softwareheritage.org/swh:1:dir:067262a26b6b51b384fe7955736135216cbbca5d.