Complete genome sequence of a novel Microbacterium sp. strain Clip185.¶

Mautusi Mitra¹ ORCID and Ana Stanescu² ORCID

¹ University of West Georgia, School of Field Investigations and Experimental Sciences
² University of West Georgia, School of Computing, Analytics, and Modeling, 1601 Maple Street, Carrollton, GA 30118, USA

Abstract¶

We have isolated a new species of Microbacterium, an Actinobacterium. We have temporarily named this bacterium as Microbacterium sp. strain Clip185 (hereafter called strain Clip185) from a contaminated Tris-Acetate-Phosphate (TAP) medium culture plate of a green micro-alga Chlamydomonas reinhardtii strain LMJ.RY0402.185141 (a Chlamydomonas Library project CLiP strain). We sequenced the whole genome of strain Clip185 using the PacBio Sequel II Continuous Long Read technology and have submitted it to NCBI along with the SRA and PacBio methylation motif data. Additionally, we have submitted the PacBio methylome to REBASE, Ref#35996. We present the whole genome sequence of this new Microbacterium species that offers insights into its coding and non-coding genes and its nearest taxonomic neighbors.

Keywords¶

Microbacterium, Actinobacterium, decaprenoxanthin, xenobiotics-degrader, heavy metal-tolerant

Introduction¶

A novel species of Microbacterium strain Clip185, subsequently referred to as Clip185 in this narrative, was isolated from a contaminated Tris Acetate Phosphate (TAP) medium culture plate of Chlamydomonas reinhardtii at the University of West Georgia; Geolocation data: Carrollton, Georgia; 1,102 ft (336 m); 33.5730 N 85.1037 W (1). Strain Clip185 has one circular chromosome with a genome size of 3.3Mb. We report the complete genome sequence of Clip185 and offer insights into its genomic coding potential.

External Data Availability¶

The whole-genome sequence along with the PacBio DNA methylation motifs has been deposited in the GenBank under the accession number GCA_028743715.1.
The PacBio methylome has been submitted to REBASE, Ref#35996.
The raw sequence reads have been deposited in the SRA under the accession number SRR23538270.
Linked publication: (1)

Background and Experimental Methods¶

Sample Collection¶

Microbacterium sp. strain Clip185 was isolated from a contaminated Tris Acetate Phosphate (TAP) medium culture plate of the green micro-alga Chlamydomonas reinhardtii strain LMJ.RY0402.18514, a Chlamydomonas Library project (CLiP) strain.

Isolation¶

Genomic DNA was isolated from the Lysogeny Broth (LB) medium-grown Microbacterium sp. strain Clip185 (colony #37) using the Qiagen Blood and Cell Culture DNA Mini Kit.

Genome Sequencing¶

After determination of genomic DNA purity and DNA quantification, the DNA sample was shipped to Georgia Genomics and Bioinformatics Core (GGBC) at the University of Georgia (Athens, GA). At GGBC, the sample was processed for preparation of the PacBio Single Molecule Real Time (SMRT) bell sequencing library according to the protocol given in the PacBio technical manual for template preparation and sequencing (please see QC section for more details). The SMRT bell sequencing library was barcoded and sequenced with two additional barcoded microbial SMRT Bell sequencing libraries in a single SMRT cell using PacBio SMRT Continuous Long Read sequencing on the PacBio Sequel II instrument. SMRT Link v9 was used as an interface to manage the workflow from sample setup to result analysis.

QC and Assembly¶

QC¶

Quantitative and qualitative QC assessment was performed on the DNA sample at GGBC using Qubit, Nanodrop, and Fragment Analyzer.
DNA was sheared using a Covaris®g-TUBE®. After shearing, the approximate size range of the fragments was determined with a Bioanalyzer® 12000 chip and the quantification of DNA was performed on a Nanodrop system.
Purification and concentration of 12kb fragment sizes was performed using use 0.45X AMPure PB beads.
DNA damages in the sheared DNA were repaired with DNA Damage Repair reagents provided by Pacific Biosciences and the PacBio Template Prep Kit was used to repair the ends of fragmented DNA. Following end repairs, DNA was purified with 0.45X AMPure PB beads.
BLUNT hairpin adapters were ligated to the DNA fragments followed by exonuclease (ExoII and ExoVII) treatments to remove failed ligation products followed by size selection and purification using three distinct and consecutive 0.45X AMPure PB bead purification steps at room temperature to adequately remove enzymes (exonucleases, ligases, etc.) and ligation products smaller than 0.4kb (e.g., adapter dimers).
SMRTbell™Library Quality assessment was performed using a Bioanalyzer® 12000 chip for sizing and was quantified via fluorescence using a Qubit® High Sensitivity kit.
Sequencing primer v4 was bound to the SMRTbell template. DNA sequencing polymerases were bound to the primer-annealed SMRTbell templates using the Sequel® II Binding Kit 2.0. AMPure® PB Purification of Polymerase Bound SMRTbell® Complexes was performed.
A dilution of the DNA Internal Control Complex (these controls are SMRTbell templates already bound with the polymerase, available from Pacific Biosciences) that had 30X DNA Internal Control Complex was added to the SMRT Bell template for independent determination of any problems that might have occurred during binding and the sequencing run.
Prior to sequencing, the SMRT Bell template-polymerase complex was loaded using MagBead loading to a 96-well sample plate with concentrations and volumes specified by the Pacific Biosciences Binding Calculator.

Genome Assembly¶

Reads were first assembled using the SMRT Link v9 software, which has inbuilt HGAP v4.0. The pipeline was run at default with a pre-specified approximately estimated genome size of 3.3Mb (based on available complete genome sizes of various Microbacterium sp. on NCBI). After assembly, the assembly metrics for each sample along with HMM predicted genes were determined by running Quast v5.02. Genome was also assembled with FLYE v2.9.1 and CANU v2.2 for statistical confidence.

Genome Statistics¶

The Clip185 genome consists of one circularized chromosome comprising a total of 3,305,635bp with an overall G+C content of 69.5%. Genome coverage was calculated using the formula: Number of Subread Bases (mapped)/Genome Size = 10,942,194,255/3,305,635 = 3,310X. Genome coverage (based on hgap.depth_coverage_mean in the PacBio coverage report): 3193.6X.

GenBank	Topology	Size (bp)	GC Content (%)
CP117996.1	Circular	3,305,635	69.5

Import and Annotation¶

The Clip185 genome was imported into KBase using the Import from Staging Area application. More specifically, chromosome CP117996.1 was imported using the Type Genome and the NCBI Tax ID 51671: Microbacterium sp.
The genome was annotated in KBase using the microbial Annotate Genome/Assembly with RASTtk - v1.073 application with default parameters.
The circular genome was visualized using the KBase Circular Genome Visualization Tool with default parameters, except for Linear, which remained unchecked.
The quality of the genome was assessed by the KBase Assess Genome Quality with the CheckM - v1.0.18 application.

Taxonomic Classification¶

Taxonomic identification was performed using the KBase Classify Microbes with GTDB-Tk - v2.3.2 application on a GenomeSet generated with the Build GenomeSet-v1.7.6 application.
A phylogenetic tree was constructed using the KBase Insert Genome Into Species Tree - v2.2.0 application with parameters: Neighbor Public Genome Count = 200.
Another phylogenetic tree based on the 16S rRNA gene was constructed using the KBase Build Phylogenetic Tree from MSA using FastTree2-v2.1.11 application with parameters: top 50 sequences obtained from NCBI using BLASTN and aligned using MUSCLE in MEGA 11.

References¶

Mitra M, Nguyen KMAK, Box TW, et al. Isolation and characterization of a heavy metal- and antibiotic-tolerant novel bacterial strain from a contaminated culture plate of Chlamydomonas reinhardtii, a green micro-alga. F1000Research 2021, 10:533. https://f1000research.com/articles/10-533

from biokbase.narrative.jobs.appmanager import AppManager
AppManager().run_app_batch(
    [{
        "app_id": "kb_uploadmethods/import_fasta_as_assembly_from_staging",
        "tag": "release",
        "version": "5b9346463df88a422ff5d4f4cba421679f63c73f",
        "params": [{
            "staging_file_subdir_path": "GCF_028743715.1_ASM2874371v1_genomic.fna",
            "assembly_name": "GCF_028743715.1_ASM2874371v1_genomic.fna_assembly"
        }],
        "shared_params": {
            "type": "draft isolate",
            "min_contig_length": 500
        }
    }],
    cell_id="ba02057d-8da4-4709-805b-35ed1caf1972",
    run_id="64b02c3c-dcca-489b-a771-bee8ed06af48"
)

Created Object Name	Type	Description
GCF_000202635.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_000633215.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_001262495.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_001314225.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_001427145.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_001427525.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_001428485.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_001552475.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_001592125.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_001652465.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_001887285.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_900104345.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_000746195.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
Clip185_Annotation_RASTtk-v1.073	Genome	Taxonomy and taxon_assignment updated with GTDB
GCF_000799385.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_000802305.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_000956415.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_000956465.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_000956475.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_000956535.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
GCF_000956575.1	Genome	Taxonomy unchanged, taxon_assignment added GTDB
Clip185_Output_Genome_Set	GenomeSet	Taxonomy and taxon_assignment updated with GTDB