Whole genome sequencing of Microbacterium sp. AISO3 from polluted San Jacinto River sediment reveals high bacterial mobility, metabolic versatility and heavy metal resistance.

The genus Microbacterium is composed of high GC content, Gram-positive bacteria of the phylum Acintobacteria known for their antibiotic production. Microbacterium species commonly colonize agricultural rhizospheres and more infrequently have been found to colonize and infect human tissues as well. Here we report the 3,696,310 bp draft genome (chromosome and plasmids) sequence assembled at the scaffold level from 232 contigs of Microbacterium sp. strain AISO3, isolated from polluted San Jacinto River sediment in Channelview, Texas. The nucleotide sequence of this genome was deposited into NCBI GenBank under the accession NHRF00000000.

Direct link to deposited data

https://www.ncbi.nlm.nih.gov/nuccore/NHRF00000000.

Experimental design, materials and methods

Polluted sediment containing a heterogenous mixture of polychlorinated dioxins, furans, biphenyls, petroleum hydrocarbons, and agricultural waste was collected from the Battleship Texas Historic Site in Channelview, Texas along the banks of the San Jacinto River [1]. Selective media was prepared to screen for microorganisms within this sample with the capacity to tolerate, degrade and/or metabolize organophosphate compounds. For this purpose utilized Carbon Selective Media (CSM) which has a composition of 2 mM NTA, 0.8 mM MgSO4·7H2O, 0.17 mM CaNO3, 0.018 mM FeSO4·7H2O, 20% v/v Phosphate Buffer. A total of 5 mL of CSM media was aliquoted into culture tubes with 100 μg/mL ethyl paraoxon as a screening agent. These tubes were prepared fresh each week for each new subculture set for a period of five weeks. The culture was then diluted into minimal media with glycerol added as a supplementary carbon source and plated onto an agar plate with 100 μg/mL ethyl paraoxon. A single orange colored bacterium was isolated from the agar plate and shipped to Genewiz (South Plainfield, NJ), where library construction and whole genome sequencing of the bacterium was performed as described below.

Samples were visually inspected upon receipt and genomic DNA was extracted from bacterial colonies using the PureLink Genomic DNA extraction kit as per manufacturer's protocols. The resulting genomic DNA was quantified using both the Nanodrop and the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). A total of 50–60 ng of each sample was run on a 0.6% agarose gel to check for quality. The Illumina Nextera XT library preparation, clustering, and sequencing reagents were used throughout the process following the manufacturer's recommendations (Illumina, San Diego, CA, USA). DNA libraries were analyzed on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA) and quantified using the Qubit 2.0 Fluorometer.

The DNA libraries were quantified by real time PCR (Applied Biosystems, Carlsbad, CA, USA), and multiplexed in equal molar mass. DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to manufacturer's instructions (Illumina, San Diego, CA, USA). Sequencing was performed using a 2 × 250 paired-end (PE) configuration (Table 1). Sequence reads were checked for quality using Fastqc [2] and filtered using BBTools [3] with minimum Phred score of 20. Paired-end reads were assembled into contigs with the Spades 3.10.1 program [4]. The Mash program [5] was used for species identification using k = 21 and sketch size of 1000 against the Mash Refseq (release 70) database. Fasta files for the five top bacterial hits sorted by distance were downloaded from RefSeq database and used to calculate the Mash distance. The resulting distance file from the previous step was imported into R using the reader package [6]. Finally, the Ggdendrogram [7] package was used to create a dendrogram plot through hclust function output using UPGMA method (Fig. 1). The Quast program [8] was used to calculate assembly statistics using scaffold mode. Preliminary reference based annotation using PATRIC [9] web resources was carried out to identify conserved pathways. Final de novo annotation was performed through the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html) and the Rapid Annotation System Technology (RAST) server [10], [11].

Table 1

Microbacterium sp. AISO3 genome statistics.

Assembly statistics
platform	Illumina MiSeq (2*250) paired end
total raw reads	3,125,578
total filtered reads	2,847,047
genome size (bp)	3,696,310
Contigs (≥ 200 bp)	230 (chromosome) + 2 (plasmids)
Contigs ≥ 500 bp)	55
Largest contig	598,210
N50	294,906
L50	4
Average coverage	282.44
Annotation statistics
GC content (%)	69.82 (chromosome) + 68.33(plasmid)
total genes	3623
coding genes	3563
rRNAs	8
tRNAs	48

Fig. 1

Dendogram of Microbacterium sp. AISO3 and the five closest neighboring genomes.

Data description

Microbacterium species are versatile, ubiquitous microorganisms capable of thriving in both endaphic and aquatic environments. More commonly known for their role in plant promotion, Microbacterium has also recently emerged as a rare opportunistic human pathogen [12], [13], [14], [15], [16]. The draft genome of Microbacterium sp. AISO3 includes 232 contigs with a GC content of 69.82%, consists of 3,478,976 bp and 2 plasmids totaling 217,334 bp. Combined, the draft genome and plasmid of AISO3 contains approximately 3623 gene sequences, 3563 coding sequences including 62 pseudogenes, 8 rRNA genes, 48 tRNAs, and 4 noncoding RNA (ncRNA) genes. The closest neighboring genome to strain AISO3 is Microbacterium sp. TS-1 (See Fig. 1). An overview of genome subsystem features (Fig. 2) shows a microorganism that has not only retained high metal resistance similar to other Acintobacteria and plant promoting rhizosphere bacteria, but also the capacity for opportunistic pathogenicity through a combination of virulence and antibiotic resistance factors including multidrug efflux, β-lactamase activity, Type VII secretion systems, and a Mycobacterium-like virulence operon. In addition, Microbacterium sp. AISO3 notably encodes for a greater number of motility, chemotaxis, and carbohydrate metabolism genes than other representative Microbacterium strains (Table 2) [17], [18].

Fig. 2

Subsystem category distribution of major protein coding genes of Microbacterium sp. strain AISO3 as annotated by the RAST annotation server. The bar chart shows the subsystem coverage shows the distribution of the 27 most abundant subsystem categories.

Table 2

Number of genes identified in representative Microbacterium species. Categories in which the greatest number of genes was identified in strain AISO3 are indicated in bolded text.

Category	Strain AISO3	Strain BH-3-3-3	Strain TS-1
Cell wall/capsule	72	70	52
Virulence	51	35	47
Amino acids	321	313	232
Cofactors/vitamins	175	188	143
Nucleosides/nucleotides	95	99	59
Carbohydrates	392	355	325
Fatty acids/lipids	135	136	115
Sulfur	22	8	18
Phosphorus	36	31	35
Protein metabolism	190	225	143
DNA metabolism	88	69	56
RNA metabolism	86	78	55
Aromatic metabolism	17	51	16
Membrane transport	132	100	121
Cell signaling	22	31	16
Cell mobility/chemotaxis	49	8	42
Stress response	74	69	72
Respiration	40	48	40
Cell division/cell cycle	23	22	20
Miscellaneous	30	32	27

Specifications
Organism/cell line/tissue	Microbacterium sp.
Strain	AISO3
Sequencer or array type	Illumina Miseq
Data format	Draft genome (Scaffolds)
Experimental factors	Bacterial strain
Experimental features	Whole genome analysis and gene annotation of AISO3
Sample source location	San Jacinto River sediment near the Battleship Texas Historic Site in Channelview, Texas
GPS coordinates	29° 45′ 20.916″ N, 95° 5′ 25.2564″ W