Draft Genome Sequences of New Isolates and the Known Species of the Family Microbacteriaceae Associated with Plants.

Draft genome sequences of 11 bacteria belonging to the family Microbacteriaceae were obtained using Illumina technology. The genomes of these strains have sizes from 3.14 to 4.30 Mb with their genomic DNA characterized as having high G+C contents (above 65%). These genomic data will be useful for natural taxonomy and comparative genomic studies of bacterial strains of the family Microbacteriaceae.

ANNOUNCEMENT

The family Microbacteriaceae Park et al. 1995 emend. Stackebrandt et al. 1997 emend. Zhi et al. 2009 comprises a large group of predominantly aerobic Gram-positive bacteria with high G+C contents in their genomic DNA that are distinguished from other actinobacteria by a combination of their unusual group B cell wall peptidoglycan and unsaturated respiratory menaquinones (1, 2). Currently, the family comprises 51 recognized genera and about 275 recognized species (http://www.bacterio.net/microbacteriaceae.html). There are about 80 sequenced genomes for type strains of the family Microbacteriaceae (https://www.ezbiocloud.net/taxon?tn=Microbacteriaceae). Efforts to obtain complete genome sequences of all type strains belonging to the family Microbacteriaceae are critical to the future microbial systematics of this group and for constructing the unified tree of life.

The type strains of six known species of the genera Agreia, Clavibacter, Okibacterium, Plantibacter, and Rathayibacter were isolated from various sources associated with plants (Table 1) (3–7). In addition, the novel strains Agreia sp. VKM Ac-1783, Agreia sp. VKM Ac-2052, Plantibacter sp. VKM Ac-1784, Plantibacter sp. VKM Ac-1787, and Rathayibacter oskolensis VKM Ac-2121T were isolated from plant galls induced by nematodes and from the healthy plant without any symptoms of disease (Table 1) (8–10). All strains were deposited in the All-Russian Collection of Microorganisms (http://www.vkm.ru/Catalogue.htm).

TABLE 1

Genome sequence statistical information and DDBJ/ENA/GenBank accession numbers

Organism	Plant source	Nematode	Total no. of reads post-trimming	Total size post-trimming(Mb)	No. of contigs (scaffolds)	Coverage (×)	N50 (bp)	Genome size (Mb)	G+C content (%)	No. of proteins	SRA no.	GenBank accession no.
A. pratensis VKM Ac-2510T	Phyllosphere of grasses	No	5,153,882	773.1	10	214	860,306	3.58	65.4	3,418	SRR5832269	FXAY00000000
Agreia sp. VKM Ac-1783	Elymus repens	Anguina agropyri	5,567,834	835.2	7	220	1,002,356	3.76	65.8	3,551	SRR5832516	FXWI00000000
Agreia sp. VKM Ac-2052	Calamagrostis neglecta	Heteroanguina graminophila	7,310,900	1,096.6	19	280	442,588	3.88	65.2	3,654	SRR5832511	FUYG00000000
C. michiganensis subsp. michiganensis VKM Ac-1403T	Lycopersicon esculentum	No	6,669,630	993.8	14 (13)	300	2,018,315	3.30	72.7	3,045	SRR5216688	FVZG00000000
O. fritillariae VKM Ac-2059T	Fritillaria ruthenica	No	5,278,668	791.8	4	250	2,068,692	3.14	67.8	2,808	SRR5832512	FUZP00000000
P. flavus VKM Ac-2504T	Phyllosphere of grasses	No	4,884,328	732.6	12 (11)	169	544,526	4.30	69.1	4,002	SRR5832515	FXAP00000000
Plantibacter sp. VKM Ac-1784	E. repens	A. agropyri	5,096,142	764.4	8	184	674,883	4.12	69.2	3,829	SRR5832517	FXWJ00000000
Plantibacter sp. VKM Ac-1787	Cousinia onopordioides	Mesoanguina picridis	5,710,806	850.9	5	211	2,657,854	4.02	69.5	3,719	SRR5832510	FUZO00000000
R. rathayi VKM Ac-1601T	Dactylis glomerata	Anguina sp.	5,193,410	779.0	46 (45)	244	321,699	3.22	69.3	3,126	SRR5832513	OCNL00000000
R. iranicus VKM Ac-1602T	Triticum aestivum	A. tritici	5,315,024	797.3	50 (49)	236	188,097	3.38	67.2	3,268	SRR5832514	QGDV00000000
R. oskolensis VKM Ac-2121T	Androsace koso-poljanskii	No	5,903,488	879.6	5	222	1,280,863	3.95	71.6	3,628	SRR5832669	FXBM00000000

Biomass for DNA extraction was obtained after growth in liquid peptone-yeast medium (11) at 28°C for 18 to 20 h on a rotary shaker. The genomic DNA was extracted using guanidinium thiocyanate and Triton X-100, followed by purification with Cleanup Standard BC022 (Evrogen, Russia). The draft genome sequences of the strains were generated at the DOE Joint Genome Institute (JGI) (Walnut Creek, CA, USA). The Illumina shotgun libraries with an insert size of 262 to 287 bp were constructed and sequenced using the Illumina HiSeq 2000 platform to produce 2 × 150-bp paired-end reads. All raw Illumina sequence data were filtered using BBDuk (http://sourceforge.net/projects/bbmap), which removes known Illumina artifacts and PhiX sequences. Reads with more than one N, quality scores (before trimming) averaging less than 8, or reads shorter than 51 bp (after trimming) were discarded. The remaining reads were mapped to masked versions of human, cat, and dog references using BBMAP (http://sourceforge.net/projects/bbmap) and discarded if their identities exceeded 95%. Sequence masking was performed with BBMask (http://sourceforge.net/projects/bbmap). The following steps were then performed for assembly: (i) artifact-filtered Illumina reads were assembled using SPAdes version 3.6.2 (with the parameters “--cov-cutoff auto --phred-offset 33 -t 8 -m 40 --careful -k 25,55,95 --12”) (12); and (2) assembly contigs with lengths of <1 kb were discarded. The genome was annotated using the JGI Microbial Genome Annotation Pipeline (13). Statistical information for the draft genome sequences is given in Table 1.

Further genome-wide comparative analyses of these strains and phylogenetically closely related bacteria belonging to the family Microbacteriaceae will enhance an understanding of the borderlines of prokaryotic species and facilitate insight into the molecular mechanisms involved in interactions between plants and bacteria.

Data availability.

The whole-genome shotgun projects reported here have been deposited in DDBJ/ENA/GenBank under the accession numbers listed in Table 1. The versions reported here are the first versions.